UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS BIOLÓGICAS EFECTOS DE LA DEGRADACIÓN DEL HABITAT Y EL RIESGO DE DEPREDACIÓN EN EL COMPORTAMIENTO Y EL ESTADO DE SALUD DE LAS LAGARTIJAS MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Luisa Amo de Paz Bajo la dirección de los doctores José Martín Rueda Pilar López Martínez Madrid © Luisa Amo de Paz, 2005 Efectos de la degradación del hábitat y el riesgo de depredación en el comportamiento y el estado de salud en lagartijas Luisa Amo de Paz Tesis doctoral 2005 Departamento de Fisiología Facultad de Ciencias Biológicas. Universidad Complutense de Madrid Museo Nacional de Ciencias Naturales Consejo Superior de Investigaciones Científicas Departamento de Fisiología Facultad de Ciencias Biológicas. Universidad Complutense de Madrid Efectos de la degradación del hábitat y el riesgo de depredación en el comportamiento y el estado de salud en lagartijas Memoria presentada por la Licenciada Dña. Luisa Amo de Paz para optar al grado de Doctor en Ciencias Biológicas, dirigida por los Investigadores Dr. José Martín Rueda y Dra. Pilar López Martínez, del Museo Nacional de Ciencias Naturales-CSIC Madrid, Junio de 2005 El Doctorando Luisa Amo de Paz V.º B.º de los Directores José Martín Pilar López V.º B.º del Tutor Rafael Hernández Tristán 1 Efectos de la degradación del hábitat y el riesgo de depredación en el comportamiento y el estado de salud en lagartijas Contenidos Agradecimientos 5 Capítulo 1: Introducción general Antecedentes y estado actual del tema 9 Objetivos 22 Métodos generales 23 Resultados y discusión general 35 Capítulo 2 Relaciones parásitos-lagartijas: consecuencias para el mantenimiento de las poblaciones de lacértidos: 75 • Prevalence and intensity of haemogregarinid blood parasites in a population of the Iberian rock lizard, Lacerta monticola 77 • Prevalence and intensity of blood and intestinal parasites in a field population of a Mediterranean lizard, Lacerta lepida 89 • Prevalence and intensity of haemogregarine blood parasites and their mite vectors in the common wall lizard, Podarcis muralis 105 Capítulo 3 Efectos de la modificación de la vegetación natural sobre las poblaciones de lacértidos: 119 • Natural oak forest vs. ancient pine plantations: effects of traditional forest management on distribution and conservation of Iberian lizards 121 • Increased predation risk due to habitat deterioration affects body condition of lizards: a behavioural approach with Lacerta monticola lizards inhabiting ski resorts 139 • Habitat deterioration affects body condition and parasite load of female lizards Psammodromus algirus through forced changes in antipredatory behavior 167 2 Capítulo 4 Efectos del ecoturismo en las estrategias de escape, el uso de refugios y la condición y estado de salud de lacértidos: 193 • Ecotourism as a form of predation risk affects body condition and health state of Podarcis muralis lizards 195 • Flexibility in refuge use helps Lacerta monticola lizards to cope with different levels of predation risk without incurring loss of body condition 217 Capítulo 5 Efectos del ecoturismo en el incremento en el riesgo de depredación debido a múltiples depredadores: 251 • Risk level and thermal costs affect the choice of escape strategy and refuge use in the wall lizard, Podarcis muralis 253 • Wall lizards combine chemical and visual cues of ambush snake predators to avoid overestimating risk inside refuges 269 • Chemical assessment of predation risk in the wall lizard, Podarcis muralis, is influenced by time exposed to chemical cues of ambush snakes 289 Capítulo 6 Conclusiones y perspectivas 305 3 Esta tesis está basada en los siguientes artículos: • Amo L, López P and Martín J. 2004. Prevalence and intensity of haemogregarinid blood parasites in a population of the Iberian rock lizard, Lacerta monticola. Parasitology Research 94: 290–293. • Amo L, Fargallo JA, Martínez-Padilla J, Millán J, López P and Martín J. 2005. Prevalence and intensity of blood and intestinal parasites in a field population of a Mediterranean lizard, Lacerta lepida. Parasitology Research. En prensa. • Amo L, López P and Martín J. Prevalence and intensity of haemogregarine blood parasites and their mite vectors in the common wall lizard, Podarcis muralis. Parasitology Research. En prensa. • Amo L, López P and Martín J. Natural oak forest vs. ancient pine plantations: effects of traditional forest management on distribution and conservation of Iberian lizards. Biodiversity and Conservation. En revision. • Amo L, López P and Martín J. Increased predation risk due to habitat deterioration affects body condition of lizards: a behavioural approach with Lacerta monticola lizards inhabiting ski resorts. Journal of Applied Ecology. En revision. • Amo L, López P and Martín J. Habitat deterioration affects body condition and parasite load of female lizards Psammodromus algirus through forced changes in antipredatory behaviour. Animal Behaviour. En revision. • Amo L, López P and Martín J. Ecotourism as a form of predation risk affects body condition and health state of Podarcis muralis lizards. Biological Conservation. En revisión. • Amo L, López P and Martín J. Flexibility in refuge use helps Lacerta monticola lizards to cope with different levels of predation risk without incurring loss of body condition. Oecologia. En revisión. • Amo L, López P and Martín J. 2003. Risk level and thermal costs affect the choice of escape strategy and refuge use in the wall lizard, Podarcis muralis. Copeia 2003: 899–905. • Amo L, López P and Martín J. 2004. Wall lizards combine chemical and visual cues of ambush snake predators to avoid overestimating risk inside refuges. Animal Behaviour 67: 647-653. • Amo L, López P and Martín J. 2004. Chemical assessment of predation risk in the wall lizard, Podarcis muralis, is influenced by time exposed to chemical cues of ambush snakes. Herpetological Journal 15: 21-25. 5 Agradecimientos Son muchas las personas que han hecho posible esta tesis, ayudándome durante todo el proceso, a las que estoy muy agradecida. En primer lugar, quería agradecer a Pilar López y José Martín, porque sin ellos esta tesis no habría sido ni mucho menos lo que es, y también por haberme dado la oportunidad de hacer la tesis, por enseñarme qué es esto del mundo científico, a aprovechar bien las primaveras y los veranos y sobre todo por estar siempre dispuestos a resolver cualquier cosa en todo momento. Muchas gracias también por todos los ‘san juanes’ de estos años. Siguiendo con herpetólogos, quería agradecer a Kevin Pilz el haberme enseñado a sacar sangre, aunque las lagartijas no creo que le estén muy agradecidas, ha creado un monstruo… A Pedro Moreira, por enseñarme todo acerca del cuidado y mantenimiento de hembras gestantes y la incubación de los huevos, por las conversaciones sobre cópulas y por esas cervecitas en el Felipe. A Carlos y a Adega, por ayudarme a coger bichos, por las fotos y los cafés del Ventorrillo. A Pedro Aragón, por las charlas relajadas en la praderita. Aquí también entran Juanto y Jesús, que aunque teóricamente no son herpetólogos, sino alimañologos, lo fueron, y espero que lo sigan siendo, estudiando los pobres lagartos de Campo Azálvaro. A Lis, por esos cafecitos en el Ventorrillo y las risas en los momentos de agobio con las becas. A Nino, por toda su ayuda a lo largo de la tesis, por tapar y destapar a las lagartijas millones de veces, por cuidar a la Curra, por los ratos relajados en el Felipe, por cogerme culebras! y sobre todo por su buen humor y paciencia durante estos años. A mis compañeros de beca, David y Elena, por las largas charlas sobre esto de la biología. Al resto de compañeros del museo: Judith, Elisa, Josué, Oscar, Isa, Noemí, Luis, Leticia, Aurelio, Ana, Cristina, Mª José, Natalia, Raquel, Juan, Tere, … gracias a todos por las comidas y sobremesas que sin ellas habrían sido muy aburridos los días de museo. También quería agradecer la ayuda con los frotis, primero a Santi Merino, por enseñarme a contar parásitos cuando era ayudante, y luego por resolver, junto con Gustavo, Elena, Judith y Elisa, cualquier duda de cosas ‘raras’ que aparecían. A Mª Luisa y Carolina por echarme una mano en el campo, a Ana y Vanesa por liberarme de 6 frotis y a Arancha por esas cintas de tongue-flicks que escuchó. A toda la gente que ha pasado por el ventorrillo que han hecho la beca mucho más agradable: Karin, Chris, Dror, Susanne, Alberto, Emilio, Iván, Pati, Montse, Kinga, Janien, Pablo, Nuria, Irene… Y en la sección de bichos: A la Curra, por acompañarme al campo y por sus gruñidos de aviso por los cotillas. A las vacas, por hacerme sentir el riesgo de depredación y ayudarme a entender los conflictos entre las estrategias antidepredatorias y otros requerimientos, como hacer la tesis… A todos los bichos que han participado en este estudio, sobre todo a las lagartijas a las que dado el tema de esta tesis no he parado de molestar todas estas temporadas de campo… Esta tesis ha sido financiada por los proyectos del Ministerio de Ciencia y Tecnología BOS 2002-00598 de Pilar López y BOS 2002-00547 de José Martín, y yo he disfrutado de una beca predoctoral de El Ventorrillo-CSIC. Y dejo para el final la parte más personal, pero no menos importante, porque el apoyo de estas personas ha sido fundamental para empezar, seguir y acabar esta tesis: En primer lugar quería agradecer a mis padres, Guillermo y Luisa, su apoyo, en todos los sentidos, durante esta tesis, desde animarme a hacer lo que quería al principio cuando no tenía beca, hasta hacerse unos expertos en bichos rastreros. Muchas gracias! A mi hermano, Guillermo, por ayudarme a coger lagartijas, por la supercámara, las fotos y por enseñarme tanto de líquenes, que no todo en el mundo son bichos. A mis amigos, a Paz, por toda su ayuda con el inglés a horas intempestivas, a Eva la conseguí llevar a coger lagartijas (gracias por esos Psammodromus!), al resto no lo conseguí, pero han estado allí siempre, muchas gracias por vuestro apoyo Ana, Yolanda, Luisi, María, Vicky, Cristina, Alberto, Carlos e Iván Y sobre todo, a Gustavo, porque todo cabe, porque sus viene tarde y se va pronto me han hecho currar mucho más de lo que tenía intención, por las lagartijas, los frotis bien hechos, por la ética en estado puro, por aguantar y entender los agobios de las épocas de campo, por las puas y los tips de los veranos, las migas y el vino de teja, por Europa y América del Sur, y por su apoyo siempre, y sobre todo por su risa, su buen humor, y su forma tan buena de ver la vida, ohmmm. Gracias maño! 7 Capítulo 1 Capítulo 1 Introducción general 9 INTRODUCCIÓN GENERAL Efectos de la degradación del hábitat y el riesgo de depredación en el comportamiento y el estado de salud en lagartijas 1.- ANTECEDENTES Y ESTADO ACTUAL DEL TEMA El riesgo de depredación está considerado como una de las mayores fuerzas selectivas en la evolución de numerosas características morfológicas y comportamentales de los animales (Lima y Dill 1990; Lima 1998). Para hacer frente a un incremento en el riesgo de depredación, los animales muestran cambios comportamentales que se conocen como estrategias antidepredatorias. Estas estrategias se pueden clasificar en dos tipos: preventivas y de escape. Las estrategias preventivas se llevan a cabo cuando el animal percibe un incremento en su susceptibilidad a ser capturado en el caso de que apareciera un depredador. Por ejemplo, al desplazarse por una zona abierta, con escasez de refugios, los animales percibirían un incremento del riesgo a ser capturados y modificarían su comportamiento para minimizar ese riesgo. Por el contrario, las estrategias de escape se realizan sólo cuando el depredador está presente realmente y ha lanzado, o es probable que lanze, un ataque. Sin embargo, ya que los animales tienen que cumplir otros requerimientos de gran importancia para su eficacia biológica además de evitar a los depredadores, como la alimentación o la reproducción (Lima y Dill 1990; Lima 1998), deben compaginar su comportamiento antidepredatorio con otros requerimientos. Una de las primeras estrategias preventivas de las presas para hacer frente al riesgo de depredación es la selección de hábitats seguros. De esta forma, los animales pueden optimizar sus requerimientos mientras minimizan el riesgo de depredación (e.g. Lima 1998; Amat y Masero 2004). Muchas especies valoran el riesgo mediante la distancia a un refugio que ofrezca Capítulo 1 Introducción general 10 protección frente a los depredadores (Lima 1993; Arenz y Leger 2000) ya que esta distancia puede determinar sus probabilidades de escapar con éxito frente a un eventual ataque. De esta forma, diferentes localizaciones espaciales de las presas, con sus consiguientes distancias a refugios, implican niveles de riesgo diferentes que los individuos son capaces de valorar en cada momento (Thorson et al. 1998; Lima y Bednekoff 1999). Por tanto, los animales seleccionan lugares que les ofrezcan protección frente a los depredadores a través, por ejemplo, de una disminución de su conspicuidad o bien manteniéndose cerca de refugios en los que esconderse ante el eventual ataque de un depredador (Arthur et al. 2004). La selección de hábitat es un proceso mediante el cual las especies ocupan diferencialmente su entorno atendiendo no sólo a la defensa frente a la depredación sino también a limitaciones morfofuncionales, fisiológicas, competitivas con otras especies, y considerando requerimientos tróficos y reproductivos (véase Pleguezuelos 1997 para una presentación general de este aspecto en reptiles ibéricos). Además de la selección de hábitats seguros, los animales presentan otra serie de estrategias antidepredatorias preventivas como la modificación de la actividad y los patrones de locomoción (McAdam y Kramer 1998). Una respuesta generalizada de las presas ante un incremento en el riesgo de depredación es la disminución de la actividad para evitar los encuentros con los depredadores o para evitar el ataque de depredadores que puedan localizar a las presas por su movimiento. Este comportamiento se ha observado en un amplio número de taxones. Por ejemplo, las pulgas de agua, Daphnia evitan el encuentro con depredadores como Chaoborus disminuyendo la velocidad de natación (Weber y Van Noordwijk 2002). Esta disminución de actividad se ha descrito también en peces (Vehanen 2003), anfibios (Laurila et al. 2004), reptiles (Hecnar y M'Closkey 1998; Downes 2001), aves (Mougeot y Bretagnolle 2000) y mamíferos (Jensen et al. 2003; Orrock y Danielson 2004). Sin embargo, cuando el movimiento es necesario debido a requerimientos alimentarios o reproductivos, los animales deben modificar sus patrones de locomoción para disminuir su Capítulo 1 Introducción general 11 vulnerabilidad a ser capturados mientras se desplazan en situaciones de alto riesgo de depredación (McAdam y Kramer 1998). En este caso, la habilidad de desarrollar una velocidad de carrera óptima parece determinante, no sólo ante el ataque de un depredador, sino también como estrategia preventiva para disminuir el tiempo expuestos a los depredadores cuando tienen que recorrer una distancia determinada (López y Martín 2002; Miles 2004). Sin embargo, y aunque las actividades locomotoras juegan un papel importante en la ecología de los animales (Turchin 1998), los estudios de los patrones de locomoción se han centrado fundamentalmente en las capacidades funcionales, dejando de lado a menudo el contexto ecológico en el que la locomoción es usada (Irschick 2000; pero ver también Martín y López 1995; Irschick y Losos 1998; Jayne y Ellis 1998; Van Damme et al. 1998; Miles 2004). La selección de hábitat seguros y la modificación de los patrones de locomoción son estrategias para minimizar el riesgo, pero cuando un depredador detecta a la presa, ésta debe realizar una adecuada respuesta de escape para evitar ser capturada. Aunque el riesgo de depredación se ha considerado generalmente en el contexto de probabilidad de mortalidad en el futuro inmediato, ya que ser capturado por un depredador supone una disminución drástica de la eficacia biológica (la muerte), las estrategias antidepredatorias implican una serie de costes, por lo que las decisiones antidepredatorias deben estar basadas en las consecuencias para la eficacia biológica a largo plazo (Clark 1994; Martín y López 2000a). De esta forma, los animales deberían valorar exactamente el riesgo de depredación y los costes asociados a esa respuesta. Tanto modelos teóricos como evidencias experimentales sugieren que las presas no exhiben una respuesta de escape hasta que el depredador se acerca tanto que los costes de depredación son mayores a los costes de la respuesta (Ydenberg y Dill 1986). De modo que, en cada situación, existiría una distancia óptima de huida entre el depredador y la presa. Si se incrementan los costes de huida, la distancia disminuye (la presa debería huir más tarde). Mientras que si se incrementa el riesgo de ser capturado la distancia aumenta (la presa debería Capítulo 1 Introducción general 12 huir antes). Varios factores pueden afectar tanto al riesgo de depredación percibido como a los costes de la respuesta, y modificar, por tanto, la distancia de huida. Por ejemplo, una gran distancia al refugio más cercano en el que esconderse aumentará el riesgo y por tanto, aumentará la distancia de aproximación (Dill y Houtman 1989; Dill 1990; Bulova 1994; Bonenfant y Kramer 1996; Cooper 1997; Martín y López 2000a). Otro factor que puede incrementar el riesgo para un animal ectotermo como una lagartija es la temperatura corporal, ya que a temperaturas corporales bajas las lagartijas no pueden desarrollar una velocidad máxima de carrera (Avery et al. 1982). De forma que, a temperaturas corporales más bajas, las lagartijas tendrán distancias de aproximación mayores (Rand 1964; Smith 1997; Cooper 2000; Martín y López 2000a). En cuanto a los costes de la respuesta, los requerimientos alimentarios pueden hacer que los animales asuman más riesgos y, por tanto, presenten distancias de aproximación menores (Martín y López 2000a). La distancia de aproximación sería menor también cuando el animal tiene que cumplir otros requerimientos, como la reproducción (Díaz-Uriarte 1999; Martín y López 1999c). De la misma forma que los animales valoran la distancia que permiten acercarse al depredador antes de escapar, también valoran la distancia que recorren durante el escape. Una estrategia antidepredatoria ampliamente empleada es el uso de refugios (Sih et al. 1992; Martín y López 1999a, b). De la misma forma que a la hora de decidir cuándo escapar de un depredador, los modelos teóricos del uso de refugios sugieren que los animales deben ajustar el tiempo que pasan escondidos en un refugio de forma que el tiempo óptimo de emergencia del refugio es cuando los costes del refugio superan a los costes de abandonarlo (Sih 1992, 1997; Martín y López 1999b; Polo et al. 2005). Por lo tanto, la decisión de cuándo salir de un refugio debe ser optimizada considerando la disminución del riesgo de depredación en el exterior con el transcurso del tiempo pero también considerando los costes del uso del refugio. En reptiles y otros animales ectotermos, uno de estos costes es el tiempo pasado a bajas temperaturas, ya que los refugios suelen estar en microhábitats con sombra y bajas Capítulo 1 Introducción general 13 temperaturas como grietas de rocas. La temperatura de una lagartija dentro de un refugio disminuye hasta niveles subóptimos después de un tiempo. Esto es especialmente importante para lagartijas de pequeño tamaño, con poca inercia térmica porque puede resultar en una disminución de la temperatura corporal en sólo unos pocos minutos. El mantenimiento de una temperatura corporal óptima es esencial en animales ectotermos como las lagartijas para maximizar numerosos procesos fisiológicos (Huey 1982; Stevenson et al. 1985) y comportamientos con importantes consecuencias para la eficacia biológica futura, como la velocidad de carrera o la eficiencia en la alimentación (Avery et al. 1982; Bennett 1980). Por lo tanto, los animales deberían ajustar su comportamiento antidepredatorio y uso de refugios para hacer frente al incremento en el riesgo de depredación sin incurrir en grandes costes de este comportamiento (Sih 1992, 1997; Dill y Fraser 1997; Martín y López 1999b; Martín et al. 2003a, b). En general los estudios acerca de los costes asociados a la realización de una respuesta antidepredatoria se han centrado fundamentalmente en la pérdida de tiempo para realizar otras actividades, como una pérdida de oportunidades de alimentación (Koivula et al. 1995; Dill y Fraser 1997; Martín et al. 2003a; Cooper y Pérez-Mellado 2004) o de reproducción (Crowley et al. 1991; Sih et al. 1990; Martín et al. 2003b). Sin embargo, las estrategias antidepredatorias, como el incrementar la velocidad en desplazamientos por zonas inseguras, el realizar carreras de escape ante un ataque o el uso de refugios, son comportamientos que implican un coste no sólo en relación al tiempo perdido para realizar otras actividades sino también en términos más fisiológicos, de pérdida de condición corporal (Martín y López 1999a; Pérez-Tris et al. 2004). Por ejemplo, se ha observado que las lagartijas roqueras Podarcis muralis sometidas experimentalmente a un elevado riesgo de depredación responden incrementando el uso de refugios, lo que conlleva una pérdida de condición corporal (Martín y López 1999a). También, las lagartijas colilargas, Psammodromus algirus sometidas a un elevado riesgo de depredación sufren una pérdida de condición corporal, no sólo porque deben interrumpir Capítulo 1 Introducción general 14 frecuentemente la alimentación sino por el estrés asociado al riesgo per se (Pérez- Tris et al. 2004). Como causas potenciales de la pérdida de condición corporal debido al uso de refugios se han apuntado una disminución del tiempo destinado a la alimentación, lo que conllevaría una reducción en la ingestión de comida (Dill y Fraser 1997; Godin y Sproul 1988; Koivula et al. 1995); una baja eficiencia de la digestión debido a las bajas temperaturas en el interior de los refugios (Huey 1982; Stevenson et al. 1985), y por tanto, una disminución en la energía disponible para almacenarse en forma de reservas grasas (Harlow et al. 1976; Harwood 1979); y los costes energéticos asociados a las secuencias de escape hasta alcanzar el refugio (Martín y López 1999a). Sin embargo, no se conoce cuál de estos factores está realmente contribuyendo a la disminución de la masa corporal. En resumen, una excesiva asignación de tiempo y energía para realizar estrategias antidepredatorias puede conllevar a una pérdida de condición corporal, lo que tiene consecuencias importantes para la eficacia biológica de los individuos a corto y largo plazo. Por ejemplo, la pérdida de condición corporal puede implicar una disminución de los recursos destinados a la defensa contra infecciones parasitarias, porque el estado nutricional puede influir en la capacidad de las lagartijas para desarrollar una respuesta inmune a una infección (Cooper et al. 1985; Smallridge y Bull 2000). El sistema inmunitario supone la defensa más efectiva para la defensa del organismo frente a parásitos (Wakelin 1996). Los efectos deletéreos del parasitismo afectan a diversos aspectos de la ecología y evolución de los hospedadores (Smallridge y Bull 2000; Eisen 2001), como el crecimiento poblacional (Hudson et al. 1998), la distribución espacial (Price 1980), el éxito reproductor (Schall 1996; Oppliger et al. 1997), afectando incluso a la selección sexual de los individuos hospedadores (Hamilton y Zuk 1982; Møller et al. 1999). Dentro del organismo, los parásitos, por ejemplo los parásitos sanguíneos intraeritrocíticos, pueden ocasionar anemia (Caudell et al. 2002; O’Dwyer et al. 2004) y daños en órganos internos (Svahn 1974; Veiga et al. 1998). Sin embargo, en el caso de las especies de lagartijas ibéricas, no se dispone de mucha información sobre las Capítulo 1 Introducción general 15 prevalencias e intensidades de infecciones parasitarias, ni se conocen apenas los efectos deletéreos que pudieran tener los parásitos sobre la condición corporal. Para reducir los posibles daños que puedan ocasionar los parásitos, los hospedadores han desarrollado una elaborada defensa antiparasitaria mediada por el sistema inmune (Sheldon y Verhulst 1996). Sin embargo, el mantenimiento del sistema inmune requiere una serie de nutrientes y energía que el hospedador puede necesitar para el crecimiento o la reproducción, por lo que a veces existe un conflicto entre el mantenimiento del sistema inmune para una defensa adecuada frente a los parásitos y otros requerimientos (Sheldon y Verhulst 1996; Møller et al. 1999). Este conflicto puede ser mucho mayor cuando además otros factores influyen en la condición corporal de los individuos. Por ejemplo, los animales que vivan en zonas con un alto riesgo de depredación, deberían realizar frecuentemente estrategias antidepredatorias por lo que podrían sufrir una disminución de masa corporal, y, por tanto, no podrían destinar suficientes recursos al sistema inmune para la lucha antiparasitaria. Por ello, los efectos deletéreos del parasitismo serían mayores, lo que podría afectar a la eficacia biológica de los individuos, y por tanto, al mantenimiento de las poblaciones de reptiles. Sin embargo, y pese a su importancia para el mantenimiento de las poblaciones, el efecto de un incremento en el riesgo de depredación, con el consiguiente aumento de los comportamientos antidepredatorios y sus consecuencias para la condición corporal y estado de salud de los individuos no se ha estudiado. Las estrategias antidepredatorias pueden tener otro coste que raramente ha sido considerado, y es un incremento en el riesgo de ser capturado por otro tipo de depredador. Este fenómeno tiene lugar cuando dos depredadores actúan simultáneamente y las presas presentan estrategias antidepredatorias conflictivas ante estos dos depredadores, es decir, la respuesta a un depredador supone un incremento en el riesgo de ser capturado por el otro depredador (Soluk y Collins 1988; Losey y Denno 1998). Esto puede dar lugar a un fenómeno denominado facilitación de la depredación, en el que dos depredadores con distintos métodos Capítulo 1 Introducción general 16 de caza actuando simultáneamente obtendrían ventajas, incrementando el riesgo de depredación para las presas (Soluk 1993; Sih et al. 1998). A pesar de que las presas viven en comunidades con varios tipos de depredadores, este fenómeno ha sido escasamente estudiado (Sih et al. 1998), especialmente en el caso de reptiles. Muchos reptiles responden ante la presencia humana como a la de un depredador, exhibiendo respuestas antidepredatorias como el uso de refugios (Martín y López 1999a, b). Sin embargo, el uso de refugios puede exponer a las presas a otro tipo de depredadores que cazan al acecho en el interior de grietas de rocas que los reptiles utilizan como refugio, como algunas culebras saurófagas. Por lo tanto, un incremento en la presencia humana, como por ejemplo, un incremento en el ecoturismo, podría incrementar el riesgo de depredación por culebras. Para evitar este efecto de múltiples depredadores, los reptiles deben valorar adecuadamente no sólo el riesgo de depredación ejercido por los depredadores de búsqueda activa del alimento, como aves o mamíferos, o el riesgo potencial ejercido por personas, sino también el riesgo de encontrar una culebra saurófaga en el interior de un refugio. Para ello muchas especies han desarrollado la habilidad de detectar las señales químicas de sus depredadores (Cooper 1990; Van Damme et al. 1995; Downes y Shine 1998; Van Damme y Quick 2001). La quimiodetección de los depredadores, así como una flexibilidad a la hora de llevar a cabo una respuesta antidepredatoria puede ayudar a los individuos a evitar la facilitación de la depredación. De esta forma, las lagartijas pueden valorar la posibilidad de encontrar a una culebra en el interior de un refugio antes de esconderse y ante el ataque de un depredador de búsqueda activa, utilizar estrategias alternativas al uso de refugios, como la huida, para así evitar entrar en refugios no conocidos que pueden ocultar a una culebra saurófaga. Sin embargo, aunque las señales químicas proporcionan una fuerte indicación de riesgo (Kats y Dill 1998), estas señales pueden persistir una vez que el depredador ha abandonado la zona, por lo que una respuesta de evitación a estas señales podría suponer una sobreestimación del riesgo de depredación (Kats y Dill 1998). Por lo tanto, las lagartijas deberían también ser Capítulo 1 Introducción general 17 capaces de valorar el tiempo que las señales llevan depositadas en el interior del refugio y la presencia real de la culebra, empleando para ello otro tipo de señales, como por ejemplo, las señales visuales. Las señales visuales aportan información muy específica acerca del estado de motivación para cazar del depredador, y por tanto, del nivel de riesgo real (Smith y Belk 2001). La hipótesis de sensibilidad al riesgo propone que los animales deben valorar adecuadamente el riesgo de depredación y responder de manera gradual en concordancia con el nivel de riesgo impuesto por el depredador (Helfman 1989). Por lo tanto, esta hipótesis asume que señales múltiples de un depredador deben contribuir de manera aditiva a la determinación del grado de comportamiento sensible al riesgo de las presas (Helfman 1989; Smith y Belk 2001). De esta forma, las lagartijas deberían ser capaces de utilizar varias señales de un depredador para valorar adecuadamente el nivel de riesgo de depredación, presentando una mayor respuesta de evitación cuando más de una señal esté presente en el interior de un refugio (McCarthy y Fisher 2000). De esta forma, valorarían adecuadamente el riesgo de depredación y así minimizarían los efectos de facilitación causados por los dos tipos de depredadores actuando simultáneamente. Las estrategias antidepredatorias de los animales han sufrido a lo largo del tiempo evolutivo una fuerte selección natural de forma que en la actualidad los animales deberían poder evitar la depredación sin incurrir en grandes costes de esta respuesta. Estas estrategias se han seleccionado en relación a la presión de depredación que los animales sufren en su medio natural. Sin embargo, la actividad humana está ocasionando cambios drásticos en el medio a una velocidad vertiginosa en relación al tiempo evolutivo, que pueden afectar al riesgo de depredación percibido por las presas (Whittingham y Evans 2004), a las estrategias antidepredatorias y a los costes de estas, que, como se ha comentado anteriormente podrían afectar al mantenimiento de las poblaciones. Las presas son capaces de estimar su vulnerabilidad frente a la depredación sin la presencia efectiva de un depredador; a través de las características del hábitat. Por ejemplo, se ha comprobado que las lagartijas que viven en hábitats abiertos con cobertura Capítulo 1 Introducción general 18 escasa, tienden a huir más y recorrer grandes distancias en su huida (Bulova 1994; Martín y López 2003) mientras que lagartijas en hábitats más complejos confían más en el uso de refugios (Martín y López 1999b, 2000a). Las lagartijas también responden a cambios estacionales en la vegetación, modificando sus respuestas de escape (Martín y López 1995, 1998). Por lo tanto, parece que las características de la vegetación juegan un papel importante y pueden determinar el tipo de estrategia antidepredatoria empleada. Los cambios antropogénicos del medio pueden hacer que los animales sean más conspicuos y, por tanto, más vulnerables a los depredadores, así como limitar el número de refugios (Hecnar y M'Closkey 1998; Martín y López 1998). El adehesamiento de un bosque o la construcción de una pista de esquí son dos ejemplos de cambios antropogénicos en la vegetación que conllevan una disminución de la cobertura vegetal, y, en el caso de la pista de esquí, también de la cobertura de rocas. En ambos casos, los animales no sólo serían más conspicuos al desplazarse por estas zonas sin vegetación sino que además sufrirían una disminución en el número de refugios disponibles en los que poder protegerse ante el ataque de un depredador. A pesar de que todas las evidencias sugieren que la degradación de la vegetación puede incrementar el riesgo de depredación para los animales, en el caso de los reptiles no se ha estudiado en profundidad el efecto que los cambios antropogénicos del medio pueden tener sobre el comportamiento antidepredatorio y sus consecuencias para la condición corporal y de salud de los individuos, factor muy importante a tener en cuenta para estudiar este posible efecto en el mantenimiento de las poblaciones de reptiles. Además estos cambios en la estructura del hábitat pueden conllevar una pérdida de hábitat, con la consiguiente desaparición de unas especies o aparición de otras. Para poder evaluar el efecto que los cambios en el medio tienen sobre las poblaciones de animales es necesario conocer cómo se distribuyen las especies en el espacio y qué factores ambientales determinan su distribución, y esto está íntimamente ligado con fenómenos relacionados con las preferencias de hábitat. Para entender cómo la fauna responde a los cambios en la vegetación se pueden usar modelos Capítulo 1 Introducción general 19 que relacionan la abundancia de las especies estudiadas con variables que describen la estructura del hábitat. Estos modelos son de considerable valor ya que permiten predicciones sobre la respuesta de las especies a cambios de la vegetación, tanto naturales como artificiales, como por ejemplo las repoblaciones con especies vegetales no autóctonas (Martín y López 2002). En el caso de las aves, hay numerosos estudios que muestran el efecto de drásticos cambios en la vegetación, como las repoblaciones, en las poblaciones de aves (Potti 1985; Carrascal y Tellería 1990; Díaz et al. 1998; Goldstein et al. 2003). Sin embargo, pese a que numerosas especies de lagartijas presentan preferencias hacia determinadas características estructurales del hábitat, por lo que cambios drásticos en esta estructura podrían afectar al mantenimiento de las poblaciones (Heatwole 1977; Martín y López 1998, 2002), los efectos de estos cambios han sido escasamente estudiados en el caso de los reptiles (pero ver también Santos y Tellería 1989; Díaz et al. 2000; Martín y López 2002). Esto es de gran importancia porque, pese a la amplia y variada riqueza de reptiles de la Península Ibérica, se ha observado que en las últimas décadas ha habido una manifiesta regresión de algunas de las especies (Pleguezuelos 1997; Pleguezuelos et al. 2002). Como causa principal de dicho declive se apunta la influencia humana que ha hecho disminuir y/o transformado los hábitats naturales. Sin embargo, los efectos de los cambios antropogénicos de la vegetación en las poblaciones de reptiles no se han estudiado en profundidad. La Sierra de Guadarrama ofrece un sistema excelente para estudiar cómo cambios profundos en la vegetación han podido afectar a la distribución y abundancia de las especies. A bajas e intermedias altitudes (1200-1700 m) la vegetación natural está dominada por bosques caducifolios de melojos, Quercus pyrenaica, mientras que a mayor altitud (1700-1900 m) la vegetación natural consiste en bosques de pino silvestre, Pinus sylvestris (Rivas- Martínez et al. 1987). Desde tiempos antiguos, los bosques de roble se han usado para la producción de leña y carbón vegetal y para pastoreo extensivo por lo que han sido poco a poco deforestados, y posteriormente se Capítulo 1 Introducción general 20 repoblaron con plantaciones de pino silvestre, que permiten una mayor explotación maderera. Esto ha dado lugar a que hoy en día amplias plantaciones de pinos se extiendan cubriendo todo el rango altitudinal de la zona, junto con los bosques naturales de pino presentes a mayor altitud, mientras que el robledal ha sido relegado a áreas más pequeñas (Izco 1984). Estos cambios drásticos en la vegetación podrían afectar la distribución y abundancia de las especies de reptiles. Así mismo, el sobrepastoreo y la explotación maderera siguen afectando a un gran número de robledales, creando zonas dentro de estos bosques con distintos niveles de degradación de la vegetación. Por otro lado, los pinares naturales, así como los piornales y enebrales situados a gran altitud sufren fuertes modificaciones del hábitat debido a las infraestructuras asociadas a la práctica del esquí. Estos cambios en la estructura del hábitat podrían afectar al mantenimiento de las poblaciones de reptiles que viven en estas zonas a través de un incremento en el riesgo de depredación y sus costes asociados. Otro factor que implica un incremento en el riesgo de depredación para los animales es la presencia humana, ya que muchos animales responden a las personas como a depredadores (Frid y Dill 2002; Beale y Monaghan 2004b). Aunque el ecoturismo se entiende en muchos casos como una forma de salvaguardar áreas naturales, y por tanto, de contribuir a la conservación de la diversidad (Munn 1992; Ceballos-Lascuráin 1996; Gössling 1999; Tisdell y Wilson 2002; pero ver también López-Espinosa de los Monteros 2002), en las últimas décadas ha experimentado un gran crecimiento y sus consecuencias para muchos taxones de animales no han sido analizadas en detalle (pero ver por ejemplo, Romero y Wikelski 2002; Müllner et al. 2004). Se conocen varios efectos negativos del ecoturismo sobre las poblaciones de animales, como una disminución del éxito reproductor (Giese 1996; Beale y Monaghan 2004a; McClung et al. 2004), una pérdida de áreas de alimentación (Sutherland y Crockford 1993; Gander y Ingold 1997; Fernández-Juricic y Tellería 2000; pero ver también Nevin y Gilbert 2005), una disminución de las tasas de alimentación (Duchesne et al. 2000), pérdida de hábitat óptimo (Burton et al. 2002), e incluso un incremento en Capítulo 1 Introducción general 21 las tasas de mortalidad (Feare 1976; Wauters et al. 1997; Müllner et al. 2004). En los reptiles los efectos deletéreos del ecoturismo no son muy conocidos (pero ver Wilson y Tisdell 2001 para tortugas marinas, y Romero y Wikelski 2002 para iguanas marinas). Sin embargo, muchas especies de lagartijas responden a las personas escondiéndose en refugios (Martín y López 1999a, b). Por lo tanto, un incremento en el ecoturismo de una zona puede suponer un incremento en el riesgo de depredación, con el consiguiente incremento en el uso de estrategias antidepredatorias, como secuencias de escape y uso de refugios. Esto podría conllevar un incremento en los costes de estas estrategias antidepredatorias, con una pérdida de condición corporal asociada (Martín y López 1999a) y también un incremento en el riesgo de ser capturados por otros tipos de depredadores, como las culebras saurófagas. En resumen, los cambios antropogénicos en el medio así como el ecoturismo son dos factores que pueden estar incrementando el riesgo de depredación para los animales, con el consiguiente efecto deletéreo de los costes de los comportamientos antidepredatorios para la condición física y el estado de salud de los individuos, afectando así al mantenimiento de las poblaciones de reptiles. Capítulo 1 Introducción general 22 2.- OBJETIVOS El objetivo principal de esta tesis doctoral es identificar los factores antropogénicos que puedan estar afectando al mantenimiento de las poblaciones de lagartijas de la Sierra de Guadarrama. A continuación se detallan los objetivos concretos de este estudio: • Estudiar las prevalencias e intensidades de infección de parásitos en varias especies de lagartijas para obtener un conocimiento previo de los efectos deletéreos que los parásitos pueden tener en la condición corporal de los individuos, y, por tanto, en el mantenimiento de las poblaciones de lagartijas (capítulo 2). • Examinar los efectos de los cambios en la estructura de la vegetación, en el mantenimiento de las poblaciones de lagartijas. Los objetivos concretos han sido: Estudiar el efecto de cambios drásticos en la vegetación (repoblaciones de pinares en bosques naturales de robles) en la distribución y abundancia de varias especies de lagartijas (capítulo 3). Analizar el efecto de la degradación de la vegetación en el comportamiento antidepredatorio y sus consecuencias para la condición física y estado de salud de los individuos (capítulo 3) • Estudiar el efecto del ecoturismo en las estrategias de escape, y examinar los costes del uso de refugios, así como analizar sus efectos en la condición física y estado de salud de los individuos (capítulo 4). • Analizar el efecto de múltiples depredadores debido al ecoturismo y estudiar las estrategias antidepredatorias de las presas para hacer frente a este incremento en el riesgo (capítulo 5). Capítulo 1 Introducción general 23 3.- MÉTODOS GENERALES Área de estudio El trabajo de campo de este estudio se realizó durante los meses de primavera y verano principalmente entre los años 2001 a 2004. El área de estudio comprende varias localidades situadas principalmente en la vertiente madrileña de la Sierra de Guadarrama (40º49’N, 3º56’E), en el Sistema Central español, aunque también se capturaron lagartos ocelados, Lacerta lepida, en un pastizal de la región de “Campo Azalvaro” (40°40’N, 4°20’W) a 1300 m. s.n.m.; en la vertiente noroeste de la Sierra de Guadarrama, en las provincias de Ávila y Segovia, para examinar la prevalencia e intensidad de parásitos sanguíneos e intestinales en esta población. En relación a la vertiente sur de la Sierra de Guadarrama, Madrid, los distintos estudios que forman parte de esta tesis doctoral se han realizado en diversas áreas que comprenden los pisos bioclimáticos supramediterráneo (1200- 1600 m s.n.m.) y oromediterráneo (1600-2000 m. s.n.m.). En el piso supramediterráneo, la vegetación potencial de la Sierra de Guadarrama está constituida por bosques de roble melojo, Quercus pyrenaica. Sin embargo, parte de estos bosques han sido sustituidos hace años por repoblaciones de pino silvestre, Pinus sylvestris. En estos dos tipos de bosque se realizaron muestreos para determinar el efecto de las antiguas repoblaciones de pinos en zonas de robledal en la distribución y abundancia de las especies de lagartijas. En robledales de Q. pyrenaica se estudió el efecto de la degradación de la vegetación en las estrategias de escape y la condición física y de salud de la lagartija colilarga, Psammodromus algirus. La vegetación en las zonas en buen estado está compuesta por matorrales de Juniperus communis, Genista florida, Crataegus monogyna, Cytisus scoparius, Rosa pouzini, Rubus ulmifolius y Lonicera periclymenum así como herbáceas como la característica Paeonia broteroi (Izco 1984). Este estudio se realizó en dos bosques, un robledal bien desarrollado, con robles grandes, situado cerca de la localidad de Miraflores de la Sierra (“Miraflores”), y otro robledal constituido por árboles más jóvenes, situado cerca de la localidad de Navacerrada (“La Golondrina”). En estos dos bosques se estudió la selección de hábitat de la lagartija colilarga en Capítulo 1 Introducción general 24 zonas con distinta degradación de la vegetación, y en el bosque de “La Golondrina” se estudió el comportamiento antidepredatorio de esta especie, y se capturaron individuos para examinar su condición corporal en relación al nivel de degradación de la vegetación. Las zonas con vegetación degradada estaban caracterizadas por grandes extensiones de jaras (Cistus ladanifer) o por pastizales sin apenas vegetación arbustiva. En pinares de Pinus sylvestris se capturaron ejemplares de lagartija roquera, Podarcis muralis, para estudiar la prevalencia e intensidad de infección de parásitos sanguíneos. En esta zona los pinares aparecen acompañados de arbustos como Juniperus communis, Cytisus scoparius y Crataegus monogyna así como del característico helecho aguila Pteridium aquilinum (Izco 1984). También se estudió el efecto del ecoturismo en el mantenimiento de las poblaciones de la lagartija roquera para lo cual se estudió el comportamiento antidepredatorio y la condición corporal de individuos de esta especie en zonas con distinto nivel de afluencia humana. Las zonas con nivel alto de ecoturismo estaban situadas colindantes con caminos y pistas forestales ampliamente transitadas mientras que las zonas con bajo nivel de ecoturismo estaban alejadas de caminos y pistas. Así mismo en una pared de piedra de las “Dehesas” cerca de la localidad de Cercedilla se realizó un estudio sobre el comportamiento antidepredatorio de las lagartijas en relación al nivel de riesgo ejercido por el depredador y los costes del uso de refugios (costes de termorregulación y riesgo de depredación por culebras saurófagas). Cerca de esta zona se capturaron ejemplares para examinar la capacidad de las lagartijas para utilizar distintas señales de las culebras para valorar adecuadamente el nivel de riesgo de depredación en el interior de los refugios, así como para examinar la capacidad de las lagartijas para evaluar el nivel de riesgo de depredación en relación al tiempo de exposición a las señales químicas de las culebras. Capítulo 1 Introducción general 25 Detalles de las zonas de estudio dónde se observa distinta afluencia humana en pinares, y distinto grado de degradación de la vegetación, tanto en robledales como en pinares y piornales de alta montaña. Capítulo 1 Introducción general 26 A mayor altitud (1900-2200 m.) se realizaron distintos estudios sobre la lagartija serrana, Lacerta monticola cyreni. Estos estudios se llevaron a cabo en zonas de matorral de alta montaña, donde la vegetación potencial, piornos (Cytisus oromediterraneus) y enebros (Juniperus communis) se mezclan con algún pino silvestre aislado y con roquedos y canchales de granito (Martín y Salvador 1997). En estas zonas se capturaron lagartijas serranas para determinar la prevalencia e intensidad de infección por parásitos sanguíneos en estas poblaciones. Así mismo se estudió el efecto de la degradación de la vegetación debido a las pistas de esquí en la selección de hábitat y uso del espacio, y su influencia en la condición y estado de salud de las lagartijas. También se estudió el comportamiento antidepredatorio y la condición corporal de las lagartijas en relación con el nivel de degradación del hábitat y el nivel de ecoturismo de la zona. En estas zonas se capturaron individuos para realizar experimentos para evaluar el efecto de la velocidad de carrera así como los distintos costes del uso de refugios en la condición corporal y el estado de salud de los individuos. Especies de estudio A continuación se describen brevemente las especies de reptiles que se han estudiado en esta tesis doctoral. La lagartija serrana, Lacerta monticola cyreni, es una lagartija de pequeño tamaño (longitud cabeza-cloaca de los adultos comprendida entre 65 y 90 mm.). Su periodo de actividad comprende únicamente desde Abril a Septiembre debido a las temperaturas ambientales limitantes. Las lagartijas se aparean en Mayo-Junio y las hembras producen una única puesta en Julio (Elvira y Vigal 1985; Salvador 1984; Pérez-Mellado 1998a). Es un endemismo ibérico. En el Sistema Central habita a altitudes superiores a 1800 m, ocupando zonas rocosas de altitud. Se encuentra catalogada como “Vulnerable” en el Atlas y Libro Rojo de los anfibios y reptiles de España. Como causas de amenaza se apuntan las actuaciones humanas en medios de alta montaña, como las infraestructuras asociadas a la práctica del esquí y el impacto del turismo (Pérez-Mellado 2002a) El lagarto ocelado, Lacerta lepida, es un lagarto de gran tamaño (longitud cabeza-cloaca de los adultos comprendida entre 140 y 170 mm.). Se Capítulo 1 Introducción general 27 reproducen durante Abril y Mayo y producen una única puesta en Junio- Julio (Pérez-Mellado 1998b). Es una especie estrechamente asociada a ecosistemas mediterráneos, donde se encuentra en zonas secas y abiertas con escaso relieve y acúmulos rocosos, o en zonas arbustivas dispersas (Pérez- Mellado 1998b). Se encuentra catalogada como “De preocupación menor” y como factores de amenaza se citan la destrucción de hábitat y la utilización de venenos en áreas de caza (Mateo 2002). La lagartija roquera, Podarcis muralis, es una lagartija pequeña (longitud cabeza-cloaca de los adultos: 60-76 mm) distribuida por Europa Central, aunque en la Península Ibérica se encuentra restringuida a áreas montañosas de la mitad norte donde ocupa taludes y muros rocosos en zonas sombreadas de los bosques (Martin- Vallejo et al. 1995). En la Sierra de Guadarrama se reproducen en Abril- Mayo y producen una única puesta en Junio (Pérez-Mellado 1998c). Se encuentra catalogada como “De preocupación menor” y como factores de amenaza se han descrito la modificación de los paisajes, la destrucción del hábitat y la eliminación de los refugios naturales (Pérez-Mellado 2002b). La lagartija ibérica, Podarcis hispanica, es una lagartija de pequeño tamaño (longitud cabeza-cloaca de los adultos comprendida entre 37 y 70 mm) de amplia distribución por la Península Ibérica, y en la Sierra de Guadarrama se encuentra prácticamente en todos los niveles altitudinales, llegando a alcanzar el piso oromediterráneo como L. monticola. En la Sierra de Guadarrama se reproduce en Abril y produce una puesta en Junio (Pérez-Mellado 1998d). Es una especie típicamente rupícola (Pérez-Mellado 1998d) y cuando convive con P. muralis ocupa las zonas más rocosas y soleadas (Martín-Vallejo et al. 1995; Sá Sousa y Pérez-Mellado 2002). Se encuentra catalogada como “De preocupación menor” y como amenazas se apuntan la destrucción de los refugios rocosos, el ecoturismo y la introducción de depredadores asociados al hombre como los gatos domésticos (Sá Sousa y Pérez-Mellado 2002). La lagartija colilarga, Psammodromus algirus, es una lagartija de tamaño medio (longitud cabeza- cloaca de los adultos comprendida entre 55 y 90 mm) que vive tanto en bosques Capítulo 1 Introducción general 28 mediterráneos perennifolios como caducifolios de la Península Ibérica y del noreste de África (Martín y López 1998; Pérez-Mellado 1998e). Ocupa zonas con cobertura arbustiva densa y sustratos de hojarasca. En la Sierra de Guadarrama se reproduce en Abril-Mayo y produce una puesta en Junio (Pérez-Mellado 1998e). Está catalogada como “De preocupación menor”. Los factores que pueden afectar a las poblaciones son la transformación del hábitat para agricultura intensiva, regadios, o destrucción de matorrales para pastoreo, así como la urbanización y la presión turística (Carretero et al. 2002). La culebra lisa europea, Coronella austriaca, es una culebra de pequeño tamaño (longitud media de 500 a 600 mm.) propia de la región Eurosiberiana de la Península Ibérica. En la región Mediterránea ocupa los macizos montañosos, en el piso supramediterráneo y principalmente en el oromediterráneo en zonas de clima fresco y húmedo. Es una especie ovovivípara que se reproduce en Junio- Julio y el parto se produce en Agosto (Galán 1998). Sus poblaciones son escasas y aisladas. Se encuentra catalogada como “De preocupación menor”. Es muy sensible a la modificación del medio, por lo que la ganadería intensiva de alta montaña y las infraestructuras asociadas a la práctica del esquí podrían afectar a sus poblaciones (Galán 2002). Capítulo 1 Introducción general 29 Especies de estudio Capítulo 1 Introducción general 30 Características del hábitat Para el estudio de la distribución y abundancia de las especies de reptiles en robledales y en pinares de repoblación se ha utilizado el método de los transectos, lo que permite estimar densidades relativas de las especies, comparando la distribución y abundancia estimadas entre los distintos microhábitats presentes en la zona de estudio. Para ello se realizaron recorridos lineales a pie y a velocidad constante en lugares considerados fisionómicamente homogéneos en base principalmente al tipo y estructura de la vegetación existente. Los transectos tenían una longitud de 200 m, con una anchura de banda de 4 m (2 m a cada lado del observador). Por cada individuo observado en los transectos se anotó la especie, sexo y edad (si era conocida). La actividad de los reptiles depende en gran manera de su tempertura corporal, que a su vez depende en gran medida de la temperatura ambiental y condiciones climáticas. Solamente cuando alcanzan un margen de temperaturas óptimas están activos y son visibles. Por lo tanto, el número de individuos activos en una población varía mucho en función de la temperatura, grado de insolación, y época del año (por ej. reproducción vs. etapa postreproductiva). Por lo que los muestreos se realizaron siempre cuando hacía buen tiempo (sol presente y temperatura del aire superior a 15° C). Para caracterizar la vegetación de la zona se tomaron tres puntos de muestreo en cada transecto. En cada punto se definían dos lineas perpendiculares de 1 m de longitud que se cruzaban en dicho punto. Utilizando una barra colocada verticalmente, anotamos a intervalos de 50 cm a lo largo de las lineas los contactos con la barra a nivel de sustrato de vegetación herbácea, hojarasca, rocas y suelo desnudo con arena o tierra. Usando el mismo procedimiento anotamos los contactos con vegetación arbustiva. De esta forma anotamos la altura a la que la planta tocaba el palo para determinar el posible uso como refugio de esa planta y el tipo de planta. También se anotó la cobertura de vegetación árborea por encima del punto. Este procedimiento produce una muestra de 9 datos por cada punto de muestreo en el transecto, que permiten calcular las coberturas de sustrato y vegetación, y el número de contactos con cada tipo de vegetación. Para cada Capítulo 1 Introducción general 31 observación de un reptil se midieron las mismas variables de hábitat a partir del punto en el que era por primera vez observado u oído. El método es similar al del punto-central (“point-centered method”) empleado en otros estudios de selección de hábitat de aves y reptiles (Martín y López 1998, 2002). Este método de los transectos se utilizó también en el estudio de la selección de hábitat de la lagartija colilarga, P. algirus, en zonas con distinto nivel de degradación de la vegetación de robledales. En el caso de la selección de hábitat de la lagartija serrana, L. monticola, en zonas con distinto nivel de degradación de la vegetación debido a las pistas de esquí, no se realizaron transectos, pero se caracterizó la vegetación en puntos elegidos al azar así como en localizaciones de individuos siguiendo el mismo procedimiento. Comportamiento antidepredatorio Para estudiar el comportamiento antidepredatorio de las lagartijas en condiciones naturales se buscaban lagartijas en horas de máxima actividad y condiciones ambientales óptimas (días soleados). Cuando se localizaba una lagartija, se realizaba un ataque simulado caminando directamente hacia la lagartija a una velocidad elevada (aproximadamente 140 m/min) hasta que la lagartija huía. Con este procedimiento se simulaba el ataque directo de un depredador atacando desde el suelo, como un felino o un pájaro atacando de las partes bajas de un matorral. La misma persona, vistiendo siempre la misma ropa, realizaba todos los ataques de manera similar para evitar efectos que pudieran afectar al riesgo de depredación percibido por las lagartijas (Burger y Gochfeld 1993; Cooper 1997) y anotaba el comportamiento de escape. Se anotó el tipo de estrategia usada distinguiendo entre lagartijas que permanecían inmóviles, lagartijas que se escondían en un refugio, y lagartijas que huían sin esconderse. También se anotaron la distancia de aproximación (distancia entre la lagartija y el observador cuando la lagartija empezaba una respuesta de escape), la distancia de huída (distancia que recorría la lagartija durante un episodio de escape), y la distancia desde la localización inicial de la lagartija al refugio más cercano y al refugio usado. Cuando se examinaba el uso de refugios, se anotaba el tiempo que la lagartija Capítulo 1 Introducción general 32 tardaba en sacar la cabeza (tiempo de aparición) y el tiempo que tardaba en salir completamente del refugio (tiempo de emergencia), observando desde una posición alejada unos 5-7 m para no molestar a los individuos. Tanto el comportamiento de escape como el uso de refugios dependen de la temperatura corporal de las lagartijas y de las condiciones térmicas en el interior del refugio (por ejemplo Hertz et al. 1982; Smith 1997, Martín y López 1999a, b). Sin embargo, este diseño experimental para examinar el comportamiento de escape no nos permitió capturar a las lagartijas inmediatamente antes de que se escondieran para medir su temperatura corporal, por lo que inmediatamente después de que las lagartijas abandonaran el refugio se midió con un termómetro digital la temperatura del aire en el lugar que fue observada por primera vez y la temperatura en el interior del refugio. Asumimos que estas medidas son una aproximación a la temperatura corporal de las lagartijas dada la gran dependencia de éstas de las condiciones térmicas ambientales (Braña 1991; Carrascal et al. 1992; Martín y Salvador 1993; Martín- Vallejo et al. 1995; Martín et al. 1995). Medidas de condición corporal, inmunocompetencia y carga parasitaria Las lagartijas eran capturadas con el método del lazo corredizo, que no conlleva ningún daño físico a los animales. Inmediatamente después de la captura, eran medidas y pesadas, y se determinó la prevalencia e intensidad de infección por ectoparásitos como ácaros y garrapatas. Posteriormente las lagartijas se llevaban a la Estación Biológica de “El Ventorrillo” para tomar el resto de medidas. Allí se alojaban en terrarios individuales con agua y comida (Tenebrio molitor) ad libitum. Para la detección y cuantificación de parásitos intestinales de L. lepida se tomó una muestra fecal de cada individuo. Estas muestras se analizaron para cuantificar la excrección de propágulos de párasitos mediante la técnica clásica de flotación y conteo en cámara MacMaster. Los propágulos se identificaron siguiendo el protocolo de Melhorn y colaboradores (1992). La detección de parásitos sanguíneos se realizó en frotis realizados a partir de una pequeña gota de sangre extraída con un microcapilar heparinizado del seno post-orbital del ojo derecho. Los frotis se Capítulo 1 Introducción general 33 secaron al aire y se fijaron con etanol absoluto durante 10 min. Posteriormente se tiñieron con tinción de Giemsa con una dilución 1:9 en tampón de fosfato salino (pH 7.2) durante 40 min. Una mitad longitudinal del frotis sanguíneo se examinó en su totalidad con el objetivo de 200 aumentos para determinar la prevalencia de parásitos extraeritrocíticos. En la otra mitad del frotis, se examinaron 20 campos a 400 aumentos para detectar la presencia de parásitos intraeritrocíticos como Hemogregarinas. Se determinó la prevalencia de infección como una variable categórica (presencia/ausencia). La intensidad de infección por parásitos intraeritrocíticos se determinó examinando el frotis a 1000 aumentos y determinando el número de células infectadas por cada 2000 eritrocitos (Merino y Potti 1995). Después de la extracción de sangre para la detección de parásitos sanguíneos se realizó la prueba de la inyección dérmica de fitohemaglutinina (PHA) que permite obtener una estima de la capacidad de respuesta del componente celular del sistema inmunitario adquirido. Proporciona una medida de la respuesta proliferativa de los linfocitos T circulantes frente a la inyección del mitógeno PHA. La principal respuesta celular observada consiste en una prominente acumulación perivascular de linfocitos T en la zona de inyección, seguida por infiltración de macrófagos (Goto et al. 1978). Esta prueba ha sido ampliamente utilizada en estudios ecológicos en aves silvestres (Saino et al. 1997; Moreno et al. 1999; Martin et al. 2001; Fargallo et al. 2002) y en lagartijas (Merino et al. 1999; Svensson et al. 2001; Belliure et al. 2004). Se ha demostrado en estudios de campo en aves que la prueba de la PHA no provoca alteraciones en los niveles de proteínas de estrés u otros parámetros hematológicos (Merino et al. 1999) y la hipercelularidad estimulada por la PHA desaparece generalmente a las 48 h tras la inyección. Para estimar la respuesta a la inyección de PHA seguimos el protocolo de Smits y colaboradores (1999) en el que no se emplea la inyección control de PBS, reduciendo así no sólo errores de medida sino también disminuyendo el tiempo de manejo del animal. La solución de PHA se preparó con 50 mg. de PHA-P (Sigma) disueltos en 10 ml. de PBS. Inyectamos en la mitad de la planta del pie de la pata Capítulo 1 Introducción general 34 posterior derecha de las lagartijas 0.02 ml de la disolución de PHA y medimos el espesor de la planta antes y 24 horas después de la inyección con un espesímetro digital de presión constante (Mitutoyo 7/547, Tokio, Japón) con una precisión de 0.01 mm. La medida de inmunocompetencia mediada por linfocitos T se estimó como la diferencia entre las medidas iniciales y finales de espesor de la planta de la pata. Una mayor inflamación de la zona indica una mayor respuesta ante la inyección del antígeno. A las 24 horas de ser medida la respuesta a la inyección del antígeno, los individuos eran liberados en el mismo lugar de captura. Experimentos de laboratorio Las lagartijas se capturaron con el método del lazo corredizo y se les tomaron las medidas biométricas y de salud correspondientes en cada caso (ver descripción anterior). Posteriormente las lagartijas se alojaron individualmente en la Estación Biológica de “El Ventorrillo” en terrarios de PVC (60 x 40 cm) con agua ad libitum y, en la mayoría de los casos, con comida (Tenebrio molitor) también ad libitum. Cada día se daban 2 o 3 gusanos a cada lagartija, cantidad suficiente ya que no siempre se comían todos. De vez en cuando se suministraban gusanos impregnados con vitaminas para reptiles. Cada terrario disponía de una zona de sombra y otra de sol, y un refugio situado a la sombra. Las condiciones de temperatura y fotoperiodo eran las ambientales de la zona. Las culebras lisas europeas empleadas en algunos de los experimentos eran capturadas a mano en el campo y alojadas individualmente en el Ventorrillo en terrarios de cristal, con serrín o papel absorbente en el sustrato para obtener su olor. Disponían de agua ad libitum y un refugio. Se alimentaban con trozos de carne impregnados con secreciones de lagartijas. Antes de las pruebas de cada día las lagartijas se dejaban termorregular durante 2 horas para que alcanzaran una temperatura corporal óptima. Los terrarios estaban suficientemente separados entre sí para no molestar a otros individuos cuando se realizaba alguna prueba a otra lagartija. La misma persona realizaba los experimentos para evitar diferencias en el riesgo de depredación percibido por las lagartijas. En los casos en los que se realizó un diseño de medidas repetidas, cada Capítulo 1 Introducción general 35 lagartija realizó una única prueba al día. Después de los experimentos las lagartijas fueron liberadas en el mismo lugar de captura. En cada capítulo se describen detalladamente los métodos usados en cada experimento. 4.- RESULTADOS Y DISCUSIÓN GENERAL Relaciones parásitos-lagartijas: consecuencias para el mantenimiento de las poblaciones de lacértidos Los resultados de estos estudios (capítulo 2) aportan datos novedosos que incrementan el escaso conocimiento de las relaciones parásito-hospedador en las especies estudiadas. En relación a los parásitos sanguíneos, en las tres especies se encontraron hemogregarinas, que pertenecen a la familia Haemogregorinidae, suborden Adeleorina, subclase Coccidiasina, Phylum Apicomplexa (Barnard y Upton 1994). Prevalencia e intensidad de infección de parásitos sanguíneos hemogregarinas en una población de lagartija serrana, Lacerta monticola La prevalencia e intensidad de infección por hemogregarinas fue mayor en adultos que en juveniles de lagartija serrana, como se ha observado en otras especies (Smallridge y Bull 2000), probablemente debido a que las lagartijas adquieren garrapatas o ácaros, los vectores transmisores de las hemogregarinas, cuando comparten lugares favorables para tomar el sol o refugios. Los juveniles, al no compartir estos lugares, porque son expulsados por los adultos a áreas subóptimas, seguramente no están en contacto con los vectores y, por tanto, con la infección. La prevalencia de infección fue mayor en los adultos de mayor tamaño corporal, y por tanto, más viejos, probablemente porque han tenido más oportunidades en su vida de ser infectados debido a su mayor edad. Durante la estación reproductora, la intensidad de infección fue mayor en machos que en hembras, debido a los efectos inmunosupresores de la testosterona (Salvador et al. 1996; Capítulo 1 Introducción general 36 Olsson et al. 2000). Sin embargo, en el período post-reproductor no hubo diferencias entre sexos en la intensidad de infección. La intensidad de infección de las hembras aumentó en verano, probablemente debido al esfuerzo reproductor, que limita los recursos destinados a la defensa frente a la infección (Gustafsson et al. 1994; Oppliger y Clobert 1997; Oppliger et al. 1997; Fargallo y Merino 2004). Una explicación alternativa sería que si la infección reduce la supervivencia, los individuos más infectados, probablemente los machos, habrían muerto durante la estación reproductora, como se ha observado en algunos mamíferos (Schuster y Schaub 2001). En relación a la condición corporal, los resultados muestran que la carga parasitaria afecta a la condición corporal. Durante el período reproductor, hubo una relación negativa entre la condición y la intensidad de infección, mientras que en el período post-reproductor esta relación fue positiva. La inversión en reproducción puede hacer que los animales no puedan invertir recursos para el sistema immune y, por tanto, presenten mayor intensidad de infección. En cambio, una vez que el período reproductor ha terminado ya pueden invertir más recursos en la defensa contra los parásitos. Estos resultados sugieren que las relaciones parásito- hospedador no son estables en esta población, y los efectos de la parasitemia podrían ser peores si un factor adicional, como un incremento en el riesgo de depredación afectara a la condición corporal de los individuos. Prevalencia e intensidad de parásitos sanguíneos e intestinales en una población natural de una lagartija mediterránea, Lacerta lepida Los resultados acerca de las prevalencias de hemogregarinas en el lagarto ocelado son similares a los obtenidos con la lagartija serrana ya que la prevalencia fue menor en juveniles que en adultos, y también menor en adultos de menor tamaño (más jóvenes). Sin embargo, la prevalencia y la intensidad de infección fueron similares en ambos sexos, por lo que en esta población de esta especie tanto los machos como las hembras parecen invertir por igual en la reproducción en términos de disminución de la defensa contra parásitos o bien los machos no Capítulo 1 Introducción general 37 sufren los efectos inmunosupresores de la testosterona tan acusadamente como en otras especies. En relación a los parásitos intestinales, se encontraron nematodos oxiúridos que no se pudieron identificar a nivel de especie, aunque se han descrito dos especies de oxiúridos parasitando a esta especie en la Península Ibérica: Parapharyngodon bulbosus (Linstow 1899) Teixeira de Freitas 1957; y Spauligodon extenuatus (Rudolphi 1819) Skrjabin, Schihobalova et Lagodovskaya, 1960 (Cordero del Campillo et al. 1994). También se encontró un nematodo ascárido y protozoos coccidios, que no habían sido descritos en esta especie. En relación a la prevalencia de nematodos, no se encontraron diferencias entre edades ni sexos, lo que sugiere que la dieta de estas clases les hace similarmente susceptibles a la infección por nematodos. Sin embargo, los adultos infectados por coccidios eran de menor tamaño que los no infectados, probablemente porque la lucha contra la infección requiera recursos que de otra forma habrían estado destinados al crecimiento o bien porque los adultos más grandes hayan controlado la infección, disminuyéndola hasta niveles no detectables. Sin embargo, estas conclusiones son preliminares debido al limitado número muestral y al bajo nivel de significación. La condición corporal de los individuos disminuyó en el transcurso del período reproductor, debido probablemente al esfuerzo reproductor. Sin embargo ni la intensidad de infección por hemogregarinas ni la presencia de parásitos intestinales afectó a la condición de los individuos, lo que sugiere la estabilidad de las relaciones hospedador-parásito en esta población. Prevalencia e intensidad de infección de parásitos sanguíneos hemogregarinas y sus vectores los ácaros en la lagartija roquera, Podarcis muralis La intensidad de infección por hemogregarinas se mantuvo en el caso de machos adultos a lo largo del período reproductor, debido probablemente a los efectos de la testosterona (Salvador et al. 1996; Olsson et al. 2000) pero disminuyó en las hembras en el período post-reproductor. Esto parece estar apoyado por la mayor prevalencia e intensidad de infección en machos por Capítulo 1 Introducción general 38 los ácaros que transmiten las hemogregarinas. Por el contrario, las hembras, después de la puesta, podrían invertir todos sus recursos en la defensa frente a parásitos, disminuyendo así la infección después del período reproductor. Los resultados mostraron una relación positiva entre la condición corporal y la intensidad de infección por hemogregarinas, lo que sugiere que los individuos infectados con peor condición corporal podrían haber muerto a lo largo del período reproductor, mientras que sólo los individuos con buena condición habrían podido sobrevivir. Efectos de la modificación de la vegetación natural sobre las poblaciones de lacértidos Robledales vs. repoblaciones de pinos: efectos del manejo forestal sobre la distribución y conservación de lagartijas. Las lagartijas no utilizan el hábitat al azar (capítulo 3), sino que seleccionan microhábitats dónde puedan cumplir con sus requerimientos específicos, como la alimentación, la termorregulación, o evitar a los depredadores (por ejemplo, Carrascal et al. 1989; Díaz y Carrascal 1991; Castilla y Bauwens 1992; Martín y López 2002). Por eso, cambios en la estructura del hábitat pueden afectar a la distribución y abundancia de las distintas especies. De esta forma, las especies encontradas en repoblaciones de pinares difieren de las encontradas en bosques naturales de roble. Por ejemplo, Psammodromus algirus sólo aparece en robledales mientras que Podarcis muralis está presente sólo en pinares de repoblación. El análisis de la relación entre la densidad de lagartijas y las características del microhábitat sugiere que la abundancia de lagartijas está determinada por las características del microhábitat. El número de individuos depende de la presencia de sustratos de roca, baja cobertura de árboles y alta disponibilidad de refugios. Sin embargo, estos resultados difieren cuando se considera cada especie por separado. Por ejemplo, P. algirus prefiere sustratos de hojarasca, mientras que Podarcis hispanica y P. muralis prefieren sustratos rocosos, lo que podría explicar la distribución diferencial de las especies entre ambos tipos de bosques. Capítulo 1 Introducción general 39 Fig. 1 Media (+ SE) de los valores de PC para los microhábitats disponibles en reforestaciones de pinos y bosques naturales de robledal (cajas blancas), y para los microhábitat usados por P. muralis, P. hispanica and P. algirus lizards (cajas negras). La presencia de P. muralis en las repoblaciones de pino pero no en robledales situados a la misma altitud sugiere una estrecha relación de la distribución de esta especie con los pinares. Las reforestaciones de pinos a bajas altitudes probablemente presentan condiciones microclimáticas favorables que han favorecido la colonización por esta especie (Guisan y Hofer 2003), que es propia de la región Eurosiberiana (Pérez- Mellado 1998; Diego-Rasilla Capítulo 1 Introducción general 40 2004). Por tanto, las repoblaciones de pinos han podido contribuir a la expansión del límite meridional y altitudinal de la distribución de esta especie en la Península Ibérica, en zonas que de otra forma estarían ocupadas por especies más mediterráneas como P. algirus. Este estudio tiene implicaciones para el manejo del bosque. Las áreas abiertas, sin árboles pero con matorral y rocas en el interior de los bosques contribuirían a mantener las poblaciones de lagartijas y la diversidad de especies, como se ha observado anteriormente (Enge y Marion 1986; Goldingay et al. 1996; Renken et al. 2004). Por tanto, de acuerdo con la “Hipótesis de la Perturbación Media”, las perturbaciones de baja intensidad pueden promover la coexistencia de especies y promover una mayor diversidad (Grime 1974; Connell 1978; Pickett y White 1985; Huston 1994). Desde el punto de vista de la conservación y el manejo de las poblaciones de lagartijas, las repoblaciones antiguas de pinos no contribuyen mucho a la diversidad de especies, y sólo favorecen a las lagartijas roqueras, P. muralis. Por lo tanto, un adecuado manejo sería permitir la recolonización por parte de los robles dentro de estas repoblaciones. Además, se deberían evitar algunas prácticas contra incendios como los aclarados y la eliminación de matorral y la vegetación muerta que no sólo perjudican a las características del suelo (Schmitz et al. 1998) sino a la calidad del hábitat para las lagartijas (Ryan et al. 2002; James y M’Closkey 2003). Por tanto, el mantenimiento y favorecimiento de la heterogeneidad del hábitat ayudaría a mantener una mayor diversidad, no sólo de lagartijas sino de otros taxones gracias a la creación de un mayor número de nichos (Potti 1985; Carrascal y Tellería 1990; Fischer et al. 2004). El incremento en el riesgo de depredación debido a la degradación del habitat afecta a la condición corporal de las lagartijas: una aproximación experimental con la lagartija serrana, Lacerta monticola en pistas de esquí. La modificación del medio debido a factores antropogénicos también parece provocar, no sólo un cambio en las especies presentes en un determinado tipo de medio sino un incremento en el riesgo de depredación para los Capítulo 1 Introducción general 41 individuos de estas especies (capítulo 3). La construcción de infraestructuras para la práctica del esquí supone una degradación del hábitat característico de la alta montaña, con un incremento del riesgo de depredación asociado a esta degradación. Para hacer frente a este incremento en el riesgo, los animales llevan a cabo estrategias antidepredatorias preventivas, como la selección de hábitats seguros. De esta forma, aunque hay diferencias en los microhábitats disponibles entre las pistas de esquí y las zonas de vegetación natural, los machos de lagartija serrana, Lacerta monticola, seleccionan microhábitats similares en ambas zonas. Las lagartijas seleccionan microhábitats con poca cobertura de árboles y arbustos, y con sustratos rocosos cerca de áreas despejadas dónde termorregular adecuadamente (Hertz y Huey 1981; Carrascal et al. 1992) mientras minimizan el riesgo de depredación al estar cerca de rocas que les sirven de refugio (Martín y Salvador 1995). Sin embargo, las pistas de esqui tienen una escasa cobertura de vegetación y de rocas, lo que implica una limitación en la disponibilidad de refugios para las lagartijas. Por lo tanto, aunque seleccionan microhábitats seguros, cuando necesitan desplazarse por todo su territorio, las lagartijas deben moverse frecuentemente a través de áreas inseguras, lejos de refugios, por lo que están más expuestas a los depredadores en las pistas de esquí que en las zonas no degradadas. Sin embargo, las lagartijas son capaces de percibir este incremento en el riesgo de depredación y presentan estrategias antidepredatorias preventivas para reducir el riesgo, modificando sus patrones de locomoción al moverse por áreas inseguras. Las lagartijas se desplazan a mayor velocidad en zonas degradadas que en zonas naturales, de forma que así disminuyen el tiempo expuestos a los depredadores cuando tienen que atravesar áreas lejos de refugios. Estudios anteriores han demostrado la habilidad de las presas para modificar sus patrones de locomoción para minimizar el riesgo de depredación (McAdam y Kramer 1998; Bakker y van Vuren 2004; Amo et al. 2005). Capítulo 1 Introducción general 42 Fig. 2 Media (+ SE) de la velocidad (cm/seg) de machos adultos de Lacerta monticola mientras se movían en zonas naturales o degradadas por las pistas de esquí. Sin embargo, moverse a gran velocidad es costoso (Kramer y Mclaughlin 2001; Gleeson y Hancock 2002) y los resultados muestran que las lagartijas que viven en pistas de esquí sufren una pérdida de condición corporal a lo largo del período de actividad. Esta pérdida de condición podría ser debida a otros factores, pero los resultados de un experimento de laboratorio muestran que los machos que tuvieron que correr frecuentemente disminuyeron su condición corporal, por lo que se puede concluir que, en condiciones naturales, moverse frecuentemente a gran velocidad supone una pérdida de condición corporal. Además, los resultados de laboratorio muestran que la velocidad de carrera no depende de la condición corporal, por lo que este estudio proporciona una nueva evidencia de que las estrategias comportamentales preventivas para minimizar el riesgo de depredación afectan negativamente a la condición de los individuos, como se ha demostrado anteriormente (Pérez-Tris et al. 2004). La pérdida de condición corporal de los machos que viven en pistas de esquí podría tener consecuencias importantes porque puede provocar una disminución en los recursos destinados al sistema inmune para la defensa contra los parásitos (Cooper et al. 1985; Smallridge y Bull 2000). Los resultados no muestran diferencias significativas en la respuesta inmune mediada por linfocitos T ni en la intensidad de infección por hemogregarinas en relación al nivel de degradación del habitat. Sin embargo, nuestros resultados no son concluyentes debido la pequeña muestra tomada. Capítulo 1 Introducción general 43 Fig. 3 Media (+ SE) del peso (g) de machos adultos de Lacerta monticola durante el período reproductor (primavera) y después del período reproductor (verano) en áreas con vegetación natural (cajas negras) y en áreas deterioradas (cajas blancas). Este estudio tiene implicaciones para el diseño de planes de conservación para esta especie y, ya que en general todas las especies de lagartijas seleccionan distancias a refugios menores de lo esperado por su disponibilidad, se podría aplicar a otras especies. Dado que la degradación del hábitat en las pistas de esquí provoca una pérdida de condición corporal, con importantes consecuencias para la eficacia biológica de los individuos, y por tanto, para el mantenimiento de las poblaciones, se deberían llevar a cabo estudios de impacto ambiental a la hora de abrir nuevas pistas de esquí (Stumpel et al. 1992; Martín y Salvador 1995, 1997). Estos estudios deberían tener en cuenta no sólo la presencia o abundancia de individuos de una especie, sino que también deberían examinar la condición corporal y el estado de salud de los individuos de las poblaciones afectadas. Además, este estudio pone de manifiesto la importancia de una alta disponibilidad de refugios para el mantenimiento de las poblaciones de lagartija serrana, como se ha observado en otras especies de reptiles (Schlesinger y Shine 1994; Hecnar y M'Closkey 1998). Por lo tanto, una manera efectiva de disminuir los efectos negativos de la degradación del hábitat en las pistas de esquí sería la restauración artificial de refugios para crear corredores seguros que comuniquen áreas no degradadas. De esta forma, las lagartijas no tendrían que desplazarse a tanta velocidad porque tendrían cerca refugios en los que esconderse y por tanto, no sufrirían los costes de las carreras sobre su condición corporal. El empleo de refugios artificiales ha demostrado su eficacia en la restauración y mantenimiento de otras especies de reptiles amenazadas (Hecnar y M'Closkey 1998; Webb y Shine 2000; Souter et al. 2004). Capítulo 1 Introducción general 44 La degradación del habitat afecta a la condición corporal y a la carga parasitaria en las hembras de lagartija colilarga, Psammodromus algirus, debido a cambios forzados en el comportamiento antidepredatorio La degradación de la vegetación, y el consiguiente riesgo de depredación no sólo afectan a las estrategias antidepredatorias preventivas, sino que este incremento en el riesgo puede hacer que los animales sean más conspicuos y, por tanto, deban realizar estrategias de escape frecuentemente, sufriendo así los costes de estas estrategias. Los resultados de un estudio en el que se examinó la selección de hábitat, el comportamiento antidepredatorio, y la condición física y de salud de individuos de P. algirus en áreas con distinto nivel de degradación de la vegetación se muestran a continuación (capítulo 3). Igual que en el experimento anterior, aunque las zonas con distinto nivel de degradación de la vegetación presentan una disponibidad de microhábitats distinta, las lagartijas seleccionan lugares similares en ambas zonas. Sin embargo, esto no parece evitar una distinta detectabidad de los individuos en ambas zonas, concretamente las hembras fueron detectadas a distancias mayores en zonas con vegetación degradada que en zonas con vegetación natural, como se ha observado con anterioridad en camaleones (Cuadrado et al. 2001). La falta de diferencias en la detectabilidad de los machos entre áreas se podría explicar porque los machos se mueven más que las hembras durante el período reproductor (Salvador et al. 1995, 1996; Martín y López 1999c) y por su coloración más vistosa (Díaz 1993; Salvador et al. 1995) que les hace probablemente muy conspicuos en ambas zonas. Las hembras parecen compensar su mayor detectabilidad en zonas degradadas presentando mayores distancias de aproximación frente a los depredadores que en zonas naturales. Este resultado concuerda con modelos teóricos de escape (Ydenberg y Dill 1986). Por lo tanto, las hembras comienzan a escapar antes, como se ha observado en estudios previos con ésta (Martín y López 1995, 1999c, 2000b) y otras especies (Cooper 1998, 2003). Sin embargo, y al contrario de lo que predicen los modelos teóricos (Dill 1990; Bonenfant y Kramer 1996) y resultados anteriores con esta especie Capítulo 1 Introducción general 45 (Martín y López 2000b), los resultados no muestran ninguna relación entre la distancia de aproximación y la distancia al refugio, por lo que parece que el incremento en la probabilidad de detección por los depredadores, debido a la degradación de la vegetación, afecta más a las decisiones de escape que la distancia a refugios. Sin embargo, la distancia al refugio más cercano sí que afecta al tipo de estrategia de escape empleada y a las distancias de huida. Cuando las lagartijas estaban lejos de refugios, huían sin esconderse y presentaban recorridos más largos durante su huída. Mientras que cuando los refugios estaban cerca escapaban para esconderse en un refugio. Fig. 4 Media (+ SE) de la distancia de aproximación de machos (cajas negras) y hembras (cajas blancas) adultos de Psammodromus algirus en zonas naturales y degradadas de robledales. Sin embargo, llevar a cabo una estrategia de escape es costoso en términos energéticos (McNamara y Houston 1990) y los resultados muestran que las hembras de zonas degradadas, que exhibían distancias de aproximación mayores ante el ataque de un depredador tenían menor condición corporal. Esto puede disminuir la respuesta inmune y aumentar la carga parasitaria, lo que conllevaría costes fisiológicos mayores. Los resultados mostraron que, aunque la intensidad de garrapatas, el vector que transmite las hemogregarinas es mayor en zonas naturales, las intensidades de parásitos sanguíneos, hemogregarinas, son mayores en zonas degradadas. Además, se observó una relación negativa entre la intensidad de hemogregarinas y la condición corporal, fundamentalmente en hembras, mientras que en zonas naturales no hubo tal relación. Estos resultados muestran que los efectos deletéreos de los parásitos son más evidentes cuando otro factor, como la degradación del hábitat influye en la condición de las hembras, en concordancia con otros estudios previos realizados en condiciones seminaturales (Oppliger et al. 1998). Capítulo 1 Introducción general 46 Fig. 5 Media (+ SE) del peso (g) de machos (cajas negras) y hembras (cajas blancas) de Psammodromus algirus en áreas naturales y degradadas de robledales. La pérdida de condición corporal de las hembras puede tener efectos negativos en su eficacia biológica, ya que se ha observado que hembras con baja condición corporal producen descendencia de menor tamaño (Shine y Downes 1999, pero ver también Gregory y Skebo 1998). Además, las hembras con intensidades altas de infecciones sanguíneas producen puestas con un menor número de huevos (Schall 1983). Ya que el tamaño de los recién nacidos afecta a su probabilidad de supervivencia (p.e. Ferguson y Fox 1984; Sinervo et al. 1992), el efecto negativo de la degradación del hábitat en la condición corporal y estado de salud de las hembras podría poner en peligro la continuidad de estas poblaciones que viven en hábitats degradados. Esto podría explicar la desaparición de las poblaciones de esta especie en hábitats fragmentados que deben mantener una alta presión de depredación (Díaz et al. 2000). Otros factores podrían estar afectando a la condición de las hembras además del incremento en el riesgo asociado a la degradación del medio, como por ejemplo, que la escasa cobertura de matorral en zonas degradadas pudiera empobrecer el ambiente térmico (Díaz y Carrascal 1991). Esto podría implicar más costes de termorregulación para las lagartijas (Díaz 1997), que no podrían invertir tanto tiempo y energía en crecimiento y defensa antiparasitaria, mostrando entonces peor condición corporal. Dado que las hembras están más confinadas a un área más pequeña que los machos, no podemos rechazar esta hipótesis, por lo que son necesarios más estudios para valorar más finamente el efecto de la degradación de la vegetación de Capítulo 1 Introducción general 47 robledales en estas poblaciones de lagartijas. Efectos del ecoturismo en las estrategias de escape y uso de refugios y su efecto en la condición y estado de salud de lacértidos Ecoturismo como una forma de riesgo de depredación afecta a la condición corporal y estado de salud en lagartijas roqueras, Podarcis muralis El ecoturismo puede suponer, al igual que la degradación de la vegetación, un incremento en el riesgo de depredación (Frid y Dill 2002). Los resultados de este estudio realizado con la lagartija roquera (capítulo 4) en áreas con distinta afluencia de visitantes muestran que, en general las lagartijas respondieron a las aproximaciones del experimentador como a las de un depredador, exhibiendo respuestas antidepredatorias, como se había descrito previamente (Martín y López 1999a). Sin embargo, no presentaron diferencias en su respuesta antidepredatoria en función del nivel medio de ecoturismo de la zona. Así, en relación a estrategias preventivas, no hubo diferencias entre zonas en la selección de microhábitats para minimizar el riesgo, es decir, presentaron distancias al refugio más cercano similares entre zonas, lo que se corresponde con estudios previos (Diego-Rasilla 2003). Además a la hora de realizar una estrategia de escape, las lagartijas respondieron ante el nivel de riesgo de depredación ejercido por el experimentador y no al nivel habitual de depredación de la zona. Mostraron distancias de aproximación y huída, y tipos de estrategias similares entre zonas con distintos niveles de ecoturismo. Sin embargo, estas estrategias antidepredatorias son costosas en términos de condición corporal y los resultados sugieren que las lagartijas que viven en zonas con una alta afluencia de visitantes muestran peor condición corporal que las lagartijas que viven en zonas sin ecoturismo, especialmente en el transcurso del período de actividad anual, debido probablemente a la alta frecuencia de comportamientos antidepredatorios que las lagartijas deben realizar en respuesta a los transeúntes. Una respuesta alternativa sería que las lagartijas en peor condición corporal no pudieran realizar una mayor Capítulo 1 Introducción general 48 respuesta antidepredatoria en zonas turísticas porque no pudieran afrontar los costes de esta respuesta (Beale y Monaghan 2004b). Sin embargo, nuestra explicación está de acuerdo con un estudio previo que muestra que las lagartijas sometidas a una alta frecuencia de aproximaciones de una persona incrementaron el uso de refugios y sufrieron una pérdida de condición corporal (Martín y López 1999a). Esta pérdida de peso puede tener consecuencias importantes ya que las actividades reproductivas también provocan una pérdida de peso durante la estación reproductora (Amo et al. 2004). Sin embargo, una vez que el período reproductor ha acabado, las lagartijas deben recuperar peso para afrontar el período invernal. Por lo tanto, si el comportamiento antidepredatorio impide a las lagartijas recuperar la condición corporal, puede que no sobrevivan a la hibernación, con el consiguiente incremento de la mortalidad y su efecto para el mantenimiento de estas poblaciones. Además los resultados sugieren que la respuesta inmune mediada por linfocitos T está positivamente relacionada con la condición corporal de las lagartijas y negativamente correlacionada con la intensidad de infección por parásitos sanguíneos, hemogregarinas. Fig. 6. Media + SE del peso (g) de individuos de Podarcis muralis en areas con bajo (cajas negras) y alto nivel de ecoturismo (cajas blancas) en primavera y verano. Por tanto, una pérdida de condición corporal podría implicar una disminución de la respuesta inmune y por tanto, exponer a las lagartijas a los efectos deletéreos de los parásitos. Esto podría explicar la mayor intensidad de infección por ácaros en zonas turísticas. Sin embargo, los resultados no muestran ningún efecto de los ácaros en la condición corporal. Un resultado interesante es que las lagartijas de mayor tamaño muestran mayores niveles de Capítulo 1 Introducción general 49 hemogregarinas en las zonas turísticas que en las zonas naturales al final de la estación reproductora pero no durante esta. Esto podría explicarse porque en las áreas sin ecoturismo, las lagartijas más grandes, y probablemente más viejas deben tener una condición lo suficientemente buena para hacer frente y disminuir la infección, mientras que en áreas con ecoturismo, la pérdida de condición corporal debe conllevar una mayor intensidad de infección. Además este efecto puede incrementarse debido a la mayor intensidad de vectores transmisores de estos parásitos sanguíneos. Fig. 7 Relación entre la respuesta inmune celular mediada por linfocitos T y el peso (g.) Por tanto, el ecoturismo puede afectar al mantenimiento de las poblaciones de lagartijas, y esto debería ser considerado a la hora de diseñar caminos en áreas protegidas para conservar la biodiversidad de lagartijas. Además, este estudio aporta evidencias de que, independientemente de que las lagartijas muestren estrategias comportamentales de escape similares en áreas con y sin turistas, la condición corporal de los individuos debe ser examinada para valorar adecuadamente el posible efecto real del ecoturismo sobre las poblaciones de lagartijas. Aunque este estudio sugiere un efecto deletéreo del ecoturismo, estudios a largo plazo son necesarios para un mejor entendimiento de este efecto en las poblaciones de reptiles. La flexibilidad en el uso de refugios ayuda a la lagartija serrana, Lacerta monticola, a enfrentarse al riesgo de depredación sin incurrir en una pérdida de condición corporal En este estudio se analizaron las estrategias antidepredatorias de las lagartijas en relación no sólo al nivel de ecoturismo reflejado por la cercanía a los caminos sino también en relación a los cambios en la vegetación debido a las infraestructuras del esquí (capítulo 4). Capítulo 1 Introducción general 50 En relación al nivel de ecoturismo (estimado a partir de la distancia al camino más cercano), no se observaron diferencias en las estrategias preventivas, como la distancia al refugio, ni en las estrategias de escape de las lagartijas. Además, y al contrario de los resultados mostrados en el experimento anterior, tampoco se observaron diferencias en la condición corporal de las lagartijas en relación al nivel de ecoturismo. Sin embargo, esta falta de diferencias en la condición corporal podría deberse a que este experimento se realizó sólo durante el período reproductor de esta especie. Los resultados del experimento anterior sugirieron que la pérdida de condición corporal de las lagartijas es más evidente en el período post-reproductor, cuando los individuos han tenido que realizar, a lo largo de todo el período reproductor, estrategias de escape frecuentes. Por lo tanto, sería necesario repetir este experimento al finalizar el período reproductor para poder establecer unas conclusiones más firmes. Los resultados de este estudio muestran que las lagartijas modifican su comportamiento antidepredatorio, tanto preventivo como de escape en relación al riesgo de depredación impuesto por la degradación de la vegetación, de forma que esta flexibilidad en el comportamiento antidepredatorio les permitiría afrontar el riesgo y disminuir los costes del uso de los refugios, manteniendo así su condición corporal a pesar de vivir en zonas con vegetación degradada. Así, las lagartijas en zonas con alto riesgo de depredación debido a la degradación de la vegetación seleccionan hábitats cerca de refugios. De esta forma, minimizan la distancia de huída que, como se ha demostrado en este y estudios anteriores, depende de la distancia al refugio (Dill 1990; Bulova 1994; Bonenfant y Kramer 1996; Cooper 1997; Martín y López 2000a, b). Así minimizan los costes de moverse a gran velocidad hasta alcanzar el refugio que previamente se ha mostrado que influyen en la condición corporal. Sin embargo, las distancias de aproximación no difirieron entre áreas con distinto nivel de degradación de la vegetación, aunque el uso de refugios si difirió. Tanto el tiempo de aparición como de emergencia del refugio varió en relación al nivel de degradación de la vegetación. Las lagartijas aparecieron y emergieron antes del refugio en zonas degradadas que en zonas naturales. Capítulo 1 Introducción general 51 Fig. 8. Media (+ SE) del tiempo de emergencia de lagartijas Lacerta monticola en áreas naturales o áreas con la vegetación degradada. Aunque el riesgo de depredación habitual debe ser mayor en zonas degradadas, el riesgo impuesto por el experimentador era similar en ambas áreas, por lo que un menor tiempo en el refugio podría ser una estrategia para minimizar los costes asociados al uso de refugios en áreas en las que las lagartijas deben esconderse en los refugios más frecuentemente que en áreas naturales. Esto parece ser muy importante porque los resultados de los experimentos de laboratorio muestran que la pérdida de oportunidades de alimentación, y la disminución de la tasa de ingestión, asociados a un incremento en el uso de refugios, provocan una pérdida de condición corporal. Por el contrario, la falta de oportunidades de termorregulación o la disminución potencial de la eficacia de la digestión a bajas temperaturas dentro de los refugios no afectó a la condición física de los individuos. Por lo tanto, aunque la disminución de la eficiencia en la digestion se había sugerido como un coste potencial del uso de refugios (Martín y López 1999a), estos resultados no apoyan esta asunción. Por el contrario, la falta de oportunidades de termorregulación sí parece ser un coste importante del uso de refugios para hembras preñadas, de forma que hembras que no pudieron termorregular no ganaron tanto peso como las hembras que pudieron mantener una temperatura corporal óptima. Además, las hembras que no pudieron termorregular experimentaron una menor respuesta inmune que las hembras controles. Sin embargo, no se encontraron diferencias en la intensidad de infección por hemogregarinas, aunque esto puede ser debido a que hubo diferencias iniciales en la intensidad entre grupos. Además, la poca ganancia de condición corporal en las hembras que no termorregularon podría deberse a esta mayor intensidad de infección, por lo que no podemos concluir que las bajas Capítulo 1 Introducción general 52 temperaturas per se causaran un efecto directo en la condición corporal. Muchos procesos fisiológicos de las lagartijas dependen de una temperatura corporal óptima (Huey 1982; Stevenson et al. 1985), por lo que probablemente las bajas temperaturas impliquen también una disminución de la respuesta inmune, lo que podría incrementar los efectos deletéreos de los parásitos en la condición de las hembras. Nuestros datos no permiten examinar esta hipótesis, por lo que son necesarios más estudios para revelar otros posibles costes fisiológicos del uso de refugios que no habían sido considerados hasta la fecha. La peor condición de las hembras puede tener efectos importantes para su eficacia biológica como se ha señalado anteriormente (Shine y Downes 1999, pero ver también Gregory y Skebo 1998). Fig. 9 Media (+ SE) de a) peso (g) de machos de Lacerta monticola, antes y después de un experimento diseñado para analizar los efectos de la pérdida de oportunidades de alimentación debida al uso de refugios; b) Media (+ SE) del tiempo de emergencia del refugio (seg.) de estos machos al sufrir dos ataques sucesivos al final del experimento. Capítulo 1 Introducción general 53 Además los resultados de los experimentos de laboratorio muestran que las lagartijas parecen ser capaces de usar los refugios de forma que disminuyen el riesgo de depredación pero sin incurrir en los costes del uso, pero esto depende de su condición corporal. Las lagartijas en peor condición corporal no parecieron poder afrontar los costes del uso de refugios y disminuyeron el tiempo en el refugio ante un ataque, mientras que las lagartijas con buena condición permanecieron más tiempo escondidas. El uso de refugios en función del estado nutricional ha sido predicho por modelos teóricos (Sih 1992, 1997; Dill y Fraser 1997; Martín y López 1999b) y se ha observado también en percebes (Dill y Gillett 1991), peces (Krause et al. 1998, pero ver también Dowling y Godin 2002), y paseriformes (Koivula et al. 1995). En el caso de los machos de lagartija, las diferencias en el tiempo de emergencia del refugio fueron más notables después del segundo ataque, mientras que en el caso de hembras preñadas estas diferencias fueron notables también durante el primer ataque. Los costes del uso de refugios aumentan a medida que aumenta el tiempo en el refugio (Martín y López 1999b), por lo que los resultados sugieren que el mantenimiento de una condición corporal adecuada es más importante para las hembras preñadas que para los machos. Además, durante la gestación, las hembras necesitan mantener temperaturas corporales constantes para maximizar la tasa de desarrollo de los embriones (Mathies y Andrews 1997; Shine 2004). De esta forma, las hembras reducen los períodos de incubación y, por tanto, los costes de la reproducción (Shine 1980, 1983; Seigel y Fitch 1984; Shine y Downes 1999). Además, los períodos de incubación cortos pueden incrementar la eficacia biológica de la descendencia en zonas con temperaturas ambientales limitantes porque los juveniles tendrán más tiempo para alimentarse y crecer antes de la hibernación (Mathies y Andrews 1997, pero ver también Shine y Olsson 2003). Por lo tanto, al emerger antes del refugio, las hembras no sólo reducen los costes para ellas sino para su descendencia. En resumen, la degradación de la vegetación afecta a las estrategias antidepredatorias, pero, al contrario de lo Capítulo 1 Introducción general 54 observado en la lagartija colilarga, P. algirus, la lagartija serrana modifica estas estrategias de forma que disminuye el uso de refugios en zonas con vegetación degradada, disminuyendo así los costes deletéreos del uso de refugios. Las diferencias en la flexibilidad del comportamiento antidepredatorio entre ambas especies pueden deberse a las distintas condiciones ambientales de las zonas en las que viven, ya que en el caso de la lagartija serrana, las temperaturas ambientales son en muchos casos limitantes debido a que vive a mayor altitud, por lo que el uso de refugios debe ser mucho más costoso en términos fisiológicos. Esta presión probablemente ha favorecido una mayor flexibilidad en las estrategias antidepredatorias en esta especie. Efectos del ecoturismo en el incremento en el riesgo de depredación debido a múltiples depredadores Un incremento en el nivel de ecoturismo puede suponer un mayor empleo de las estrategias antidepredatorias, y por tanto, las lagartijas pueden sufrir sus costes, de pérdida de condición corporal, como se ha mostrado anteriormente, pero también el incremento del riesgo de ser capturadas por culebras saurófagas que cazan al acecho en el interior de los refugios. Sin embargo, la flexibilidad en el comportamiento antidepredatorio puede ayudar a las lagartijas a hacer frente al riesgo de depredación debido al ecoturismo sin incurrir en los costes de depredación dentro de los refugios (capítulo 5). Capítulo 1 Introducción general 55 El nivel de riesgo y los costes térmicos afectan la elección de estrategia de escape y el uso de refugios en la lagartija roquera, Podarcis muralis Los resultados de este estudio muestran que las lagartijas roqueras fueron capaces de ajustar su respuesta antidepredatoria valorando el nivel de riesgo de depredación y los costes del uso de refugios (capítulo 5). Las lagartijas emplearon dos respuestas de escape secuenciales de creciente intensidad. Primero, intentaron evitar su detección, permaneciendo inmóviles, y si la detección ocurría, intentaron escapar tanto escondiéndose en un refugio como huyendo sin esconderse. El tipo de respuesta inicial dependió del nivel de riesgo ejercido por el depredador. Por lo tanto, cuando el depredador lanzaba un ataque directo, las lagartijas siempre huían, mientras que cuando el depredador se aproximaba indirectamente, la respuesta de la lagartija dependía de su valoración del riesgo de ser capturada, de forma que las lagartijas que estaban a mayor altura en el muro de piedra dónde se realizó el experimento, valoraban un menor riesgo de ser capturadas y permanecían quietas. Además, en una situación de bajo riesgo de depredación, los costes térmicos del refugio parecieron influir la decisión de escapar o confiar en la cripsis. Cuando la temperatura del refugio era baja, las lagartijas confiaron más en la cripsis que en el uso de refugios fisiológicamente costosos. La temperatura corporal, estimada por medio de la temperatura del aire, así como la distancia al refugio más cercano no influyeron en la respuesta de las lagartijas. El riesgo inicial de depredación tampoco influyó a la estrategia de escape una vez que las lagartijas habían empezado a escapar. Sin embargo, el tipo de estrategia pareció estar influido por la percepción de las lagartijas a ser capturadas mientras escapaban. Las lagartijas huyeron sin esconderse cuando percibieron un riesgo bajo, a mayor altura en el muro, y a mayor temperatura corporal. Por tanto, aunque la temperatura corporal no determinó la respuesta inicial (cripsis o escape), influyó en la decisión de esconderse en el refugio más cercano o huir del depredador sin esconderse. Además, cuando los costes térmicos del uso de refugios eran altos, las Capítulo 1 Introducción general 56 lagartijas huyeron del depredador sin esconderse, lo que indica que las decisiones de escape están basadas también en las consecuencias para la eficacia biológica a medio y largo plazo (Martín y López 2000a). Otro coste del uso de refugios que pudo afectar a las decisiones de escape es el riesgo de depredación por culebras saurófagas. Esto explicaría que la elección de la estrategia de escape estuviera determinada por la actividad inicial de las lagartijas y por la distancia al refugio más cercano. Una lagartija tomando el sol cerca de un refugio puede valorar si el refugio contiene señales químicas que indiquen la presencia de una culebra (Downes y Shine 1998). Por lo tanto, estas lagartijas probablemente tienen más información acerca del nivel de seguridad del refugio que las lagartijas que al estar moviéndose por el muro no necesariamente tienen información acerca de los refugios cercanos, por lo que emplean otra estrategia de escape, huir sin esconderse. Con esta flexibilidad en las estrategias de escape, las lagartijas pueden evitar el riesgo de depredación por dos tipos de depredadores (Sih et al. 1998). El riesgo inicial de depredación (alto vs bajo) afectó el uso de refugios de las lagartijas. Las lagartijas incrementaron el tiempo de aparición de los refugios cuando el riesgo de depredación era alto. Sin embargo, una vez que salieron del refugio reanudaron la actividad en un intervalo de tiempo independiente del nivel de depredación. Los costes térmicos del uso de refugios afectaron más a las lagartijas en la situación de alto riesgo de depredación, como se ha observado en estudios anteriores (Martín y López 1999b). Esto se debe a que cuando la lagartija entra en un refugio como estrategia preventiva, los costes del refugio afectan menos porque probablemente salga antes de que los costes se lo exigan. En conclusión, las estrategias antidepredatorias de las lagartijas roqueras estuvieron influidas no sólo por la probabilidad inmediata de mortalidad, estimada por el riesgo inicial impuesto por el depredador y los factores que afectaron a la percepción de las lagartijas de su susceptibilidad sino también por las consecuencias a largo plazo para su eficacia biológica, como los costes fisiológicos del uso de refugios y el riesgo de depredación por culebras. Capítulo 1 Introducción general 57 Las lagartijas roqueras combinan las señales químicas y visuales de culebras depredadoras que cazan al acecho para evitar sobreestimar el riesgo de depredación en el interior de los refugios Las lagartijas roqueras son capaces de discriminar las señales químicas de las culebras saurófagas Coronella austriaca, como lo demostró una mayor tasa de extrusiones linguales y menor latencia a la primera extrusión en respuesta a algodones impregnados con estas señales. Por tanto, esta habilidad puede permitir a las lagartijas evitar entrar en refugios inseguros (capítulo 5). Fig. 10 Media + SE del número total de extrusiones linguales (TF) en respuesta a estímulos de agua, colonia, lagartija serrana y culebra lisa presentados a individuos de Podarcis muralis en aplicadores de algodón. Además las lagartijas respondieron a la presentación de estas señales huyendo rápidamente del algodón, lo que sugiere que en una situación natural la primera respuesta de las lagartijas cuando detectan señales de una culebra sería huir rápidamente de la zona. Los resultados del segundo experimento sugirieron que las lagartijas son capaces de usar tanto las señales químicas como las visuales para valorar el riesgo de depredación en el interior de un refugio. El tiempo que tardaron en esconderse no dependió del nivel de riesgo del refugio, lo que sugiere que el riesgo de ser capturado por el depredador en el exterior es más importante que el riesgo eventual de encontrar una culebra, o bien que las lagartijas no tuvieron tiempo de valorar el riesgo dentro del refugio ante el ataque del depredador en el exterior. Esto sugeriría un caso de facilitación de la depredación. Sin embargo, el tiempo dentro del refugio estuvo relacionado con las señales de la culebra que encontraron en su interior. El tiempo de aparición después del segundo ataque fue mayor cuando las lagartijas encontraron señales visuales que químicas, lo que sugiere que las Capítulo 1 Introducción general 58 lagartijas discriminan más rápido este tipo de señales que las visuales ya que las culebras son muy crípticas en el interior de las grietas y sus señales químicas deben proporcionar signos evidentes de su presencia (Van Damme et al. 1995; Kats y Dill 1998). Una explicación alternativa sería que las lagartijas necesitan más tiempo para mirar a través de la pared de cristal del terrario que para realizar una extrusión lingual. También podrían haber detectado que la culebra estaba realmente fuera del refugio (W. E. Cooper, Jr, comunicación personal). Los resultados del primer experimento sugieren que las lagartijas pueden detectar la presencia de la culebra empleando únicamente señales químicas, sin embargo, una vez que aparecen del refugio, las lagartijas esperan menos tiempo antes de abandonarlo cuando el refugio contiene señales químicas y visuales que sólo cuando estuvieron presentes las señales químicas. Esto se debe a que la detección química de la culebra indica a las lagartijas que el refugio era arriesgado en un determinado momento, pero no indica necesariamente un riesgo actual, por tanto las lagartijas abandonaron el refugio rápidamente sólo cuando vieron a la culebra. Por tanto, los resultados indican que las señales visuales son importantes para confirmar el nivel de riesgo potencial que implican las señales químicas. Nuestros resultados confirman la asunción de la hipótesis de sensibilidad al riesgo ya que la respuesta antidepredatoria de las lagartijas fue mayor cuando estuvieron expuestas a señales químicas y visuales que cuando sólo había una señal en el refugio. Las lagartijas aparecieron y emergieron antes del refugio cuando ambas señales estaban presentes simultáneamente. Resultados similares se han observado en peces (Smith y Belk 2001), aunque también se ha observado que otras especies como Pimephales promelas, responden más a las señales químicas en ausencia de las visuales y cuando el riesgo percibido es alto (Hartman y Abrahams 2000). Además las larvas de la salamandra Notophthalmus lousianensis, distinguían entre especies depredadoras y no depredadoras sólo cuando disponían de señales químicas pero cuando las señales visuales están presentes evitan a ambas especies (Mathis y Vincent 2000). Por el contrario, los peces Cottus cognatus, Capítulo 1 Introducción general 59 muestran un comportamiento de evitación sensible al riesgo sólo cuando están expuestos a señales visuales pero no a señales químicas (Chivers et al. 2001). Las diferencias entre especies en la respuesta comportamental a señales químicas y visuales parecen depender de las condiciones ambientales. Por ejemplo, las lagartijas confían más en las señales químicas porque la visibilidad dentro de los refugios donde pueden encontrar a las culebras depredadoras es muy limitada. Además, la respuesta de las presas a diferentes niveles de información del depredador parece depender de las condiciones ambientales (van der Veen 2002). Fig. 11 Media + SE de las diferencias en el tiempo de espera de P. muralis en refugios control, y refugios que contenían señales químicas, visuales y ambas después de dos ataques sucesivos (cajas blancas: primer ataque; cajas negras: segundo ataque). Los depredadores de búsqueda activa en el exterior pueden forzar a las lagartijas a incrementar su uso de refugios incluso cuando el riesgo de depredación por culebras que cazan al acecho es alto. Por otro lado, la presencia de las culebras en el refugio hace que las lagartijas disminuyan su uso de refugios, exponiéndose al riesgo de depredación en el exterior. Esto podría causar un incremento en el riesgo para la presa, como se ha observado en otras especies (Soluk 1993; Korpimäki et al. 1996). Los resultados de este estudio apoyan esta idea. Las lagartijas probablemente no puedan eludir al depredador en el exterior sin esconderse en un refugio y no pueden modificar el tiempo que tardan en entrar al refugio en relación al riesgo en el interior de este. Sin embargo, los resultados muestran que la habilidad de identificar varias señales de las culebras puede mejorar la valoración del riesgo de depredación en el interior del refugio. Esta habilidad puede ayudar a las lagartijas a reducir el incremento en el riesgo causado por el efecto de la actuación simultánea de dos depredadores que requieren respuestas conflictivas. Capítulo 1 Introducción general 60 La valoración química del riesgo de depredación está influida por el tiempo de exposición a las señales químicas de culebras que cazan al acecho en la lagartija roquera, Podarcis muralis. Los resultados de este estudio también sugieren que las lagartijas fueron capaces de detectar las señales químicas de las culebras y usarlas a corto plazo para valorar el riesgo de depredación potencial dentro de un refugio, pero al cabo de un tiempo las lagartijas valoraban de nuevo el riesgo para detectar si la culebra estaba realmente presente en el interior del refugio y modificaban su respuesta (capítulo 5). Para evitar el riesgo por culebras, las lagartijas modifican su comportamiento y el uso de refugios potencialmente inseguros. Durante los primeros minutos las lagartijas usaron por igual los refugios con señales químicas que los refugios control, probablemente porque estuvieron investigando los olores de los refugios. Sin embargo, después de este tiempo y cuando probablemente habían identificado la fuente del olor, disminuyeron el tiempo pasado en los refugios con señales químicas de una culebra mientras que aumentaron el uso de los refugios seguros, lo que concuerda con resultados anteriores (Downes y Shine 1998; Downes y Bauwens 2002). Fig. 12 Porcentaje de tiempo (media + SE) pasado en el refugio experimental en relación al tiempo total pasado en el area experimental del terrario, cuando el refugio era “control” (cajas blancas) o tenía señales químicas de una culebra “depredador” (cajas negras). Las lagartijas también modificaron sus patrones de locomoción en el tratamiento con olor de culebra, aumentando el comportamiento de escape, como se ha observado en ésta (Amo et al. 2005) y otras especies (Downes y Bauwens 2002). 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Por lo tanto el objetivo de este capítulo es profundizar en el conocimiento de las relaciones parásito-hospedador en reptiles, para lo cual se han estudiado diversas especies de parásitos que afectan a varias especies de lacértidos de la Sierra de Guadarrama. Los resultados de estos estudios se recogen en tres publicaciones que se muestran a continuación. Capítulo 2 Relaciones hospedador-parásito 77 Prevalence and intensity of haemogregarinid blood parasites in a population of the Iberian rock lizard, Lacerta monticola RESUMEN El estudio del efecto de los parásitos en las poblaciones de sus hospedadores es esencial para entender su papel en la dinámica de poblaciones y ecología del hospedador. En este estudio, describimos la biología general de las hemogregarinas en una población de la lagartija serrana, Lacerta monticola, y examinamos los factores determinantes de la prevalencia e intensidad de infección. Los resultados muestran que tanto la prevalencia como la intensidad de infección fueron mayores en adultos que en juveniles. La tasa de prevalencia fue mayor en lagartijas de mayor tamaño, probablemente porque las lagartijas de mayor tamaño son también más viejas y por tanto, han estado más a menudo en contacto con parásitos durante su vida que las lagartijas más pequeñas y jóvenes. Durante la estación reproductora, la intensidad de infección fue mayor en machos que en hembras, debido probablemente a los efectos inmunosupresores de la testosterona. La intensidad de la infección tuvo un efecto negativo en la condición corporal de los individuos durante la época reproductora. Estos resultados sugieren que las interacciones entre parásitos y hospedadores no son estables en esta población de lagartijas. Capítulo 2 Relaciones hospedador-parásito 79 Prevalence and intensity of haemogregarinid blood parasites in a population of the Iberian rock lizard, Lacerta monticola Abstract The study of the effect of parasites on their host populations is essential for understanding their role in host population dynamics and ecology. We describe the general field population biology of haemogregarines in the Iberian rock lizard, Lacerta monticola, examining the factors that determine the prevalence and intensity of infection. Prevalence and infection intensity were higher in adults than in juvenile lizards. The prevalence rate was higher in larger lizards, probably because they were also older and had been more often in contact with parasites during their lifetime. During the mating season, the intensity of parasite infection was greater in males than in females, probably due to immunosuppressive effects of testosterone. The parasite load had a negative effect on the body condition during the reproductive season. The results suggest that the interactions between parasites and hosts are not stable in this lizard population. Introduction The prevalence and intensity of a parasitic infection provide a first approach to understand the parasite’s impact on a natural population (Smallridge and Bull 2000). However, this remains little known for wildlife, natural populations, especially in the case of reptiles. This is important because parasitism causes deleterious effects on several aspects of the ecology and evolution of parasite hosts (Smallridge and Bull 2000; Eisen 2001), such as host population growth (Hudson et al. 1998), spatial distribution (Price 1980), reproductive success (Schall 1996; Oppliger et al. 1997), and sexual selection (Hamilton and Zuk 1982; Møller et al. 1999). The haemogregarines (family Haemogregorinidae, suborder Adeleorina, subclass Coccidiasina, phylum Apicomplexa; Barnard and Upton 1994) have an indirect life cycle involving definitive invertebrate hosts, Capítulo 2 Relaciones hospedador-parásito 80 and vertebrate hosts such as lizards, snakes or frogs (e.g. Smith et al. 1994; Caudell et al. 2002; Lainson et al. 2003). The zygote is active (ookinete), and the life cycle is heteroxenous, involving merogonous development and the formation of gametocytes in the circulatory system and/or visceral tissues of a vertebrate, and gamogony proper and sporogony within the gut of an invertebrate vector (Barnard and Upton 1994). Transmission to the vertebrate host occurs when this ingests a mite or a tick infected with this parasite, or by the bite of an arthropod or leech. The effects of haemogregarines on their hosts are poorly known. Haemogregarines take up over half of the volume of an infected erythrocyte and destroy this blood cell, causing anemia (Caudell et al. 2002; O’Dwyer et al. 2004). The presence of gametocytes in the erythrocytes indicates the existence of schizonts in internal organs, where they can cause additional damage (Svahn 1974; Veiga et al. 1998). To reduce these costs, hosts have evolved an elaborate antiparasite defense via the immune system (Sheldon and Verhulst 1996). However, there is often a trade- off between the immune response and other demands such as growth or reproduction (Sheldon and Verhulst 1996; Møller et al. 1999). To have a first approach in the understanding of the ecological interactions in the relationships between haemoparasites and lizards, we aimed to describe the general field population biology of haemogregarines in the Iberian rock lizard, Lacerta monticola. This is a small, insectivorous lacertid lizard endemic to some high mountains of the Iberian Peninsula (Salvador 1984; Pérez-Mellado 1998a). We specifically examined whether specific subpopulations of lizards were at more risk of infection than others, and whether some host parameters (e.g. sex, age or body condition) are important predictors of the degree of infection. Because the haemogregarina genus can only be characterized by oocyst stages within the invertebrate host (Levine 1982; Barnard and Upton 1994), at this moment we can only identify these parasites as haemogregarines, until the parasite life cycles are more fully known. Capítulo 2 Relaciones hospedador-parásito 81 Materials and methods We performed the study in the Guadarrama Mountains (Madrid Prov., central Spain) at an elevation range of 1,900–2,200 m. Granite rock boulders and screes, interspersed with shrubs (Cytisus oromediterraneus and Juniperus communis) and meadows, predominate at the study site (Martín and Salvador 1997). In this area, L. monticola (snout-to-vent length, SVL, of adult lizards ranges between 65 and 90 mm) lizards mate in May– June, and produce a single clutch in July (Salvador 1984; Pérez-Mellado 1998a). Lizards (n=119) were collected by noosing from May to September 2002, during the entire annual active period. Sampling was representative of the population of lizards. For instance, it is unlikely that infected lizards were easier to catch, because we were able to capture nearly every lizard we detected. We subdivided the study period into two seasons: ‘spring’ (May–June, i.e. from the beginning of the period of activity of lizards, and coinciding with their mating season) and ‘summer’ (July–September, i.e. after the mating period had finished). Each captured lizard was individually marked by toe clipping, sexed, weighed and measured for its snout–vent length (SVL). Lizards were classified as non-matured ‘juveniles’ (SVL<60 mm). A smear was made on a microscope slide from blood taken from the postorbital sinus by using a 9-µl heparinized hematocrit tube. The lizard was then released at the point of capture. Blood smears were air-dried. In the laboratory, the smears were fixed in absolute methanol for 10 min, and then stained in Giemsa diluted 1:9 with phosphate buffer (pH 7.2) for 40 min before their examination for parasites. On mounted slides, half a smear, chosen at random, was scanned entirely at 200·x along the length of the slide, looking for extraerythrocytic protozoa (Merino and Potti 1995). Numbers of intraerythrocytic parasites were estimated at 400 x by counting the number of parasites per 2,000 erythrocytes. The only haemoparasites found were haemogregarines. Parasite prevalence was estimated as the percentage of infected lizards. Parasite intensity was estimated for each infected host as the percentage of infected red blood cells found in approximately 2,000 cells. Capítulo 2 Relaciones hospedador-parásito 82 To compare the prevalence of infection between age classes, we used a chi-square test. To determine changes in prevalence between seasons, sex and body size (SVL) of adult lizards, we used generalized linear models (GLZM), with the parasite prevalence as the dependent variable, following a binomial distribution, and including the interactions between sex and size, and between sex and season in the model. Comparisons of the mean infection intensity of lizards between age classes are based on one-way ANOVA. Among adult lizards, we compared the infection intensity in relation to the SVL and sex of the lizard and the season by using general linear models (GLM), including the interaction between sex and season in the model. To examine the effect of sex, season and intensity of infection on body condition, we used the body mass of adult lizards in the GLM, correcting by means of the SVL and including the interactions between SVL, sex, season and intensity of infection in the model. Lizards can autotomize the tail when captured by a predator. This could affect their body condition because they have to allocate resources in the regeneration of the tail. Therefore, we compared initially the body mass of adult lizards with an analysis of covariance using the SVL as a covariate, and taking into account the tail condition (original complete vs. complete but regenerated vs. incomplete and in process of regeneration) as a categorical independent factor. There was a significant positive correlation between body mass and SVL (GLM, r = 0.74, F1,102 = 119.35, P < 0.0001), but there were no significant differences in relation to tail condition (F2,102 = 1.80, P = 0.17). Thus, we did not include tail condition in subsequent analyses. Results Overall population data The prevalence of infection of the surveyed population of L. monticola by haemogregarines was 77.3% (92 of 119 individuals). The intensity of infection ranged from 0 to 48.2 infected cells in 2,000 erythrocytes (X ± SE=4.4 ± 0.7). Among the 92 infected lizards, 78 showed an infection intensity of 10 or less infected cells in 2,000 erythrocytes, seven lizards displayed an intensity of 10–20 infected cells, five exhibited an Capítulo 2 Relaciones hospedador-parásito 83 infection of 20–30 infected cells, and only two lizards showed an intensity of >30 infected cells. Effects of season and of host age, sex and size The prevalence of infection was significantly higher in adults than in juveniles (infected/non-infected: 88/18 adults vs. 4/9 juveniles; χ2 = 18.02, d f= 1, P < 0.0001). The prevalence of infection in adult lizards did not significantly differ either between sexes (GLZM, Wald’s χ2 < 0.01, df = 1, P = 0.99), or between seasons (Wald’s χ2 < 0.01, df = 1, P = 0.99), but there were significant differences in relation to the body size of lizards (Wald’s χ2 = 5.84, df = 1, P = 0.02). Thus, the prevalence rate was higher in larger lizards (SVL, X ± SE = 74 ± 1 mm) than in smaller ones (70 ± 1 mm). The interactions between sex and season (Wald’s χ2 < 0.01, df = 1, P = 0.99), and between sex and size were not significant (Wald’s χ2=0.29, df=1, P=0.59). Infection intensity was significantly higher in adult lizards than in juveniles (adults: X ± SE = 4.7 ± 0.7 infected cells/2,000 erythrocytes; juveniles: 1.7 ± 1.3; one-way ANOVA, F1,117 = 7.77, P = 0.006). The infection intensity among adult lizards did not significantly differ on average between sexes, or between seasons, nor in relationship to body size, and these variables were removed from the GLM, but the effect of the interaction between sex and season was significant (GLM, r = 0.27, F1,104 = 8.05, P = 0.005). Thus, males showed significantly higher levels of infection intensity than females during spring (males: X ± SE = 7.7 ± 2.3; females: 2.6 ± 1.3 infected cells/2,000 erythrocytes; Tukey’s test, P = 0.04), but not in summer (males: 4.1 ± 1.3; females: 5.0 ± 1.0 infected cells/2,000 erythrocytes; Tukey’s test, P =0.24). Effects of host body condition After removing the effect of covariation of body mass with SVL (GLM, r = 0.82, F1,101 = 147.36, P < 0.0001), the body condition of males was significantly higher than that of females (F1,101 = 23.68, P < 0.0001), and all lizards had a significantly lower body condition in summer (F1,101 = 14.16, P = 0.0003) but, on average, the intensity of infection did not significantly affect body condition, and was removed from the model. However, the interaction Capítulo 2 Relaciones hospedador-parásito 84 between season and parasite load was significant (F1,101 = 5.81, P = 0.02). Thus, during spring there was a negative relationship between body condition and parasite load, whereas in the summer the relationship tended to be positive. The rest of the interactions were non- significant, and were removed from the model. Discussion More adults than juveniles were infected, and adults showed higher levels of parasite load than juveniles. A similar situation was found in the skink Tiquila rugosa (Smallridge and Bull 2000), and in another Mediterranean lacertid lizard, Lacerta lepida (Amo et al., unpublished data). Lizards may acquire mites, and thus be more exposed to haemogregarine infection, when they share favorable places such as basking spots or refuges, in which mites have detached from a previous occupant. Juvenile lizards may be less exposed to mites because they are not physically associated with their parents or siblings, and are often relied to marginal areas by older lizards. Prevalence was probably higher in larger, and thus older, lizards, because these have been more often in contact with parasites during their lifetime. During the mating season, the intensity of parasite infection was greater in males than in females. Testosterone is considered as an immune suppressor (Salvador et al. 1996; Olsson et al. 2000). During the mating period, males maintain high levels of this hormone, which renders them more aggressive. Agonistic interactions increase, and this social stress may increase parasite load, as has been observed in mice (Schuster and Schaub 2001). Males with greater levels of testosterone may also show higher mobility looking for females (Salvador et al. 1996; Olsson et al. 2000), but this can also lead to an increase in exposure to parasites of infected conspecifics (Salvador et al. 1996; Veiga et al. 1998). Furthermore, the immune suppression effects of testosterone, and the need of allocating resources in reproduction may cause that males could not allocate enough resources in defense against parasites. However, there were no intersexual differences in the intensity of infection after the mating period. The intensity of infection in females seemed to increase in summer. Previous studies have Capítulo 2 Relaciones hospedador-parásito 85 demonstrated that an increase in reproductive effort decreases parasite defense and, thus, increases parasite load (Gustafsson et al. 1994; Oppliger and Clobert 1997; Oppliger et al. 1997; Fargallo and Merino 2004). Therefore, females may need to reallocate most of resources in pregnancy, and assume the cost of a higher parasite infection during this period. Alternatively, if infection reduces survival, the most infected individuals, especially males, may have died during the mating season, as have been observed in mammals (Schuster and Schaub 2001). In relation to the host body condition, our results suggest that the body mass condition of adult lizards differed significantly in relation to parasite intensity. During the spring, there was a negative relationship between body condition and parasite load, whereas in the summer this relationship was positive. Haemogregarines cause a depression of hematocrit levels (Wintrobe 1991). Thus, infected lizards may have reduced hemoglobin concentrations, and reduced the capacity for oxygen transportation to muscle tissue (Oppliger et al. 1996; Veiga et al. 1998) involved in various aspects of lizard physiology and behaviour, such as foraging efficiency or sprint speed (Caudell et al. 2002), which should also affect body condition. Animals should allocate resources to try to reduce parasitemia, but during the mating period, both male and female lizards must also allocate resources in reproduction. This trade-off may explain the inverse relationship between body mass and parasitic infection. However, after the mating period, lizards can allocate all the metabolites to the immune function. In conclusion, the lack of constancy of prevalence and intensity of infection by haemogregarines between seasons, ages and sexes and among body size classes, and the negative effect of parasitemia on the body condition of lizards suggest that the interactions between parasites and hosts are not stable in this lizard population. Furthermore, these results suggest that the deleterious effects of parasitemia may be more evident when additional factors influence the lizard’s condition. For example, habitat deterioration may result in an increase in predation pressure, with associated physiological costs such as a decrease in body mass Capítulo 2 Relaciones hospedador-parásito 86 condition or increased stress (Martín and López 1999), which may render lizards unable to allocate resources to parasite defense (Oppliger et al. 1998; Schuster and Schaub 2001). This is an endangered lizard species, potentially threatened by changes in habitat conditions (Martín and Salvador 1997). Therefore, knowing the effects of parasites could have important implications for the conservation of this lizard. Acknowledgements We thank two anonymous reviewers for helpful comments, and ‘El Ventorrillo’ MNCN Field Station for use of their facilities. Financial support was provided by the MCYT projects BOS 2002-00598 and BOS 2002-00547, and to L. Amo by an ‘El Ventorrillo’ C.S.I.C. grant. This study was performed Ander license of the ‘Consejería de Medio Ambiente de la Comunidad de Madrid’. The experiments comply with the current laws of Spain where the experiments were performed. References Barnard SM, Upton SJ (1994) A veterinary guide to the parasites of reptiles, vol 1. Protozoa. Krieger, Malabar, Florida Caudell JN, Whittier J, Conover MR (2002) The effects of haemogregarine-like parasites on brown tree snakes (Boiga irregularis) and slatey-grey snakes (Stegonotus cucullatus) in Queensland, Australia. Int Biodet Biodegrad 49: 113– 119 Eisen RJ (2001) Absence of measurable malaria-induced mortality in western fence lizards (Sceloporus occidentalis) in nature: a 4-year study of annual and over-winter mortality. Oecologia 127: 586–589 Fargallo JA, Merino S (2004) Clutch size and haemoparasite species richness in adult and nestling blue tits. Ecoscience 11: 168–174 Gustafsson L, Nordling D, Andersson MS, Sheldon BC, Qvarnström A (1994) Infectious diseases, reproductive effort and the cost of reproduction in birds. Philos Trans R Soc Lond B 346: 323– 331 Hamilton WD, Zuk M (1982) Heritable true fitness and bright birds: a role for parasites? Science 218: 384–387 Hudson PJ, Dobson AP, Newborn D (1998) Prevention of population cycles by parasite removal. Science 282: 2256– 2258 Lainson R, de Souza MC, Franco CM (2003) Haematozoan parasites of the lizard Ameiva ameiva (Teiidae) from Amazonian Brazil: a preliminary note. Mem Inst Oswaldo Cruz 98: 1067– 1070 Levine ND (1982) Some corrections in haemogregarine (Apicomplexa: Protozoa) nomenclature. J Protozool 29: 601–603 Martín J, López P (1999) An experimental test of the costs of antipredatory refuge use in the wall lizard, Podarcis muralis. Oikos 84: 499–505 Martín J, Salvador A (1997) Microhabitat selection by the Iberian rock lizard Lacerta monticola: effects on density and spatial distribution of individuals. Biol Conserv 79: 303–307 Merino S, Potti J (1995) High prevalence of hematozoa in nestings of a passerine species, the pied flycatcher (Ficedula hypoleuca). Auk 112: 1041–1043 Møller AP, Christe P, Lux E (1999) Parasitism, host immune function, and sexual selection. Q Rev Biol 74: 3–20 Capítulo 2 Relaciones hospedador-parásito 87 O’Dwyer LH, MoÇo TC, da Silva RJ (2004) Description of the gamonts of a small species of Hepatozoon sp. (Apicomplexa, Hepatozoidae) found in Crotalus durissus terrificus (Serpentes, Viperidae). Parasitol Res 92: 110–112 Olsson M, Wapstra E, Madsen T, Silverin B (2000) Testosterone, ticks and travels: a test of the immunocompetence-handicap hypothesis in free-ranging male sand lizards. Proc R Soc Lond B 267: 2339– 2343 Oppliger A, Clobert J (1997) Reduced tail regeneration in the common lizard, Lacerta vivipara, parasited by blood parasites. Funct Ecol 11: 652–655 Oppliger A, Celerier ML, Clobert J (1996) Physiological and behaviour changes in common lizards parasitised by haemogregarines. Parasitology 113: 433–438 Oppliger A, Christe P, Richner H (1997) Clutch size and malarial parasites in female great tits. Behav Ecol 8: 148–152 Oppliger A, Clobert J, Lecomte J, Lorenzon P, Boudjemadi K, John-Alder HB (1998) Environmental stress increases the prevalence and intensity of blood parasite infection in the common lizard Lacerta vivipara. Ecol Lett 1: 129–138 Pérez-Mellado V (1998) Lacerta monticola Boulenger, 1905. In: Salvador A (ed) Reptiles, fauna Ibérica, vol 10. Museo Nacional de Ciencias Naturales, Madrid, pp 207–215 Price PW (1980) Evolutionary biology of parasites. Princeton Univ Press, Princeton Salvador A (1984) Lacerta monticola Boulenger, 1905. Iberische Gebirgseidechse. In: Böhme W (ed) Handbuch der Reptilien und Amphibien Europas, vol 2/1. Aula Verlag, Wiesbaden, pp 276–289 Salvador A, Veiga JP, Martín J, López P, Abelenda M, Puerta M (1996) The cost of producing a sexual signal: testosterone increases the susceptibility of male lizards to ectoparasitic infestation. Behav Ecol 7: 145–150 Schall JJ (1996) Malarial parasites of lizards. Adv Parasitol 37: 255– 333 Schuster JP, Schaub GA (2001) Experimental Chagas disease: the influence of sex and psychoneuroimmunological factors. Parasitol Res 87: 994–1000 Sheldon BC, Verhulst S (1996) Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology. Trends Ecol Evol 11: 317–321 Smallridge CJ, Bull CM (2000) Prevalence and intensity of the blood parasite Hemolivia mariae in a field population of the skink Tiquila rugosa. Parasitol Res 86: 655–660 Smith TG, Desser SS, Martin DS (1994) The development of Hepatozoon sipedon n. sp. (Apicomplexa: Adeleina: Hepatozoidae) in its natural host, the Northern water snake (Nerodia sipedon sipedon), the culicine vectors, Culex pipiens and Culex territans, and an intermediate host, Northern leopard frog (Rana pipiens). Parasitol Res 80: 559– 568 Svahn K (1974) Incidence of blood parasites of the genus Karyolysus (Coccidia) in Scandinavian lizards. Oikos 25: 43–53 Veiga JP, Salvador A, Merino S, Puerta M (1998) Reproductive effort affects immune response and parasite infection in a lizard: a phenotypic manipulation using testosterone. Oikos 82: 313–318 Wintrobe MM (1991) Clinical haematology. Lea and Feiberger, Philadelphia Capítulo 2 Relaciones hospedador-parásito 89 Prevalence and intensity of blood and intestinal parasites in a field population of a Mediterranean lizard, Lacerta lepida RESUMEN En este estudio describimos los parásitos sanguíneos e intestinales en el lagarto ocelado, Lacerta lepida, examinando los factores que determinan la prevalencia e intensidad de la infección por hemogregarinas así como la prevalencia de coccidios y nematodos. En relación a las hemogregarinas, no se detectó ningún juvenil infectado mientras que el 71.7 % de los adultos estaban infectados. La prevalencia de infección estuvo positivamente relacionada con el tamaño de los adultos. No hubo diferencias entre estaciones o sexos en la prevalencia o intensidad de la infección en adultos. No hubo diferencias en la prevalencia de infección por nematodos entre edades o sexos, ni en relación al tamaño de los adultos, pero los adultos que excretaron oocistes de coccidios parecieron ser más pequeños. Durante el período reproductor, las actividades reproductivas conllevan una disminución de la condición corporal. Sin embargo, ni la intensidad de infección por hemogregarinas ni la prevalencia de parásitos intestinales estuvieron relacionados con la condición corporal de los lagartos. Capítulo 2 Relaciones hospedador-parásito 91 Prevalence and intensity of blood and intestinal parasites in a field population of a Mediterranean lizard, Lacerta lepida Abstract We describe the blood and intestinal parasites in the Ocellated lizard, Lacerta lepida, examining the factors that determine the prevalence and intensity of infection of haemogregarines, and the prevalence of coccidia and nematodes. In relation to haemogregarines, no juveniles were detected as being infected whereas the 71.7 % of adults were infected. The prevalence of infection was positively related to the size of adults. There were not differences between seasons or sexes in the prevalence or intensity of infection in adults. There were not significant differences in the prevalence of infection by nematodes between ages or sexes, nor in relation to the size of adult lizards, but adult lizards excreting coccidian oocysts tended to be smaller. During the mating period reproductive activities lead to a decrease in the body condition. However, neither the intensity of haemogregarine’s infection nor the prevalence of intestinal parasites were related to the lizards’ body condition. Introduction The study of the relations between parasites and their host populations is essential for understanding their role in host’s population dynamics and ecology. Although the effect of virulent parasites species on humans and domestic animals is relatively well understood, it is however little known the effect of less virulent parasites causing subtle but chronic diseases in natural populations, especially in reptiles. Parasites compete with the host for resources; it has been evidenced that parasites affect host population growth and regulation (Holmes 1995; Krebs 1995; Hudson et al. 1998), spatial distribution (Price 1980; van Riper et al. 1986), individual reproductive success (Schall 1996; Pacejka et al. 1998), and sexual selection (Hamilton and Zuk 1982; Møller et al. 1999). Prevalence and intensity of a parasitic infection provides a first approach to understand the parasite Capítulo 2 Relaciones hospedador-parásito 92 impact on a natural population (Smallridge and Bull 2000). Lizards are one of the more prominent groups of animals in Mediterranean climates. The Ocellated lizard (Lacerta lepida) is the largest European lizard distributed in Mediterranean habitats of the Iberian Peninsula, southern France, and northern Italy (Pérez-Mellado 1998b), from sea level to high mountains, reaching the highest densities in Mediterranean forests and shrublands (Castilla and Bauwens 1992). Althought this especies is strictly protected, its populations are decreasing mainly due to habitat destruction (Pérez-Mellado 1998b). Therefore, it is especially important for conservation the knowledge of factors, such as parasites, that can be affecting the maintenance of populations. Anecdotal records have indicated that Ocellated lizards are infected by blood and intestinal parasites (Roca et al. 1986; Pérez-Mellado 1998b), but neither the prevalence of a population nor the intensity of infection of individuals have been examined. We aimed to describe the prevalence and intensity of infection of haemo- and endoparasites in a montane population of Ocellated lizards. We also aimed to analyse which factors related to the reproductive period or the lizard’s characteristics (age, sex, size, body condition) determined the parasite status of lizards (infected vs. uninfected) and the intensity of infection. Materials and methods We performed the study in an extensive montane grassland in the region of Campo Azálvaro (40°40’N, 4°20’W; 1300 m. a.s.l.; Ávila and Segovia provinces, central Spain, see Fargallo et al. 2001). In this area L. lepida lizards mate in May and produce a single clutch in June (Pérez-Mellado 1998b). Lizards were collected with Sherman traps in two trapping seasons among the mating season, in spring (June) and in summer (July-August). Traps were operating during three consecutive days. Each trapping session consisted in 10 trapping plots, each consisting in 10 Sherman traps following a line, with traps spaced 15 m. Traps were baited with a mixture of tuna, flour and oil and with some small pieces of apple, and were set under the cover of herbs to provide camouflage and thermal Capítulo 2 Relaciones hospedador-parásito 93 insulation. Traps were checked twice daily at 1400 and 1900 h. and closed daily at the last revision to avoid the capture of small mammals at night. Each captured lizard (n = 56) was individually marked by toe clipping, sexed, weighed, and measured for its snout-vent length (SVL). Lizards were classified as non-matured juveniles (SVL < 120 mm) or sexually mature adults (SVL > 120 mm) (Pérez-Mellado 1998b). A smear was made on a microscope slide from blood taken from the postorbital sinus by using one 9 µl heparinised hematocrit tube. Lizards were released at the point of capture. Blood smears were air-dried and fixed in the laboratory with absolute ethanol for 10 min and then stained in Giemsa diluted 1:9 with phosphate buffer (pH 7.2) for 40 min before their examination for parasites. On mounted slides, half a smear, chosen at random, was scanned entirely at 200 x along the longitudinal of slide, looking for extraerythrocytic protozoa (Merino and Potti 1995). Number of intraerythrocytic parasites was estimated at 400 x by counting the number of parasites per 2000 erythrocytes. The only haemoparasites found were haemogregarines. Because haemogregarina genus can only be characterized by oocyst stages within the invertebrate host (Levine 1982; Barnard and Upton 1994), we could only identify these parasites as haemogregarines. We also take samples of faeces from 25 lizards captured in summer, to determine the prevalence and intensity infection of intestinal parasites in the lizard population. We analysed faecal samples in order to quantify the excretion of parasite propagules by the classical technique of flotation and counting in MacMaster chamber. Propagules were identified following Melhorn et al. (1992). We found coccidia oocysts and two types of nematode eggs (Families Oxyuridae and Ascaridiidae). To compare the prevalence of different parasites between lizard ages we used chi-square tests. We calculated logitic regression by using Generalized Linear Models (GLZM; logit link function, binomial distribution) in STATISTICA Software (StatSoft Inc. 1984-2001, Tulsa, OK, USA) to assess what variables better explain the variation in the infection (presence vs. absence) by haemogregarines, coccidia or nematodes. As potential explanatory Capítulo 2 Relaciones hospedador-parásito 94 variables we used sex, season (factors) and body size (continuous). Differences in the intensity of haemogregarind`s infection among adult lizards in relation to their sex, size and season were analysed by using General Linear Models (GLM), including all the interactions between these three variables. To examine the variation of body condition of adult lizards in relation to parasites we used General Linear Models (GLM) in which body mass was included as response variable, sex, season and intensity of infection by haemogregarines as factors and body size (SVL) as a covariate. We also analysed variation of body condition of adult lizards in relation to the presence or absence of intestinal parasites (coccidia and nematodes) by using GLM, with body mass as the response variable, sex and parasite status as factors and body size as a covariate. Lizards can autotomize the tail. This could affect the body condition. Therefore, we initially compared the body mass of adult lizards with an analysis of covariance using SVL as a covariate and taking into account the tail condition (original complete, complete but regenerated, incomplete and in process of regeneration) as a categorical independent factor. There was a significant positive correlation between body mass and SVL (GLM, r = 0.89, F2,43 = 99.71, P < 0.0001), but there were not significant differences in relation to tail condition (F2,43 = 0.52, P = 0.60). Thus, we did not include tail condition in subsequent analyses. Results Overall population data Thirty-three of 56 lizards (58.9%) were found infected by haemogregarines (protozoa) (see Table 1). In relation to intestinal parasites, we found coccidia (protozoa) and oxyurides (nematoda). The prevalence of coccidia was 28% (n = 25) and the prevalence of oxyurides was 40.0 % (n = 25) (Table 1). Only one lizard was found infected with Ascaridiidae (nematoda) eggs. Parasite infection There were significant differences in the prevalence of haemogregarines between adults and juveniles (χ2 = 17.47, df = 1, P < 0.0001, Table 1). Thus, only Capítulo 2 Relaciones hospedador-parásito 95 adult lizards were detected as being infected by haemogregarines. The prevalence of haemogregarines did not differ significantly between sexes (GLZM, Wald’s χ2 = 0.44, df = 1, P = 0.51) (Fig. 1), or seasons (Wald’s χ2 = 0.28, df = 1, P = 0.60) (Fig. 1). Infected lizards were significantly larger than uninfected ones (Wald’s χ2 = 4.23, df = 1, P = 0.04). The interactions between sex and season and between sex and body size were not statistically significant (both P > 0.3). Fig. 1 Number of male and female adult Lacerta lepida lizards infected (black bars) or uninfected (open bars) with Haemogregarines during the mating season (Spring) or after the reproductive season (Summer). The intensity of infection by haemogregarines in adult lizards did not differ significantly between sexes (GLM, F1,41 = 1.27, P = 0.27), or seasons (F1,41 = 0.001, P = 0.97), and was not correlated with body size (F1,41 = 0.001, P = 0.97). The interaction between season and sex was non significant (F1,41 = 0.08, P = 0.78; Table 1). In the case of coccidia, there was not significant difference in the prevalence of oocyst excretion of juveniles and adults (χ2 = 0.11, df = 1, P = 0.74; Table 1). There were not significant differences in the prevalence of coccidia between adult males and females (GLZM, Wald’s χ2 = 0.33, df = 1, P = 0.57). The prevalence was correlated to the body size of adult lizards (Wald’s χ2 = 3.82, df = 1, P = 0.05). Infected lizards tended to be smaller. There were not significant differences in the prevalence of nematodes between adults and juveniles (χ2 = 0.36, df = 1, P = 0.55; Table 1), nor between sexes with adults (GLZM, Wald’s χ2 = 0.04, df = 1, P = 0.84), and nor was correlated with body size (Wald’s χ2 = 0.42, df = 1, P = 0.51). Capítulo 2 Relaciones hospedador-parásito 96 Table 1 Parasitic status (presence vs. absence) and intensity of infection by blood (Haemogregarines) and intestinal (Coccidia and Nematodes) parasites in the Ocellated lizard, Lacerta lepida in relation to age (juveniles vs. adults) and sex of adult lizards, and to the season (spring vs. summer) for blood parasites. Haemogregarines Infected vs. non infected Infection intensity (mean + SE) Juveniles 0/10 0 Adults 33/13 12.4 + 3.3 Adult males 15/10 12.5 + 5.2 Adult females 18/3 12.3 + 3.7 Adults in Spring 14/4 9.5 + 3.4 Adults in Summer 19/9 14.3 + 4.9 Males in Spring 7/3 6.7 + 3.2 Males in Summer 8/7 16.4 + 8.4 Females in Spring 7/1 13.0 + 6.5 Females in Summer 11/2 11.9 + 4.7 Coccidia Infected vs. non infected Infection intensity (mean + SE) Juveniles 2/4 54.1 + 34.6 Adults 5/14 49.6 + 26.9 Adult Males 3/8 52.3 + 35.2 Adult Females 2/6 45.5 + 44.5 Nematodes Infected vs. non infected Infection intensity (mean + SE) Juveniles 2/4 667.8 + 666.4 Adults 9/10 1504.4 + 1204.2 Adult Males 6/5 2541.8 + 2061.5 Adult Females 3/5 78.0 + 76.0 Capítulo 2 Relaciones hospedador-parásito 97 Body condition Males showed a higher body condition than females (F1,42 = 17.16, P = 0.0002), and all lizards had a significantly lower body condition in summer (F1,42 = 7.53, P = 0.009), but the intensity of infection by haemogregarines was not significantly related to body condition (F1,37 = 0.00, P > 0.99). Similarly, the presence of coccidia or nematodes was not significantly related to the body condition of lizards (GLM, F1,16 = 1.12, P = 0.72; F1,16 = 0.74, P = 0.40, respectively). Discussion No juvenile lizards were observed infected by haemogregarines. A similar result was found in the skink Tiquila rugosa (Smallridge and Bull 2000) and in the alpine lacertid lizard, Lacerta monticola (Amo et al. 2004). In addition, the prevalence of infection was positively correlated with adult size. Since lizard size increases with age, these two results point out to a positive association between haemogregarine prevalence and age. Lizards may acquire mites or ticks (haemogregarine vectors, Paperna et al. 2002) when they share favourable places such as basking spots or refuges, in which mites had detached from a previous occupant. Thus, it is expected adults to be more infected because they occupy more frequently these places and interact more often with other adults. On the contrary juveniles are often relied to suboptimal areas by dominant older male lizards. Therefore, they may be less exposed to vectors which can explain the different prevalence we found. There were no seasonal differences in the prevalence or intensity of infection in adults. This suggests that infections occur early in the breeding season, when lizards are more active. This consistency, in prevalence and intensity of infection may reflect the stability of the parasite- host interactions in this population. These results are similar to those found in the skink T. rugosa (Smallridge and Bull 2000). An interesting result is that males and females seem to be similarly susceptible to parasite’s infection, as the prevalence and intensity of infection were similar in both sexes. Recent studies in lizards (Salvador et al. 1996; Olsson et al. 2000; Klukowski and Capítulo 2 Relaciones hospedador-parásito 98 Nelson 2001; Uller and Olsson 2003) and other organisms (see Zuk 1996) have found that males are more susceptible to parasite’s infection probably due to the immune suppressive effects of testosterone at least during the reproductive period (Roberts et al. 2004). Early in the mating period males maintained high levels of this hormone (Tokarz et al. 1998), thanks to which they are more aggressive, thus, more able to obtain and maintain a territory. Our findings agree with a previous study with the lizard Psammodromus algirus, in which there were not differences in haemogregarine load between males with a testosterone implant and control males (Veiga et al. 1998). In pregnant females, the development of eggs requires a great amount of energy and metabolites, which could not be allocated to defence against parasites. Therefore, both sexes seem to invest more in reproduction than in defence against parasites. In relation to nematodes, we could not reach the especie level when identifying oocysts in faeces, however, two species of oxyurid has been cited parasitising Lacerta lepida in the Iberian peninsula: Parapharyngodon bulbosus (Linstow 1899) Teixeira de Freitas 1957; and Spauligodon extenuatus (Rudolphi 1819) Skrjabin, Schihobalova et Lagodovskaya, 1960 (Cordero del Campillo et al. 1994). Conversely, no ascarid species has been cited, although an Heterakidae (whose eggs are sligthly similar to those of ascarids) has been reported in a Lacertidae species in Madeira Island (Portugal) (Sanchez- Gumiel et al. 1991). There are not previous reports of oocyst faecal excretion in lizards in the Iberian Peninsula. Helminth acquisition appears to be related with the diet of saurian reptiles (Sanchis et al. 2000), for which it would be expected, as our results support, no differences between sexes and age classes. Therefore, even thought juveniles feed upon a restricted number of prey types, which were small in size, and adults consume a large number of prey taxa (Castilla et al. 1991), adults and juveniles may have similar diets and food habitat that make them both similarly vulnerable to helminth’s infection. On contrast, even thought there were no differences in the prevalence of coccidia oocyst excretion between adults and juveniles, adult Capítulo 2 Relaciones hospedador-parásito 99 infected lizards tended to be smaller. Thus, it seems that smaller lizards are more vulnerable to coccide’s infection, or that larger lizards have infection intensities under undetectable levels. An alternative hypothesis might be that, since the parasited lizards might allocate resources to fight against infection, they suffered a decrease in their growth rate, and therefore, they showed smaller body sizes. However, we can not obtain reliable conclusions due to our limited sample and the low level of significance. The body condition of lizards decreased over the activity season. During the mating period reproductive activities lead to a decrease in the body condition. However, the intensity of infection by haemogregarines did not affect body condition of lizards. Therefore, these reproductive costs seem to be more determinant to body condition than costs associated to parasite load. Haemogregarines destroy erythrocytes, which should result in depressed haematocrit levels (Wintrobe 1991; O’Dwyer et al. 2004). Thus, parasited lizards may have reduced haemoglobin concentrations, and reduced capacity for oxygen transportation (Salvador et al. 1996; Oppliger et al. 1996; Veiga et al. 1998). The reduced ability to transport oxygen to the muscle tissue may affect the lizard different aspects of lizard’s physiology and behaviour with important fitness consequences, such as foraging efficiency or sprint speed (Caudell et al. 2002), which should also affect body condition (Wozniak et al. 1996). However, lizards could compensate this for the loss of their oxygen transport ability by increasing the production of erythrocytes, as it has been observed in American kestrels (Falco sparverius) infected with blood parasites (Dawson and Bortolotti 1997). Therefore, additional research should be conducted to examine the physiological mechanisms implicated on the compensation of the deleterious effects of haemogregarine infection. In general, parasites that have evolved with a species will not overly detrimental to that species (Caudell et al. 2002). Thus, most of these parasites do not produce clinical disease unless the host is highly infected or stressed (Oppliger et al. 1996, 1998; Campbell 1996; Lane and Mader 1996). It has been shown that stress can often magnify the deleterious Capítulo 2 Relaciones hospedador-parásito 100 effects of normally benign parasites (Caudell et al. 2002). For example, the blood parasite, Plasmodium mexicanum, contributed to mortality of Sceloporus occidentalis lizards under laboratory, and probably stressful conditions, whereas there was no evidence of mortality induction under natural conditions (Eisen 2001). The lack of effect of infection by haemogregarines and intestinal parasites on body condition suggests the stability of the parasite-host interactions in this lizard population. However, this equilibrium in host-parasite interaction may be broken if any additional factor influence the lizard’s condition. For example, an increase in predation pressure, with the subsequent increase in the use of refuges or loss of time to forage has physiological costs, such as a decrease in body mass condition (Martín and López 1999; Perez-Tris et al. 2004). This could imply that lizards with decreased body condition may not be able to allocate resources to parasite defence. For example, Lacerta vivipara lizards submitted to high stress conditions due to a low habitat quality have higher levels of haemogregarines than non-stressed individuals (Oppliger et al. 1998). Therefore, if the stability of host-parasite interactions is modified by external factors, such as habitat disturbance or predation pressure, probably the deleterious effects of parasitism may be more evident. This could have important implications related to conservation of lizards. Acknowledgements We thank "El Ventorrillo" MNCN Field Station for use of their facilities. Financial support was provided to L. Amo by an “El Ventorrillo” C.S.I.C. grant, to P. López by the MCYT project BOS 2002-00598, and to J. Martín by the MCYT project BOS 2002- 00547. The experiment complies with the current laws of Spain. References Amo L, López P, Martín J (2004) Prevalence and intensity of Haemogregarinid blood parasites in a population of the Iberian rock lizard, Lacerta monticola. Parasitol Res 94: 290-293 Barnard SM, Upton SJ (1994) A veterinary guide to the parasites of reptiles, vol. 1, Protozoa. Krieger, Malabar, Florida Campbell TW (1996) Haemoparasites. In Mader DR (ed) Reptile medicine and Surgery. WB Saunders, Philadelphia, PA, pp 379-381 Castilla AM, Bauwens D, Llorente GA (1991) Diet composition of the lizard Lacerta lepida in central Spain. 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Parasitol Res 92: 110–112 Olsson M, Wapstra E, Madsen T, Silverin B (2000) Testosterone, ticks and travels: a test of the inmunocompetence-handicap hypothesis in free-ranging male sand lizards. Proc R Soc Lond B 267: 2339- 2343 Oppliger A, Celerier ML, Clobert J (1996) Physiological and behaviour changes in common lizards parasited by haemogregarines. Parasitol 113: 433- 438 Oppliger A, Clobert J, Lecomte J, Lorenzon P, Boudjemadi K, John-Alder HB (1998) Environmental stress increases the prevalence and intensity of blood parasite infection in the common lizard Lacerta vivipara. Ecol Let 1: 129-138 Capítulo 2 Relaciones hospedador-parásito 102 Pacejka AJ, Gratton CM, Thompson CF (1998) Do potentially virulent mites affect house wren (Troglodytes aedon) reproductive success? Ecology 795: 1797-1806 Paperna I., Kremer-Mecabell T, Finkelman S (2002) Hepatozoon kisrae n. sp infecting the lizard Agama stellio is transmitted by the tick Hyalomma cf. Aegyptium. 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Rev Ib Parasitol 46: 129-136 Salvador A, Veiga JP, Martín J, López P, Abelenda M, Puerta M (1996) The cost of producing a sexual signal: testosterone increases the susceptibility of male lizards to ectoparasitic infestation. Behav Ecol 7: 145-150 Sanchez-Gumiel N, Zapatero Ramos LM, Castano Fernandez C, Gonzalez Santiago PM (1991) Description of Spinicauda dugesii sp. n. (Nematoda: Heterakidae) of Podarcis dugesii (Reptilia: Lacertidae) from Madeira Island. Folia Parasitol 38: 183-186 Sanchis V, Roig JM, Carretero MA, Roca V, Llorente GA (2000) Host-parasite relationships of Zootoca vivipara (Sauria: Lacertidae) in the Pyrenees (North Spain). Folia Parasitol 47: 118- 122 Schall JJ (1996) Malarial parasites of lizards. Adv Parasitol 37: 255-333 Smallridge CJ, Bull CM (2000) Prevalence and intensity of the blood parasite Hemolivia mariae in a field population of the skink Tiquila rugosa. Parasitol Res 86: 655-660 Tokarz RR, Mcmann S, Seitz L, John-Alder H (1998) Plasma corticosterone and testosterone levels during the annual reproductive cycle of male brown anoles (Anolis sagrei). Physiol Zool 71: 139- 146 Uller T, Olsson M (2003) Prenatal exposure to testosterone increases ectoparasite susceptibility in the common lizard (Lacerta vivipara). Proc R Soc Lond B 270: 1867-1870 Van Riper CIII, Van Riper SG, Goff ML, Laird M (1986) The epizootiology and ecological significance of malaria in Hawaiian land birds. Ecol Monog 56: 327-344 Veiga JP, Salvador A, Merino S, Puerta M (1998) Reproductive effort affects immune response and parasite infection in a lizard: a phenotypic manipulation using testosterone. Oikos 82: 313-318 Wintrobe MM (1991) Clinical haematology. 8th ed, Lea and Feiberger, Philadelphia, PA Wozniak EJ, Kazacos KR, Telford SR, Mclaughlin GL (1996) Characterization of the clinical and anatomical pathological changes associated with Hepatozoon mocassini infections in unnatural reptilian hosts. Inter J Parasitol 26: 141-146 Capítulo 2 Relaciones hospedador-parásito 103 Zuk M (1996) Disease, endocrine-immune interactions, and sexual selection. Ecology 77: 1037-1042 Capítulo 2 Relaciones hospedador-parásito 105 Prevalence and intensity of haemogregarine blood parasites and their mite vectors in the common wall lizard, Podarcis muralis RESUMEN En este estudio describimos la biología general de las hemogregarinas y ácaros en la lagartija roquera, Podarcis muralis, y examinamos los factores que determinan la prevalencia y la intensidad de infección. La intensidad de infección por hemogregarinas en las hembras fue ligeramente menor en verano que en primavera, mientras que los machos tuvieron niveles similares de intensidad a lo largo de las estaciones, debido probablemente a los efectos inmunosupresores de la testosterona. La prevalencia e intensidad de infección por ácaros fue mayor en machos que en hembras, lo que apoya la explicación anterior. La carga parasitaria fue mayor en las lagartijas en mejor condición corporal, lo que podría reflejar la mortalidad de las lagartijas en peor condición corporal. Capítulo 2 Relaciones hospedador-parásito 107 Prevalence and intensity of haemogregarine blood parasites and their mite vectors in the common wall lizard, Podarcis muralis Abstract We describe the general field population biology of haemogregarines and mites in the wall lizard, Podarcis muralis, examining the factors that determine the prevalence and intensity of infection. The intensity of infection by haemogregarines in females was slightly lower in summer than in spring, whereas males maintained similar levels of intensity among all the season, probably due to immunosuppressive effects of testosterone. This is also supported because the prevalence and the infection intensity by mites were higher in males than in females. Parasite load was higher in lizards with better body condition, which could be reflecting the mortality of infected lizards with poor condition. Introduction Prevalence and intensity of parasitic infections in reptiles remains nowadays little known, despite this is important because parasitism causes deleterious effects on several aspects of the ecology and evolution of parasite hosts (Smallridge and Bull 2000; Eisen 2001), such as host population growth and regulation (Holmes 1995; Hudson et al. 1998), spatial distribution (Price 1980; van Riper et al. 1986), reproductive success (Schall 1996; Pacejka et al. 1998), and sexual selection (Hamilton and Zuk 1982; Møller et al. 1999). The haemogregarines (family Haemogregorinidae, suborder Adeleorina, subclass Coccidiasina, Phylum Apicomplexa) (Barnard and Upton 1994) have an indirect life cycle involving definitive invertebrate hosts, and vertebrate hosts, such as lizards, snakes or frogs (e.g. Smith et al. 1994; Caudell et al. 2002; Lainson et al. 2003; Amo et al. 2004). The zygote is active (ookinete) and the life cycle is heteroxenous, involving merogonous development and the formation of gametocytes in the circulatory system and/or visceral tissues of a vertebrate, Capítulo 2 Relaciones hospedador-parásito 108 and gamogony proper and sporogony within the gut of an invertebrate vector (Barnard and Upton 1994). Transmission to the vertebrate host occurs when this ingests a mite or a mite infected with this parasite, or by the bite of an arthropod or leech. The effects of haemogregarines on their hosts are poorly known. Haemogregarines take up over half of the volume of an infected erythrocyte and destroy this blood cell (Caudell et al. 2002; O’Dwyer et al. 2004) causing anemia. Therefore, the presence of gametocytes in the erythrocytes indicates the existence of schizonts in internal organs (Svahn 1974), where they can cause additional damage (Veiga et al. 1998). As a result, hosts have evolved an elaborate antiparasite defence via the immune system in order to reduce the fitness costs of parasitism (Sheldon and Verhulst 1996). However, immune response requires resources that the host may also need to supply other demands such as growth or reproduction. Thus, there is often a trade off between antiparasite defence and other demands (Sheldon and Verhulst 1996). To have a first approach in the understanding of the ecological interactions in the relationships between haemoparasites, mites and adult lizards, we aimed to describe the general field population biology of haemogregarines and mites parasitizing common wall lizards, Podarcis muralis. We specifically examined whether specific sub-populations of lizards were at more risk for infection than others, and whether some host parameters (e.g. sex, size or body condition) are important predictors of the degree of infection. This species is a small lacertid lizard, restricted to the northern area of the Iberian Peninsula, and Guadarrama Mountains (Central Spain, Madrid Prov.), which represent the southernmost limit of its distribution (Pérez-Mellado 1998c). Lizards are preferently found on rock walls, rock piles and talus in forests. This lizard is protected by law, and is susceptible to habitat degradation, which may affect lizard’s body and health condition. Therefore, knowing the effects of parasites could have important implications for conservation of this especies. Because haemogregarina genus can only be characterized by oocyst stages within the invertebrate host (Levine 1982; Barnard and Upton 1994), at this moment we can only identify these parasites as haemogregarines until Capítulo 2 Relaciones hospedador-parásito 109 the parasite life cycles were more fully known. Materials and methods We performed the study in the Guadarrama Mountains (Madrid Prov., Central Spain) at an elevation range of 1200-1800 m. Adult lizards were captured in rocks and talus inside a Pinus sylvestris forest. In this area, lizards are active from March to September, mate in April-May, and produce one single clutch during July (Pérez-Mellado 1998c). Females can produce more than a clutch (Pérez- Mellado 1998c). Lizards (n=74) were collected by noosing from June to August 2002. Sampling was representative of the population of adult lizards. For instance, it is unlikely that infected lizards were easier to catch because we were able to capture nearly every lizard we detected. We divided the study period in two seasons: ‘spring’ (June; i.e., during the mating season) and ‘summer’ (July-September; i.e., after the mating period had finished). Each captured lizard was individually marked by toe clipping, sexed, weighed, and measured for its snout-vent length (SVL). We also noted the number of mites observed on the body immediately after capture. Thereafter, a smear was made on a microscope slide from blood taken from the postorbital sinus by using one 9 µl heparinised hematocrit tube. The lizard was then released at the point of capture. Blood smears were air-dried. In the laboratory the smears were fixed in absolute methanol for 10 min and then stained in Giemsa diluted 1:9 with phosphate buffer (pH 7.2) for 40 min before their examination for parasites. On mounted slides, half a smear, chosen at random, was scanned entirely at 200 x along the longitudinal of slide, looking for extraerythrocytic protozoa (Merino and Potti 1995). Numbers of intraerythrocytic parasites were estimated at 400 x by counting the number of parasites per 2000 erythrocytes. The only haemoparasites found were haemogregarines. Parasite prevalence was estimated as the percentage of infected lizards. Parasite intensity was estimated for each infected host as the percentage of infected red blood cells found in approximately 2000 cells. To determine changes in prevalence of mites and haemogregarines between Capítulo 2 Relaciones hospedador-parásito 110 seasons, sex and body size (SVL) of lizards, we used generalized linear models (GLZM), with the parasites prevalences as the dependent variable following a binomial distribution, and including the interactions between the three variables in the model. We compared the infection intensity of mites and haemogregarines in relation to SVL, sex and season by using General Linear Models (GLM), including the interaction between sex and season in the model. To examine the effect of sex, season and intensity of infection by mites and haemogregarines on body condition we analysed the body mass of adult lizards by using GLM, correcting by the SVL and including the interactions between SVL, sex, season and intensity of infection of both parasites in the model. Lizards can autotomize the tail when captured by a predator. This could affect their body condition because lizards have to allocate resources in regeneration of tail. Therefore, we compared initially the body mass of adult lizards with an analysis of covariance using SVL as a covariate and taking into account the tail condition (original complete vs complete but regenerated vs incomplete and in process of regeneration) as a categorical independent factor. There was a significant positive correlation between body mass and SVL (GLM, r = 0.78, F1,70 = 102.59, P < 0.0001), but there were not significant differences between tail conditions (F2,70 = 1.29, P = 0.28). Thus, we did not include tail condition in subsequent analyses. Results Overall population data Prevalence of infection by mites in the surveyed population of P. muralis was 29.7 % (22 of 74 individuals). The intensity of infection ranged from 0 to 13 mites per host (mean + SE = 0.8 + 0.2). Among the 22 infected lizards, 12 showed an infection intensity of 1-2 mites, six lizards displayed an intensity of 3–4 mites, and four showed an intensity of 6-14 mites. Prevalence of infection by haemogregarines (Fig. 1) was 58.1 % (43 of 74 individuals). Intensity of infection ranged from 0 to 55.9 infected cells in 2000 erythrocytes (mean + SE = 2.1 + 0.8). Among the 43 infected lizards, 41 showed an infection intensity of 10 or less infected cells in 2000 Capítulo 2 Relaciones hospedador-parásito 111 erythrocytes, and only one lizard displayed an intensity of 10–20 infected cells, and another one showed an intensity of 50-60 infected cells. Fig. 1 Gametocytes of Haemogregarine parasites inside erythrocytes of the blood of Podarcis muralis lizards. Arrows indicate parasites. Bars = 10 µm for all figures. Effects of season and of host sex and size Prevalence of mite’s infection was significantly higher in male than in female lizards (infected vs. non infected, males: 16/22, females: 6/30; GLZM, Wald’s χ2 = 5.64, df =1, P = 0.02), but there were not significant differences in relation to the size of lizards (Wald’s χ2 = 0.19, df = 1, P = 0.66) nor between seasons (Wald’s χ2 = 0.45, df = 1, P = 0.50). All interactions were not significant and were removed from the model. Infection intensity of mites was also significantly higher in male than in female lizards (male: X + SE = 1.18 + 0.38 mites/lizard, females: X + SE = 0.47 + 0.23; GLM, r = 0.26, F1,70 = 4.62, P = 0.04), but there were no significant differences in relation to body size (F1,70 = 0.30, P = 0.58), nor between seasons (F1,70 = 0.71, P = 0.40). The interactions Capítulo 2 Relaciones hospedador-parásito 112 between these variables were no significant and were removed from the model. Prevalence of haemogregarine’s infection in lizards did not significantly differ either between sexes (GLZM, Wald’s χ2 = 0.05, df =1, P = 0.83), or in relation to body size (Wald’s χ2 = 1.48, df = 1, P = 0.22), or between seasons (Wald’s χ2 = 2.90, df = 1, P = 0.09). All interactions were no significant, and were removed from the model. Infection intensity by haemogregarines did not significantly differ between sexes, or between seasons, nor in relationship to body size, but the effect of the interaction between sex and season was significant (GLM, r = 0.30, F1,69 = 5.12, P = 0.03). Thus, in males parasite load was similar in spring and in summer (Tukey’s test, P = 0.95), whereas in females parasite load tend to be higher in spring than in summer, although differences were only marginally significant (P = 0.07). Effects of host body condition After removing the effect of covariation of body mass with SVL (GLM, r = 0.90, F1,68 = 164.59, P < 0.0001), body condition of males was significantly higher than that of females (F1,68 = 58.37, P < 0.0001), and lizards with greater body mass showed higher levels of infection by haemogregarines (F1,68 = 6.41, P = 0.01). There were not significant differences in body mass between seasons (F1,68 = 1.92, P = 0.17), or in relation to the intensity of mite’s infection (F1,68 = 0.10, P = 0.76). The interactions between these variables were not significant and were removed from the model. Discussion The intensity of haemogregarine’s infection was maintained across seasons in males whereas it decreased in females. Testosterone is considered as an immune suppressor (Salvador et al. 1996; Olsson et al. 2000). During the mating period males maintained high levels of this hormone (Tokarz et al. 1998), which renders males more aggressive. Therefore, males frequently interact with conspecifics, not only during copulations with females, but also during fights with other males. This social stress (Schuster and Schaub 2001) might maintain parasite load across the seasons. What is more, males with greater levels of testosterone also show higher mobility Capítulo 2 Relaciones hospedador-parásito 113 that could result in more encounters with females (Salvador et al. 1996; Olsson et al. 2000), but that can also led to an increase in exposure to mites of infected conspecifics, and thus, to haemogregarines’ infection (Salvador et al. 1996; Veiga et al. 1998). The higher prevalence and intensity of infection by mites in males seems to support this hypothesis. Furthermore, the immune suppression effects of testosterone and the need of allocating all resources in reproduction may cause that males could not allocate resources in defence against parasites. Therefore, in the course of seasons males could not decrease the parasite load. On contrast, females may allocate all resources in defence against parasites after laying, and therefore, they could decrease their parasite load at the end of the breeding season. Haemogregarines cause a depression of hematocrit levels (Wintrobe 1991). Thus, infected lizards may have reduced haemoglobin concentrations, and reduced capacity for oxygen transportation (Oppliger et al. 1996; Veiga et al. 1998) to the muscle tissue that may affect different aspects of lizard’s physiology and behaviour, such as foraging efficiency or sprint speed (Caudell et al. 2002), which should also affect body condition. In contrast, our results suggest a positive correlation between body mass and parasite load. Since the defense against parasites requires allocation of resources to immune system, an explanation to our results might be that if infection reduces survival, only individuals in good condition could survive and therefore, individuals in poor condition might have died before we sampled it, as have been observed in mammals (Schuster and Schaub 2001). The lack of constancy in prevalence or infection intensity between sexes across the season suggests that the interactions between parasites and hosts are not stable in this lizard population. However, more work is needed to examine the effects of parasitemia in body condition in this species. Capítulo 2 Relaciones hospedador-parásito 114 Acknowledgements We thank "El Ventorrillo" MNCN Field Station for use of their facilities. We especially thank Santiago Merino for the photographs. Financial support was provided to L. Amo by an “El Ventorrillo” C.S.I.C. grant, to P. López by a the MCYT project BOS 2002-00598, and to J. Martín by the MCYT project BOS 2002-00547. This study was performed under license of “Consejería de Medio Ambiente de la Comunidad de Madrid”. The experiments comply with the current laws of Spain where the experiments were performed. References Amo L, López P, Martín J (2004) Prevalence and intensity of Haemogregarinid blood parasites in a population of the Iberian Rock Lizard, Lacerta monticola. Parasitol Res 94: 290-293 Barnard SM, Upton SJ (1994) A veterinary guide to the parasites of reptiles, vol. 1, Protozoa. Krieger, Malabar, Florida Caudell JN, Whittier J, Conover MR (2002) The effects of haemogregarine-like parasites on brown tree snakes (Boiga irregularis) and slatey-grey snakes (Stegonotus cucullatus) in Queensland, Australia. Int Biodet Biodegrad 49: 113- 119 Eisen RJ (2001) Absence of measurable malaria-induced mortality in western fence lizards (Sceloporus occidentalis) in nature: a 4-year study of annual and over-winter mortality. Oecologia 127: 586-589 Hamilton WD, Zuk M (1982) Heritable true fitness and bright birds: a role for parasites? Science 218: 384-387 Holmes J (1995) Population regulation: a complex of interactions. Wildl Res 22: 11-19 Hudson PJ, Dobson AP, Newborn D (1998) Prevention of population cycles by parasite removal. Science 282: 2256- 2258 Lainson R, de Souza MC, Franco CM (2003) Haematozoan parasites of the lizard Ameiva ameiva (Teiidae) from Amazonian Brazil: a preliminary note. Mem Inst Oswaldo Cruz 98: 1067-1070 Levine ND (1982) Some corrections in haemogregarine (Apicomplexa: Protozoa) nomenclature. J. Protozool 29: 601-603 Merino S, Potti J (1995) High prevalence of hematozoa in nestings of a passerine species, the pied flycatcher (Ficedula hypoleuca). Auk 112: 1041-1043 Møller AP, Christe P, Lux E (1999) Parasitism, host immune function, and sexual selection. Q Rev Biol 74: 3-20 O’Dwyer LH, Moço TC, da Silva RJ (2004) Description of the gamonts of a small species of Hepatozoon sp. (Apicomplexa, Hepatozoidae) found in Crotalus durissus terrificus (Serpentes, Viperidae). Parasitol Res 92: 110–112 Olsson M, Wapstra E, Madsen T, Silverin B (2000) Testosterone, mites and travels: a test of the immunocompetence-handicap hypothesis in free-ranging male sand lizards. Proc R Soc Lond B 267: 2339- 2343 Oppliger A, Celerier ML, Clobert J (1996) Physiological and behaviour changes in common lizards parasited by haemogregarines. Parasitology 113: 433-438 Pacejka AJ, Gratton CM, Thompson CF (1998) Do potentially virulent mites affect house wren (Troglodytes aedon) reproductive success? Ecology 795: 1797-1806 Pérez-Mellado V (1998) Podarcis muralis Laurenti, 1768. In Salvador A (ed) Reptiles. Fauna Ibérica, Vol. 10. Museo Nacional de Ciencias Naturales, Madrid, pp 283-294 Capítulo 2 Relaciones hospedador-parásito 115 Price PW (1980) Evolutionary biology of parasites. Princenton University Press, Princenton Salvador A, Veiga JP, Martín J, López P, Abelenda M, Puerta M (1996) The cost of producing a sexual signal: testosterone increases the susceptibility of male lizards to ectoparasitic infestation. Behav Ecol 7: 145-150 Schall JJ (1996) Malarial parasites of lizards. Adv Parasitol 37: 255-333 Schuster JP and Schaub GA (2001) Experimental Chagas disease: the influence of sex and psychoneuroimmunological factors. Parasitol Res 87: 994-1000 Sheldon BC, Verhulst S (1996) Ecological inmunology: costly parasite defences and trade-offs in evolutionary ecology. Trends Ecol Evol 11: 317-321 Smallridge CJ, Bull CM (2000) Prevalence and intensity of the blood parasite Hemolivia mariae in a field population of the skink Tiquila rugosa. Parasitol Res 86: 655-660 Smith TG, Desser SS, Martin DS (1994) The development of Hepatozoon sipedon n. sp. (Apicomplexa: Adeleina: Hepatozoidae) in its natural host, the Northern water snake (Nerodia sipedon sipedon), the culicine vectors, Culex pipiens and Culex territans, and an intermediate host, Northern leopard frog (Rana pipiens). Parasitol Res 80: 559- 568 Svahn K (1974) Incidence of blood parasites of the genus Karyolysus (Coccidia) in Scandinavian lizards. Oikos 25: 43-53 Tokarz RR, McMann S, Seitz L, John-Alder H (1998) Plasma corticosterone and testosterone levels during the annual reproductive cycle of male brown anoles (Anolis sagrei). Physiol Zool 71: 139- 146 van Riper CIII, van Riper SG, Goff ML, Laird M (1986) The epizootiology and ecological significance of malaria in Hawaiian land birds. Ecol Monog 56: 327-344 Veiga JP, Salvador A, Merino S, Puerta M (1998) Reproductive effort affects immune response and parasite infection in a lizard: a phenotypic manipulation using testosterone. Oikos 82: 313-318 Wintrobe MM (1991) Clinical haematology. Lea and Feiberger, Philadelphia, PA 117 Capítulo 3 Capítulo 3 Efectos de la degradación del medio 119 Efectos de la modificación de la vegetación natural sobre las poblaciones de lacértidos La transformación del medio natural debido a la influencia humana se apunta como causa principal del declive de muchas poblaciones de reptiles. Sin embargo, los efectos de los cambios antropogénicos de la vegetación en las poblaciones de reptiles no se han estudiado en profundidad. Estos cambios pueden conllevar una pérdida de hábitat, con la consiguiente desaparición de unas especies o aparición de otras. Además, los cambios antropogénicos del medio pueden hacer que los animales sean más conspicuos y, por tanto, más vulnerables a los depredadores, así como limitar el número de refugios. Por lo tanto, los cambios del medio pueden hacer que las lagartijas tengan que incrementar la frecuencia de las estrategias antidepredatorias. Dado que estas estrategias son costosas y pueden llevar una disminución de la condición corporal, el incremento del riesgo asociado a los cambios en el medio puede afectar al mantenimiento de las poblaciones de lagartijas. Por lo tanto, el objetivo de este capítulo es estudiar cómo los cambios en el medio afectan a la distribución y abundancia de algunas especies y al comportamiento antidepredatorio y a la condición física de los individuos de poblaciones de lagartijas. Capítulo 3 Efectos de la degradación del medio 121 Natural oak forest vs. ancient pine plantations: effects of traditional forest management on distribution and conservation of Iberian lizards RESUMEN Hoy en día la vegetación en Europa aparece profundamente modificada debido a las actividades humanas. En la Sierra de Guadarrama, las reforestaciones de pino silvestre, Pinus sylvestris, han reemplazado desde hace años a los bosques de melojo originales, Quercus pyrenaica. Sin embargo, no se han realizado suficientes estudios para analizar el efecto de las reforestaciones en la fauna, especialmente de reptiles. En este estudio describimos los patrones de selección de microhábitat de varias especies de lacértidos, y examinamos si la modificación de la vegetación original afectó a la distribución y a las densidades poblacionales de las lagartijas. Se encontraron lagartijas tanto en robledales como en plantaciones de pinos, pero las especies encontradas fueron diferentes en ambos tipos de bosques. Los resultados muestran que las lagartijas no usan el hábitat al azar. La mayoría de las lagartijas seleccionaron hábitats con rocas y poca cobertura de árboles, así como pocas distancias a refugios. Así mismo, el análisis de la relación entre la densidad relativa de lagartijas y el microhábitat mostró que la abundancia de lagartijas está determinada por las características de la vegetación. Los dos tipos de bosque (robledal y pinar) difirieron en algunas características estructurales de la vegetación, lo que probablemente está ligado a las especies de lagartijas encontradas. Sin embargo, desde un punto de vista de conservación y manejo de las poblaciones de lagartijas, las plantaciones de pinos no parecen contribuir mucho a la diversidad de las especies de lagartijas. Este estudio tiene implicaciones para el manejo forestal. Encontramos que áreas abiertas dentro de los bosques, sin árboles pero con matorral denso y rocas podrían contribuir al mantenimiento de las poblaciones de lagartijas. Capítulo 3 Efectos de la degradación del medio 123 Natural oak forest vs. ancient pine plantations: effects of traditional forest management on distribution and conservation of Iberian lizards Abstract Natural vegetation in Europe appears nowadays deeply modified by human activities. In the Guadarrama Mountains (Central Spain), ancient reforestations with Scots pines, Pinus sylvestris, replaced original deciduous pyrenean oak, Quercus pyrenaica, forests. However, the effect of reforestations on fauna remains little known, especially in reptiles. We described patterns of microhabitat selection in several species of Lacertid lizards, and analyzed whether the modification of the original vegetation affected distribution and population densities of lizards. Lizards were found either in oak forests or in pine plantations, but species found differed between forests types. Results showed that lizards do not use habitat at random. Most lizards selected habitats with rocky outcrops and with low cover of trees, and close to refuges. Furthermore, the analysis of the relation between relative lizard density and microhabitat showed that the abundance of lizards is determined by vegetation characteristics. The two types of forest (oak vs. pine) differed in some vegetation structure characteristics, which probably are linked to the lizards’ species found. However, from the perspective of conservation and management of lizards, pine plantations seem not to contribute too much to the diversity of lizards’ species. This study has implications for forest management. We found that open areas, without trees but with dense shrubs and rocks inside forests would contribute to maintain lizards’ populations. Introduction Natural vegetation in Europe appears nowadays deeply modified by human activities. Reforestations with Pinus spp. are widely extended all over the Iberian Peninsula, sometimes due to new agriculture policies, which aim to decrease food production and restore the environmental diversity previously lost through agricultural intensification (Diaz et al. 1998; Schmitz et al. 1998). However, changes are very often due to ancient traditional forest management, Capítulo 3 Efectos de la degradación del medio 124 with the substitution of original forest species by others with faster growth used for lumber exploitation. For example, the Guadarrama Mountains (Central Spain) are covered with two different types of natural forests: at low and intermediate altitude levels (1200-1700 m) natural vegetation is dominated by forests of deciduous Pyrenean oak, Quercus pyrenaica, whereas at high altitude (1700-1900 m) the natural vegetation consists of forests of Scots pine, Pinus sylvestris (Rivas-Martínez et al. 1987). Since ancient historical times, oak forests were used for production of charcoal and for extensive livestock grazing, whereas pine forests were traditionally managed for lumber exploitation. Oak forests were progressively deforested, but later reforested with pine plantations. Thus, nowadays, broad plantations of pines extend covering any altitudinal range through all the area, together with the natural pine forests still present at high altitude, whereas the oak forest has been relegated to certain smaller areas. Pine reforestations are now older enough so that they apparently resemble the natural pine forests found at high altitude. Nevertheless, subarboreal and herbaceous vegetation associated to these old pine plantations are typical from the original oak forest, although at least diversity of annual herbs has decreased and structure of vegetation has been modified (Izco 1984; Rivas- Martínez et al. 1987). To understand how the fauna respond to these changes on vegetation, we can use models that relate the abundance of the study species to variables describing the structure of the habitat. They can have considerable value, as they allow predictions to be made about a species’ response to artificial and natural habitat changes (Martín and López 2002). However, the evaluation of the effects of forest reforestation on animal communities has been mainly focused on the effects of recent plantations on bird populations (Potti 1985; Carrascal and Tellería 1990; Díaz et al. 1998; Goldstein et al. 2003). The long-term effects of reforestation have been rarely studied, and especially there is a general lack of studies focusing on other animal groups such as reptiles. This is, however, important in Mediterranean climates because reptiles are one of the more prominent groups of animals. The Iberian geographical area Capítulo 3 Efectos de la degradación del medio 125 includes many endemic species with populations reaching higher densities than in Central Europe (Corbett 1989). Furthermore, lizards are important keys in the tropic chains of Mediterranean ecosystems (e.g. Martín and López 1996) and changes in their populations should affect other taxa, suchs as snakes, raptors or some mammals. Numerous species of lizards show preferences for specific structural features of the habitat (Heatwole 1977; Martín and López 1998, 2002). Some studies have identified habitat requirements of some of these Mediterranean lizards occupying “optimal” areas (e.g. Carrascal et al. 1989; Díaz and Carrascal 1991; Castilla and Bauwens 1992). Lizards show some specific characteristics that make them vulnerable to changes in the structure of vegetation, such as their thermal requirements, which make them dependent on availability of sunny places where to bask (Martín and López 2002; Scheers and Van Damme 2002; Sabo 2003). Also, lizard dependence on habitat structure for finding refuges against predators seems to be an important determinant of their survival (Milne and Bull 2000; Webb and Shine 2000; Souter et al. 2003). Lower mobility and dispersal abilities of lizards, compared to birds or mammals, may also increase their vulnerability to local extinction to a greater extent than for other groups (Diaz et al. 2000; MacNally and Brown 2001). However, the effects of habitat modification on lizards’ populations have not been extensively analysed in Europe (but see Santos and Tellería 1989; Díaz et al. 2000; Martín and López 2002). Microhabitat diversity and structure has often been observed to be more important than macrohabitat type as a predictor for composition of lizard communities (Pianka 1967; Szaro and Belfit 1986; Menke 2003). Thus, it remains possible that lizards populations would have adapted to old changes in macrohabitat (i.e. from oak to pine forests) provided that microhabitat structure remained similar. Alternatively, changes in macrohabitat type distribution might be parallel to changes in distribution of lizard species associated with each habitat type. In this study, we described microhabitat selection in several species of lizards, and specifically analyzed whether modifications of the original vegetation Capítulo 3 Efectos de la degradación del medio 126 in the Guadarrama Mountains may have affected distribution and population densities of several lizard species. We further analysed whether the number of lizards (i.e. relative abundance) of each species observed in each forest patch was dependent on modifications of the physical structure of the habitat. Methods Study area The study was conducted in the Guadarrama Mountains (Madrid province, Central Spain). We limited the study to the altitudinal range of 1200- 1600 m. In this area and altitudinal range, there is a Mediterranean climate with a mean annual temperature ranged between 8 and 12 ºC (Izco 1984), and the natural vegetation should be dominated by Pyrenean oak, Q. pyrenaica, forests. However, the area is nowadays covered with both remains of the original oak forests, and also extensive ancient plantations of Scots pine, P. sylvestris. Lizard censuses and vegetation sampling Lizard censuses were conducted from April to July 2002, coinciding with the mating season of lizards, when they were particularly active. We marked 81 line transects 200 m in length distributed more or less evenly throughout the study area, at least 1-2 km apart, and chosen to cover homogeneous patches of forests of both types through all the altitudinal gradient (between 1200-1600 m). We identified and counted lizards flushed or observed in each transect and in a 10 m- wide belt, 5 m on each side of the survey line. Transect census were made in days with favourable climate conditions (warm sunny days) and between 09:00 and 13:00 GMT, when lizards were more active. This method provides relative abundance of lizards for each transect (Martín and López 2002). We also conducted additional surveys in the area, lifting up stones and logs in order to confirm the presence of secretive semifossorial skinks and fossorial amphisbaenians in each type of forest. When we detected a lizard in a transect, we marked the point and, when the census had been completed, we took four 1 m transects, one at each of the four cardinal orientations radiating from Capítulo 3 Efectos de la degradación del medio 127 the point where each individual was first sighted. We used a scored stick standing vertically at nine sample points (two points at 50 and 100 cm in each of the four transects, and the central point), and recorded the type of substrate found at each point (grass, leaf litter, bare soil, or rocks). We also noted whether there were tree cover above the sample point, the type of tree (oak vs. pine), and the subarboreal vegetation at each point. We classified this subarboreal vegetation in two types according to their height and characteristics: herbaceous perennials (< 50 cm height; including species such as Paeonia broteroi or Pteridium aquilinum), and woody shrub species (> 50 cm height; including Juniperus communis, Genista florida, Crataegus monogyna, Cytisus scoparius, Rosa pouzini, Rubus ulmifolius, and Lonicera periclymenum). We noted the type of vegetation (herbaceous vs. shrub) and the height from the ground to the first contact of leaves with the stick. This variable provided an indication of the potential utility of this subarboreal vegetation as a refuge by lizards (Martín and López 1998). Thus, a low height indicated that vegetation was close to the ground, and thus, provided a narrow refuge where to hide. Previous studies have shown the importance of plant cover at the ground level for lizards (Carrascal et al. 1989; Martín and López 1998). Also, for these lizard species, the subarboreal vegetation total height was not considered important because lizards move on the ground and below vegetation. We calculated percent cover values for each habitat variable in the area surrounding each lizard (i.e. % contacts with each substratum and vegetation type, and height of potential refuges; for a similar sampling methodology see Martín and López 1998, 2002). We also noted the distances of each point to the nearest potential refuge and to the nearest sunny spot where lizards could bask. Given the large size of the area surveyed and the high lizard density, and because we made each transect only once, the probability of repeated sampling of the same individual was very low. We therefore treated all measurements as independent. To estimate the availability of microhabitats along each transect, we recorded the same variables than above at three points per transect (at 70, 140 and 200 m along the progression line). Capítulo 3 Efectos de la degradación del medio 128 Data analysis We used principal component analysis (PCA) to reduce all the habitat variables to a smaller number of independent components. We performed a PCA on the points describing microhabitats available in both types of forests and in the lizard-observed microhabitat points. Original data (number of contacts) were normalised by means of square root transformation. The initial factorial solutions were rotated by the Varimax procedure (Nie et al. 1975). Thereafter, we used General Linear Models (GLM) to compare PC scores describing microhabitat characteristics of the two types of forests (pine vs. oak forest), and those used by each lizard species. Thus, we determined whether lizards used available microhabitats in a non-random fashion (for a similar procedure see Martín and López 1998, 2002). We also used these PC scores to obtain a predictive model for the relative number of lizards observed in a transect from average microhabitat characteristics in that transect (Maurer 1986; Verner et al. 1986; Rubio and Carrascal 1994; Martín and López 2002). Thus, we examined the relationships between PC scores (independent variables) and number of lizards of all species censused in each transect (dependent variable) using forward stepwise General Regression Models (GRM). We also made separate similar comparisons between PC scores and number of lizards of each species. Results Lizard species and distribution The type of forest per se did not influence the presence in transects of some lizards in general (GLZM, Wald’s χ2 = 0.22, df = 1, P = 0.64). Lizards were found both in oak forests and in pine plantations. However, the lizard species found were different. In oak forests, we found two dominant species of lacertid lizards: large psammodromus (Psammodromus algirus) and Iberian wall lizards (Podarcis hispanica). There were also some occelated lizards (Lacerta lepida), which were more abundant in lowland holm oak forests (unpublished data), and, therefore, occupied marginal habitats in this area. We also found a few Schreiber’s green lizards (Lacerta schreberi), but they were rather linked to riverine vegetation Capítulo 3 Efectos de la degradación del medio 129 habitats and not to forests. Also, in additional surveys in oak forests we found other less abundant lizard-like species: one skink (Chalcides bedriagai), and an amphisbaenian (Blanus cinereus), which have fossorial or semifossorial habits and were not represented in transects. In contrasts, pine forests at the same altitudinal range were predominantly occupied by common wall lizards (Podarcis muralis), which were widespread, and a small number of Iberian wall lizards (P. hispanica), limited to some particular locations. We also found a few L. schreberi here, but also linked to riverine vegetation and not to pine forests. No other lizard species was found in pine forests even during additional surveys. Microhabitat selection by lizards The PCA for microhabitats available and those used by lizards produced four components that together accounted for the 73.1 % of the variance (Table 1). The first PC (PC-1) was negatively correlated with the cover of rocky outcrops, and positively correlated with substrates with grass and leaf litter, with the cover of trees at the canopy level, and with distance to the nearest refuge. The second PC was negatively correlated with the cover of shrubs, and with the distance to the nearest open area. The third PC (PC-3) was negatively correlated with cover and minimal height from the ground of perennial herbaceous vegetation. The fourth PC (PC-4) was positively correlated with bare soil cover. Overall, microhabitats available in both types of forest and those used by lizards were significantly different (GLM, Wilks χ2 = 0.31, F4,16 = 32.54, P < 0.0001; Fig. 1). Both types of forests differed only in the cover of bare soil, which was significantly higher in pine forests (PC-4), but not in other characteristics (PC-1 to PC-3, see below). In both forests, available microhabitats had a high cover of trees, and substrates with high cover of grass and leaf litter far from refuges, but lizards selected areas with less trees and more rock substrates, and close to refuges (PC-1: F4,370 = 149.30, P < 0.0001; Tukey’s test, P < 0.0001 in all cases). Furthermore, there were significant differences between microhabitats selected by P. algirus lizards and the two wall lizards (Tukey’s Capítulo 3 Efectos de la degradación del medio 130 test, P < 0.0001), which did not differ between them (P > 0.11) in selecting microhabitats with the highest cover of rocks (Fig. 1). Table 1 Principal components analysis for available and lizard microhabitat data. Emboldened values indicated correlations of variables with the principal components greater than 0.55. Also, P. algirus selected microhabitats with a high cover of shrubs and far from open areas (PC-2: F4,370 = 6.76, P < 0.0001; Tukey’s tests: P < 0.0002 in all cases), whereas cover of shrubs in microhabitats selected by both wall lizard species did not differ from those available in both types of forest (P > 0.40 in all cases) (Fig. 1). Finally, all lizard species selected microhabitats with low cover of bare soil (PC-4: F4,370 = 8.44, P < 0.0001), which PC-1 PC-2 PC-3 PC-4 Substrate: Rocks -0.87 0.16 0.01 -0.07 Bare soil -0.03 -0.01 0.01 0.90 Grass 0.78 0.19 0.18 -0.16 Litter 0.74 -0.26 -0.18 -0.10 Vegetation: Shrub cover 0.05 -0.91 0.07 -0.04 Shrub minimal height 0.12 -0.22 -0.01 -0.21 Herbaceous cover 0.13 -0.01 -0.68 -0.23 Herbaceous minimal height -0.04 0.01 -0.80 0.10 Tree cover 0.66 0.03 -0.25 0.07 Distance to refuge 0.56 0.34 0.07 0.42 Distance to open 0.03 -0.86 -0.08 0.03 Eigenvalue 2.69 1.86 1.25 1.14 % Var. 24.43 26.94 11.38 10.36 Capítulo 3 Efectos de la degradación del medio 131 was similar to available in oak forests (Tukey’s test, P > 0.92 in both cases), but not in pine forests, where substrates had a higher proportion of bare soil (P < 0.002 in all cases) (Fig. 1). The cover of herbaceous vegetation (PC-3: F4,370 =1.87, P = 0.12) did not influence habitat selection by lizards. Lizard-habitat relationships A forward stepwise GRM showed a relationship between relative lizard density of all species and characteristics of microhabitats defined by the average PC scores in a transect (GRM, R2 = 0.15, F1,79= 14.31, P = 0.0003). Thus, without considering the species, more lizards were found in transects when there were rocks rather than grass or leaf litter, when the distance to a refuge was low, and when the cover of trees was scarce (PC-1), whereas other habitat variables (PC-2, PC-3 or PC- 4) were not significantly related to overall lizard abundance. This relationship was similar for the number of individuals of common wall lizards, P. muralis, censused in each transect (GRM, R2 = 0.20, F1,79 = 20.55, P < 0.0001). However, in the case of Iberian wall lizards, P. hispanica, the number of individuals differed in relation to the vegetation characteristic defined by PC- 1 and PC-4 (GRM, R2 = 0.14, F2,78= 6.10, P = 0.003). Thus, Iberian wall lizards were more abundant in transect with rocky outcrops, low distance to the nearest refuge, and low cover of trees rather than in substrates of grass, leaf or bare soil. On contrast, the number of P. algirus individuals did not seem to depend on vegetation characteristics defined by PC scores. Capítulo 3 Efectos de la degradación del medio 132 Fig. 1 Means (+ 1 SE) of PC scores for microhabitat available in pine plantations and original oak forests (open boxes), and for microhabitat used by P. muralis, P. hispanica and P. algirus lizards (black boxes). Capítulo 3 Efectos de la degradación del medio 133 Discussion Microhabitat selection and lizard- habitat relationships Our surveys showed striking differences in lizard species found in oak forests and in pine plantations. This was very likely explained because pine and oak forests differed in the characteristics and structure of microhabitats, because lizards did not use habitat at random, and because each lizard species has some specific microhabitat requirements. The two wall lizards, P. muralis and P. hispanica, selected habitats with rocky outcrops and with a low cover of trees. In contrast, P. algirus lizards preferred microhabitats with a high cover of dense shrubs, far from open areas. The reasons for these patterns have been discussed in other studies. For example, lizards select microhabitats where they can optimise their thermoregulatory, antipredator and foraging requirements (e.g. Carrascal et al. 1989; Díaz and Carrascal 1991; Castilla and Bauwens 1992; Martín and López 2002). In places with dense tree canopy, it must be very difficult to find good spots where to bask. Thus, thermoregulation seems to be a determinant factor affecting lizard distribution and abundance. Antipredator requirements seem also to affect lizard abundance, since lizards were more abundant in areas with rocks or low bushes, which can be used as refuges where hiding from predators (Martín and López 1998). Furthermore, the analysis of the relation between relative lizard density and microhabitat characteristics showed that the abundance of lizards is determined by microhabitat characteristics. Although each species has some specific habitat requirements, the general patterns of abundance in relation to habitat structure were very similar between species. Thus, the number of individual lizards depended on the presence of rocky substrates, low cover of canopy of trees, and the availability of refuges. These results are similar to those from a study of the effects of miming and fire on lizard’s diversity in Australian forests (Taylor and Fox 2001). However, when considering each species separately, we obtained different results which agreed with their microhabitat selection patterns (see above), and explained the differential distribution of species between habitats. Capítulo 3 Efectos de la degradación del medio 134 The presence of P. muralis in pine plantations but not in oak forests located at similar altitude suggests a narrow link between the distribution of this species and pine forests. Pine plantations at low altitude probably present particularly favourable microclimate conditions that might have favoured the colonization by P. muralis (Guisan and Hofer 2003), a species usually found at higher altitude in natural pine forests closely related to the Euro-Siberian climatic region (Pérez- Mellado 1998; Diego-Rasilla 2004). Therefore, pine plantations might have contributed to the expansion of the southern and altitudinal limits of P. muralis distribution in the Iberian Peninsula, in areas otherwise occupied by more Mediterranean lizard species, such as P. algirus. Implications for the forest management This study has implications for forest management. We found that open areas, without trees but with dense shrubs and rocks, inside forests contribute to maintain lizards’ populations and lizard species diversity. Thus, management actions (for example, lumber exploitation) to create or maintain open areas in the forests, with shrubs, consequence of the degradation of the potential vegetation of the forests should contribute to maintain and favour the populations of lizards. Shrubs may provide refuge for lizards, as well as insects for feeding. Furthermore, dense tree canopy do not allow sunlight to reach the ground. Thus, open areas may allow lizards to bask, which may benefit lizards’ populations. Other studies have reported an increase in lizard abundance within deforested areas (Enge and Marion 1986; Goldingay et al. 1996; Renken et al. 2004) or at forest edges (Schlaepfer and Gavin 2001), which may be explained by the abundance of basking places within these deforested areas (Renken et al. 2004). Thus, according to the “intermediate disturbance hypothesis”, these results suggested that disturbances can promote the coexistence of species (Grime 1974; Connell 1978; Pickett and White 1985; Huston 1994) and promote high diversity of lizards’ species. For example, more lizards were captured in open deforested areas than in Australian forests (Taylor and Fox 2001), and the number of individuals of some desert species also increased due to the grazing-induced reduction in vegetation cover (Read Capítulo 3 Efectos de la degradación del medio 135 2002). Moreover, worldwide patterns in lizard abundance show that hotter, drier areas support more lizard species and individuals than cooler forested areas (Pianka 1988). From the perspective of conservation and management of lizards, pine plantations seem not to contribute so much to the diversity of lizards’ species. Thus, reforestation with pines does not benefit lizards’ populations, but in the particular potential expansion of P. muralis lizards. In the case of old pine plantations in the original area occupied by oaks, oaks tend to recolonize the area below the pine forest, and pines are exclusively maintained by human activities. Thus, a good method to maintain original lizard’s populations should be to allow the recolonization of some areas of the pine plantation by oaks. This measure may also be a preventive strategy against fire (Calvo et al. 2003). Pines contain resins, which have a high inflammability. In order to prevent fires in the pine forests, the clear cutting techniques are often employed to create areas without trees to prevent the fire expansion. These open areas without any shruby vegetation not only prejudice soil characteristics (Schmitz et al. 1998) but also decrease habitat quality for lizards (Ryan et al. 2002). Furthermore, removing of dead vegetation and clearing of underbrush to prevent fires often eliminate potential refuges available to lizards (James and M’Closkey 2003). Similar results were obtained when comparing beetles species in clear cuts and following stages of vegetation succession (Similä et al. 2002). On contrast, oaks do not have resins and recover quicker from the fire than pines. Therefore, the maintenance of oak stands inside pine plantations, with all the shrubs associated to this type of forest, should favour the maintenance of diversity and abundance of lizard’s populations. This agrees with previous results that found reptiles to be more abundant in mixed forests that in clear- cuts and pine plantations (Ryan et al. 2002). Similar results indicated the impoverishment of birds’ species in pine plantations in relation to the native vegetation of a concrete area (Sekercioglu 2002). The clearing of some areas of the pine plantations with the encouragement of deciduous trees such as oaks and undergrowth are also forest managements proposed to favour also the bird’s abundance and richness Capítulo 3 Efectos de la degradación del medio 136 (Potti 1985; Carrascal and Tellería 1990). Thus, maintaining and favouring the habitat heterogeneity may help maintain a high diversity, not only of lizards but also of other taxa, by providing a large number of different niches (Fischer et al. 2004). Acknowledgements We thank "El Ventorrillo" MNCN Field Station for use of their facilities. Financial support was provided to L. Amo by an “El Ventorrillo” C.S.I.C. grant, to P. 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J Wild Manag 50: 752–761 Taylor JE, Fox BJ (2001) Disturbance effects from fire and mining produce different lizard communities in eastern Australian forests. Austral Ecol 26: 193– 204 Verner J, Morrison ML, Ralph CJ (1986) Wildlife 2000. Modeling Habitats Relationships of Terrestrial Vertebrates. University of Wisconsin Press, Madison, Wisconsin. Webb JK, Shine R (2000) Paving the way for habitat restoration: can artificial rocks restore degraded habitats of endangered reptiles? Biol Conserv 92: 93-99 Capítulo 3 Efectos de la degradación del medio 139 Increased predation risk due to habitat deterioration affects body condition of lizards: a behavioural approach with Lacerta monticola lizards inhabiting ski resorts RESUMEN Una de las primeras estrategias antidepredatorias de las presas para minimizar el riesgo de depredación es la selección de hábitats seguros, que pueden disminuir la conspicuidad de las presas o proveerlas de refugios en los que esconderse. Sin embargo, la vegetación natural se encuentra hoy en día profundamente modificada debido a las actividades humanas, y este cambio en la estructura del medio puede incrementar el riesgo de depredación. Por ejemplo, la construcción de pistas de esquí en las montañas deteriora el medio natural, lo que implica una pérdida de cobertura y de refugios potenciales para algunos taxones. La lagartija serrana es un endemismo amenazado, pero su hábitat preferido está a menudo degradado por instalaciones de esquí. En este estudio analizamos si las lagartijas modificaron su comportamiento en áreas afectadas por estos cambios antropogénicos en la estructura del microhábitat, y si esos cambios tenían consecuencias para la condición corporal y el estado de salud de las lagartijas. Concretamente examinamos en el campo en áreas con distinto nivel de degradación del medio (y distinto riesgo de depredación): a) la selección de microhábitats; b) los patrones de locomoción espontánea; c) la condición corporal, la respuesta inmune y la carga parasitaria de machos de lagartijas. Los resultados sugirieron que la degradación del medio no sólo implicó una pérdida de hábitat para las lagartijas, sino que también provocó un incremento en el riesgo de depredación percibido por estas. Los machos parecieron ajustar sus patrones de movimiento de acuerdo con estas diferencias en el riesgo, aumentando la velocidad durante sus desplazamientos a lo largo de áreas degradadas, pero, como consecuencia, sufrieron una pérdida de condición corporal. También realizamos dos experimentos de laboratorio para analizar a) el efecto de las carreras a gran velocidad en la condición corporal y el estado de salud de lagartijas, y b) el efecto de la condición corporal en la velocidad de las lagartijas. Los experimentos de laboratorio apoyaron los resultados obtenidos en el campo. Por lo tanto, nuestro estudio Capítulo 3 Efectos de la degradación del medio 140 proporciona una nueva evidencia de que las estrategias comportamentales para hacer frente a un incremento en el riesgo pueden afectar a la condición corporal de las lagartijas. Capítulo 3 Efectos de la degradación del medio 141 Increased predation risk due to habitat deterioration affects body condition of lizards: a behavioural approach with Lacerta monticola lizards inhabiting ski resorts Abstract One of the first strategies of prey to minimise predation risk is the selection of safe habitats, which can decrease conspicuousness of prey or provide them with refuges where to hide. However, natural vegetation appears nowadays deeply modified by human activities, and this change in habitat structure may increase predation risk. For example, the construction of ski slopes in mountains deteriorates the natural habitat, which implies a loss of cover and potential refuges for some taxa. The Iberian rock lizard is an endangered endemic species restricted to some high mountains of the Iberian Peninsula, but its preferred habitat is often occupied by ski resorts. We analysed whether lizards modified their behaviour in areas affected by these anthropogenic-changes in microhabitat structure, and whether these changes had consequences for the body condition and health state of lizards. Concretely we examined in the field in areas with different level of deterioration of the habitat due to the presence of ski slopes (i.e. different predation risk): a) microhabitat selection; b) spontaneous movement patterns, c) body condition, immune response and parasite load of male lizards. Results suggested that habitat deterioration not only implied a loss of optimal habitat for lizards, but also led to an increase in perceived risk. Males seemed to adjust their movement patterns accordingly to differences in risk, increasing speed during their displacements across risky areas within deteriorated habitats, but as a consequence, they incurred loss of body condition. We also performed two laboratory experiments to analyse a) whether fleeing at high speed affects body condition and health state of lizards, and b) whether changes in body mass affect sprint speed of lizards. The results supported our findings in the field. Therefore, our study provides new evidence that behavioural strategies to cope with increased predation risk due to human-induced habitat deterioration may affect body condition of lizards. Capítulo 3 Efectos de la degradación del medio 142 Introduction Predation risk is a major selective force in the evolution of several morphological and behavioural characteristics of animals (Lima and Dill 1990; Lima 1998). However, prey should optimize their antipredatory response by balancing antipredatory demands with other requirements (Lima and Dill 1990; Lima 1998). One of the first strategies of prey to cope with predation risk is the selection of safe habitats where they can attend their requirements while minimising risk (e.g. Lima 1998; Amat and Masero 2004). Habitat selection may decrease conspicuousness of prey, and provide refuges where to hide under the attack of a predator (Arthur et al. 2004). However, natural vegetation appears nowadays deeply modified by human activities, and this change in habitat structure may increase perceived risk of predation (Whittingham and Evans 2004). For example, deterioration of vegetation may make animals more conspicuous and thus, more vulnerable to predators (Martín and López 1998), which will require animals to increase the frequency of antipredatory behaviours. One of these antipredatory behaviours is the modification of activity and locomotor patterns (Martín and Salvador 1997; McAdam and Kramer 1998). A generalized response to increased predation risk is the decrease in activity to avoid encounters with predators or the attack of predators that localize their prey by their movement. For example Daphnia water fleas avoided encounters with their predators, Chaoborus phantom midge larvae, by decreasing swimming speed (Weber and Van Noordwijk 2002). Similarly Rana temporaria tadpoles decreased their activity in the presence of the predatory Aeshna dragonfly larvae (Laurila et al. 2004). This decrease in activity has also been observed in other taxa, such as reptiles (Hecnar and M'Closkey 1998; Downes 2001), fishes (Vehanen 2003), mammals (Jensen et al. 2003; Orrock and Danielson 2004) and birds (Mougeot and Bretagnolle 2002). However, when movement is required due to foraging or reproductive requirements, animals may modify their locomotor patterns in order to decrease their vulnerability while moving under high risk of predation (Martín and Salvador 1997; McAdam and Kramer 1998). In this case, the Capítulo 3 Efectos de la degradación del medio 143 ability to develop an optimal speed seems to be determinant not only to escape from predators, but also to decrease time exposed to potential predators when moving undisturbed (López and Martín 2002; Miles 2004). Nevertheless, and although locomotor activities play an important role in the ecology of animals (Turchin 1998), studies of locomotor patterns have mainly focused on the functional capacities of animals, often ignoring the behavioural context in which locomotion is used (Irschick 2000, but see Martin and Lopez 1995; Irschick and Losos 1998; Jayne and Ellis 1998; Van Damme et al. 1998; Miles 2004). Antipredatory behaviours such as fleeing at high speed and refuge use are costly. For example, Podarcis muralis lizards experimentally submitted to a high predation pressure increased refuge use, which led to a loss of body mass (Martín and López 1999a). Similarly, Psammodromus algirus lizards submitted to attacks lose mass not only because they interrupted feeding, but also because of stress of predation risk per se (Pérez-Tris et al. 2004). Therefore an excessive allocation of time and energy to antipredatory strategies can decrease body condition, with important consequences for short and long term fitness. Furthermore, the loss of body condition could also decrease the ability to invest in defence against parasites, because the nutritional status can influence the capacity of a lizard to mount an immune response to infection (Cooper et al. 1985; Smallridge and Bull 2000). This may influence host-parasites relationships, and increase the negative effects of parasites on their host, which, in turn, can affect the maintenance of lizard’s populations. However, there are no ecological studies about the costs, in terms of body condition and health state, of changes in movement patterns in response to increased predation risk. The Iberian rock lizard, Lacerta monticola, offers an excellent system to study the changes in movement patterns, and the consequences of these changes, under different levels of perceived risk. This species is an endemic endangered small diurnal lacertid found mainly in rocky habitats in some high mountains of the Iberian Peninsula. In its natural habitat this lizard suffers a high predation risk as shown by the high rate of regenerated tails (Martín and Salvador 1997). Lizards responded to predators by Capítulo 3 Efectos de la degradación del medio 144 running rapidly for cover into the nearest refuge (Martín and López 1999c). This species suffers a loss of optimal habitat due to the construction of ski infrastructures (Martín and Salvador 1995; Pérez-Mellado 2003). The natural vegetation of the ski slopes is not only badly affected, but also “obstacles” (rocks) in the slopes are continuously removed, causing extreme disturbance to the habitat (Martín and Salvador 1995). Such drastic changes have a considerable impact on the whole ecosystem (Haslett 1991; Illich and Haslett 1994; Wipf et al. 2005). The lack of vegetation and rocks causes a loss of cover and potential refuges for lizards, and thus, it creates areas with different level of predation risk. Males defend territories by performing continuous movements across their home range searching for females and expelling intruder males (Martín and López 2000; Aragón et al. 2001). Therefore, males may be more susceptible than females to predation because they have higher movement rates and are more conspicuous (Martín and Salvador 1997; Martín and López 1999b, 2000). Here we analysed whether male lizards modified their microhabitat use and locomotor patterns under different level of perceived predation risk, induced by anthropogenic-changes in microhabitat structure (i.e. construction of ski slopes), and whether these changes had consequences on their body condition and health state. Concretely we first examined in the field in areas with different level of deterioration of the habitat (i.e. different predation risk): a) microhabitat selection, b) spontaneous movement patterns; and c) body condition, immune response, and parasite load of male lizards. We predicted that males might not use microhabitat at random, and therefore, regardless of the scarce cover of shrubs or rocks in deteriorated areas, we expected that lizards decreased predation risk by using less risky areas (i.e. those close to refuges such as shrubs or rocks). However, because males need to patrol their entire home ranges, we also predicted that males might assess that predation risk is higher while moving in areas with low vegetation cover and low availability of refuges. Thus, we expected that, in deteriorated areas, males would increase speed when crossing unsafe areas far from refuges to reduce time exposed to predators. Capítulo 3 Efectos de la degradación del medio 145 However, since moving at high speed is more costly than moving slower (Kramer and Mclaughlin 2001; Gleeson and Hancock 2002), we expected that these males would suffer a loss of body condition, which might also affect their health state. In addition, as in the field many other factors rather than speed during displacements may be affecting body condition of males, we performed a laboratory study to analyse the effect of fleeing at high speed on body condition and health state of lizards. We expected that lizards that were forced to run by a simulated predator and did not have a close refuge for hiding might suffer a loss of body mass after several attacks, whereas lizards that did not need to run for longer, because they had a close refuge, would not decrease their body mass. Immediately after this experiment, we also measured sprint speed of lizards when they were forced to flee to test whether differences in body condition influenced escape speed. As a previous study showed (Pérez-Tris et al. 2004), we did not expect differences in escape speed of lizards of similar body size regardless of their body condition. Methods Study area and species We performed the study in the Guadarrama Mountains (Madrid Prov., Central Spain) at an elevation range of 1900-2200 m. Natural landscape at this altitudinal range is characterised by granite rock boulders and screes interspersed with shrubs (Cytisus oromediterraneus and Juniperus communis), meadows of Festuca and other grasses, and a few dispersed Scots pines (Pinus sylvestris), which form extensive forest at lower altitudes (Martín and Salvador 1997). This area is characterised by the presence of several ski resorts and associated infrastructures. In anthropogenic-induced deteriorated areas, mostly in ski slopes, there is no cover of shrubs nor even grasses, and rocks boulders are scarce or have been eliminated. In this region, L. monticola (snout-to-vent length, SVL, of adult lizards ranges between 65 mm and 90 mm) is active from May to September due to limiting environmental temperatures. Lizards mate in May-June and produce a single clutch in July Capítulo 3 Efectos de la degradación del medio 146 (Elvira and Vigal 1985; Salvador 1984; Pérez-Mellado 1998). Microhabitat use by lizards To evaluate habitat characteristics of areas with different level of deterioration due to ski infrastructures, and microhabitat use by lizards in these areas, we walked haphazardly during May-June 2003 in days with favourable climate conditions (warm sunny days) and between 09:00 and 13:00 GMT, when lizards were more active. When we detected an adult male lizard (n = 103), we marked the point where it was first observed. For each point we took four 1 m transects, one at each of the four cardinal orientations radiating from the point where each individual was sighted. We used a scored stick standing vertically at nine sample points (two points at 50 and 100 cm in each of the four transects, and the central point), and recorded the type of substrate found at each point (grass, leaf litter, bare soil, or rocks). We noted the presence of canopy tree cover (Scots pines) above each sample point, and the cover and the height from the ground to the first contact with the stick of leaves of subarboreal vegetation at each point. This later variable provided an indication of the potential utility of vegetation as a refuge by lizards (Martín and López 1998). Thus, a low vegetation height indicated that vegetation is close to the ground, providing a narrow refuge where to hide. Previous studies have shown the importance of plant cover at the ground level for lizards (Carrascal et al. 1989; Martín and López 1998). Also, for this lizard species, the subarboreal vegetation total height was not considered important because lizards move only on the ground or rocks and below vegetation. We also noted the distance to the nearest available refuge, and to the nearest open sunny spot where lizards could bask. We calculated percent cover values for each habitat variable in the area surrounding each lizard (i.e. % contacts with each substratum and vegetation type), and average distances to refuges and to sunny spots, and height of potential refuges (for a similar sampling methodology see Martín and López 1998, 2002). Given the large size of the area surveyed and the high lizard density, and because we avoided sampling the same area twice, the probability of repeated sampling of the Capítulo 3 Efectos de la degradación del medio 147 same individual was very low. We therefore treated all measurements as independent. To estimate the availability of microhabitats we recorded the same variables than above at 128 points randomly chosen through all the sampled areas. We used principal component analysis (PCA) to reduce all the habitat variables to a smaller number of independent components. We performed a PCA on the points describing available microhabitats and the lizard-observed microhabitat points. Original data (number of contacts) were normalised by means of square root transformation. The initial factorial solutions were rotated by the Varimax procedure (Nie et al. 1975). We used General Linear Models (GLM) to compare PC scores describing microhabitat characteristics in relation to the level of deterioration of the habitat (natural vs. deteriorated), and the type of point (available vs. used by lizards) to determine whether lizards used available microhabitats in a non- random fashion. We included the interaction between level of deterioration and type of point in the model to test for differences in microhabitat selection of lizards in areas with different level of deterioration (Martín and López 1998, 2002). Movement patterns We performed this study in the same area described above during May-June to determine whether male lizards changed their spontaneous movement patterns in relation to the level of deterioration of the habitat. We haphazardly walked both natural areas and areas where habitat was deteriorated due to ski infrastructures, between 1000 and 1600 h, when lizards were fully active. When a lizard was sighted, we initiated focal observations using binoculars and at a distance of 7-10 m to avoid disturbing the lizard. We chose adult males (n = 45) of similar body size in both areas to avoid the potential effect of lizards’ size on locomotor patterns. We recorded for approximately 10 min the time spent in movement, the distance moved, and the duration of the observation. Since not all observations could have the same exact duration because some lizards were lost or unintentionally disturbed, we calculated the time in movement and the distance moved in relation to the total duration of the observation. We calculated average ‘burst speed’ by dividing the distance Capítulo 3 Efectos de la degradación del medio 148 moved by the time spent in movement. Since temperature may affect movement patterns (e.g. Zhang and Ji 2004), and because it was difficult to measure body temperature of lizards immediately after the trial, we measured air temperature with a digital thermometer to the nearest 0.1 ºC which can give an indication of body temperature in this species (Martín and Salvador 1993). We used analyses of covariance (ANCOVA) to test for differences in time spent in movement, distance moved, or average burst speed (dependent variables) in relation to the level of deterioration of the area, with air temperature as a covariant. Measurement of body condition and parasite load of lizards We captured by noosing adult male lizards (n = 86) in areas with different level of deterioration of the habitat, during the mating period (May-middle of June) and after the mating period has finished (middle of June-July) to examine their body condition and health state (parasite load and immune response). We weighed and measured SVL of lizards immediately after capture. Then, we took captured lizards for data collection (see below) to "El Ventorrillo" Field Station, 5 km from the capture site, where lizards were individually housed in 60 x 40 cm PVC outdoor terraria. They were fed mealworm larvae (Tenebrio molitor), and water was provided ad libitum. The photoperiod and ambient temperature were that of the surrounding region. All lizards were healthy and were returned to their exact capture sites 48 h after capturing. To assess blood parasite load, we made a smear on a microscope slide from blood taken from the postorbital sinus by using one 9 µl heparinized hematocrit tube. Blood smears were air- dried, fixed in absolute methanol for 10 min and then stained in Giemsa diluted 1:9 with phosphate buffer (pH 7.2) for 40 min before their examination for parasites. On mounted slides, half a smear, chosen at random, was scanned entirely at 200 x along the longitudinal of slide, looking for extraerythrocytic protozoa (Merino and Potti 1995; Amo et al. 2004). Number of intraerythrocytic parasites (Haemogregarines, the only parasite found) was estimated at 1000 x by counting the number of parasites per 2000 erythrocytes. Capítulo 3 Efectos de la degradación del medio 149 We measured T-cell mediated immune (CMI) responsiveness of 32 male lizards in summer by using a delayed-type hypersensitivity test. This test is a reliable measure of T-cell- dependent immunocompetence in vivo (Lochmiller et al. 1993), and it has been used in many studies of animals including lizards (Merino et al. 1999; Svensson et al. 2001; Belliure et al. 2004). We estimated CMI on the basis of quantification of the swelling response to intradermally injected phytohaemagglutinin (Smits et al. 1999). We injected the lizard’s footpad of the right hind limb with 0.02 ml of phytohaemagglutinin solution (PHA-P, Sigma), and measured the swellings with a pressure sensitive spessimeter (to the nearest 0.01 mm) before and 24 h after the injection (Smits et al. 1999). Results of previous studies showed that repeatability of this measure, calculated as the intraclass correlation coefficient (Lessells and Boag 1987) is high (r>0.95, L. Amo, unpublished data). We used analyses of covariance (ANCOVA) to analyse differences in blood parasite load between seasons and levels of deterioration of the habitat, including the interaction between season and level of deterioration, and SVL as a covariant. We also used ANCOVA to analyse differences in body mass between seasons and levels of deterioration, including the interaction between season and level of deterioration, and SVL and blood parasite load as covariants. Finally, we used ANCOVA to analyse the effect of level of deterioration on CMI of lizards, including SVL, body mass and blood parasite load as covariants. Effects of fleeing at high speed on body condition In July we captured by noosing 29 adult male lizards in a nearby area to determine whether increasing the frequency of displacements at high speed to reach safe refuges influenced body condition, and health state (parasite load and immune response) of lizards. Lizards were taken to “El Ventorrillo” Field Station and maintained in the same initial conditions than described above. All lizards were measured, and we extracted a drop of blood for parasite measurements (see above). These measures were taken again immediately after the experiment finished. At the end of the experiment we also performed the Capítulo 3 Efectos de la degradación del medio 150 PHA test to assess CMI (see above). To avoid changes in body condition and parasite load of lizards due to captivity per se, they were held in captivity only one week before testing to allow acclimation to laboratory conditions. Lizards were healthy and were released at the end of the experiment at the exact place of capture. The experiments were performed under license from the Consejería del Medio Ambiente de la Comunidad de Madrid (Spain). Terraria were placed in an open sunny location while shade was provided by one of the terrarium walls and the refuge (flat stones of similar size and shape). Thus, we allowed lizards to thermoregulate and attain their preferred body temperatures for at least 2 h before and during the trials (Martín and Salvador 1993). Lizards were assigned to one of three treatments. In the ’control’ treatment lizards were individually housed in outdoor terraria with a refuge and they were not disturbed during the course of the experiment. In the two experimental treatments, lizards were submitted to simulated repeated persistent attacks of 15 s of duration. We performed an attack every 10 min each day from 1100 to 1700 h GMT, when lizards were fully active, across 8 days. The experimenter simulated a predatory attack by rapidly approaching the terraria and tapping lizards close to the tail with a brush to stimulate them to perform a fleeing response. With this procedure we simulated an attack from an avian predator coming from above the lizard. The same person performed all predatory attacks in a similar way. In the ‘refuge’ treatment lizards had a refuge in their terraria in which they could hide during the attacks. In the ’fleeing’ treatment the experimenter removed the refuge just before the daily session of attacks. Thus, because lizards could not hide, they had to run all the time during the attacks. The refuge was replaced at 1700 h, after the session of attacks finished. Terraria were placed separately from each other, such that our approaches to a terrarium did not influence lizards in other terraria. We used repeated-measures analysis of variance (ANOVA) to analyse changes in body mass or intensity of blood parasites between the beginning and the end of the experiment (within subject factor) and between treatments (between subject factor). We included the interaction to analyse whether Capítulo 3 Efectos de la degradación del medio 151 changes in body mass or parasite load across time varied between treatments. We used analyses of covariance (ANCOVA) to analyse differences in CMI of lizards at the end of the experiment (dependent variable) between treatments, and taking into account final body mass, and final blood parasite load as covariants. Immediately after the experiment finished we measured sprint speed of lizards to examine whether changes in body condition, resulting from the different experimental treatments, affected escape performance of lizards. Lizards were individually tested in a linear terrarium (80 x 30 cm) with a carpeted floor, which provided excellent traction, and where all potential obstacles had been removed. The spatial scale of the experimental terrarium was realistic in that its length approximates to that of an escape attempt under natural conditions, in which L. monticola individuals ran rapidly to cover into the nearest refuge (Carrascal et al. 1992; López and Martín 2002). Individuals were allowed to bask for at least 2 h before trials to attain a body temperature within the activity temperature range of the species (Martín and Salvador 1993). Lizards were induced to flee at high speed by tapping them close to the tail with a brush (López and Martín 2002). The escape sequences were performed during 1 min to avoid differences in speed if lizards became fatigued. Lizards passed all the trials without apparent signs of stress. Experiments were recorded on videotape using a video- camera (Hi-8 format, 25 frames s−1) aligned perpendicularly over the centre of the arena. From each individual, we selected two escape sequences. We analysed the sequences frame-by-frame to determine sprint speed of lizards. Measurements were based on calibrated distances measured (to the nearest 1 mm) from the video monitor using the tip of the snout as a position reference (Martín and Avery 1998). For each sequence we measured the distance between the initial position (lizard paused) of the lizard’s snout mark and the final position in the first pause after fleeing (escape distance), and the time interval between the initial and final position (escape duration). From these data, we calculated the average ‘escape speed’ (distance moved divided by time taken; Martín and Avery 1998). We also recorded the ‘maximal speed’ within Capítulo 3 Efectos de la degradación del medio 152 each sequence (the greatest speed recorded in a frame, 40 ms, during the escape response). For each individual, an average value was determined from the two sequences that were analysed. We used ANCOVAs to analyse differences in escape or maximal speed (dependent variables) between treatments, with final body mass of lizards as a covariant. Results Microhabitat selection by lizards The PCA for microhabitats available and those used by lizards produced three components that together accounted for the 72 % of the variance (Table 1). Table 1 Principal components analysis for available and lizard microhabitat data in areas with natural vegetation and areas with habitat deteriorated by ski infrastructures. Emboldened values indicated correlations of variables with the principal components greater than 0.70 PC-1 PC-2 PC-3 Substrate: Rocks 0.47 0.79 -0.27 Bare soil 0.23 -0.88 -0.22 Grass 0.05 -0.05 0.83 Litter -0.82 0.02 0.36 Vegetation: Shrub cover -0.89 0.16 -0.15 Shrub minimal height -0.54 0.20 0.11 Tree cover -0.06 -0.05 0.79 Distance to refuge 0.37 -0.77 0.17 Distance to open -0.76 0.003 -0.11 Eigenvalue 2.94 2.10 1.41 % Var. 32.71 23.36 15.70 Capítulo 3 Efectos de la degradación del medio 153 The first PC (PC-1) was negatively correlated with substrates of leaf litter, with cover of shrubs, and with distance to a sunny open area. The second PC (PC-2) was positively correlated with cover of rocky outcrops, and negatively correlated with substrates of bare soil and distance to refuges. The third PC (PC-3) was positively correlated with substrates of grass and tree canopy. There were significant differences in relation to all PCs between levels of deterioration (GLM, Wilks χ2 = 0.87, F3,225 = 11.60, P < 0.001) and between types of microhabitat points (available vs. used by lizards) (Wilks χ2 = 0.54, F3,225 = 62.62, P < 0.001). The interaction between level of deterioration and type of point was significant (Wilks χ2 = 0.86, F3,225 = 12.00, P < 0.001; Fig. 1). The general model showed significant overall differences for all PCs (PC-1: R2 = 0.20, F3,227 = 18.44, P < 0.001; PC-2: R2 = 0.37, F3,227 = 44.99, P < 0.001; PC-3: R2 = 0.11, F3,227 = 10.48, P = 0.001). There were significant differences between deteriorated and natural areas in PC-1 and PC-2 scores (Tukey’s tests, P < 0.02 in both cases), but not in PC-3 (P = 0.17). Thus, in deteriorated areas, there was a lower cover of shrubs and of substrates with leaf litter or rocks, and higher cover of bare soil substrates. Also in deteriorated areas points were closer to open areas and farther from refuges than in natural areas. Regardless of these differences in availability, lizards selected similar microhabitats in both areas, i.e., there were not significant differences between areas in the PC scores of microhabitat used by lizards (P > 0.87 in all cases). Thus, in both areas lizards selected microhabitats with low cover of trees and shrubs, with substrates of rocks rather than bare soil, grass or leaf litter, and close to sunny areas and to refuges. Capítulo 3 Efectos de la degradación del medio 154 Fig. 1 Means (+ 1 SE) of PC scores for microhabitat available (black boxes) and microhabitat used by adult male Lacerta monticola lizards (open boxes) in natural areas and areas deteriorated by ski infrastructures. Capítulo 3 Efectos de la degradación del medio 155 Movement patterns Lizards decreased the time spent in movement when air temperature was higher (ANCOVA, model: R2 = 0.11, F2,43 = 2.77, P = 0.07; temperature effect: F1,43 = 5.33, P = 0.03), but there were no significant differences between levels of deterioration (mean + SE, natural: 31,2 + 4.1, deteriorated: 30 + 2.6 % time in movement; F1,43 = 0.04, P = 0.85). Neither, there were significant differences (ANCOVA, model: R2 = 0.10, F2,43 = 2.45, P = 0.10, air temperature effect: F1,43 = 3.07, P = 0.09) in the distance moved during the trial between areas (natural: 1.48 + 0.23, deteriorated: 1.93 + 0.25 m/min, F1,43 = 3.16, P = 0.08). Lizards tended to moved greater distances in deteriorated than in natural areas. However, lizards moved at greater speeds in deteriorated than in natural areas (ANCOVA, model: R2 = 0.18, F2,43 = 4.79, P = 0.01; risk effect: F1,43 = 9.40, P = 0.004; air temperature effect: F1,43 = 0.18, P = 0.67) (Fig. 2). Fig. 2 Mean (+ 1 SE) average burst speed (cm/s) of adult male Lacerta monticola lizards while moving undisturbed in natural areas and in areas deteriorated by ski infrastructures. Parasite load and body condition of lizards Larger lizards showed greater blood parasite loads (ANCOVA, model: R2 = 0.08, F4,81 = 1.77, P = 0.14; body size effect: F1,84 = 4.79, P = 0.03), but there were no significant differences between deteriorated and natural areas (mean + SE, natural: 0.85 + 0.13 parasites/ 2000 erythrocytes, deteriorated: 0.76 + 0.25; F1,81 = 1.16, P = 0.28) nor between seasons (spring: 0.85 + 0.19, summer: 0.75 + 0.14; F1,81 = 0.05, P = 0.83). The interaction between season and level of deterioration was not significant (F1,81 = 0.80, P = 0.37). Capítulo 3 Efectos de la degradación del medio 156 Body mass of lizards (ANCOVA, model: R2 = 0.78, F4,81 = 70.39, P < 0.0001) was positively correlated to SVL (F1,81 = 251.42, P < 0.001). There were no significant overall differences between natural and deteriorated areas (mean + SE, 7.9 + 0.1 g, deteriorated: 7.9 + 0.2 g; F1,81 = 0.96, P = 0.33) nor between seasons (spring: 7.2 + 0.1 g, summer: 6.8 + 0.2 g; F1,81 = 0.45, P = 0.50), but the interaction was significant (F1,81 = 5.42, P = 0.02; Fig. 3). Thus, in natural areas, there were no significant differences in relative body mass between seasons (Tukey’s test, P > 0.99), but in deteriorated areas lizards showed lower relative body mass in summer than in spring (P = 0.0002). During spring, there were no significant differences in relative body mass between lizards of both areas (P = 0.39), whereas in summer, lizards inhabiting deteriorated areas had lower relative body mass than lizards in natural areas (P < 0.001). The effect of blood parasite load was not significant and it was removed from the final model. Fig. 3 Mean (+ 1 SE) body mass (g) of adult male Lacerta monticola lizards during the mating period (spring) and after the mating period had finished (summer) in natural areas (black bars) and areas deteriorated by ski infrastructures (open bars). There were no significant differences in the CMI of lizards during summer (ANCOVA, model: R2 = 0.24, F4,29 = 2.23, P = 0.09) between areas (mean + SE, natural: 0.38 + 0.05 mm, deteriorated: 0.60 + 0.07 mm; F1,29 = 3.48, P = 0.07) nor in relation to the SVL (F1,29 = 1.70, P = 0.20), body mass (F1,29 = 2.04, P = 0.16) or blood parasite load (F1,29 = 0.006, P = 0.94). Capítulo 3 Efectos de la degradación del medio 157 Costs of fleeing to refuges on body condition There were not overall significant differences between the initial and the final body mass of lizards (repeated- measures ANOVA, F1,27 = 0.22, P = 0.64) nor between treatments (F2,27 = 0.32, P = 0.73). However, the interaction was significant (F2,27 = 3.47, P < 0.05; Fig. 4). Although post-hoc comparisons did not show significant differences (Tukey’s test, P > 0.25 in all cases), control lizards tended to increase their body mass, lizards that were attacked but could hide in a refuge tended to maintain their body mass, and lizards that were attacked but could not hide and, thus, were forced to flee, tended to decrease their body mass at the end of the experiment. All lizards tended to decrease their blood parasite load in the course of experiment. (mean + SE, 4.2 + 1.0 vs. 3.3 + 0.9 parasites/ 2000 erythrocytes; repeated-measures ANOVA, F1,27 = 3.94, P = 0.06), but there were no significant differences between treatments (control: 3.4 + 1.6, refuge: 6.0 + 1.4, fleeing: 1.8 + 1.5; F2,27 = 1.82, P = 0.18), and the interaction was not significant (F2,27 = 1.54, P = 0.23). Fig. 4 Changes in mean (+ 1 SE) body mass (g) of adult male Lacerta monticola lizards after suffering three treatments: a) undisturbed lizards (‘control’, open bars); b) lizards that could hide in refuges to avoid simulated attacks (‘refuge’, lined bars); c) lizards that have to run continuously to avoid simulated attacks as there were not available refuges (‘fleeing’, black bars). Attacks were performed each 10 min during 4 h each day across 8 days. The CMI of lizards (ANCOVA, R2 = 0.22, F4,25 = 1.81, P = 0.16) did not differ between treatments (mean + SE, control: 0.66 + 0.09 mm, refuge: 0.54 + 0.06, fleeing: 0.49 + 0.05; F2,25 = 1.18, P = 0.32), and was not related to the final body mass (F1,25 = 2.12, P = 0.16) or the final blood parasite load (F1,25 = 1.81, P = 0.19). Capítulo 3 Efectos de la degradación del medio 158 Fig. 5 Mean (+ 1 SE) average escape speed (cm/s) of adult male Lacerta monticola lizards forced to run in a racetrack, after having been exposed for 8 days in their terraria to three treatments: a) undisturbed lizards (‘control’); b) lizards that could hide in refuges to avoid simulated attacks (‘refuge’); c) lizards that have to run continuously to avoid simulated attacks as there were not available refuges (‘fleeing’). Average escape speed of lizards was not affected by their body mass (ANCOVA, model: R2 = 0.12, F3,26 = 1.15, P = 0.35; body mass effect: F1,26 = 2.84, P = 0.10), and there were not significant differences between treatments (F2,26 = 0.42, P = 0.66; Fig. 5). Neither, the maximal speed was affected by body mass (ANCOVA, model: R2 = 0.02, F3,26 = 0.16, P = 0.92; body mass effect: F1,26 = 0.38, P = 0.54), nor there were differences between treatments (mean + SE, control: 147 + 11 cm/s, refuge: 145 + 7, fleeing: 149 + 6; F2,26 = 0.08, P = 0.92). Thus, regardless of their body mass or treatment, lizards had similar escape and maximal speed when they were attacked and forced to run. Discussion To our knowledge, this is the first study showing a negative relationship between changes in movement patterns of male lizards, forced by the increased predation risk in human-induced deteriorated habitats, and their body condition. In relation to habitat selection, our results show that lizards did not use habitat at random, as was previously shown in this and other lacertid species (e.g. Martín and Salvador 1995; Martín and López 2002). Although there were differences in characteristics of available microhabitats between areas with different level of deterioration, lizards selected similar microhabitats in both areas (i.e. those with low cover of trees and shrubs, and with substrates of rocks close to open areas, rather than bare soil, grass or leaf litter). In this, and other alpine lizards, the availability of basking sites may be a potential limiting factor and rocks offer a high availability and diversity of such sites (Hertz and Huey Capítulo 3 Efectos de la degradación del medio 159 1981; Carrascal et al. 1992). Rocky areas also provide a high availability of refuges (Martín and Salvador 1995). Thus, in these areas lizards can face the conflicting demands of avoiding predation risk while attending other activities (Lima and Dill 1990; Carrascal et al. 1992). However, in ski slopes, the scarce cover of vegetation, and even of rock substrates, implies a low availability of refuges. Thus, when lizards moved through their territories, they may often need to move through risky areas far from refuges, where they would be more exposed to predators. However, lizards seemed to perceive this increase in risk, and responded by modifying their locomotor patterns when moving through more risky areas. Lizards typically moved in a discontinuous way, alternating short periods of locomotor activity with short pauses (Avery et al. 1987a, b; Braña 2003). Our results suggest that lizards modified their locomotor patterns in accordance to perceived risk; lizards moved at faster average speed in deteriorated areas, thus decreasing time exposed to potential predators. Furthermore, results also suggest that lizards tended to moved higher distances in deteriorated areas, probably because they may be forced to cross large unsafe areas to reach safer areas close to refuges. This might also aggravate costs of fleeing. Similarly, previous studies have shown the ability of prey to modify their locomotor patterns in order to decrease predation risk in several contexts (McAdam and Kramer 1998; Bakker and van Vuren 2004; Amo et al. 2005). For example, some rodents increased the frequency of pauses when leaving forest cover to move to open areas probably to increase vigilance levels while moving in these risky areas (McAdam and Kramer 1998). Similarly, common wall lizards modified their patterns of locomotion when resuming activity after being hidden in a refuge after a predatory attack; lizards decreased the frequency of short pauses when moving in the open, and hence, they decreased time exposed to visually guided predators (Amo et al. 2005). Lizards also increased time spent in escape sequences when they found chemical cues of a predatory snake on the ground (Downes and Bauwens 2002; Amo et al. 2005). Also larval Ambystoma salamanders exposed to a high predation risk, indicated by chemical signals of a Capítulo 3 Efectos de la degradación del medio 160 predator, increased movement in an effort to reach a refuge (Sih and Kats 1991). However, in reptiles, the cost of locomotion is a significant component of the daily energy expenditure (Christian et al. 1997) and to perform frequently fleeing sequences at high speed is costly (Kramer and Mclaughlin 2001; Gleeson and Hancock 2002). Our results showed that male lizards inhabiting areas with high predation risk, where they probably needed to move faster more often, suffered a loss of body condition in the course of season. Nevertheless, other factors rather, or in addition, than increasing speed may be affecting body condition in the field. For example, the lower cover in deteriorated areas may make lizards more conspicuous and, thus, more frequently attacked than in natural areas. Thus, lizards may not only suffer costs associated to move faster when undisturbed, but also may suffer the costs associated to increasing escape responses and refuge use (Martín and López 1999a; Pérez-Tris et al. 2004). Hormones such as corticosterone released during environmental stressful situations that prepare the body to perform an antipredatory response (Wingfield et al. 1998; Belliure and Clobert 2004; Belliure et al. 2004) may also influence body condition (Assenmacher 1973; Axelrod and Reisine 1984; Pérez-Tris et al. 2004). However, results of the laboratory experiment seem to confirm that costs associated to continuous fleeing episodes can cause per se a decrease in body condition of lizards. Lizards that were attacked and that could not hide and, thus, were forced to run tended decrease their body mass. In contrast, lizards that were attacked but could hide in a refuge tended to maintain their body mass. Although previous studies have shown that an increase in use of cold refuges may lower body condition (Martín and López 1999a), our experimental design minimised the costs associated to temperature impairment inside refuges, and, therefore, lizards could have maintained their body mass even if they increased refuge use. The escape sprint speed that lizards were able to attain after having suffering the experimental treatments did not differ between conditions. Therefore, our study provides new evidence that behavioural strategies to cope with increased predation risk affected body Capítulo 3 Efectos de la degradación del medio 161 condition of lizards, and that sprint speed does not depend on body mass, according to previous results with the lizard P. algirus (Pérez-Tris et al. 2004). The loss of body mass of lizards inhabiting ski slopes during the breeding period may have deep consequences because lizards in poor body condition could not allocate so many energy and resources in immune defence against parasites (Cooper et al. 1985; Smallridge and Bull 2000). Defence against blood parasites seems to be very important because haemogregarines have many deleterious effects (Wintrobe 1991; Oppliger et al. 1996; Veiga et al. 1998), which may affect different aspects of lizard’s physiology and behaviour (Caudell et al. 2002), and which should also affect body condition, as has been previously found in this lizard species (Amo et al. 2004). However, our results did not show any effect of habitat deterioration neither on CMI response of lizards, nor in their intensity of haemogregarines’ infection. However, as CMI response, assessed by PHA test is not well known in wild animals (Adamo 2004), and especially in reptiles, and our sample was rather small, we should be cautious in the interpretation of such results. Therefore, further research is needed to analyse the effect of habitat deterioration on CMI response and intensity of infection by parasites. Our results have applications for the design of conservation plans for this endangered species because the deleterious effect of habitat deterioration in ski slopes on body condition of lizards might affect their fitness and cause a reduction of the population. Reduction of population size can negatively affect demographic and genetic structure which increases the probability of local extinction (Gilpin and Soulé 1986; Hecnar and M'Closkey 1998). Therefore, environmental impact studies should be performed before opening new ski slopes (Stumpel et al. 1992; Martín and Salvador 1995, 1997), but taking into account not only the presence of lizards but also their body condition to ensure the correct treatment of this lizard species. Our results also highlight the importance of a high availability of refuges for the maintenance of lizards’ populations, as has previously found in other lizard species such as skinks (Hecnar and M'Closkey 1998) or geckos (Schlesinger and Shine 1994). Therefore, an effective way to decrease the Capítulo 3 Efectos de la degradación del medio 162 deleterious effects of habitat loss, with the subsequent decrease in availability of refuges, may be the artificial restoration of some refuges to create safe corridors that communicate good areas for lizards. In this way, lizards would not need to move at high speed because they will be close to refuges, and would not incur loss of body condition associated to this high speed. Therefore, this measure may minimize the deleterious effect of ski resorts on this lizard species. Refuges may not necessarily be large rocks; small irregular rocks may be enough to refuge lizards. The use of artificial refuges have reported good results in the restoration and maintenance of other endangered reptile species such as five-lined skinks, Eumeces fasciatus (Hecnar and M'Closkey 1998), blue tongue lizards, Tiliqua adelaidensis (Souter et al. 2004), and velvet geckos, Oedura lesueurii, and their predators, the broad-headed snakes, Hoplocephalus bungaroides (Webb and Shine 2000). In summary, to our knowledge, our study is one of the firsts in relating the effects of habitat deterioration on movement patterns, body condition and parasite load of male lizards. Habitat deterioration not only implied a loss of habitat for lizards, but also led to an increase in perceived predation risk. Males seemed to adjust their movement patters accordingly to differences in risk levels by increasing speed during their normal displacement across more risky deteriorated areas, but as a consequence, they suffered a loss of body condition. The laboratory experiment supported our findings in the field. Therefore, our study provides new evidence that behavioural strategies to cope with increased predation risk due to human- induced habitat deterioration may affect body condition of lizards. Acknowledgements We thank "El Ventorrillo" MNCN Field Station for use of their facilities. Financial support was provided to L. Amo by an “El Ventorrillo” C.S.I.C. grant, to P. López by a the MCYT project BOS 2002-00598, and to J. Martín by the MCYT project BOS 2002- 00547. References Adamo SA (2004) How should behavioural ecologists interpret measurements of immunity? 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J Therm Biol 29: 45–53 Capítulo 3 Efectos de la degradación del medio 167 Habitat deterioration affects body condition and parasite load of female lizards Psammodromus algirus through forced changes in antipredatory behavior RESUMEN Los cambios antropogénicos en el medio pueden afectar al comportamiento antidepredatorio de muchas especies de lagartijas. La deforestación de bosques puede hacer que las lagartijas sean más conspícuas así como limitar el número de refugios adecuados para evitar a los depredadores, lo que requeriría que las lagartijas incrementaran la magnitud de sus respuestas de escape. Sin embargo, la destinación de tiempo y energía a estrategias antidepredatorias puede conllevar una pérdida de condición corporal, y una disminución en la capacidad de las lagartijas de invertir en la lucha frente a infecciones, lo que puede tener consecuencias importantes para la eficacia biológica a corto y largo plazo. En este estudio analizamos si las lagartijas colilargas, Psammodromus algirus, presentan distinto uso del medio, estrategias antidepredatorias y condición corporal en robledales con distinto nivel de degradación de la vegetación. Los resultados sugieren que las lagartijas seleccionan microhábitats similares en áreas naturales o con la vegetación degradada. Sin embargo, la degradación del medio pareció incrementar el riesgo de depredación para las hembras, que fueron más fácilmente detectables en áreas deterioradas. Las hembras parecieron ajustar su comportamiento antidepredatorio de acuerdo a este incremento en el riesgo, y presentaron mayores distancias de aproximación. Esto podría explicar que las hembras en áreas degradadas presentaran menor condición corporal y mayores niveles de parasitación, lo que puede tener consecuencias negativas para los individuos y, por tanto, afectar al mantenimiento de estas poblaciones. Capítulo 3 Efectos de la degradación del medio 169 Habitat deterioration affects body condition and parasite load of female lizards Psammodromus algirus through forced changes in antipredatory behavior Abstract Human-induced deterioration of vegetation may influence antipredatory behaviour of many prey species. Deforestation may make prey more conspicuous to potential predators and limit the number of refuges suitable to avoid predators, which will require prey to increase the magnitude of escape responses. However, an excessive allocation of time and energy to antipredatory strategies might lead to a loss of body mass, and a decrease in the ability to invest in defence against parasites, which may have important consequences for short and long term fitness. We analyzed whether Psammodromus algirus lizards inhabiting patches with different level of deterioration of the vegetation within the same oak forest habitat differed in microhabitat use, antipredatory strategies, and health state. Results suggested that lizards selected similar microhabitats regardless of the level of deterioration of the vegetation. However, habitat deterioration seemed to increase predation risk, at least for females, which were easily detected in deteriorated areas. However, females seemed to adjust their antipredatory behaviour accordingly by increasing approach distances allowed to predators. This might explain that females in deteriorated habitats suffered a loss of body condition and had greater blood parasite loads, which might have important deleterious consequences, and, therefore, affect the maintenance of lizards’ populations. Introduction Predation risk is a major force in the evolution of several morphological and behavioural characteristics of animals (Lima and Dill 1990). As failing to avoid predation leads to a drastic decrease in fitness, i.e. the death, animals have been submitted along the evolutionary time to a strong selection in favour to behavioural strategies to currently assess the risk of predation and to cope with it Capítulo 3 Efectos de la degradación del medio 170 (Lima and Dill 1990; Lima 1998). One of the first antipredatory strategies that animals use to minimize predation risk is the selection of safe habitats where to cope with their requirements such as thermoregulation (Martín and López 2002; Scheers and Van Damme 2002; Sabo 2003) or foraging (Díaz and Carrascal 1991; Yasué et al. 2003) while minimizing risk (Pitt 1999). In this way prey select microhabitats where they can decrease their conspicuousness (Merilaita and Tullberg 2005) and find refuge in case of being attacked (Milne and Bull 2000; Webb and Shine 2000; Souter et al. 2003). Thus, microhabitat characteristics may influence risk perceived by prey, and their antipredatory behaviour once a predator has launched an attack. Theoretical models and empirical evidence suggest that prey should wait to perform an escape response until costs of not responding are higher than costs of such response (Ydenberg and Dill 1986; Dill and Houtman 1989; Bonenfant and Kramer 1996). Therefore, when prey perceive an increase in predation risk, mediated by habitat characteristics (e.g. a low availability of refuges), they may begin to escape sooner than when prey perceive that risk of capture is lower. Factors such as the extent of reliance on refuges for avoiding predators, and other components of risk such as the probability of detection by predators may strongly affect escape decisions (Lima and Dill 1990). Previous studies have shown different escape responses in relation to habitat characteristics. For example, lizards living in open habitats with sparse cover rely more on speed and running long distances than on using refuges (Bulova 1994; Martín and López 2003). Also, lizards may respond to seasonal changes in vegetation structure, which affect predation risk, by modifying their escape responses (Martín and López 1995). Therefore, as microhabitat characteristics can affect predation risk levels and, thus, influence the type of escape strategy, human- induced changes in habitat characteristics may affect perception of risk by prey and force changes in escape behaviour. For example, deterioration of natural vegetation could decrease the availability of vegetation cover that some prey use as refuge, which would increase risk. Thus, prey might need to respond by increasing the magnitude of Capítulo 3 Efectos de la degradación del medio 171 their escape response (e.g. greater approach distances) in deteriorated areas. However, antipredatory behaviours such as escape sequences or refuge use are costly not only in terms of losing time to perform other activities such as foraging (Koivula et al. 1995; Dill and Fraser 1997; Martín et al. 2003; Cooper and Pérez-Mellado 2004) or reproduction (Sih et al. 1990; Crowley et al. 1991; Martín and López 2003), but also in terms of body condition (Martín and López 1999a; Pérez-Tris et al. 2004). For example, Podarcis muralis lizards experimentally submitted to a high predation pressure increased refuge use, which led to a loss of body mass (Martín and López 1999a). Similarly, Psammodromus algirus lizards submitted to a higher risk of predation caused alarmed lizards a loss of mass not only because they interrupted feeding, but also because of stress in relation to predation risk per se (Pérez-Tris et al. 2004). Therefore, an excessive allocation of time and energy to antipredatory strategies can affect body condition, which has important consequences for short and long term fitness. Moreover, the loss of body condition could also lead to a decrease in the ability to invest in defence against parasites because the nutritional status can influence the capacity of a lizard to mount an immune response to infection (Cooper et al. 1985; Smallridge and Bull 2000). This may influence host-parasites relationships, and may increase the negative effects of parasites on their host, which, in turn, could affect the maintenance of lizard’s populations. However, the effect of habitat deterioration on predation risk, escape behaviour, body condition and parasite load of lizards has scarcely been analysed in natural conditions (but see Oppliger et al. 1998 for experiments in seminatural enclosures). The lizard P. algirus, a medium- sized lacertid lizard inhabiting Mediterranean forests of the Iberian Peninsula, usually escapes by fleeing into patches of tree leaf litter under cover of shrubs, a non-entirely safe refuge (Martín and López 1995, 2000). Nowadays this species suffers a loss of habitat due to human activities (Carretero et al. 2002). In the Iberian Peninsula, montane forests of deciduous Pyrenean oaks, Quercus pyrenaica, where this species inhabits, have been traditionally used for timber production Capítulo 3 Efectos de la degradación del medio 172 and for extensive livestock grazing, being progressively deforested in some areas. Deforestation may increase predation risk because it may make lizards more conspicuous and limit the number of refuges suitable to avoid predators (Martín and López 1998), which will require lizards to increase the magnitude of escape responses. Thus, this species offers a good model to analyse the possible effects of changes in vegetation structure, due to habitat deterioration, on antipredatory behaviour of lizards, and their consequences for body condition and health state. In this study, we analyzed whether P. algirus lizards inhabiting areas with different level of deterioration of the vegetation within the same oak forest habitat differed in microhabitat use, antipredatory strategies, and health state. Concretely, we first examined 1) the characteristics of microhabitats available and selected by lizards, and 2) the conspicuousness of lizards in the microhabitats where they were initially located, which may reflect the probability of being detected by predators. Then, we simulated predator attacks and analyzed: 3) the escape strategies, and approach and flight distances. Finally, we captured lizards in different areas to measure their 4) body condition and 5) parasite load (ectoparasites and blood parasites). We hypothesized that since microhabitats and refuges optimal for lizards may be limited and dispersed in deteriorated areas, lizards may be more conspicuous and vulnerable to predators, and, thus, need to run early, for longer and more often to avoid predators. However, since antipredatory behaviour may be costly, lizards in these areas will suffer stress and energetic costs, which should negatively affect their body condition. Furthermore, lizards with decreased body condition might not be able to allocate enough resources to parasite defence. Therefore, the deleterious effects of parasitemia should be more evident in deteriorated areas. Alternatively, lizards might adjust their antipredatory behaviour within each habitat according to local microhabitat characteristics, minimizing the negative effects of antipredatory behaviour, thus, maintaining a good body condition and a low parasite load regardless of levels of habitat deterioration. Capítulo 3 Efectos de la degradación del medio 173 Methods Study area and study species The study was performed during three consecutive years (2002-2004) in the Guadarrama Mountains (Madrid Province, Central Spain) within two large oak forests that included several areas with different level of deterioration of the vegetation. One of the forest areas was located near Miraflores de la Sierra village (‘Miraflores’ hereafter), and the other one near Cercedilla village (‘Golondrina’). In both forests, vegetation in natural areas included primarily trees and small saplings of the deciduous Pyrenean oak, Q. pyrenaica, as well as less abundant and dispersed subarboreal perennial shrubs with dominant Cistus laurifolius, and less frequent and disperse Rosa pouzini, Rubus ulmifolius Genista florida, Crataegus monogyna, and Cytisus scoparius. Oak-leaf litter is very abundant on the ground year-round. The lack of oak trees was the most important and obvious characteristic of deforested areas, which showed a mix of areas with abundant presence of C. laurifolius shrubs and extensive open grassy areas. Thus, we easily classified areas as ‘natural’ or ‘deteriorated’ according to this criterion. Microhabitat use by lizards To evaluate microhabitat characteristics of areas with different level of deterioration, and microhabitat use by lizards in these areas, we marked 52 line transects of 200 m length, evenly distributed throughout the study areas, at least 1-2 km apart, and chosen to cover homogeneous patches of forest with different level of deforestation (natural vs. deteriorated). During April and May 2002, we walked each transect once in days with favourable climate conditions (warm sunny days) and between 09:00 and 13:00 GMT, when lizards were more active. When we detected an adult lizard, we marked the point where the lizard was first sighted. For each point we took four 1 m transects, one at each of the four cardinal orientations radiating from the point. We used a scored stick standing vertically at nine sample points (two points at 50 and 100 cm in each of the four transects, and the central point), and recorded the type of substrate found at each point (grass, leaf litter, bare soil, or rocks). We noted the presence of Capítulo 3 Efectos de la degradación del medio 174 canopy tree cover above each sample point, and the cover and the height from the ground to the first contact of leaves with the stick of subarboreal vegetation at each point. This later variable provided an indication of the suitability of vegetation as a refuge for these lizards (Martín and López 1998). Thus, a low vegetation height indicated that vegetation was close to the ground, providing a narrow refuge where to hide. Previous studies have shown the importance of plant cover at the ground level for lizards (Carrascal et al. 1989; Martín and López 1998). Also, for this lizard species, the subarboreal vegetation total height was not considered important because lizards move on the ground and below vegetation. We also noted the distance to the nearest available refuge, and to the nearest sunny spot where lizards could bask. We calculated percent cover values for each habitat variable in the area surrounding each lizard (i.e. % contacts with each substratum and vegetation type), the average distances to refuges and sunny spots, and average height of potential refuges (for a similar sampling methodology see Martín and López 1998, 2002). Given the large size of the area surveyed and the high lizard density, and because we avoided sampling the same area twice, the probability of repeated sampling of the same individual was very low. We therefore treated all measurements as independent. To estimate the availability of microhabitats along each transect, we recorded the same variables than above at three points per transect (at 70, 140 and 200 m along the progression line). We used principal component analysis (PCA) to reduce all the habitat variables to a smaller number of independent components. We performed a PCA on the points describing available microhabitats and the lizard-observed microhabitat points. Original data (number of contacts) were normalised by means of square root transformation. The initial factorial solutions were rotated by the Varimax procedure (Nie et al. 1975). We used General Linear Models (GLM) to analyse differences in the PC scores reflecting characteristics of microhabitat selected in relation to the level of deterioration of the vegetation (natural vs. deteriorated), and to determine whether male and female lizards used available microhabitats in a non-random fashion (available vs. males Capítulo 3 Efectos de la degradación del medio 175 vs. females) (Martín and López 1998, 2002). We included in the model the interaction between level of deterioration and type of microhabitat to examine whether microhabitat use by lizards changed with deterioration. Escape behaviour During April 2003 and 2004 we simulated predatory attacks to adult lizards, recording their escape behaviour, and the microhabitat used before the attack in areas with different level of deforestation within the “Golondrina” forest area (see above). We walked haphazardly in days with favourable climate conditions (warm sunny days) and between 09:00 and 13:00 GMT until a lizard was detected. We noted the distance at which the lizard was detected by the observer (‘detection distance’), and how the lizard was first detected (i.e. whether we saw it or we heard it). Noisy escape responses are especially notorious in this lizard species (Martín and López 2001) and some lizards were not detected but after they were heard when started to move. Then, we attempted to approach the lizard directly. The same person performed all approaches, walking at the same moderate speed (about 40 m/min) and wearing the same clothing, to avoid confounding effects that may have affected lizards’ risk perception (e.g., Burger and Gochfeld 1993; Cooper 1997). We noted the ‘approach distance’ as the distance between the lizard and the observer when the lizard started escape (a straight line measured to the nearest 0.1 m), and the ‘flight distance’ as the distance that the lizard ran during an episode of escape. We noted the ‘escape strategy’ used, distinguishing between a) lizards that fled to hide in the nearest available refuge, b) lizards that fled to hide in another refuge, and c) lizards that fled but stopped outside of refuges. We noted the distance from the initial location of the lizard to the nearest available refuge, and the distance to the refuge actually used. Once the escape episode had finished we used the same methodology than above to record the microhabitat characteristics at the point where the lizard was first sighted. We used backward stepwise General Regression Models (GRM) to analyze the differences in detection, approach or flight distances (dependent variables), in relation to the level of deterioration of the vegetation (natural vs. deteriorated), Capítulo 3 Efectos de la degradación del medio 176 the PC scores describing microhabitat characteristics at the initial location of lizards, the distance to the nearest refuge, and the sex of lizards, including in the initial model the interaction between level of deterioration and sex. We used Generalized Non Linear Models (GLZ) to examine the effects of the level of deterioration, the PC scores defining microhabitat characteristics, and sex on the type of detection (dependent variable), with this variable following a binomial distribution (lizards detected by seeing or by hearing). We included the interaction between sex and level of deterioration in the model. We also used GLZ to assess the effect of level of deterioration, microhabitat characteristics, distance to the nearest refuge, and sex on the type of escape strategy used by lizards (dependent variable), with this variable considered as a multinomial ordinal variable (classifying lizards that hid in the nearest refuge, lizards that fled to another refuge, and lizards that fled but did not hide). Measurement of body condition and parasite load of lizards During April 2003 and 2004 we captured 48 adult lizards by noosing in areas with different level of deterioration of the vegetation within the “Golondrina” forest. Immediately after capture we weighed and measured snout- vent-length (SVL) of lizards, and noted the number of ticks (Ixodes ricinus) observed fixed on their body, usually on skin pockets (Salvador et al. 1996, 1999). To assess blood parasite load, we made a smear on a microscope slide from blood taken from the postorbital sinus by using one 9 µl heparinized hematocrit tube. Blood smears were air- dried, fixed in absolute methanol for 10 min and then stained in Giemsa diluted 1:9 with phosphate buffer (pH 7.2) for 40 min before their examination for parasites. On mounted slides, half a smear, chosen at random, was scanned entirely at 200 x along the longitudinal of slide, looking for extraerythrocytic protozoa (Merino and Potti 1995; Amo et al. 2004). Number of intraerythrocytic parasites (i.e. Haemogregarines, the only parasite found) was estimated at 400 x by counting the number of parasites per Capítulo 3 Efectos de la degradación del medio 177 2000 erythrocytes. Lizards were released at the exact place of capture. The experiments were performed under license from the Consejería del Medio Ambiente de la Comunidad de Madrid (Spain). We used backward stepwise GLM to analyse differences in the intensity of tick’s infection in relation to sex and body size (SVL) of lizards, and to the level of deterioration of the vegetation. The interactions between sex and level of deterioration, and between these two variables and SVL were included in the initial model. We also used backward stepwise GLM to analyse differences in the intensity of haemogregarines’ infection in relation to the sex, SVL, tick load, and level of deterioration, including in the initial model the interactions between sex and level of deterioration, and between each of these variables with SVL and tick load. Backward stepwise GLM were also used to analyse the effect of sex, size, intensity of infection by ticks and by haemogregarines, and level of deterioration on body mass, including in the initial model the interactions between the categorical and the continuous variables. Results Microhabitat selection by lizards The PCA for microhabitats available and those used by lizards produced three components that together accounted for the 59.9 % of the variance (Table 1). The first PC (PC-1) was negatively correlated with the cover of shrubs and with the distance to a sunny area where lizards could bask, and it was positively correlated with distance to the nearest refuge and with the cover of grass substrates. The second PC (PC-2) was positively correlated with substrates of leaf litter and with the cover of trees. The third PC (PC-3) was positively correlated with the cover of rocky outcrops. Capítulo 3 Efectos de la degradación del medio 178 Table 1 Principal components analysis for available and lizard microhabitat data in 52 transects made in natural and deteriorated areas within oak forests. Emboldened values indicated correlations of variables with the principal components greater than 0.60 The model obtained by GLM analysis about vegetation characteristics defined by PC scores showed significant differences respect to PC-1 (R2 = 0.43, F5,285 = 42.46, P < 0.0001) and PC-2 (R2 = 0.28, F5,285 = 21.96, P < 0.0001), but not in relation to PC-3 (R2 = 0.04, F5,285 = 2.09, P = 0.07). Concretely, vegetation characteristics defined by the PC scores differed significantly between levels of deterioration of the vegetation (GLM, Wilks χ2 = 0.86, F3,283 = 15.31, P < 0.0001; Fig. 1) and between available microhabitats and those selected by PC-1 PC-2 PC-3 Substrate: Rocks 0.01 -0.05 0.95 Bare soil 0.02 -0.45 0.07 Grass 0.71 -0.05 -0.37 Litter -0.26 0.73 -0.28 Vegetation: Shrub cover -0.90 0.03 -0.13 Shrub minimal height 0.34 0.44 0.04 Tree cover 0.32 0.75 0.11 Distance to refuge 0.75 0.20 -0.09 Distance to sunny areas -0.67 0.19 -0.17 Eigenvalue 2.62 1.58 1.19 % Var. 29.10 17.54 13.26 Capítulo 3 Efectos de la degradación del medio 179 lizards (Wilks χ2 = 0.50, F6,566 = 38.91, P < 0.0001). The interaction between these factors was not significant (Wilks χ2 = 0.97, F6,566 = 1.68, P = 0.12), thus, lizards always selected similar microhabitats regardless of the level of deterioration of an area. Fig. 1 Means (+ 1 SE) of PC scores for microhabitats available (black boxes) and used by male (open boxes) and female (hatched boxes) adult Psammodromus algirus lizards in natural and deteriorated areas within oak forests. Capítulo 3 Efectos de la degradación del medio 180 Microhabitats available in deteriorated areas had less cover of trees and leaf litter than those available in natural areas (PC-2; Tukey’s test, P < 0.0001), but did not differ respect to other characteristics (P > 0.09 in all cases). There were no significant differences in the microhabitats selected by males and females (Tukey’s test, P > 0.59 in all cases). There were significant differences in PC-1 and PC-2 between available microhabitats and those selected by male and female lizards (P < 0.001 in all cases). However, differences in PC-3 between available microhabitats and those selected by lizards were significant only in females (P = 0.04) but not in males (P = 0.11). Thus, male and female lizards preferred substrates with low cover of grass, leaf litter and tree canopy, high cover of shrubs, far from open areas and close to refuges (PC-1 and PC-2), but females also preferred substrates of rocks (PC-3). Escape behaviour There were significant overall differences (GRM, R2 = 0.15, F1,81 = 7.27, P = 0.001) in the distance at which lizards were detected by the observer between levels of deterioration of the vegetation (F1,81 = 8.93, P = 0.004), but the interaction between level of deterioration and sex was significant (F1,81 = 4.26, P = 0.04, Fig. 2). Thus, females were detected at longer distances in deteriorated areas (Tukey’s test, P = 0.005), but males were detected at similar distances in both areas (P = 0.74). Microhabitat characteristics at the initial location did not significantly influence detection distances and were removed from the final model. The type of detection differed in relation to microhabitat characteristics. Thus, when the percentage of rocky outcrop increased, lizards were more often detected by seeing than by hearing (PC-3; GLZ, Wald’s χ2 = 9.14, df = 1, P = 0.002), whereas there were no significant differences when other microhabitats changed (PC-1 and PC-2, P > 0.23 in both cases). There were no significant differences between levels of deterioration (P > 0.23), nor between sexes (P > 0.99), and the interaction between sex and level of deterioration was not significant (P > 0.99). Approach distances (GRM, R2 = 0.29, F4,78 = 7.89, P < 0.0001) differed between levels of deterioration (F1,78 = 12.47, P = 0.0007), but the interaction Capítulo 3 Efectos de la degradación del medio 181 between sex and level of deterioration was significant (F1,78 = 4.64, P = 0.03, Fig. 2). Thus, approach distances of females were longer in deteriorated areas (Tukey’s test, P = 0.005), whereas there were no significant differences in males (P = 0.91). Microhabitat characteristics also affected approach distances of lizards, which were longer when the cover of trees was greater and when substrates had more leaf litter (PC-2; F1,78 =9.35, P = 0.003), and when there was a low cover of rocky outcrops (PC- 3; F1,78 = 7.95, P = 0.006). Other variables did not significantly affect approach distances and were removed from the final model. The escape strategy of lizards was related to the distance to the nearest refuge (Wald’s χ2 = 5.60, df = 1, P = 0.02). When lizards were far from refuges, they escaped by fleeing without using refuges, whereas when the nearest refuge was closer lizards escape by hiding in this or another refuge. There were no significant differences in relation to the level of deterioration, sex, or microhabitat characteristics defined by PC scores (P > 0.11 in all cases), and the interaction between sex and level of deterioration was not significant (P = 0.29). Fig. 2 Mean (+ 1 SE) a) detection and b) approach distances (cm) of male (black boxes) and female (open boxes) adult Psammodromus algirus lizards in natural and deteriorated areas within oak forests. Flight distances were positively correlated to the distance to the nearest refuge (GRM, R2 = 0.30, F1,82 = 35.15, P < 0.0001), but they were not dependent on microhabitat characteristics, level of deterioration, or sex. Capítulo 3 Efectos de la degradación del medio 182 Parasite load and body condition of lizards Males showed significantly higher intensities of infection by ticks (GLM, R2 = 0.46, model: F2,44 = 19.05, P < 0.0001; sex: F1,44 = 19.30, P < 0.0001), and lizards of similar body size showed significantly higher tick loads in natural than in deteriorated habitats (size x deterioration; F1,44 = 10.55, P = 0.002). Other variables and interactions were not significant and were removed from the final model. In contrast, the intensity of infection by haemogregarines in blood was significantly lower in natural than in deteriorated areas (GLM, R2 = 0.27, F1,45 = 16.28, P = 0.0002, Fig. 3a). Other variables and interactions were not significant and were removed from the model. Body mass of lizards (GLM, R2 = 0.93, model: F8,38 = 61.11, P < 0.0001) covaried with SVL (F1,38 = 287.53, P < 0.0001). Males were heavier than females (F1,38 = 4.66, P = 0.04), and, moreover, males were heavier than females of equivalent SVL (sex x SVL: F1,38 = 7.60, P = 0.009). Females of equivalent SVL, but not males, were heavier in natural habitats (sex x deterioration: F1,38 = 6.32, P = 0.02, Fig. 3b). Fig. 3 Means (+ 1 SE) a) intensity of Haemogregarines’ infection and b) body mass of male (black boxes) and female (open boxes) adult Psammodromus algirus lizards in natural and deteriorated areas within oak forests. Also, in deteriorated areas there was a negative relationship between body mass and the intensity of infection by haemogregarines, whereas in natural areas there was not such a relationship Capítulo 3 Efectos de la degradación del medio 183 (blood parasites x deterioration: F1,38 = 8.27, P = 0.007). However, this effect of blood parasites on body mass was noted mainly in females but not in males (sex x blood parasites: F1,38 = 8.91, P = 0.005). The rest of variables and interaction were not significant and were removed from the final model. Discussion To our knowledge, this is the first study showing a relationship between levels of human-induced habitat deterioration and antipredatory behaviour, parasite load and body condition of lizards under natural conditions. In relation to habitat selection, our results show that lizards do not use habitat at random, as has previously shown in this and other lacertid species (e. g. Castilla and Bauwens 1992; Martín and López 2002). Although there were differences in microhabitat characteristics between natural and deteriorated areas, lizards selected similar places in both areas. However, regardless that there were not important sexual differences in microhabitat use between areas with different level of deterioration, there were sexual differences in exposure of lizards to predation. The distance at which females were detected was higher in deteriorated areas, as has been previously observed in the common chamaleon (Cuadrado et al. 2001). This may increase predation risk for females. However, females in deteriorated habitats seemed to compensate their higher detectability with escape responses of higher magnitude. Females had greater approach distances when attacked by a simulated predator in deteriorated areas. Thus, females seemed able to assess their higher conspicuousness in deteriorated areas and began to escape earlier, accordingly to theoretical models of escape behaviour (Ydenberg and Dill 1986) and previous results with this (Martín and López 1995, 1999b, 2000) and other lizards species (Cooper 1998 2003). This is also supported because males, with a similar conspicuousness in natural and deteriorated areas showed similar approach distances in both areas. The lack of differences in the distance of detection of males between natural and deteriorated habitats might be explained because males have higher movement rates during the mating period, searching for females or patrolling their territory to Capítulo 3 Efectos de la degradación del medio 184 defend them from intruder males (Salvador et al. 1995, 1996), and also because males’ bright nuptial coloration (see Díaz 1993; Salvador et al. 1995; Martín and López 1999b) could make them similarly conspicuous in both areas. Distance to available refuges may influence approach distances (Dill 1990; Bonenfant and Kramer 1996), because prey that are farther from a refuge may perceive an increase in risk. However, our results did not show any relationship between approach distance and distance to the nearest refuge, in contrast to previous results with this species (Martín and López 2000). Thus, it seems that increased probability of detection by predators, mediated by the deterioration of the vegetation, affected more strongly escape decisions than distances to refuges. Nevertheless, distance to the nearest refuge still affected the type of escape strategy and flight distances. When lizards were far from refuges, they escaped by fleeing without using refuges and had longer flight distances, whereas when refuges were closer lizards escaped by hiding in the nearest or other refuge. The greater approach distances of lizards when the cover of trees and leaf litter increased could be attributed to a higher detectability of lizards in such areas. Results of a previous study suggest that P. algirus lizards often showed noisy escape responses, by which they might be signalling their alertness and ability to escape to avoid being chased (Martín and López 2001). Thus, leaf littler substrates could favour this strategy, and therefore, farther lizards on leaf litter could be detected easily than closer lizards on grass or rocks substrates, which would explain the positive relationship between leaf litter cover and approach distances. To perform an escape response is costly in terms of energy expenditure (McNamara and Houston 1990) and loss of time to perform other activities (Carrascal et al. 2001; Martín and López 1999b). Furthermore, the energetic requirements of antipredatory behaviours such as fleeing sequences or refuge use can also lead to a loss of body mass (Martín and López 1999a; Pérez-Tris et al. 2004). Our results suggest that females submitted to high predation pressure (i.e., those inhabiting deteriorated habitats) suffered a loss of body mass, as previously observed in Capítulo 3 Efectos de la degradación del medio 185 this species under laboratory conditions (Pérez-Tris et al. 2004). This loss of body condition may cause a decrease in the allocation of resources to the immune function and therefore, a decrease in parasite defence (Cooper et al. 1985; Smallridge and Bull 2000). This is very important because lizards are submitted to infection by ticks that not only are known to have adverse effects on populations of lizards (Salvador et al. 1996; Main and Bull 2000; but see also Bull and Burzacott 1993) but also are the vector of blood parasites such as haemogregarines (Veiga et al. 1998). Our results showed that males supported higher intensity of infection by ticks than females, probably because their higher movement rate makes them more susceptible to encounter ticks, as well as due to the immunosuppresor effects of testosterone (Salvador et al. 1996; Klukowski and Nelson 2001; Olsson et al. 2000). An interesting result is that tick load was higher in natural than in deteriorated areas. Hence, habitat degradation may influence the prevalence and intensity of parasitic arthropod through changes in microclimatic conditions, as has being previously observed in birds (Merino and Potti 1996). However, despite this higher intensity, we did not find a negative effect of tick load on body condition of lizards, as previously found in other lizard species (Olsson et al. 2000). In contrast, intensity of infection by haemogregarines was higher in deteriorated than in natural areas. Hence, in habitats of good quality, lizards supported higher tick load but lower levels of blood parasite load. An explanation could be that lizards in natural habitats had a better condition and, therefore, they could mount an immune response to decrease blood parasite load, avoiding its deleterious effects. On contrast, in deteriorated areas, lizards had a poorer body condition and, thus, they suffered higher blood parasite intensities even if ticks were less abundant here. This seems to be confirmed because our results also show that the intensity of haemogregarines negatively influenced the body condition of lizards in deteriorated habitats whereas in natural areas there was not such a relationship. Furthermore, this effect was higher in females than in males. Capítulo 3 Efectos de la degradación del medio 186 Haemogregarines destroy erythrocytes, which results in depressed hematocrit levels (Wintrobe 1991) and reduced haemoglobin concentrations, which reduced capacity for oxygen transportation (Oppliger et al. 1996; Veiga et al. 1998). This may affect different aspects of lizard’s physiology and behaviour, such as foraging efficiency or sprint speed (Caudell et al. 2002), which also affect body condition, as previously observed in other lizard species (Amo et al. 2004). Our results suggest that the deleterious effects of parasitemia are more evident when other additional factor, such as habitat deterioration, influences the female’s condition. This agrees with previous results made in seminatural experimental enclosures where Lacerta vivipara lizards showed higher levels of parasitemia in poor quality habitats (Oppliger et al. 1998). The poor body condition of female lizards in deteriorated habitats may have deep effects on their fitness. Previous results showed that females in poor body condition produced offspring of small size (Shine and Downes 1999, but see also Gregory and Skebo 1998). Furthermore, females with blood parasites had reduced fat stores and produced smaller clutches (Schall 1983). Body size of neonate lizards can affect their probability of survival (e.g. Ferguson and Fox 1984; Sinervo et al. 1992). Therefore, the possible deleterious effect of habitat deterioration on body condition of females may endanger P. algirus populations inhabiting these areas. Moreover, the increase in lizards’ vulnerability to predators due to change in vegetation characteristics and its consequences may explain the disappearance of P. algirus populations in fragmented habitats, which maintain higher predation pressure (Díaz et al. 2000). Nevertheless, other factors may be also affecting body condition of females rather than just the increased predation risk, such as the scarce cover of shrubs in deteriorated habitats that may impoverish thermal environment (Díaz and Carrascal 1991). This may imply more thermoregulatory costs to lizards (Díaz 1997). Hence, females might not invest so much time and energy in growth and parasite defence, and therefore, show a poor body condition. As females seemed to be highly confined to a determined small area compared Capítulo 3 Efectos de la degradación del medio 187 with males, we can not refuse this hypothesis. In summary, to our knowledge, our study is one of the firsts in relating the effects of habitat deterioration on antipredatory behaviour, body condition and parasite load of lizards. Habitat deterioration not only implies a loss of habitat for P. algirus lizards, but also leads to an increase in perceived risk of predation, at least for females. Females seemed to adjust their antipredatory behaviour accordingly to increased predation risk in areas with deteriorated vegetation, but this change in antipredatory behaviour may infer a cost in terms of a loss of body condition and greater blood parasite loads. Acknowledgements We thank "El Ventorrillo" MNCN Field Station for use of their facilities. Financial support was provided to L. Amo by an “El Ventorrillo” C.S.I.C. grant, to P. López by a the MCYT project BOS 2002-00598, and to J. 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Adv Stud Behav 16: 229–249 191 Capítulo 4 Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 193 Efectos del ecoturismo en las estrategias de escape, el uso de refugios y la condición y estado de salud de lacértidos Dado que muchas lagartijas responden a la presencia humana como a la de un depredador, un incremento en el ecoturismo de una zona puede suponer un incremento en el riesgo de depredación percibido por las lagartijas, con el consiguiente aumento en las estrategias antidepredatorias como el uso de refugios. El uso de refugios es costoso y resultados anteriores muestran que un uso frecuente de refugios conlleva una pérdida de condición corporal. Como causas de esta pérdida de condición corporal se han sugerido varios factores pero realmente no se conoce cuál de ellos afecta a la condición de los individuos. Además, también se desconoce la flexibilidad en el uso de refugios de las lagartijas para hacer frente al riesgo de depredación pero sin incurrir en los costes de éstos. En este capítulo se recogen dos estudios para analizar los efectos del ecoturismo sobre las estrategias antidepredatorias y la condición corporal de las lagartijas. Así mismo se examinan los factores que se han sugerido como responsables de la pérdida de condición corporal por el uso de refugios y las estrategias de las lagartijas para minimizar estos costes, de acuerdo con su condición corporal. Capítulo 4 Ecoturismo, escape, uso de refugios y condición corporal 195 Ecotourism as a form of predation risk affects body condition and health state of Podarcis muralis lizards RESUMEN El ecoturismo ha experimentado un gran crecimiento, y aunque puede tener efectos negativos para muchas especies, sus consecuencias para muchos taxones no se han explorado. Muchas especies de lagartijas están en peligro de extinción y el ecoturismo ha sido propuesto como un posible factor responsable del declive de algunas poblaciones, pero no se ha examinado el efecto del ecoturimo en el comportamiento y la condición corporal y estado de salud de las lagartijas. Muchas especies responden a la presencia de una persona como a la de un depredador, escapando a refugios. Sin embargo, un incremento en las estrategias antidepredatorias puede conllevar una pérdida de condición corporal, lo que puede tener consecuencias importantes para la eficacia biológica a corto y largo plazo. Por ejemplo, puede provocar una disminución de la capacidad de luchar contra parásitos. En este estudio analizamos el efecto del ecoturismo en el comportamiento antidepredatorio de las lagartijas roqueras, Podarcis muralis, así como en la condición corporal y el estado de salud (ectoparásitos, parásitos sanguíneos y respuesta inmune celular mediada por linfocitos T). Los resultados mostraron que las lagartijas no modifican su comportamiento de escape en relación a la presión turística. Las lagartijas tuvieron distancias de aproximación y huida así como estrategias de escape similares. Sin embargo, las lagartijas de áreas con una alta presión turística, donde probablemente tenían que llevar a cabo estos comportamientos de escape frecuentemente presentaron menor condición corporal y mayor intensidad de infección por garrapatas. Las lagartijas en peor condición corporal tuvieron menor respuesta inmune. Este efecto negativo del ecoturismo debería ser tenido en cuenta a la hora de diseñar caminos en áreas protegidas. Este estudio aporta evidencias de que, a pesar de que las lagartijas muestren comportamientos de escape similares en áreas turísticas o no turísticas, la condición corporal de los individuos debería ser tenida en cuenta para examinar adecuadamente los efectos reales del ecoturismo en las poblaciones de lagartijas. Capítulo 4 Ecoturismo, escape, uso de refugios y condición corporal 197 Ecotourism as a form of predation risk affects body condition and health state of Podarcis muralis lizards Abstract Ecotourism has experienced a greater increase, and, even although it might have deleterious effects for many species, its consequences for wildlife remains little explored. Many lizard species are endangered and ecotourism has been proposed as a potential factor responsible of the decline of several lizards’ populations, but no study has examined the effect of ecotourism on lizards’ behaviour, body condition and health state. Many lizards respond to people as if they were predators, by readily escaping to refuges. However, an increase in the frequency of these antipredatory strategies can lead to a loss of body condition, which may have important consequences for short and long term fitness. For example, it might decrease the ability to invest in defence against parasites. We analysed the effects of ecotourism on escape behaviour of common wall lizards, Podarcis muralis, as well as on their body condition and health state (ectoparasites, blood parasites, and T-cell mediated immune response). Results showed that lizards did not modify their escape behaviour in response to ecotourism. Lizards had similar approach and flight distances, and escape strategies regardless of the level of ecotourism pressure. However, lizards inhabiting areas with high ecotourism levels, where they presumably needed to perform antipredatory behaviours more often, showed lower body condition and higher intensity of infection by ticks. Moreover, lizards with poorer body condition had lower T-cell mediated immune responses. Therefore, ecotourism seems to have deleterious effects on body condition and on host-parasite relationships in this lizard species. These effects should be taking into account when designing walking paths in protected areas. Our study reports evidence that regardless lizards showed similar escape behaviour in tourist than in natural areas, their body condition and health state should be also examined to accurately assess the actual effects of ecotourism on lizards’ populations. Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 198 Introduction Ecotourism is largely perceived to safeguard natural areas and thereby to contribute to the conservation of biodiversity (Munn 1992; Ceballos- Lascuráin 1996; Gössling 1999; Tisdell and Wilson 2002; Lindsey et al. 2005; but see López-Espinosa de los Monteros 2002). In the last decades, ecotourism has experienced a greater increase, but its consequences to many taxa has not been so rapidly assessed (see Frid and Dill 2002 for a general review). Known deleterious effects of ecotourism are decreased reproductive success (Giese 1996; Beale and Monaghan 2004a; McClung et al. 2004), loss of feeding areas (Sutherland and Crockford 1993; Gander and Ingold 1997; Fernández- Juricic and Tellería 2000; but see Nevin and Gilbert 2005), decreased foraging rates (Duchesne et al. 2000), loss of optimal habitat (Burton et al. 2002), and even greater mortality rates (Feare 1976; Wauters et al. 1997; Müllner et al. 2004). Changes in behaviours such as escape responses have often been considered the most sensitive measure of the effects of human disturbance on animals, and it has frequently used as an index of disturbance effects (Carney and Sydeman 1999). Thus, it has been assumed that an increased avoidance response of animals to human presence in tourist areas means a deleterious effect of ecotourism on those animal populations (see for example, Ikuta and Blumstein 2003). This may be true because antipredatory behaviours are costly in terms of losing time for other activities such as foraging (Koivula et al. 1995; Dill and Fraser 1997; Martín et al. 2003; Cooper and Pérez-Mellado 2004) or reproduction (Sih et al. 1990; Crowley et al. 1991; Martín and López 2003). Moreover, predator avoidance can also lead to physiological costs for prey such as a decrease in body condition (Martín and López 1999a; Pérez-Tris et al. 2004). For example, Podarcis muralis lizards experimentally submitted to a high predation pressure increased refuge use, which led to a loss of body mass (Martín and López 1999a). Also, Psammodromus algirus lizards submitted to a higher risk of predation caused alarmed lizards a loss of mass not only because they interrupted feeding, but also because of stress in relation to Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 199 predation risk per se (Pérez-Tris et al. 2004). However, measuring escape responses may be not enough for assessing the effects of ecotourism on wildlife (Higham 1998). On one hand, if animals inhabiting areas with high ecotourism pressure do not respond differently to the current approach of a human from those inhabiting areas with no ecotourism pressure, the conclusion could be that those individuals are not affected by tourism. However, if animals inhabiting tourist areas perform these antipredatory behaviours with the same characteristic but more frequently, they may suffer the costs of these behaviours as for example a loss of time to perform other activities (Lima and Dill 1990) and/or a decrease in body condition (Martín and López 1999a; Pérez-Tris et al. 2004). On the other hand, the antipredatory behaviour seems to be dependent on body condition of animals (Beale and Monaghan 2004b). Therefore, animals may not differ in their escape response because animals that inhabit areas with high ecotourism pressure might have poor body condition for different reasons, and thus they might not be able to afford the costs of these behaviours. This is because, although predation is a major selective force, prey should optimize their antipredatory response by balancing antipredatory demands with other requirements (Lima and Dill 1990; Lima 1998). Hence, prey may wait to perform an escape response until costs of not responding are higher than costs of such response (Ydenberg and Dill 1986; Dill and Houtman 1989; Bonenfant and Kramer 1996). Thus, they could accurately adjust their antipredatory behaviour to cope with increased predation risk without incurring excessive costs (Sih 1992, 1997; Dill and Fraser 1997; Martín and López 1999b; Martín et al. 2003). A decrease of body condition may have important consequences for short and long term fitness of individuals. For example, the loss of body condition could lead to a decrease in the ability to invest in defence against parasites as the nutritional status can affect the capacity of a lizard to mount an immune response to infection (Cooper et al. 1985; Smallridge and Bull 2000). This may influence host-parasites relationships, and may increase the negative effects of parasites on their host, which, in turn, Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 200 can affect the maintenance of animals’ populations. Ecotourism may have deleterious effects on reptiles because many species respond to humans as if they were potential predators, by readily escaping to refuges (e.g. Martín and López 1999a,b; Amo et al. 2003), and an increase in antipredatory behaviours may be costly for lizards (Martín and López 1999a; Pérez-Tris et al. 2004). However, deleterious effects of ecotourism have only being described for sea turtles, with a decrease in nestling success associated to tourist areas (see Wilson and Tisdell 2001 and references therein) and in Galápagos marine iguanas (Amblyrhynchus cristatus), which showed different stress responses (i.e. corticosterone levels) than iguanas from non tourist areas (Romero and Wikelski 2002). Therefore, much more research is needed to examine the effect of ecotourism on reptiles’ populations. This effect may be especially important because in the last decades many populations of several lizards’ species are in decline in Europe (Corbett 1989; Pleguezuelos 1997; Pleguezuelos et al. 2002). For some of them, ecotourism has been suggested as possible factor of this decline (Pérez-Mellado 2002; Sá Sousa and Pérez-Mellado 2002; Carretero et al. 2002), but no studies have analysed the effect of ecotourism on body and health condition of lizards. Here we analysed the effects of ecotourism on common wall lizards, Podarcis muralis, taking into account not only the escape behaviour but also the body condition and health state of lizards. This is a small lacertid lizard (60-76 mm adult snout-to-vent length) widespread in Central Europe, although in the Iberian Peninsula it is restricted to mountain areas of the northern half, where they occupy soil dwellings, talus and walls in shaded zones in forests (Martin-Vallejo et al. 1995). Concretely, we examined in areas with different level of ecotourism pressure within the same habitat whether lizards differed in (a) the distance to the nearest refuge, (b) the escape strategy used when attacked by a simulated predator, (c) approach and flight distances, (d) body condition, and (e) health state of lizards (T-cell mediated immune response, and ectoparasite and blood parasite load). Since lizards are known to accurately assess the risk of predation and respond accordingly, we expected that lizards Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 201 might not show differences in antipredatory behaviour if they responded to the current level of predation risk of each attack (Martín and López 2005) regardless of the average levels of risk in an area. Hence, lizards in areas with high ecotourism pressure might suffer the costs associated to perform antipredator behaviours more frequently, with the subsequent deleterious effects for their body and health condition. Furthermore, lizards with decreased body condition might not be able to allocate resources to parasite defence, and, therefore, the deleterious effects of parasitemia should be more evident in tourist areas. Alternatively, lizards submitted to a higher predation pressure might be able to modify their escape behaviour to cope with the average predation risk of the area without incurring in excessive costs of antipredatory behaviours. Therefore, if they had shorter distances to refuges, and shorter approach and flight distances, we would not expect differences between areas with different level of ecotourism pressure in the body condition and health state of lizards. Methods Study area The study was performed during spring and summer 2003 at a large pine forest area in the Guadarrama mountains (40°44’N, 4°02’W; Madrid province, Spain). The dominant vegetation consists of Pinus sylvestris forest, with shrubs such as Juniperus communis and Cytisus scoparius. These mountains are the traditional recreational area of Madrid City, and hence, they suffer a high level of ecotourism, with a great number of pedestrians, especially during the weekends and during the period of activity of lizards (i.e., in spring and summer). For this study, we classified sampling areas according to two levels of ecotourism pressure: 1) areas with ‘high’ level of ecotourism, located close (within 5 m) to frequently transited walking paths, or in recreational and picnic areas, and 2) areas with ‘low’ level of ecotourism, far from walking paths (more than 30 m), and where people was rarely observed. Except for the presence of walking paths, both types of areas were similar respect to microhabitat or microclimate characteristics. Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 202 Escape behaviour During April we simulated predatory attacks to adult lizards (n = 70), recording their escape behaviour in areas with different level of ecotourism within the study area. We walked haphazardly in days with favourable climate conditions (warm sunny days) and between 09:00 and 13:00 h GMT until an adult lizard was sighted with binoculars, whereupon we attempted to approach it directly. The same person performed all approaches, walking at the same moderate speed (about 40 m/min) and wearing the same clothing, to avoid confounding effects that may have affected lizards’ risk perception (e.g., Burger and Gochfeld 1993; Cooper 1997; Martín and López 2005). For each approach, we noted: a) the distance at which the lizard was detected, b) the distance from the initial location of the lizard to the nearest available refuge, c) the ‘approach distance’ as the distance between the lizard and the observer when the lizard first moved (a straight line measured to the nearest 0.1 m), d) the ‘escape strategy’ used, distinguishing between lizards that remained stationary without hiding, lizards that fled to hide in the nearest refuge, and lizards that fled without hiding (Amo et al. 2003), and e) the ‘flight distance’ as the distance that the lizard ran during an episode of escape until it hid or stopped for first time. We used two-way analyses of variance (ANOVAs) to compare detection distance or distance to the nearest refuge (dependent variables) between areas with different levels of ecotourism, and between sexes of lizards, including the interaction between sex and level of ecotourism. We used two-way analyses of covariance (ANCOVAs) to compare approach or flight distances (dependent variables) between areas with different levels of ecotourism, and between sexes, and their interaction, with the distance to the nearest refuge as a covariant. We used Generalized Non Lineal Models (GLZ) to assess the effect of level of ecotourism and distance to the nearest refuge on the type of escape strategy used by lizards as a dependent variable following a multinomial distribution classifying lizards that did not flee, lizards that hid in the nearest refuge, and lizards that fled without hiding. As there were some cases where there were no data for both sexes, we Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 203 could not perform the analysis including the sex in the model. For example, no male escaped by fleeing without using refuges in tourist areas, and no female used refuges in non-tourist areas. Although results of a previous study did not show sexual differences in the escape strategy of wall lizards (Amo et al. 2003), we used Chi-squared tests to compare the frequency of each type of escape strategy between males and females and between tourist and non- tourist areas. Body condition, health state, and parasite load of lizards During the mating period (June) and after the mating period had finished (July-August) we captured by noosing 78 adult lizards to examine their body condition, health state, and parasite load in areas with different level of ecotourism. We weighed lizards, measured their snout-vent-length (SVL), and noted the number of ectoparasites (ticks) observed on their body immediately after capture. Then, we took captured lizards for data collection (see below) to "El Ventorrillo" Field Station, 5 km from the capture site. Lizards were individually housed in 60 x 40 cm PVC outdoor terraria. They were fed mealworm larvae (Tenebrio molitor), and water was provided ad libitum. The photoperiod and ambient temperature were that of the surrounding region. All lizards were healthy and were returned to their exact capture site 48 h after capturing. The experiments were performed under license from the Consejería del Medio Ambiente de la Comunidad de Madrid (Spain). To assess blood parasite load, we made a smear on a microscope slide from blood taken from the postorbital sinus by using one 9 µl heparinized hematocrit tube. Blood smears were air- dried, fixed in absolute methanol for 10 min and then stained in Giemsa diluted 1:9 with phosphate buffer (pH 7.2) for 40 min before their examination for parasites. On mounted slides, half a smear, chosen at random, was scanned entirely at 200 x along the longitudinal of slide, looking for extraerythrocytic protozoa (Merino and Potti 1995; Amo et al. 2004, in press). Number of intraerythrocytic parasites (haemogregarines) was estimated at 400 x by counting the number of parasites per 2000 erythrocytes. Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 204 We measured T-cell mediated immune (CMI) responsiveness of lizards captured in summer by using a delayed- type hypersensitivity test. We estimated CMI on the basis of quantification of the swelling response to intradermally injected phytohaemagglutinin (Smits et al. 1999). This test is a reliable measure of CMI in vivo (Lochmiller et al. 1993), and it has been used in many studies of animals including lizards (Merino et al. 1999; Svensson et al. 2001; Belliure et al. 2004). We injected the lizard’s footpad of the right hind limb with 0.02 ml of phytohaemagglutinin solution (PHA-P, Sigma), and measured the swelling with a pressure sensitive spessimeter (to the nearest 0.01 mm) before and 24 h after the injection (Smits et al. 1999). Results of previous studies showed that repeatability of this measure, calculated as the intraclass correlation coefficient (Lessells and Boag 1987) was high (r>0.95, L. Amo, unpublished data). We used backward general lineal models (GLM) to analyze differences in intensity of ticks’ infection (dependent variable) in relation to sex, size, season (spring vs. summer) and level of ecotourism of the area, including the interactions between these variables in the initial model. Similar backward GLMs were used to analyse differences in blood parasite load (dependent variable) in relation to all the above independent variables plus the intensity of ticks’ infection, and to analyze differences in body mass (dependent variable) in relation to all the above variables plus ectoparasite and blood parasite loads. Finally, we also used backward GLMs to analyze differences in CMI response (dependent variable) in relation to the sex, size, mass and level of ecotourism, and ectoparasite and blood parasite loads. Results Escape behaviour The distance of detection did not differ between areas with different levels of ecotourism (ANOVA, F1,65 = 0.47, P = 0.49) or between sexes (F1,65 = 1.42, P = 0.24), and the interaction was not significant (F1,65 = 2.29, P = 0.14) (Table 1). There were neither significant differences in the distance to the nearest refuge at which lizards were located before the attack between levels of ecotourism (ANOVA, F1,66 = 0.01, P = Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 205 0.98) nor between sexes (F1,66 = 0.56, P = 0.46), and the interaction was not significant (F1,66 = 0.38, P = 0.54) (Table 1). Approach distances were not related to the distance to the nearest refuge (ANCOVA, F1,65 = 1.28, P = 0.26), did not differ significantly between levels of ecotourism (F1,65 = 0.44, P = 0.51), or between sexes (F1,65 = 1.80, P = 0.18), and the interaction was not significant (F1,65 = 0.10, P = 0.75) (Table 1). Similarly, flight distances were not related to the distance to the nearest refuge (ANCOVA, F1,64 = 0.60, P = 0.44), there were no significant differences between levels of ecotourism (F1,64 = 0.93, P = 0.34), or between sexes (F1,64 = 1.36, P = 0.25), and the interaction was not significant (F1,64 = 0.48, P = 0.49) (Table 1). The escape strategy of lizards was not related to the distance to the nearest refuge (Wald’s χ2 = 3.43, df = 2, P = 0.18) nor to the level of ecotourism of the area (Wald’s χ2 = 1.96, df = 2, P = 0.37). There were no significant differences between males and females when comparing each type of escape strategy in tourist or non tourist areas (Chi-squared test, χ2 < 1.05, df = 1, P > 0.30 in all cases; Table 1), neither there were significant differences between tourist or non tourist areas when comparing each type of escape strategy in males or females (Chi-squared test, χ2 < 2.30, df = 1, P > 0.13 in all cases; Table 1). Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 206 Table 1 Mean + SE of variables describing escape behaviour of male and female Podarcis muralis lizards in areas with low or high level of ecotourism. Low level of ecotourism Males Females High level of ecotourism Males Females Distance to the nearest refuge (cm) 31 + 16 16 + 3 24 + 10 23 + 7 Distance of detection (cm) 389 + 36 400 + 43 418 + 36 324 + 22 Approach distance (cm) 81 + 31 38 + 27 112 + 44 53 + 24 Flight distance (cm) 6 + 3 0 + 0 20 + 15 3 + 3 Number of lizards stationary 12 11 11 13 Number of lizards fleeing without hiding 1 2 0 3 Number of lizards hiding in a refuge 5 0 9 3 Parasite load, body condition, and health state of lizards The intensity of infection by ticks (GLM, R2 = 0.15, F2,71 = 6.51, P = 0.003) was higher in males than in females (F1,71 = 5.50, P = 0.02; Fig. 1), and higher in areas with high level of ecotourism (F1,71 = 8.27, P = 0.005; Fig. 1). Other variables and interactions were not significant and were removed from the final model. Fig. 1 Mean (+ SE) intensity of infection by ticks in male (black bars) and female (open bars) Podarcis muralis lizards in areas with low and high level of ecotourism. Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 207 Blood parasite load (GLM, R2 = 0.16, F5,68 = 2.59, P = 0.03) did not change in the course of seasons in areas with low level of ecotourism, whereas it decreased in areas with high level of ecotourism (season x ecotourism level: F1,68 = 8.78, P = 0.004). The interaction between season, level of ecotourism and size of lizards was significant (F1,68 = 8.38, P = 0.005). Thus, during the breeding period there was no relationship between body size and intensity of haemogregarines’ infection in any area. However, after the breeding period the relationship between body size and intensity of blood parasites was negative in areas with low ecotourism but positive in areas with high ecotourism pressure. Other variables and interactions were not significant and were removed from the final model. Body mass of lizards (GLM, R2 = 0.83, F6,67 = 53.10, P < 0.0001) was positively correlated with their SVL (F1,67 = 118.49, P < 0.0001). Males were significantly heavier than females of equivalent body size (sex x size: F1,67 = 5.26, P = 0.02). Lizards had relative lower body mass in areas with high level of ecotourism (F1,67 = 9,29, P = 0.003), but this relationship varied between seasons (ecotourism x season: F1,67 = 6.01, P = 0.02; Fig. 2), being significant only after the mating period (Tukey’s test, P = 0.0002), but not during the mating period (P = 0.47). Ectoparasite and blood parasite loads did not affect body mass, and were removed from the model. Fig. 2. Mean (+ SE) body mass (g) of Podarcis muralis lizards in areas with low (black bars) and high level of ecotourism (open bars) in spring and summer. Lizards with higher CMI showed greater body mass (GLM, R2 = 0.27, model: F2,37 = 6.73, P = 0.003; body mass: F1,37 = 4.89, P = 0.03; Fig. 3a) and lower blood parasite load (F1,37 = 8.38, P Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 208 = 0.006; Fig. 3b). The ectoparasite load, sex and level of ecotourism were not related to the CMI response and were removed from the model. Fig. 3. Relationship between T-cell mediated immune response (CMI) and a) body mass (g), or b) intensity of haemogregarines’ infection (log- transformed) in Podarcis muralis lizards. Discussion Our results suggested that common wall lizards responded to the current level of predation risk rather than to the average risk of predation in an area. Hence, there were no differences in their initial distance to the nearest refuge in relation to the level of ecotourism pressure of the area. Since the risk of capture is higher for prey that are farther from the refuge (Bulova 1994; Blázquez et al. 1997; Cooper 1997), we expected that lizards in areas with high ecotourism pressure should be located closer to refuges in comparison with areas with low pressure. However, lizards should accomplish more than simply avoiding predators (Lima and Dill 1990; Lima 1998), especially during the mating period, hence they should not be restricted to areas near refuges. Moreover, distance of detection of lizards was also similar in both areas. Thus, our results suggest that lizards did not modify their microhabitat use in order to locate themselves closer to refuges or be more cryptic in relation to the average risk of predation in an area, which agrees with previous results with this species (Diego-Rasilla 2003). Furthermore, when attacked, lizards showed similar antipredatory behavior regardless of the tourist pressure of the area. Lizards began to escape at similar approach distances from the predator and had similar flight distances. Neither the Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 209 type of escape strategy used differed in relation to the level of ecotourism, although it was related to the distance to the nearest refuge. Lizards that fled from the predator were at higher distances from the nearest refuge than lizards that remained stationary or those that hid in the refuge, as observed previously (Amo et al. 2003). Therefore, our results suggest that lizards respond to the current level of predation risk regardless of the average level of risk in an area. Previous results also showed that wall lizards adjusted continuously their escape decisions to the level of risk posed by the predator in each attack (Amo et al. 2003; Martín and López 2005). On contrast, results of a previous study suggested that P. muralis lizards of a population submitted to a high predation pressure had greater approach distances and greater use of refuges compared with individuals of a low predation pressure population (Diego- Rasilla 2003). However, the study populations that differed in the pressure of ecotourism also differed in altitude, with possible differences in ambient temperatures. Thus, in that study an increase in the use of refuges in the high predation pressure population (but also placed at low altitude) could be due not only to the high risk but also to the higher refuge temperature and, hence, lower costs of refuge use (Amo et al. 2003). However, antipredatory strategies are costly in term of body condition for this lizard (Martín and López 1999a), and our results suggest that lizards inhabiting areas with high ecotourism pressure showed lower body condition than lizards inhabiting less visited areas, especially in summer, probably because of the increased cumulative frequency of antipredatory behaviours that lizards should have performed in response to human disturbance during all their activity period. Alternatively, lizards in worse body condition might not be able to perform the expected greater response to humans in tourist areas if they were not able to afford the costs of antipredatory behaviour (Beale and Monaghan 2004b). However, our explanation agrees with a previous study that showed that wall lizards submitted to a high frequency of approaches by a simulated predator increased refuge use and, as a consequence, suffered a loss of body mass (Martín and López 1999a). This loss of body mass may have Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 210 important consequences because reproductive activities also decreases body mass during the mating period (Amo et al. 2004). However, once the breeding period has finished, lizards might recover body mass to afford the winter period. Thus, if antipredatory behaviour prevents lizards to increase their body mass at the end of the mating period, lizards may not be able to adequately afford the hibernation period, with the subsequent increase in mortality, and its effect for maintenance of these lizards’ populations. Furthermore, our results also suggest that CMI is positively correlated to body condition of lizards. CMI constitutes one of the main components of immunity in vertebrates (Roitt et al. 1989; Wakelin 1996) and the ability to mount a CMI response may have important fitness consequences (Gonzalez et al. 1999), because it constitutes a generalized short-term response to grafts, allergens and wounds (Belliure et al. 2004). Our results showed that the CMI was negatively correlated with blood parasite load. Therefore, if a loss of body mass implied a loss of immunocompetence, lizards may be exposed to the deleterious effects of parasites. Thus, this may explain why in areas with high influx of visitants lizards support higher levels of ticks. However, and although previous results have shown a negative effect of ectoparasites on body condition in some lizards species (Dunlap and Mathies 1993, Main and Bull 2000), our results did not show any effect of ticks on body mass of wall lizards, as previously observed in this (Amo et al. in press) and other lizard species (Bull and Burzacott 1993; Olsson et al. 2000). Also our results showed that males supported higher intensity of ticks’ infection than females, as previously observed (Salvador et al. 1999; Amo et al. in press). An interesting result is that larger lizards support higher levels of haemogregarines’ infection in tourist areas than in natural areas during the summer, whereas there were not such differences during the spring. This result can be explained because in natural areas larger, and thus, probably older lizards, may have good body condition to perform an adequate response to infection and decrease its intensity, whereas in areas with high level of ecotourism, the loss of body condition may imply a lower immune response and Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 211 thus, higher blood parasite loads. This effect may also be enhanced because in areas with high levels of ecotourism lizards may be exposed to infection more frequently than in natural areas due to the higher intensity of haemogregarines’ vectors (i.e., ticks). On conclusion, regardless of average levels of ecotourism pressure (i.e. predation risk), lizards responded similarly to the current approach of a human by usually fleeing to hide in refuges. However, lizards inhabiting areas with high ecotourism pressure, and, hence, that must escape and hide in refuges more often, suffered the costs associated to these antipredatory behaviours, showing lower body condition and higher intensity of ectoparasites’ infection. Furthermore, lizards with poor body condition had low CMI, which may aggravate the deleterious effects of antipredatory behaviour on body condition. Therefore, ecotourism seem to affect the maintenance of lizards’ populations, which should be considered when designing walking paths in protected areas to conserve lizards’ biodiversity. Similar results have being shown in hoatzin chicks Opisthocomus hoazin, with juveniles showing lower body condition in tourist areas, probably due to higher corticosterone levels in response to human disturbance and with the subsequent decrease in survival (Müllner et al. 2004). Furthermore, our study reports evidence that regardless lizards showed similar escape responses in tourist and tourist-free areas, their body condition also need to be examined to accurately assess the effects of ecotourism on lizards’ populations. Nevertheless, although this study suggests a deleterious effect of ecotourism on lizards’ populations, long- term studies are needed to better understand the effects of ecotourism on reptile populations, as previously observed in birds (Higham 1998). Acknowledgements We thank "El Ventorrillo" MNCN Field Station for use of their facilities. Financial support was provided to L. Amo by an “El Ventorrillo” C.S.I.C. grant, to P. López by a the MCYT project BOS 2002-00598, and to J. Martín by the MCYT project BOS 2002- 00547. 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Tourism Manage 22: 279-288 Ydenberg RC, Dill LM (1986) The economics of fleeing from predators. Adv Stud Behav 16: 229–249 Capítulo 4 Ecoturismo, escape, uso de refugios y condición corporal 217 Flexibility in refuge use helps Lacerta monticola lizards to cope with different levels of predation risk without incurring loss of body condition RESUMEN Las presas a menudo responden a los depredadores incrementando el uso de refugios, pero esta estrategia implica una serie de costes que conllevan una pérdida de condición corporal. Sin embargo, los factores responsables de esta pérdida de condición corporal no se conocen. Tampoco se conoce cómo las presas usan los refugios para hacer frente al riesgo de depredación sin incurrir en costes para la condición corporal, ni cómo las presas los usan de acuerdo con su condición corporal. En este estudio analizamos en el campo si los adultos de Lacerta monticola modifican su comportamiento antidepredatorio (estrategias de escape y uso de refugios) en función de la degradación de la vegetación y el ecoturismo, dos factores que representan distintos niveles de riesgo de depredación, y sus consecuencias para la condición física de los individuos. Los resultados sugieren que las lagartijas que viven en zonas degradadas permanecen cerca de los refugios, disminuyendo así la distancia de huida durante el escape. También disminuyeron el uso de refugios y, como consecuencia, presentaron una condición corporal similar a las lagartijas que vivían en áreas naturales. También realizamos dos experimentos de laboratorio para analizar cual de los presuntos costes del uso de refugios podía ser responsable de la disminución de la condición corporal en machos: a) una disminución de la eficacia de la digestión debido a bajas temperaturas en el interior de los refugios o b) una disminución del tiempo destinado a la alimentación, con la consiguiente disminución de la ingestión de alimento. Realizamos también un experimento de laboratorio para analizar si el tiempo pasado a bajas temperaturas en el interior de los refugios conllevaba una pérdida de condición corporal para hembras gestantes. Los resultados sugieren que el uso de refugios es costoso en términos de condición corporal debido a la pérdida de oportunidades de alimentación y reducción de la ingestión de alimento en machos, y debido al tiempo pasado a bajas temperaturas en Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 218 hembras gestantes. Sin embargo, las lagartijas modificaron su uso de refugios en relación a su condición corporal, de forma que las lagartijas con peor condición mostraron una disminución en el uso de refugios. Las lagartijas parecen capaces de compensar el incremento en el riesgo de depredación con estrategias antidepredatorias flexibles para hacer frente al riesgo sin incurrir en costes para la condición física. Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 219 Flexibility in refuge use helps Lacerta monticola lizards to cope with increased predation risk without incurring loss of body condition Abstract Prey often respond to increased predation risk by increasing refuge use, but this strategy is costly as it may entail a loss of body condition. However, the factors responsible of this loss of body condition remain unclear. Also how prey deal with refuge use to cope with predation risk without incurring costs of body condition, and how prey use refuges according to their initial body condition remains little know. Here we analyzed in the field whether adult Iberian rock lizards, Lacerta monticola, modified their antipredatory behaviors (escape strategies and refuge use) in areas with different levels of human-induced habitat deterioration and ecotourism pressure, which for lizards have similar consequences than predation risk, and their effects on body condition of lizards. Results showed that lizards inhabiting deteriorated areas where risk is higher remained closer to refuges, thus decreasing flight distances when they escaped, and decreased time spent hidden in refuges, and as a consequence showed similar body condition than lizards inhabiting less risky natural areas. We also performed two laboratory experiments to analyze which costs of increased refuge use could be responsible of the decrease in body condition of male lizards: a) a decrease of the efficiency of digestion due to low temperatures inside refuges and/or b) a decrease of the time available for foraging, which would lead to reduced food intake. We further performed a laboratory experiment to analyse whether increasing time spent at low temperatures inside refuges entails costs on the body condition of pregnant females. Results suggest that refuge use is costly in terms of body condition due to loss of foraging opportunities and reduced food intake in males, and due to time spent at low temperatures in pregnant females but not in males. Also, lizards modified refuge use in relation to their body condition, with lizards with worse condition decreasing time hidden after predatory attacks. On conclusion, lizards seemed able to compensate increased predation risk with flexible antipredatory strategies to cope with risk without incurring costs for body condition. Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 220 Introduction Predation risk is a major force in the evolution of several morphological and behavioural characteristics of animals (Lima and Dill 1990; Lima 1998). However, during their life time animals need to accomplish with multiple demands, and, thus, predator avoidance often implies a loss of time to cope with other requirements such as foraging (Koivula et al. 1995; Dill and Fraser 1997; Martín et al. 2003a; Cooper and Pérez-Mellado 2004) or reproduction (Sih et al. 1990; Crowley et al. 1991; Magnhagen 1991; Martín et al. 2003b). Predator avoidance can also lead to physiological costs such as a decrease in body condition (Martín and López 1999a; Pérez-Tris et al. 2004). For example, Psammodromus algirus lizards submitted to a high predation risk caused alarmed lizards a loss of mass not only because they interrupted feeding, but also because of stress in relation to predation risk per se (Pérez-Tris et al. 2004). Hence, although predation risk has been generally considered in the context of probability of mortality in the immediate future, antipredatory decisions should be also made based on consequences for long-term expected fitness (Clark 1994; Martín and López 2000). Many prey respond to predators by increasing refuge use (Sih et al. 1992; Martín and López 1999a, b). However, this antipredatory strategy implies physiological costs (Martín and López 1999a). For example, Podarcis muralis lizards experimentally submitted to a high predation pressure increased refuge use, which led to a loss of body mass (Martín and López 1999a). Factors proposed to be responsible of this decrease in body condition were: a) a decrease of the time available for foraging, which would lead to reduced food intake (Dill and Fraser 1997; Godin and Sproul 1988; Koivula et al. 1995), b) the low efficiency of digestion due to low temperatures inside refuges (Huey 1982; Stevenson et al. 1985), and, thus, a decrease in the energy available for storage (Harlow et al. 1976; Harwood 1979), or c) energetic costs due to an increased number of fleeing sequences to avoid predators (Martín and López 1999a). It remains unclear which of these factors are really contributing to the decrease in body condition. This is, however, very important because the loss of body mass may have important Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 221 consequences for short and long term fitness. For example, the loss of body condition could decrease the ability to invest in defence against parasites because the nutritional status can influence the capacity of a lizard to mount an immune response to infection (Cooper et al. 1985; Smallridge and Bull 2000). This may influence host-parasites relationships, and may increase the negative effects of parasites on their host. Therefore, although refuges may prevent prey to be captured, costs of refuge use may have consequences for long-term fitness. Theoretical models of refuge use suggest that prey should adjust time spent in a refuge so that the optimal emergence time is the time when the costs of staying exceed the costs of leaving (Sih et al. 1992; Martín and López 1999b; Polo et al. 2005). The decision of when to come out from a refuge should be optimized by considering the expected fitness effects of diminution of risk with time in the exterior, but also considering costs of refuge use. Hence, animals should accurately adjust their antipredatory behaviour and refuge use to cope with increased predation risk without incurring excessive costs (Sih 1992, 1997; Dill and Fraser 1997; Martín and López 1999b; Martín et al. 2003a,b). Previous studies have examined the ability of prey to modify their refuge use to cope with predation risk without incurring costs in terms of the loss of time to perform other activities (Sih 1997; Martín et al. 2003a,b). However, it remains little known whether prey can modify refuge use to avoid incurring costs for their body condition, and whether prey use refuges according to their body condition. These physiological costs of refuge use may be particularly important when prey are submitted to a high predation pressure and, thus, may need to use refuges frequently (Polo et al. 2005), or when prey have poor body condition. The Iberian rock lizard, Lacerta monticola is a small diurnal lacertid found mainly in rocky habitats in high mountains of the Iberian Peninsula, where temperatures are limiting for lizards. Therefore, costs of refuges associated to low temperatures may be very high for this species (Martín and López 1999b; Polo et al. 2005). These lizards responded to predators by rapidly running for cover into the nearest refuge, Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 222 usually rock crevices (Carrascal et al. 1992). However, this species suffers a loss of optimal habitat due to ski infrastructures (Martín and Salvador 1995; Pérez-Mellado 2003). Ski slopes become areas with increased risk of predation because the artificial removal of vegetation cover and rocks causes a loss of potential refuges for lizards. Furthermore, these areas are crossed by many tourist walking paths with high influx of pedestrians, especially during the reproductive period of this lizard species. As lizards and many other animals escape from humans as if they were predators (e.g. Martín and López 1999b, 2000a; Miller et al. 2001), prey may consider paths as high predation risk areas (Mainini et al. 1993; Frid and Dill 2002). Here we first analyzed in the field whether adult Iberian rock lizards modify their antipredatory strategies (escape strategies and refuge use) in areas with different levels of deterioration of the habitat and ecotourism pressure, which presumably represent different levels of predation risk, and the consequences for body condition of lizards. We expected that if lizards responded to the average level of risk in each area by adjusting their antipredatory behaviour, they might be able to decrease costs associated to antipredatory behaviours and maintain their body condition. Alternatively, lizards might respond in a similar way each time they found a potential predator or when were disturbed by humans, regardless of the average level of risk in an area. We expected that lizards inhabiting deteriorated areas and those close to paths suffered a loss of body condition because they might need to escape and use refuges more often than lizards inhabiting natural areas and far from paths. We also performed two laboratory studies to analyze which costs presumably associated to increased refuge use could be responsible of the decrease in body condition of lizards. Results of a previous study indicated that increasing the number of fleeing sequences to refuges led to a decrease in body condition of male lizards (Amo, López and Martín, manuscript in preparation). Here we examined whether a) a decrease of the time available for foraging, which would reduce food intake and/or b) a decrease of the efficiency of digestion due to low Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 223 temperatures inside refuges also influenced body condition. We expected that both potential causes may affect body condition. We also analysed whether lizards modify refuge use in relation to their current body condition. We hypothesized that, under the same levels of risk, lizards with a worse body condition might not afford the costs of refuge use, and thus, should decrease time hidden in refuges after a predator’s attack, whereas lizards with a good body condition may remain for longer inside refuges. Alternatively, if avoiding predation was more important than these associated physiological costs, initial body condition should not affect refuge use. The decrease of body temperature inside a cold refuge may be especially costly for pregnant females that may need to maintain constant body temperatures during gestation (Mathies and Andrews 1997; Shine 2004) because the developmental rates of embryos are temperature dependent (Muth 1980). Therefore, we performed a laboratory experiment to analyse whether increasing time spent at low temperatures inside refuges entails costs on the body condition of pregnant females. We expected that females forced to increase time spent at low temperatures, simulating temperatures inside cold refuges, decreased their body condition. We also analysed refuge use of these females immediately after this experiment. We expected that if females had suffered a loss of body condition, they decreased costs of refuge use by emerging sooner from refuges. Methods Study area We performed the study in the Guadarrama Mountains (Madrid Prov., Central Spain) at an elevation range of 1900-2200 m. Natural landscape is characterised by granite rock boulders and screes interspersed with shrubs (Cytisus oromediterraneus and Juniperus communis), together with meadows of Festuca and other grasses (Martín and Salvador 1995). This area is characterised by the presence of several ski resorts and associated infrastructures. In anthropogenic-induced deteriorated areas, mostly in ski slopes, there is no cover of shrubs nor even grasses, and rocks boulders are scarce or have been eliminated. Also, both natural and Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 224 deteriorated areas are crossed by many tourist walking paths with high influx of pedestrians, especially during spring and summer. In this region L. monticola (snout-to-vent length, SVL, of adult lizards range between 65 mm and 90 mm) is active from May to September due to limiting environmental temperatures. Lizards mate in May-June and produce a single clutch in July (Elvira and Vigal 1985; Salvador 1984; Pérez-Mellado 1998). Antipredatory behaviour During June 2004 we recorded the characteristics of escape behaviour of adult lizards in areas with different level of deterioration of the habitat and different ecotourism pressure. We distinguished two levels of deterioration of the habitat; ‘natural’ areas characterized by original habitat conditions (see above), and ‘deteriorated’ areas in ski slopes and associated infrastructures characterized by the low cover of vegetation and rocks. The level of ecotourism was assessed as the distance to the nearest path, because the influence of pedestrians was limited to these paths, as people usually did not cross-country walk through this rough habitat (personal observation). We walked in days with favourable climate conditions (warm sunny days) and between 09:00 and 13:00 GMT until an adult lizard was sighted with binoculars, whereupon we attempted to approach it directly. The same person performed all approaches, walking at the same moderate speed (about 40 m/min) and wearing the same clothing, to avoid confounding effects that may have affected lizards’ risk perception (e.g., Burger and Gochfeld 1993; Cooper 1997). For 124 lizards, we noted the distance from the initial location of the lizard to the nearest available refuge (a straight line measured to the nearest 0.1 m), the ‘approach distance’, as the distance between the lizard and the observer when the lizard first moved, and the ‘flight distance’ as the distance the lizard moved during an episode of escape. We also noted the ‘escape strategy’ used, distinguishing between lizards that remained stationary, lizards that fled to hide in the nearest refuge, and lizards that fled but stopped without hiding (Amo et al. 2003). When a lizard hid in a refuge we retreated to a hidden position and recorded the time the lizard Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 225 spent in the refuge until the head emerged (‘appearance time’) and the time until the lizard left the refuge (‘emergence time’). Escape behavior and emergence times of lizards may depend on their body temperature and thermal conditions inside the refuge (e.g., Hertz et al. 1982, Martín and López 1999a, b). However, measuring initial body temperature of lizards in the field was complicated because when capturing lizards they often had been hidden in cold refuges, which lowered their initial body temperature. Nevertheless, air (Ta) and substrate (Ts) temperatures can be used as a good approximation to lizards’ body temperatures in this species, given the strong dependence of these on the thermal environment (Carrascal et al. 1992; Martín and Salvador 1993; Martín et al. 1995). Thus, immediately after a lizard emerged from the refuge, we measured with a digital thermometer to the nearest 0.1 ºC the Ta at the point the lizard occupied before the attack (shaded bulb, 2 cm above the point), and the Ts inside the refuge used and/or inside the nearest refuge. Additionally, 75 of the 124 adult lizards, which escape behaviour had been recorded, were later captured by noosing after emerging from refuges to measure their body mass and body size (SVL). Thereafter, lizards were immediately released at the point of capture. We used analyses of covariance (ANCOVA) to test for differences in each of the antipredatory behaviour variables (distance to the nearest refuge, approach and flight distances, or appearance and emergence times) between sexes, and between levels of deterioration of the habitat (natural vs. deteriorated), including the interaction between sex and level of deterioration, and considering in the initial model as potential covariants the distance to the nearest path, Ta and Ts inside refuges. Non significant covariants were removed from the final models. We used Generalized Non-Linear Models (GLZ) to tests for differences in escape strategy of lizards (dependent variable following an ordinal multinomial distribution, i.e., remaining stationary vs. fleeing without hiding vs. fleeing to hide in a refuge) in relation to sex, level of deterioration, distance to the nearest path, and Ta and Ts inside refuges. We also included the interaction between sex and level of deterioration in the initial model. We used ANCOVA to test for differences in Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 226 body mass between sexes, and levels of deterioration of the habitat, with SVL and the distance to the nearest path as covariants. Costs of refuge use: diminution of digestion efficiency vs. loss of foraging opportunities Diminution of digestion efficiency In July we captured by noosing 29 adult male lizards in the study area to determine whether the presumably diminution of digestion efficiency due to low temperatures inside refuges (Harlow et al. 1976; Harwood 1979) actually influenced body condition and health state of lizards. Lizards were individually housed at “El Ventorrillo” Field Station 5 km from the capture site, in outdoor 60 x 40 cm PVC terraria containing sand substratum and rocks for cover. Water was provided ad libitum. The photoperiod and ambient temperatures were those of the surrounding region. To avoid changes in body and health condition and parasite load of lizards due to captivity, they were held in captivity only one week before testing to allow acclimation to laboratory conditions. After the experiment lizards were released at the points of capture. We weighed and measured SVL of lizards immediately after capture. To assess blood parasite load, we made a smear on a microscope slide from blood taken from the postorbital sinus by using one 9 µl heparinized hematocrit tube. Blood smears were air-dried, fixed in absolute methanol for 10 min and then stained in Giemsa diluted 1:9 with phosphate buffer (pH 7.2) for 40 min before their examination for parasites. On mounted slides, number of intraerythrocytic blood parasites was estimated at 1000 x by counting the number of parasites per 2000 erythrocytes. The only parasites found were haemogregarines (Amo et al. 2004). We measured T-cell mediated immune (CMI) responsiveness to lizards by using a delayed-type hypersensitivity test. This test is a reliable measure of T- cell-dependent immunocompetence in vivo (Lochmiller et al. 1993), and it has been used in many studies of animals including lizards (Merino et al. 1999; Svensson et al. 2001; Belliure et al. 2004). CMI responsiveness was estimated on the basis of quantification Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 227 of the swelling response to intradermally injected phytohaemagglutinin (Smits et al. 1999). We injected the lizard’s footpad of the right hind limb with 0.02 ml of phytohaemagglutinin solution (PHA-P, Sigma), and measured the swellings with a pressure sensitive spessimeter (to the nearest 0.01 mm) before and 24 h after the injection (Smits et al. 1999). Results of previous studies showed that repeatability of this measure, calculated as the intraclass correlation coefficient (Lessells and Boag 1987) was high (r>0.95, L. Amo, unpublished data). These measures were taken again immediately after the experiments finished. Although response to PHA injection is not well known in lizards, and the immune system of lizards might respond differently to the first injection of PHA than to the second injection, in any case, the second response should be similar for individuals of all treatments, and, thus, any differences between groups should be attributed to the effect of treatment. Lizards were, then, assigned to one of three treatments. In the ‘control’ treatment lizards were left undisturbed in their outdoor terraria (mean Ta = 19º C, Ts = 26.2º C), where they could bask and attain optimal body temperatures, and were fed two mealworms (Tenebrio molitor) at 13:30 GTM each day. In the ‘no thermoregulation’ treatment, lizards were maintained in their terraria but inside a cold room (Ta = 17.1º C, Ts = 17.3º C), where they could not attain optimal body temperatures, from 9:00 am to 13:00 GTM each day. After this time, terraria were replaced to outdoor conditions, and 30 min afterwards lizards were fed two mealworms. In the ‘no digestion efficiency’ treatment lizards were maintained in outdoor conditions until 12:30 GTM, when they were fed two mealworms. Lizards were observed until they ate the mealworms, and immediately after we carried lizards in their terraria inside a cold room (Ta = 17.1º C, Ts = 17.3º C) during four hours, until 16:30 GTM. Thereafter, they were returned to outdoor conditions. This procedure was repeated during ten days. We used repeated-measures two way ANOVAs to examine differences in body mass, CMI response, or parasite load between the beginning and the end of the experimental treatments (‘time’; within subject factor) and between treatments (between subject factor), including the interaction to test for Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 228 differences between treatments in the course of time. Immediately after these treatments finished, we designed an experiment to compare the time spent hidden inside refuges in response to simulated predator attacks, and the variation between two successive attacks, by lizards in relation to their body condition. The experiment was conducted in a terrarium (100 x 40 x 50 cm) with a sand substrate and a single refuge in the middle of one end of the terrarium. The refuge was built with flat rocks, which had one opening (7 x 6 cm). Air temperature inside the refuge during tests was maintained at 17.4 + 0.1° C to avoid the confounding effects of temperature differences on refuge use (Martín and López 1999b). Lizards were gently transferred to the experimental terraria, where the refuge had the door initially closed, and given two min before trials for acclimatization to a novel environment. Then, the experimenter opened the door of the refuge and simulated a predatory attack by tapping lizards close to the tail with a brush to stimulate them to run and hide in the refuge. When the lizard hid, we retreated to a hidden position and recorded the time that the lizard spent in the refuge until the lizard emerged entirely from the refuge (‘emergence time’). Immediately after the lizard resumed normal activity, we simulated another predatory attack with the same procedure and measurements as in the first attack. We used repeated measures two way ANOVAs to test for differences in emergence time from the refuge between the first and the second attack (within subject factor) and between treatments (between subject factor). We included the interaction to test for differences between treatments between attacks. Loss of foraging opportunities We performed this experiment to examine the effect of loss of foraging opportunities while lizards were hidden in refuges on their body condition, health state, and parasite load. We captured 30 male lizards in the study area that were maintained in the same initial conditions than described above. We measured lizards, took a drop of blood for parasites’ estimations, and performed the PHA test to measure CMI response (see above) before and after the experiment. Lizards were, then, assigned to one of three treatments. In the ‘control’ Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 229 treatment lizards were left undisturbed in their outdoor terraria, where they could normally bask, and were fed two mealworms at 12:30 GTM each day. In the two experimental treatments, lizards were also kept in outdoor terraria, but they were submitted to repeated persistent attacks. We performed an attack, which lasted for 15 s, every10 min within a four hours period each day, from 9:00 to 13:00 hours GMT, when lizards were fully active. In the ‘only refuge use’ treatment lizards were fed two mealworms every day after the attacks had finished. In the ‘loss of foraging opportunities’ treatment, lizards received just one mealworm every two days, to simulate that they had missed the opportunity to find food while hidden in refuges. This procedure was repeated during 10 days. Immediately after this experiment finished, we measured refuge use of lizards, with the same procedure and measurements as in the first experiment. Thereafter, lizards were released at the points of capture. Statistical analyses were conducted as in the previous experiment. Costs of time spent at low temperatures for pregnant females In July we captured by noosing 20 adult female lizards with mating scars on the belly indicating several copulations, and hence, probably pregnant, in a nearby area to determine whether costs of refuge use, in terms of time spent at low temperatures, influence body condition and health state of pregnant females. Lizards were individually housed in the same initial conditions than described above. Females were measured and weighed, and we extracted a drop of blood to assess blood parasite load, and performed PHA test to assess CMI response (see above). These measures were taken again immediately after the experiments finished. We confirmed that all females were actually pregnant by abdominal palpation at the end of the experiment. Lizards were assigned to one of two treatments. In the ‘control’ treatment lizards were maintained all day in outdoor conditions (Ta = 19º C, Ts = 26.2º C) where they could normally bask and attain optimal body temperatures. In the ’no thermoregulation’ treatment, lizards were maintained within their Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 230 terraria inside a cold room (Ta = 17.1º C, Ts = 17.3º C) during four hours, from 8:00 to 12:00 GTM every day. After this time, terraria were replaced to outdoor conditions. All lizards were fed two mealworms at 12:30 GTM each day. This procedure was repeated during ten days. Immediately after this experiment finished, we measured refuge use of lizards using the same procedure than in the previous experiments. After the experiment lizards were released at the point of capture. Statistical analyses were conducted as in the previous experiment. The experiments were performed under license from the Consejería del Medio Ambiente de la Comunidad de Madrid (Spain). Results Escape behaviour The distance from the initial location of lizards to the nearest refuge (ANCOVA, model: R2 = 0.14, F3,121 = 6.84, P < 0.0001) was shorter in females (F1,121 = 10.71, P = 0.001) and tended to be shorter in deteriorated areas (F1,121 = 3.66, P = 0.06). The interaction was not significant (F1,121 = 0.41, P = 0.52; Fig. 1a). When air temperature was lower, lizards tended to have greater approach distances, although the relationship did not reach significance (ANCOVA, model: R2 = 0.05, F4,125 = 1.56, P = 0.19, temperature effect: F1,125 = 3.75, P = 0.06), but there were no significant differences between sexes (F1,125 = 2.30, P = 0.13), nor between natural and deteriorated areas (F1,125 = 0.63, P = 0.43), and the interaction was not significant (F1,125 = 0.63, P = 0.43; Table 1). Lizards had longer flight distances when the nearest refuge was farther (ANCOVA, model: R2 = 0.20, F4,115 = 7.15, P < 0.0001, distance to refuge effect: F1,115 = 21.71, P < 0.0001), but there were no significant differences between sexes (F1,115 = 1.01, P = 0.32), nor between natural and deteriorated areas (F1,115 = 2.25, P = 0.14), and the interaction was not significant (F1,115 = 2.01, P = 0.16; Table 1). The escape strategy did not differ between sexes (GLZM, Wald’s χ2 = 0.15, df = 1, P = 0.70) nor between natural and deteriorated areas (Wald’s χ2 = 1.55, df = 1, P = 0.21), and the interaction was not significant (Wald’s χ2 = 0.51, df = 1, P = 0.47). Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 231 Lizards appeared sooner from the refuge when Ts inside the refuge was low (ANCOVA, R2 = 0.23, model: F4,52 = 3.96, P = 0.007; temperature: F1,52 = 12.16, P = 0.001) and sooner in areas with deteriorated vegetation (F1,52 = 4.47, P = 0.039). There were not differences between sexes (F1,52 = 0.77, P = 0.38), and the interaction was not significant (F1,52 = 0.005, P = 0.94; Table 1). Emergence time from the refuge (ANCOVA, R2 = 0.28, F4,53 = 5.13, P = 0.001) was significantly longer when refuge temperature was higher (F1,53 = 10.85, P = 0.002), was longer in females than in males (F1,53 = 4.09, P = 0.048), and longer in natural than in deteriorated areas (F1,53 = 8.18, P = 0.006). The interaction was not significant (F1,53 = 0.13, P = 0.72; Fig. 1b). Males were significantly heavier than females (ANCOVA, R2 = 0.71, model: F4,70 = 42.74, P < 0.0001; sex: F1,70 = 20.70, P < 0.0001), after removing the effect of covariation with SVL (F1,70 = 164.22, P < 0.0001), but there were no significant differences between natural and deteriorated areas (F1,70 = 0.09, P = 0.76), and the interaction was not significant (F1,70 = 0.19, P = 0.66; Table 1). Fig. 1. Mean (+ SE) a) distance to the nearest refuge and b) emergence time of male (black bars) and female (open bars) lizards, Lacerta monticola inhabiting natural areas or areas with deteriorated habitats. The level of ecotourism, assessed by the distance to the nearest patch, was not related to any variable describing escape behaviour and refuge use, nor to the body condition of lizards, and, thus, it was not included in any of the final models. Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 232 Table 1. Mean (+ SE) of variables describing escape behaviour and body condition of male and female Lacerta monticola lizards inhabiting natural areas or areas deteriorated by ski slopes. Natural Deteriorated Males Females Males Females Approach distance (cm) 182 + 9 168 + 12 194 + 12 170 + 14 Flight distance (cm) 25 + 6 26 + 9 20 + 8 10 + 4 Appearance time from refuge (s) 38 + 15 65 + 23 25 + 13 17 + 4 Body mass (g) 7.0 + 0.2 6.5 + 0.5 6.9 + 0.2 6.7 + 0.3 Costs of refuge use: diminution of digestion efficiency or loss of foraging opportunities Diminution of digestion efficiency All lizards increased their body mass in the course of the experiment (repeated measures ANOVA, within factor: F1,26 = 51.88, P < 0.0001), but there were no significant differences between treatments (F2,26 = 1.21, P = 0.32), and the interaction was not significant (F2,26 = 0.80, P = 0.46; Fig. 2a). There were no significant differences in blood parasite load between the beginning and the end of the experiment (repeated measures ANOVA, F1,26 = 0.63, P = 0.44) nor between treatments (F2,26 = 0.19, P = 0.83), and the interaction was not significant (F2,26 = 0.23, P = 0.80; Table 2). There were not differences in CMI response in the course of the experiment (repeated measures ANOVA, F1,26 = 1.55, P = 0.22) nor between treatments (F2,26 = 0.48, P = 0.63), and the interaction was not significant (F2,26 = 0.78, P = 0.47; Table 2). Lizards increased time until emergence from the refuge after the second attack compared to the first attack (repeated measures ANOVA, F1,20 Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 233 = 3.40, P = 0.08), but there were no significant differences between treatments (F2,20 = 1.65, P = 0.22), and the interaction was not significant (F2,20 = 0.30, P = 0.74; Fig. 2b). Table 2. Mean (+ SE) intensity of blood parasites’ infection (nº infected cells/2000 erythrocytes) and T- cell mediated immune response-CMI (mm) of male lizards, Lacerta monticola before and after an experiment testing for the effects of diminution of digestion efficiency inside refuges (see methods). Control No thermoregulation No digestion efficiency Initial Final Initial Final Initial Final Blood parasites 2.24 + 0.70 2.24 + 0.66 1.52 + 0.73 1.62 + 0.85 1.64 + 0.85 1.89 + 0.87 CMI 0.36 + 0.09 0.54 + 0.11 0.44 + 0.07 0.46 + 0.06 0.37 + 0.07 0.39 + 0.09 Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 234 Fig. 2. Mean (+ SE) a) body mass (g) of male lizards, Lacerta monticola before and after an experiment testing for the effects of diminution of digestion efficiency inside refuges (see methods). b) Mean (+ SE) emergence time (s) of lizards when suffered two simulated repeated attacks after the experimental treatments. Loss of foraging opportunities There were no significant overall differences in body mass of lizards between treatments (repeated measures ANOVA, F2,26 = 0.004, P = 0.996) nor in relation to the time (F1,26 = 0.42, P = 0.52), but the interaction was significant (F2,26 = 21.55, P < 0.0001; Fig. 3a). Thus, lizards in the control treatment increased significantly their body mass in the course of the experiment (Tukey’s test, P = 0.04), whereas lizards in the ’loss of foraging opportunities’ treatment significantly decreased it (P = 0.0002), and lizards in the ‘only refuge use’ treatment maintained their body mass in the course of the experiment (P = 0.76). Lizards suffered an increase in blood parasite load in the course of the experiment (repeated measures ANOVA, F1,27 = 5.01, P = 0.03), differences approached significance between treatments (F2,27 = 2.86, P =0.08), and the interaction was not significant (F2,27 = 0.55, P =0.58; Table 3). Thus, control lizards seemed to show lower intensity of infection than lizards in the ‘loss of foraging opportunities’ treatment (Tukey’s test, P = 0.07). There were not differences between the two experimental groups (P = 0.25) nor between the control and the ‘only refuge use’ treatments (P = 0.77). Lizards had a greater CMI response at the end of the experiment (repeated measures ANOVA, F1,27 = 5.01, P = Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 235 0.03), and differences between treatments approached significance (F2,27 = 2.86, P = 0.08). The interaction was not significant (F2,27 = 0.55, P = 0.58; Table 3). Thus, lizards in the ’loss of foraging opportunities’ treatment seemed to have greater CMI responses than control lizards (Tukey’s test, P = 0.07), but there were not differences between both experimental treatments (P = 0.25) or between the ‘only refuge use’ and the control treatments (P = 0.77). There were no significant differences in emergence time from the refuge between treatments (repeated measures ANOVA, F2,13 = 0.91, P = 0.43) nor in relation to the time (F1,13 = 1.75, P = 0.21), but the interaction was significant (F2,13 = 4.64, P = 0.03; Fig. 3b). Thus, lizards previously submitted to the ’loss of foraging opportunities’ tended to decrease their emergence time from the refuge after a second attack (Tukey’s test, P = 0.08), whereas there were no significant differences between attacks in the control (P = 0.81) or in the ’only refuge use’ treatments (P = 0.99). Fig. 3. a) Mean (+ SE) a) body mass (g) of male lizards, Lacerta monticola, before and after an experiment testing for the effects of loss of foraging opportunities inside refuges (see methods). b) Mean (+ SE) emergence time (s) of lizards when suffered two simulated repeated attacks after the experimental treatments. Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 236 Table 3. Mean (+ SE) intensity of blood parasites’ infection (nº infected cells/2000 erythrocyted) and T- cell mediated immune response-CMI (mm) of male lizards, Lacerta monticola before and after an experiment testing for the effects of loss of foraging opportunities inside refuges (see methods). Control Only refuge use Loss of foraging opportunities Initial Final Initial Final Initial Final Blood parasites 0.80 + 0.55 1.10 + 0.61 1.43 + 0.48 2.21 + 0.96 3.32 + 0.98 4.48 + 1.59 CMI 0.36 + 0.06 0.55 + 0.09 0.61 + 0.10 0.76 + 0.06 0.41 + 0.05 0.63 + 0.10 Costs of time spent at low temperatures for pregnant females There were significant differences in body mass of female lizards between the beginning and the end of the experiment (repeated measures ANOVA, F1,18 = 198.90, P < 0.0001), there were not overall differences between treatments (F1,18 = 0.57, P = 0.46), but the interaction was significant (F1,18 = 7.53, P = 0.01; Fig. 4a). All females increased their body mass in the course of experiment, but control females tended to increase their body mass more than experimental females. Blood parasite load was significantly higher at the end of the experiment in both treatments (repeated measures ANOVA, F1,18 = 13.57, P = 0.002), and experimental females tended to be more parasitized than control females (F1,18 = 3.85, P = 0.07). The interaction was not significant (F1,18 = 0.90, P = 0.36, Table 4). Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 237 Table 4. Mean (+ SE) intensity of blood parasites’ infection (nº infected cells/2000 erythrocytes) and T- cell mediated immune response-CMI (mm) of pregnant female lizards, Lacerta monticola before and after an experiment testing for the effects of costs of thermoregulation inside refuges (see methods). Control No thermoregulation Initial Final Initial Final Blood parasites 0.38 + 0.10 1.86 + 0.57 1.37 + 0.78 3.89 + 0.92 CMI 0.37 + 0.04 0.33 + 0.07 0.42 + 0.05 0.19 + 0.05 After the experiment, females showed significantly lower CMI responses than before the experiment (F1,18 = 6.49, P = 0.02), there were no significant differences between treatments (repeated measures ANOVA, F1,18 = 0.61, P = 0.45), but the interaction approached significance (F1,18 = 3.18, P = 0.09, Table 4). Control females did not significantly changed their CMI response during the experiment (Tukey’s test, P = 0.95), whereas experimental females significantly decreased their CMI (P = 0.03). In relation to refuge use, experimental females tended to emerged sooner from the refuge than control females (repeated measures ANOVA, F1,17 = 4.09, P = 0.06). There were not differences between attacks (F1,17 = 0.56, P = 0.47) and the interaction was not significant (F1,17 = 0.00, P = 0.99; Fig. 4b). Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 238 Fig. 4. a) Mean (+ SE) body mass (g) of pregnant female lizards, Lacerta monticola, before and after an experiment testing for the effects of costs of thermoregulation inside refuges (see methods). b) Mean (+ SE) emergence time (s) of lizards when suffered two simulated repeated attacks after the experimental treatments. Discussion Our results suggest that habitat deterioration lead to an increase in perceived risk of predation by lizards. However, lizards seemed to compensate the increased risk in deteriorated habitats by using flexible antipredatory strategies, thus decreasing costs of these strategies and maintaining a good body condition. On contrast, the level of ecotourism seemed not to affect predation risk because lizards did not modify their antipredatory behaviour in relation to the distance to the nearest path, and neither their body condition was affected by closeness to a path. Despite that lizards responded to people as to predators (e.g. Martín and López 1999b, 2000a), habitat deterioration seems to be much more important than ecotourism pressure for this species, at least during the mating period. Lizards might not have escaped from humans as often as we initially expected, because they might view humans on paths as less threatening than humans off paths, as previous studies with other animals have suggested that paths might make the route or behavior of humans more predictable and thus less of a risk (Mainini et al. 1993; Miller et al. 2001). Nevertheless, an effect of ecotourism might have been detected at the end of the period of activity, after lizards had needed to perform frequent antipredatory behaviours across all the season. As we performed the experiment only during the mating season, we can not refuse this Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 239 alternative hypothesis. Therefore, further research is needed to accurately examine the effects of ecotourism on this species. Deteriorated habitats can be characterized by a low cover of vegetation and rocks, and low availability of refuges (Amo, López and Martín, manuscript in preparation). As many prey assess their susceptibility to predators accordingly to the distance to refuges (Martín and López 2000), deteriorated areas may imply a higher risk of predation for lizards. However, our results suggest that lizards were able to assess this increase in risk, and minimized their exposure to predation by selecting microhabitats close to refuges in deteriorated habitats. This first antipredatory strategy seemed very important because flight distance depended on distance to the nearest refuge, as previously observed in this (Martín and López 2000) and other species (Bulova 1994; Bonenfant and Kramer 1996; Cooper 1997; Dill 1990). Thus, by being close to refuges, lizards not only minimized their susceptibility to be captured while fleeing in unsafe areas with low cover, but also decreased physiological costs associated to fleeing (Amo, López and Martín, manuscript in preparation). Moreover, females were closer to refuges than males, as previously observed in other species (Bauwens and Thoen 1981; Cooper 2003), probably because their usual lower sprint speed compared to males, especially during pregnancy, makes them more vulnerable to be captured (Shine 1980; Cooper et al. 1990; Magnhagen 1991; Downes and Shine 1999; Le Galliard et al. 2003). However, and regardless of differences in predation risk between areas with different level of deterioration, approach distances and escape strategies did not differ between areas. Thus, lizards seemed to react to the current presence of the predator rather than to the average level of risk in an area. This may also help to minimize the costs associated to antipredatory strategies. Our results also showed that lizards tended to have greater approach distances when air temperature was lower, as has being observed in previous studies (Rand 1964; Smith 1997; Cooper 2000; Martín and López 2000, but see also Martín and López 2003). Refuge use was influenced by temperature and by reproductive requirements. Therefore, accordingly to Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 240 theoretical models of refuge use and previous results with this (Martín and López 1999b, 2000) and other species (Amo et al. 2003), lizards appeared and emerged from the refuge when thermal costs of refuge use were higher than benefits of remaining inside the refuge (Martín and López 1999b). Also, males emerged sooner than females from the refuge to reduce the cost of lost opportunity to search for mates during the mating season (Martín et al. 2003b), as their reproductive success is related to the number of courted females (Aragón et al. 2001). Therefore, when reproductive and antipredatory demands conflicted, males responded by accepting some degree of risk, leaving the refuge earlier to maximize fitness (Cooper 1999; Martín et al. 2003b). One interesting result is that both appearance and emergence times from the refuge differed in relation to the level of habitat deterioration. Lizards appeared and emerged sooner in deteriorated areas, in spite that predation risk may be higher here and, thus, we expected that the benefits of using refuges for longer should be higher here than in natural areas. The current risk of predation posed by the experimenter was similar for lizards in both areas. Therefore, the decrease in time spent in the refuge in deteriorated areas may be a strategy to minimize the costs associated to refuge use in areas where lizards may need to use refuges more frequently than in natural areas. This may be very important because results of the laboratory study showed that the loss of foraging opportunities and a decrease of food ingestion associated to refuge use caused a loss of body condition. However, there were not differences in parasite load and CMI response. Nevertheless, differences in initial parasite load and CMI response between lizards of the three treatments before the experiment might be masking the effect of the treatments on these measures taken at the end of the experiment. On contrast, the lack of opportunities for thermoregulation and the potential diminution of digestion efficiency did not affect body mass, parasite load or immune responses of male lizards. Although the diminution of digestion efficiency was suggested as a potential cost of refuge use (Martín and López 1999b), our experiment did not support this assumption. Nevertheless, actual temperatures inside refuges in the Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 241 field may be much lower than in our experimental set-up, and, thus, these lower temperatures might affect physiological functions of lizards, which will explain why lizards adjusted in the field time hidden in refuges to temperature conditions (Martín and López 1999b). Nevertheless, the lack of opportunities for thermoregulation seems to be an important cost of refuge use for pregnant females. Experimental females that were maintained for longer under low temperatures, simulating the conditions inside cold refuges, did not increase their body mass as much as control females that could normally bask and attain optimal body temperatures. Furthermore, experimental females showed a lower CMI response than control ones. The ability to mount a CMI response to a mitogenic stimulus may have important fitness consequences (Gonzalez et al. 1999), because it constitutes a generalized short-term response to grafts, allergens and wounds. As CMI response may be implicated in defence against parasites, we could expect that females with low CMI response had also higher blood parasite loads. This is very important because haemogregarines are known to have adverse effects in this species (Amo et al. 2004). However, our results did not show differences between treatments in intensity of haemogregarines’ infection between the initial and final measure. This could be due to experimental females tending to be initially more parasitized than control females. Furthermore, the loss of body condition of females could also be caused by the higher parasite load, thus, we can not conclude that low temperatures per se caused a direct effect on body mass. Many physiological processes in lizards depend on optimal body temperature (Huey 1982; Stevenson et al. 1985), so probably low temperatures implied a decrease in CMI response, with the subsequent increase of deleterious effects of parasites on body condition. Our data did not allow us to test this hypothesis, and hence, further studies are needed to reveal more physiological costs of refuge use that have not being considered until now. The poor body condition of females that spent longer times at low temperatures may have deep effects on their fitness. Previous results showed that female lizards of other species in Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 242 poor body condition produced offspring of small size (Shine and Downes 1999; but see also Gregory and Skebo 1998), and body size of neonate lizards can affect their probability of survival (e.g. Ferguson and Fox 1984; Sinervo et al. 1992). Furthermore, females parasitized by blood parasites also showed reduced fat stores and produced smaller clutches (Schall 1983). In relation to refuge use our results seem to confirm the initial hypothesis; lizards were able to modulate refuge use accordingly to their body condition, thus coping with predation risk while decreasing physiological costs. Lizards with worse body condition might not afford the costs of refuge use, and thus, had to decrease time spent in refuges, whereas lizards with a good body condition remained hidden for longer. These results agree with theoretical models (Sih 1992, 1997; Dill and Fraser 1997; Martín and López 1999b) and previous studies with barnacles (Dill and Gillett 1991), fishes (Krause et al. 1998, but see also Dowling and Godin 2002), or birds (Koivula et al. 1995). In the case of males, differences in emergence times were more notorious in the second attack, whereas in pregnant females such differences were notorious since the first attack. Costs of refuge use increase when increases time spent in the refuge (Martín and López 1999b), thus, our results suggested that the loss of body condition had a greater importance for pregnant females than for males. Furthermore, during pregnancy, females may need to maintain constant body temperatures to maximize developmental rates of embryos (Mathies and Andrews 1997; Shine 2004), which are temperature dependent (Muth 1980). In this way females may shorten the incubation period and therefore decrease costs of reproduction (Shine 1980, 1983; Seigel and Fitch 1984; Shine and Downes 1999). Shorter incubation periods may increase offspring fitness in areas with limited environmental temperatures because juveniles may have more time for growing before hibernation (Mathies and Andrews 1997, but see also Shine and Olsson 2003). To summarize, refuge use is costly in terms of body condition due to loss of foraging opportunities and the subsequent reduced food intake, and due to time spent at low temperatures, at least for pregnant females. Therefore, lizards living in areas with high risk of Capítulo 4 Efectos del ecoturismo: escape, uso de refugios y condición corporal 243 predation may suffer the physiological costs of increased refuge use. However, lizards inhabiting deteriorated areas, where predation risk was high, remained closer to refuges, and decreased the flight distance during an escape episode, but they also decreased time spent hidden in refuges, probably to maintain similar body conditions than lizards inhabiting natural less risky areas. Therefore, our results suggest that lizards were able to compensate this increased predation risk with flexible antipredatory strategies, decreasing costs of antipredatory behaviours that may affect their body condition. This result seems also to be confirmed by laboratory experiments that showed that lizards modified their refuge use in relation to their body condition, with lizards with worse body condition having shorter hidden times. On contrast, ecotourism neither influence the antipredatory behaviour nor the body condition of lizards, at least during the mating period. Acknowledgements We thank "El Ventorrillo" MNCN Field Station for use of their facilities. Financial support was provided to L. Amo by an “El Ventorrillo” C.S.I.C. grant, to P. López by a the MCYT project BOS 2002-00598, and to J. Martín by the MCYT project BOS 2002- 00547. _________________________________ References Amo L, López P, Martín J (2003) Risk level and thermal costs affect the choice of escape strategy and refuge use in the wall lizard, Podarcis muralis. Copeia 2003: 899–905 Amo L, López P, Martín J (2004) Prevalence and intensity of Haemogregarinid blood parasites in a population of the Iberian Rock Lizard, Lacerta monticola. Parasitol Res 94: 290-293 Aragón P, López P, Martín J (2001) Seasonal changes in activity and spatial and social relationships of the Iberian rock lizard, Lacerta monticola. Can J Zool 79: 1965-1971 Bauwens D, Thoen C (1981) Escape tactics and vulnerability to predation associated with reproduction in the lizard Lacerta vivipara. 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Sin embargo, el uso de refugios es costoso, por lo que los animales deben ajustar la magnitud y las características de sus respuestas antidepredadorias de acuerdo a los niveles de riesgo de depredación que perciben, para lograr hacer frente al riesgo sin incurrir en costes excesivos del uso de refugios. Entre estos costes se encuentra un incremento en el riesgo debido a que los refugios pueden esconder depredadores que cazan al acecho, incrementando así el riesgo de depredación para las presas. Por todo ello, las presas deben valorar adecuadamente el nivel de riesgo ejercido por el depredador del exterior así como el riesgo potencial de encontrar un depredador en el interior del refugio. Para valorar el riesgo de depredación por culebras en el interior de los refugios, muchas especies de lagartijas han desarrollado la habilidad de detectar las señales químicas de éstas. Sin embargo, las señales químicas pueden permanecer aún cuando el depredador ya ha abandonado la zona, lo que puede suponer una sobreestimación del riesgo de depredación. Para evitar esto, pueden utilizar otro tipo de señales, como las visuales y además valorar el tiempo que las señales llevan depositadas y la presencia real de la culebra. El objetivo de este estudio es examinar la capacidad de las lagartijas para hacer frente a dos tipos de depredadores que requieren respuestas conflictivas, actuando simultáneamente. Capítulo 5 Efectos del ecoturismo: múltiples depredadores 253 Risk level and thermal costs affect the choice of escape strategy and refuge use in the wall lizard, Podarcis muralis RESUMEN Los animales deben ajustar la magnitud y las características de sus respuestas antidepredatorias de acuerdo a los niveles de riesgo de depredación que perciben, para lograr hacer frente al riesgo sin incurrir en costes excesivos. En este estudio, analizamos en condiciones naturales los factores que determinan la elección de la estrategia de escape y los patrones de uso de refugios en las lagartijas roqueras en dos niveles de riesgo de depredación simulado y en diversas condiciones ambientales que pueden afectar a la percepción de las lagartijas del riesgo de depredación y los costes del uso de refugios. Los resultados mostraron que las lagartijas ajustaron su respuesta antidepredatoria de acuerdo a varios factores. El nivel de riesgo de depredación provocado por el depredador afectó a la respuesta inicial de escape pero no las consiguientes estrategias de escape empleadas por las lagartijas. Las estrategias de escape dependieron de la vulnerabilidad de las lagartijas a ser capturadas (como la altura en el muro donde se realizó el experimento, o la temperatura del aire) y de los costes del uso de refugios (temperatura del refugio y el riesgo potencial de depredación por serpientes). Tanto el riesgo inicial provocado por el depredador como los costes de termorregulación asociados al uso de refugios afectaron a los tiempos de emergencia del refugio. Por lo tanto, el comportamiento de las lagartijas roqueras estuvo influido no sólo por la probabilidad de mortalidad en el futuro inmediato, como el riesgo de depredación inicial y la percepción de su susceptibilidad a ser capturadas, sino también estuvo influido por las consecuencias a largo plazo para su eficacia biológica, como los costes fisiológicos debidos al uso de refugios y por el eventual riesgo de mortalidad asociado al uso de refugios inseguros. Capítulo 5 Efectos del ecoturismo: múltiples depredadores 255 Risk level and thermal costs affect the choice of escape strategy and refuge use in the wall lizard, Podarcis muralis Abstract Animals should tend to adjust the magnitude and characteristics of their escape responses according to the perceived levels of predation risk to cope with risk without incurring excessive costs. We analyze in the field the factors that determine the choice of escape behavior and patterns of refuge use of wall lizards under two simulated levels of predation risk and under variable environmental conditions, which may affect risk perception and costs of refuge use. The results show that wall lizards adjusted their antipredatory response according to several factors. The threat of predation posed by the predator affected the initial type of response of lizards but not the subsequent escape strategies employed. The escape strategy depended on the vulnerability to be captured (i.e., height on the wall and air temperature) and costs of refuge use (temperature and potential predation by ambush snakes). The initial risk of predation and thermal costs of refuge use affected emergence times from the refuge. The antipredator decisions of wall lizards, therefore, were influenced not only by the probability of mortality in the immediate future, such as the initial threat of predation and perceived susceptibility but also by consequences for long-term expected fitness, such as physiological costs of refuge use, and by the eventual risk of mortality associated with the use of unsafe refuges. Introduction Although predation is a major selective force, prey should optimize their antipredatory response by balancing antipredatory demands with other requirements (Lima and Dill 1990; Lima 1998). For example, theoretical models and empirical evidence suggest that prey should not flee immediately upon detecting an approaching predator but when the predator approaches closer than the point at which predation risk is equal to escape costs (Ydenberg and Dill 1986). Approaching predators do not always pose an immediate threat, and Capítulo 5 Efectos del ecoturismo: múltiples depredadores 256 environmental variables such as microhabitat structure or temperature may also affect predation risk (Martín and López 1998, 2000a). Thus, animals should tend to adjust the magnitude and characteristics of their escape responses according to the perceived levels of predation risk (Martín and López 1995, 2000a; Cooper 1997). Prey often increase refuge use to cope with predator attacks (Sih et al. 1992; Cooper 1998). However, refuge use may be costly in terms of the loss of time available for other activities (Sih et al. 1990; Dill and Fraser 1997; Martín et al. 2003a,b), or because of physiological costs (Wolf and Kramer 1987; Martín and López 1999a,b). Furthermore, refuge use may entail an increase in the risk of predation by ambush predators that wait for their prey inside refuges (Downes and Shine 1998). For this reason, animals should adjust their refuge use and the decision of when to come out from a refuge after a predator’s unsuccessful attack by balancing antipredator demands with other requirements (Sih 1997; Dill and Fraser 1997; Martín et al. 2003a,b). Flexibility in the antipredatory responses and refuge use might help animals to cope with increased predation risk without incurring excessive costs (Sih 1992, 1997; Martín and López 1999b). Optimal emergence from refuge theory and optimal escape theory have both been very successful in predicting aspects of antipredatory behavior (Ydenberg and Dill 1986; Sih 1997; Martín and López 1999b). Tests of predictions of these theories are needed to ascertain their applicability to a wider range of prey species and refuge types, to more microhabitat and environmental conditions, and to other risk and cost factors, such as the presence of multiple types of predators. Wall lizards (Podarcis muralis) responded to simulated predatory attacks by hiding inside rock crevices, and when average predation pressure increased, lizards increased time spent in refuges (Martín and López 1999a). However, previous experiments have shown that an increase in the time spent in refuges at unfavorable temperatures led to a diminution of the efficiency of physiological functions, which resulted in loss of mass (Martín and López 1999a). Additionally, prey defenses against one predator may increase the risk of being killed by other types of Capítulo 5 Efectos del ecoturismo: múltiples depredadores 257 predators (Sih et al. 1998). By increasing refuge use, wall lizards may expose themselves to increased predation risk by smooth snakes (Coronella austriaca), which inhabit the same rock crevices ambushing for lizards (Galán 1998). Under these circumstances, we hypothesized that lizards might have different alternative escape strategies to avoid simultaneously the risk of being captured by predators that employ different foraging strategies (active vs. ambush). When choosing an antipredatory strategy, lizards might consider the thermal costs of use of a refuge, the probability of being preyed upon by ambush predators that hunt in refuge, and the probability of eluding the predator that hunts in the open. The latter should be related to the initial perceived threat of predation. In the field, we tested the factors that determine the choice of escape behavior and patterns of refuge use of wall lizards under two simulated levels of predation risk and under variable environmental conditions that may affect risk perception and costs of refuge use. We hypothesized that if lizards cannot easily elude the predatory attack without hiding, or when costs of refuge use are low, they should preferentially hide in refuges. However, lizards should employ alternative escape strategies to hiding when perceived risk is low and/or costs of refuge use are high. Material and Methods Species and study site We performed the experiment in the Guadarrama Mountains, in a pine forest at Cercedilla (Madrid Prov., Central Spain) at an elevation of 1500 m. The dominant vegetation consists of Pinus sylvestris forest, with shrubs such as Juniperus communis and Cytisus scoparius. We conducted the experiment on an old wall (120 m long x 5 m high) built a long time ago with granite rocks to hold a sandbank. The wall lizard (P. muralis) is a small lacertid lizard (60–76 mm adult snout–vent length, SVL) widespread in central Europe, although in the Iberian Peninsula it is restricted to mountainous areas of the northern half, where it occupies soil dwellings, talus and walls in shaded zones in forests (Martin- Vallejo et al. 1995). At our study site, lizards were found basking or walking on the wall and using the numerous Capítulo 5 Efectos del ecoturismo: múltiples depredadores 258 crevices between rocks as refuges. We often observed in the area active- foraging predators known to eat this lizard (Martín and López 1990), such as jays (Garrulus glandarius), magpies (Pica pica), great gray shrikes (Lanius excubitor), buzzards (Buteo buteo), short-toed eagles (Circaetus gallicus), kestrels (Falco tinnunculus), or little owls (Athene noctua), as well as abundant feral cats that frequently chased and killed these lizards in this and other populations (Boag 1973; Brown et al. 1995). In addition, smooth snakes (C. austriaca) were abundant on this wall. This snake is a saurophagous specialist that ambushes lizards hidden in rock crevices (Galán 1998). Experimental procedure We conducted the experiment in July 2001. We searched for lizards between 1000 and 1200 h by walking a track close to the wall until a lizard (8–10 m distant) was sighted using binoculars. We noted the lizard’s initial activity (basking or moving on the wall), sex, and age (juvenile vs. adult) without disturbing it. Lizards and other animals may react differentially to the approach of a predator as a function of the threat of the attack (Martín and López 1999b). Thus, we approached individual lizards in one of two ways: by walking slowly (approximately 45 m/min) near but tangentially to the lizard with a minimum bypass distance of 1.5 m, looking straight ahead and without paying attention to the lizard (low- predation risk level), or by simulating a predatory attack by walking directly and fast (approximately 140 m/min) toward the lizard but stopping at 50 cm from the lizard (high-predation risk). The order of presentation of the different treatments (low vs. high risk) was randomized. We did not sample the same wall section twice to avoid pseudoreplication. With this procedure, we simulated a predator passing by (low risk) or one actually making an attack from the ground (high risk), such as a feral cat or a bird attacking from low bushes growing in front of the wall. To avoid confounding effects that may affect risk perception of lizards (Burger and Gochfeld 1993; Cooper 1997), the same person wearing the same clothing performed all approaches in a similar way and recorded the lizard’s behavior. We noted whether lizards responded to the attack because in some Capítulo 5 Efectos del ecoturismo: múltiples depredadores 259 occasions lizards remained unresponsive and stationary, presumably cryptic, after the simulated attack. However, on most occasions, the usual escape response of the lizard was (1) to rapidly flee with either a short run to the nearest refuge and hiding entirely in it, or (2) to flee further and for a long time on the wall while passing by potential refuges without hiding. We noted whether the lizards remained stationary or fled and, if the latter, the type of escape strategy used (hiding vs. fleeing without hiding). If the lizard hid in a refuge, we started a stopwatch and retreated to a distance of 5–7 m to observe from a hidden position with binoculars. We measured the time that the lizard spent in the refuge until the head appeared from the refuge (appearance time) and the time from appearance until the lizard emerged entirely from the refuge and resumed its normal activity (waiting time). At the end of the trial, we measured the distance from the initial location of the lizard before the attack to the nearest available refuge (i.e., rock crevice between rock blocks where a lizard could hide), the distance to the refuge actually used or to the point where the lizard first stopped after fleeing if the lizard did not hide, and the height on the wall at its initial and final locations. Antipredatory behavior of lizards may depend on the thermal state of individuals (i.e., their body temperature) and/or on the thermal conditions of the refuge (thermal costs of refuge use, i.e., cooling rate; Martín and López 1999a, 2000b; Cooper 2000). Thus, we also measured the air temperature at the point where the lizard was before the attack and the substrate temperature inside the nearest refuge with a digital thermometer to the nearest 0.1 ºC. Data analysis Given the high lizard density, and because we avoided sampling the same wall section twice, the probability of repeated measurements on the same individual was low. Therefore, we treated all measurements as independent. To assess differences in type of responses of lizards to the attack (fleeing vs. remaining stationary) in relation to the level of predation risk and the initial activity of lizards, we used Chi-squared tests. We further used logistic regression to test for the effects of the initial activity, risk level, several environmental variables (distance to the Capítulo 5 Efectos del ecoturismo: múltiples depredadores 260 nearest refuge, initial height on the wall, air, and refuge temperatures), sex, and age (juvenile vs. adults) on the escape strategy (hiding vs. running without hiding) used by lizards that fled. Logistic regression is suitable for dichotomous dependent variables, and can handle categorical as well as continuous independent variables (Hosmer and Lemeshow 1989). Data analyses were performed using Windows-SPSS package (SPSS, Inc; Statistical Package for the Social Sciences, 1993, Mc-Graw- Hill). The analysis provides a likelihood ratio statistic as a goodness-of-fit estimator for the model and maximum likelihood estimators and standard errors of the independent parameters. To assess the significance of the independent variables, we calculated the difference in deviance for a model with and without the variable of interest. The resulting difference in deviance between the two models follows a Chi-square distribution with one degree of freedom (Hosmer and Lemeshow 1989). Relationships between refuge temperature and time spent in the refuge (appearance and waiting times) were estimated with Pearson’s correlations for lizards that suffered a direct or an indirect attack independently. Because emergence time in lizards may also depend on temperature (Martín and López 1999a,b; see Results), we used two-way analysis of covariance (ANCOVA), with refuge temperature as a covariant, to examine differences in emergence times as a function of initial activity and risk level (Sokal and Rohlf 1995). Data were log-transformed to ensure normality (tested with Kolmogorov- Smirnov and Lilliefors’ tests) when it was required. Tests of homogeneity of variances (Levene’s test) showed that in all cases variances were not significantly heterogeneous after transformation. Results Responsiveness to the attack The decision to flee vs. remain stationary and cryptic seemed to be independent of initial activity of lizards (Chi-squared test: χ2 = 0.48, P = 0.49). Thus, lizards that were basking preferentially fled when they suffered a simulated attack (83.8%, 57 of 68), although a few remained motionless (16.2%, 11 of 68) but also lizards that were moving on the wall preferentially Capítulo 5 Efectos del ecoturismo: múltiples depredadores 261 fled (78.4%, 29 of 37), although some of them remained motionless (21.6%, 8 of 37). However, the decision to flee depended on the initial threat of predation level (Chi-squared test: χ2 = 15.47, P = 0.0001). When suffering a direct attack, all lizards fled from the observer (100%, n = 42), whereas in a low-risk situation, although most lizards fled (69.8%, 44 of 63), a high proportion of lizards remained stationary (30.2%, 19 of 63). Some environmental variables seemed to affect the type of response. Thus, restricting the analyses to the low risk situation, lizards that remained stationary were initially at significantly higher height on the wall than lizards that fled (means + SE: 116 + 20 cm vs. 53 + 8 cm; one-way ANOVA: F1,61 = 6.81, P = 0.011). However, refuge temperature was significantly lower when lizards did not flee than when lizards fled, often to hide in a refuge (means + SE: 18.0 + 0.4 C vs. 20.2 + 0.3 C; F1,53 = 14.79, P = 0.0003). There were no significant differences between lizards that did not flee and lizards that fled either in distance to the nearest refuge (means + SE: 5 + 3 cm vs. 10 + 3 cm, F1,61 = 0.80, P = 0.37), or exterior air temperatures (means + SE: 19.7 + 0.8 C vs. 20.1 + 0.4 C; F1,53 = 0.33, P = 0.57). Therefore, the height on the wall, which probably affected risk perception, and potential thermal costs of refuge use influenced the type of antipredatory response of lizards under low-risk level attacks. Choice of escape strategy The logistic regression of escape strategy (hiding vs. running without hiding) showed that some variables significantly affected this decision (Chi- squared test: χ2 = 32.18, df = 9, P = 0.0002; Table 1). The model predicted correctly the strategy of 81.9% of lizards. Thus, most lizards that hid in the nearest refuge were initially basking, at lower height on the wall, and close to refuges. Also, when lizards hid, air temperature was lower and refuge temperature higher than when lizards fled without hiding. Neither, sex, age or risk level significantly influenced the escape strategy. Capítulo 5 Efectos del ecoturismo: múltiples depredadores 262 Table 1. Results from logistic regression analysis of escape strategy (hiding vs. running without hiding) on initial activity, risk level, several environmental variables, sex, and age in Podarcis muralis. The maximum likelihood partial regression estimates (b), the standard errors (SE) of the estimates and the level of significance (P) of each independent variable are shown. Source b SE P Initial activity -1.59 0.58 0.006 Initial height 0.01 0.01 0.011 Refuge temperature -0.83 0.32 0.011 Distance to nearest refuge 0.07 0.03 0.029 Air temperature 0.36 0.17 0.035 Sex -0.73 0.50 0.15 Age -0.32 0.57 0.57 Risk level -0.16 0.50 0.75 Constant 4.83 4.16 0.25 Refuge use Initial activity of lizards did not influence refuge use. Thus, appearance time from a refuge did not differ significantly between lizards that were initially basking or walking (means + SE: 47 + 11 s vs. 37 + 15 s; two-way ANCOVA, initial activity effect: F1,51 = 3.44, P = 0.07). However, there were significant differences in appearance time in relation to the risk level; lizards spent significantly less time inside the refuge until they appeared and looked outside in the low-risk situation than after being directly attacked (means + SE: 30 + 9 sec vs. 59 + 16 sec; risk level effect: F1.51 = 8.50, P = 0.005). The interaction was not significant (F1.51 = 0.35, P = 0.56). Time from appearance until resuming activity (i.e., waiting time) did not differ between lizards that were basking or walking before the attack (means + SE: 60 +12 sec vs. 87 + 23 sec; two-way ANCOVA, initial activity effect: F1.50 = 0.21, P = 0.65), did not differ between a low- and a high-risk situation (means + SE: 56 + 14 sec vs. Capítulo 5 Efectos del ecoturismo: múltiples depredadores 263 78 + 16 sec; risk level effect: F1.50 = 0.96, P = 0.33), and the interaction was not significant (F1.50 = 0.76, P = 0.39). Thus, once lizards appeared and looked outside, they emerged entirely from the refuge and resumed their activities in a period of time that was not dependent on the initial activity or threat of predation. The effect of thermal conditions of the refuge on time spent in the refuge seemed to be dependent on risk level, probably because of the risk sensitive adjustments of refuge use. Thus, in the low-risk situation, neither the appearance time (r = 0.12, F1,29 = 0.44, P = 0.51) nor waiting time were significantly correlated with refuge temperature (r = - 0.17, F1,29 = 0.77, P = 0.39). However, in the high-risk situation, where lizards spent longer times in the refuge, although there was not a significant relationship between appearance times of lizards and refuge temperature (r = 0.25, F1,24 = 1.64, P = 0.21), waiting time was significantly and positively correlated with refuge temperatures (r = 0.60, F1,24 = 11.68, P = 0.003). Discussion The results show that wall lizards were able to adjust their antipredatory response by assessing the level of predation risk and the costs of refuge use. Lizards employed two sequential responses of increasing intensity. First, they tried to prevent detection by remaining motionless, and if detection occurred (or if a lizard assessed that this was likely), then they attempted to escape by either fleeing to a refuge, or by fleeing without hiding. The initial type of response of lizards seemed to depend mainly on the threat of predation posed by the predator. Thus, when the predator directed a specific attack to a lizard, it always fled. However, when the predator approached a lizard indirectly, the lizard’s response depended on its assessment of the risk of being preyed upon. When lizards were at higher heights on the wall, risk of predation apparently was assessed as lower. This is because lizards could not be captured by humans (or a simulated terrestrial predator) if they were on the higher parts of the wall. Thus, they remained motionless, relying on crypsis. Furthermore, in a low-risk situation, thermal costs of refuge use also seemed Capítulo 5 Efectos del ecoturismo: múltiples depredadores 264 to influence the decision about whether to flee or to rely on crypsis. When refuge temperature was low, lizards relied more on crypsis than on fleeing to costly refuges. The body temperature of the lizard, inferred from the air temperature, and the distance to the nearest refuge did not seem to influence lizard response. Although many lizards use defensive behaviors depending on their body temperature (Hertz et al. 1982; Crowley and Pietruszka 1983; Cooper 2000), in other lizards, escape decisions seem not to be directly influenced by body temperature. For example, escape distances of Psammodromus algirus were not related to environmental temperature (Martín and López 1995). This could be because flight is vital to escape predation, and, therefore, it should be less constrained by temperature than other activities (Bennett 1980). Furthermore, even if cool lizards cannot exhibit optimal sprint speed, they still might be able to perform a short run safely to the nearest refuge. The initial threat of predation (i.e., high vs. low risk), however, did not influence the escape strategy of lizards once they began their escape. However, the choice of escape strategy may be influenced by their perception of their susceptibility to be captured while escaping. Lizards fled without hiding under perceived low risk, that is, at higher heights on the wall and when the air temperature was high and, thus, presumably body temperature (see Martín-Vallejo et al. 1995) and escape performance were also high. Thus, results suggest that temperature did not limit lizards’ first response to the attack of a predator (to rely on crypsis or to flee), at least in the margins of temperature that we observed during the experiment. However, temperature seemed to influence their decisions to hide in the nearest refuge or to flee away from the predator. Also, when thermal costs of refuge use were higher, lizards fled from the predator without hiding. This indicates that escape decisions of lizards were also based on consequences for long-term expected fitness (Martín and López 2000b). When a lizard retreats into a cold refuge, its body temperature may decrease below optimum after a few minutes (Martín and López 1999b, 2000b). Previous experiments have shown that after increasing the frequency of attacks toward wall lizards, Capítulo 5 Efectos del ecoturismo: múltiples depredadores 265 experimental lizards increased the time spent in refuges, but they had lower relative body mass than control individuals. This was probably because of the loss of time available for foraging and to a diminution of the efficiency of physiological functions at unfavorable temperatures (Martín and López 1999a), which in the long term may affect a lizard’s survival and reproductive success (see references in Martín and López 1999a). Another cost of refuge use that may affect escape decisions is the subsequent risk of predation by ambush snakes hidden in refuges. This might explain why choice of escape strategy was related to the initial activity of lizards and to the distance to the nearest refuge. A lizard basking close to a refuge may assess, thanks to its vomeronasal system, whether chemical cues from a snake are present (Downes and Shine 1998; unpubl. data). Thus, those lizards that presumably had more information on refuge safety (i.e., snake absence) hid inside the known refuge. However, when distance to the refuge increased, the possibility that a lizard could assess refuge safety decreased. Therefore, lizards that were moving on the wall, which presumably had a low knowledge of the safety of the nearest refuge, employed an alternative escape strategy by fleeing without hiding. With this flexible escape strategy, a lizard might avoid the risk of being preyed upon by either of two different types of predators (Sih et al. 1998). The initial risk of predation (high vs. low) affected refuge use of wall lizards. Lizards increased emergence latencies from refuges when the risk of predation was high perhaps because lizards needed to ensure that the risk of suffering a new attack had decreased below a security margin when emerging (Sih 1992; Cooper 1998; Martín and López 1999b). However, once they emerged, they resumed their activity in an interval of time that was independent of the initial threat of predation. This result suggests that lizards acquired information on the presence of a predator when they partly emerged from the refuge; thus, they resumed their activities if the predator was not detected (for a similar result in other lizards, see Martín and López 1999b). Thermal costs of refuge use also affected emergence times from the refuge; however, thermal costs were Capítulo 5 Efectos del ecoturismo: múltiples depredadores 266 more relevant in the high predation risk situations, as has been observed in previous studies (Martín and López 1999b). When lizards retreated into the refuge as a preventative strategy (i.e., when they were not directly attacked), thermal costs of refuge use did not affect emergence time because lizards emerged after only a short wait. In contrast, in the high-risk situation, lizards spent more time inside the refuge and they consequently suffered a decrease in their body temperature. Therefore, although appearance time was not influenced by refuge temperature, time from appearance until lizards resumed activity was. This suggests that lizards have to spend more time basking after being inside a cold refuge to regain an optimal body temperature before recovering normal activity. This was an unexpected result taking into account that appearance time from a refuge was related to refuge temperature in other species (Martín and López 1999b). Differences may be attributed to differences in the magnitude of costs of refuge use in different habitats. Thus, our results suggest that for P. muralis lizards, the risk of predation on the outside was a more relevant cost than the costs of remaining inside a cold refuge, although they were able to cope with the decrease in body temperature by increasing the time spent basking before emerging entirely from a refuge after a predatory attack. In conclusion, the antipredator decisions of wall lizards were influenced not only by the immediate probability of mortality, as indicated by the initial threat of predation posed by the predator and factors that putatively affected their perceived susceptibility but also by consequences for long-term fitness, such as physiological costs of refuge use, and by the subsequent risk of mortality by another type of predator associated with the use of unsafe refuges. Acknowledgements We thank two anonymous reviewers for helpful comments, and ‘‘El Ventorrillo’’ MNCN Field Station for use of their facilities. Financial support was provided by the MCYT project BOS 2002–00547. This experiment and the care and use of animals in the field were performed under license from the ‘‘Consejería de Medio Ambiente de la Comunidad de Madrid’’ (i.e., Madrid Environmental Agency) of Spain. 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Adv Stud Behav 16: 229–249 Capítulo 5 Efectos del ecoturismo: múltiples depredadores 269 Wall lizards combine chemical and visual cues of ambush snake predators to avoid overestimating risk inside refuges RESUMEN La hipótesis de la sensibilidad al riesgo asume que varias señales de un depredador deben contribuir de una manera aditiva a determinar el nivel de comportamiento sensible al riesgo. La habilidad de usar varias señales para valorar adecuadamente el nivel actual de riesgo de depredación debería ser especialmente importante para las presas expuestas a múltiples depredadores. Las lagartijas roqueras, Podarcis muralis, responden a los ataques de depredadores como pájaros o mamíferos escondiéndose en el interior de grietas de rocas, donde pueden encontrarse con otro depredador, la culebra lisa europea, Coronella austriaca. En este estudio investigamos en el laboratorio si las señales químicas son importantes para las lagartijas para detectar a las culebras. La mayor tasa de extrusiones linguales, así como la menor latencia de la primera extrusión lingual en respuesta al olor de las culebras indican que las lagartijas son capaces de detectar las señales químicas de estos depredadores. Además analizamos el uso que las lagartijas hacen de diversas señales de culebras para detectar su presencia dentro de refugios. Realizamos ataques simulados y sucesivos para comparar la propensión de las lagartijas a entrar en el refugio, así como el tiempo pasado dentro de refugios seguros, sin depredadores; refugios que contenían señales visuales o químicas de una culebra y refugios que contenían ambas señales. La respuesta antidepredatoria de las lagartijas fue mayor cuando estuvieron expuestas a la combinación de señales visuales y químicas que cuando estuvieron expuestas a una única señal. Estos resultados apoyan la teoría de la sensibilidad al riesgo. Esta habilidad puede mejorar la precisión en la valoración del nivel real del riesgo de depredación dentro de los refugios. Esto es especialmente importante para permitir a las lagartijas hacer frente a los riesgos impuestos por dos tipos distintos de depredadores que requieren respuestas conflictivas. Capítulo 5 Efectos del ecoturismo: múltiples depredadores 271 Wall lizards combine chemical and visual cues of ambush snake predators to avoid overestimating risk inside refuges Abstract The threat sensitivity hypothesis assumes that multiple cues from a predator should contribute in an additive way to determine the degree of risk-sensitive behaviour. The ability to use multiple cues in assessing the current level of predation risk should be especially important to prey exposed to multiple predators. Wall lizards, Podarcis muralis, respond to predatory attacks from birds or mammals by hiding inside rock crevices, where they may encounter another predator, the smooth snake, Coronella austriaca. We investigated in the laboratory whether chemical cues may be important to wall lizards for detection of snakes. The greater tongue-flick rate and shorter latency to first tongue-flick in response to predator scents indicated that lizards were able to detect the snakes’ chemical cues. We also investigated the use of different predatory cues by lizards when detecting the presence of snakes within refuges. We simulated successive predator attacks and compared the propensity of lizards to enter the refuge and time spent within it for predator-free refuges, refuges containing either only visual or chemical cues of a snake, or a combination of these. The antipredatory response of lizards was greater when they were exposed to both visual and chemical cues than when only one cue was presented, supporting the threat sensitivity hypothesis. This ability may improve the accuracy of assessments of the current level of predation risk inside the refuge. It could be especially important in allowing lizards to cope with threats posed by two types of predators requiring conflicting prey defences. Introduction The ability to detect the presence of predators is an important component of antipredatory behaviour (Van Damme et al. 1995). Prey should use multiple cues of predators to assess accurately the level of predation risk (McCarthy and Fisher 2000). The importance of chemical cues for predator recognition has being documented for a number of taxa (review in Kats and Dill 1998). Capítulo 5 Efectos del ecoturismo: múltiples depredadores 272 Chemosensory cues may reliably reveal the presence of predators (Kats and Dill 1998), even in the absence of other cues (Chivers and Smith 1998; Kats and Dill 1998; Chivers et al. 2001). However, visual cues, such as predator size and activity, may provide information more temporally specific to a predator’s current motivation and threat (Smith and Belk 2001). Only a few studies have compared the relative importance of the two types of stimuli. These suggest that prey can combine information from both chemical and visual cues to make a better assessment of the level of risk under conflicting situations (Vanderstighelen 1987; Hartman and Abrahams 2000; Mathis and Vincent 2000; Chivers et al. 2001). The threat sensitivity hypothesis proposes that animals should accurately assess the risk of predation, and respond in a graded manner in accordance with the threat posed by the predator (Helfman 1989). Animals that give antipredator responses to inappropriate stimuli expend time and energy that could be used in other activities, but animals that fail to respond to a dangerous stimulus have a lower probability of survival. Thus, the threat sensitivity hypothesis assumes that multiple predator cues should contribute in an additive way to determine the degree of risk-sensitive behaviour (Helfman 1989; Smith and Belk 2001). For example, detection of chemical cues of an ambush predator may cause prey to increase vigilance to detect the predator itself. Studies have provided support for the threat sensitivity hypothesis (Mathis and Vincent 2000; Chivers et al. 2001). For example, the western mosquitofish, Gambusia affinis, responded more when confronted with visual and chemical cues of predatory fish, Lepomis cyanellus, than when only one cue was presented (Smith and Belk 2001). Prey often respond to predator presence by increasing refuge use (Sih et al. 1992; Dill and Fraser 1997). However, some types of refuges may expose prey to other types of predators (Sih et al. 1998). Thus, conflicting prey defences can cause higher predation rates than expected. For example, the mortality of a mayfly, Ephemerella subvaria, prey in the presence of both fish, Cottus bairdi, and stoneflies, Agnetina capitata, was greater than expected, because stoneflies under rocks caused mayflies to come out of hiding Capítulo 5 Efectos del ecoturismo: múltiples depredadores 273 from under rocks, thus resulting in greater exposure to fish (Soluk 1993). However, flexibility in antipredator responses may help prey to avoid the riskenhancement effects. For example, when exposed to predators that occupy different microhabitats, male water striders, Aquarius remigis, reduced predation risk by decreasing not only general activity but also mating activities that attracted the attention of predators (Krupa and Sih 1998). Theoretical models of refuge use suggest that prey should adjust the time spent in a refuge according to predation risk and cost of staying in the refuge. The optimal emergence time is the time when the costs of staying (i.e. costs of refuge use) exceed the costs of leaving (i.e. predation risk in the exterior; Sih et al. 1992; Martín and López 1999a). When a refuge contains chemical cues from an ambush predator it is likely that the predator is there or close by and, if the prey remains in the refuge, the probability that that predator detects the prey increases over time. Hence, prey hidden in an unsafe refuge should emerge sooner than from a predator-free refuge. However, this response may expose prey to another type of predator. This example may be a case of predator facilitation caused by conflicting prey defences to avoid the different types of predators acting simultaneously (Sih et al. 1998). Furthermore, although there are significant advantages for prey able to detect predators via chemical cues, particularly when other cues are unavailable, chemical assessment might lead to excessively conservative estimates of risk, because chemical cues may persist long after the predator has departed, giving an inflated indication of current risk (Kats and Dill 1998). Thus, prey could overestimate the risk of predation inside a refuge, exposing itself to the risk of predation in the exterior. Minimizing the negative effects of the trade-off between emerging too soon and remaining inside the refuge would require prey to discriminate between different predator cues inside the refuge (indicating different levels of predation risk) and to adjust their behaviour accordingly. For individuals that can match their predator avoidance responses to the level of threat, the long- term payoffs should be greater than for individuals that are less flexible (Mathis and Vincent 2000). Capítulo 5 Efectos del ecoturismo: múltiples depredadores 274 Wall lizards, Podarcis muralis, responded to predatory attacks from birds or mammals when out in the open by hiding inside rock crevices (Martín and López 1999b). However, smooth snakes, Coronella austriaca, also use these crevices to ambush their lizard prey (Rugiero et al. 1995; Galán 1998; L. Amo, P. López and J. Martín, unpublished data). Thus, P. muralis offers an excellent model for the study of prey adaptations to minimize risk in a multiple predator environment. The first antipredatory mechanism of many lizards to avoid snakes is the ability to detect their chemical trails (Cooper 1990; Van Damme et al. 1995; Downes and Shine 1998; Van Damme and Quick 2001). The detection of the scent of a sedentary ‘ambush’ predator may initially provide a strong indication of probable current danger (Kats and Dill 1998). However, chemical cues may persist after the predator has left the area, so an avoidance response to such cues may be an overestimation of the risk of predation inside the refuge. If lizards emerge quickly from a refuge containing only chemical cues, they could be exposed to the predator that hunts in the open. Therefore, according to the threat sensitivity hypothesis, wall lizards should use other cues in addition to chemical cues to assess accurately the risk of predation inside the refuge. By doing this, lizards might minimize the facilitation effects caused by both types of predators acting simultaneously. In this study, we aimed to analyse the antipredatory strategies of lizards when simultaneously confronting two types of predators with different foraging strategies. We tested in the laboratory the ability of lizards to detect the chemical cues of smooth snakes. We then simulated a system with two predators, one that searches actively for prey in the open (simulated by the experimenter) and an ambush predator that waits for prey inside refuges (the smooth snake). We compared the propensity of wall lizards to enter the refuge, time spent in it, and variation in repeated attacks between predator-free refuges and refuges containing visual cues of a snake, chemical cues or both. We hypothesized that wall lizards should be able to discriminate the chemical cues of a snake and use them to assess the presence of a snake inside a refuge. However, an estimate of risk based only on chemical cues may be excessively Capítulo 5 Efectos del ecoturismo: múltiples depredadores 275 conservative, so lizards might also need visual cues to assess risk level accurately, especially when emerging from the refuge is costly in terms of predation. According to the threat sensitivity hypothesis, we hypothesized that lizards should respond more accurately when they found more than a single cue inside the refuge. Methods Study Animals and Maintenance During March and April 2000, we captured by noosing 34 P. muralis (X + SE snout-vent length = 66 + 2 mm) at a rock wall (120 m long x 5 m high) near Cercedilla, Madrid Province, Spain. This lizard is a small lacertid lizard widespread in Central Europe. It is common in mountains of the northern half of the Iberian Peninsula, where it occupies soil dwellings, talus and walls in shaded zones in forests (Martin- Vallejo et al. 1995). The smooth snake is a specialist predator that feeds mainly on these lizards (Galán 1998). Its geographical distribution and habitat preferences overlap frequently with those of P. muralis. Smooth snakes seemed to be especially abundant on the wall at our study site. For example, during a parallel field study, we captured and marked six snakes that were often seen during the day ambushing inside crevices or occasionally basking outside very close to the crevices (L. Amo, P. López and J. Martín, unpublished data). We captured, on the same wall, two smooth snakes to be used as potential predators, and two adult male Iberian rock lizards, Lacerta monticola, to be used as sources of control scent stimuli. The Iberian rock lizard is insectivorous and thus innocuous to wall lizards, but is often found in the same microhabitats. All lizards were individually housed at ‘El Ventorrillo’ Field Station 5 km from the capture site in outdoor PVC terraria (60 x 40 cm and 50 cm high) containing sand substratum and rocks for cover. Every day, they were fed mealworm larvae, Tenebrio molitor, dusted with multivitamin powder for reptiles, and water was provided ad libitum. The photoperiod and ambient temperature were those of the surrounding region. Lizards were held in captivity at least 1 month before testing to allow acclimation to laboratory conditions. To avoid contact with the scent and visual stimuli by lizards before Capítulo 5 Efectos del ecoturismo: múltiples depredadores 276 they were tested, the Iberian rock lizards and the smooth snakes were housed separately. The smooth snakes were individually housed in glass terraria (60 x 30 cm and 20 cm high) with strips of absorbent paper fixed on the substrate to absorb snake scent. Species-appropriate food and water were provided ad libitum. To avoid using live lizards as food, we fed the snakes domestic crickets and small bits of minced lamb bearing the scent of live lizards (faeces and the secretion from femoral pores and skin of wall lizards). This feeding method did not affect the lizards, but their scent attracted the attention of the snakes to the meat. Because lamb is an artificial food, we also used multivitamin powder. We kept the snakes approximately 1 month in captivity. All animals were healthy during the trials. We did not observe behavioural or physiological changes from possible stress of experiments, and all maintained or increased their original body mass (X + SE mass increment = 0.7 + 0.1 g). All animals were returned to their exact capture sites at the end of the experiments. The experiments were performed under licence from the Madrid Environmental Agency (Consejería del Medio Ambiente de la Comunidad de Madrid). Experiment 1: Detection of Chemical Cues from Snakes We compared tongue-flick rates by lizards in response to stimuli arising from cotton applicators impregnated with scents of (1) smooth snake (predator), (2) Iberian rock lizard (reptile scent control), (3) cologne (pungency control) or (4) deionized water (odourless control) to test for differential responses to scents (Cooper and Burghardt 1990). Water was used to gauge baseline tongue-flick rates in the experimental situation. We prepared stimuli by dipping the cotton tip (1 cm) of a wooden applicator attached to a long stick (150 cm) in deionized water. Other stimuli were added by rolling the moistened cotton over the body surface of the snake or the Iberian rock lizard, or by dipping it in diluted cologne. A new swab was used in each trial. Every lizard was exposed to each stimulus in counterbalanced order. One trial was conducted per day for each animal (N = 34). Trials were conducted in outdoor conditions during May between 1100 Capítulo 5 Efectos del ecoturismo: múltiples depredadores 277 and 1700 hours when lizards were fully active. To begin a trial, the experimenter slowly approached the terrarium and slowly moved the cotton swab to a position 1 cm in front of the lizard’s snout. The number of tongue-flicks directed and not directed to the swab was recorded for 60 s, beginning with the first tongue-flick. Latency to the first tongue-flick was defined as the time from presentation of the cotton swab to the first tongue-flick directed at the swab. We also recorded the time that lizards remained close (within 1 cm) to the cotton swab. An increase in time spent fleeing from the cotton swab (i.e. running rapidly from the stimulus to the opposite side of the terrarium) indicated that lizards tried to escape from that stimulus. To examine differences in number of tongue-flicks and latency to first tongue- flick between conditions, we used repeated measures one-way analyses of variance (ANOVAs) with scent stimuli as the within-subject factor. Because lizards often moved away from the stimulus, the swab had to be continuously repositioned in front of the lizard. Thus, to analyse the number of tongue-flicks directed to the swab in relation to the actual time that lizards remained exposed to the stimulus, we used repeated measures one-way analysis of covariance (ANCOVA), with the number of repositionings of the swab as a covariate to avoid possible effects of this variable. Data were log-transformed to ensure normality (Shapiro-Wilk test). Tests of homogeneity of variances (Hartley’s Fmax test) showed that in all cases, variances were not significantly heterogeneous after transformation. Pairwise comparisons were planned using Tukey’s honestly significant difference (HSD) tests (Sokal and Rohlf 1995). Experiment 2: Relative Importance of Visual and Chemical Cues In this experiment, we compared wall lizards’ use of clean refuges with those that contained chemical or visual cues of a smooth snake. We compared the propensity of the lizard to enter a refuge, time spent in it and variation in successive attacks. Each individual (N = 20) was tested in each of four trials in a counterbalanced sequence: (1) control treatment (odourless and empty refuge); (2) chemical treatment (refuge Capítulo 5 Efectos del ecoturismo: múltiples depredadores 278 containing snake scent); (3) visual treatment (odourless refuge that allowed lizards to see but not to detect chemical cues from a smooth snake within the refuge); and (4) visual and chemical (combined) treatment (refuge that allowed lizards to see and detect chemical cues from a smooth snake; see below). The experiment was conducted in a terrarium (100 x 40 cm and 50 cm high) with a sand substrate and a single refuge in the middle of one end of the terrarium. The refuge was built with flat rocks, and had two openings (7 x 6 cm) that allowed entry. One entry was open, and the other was closed with the glass walls of a smaller adjacent terrarium (50 x 40 x 40 cm) that was used to house the smooth snake. This smaller terrarium was sealed to prevent lizards from detecting chemical cues from the snake. The refuge design ensured that lizards could see the snake only after they had entered the refuge. In the control treatment, the adjacent terrarium was empty, and we applied deionized water to a clean strip of absorbent paper fixed on the substrate of the refuge. In the chemical treatment, the adjacent terrarium was also empty, and we fixed strips of predator-scented absorbent paper moistened with deionized water to the floor of the refuge to add the predator scent. The strips of absorbent paper had been in the terrarium of the snake for at least 3 days. In the visual treatment, we used a clean strip of paper moistened with deionized water and we placed the snake into the adjacent terrarium. In the treatment with chemical and visual cues combined, we placed the snake into the adjacent terrarium and fixed predator-scented strips of absorbent paper moistened with deionized water to the floor of the refuge. We used new papers and a new refuge in each trial to avoid mixing chemical cues. After each trial, the refuges were cleaned thoroughly with water and the sand substrate was replaced. Before each trial, a lizard was gently transferred to an experimental terrarium, where the refuge entry was initially closed. After a 5-min acclimation period, during which the lizard typically moved normally through the terrarium, the experimenter opened the entry of the refuge and simulated a predatory attack by tapping the lizard close to the tail with a brush to stimulate it to run and Capítulo 5 Efectos del ecoturismo: múltiples depredadores 279 hide in the refuge. Lizards usually ran for some time and frequently passed several times close to the refuge without entering. An experimenter recorded the time from the beginning of the attack until the lizard entered the refuge. When the lizard hid, the observer retreated to a hidden position and recorded the time that the lizard spent in the refuge until the head emerged from the refuge (appearance time), and the time from appearance until the lizard emerged entirely from the refuge (waiting time). Immediately after the lizard resumed normal activity, we simulated another predatory attack with the same procedure and recorded data as in the first attack. Air temperature inside the refuge was maintained at 20 + 0.1 ºC. We used repeated measures factorial ANOVAs to assess differences in time until entering the refuge, and appearance and waiting times, between treatments and between the two attacks of each individual (both within-subject factors). We included the interaction in the models to test whether responses to the different treatments changed between the first and the second attack (Sokal and Rohlf 1995). Data were log-transformed to ensure normality (ShapiroeWilk test). Tests of homogeneity of variances (Levene’s test) showed that in all cases variances were not significantly heterogeneous after transformation (Sokal and Rohlf 1995). Results Experiment 1: Detection of Chemical Cues from Snakes All lizards responded to swabs by tongue flicking. There were significant differences between stimulus conditions in total tongue-flicks (repeated measures one-way ANOVA: F3,99 = 8:32, P < 0.0001; Fig. 1a). Chemicals from the snake elicited significantly more tongue- flicks than the other conditions (Tukey’s test: P < 0.01 in all cases). Responses to deionized water, cologne and L. monticola were not significantly different (P > 0.60 in all cases). The number of tongue-flicks directed to swabs differed significantly between treatments (repeated measures oneway ANCOVA: F3,96 = 4.15, P = 0.008; Fig. 1b), with more directed to swabs with snake scent than to other stimuli (Tukey’s tests: P < 0.01 in all cases). Responses to chemicals from other conditions were not significantly Capítulo 5 Efectos del ecoturismo: múltiples depredadores 280 different (P > 0.90 in all cases). The number of tongue-flicks not directed to the swab did not differ significantly between treatments (repeated measures one-way ANOVA: F3,99 = 1.40, P = 0.25, power = 0.40). The time that lizards remained close to the stimulus differed significantly between treatments (repeated measures one-way ANOVA: F3,99 = 4.03, P < 0.01). Lizards often fled after tongue flicking the snake stimulus. Thus, lizards spent significantly less time close to the snake stimulus (X + SE = 42 + 3 s) than to the other stimuli (water: 51 + 2 s; cologne: 50 + 2 s; L. monticola: 48 + 3 s; Tukey’s test: P < 0.05 in all cases). There were no significant differences between the other treatments (P > 0.90 in all cases). Figure 1. Mean + SE (a) total number of tongue-flicks, (b) tongue-flicks directed to swabs in relation to the time exposed to the stimulus and (c) latency (s) to the first tongue-flick by the lizard Podarcis muralis (N = 34) in response to deionized water, cologne, Iberian rock lizard, or smooth snake stimuli presented on cottontipped applicators Capítulo 5 Efectos del ecoturismo: múltiples depredadores 281 Mean latency to the first tongue-flick differed significantly between conditions (repeated measures one-way ANOVA: F3,99 = 3.12, P = 0.03; Fig. 1c). The latency in response to snake scent was significantly shorter than to the water (Tukey’s test: P = 0.02), but it was not significantly different from latency to cologne (P = 0.15) or to L. monticola (P = 0.90). There were no significant differences between the other treatments (P > 0.44 in all cases). Experiment 2: Relative Importance of Visual and Chemical Cues Latency to enter the refuge after being attacked did not differ significantly either between first and second attacks (repeatedmeasures two- wayANOVA: F1,16 = 1.57, P = 0.23, power = 0.12) or between treatments (F3,48 = 1.73, P = 0.17, power = 0.36). The interaction was not significant (F3,48 = 2.44, P = 0.07, power = 0.39; Fig. 2a). Time for the head to emerge from the refuge did not differ significantly between the first and second attacks (repeated measures two-way ANOVA: F1,16 = 2.97, P = 0.10, power = 0.24), but there were significant differences between treatments (F3,48 = 3.93, P = 0.014), and the interaction was significant (F3,48 = 3.46, P = 0.02; Fig. 2b). For first attacks, time spent in the refuge was greater in the control treatment than in the chemical and visual combined treatment (Tukey’s test: P = 0.02), but no other differences between pairs of treatments were significant (P > 0.52 in all cases). After the second attack, there were significant differences between the control and both the chemical and visual combined treatments (P = 0.0004) and the chemical treatment (P = 0.0005). Waiting time did not differ significantly between the first and the second attack (repeated measures two- way ANOVA: F1,16 = 0.61, P = 0.45, power = 0.20), but treatments differed significantly (F3,48 = 7.50, P = 0.0003). The interaction was not significant (F3,48 = 1.32, P = 0.28, power = 0.20; Fig. 2c). Lizards left the refuge significantly later when they were hidden in a predator-free refuge than when they were in a refuge containing visual cues of a snake, either alone (Tukey’s test: P = 0.04) or in combination with chemical cues (P = 0.0003), but not when it contained only chemical ones (P = 0.38). Lizards left the refuge sooner when they were hidden Capítulo 5 Efectos del ecoturismo: múltiples depredadores 282 in a refuge containing both combined visual and chemical cues than when the refuge contained only chemical cues (P = 0.02). The visual treatment did not differ significantly from the combined chemical and visual treatment or the chemical treatment (P > 0.29 in both cases). Figure 2. Mean + SE differences between (a) time to enter the refuge, (b) appearance time and (c) waiting time of the lizard Podarcis muralis (N = 20) in predator-free refuges and in refuges containing chemical cues, visual cues or both after two simulated repeated attacks (open barst: first attack; black bars: second attack) Discussion The greater tongue-flick rate and shorter latency to first tongue-flick in response to snake scents presented on cotton swabs (experiment 1) indicate that P. muralis is able to detect and discriminate the chemical cues of C. austriaca snakes. This ability is particularly important to wall lizards for three reasons: lizards are an important Capítulo 5 Efectos del ecoturismo: múltiples depredadores 283 part of the diet of C. austriaca (Rugiero et al. 1995), the snake occupies the same microhabitats and is found inside refuges (Rugiero et al. 1995), and it is extremely cryptic because it uses a sit-and-wait hunting strategy within rock crevices (Galán 1998). Hence, the ability to detect chemical cues of this snake may enable P. muralis to avoid entering hazardous crevices. Lizards exposed to predator chemical stimuli responded by rapidly fleeing away from the swab. This result suggests that, in a natural situation, the first response of wall lizards upon detection of chemical cues from a snake would be to flee, thereby avoiding crevices likely to contain smooth snakes. Results of experiment 2 suggest that lizards were able to use both chemical and visual snake cues to assess the level of predation risk inside a refuge. Time to enter a refuge by wall lizards seemed not to be influenced by the potential risk of predation inside the refuge. Regardless of the type of cue found inside the refuge, lizards sheltered quickly in both attacks. These results suggest that the actual predation risk in the open seems to be more determinant for lizards than the eventual risk of encountering a hidden snake inside the refuge. Also, assessment of risk before entering might not always be possible, especially if the lizard is fleeing from a predator. This result suggests a case of predator facilitation because predators in the exterior may force lizards to hide in potentially hazardous refuges. However, our results also suggested that time spent in the refuge was related to the snake’s cues found inside it. Appearance time in the second attack was greater when lizards found only visual cues of a snake than when there were chemical cues. This result could suggest that wall lizards discriminated the actual source of chemical cues more quickly than that of visual ones; smooth snakes are inconspicuous inside the dark crevices and their chemical stimuli may provide more important cues (Van Damme et al. 1995; Kats and Dill 1998). An alternative explanation is that lizards may have taken longer to look through the window than to tongue-flick the floor, so this result could have been caused by the cue that they encountered first. Also, lizards may have perceived that the snake was outside the refuge (W. E. Cooper, Jr, personal communication). The results of experiment 1 suggest that Capítulo 5 Efectos del ecoturismo: múltiples depredadores 284 lizards may assess the possible presence of a snake using only chemical cues. However, after appearing, lizards waited longer before resuming activity when the refuge contained chemical cues than when visual ones were present. Chemical detection of a snake may indicate to lizards that the refuge was risky at a certain moment, but it does not necessarily indicate a current risk. Thus, lizards left the refuge quickly only when they also saw the snake. Therefore, our results suggest that visual cues are important to confirm the uncertain level of risk implied by chemical cues. Our results also confirm the assumption of the threat sensitivity hypothesis that multiple cues from a predator may contribute in an additive way to determine the degree of risk- sensitive behaviour. The antipredatory response of lizards was greater when they were exposed to both visual and chemical cues of ambush snakes (i.e. they appeared and emerged sooner from the refuge) than when only one cue was presented. Similar results were obtained in a study of the mosquitofish, G. affinis, which increased avoidance behaviour when chemical and visual cues of predatory fish were presented (Smith and Belk 2001). However, fathead minnows, Pimephales promelas, were most likely to react to chemical alarm cues in the absence of visual information and when the perceived risk was high (Hartman and Abrahams 2000). Larval newts, Notophthalmus lousianensis, distinguished between predatory and nonpredatory species only when chemical cues were available, although when only visual cues were present, newts attempted to avoid both species (Mathis and Vincent 2000). In contrast, slimy sculpins, Cottus cognatus, showed threat-sensitive predator avoidance when exposed only to visual cues, but not when exposed only to chemical cues from a predator (Chivers et al. 2001). Differences between species in behavioural responses to visual and chemical predator cues should depend on ambient conditions. For example, newts and wall lizards may rely heavily on chemical cues because visibility is greatly restricted in the habitats where they may encounter their predators. Furthermore, the response of prey to different levels of information about a predator should also depend on ambient conditions. For example, yellowhammers, Emberiza citrinella, Capítulo 5 Efectos del ecoturismo: múltiples depredadores 285 that heard alarm calls only from conspecifics delayed resuming activity longer than birds that saw a sparrowhawk, Accipiter nisus, model (van der Veen 2002). In this case, birds with less complete information perceived predation risk as being higher because they could not locate the predator. Thus, they were more cautious. Actively foraging predators in the exterior may force lizards to increase refuge use even when the risk of predation from sit-and-wait snakes inside the refuge is high. The actual presence of a snake in the refuge may also force lizards to decrease their refuge use, exposing them to increased predation in the open. This response could enhance risk for the prey, as has been observed in other animals (Soluk 1993; Korpima¨ki et al. 1996). The results of this study may support this idea. Wall lizards probably could not elude the predator in the open without hiding in the refuge, and did not modify the time taken to enter the refuge in relation to the predation risk within it. However, our results also suggest that the ability to identify different cues of a predator accurately may help lizards to improve their accuracy of predation risk assessment inside the refuge. This ability may help wall lizards to reduce the risk- enhancing effects of two types of predators requiring conflicting prey defences. Acknwledgements We thank two anonymous referees andW. E. Cooper, Jr, for helpful comments and ‘El Ventorrillo’ MNCN Field Station for use of their facilities. 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Herpetol J 5: 181-188 Mathis A, Vincent F (2000) Differential use of visual and chemical cues in predator recognition and threat-sensitive predator-avoidance responses by larval newts (Notophthalmus viridescens). Can J Zool 78: 1646-1652 Rugiero L, Capula M, Filippi E, Luiselli L (1995) Food habits of the mediterranean populations of the smooth snake (Coronella austriaca). Herpetol J 5: 316-318 Sih A, Kats LB, Moore RD (1992) Effects of predatory sunfish on the density, drift and refuge use of the stream salamander larvae. Ecology 73: 1418-1430 Sih A, Englund G, Wooster D (1998) Emergent impact of multiple predators on prey. Trends Ecol Evol 13: 350-355 Smith ME, Belk MC (2001) Risk assessment in western mosquitofish (Gambusia affinis): do multiple cues have additive effects? Behav Ecol Sociobiol 51: 101- 107 Sokal RR, Rohlf FJ (1995) Biometry. 3rd edn. New York: W. H. Freeman. Soluk DA (1993) Multiple predator effects: predicting combined functional response of stream fish and invertebrate predators. Ecology 74: 219-225 Van Damme R, Quick K (2001) Use of predator chemical cues by three species of lacertid lizards (Lacerta bedriagae, Podarcis tiliguerta, and Podarcis sicula). J Herpetol 35: 27-36 Van Damme R, Bauwens D, Thoen C, Vanderstighelen D, Verheyen RF (1995) Responses of naive lizards to predator chemical cues. J Herpetol 29: 38-43 Capítulo 5 Efectos del ecoturismo: múltiples depredadores 287 Vanderstighelen D (1987) Responses of the common lizard (Lacerta vivipara) to visual and chemical stimuli of the viper (Vipera berus). In: Van Gelder JJ, Strijbosch H, Bergers PJM (eds) Proceedings of the Fourth Ordinary General Meeting of the Societas Europaea Herpetologica, Societas Europaea Herpetologica, Nijmegen, pp 425-428 van der Veen IT (2002) Seeing is believing: information about predators influences yellowhammer behavior. Behav Ecol Sociobiol 51: 466-471 Capítulo 5 Efectos del ecoturismo: múltiples depredadores 289 Chemical assessment of predation risk in the wall lizard, Podarcis muralis, is influenced by time exposed to chemical cues of ambush snakes RESUMEN Las lagartijas a menudo responden a la presencia de un depredador incrementando el uso de refugios. Sin embargo, este comportamiento puede exponer a las lagartijas a culebras saurófagas que se encuentran en los mismos refugios acechando a sus presas. Estas culebras, que no siempre son visibles, depositan señales químicas que pueden ser reconocidas por las presas. Aunque las ventajas de usar estas señales químicas de las culebras son obvias, la detección química de los depredadores puede llevar a estimas muy conservativas del riesgo de depredación. Esto es debido a que las señales químicas pueden indicar que la zona era arriesgada en un determinado momento, pero no necesariamente en la actualidad ya que el depredador ha podido abandonar la zona. En este estudio examinamos si las lagartijas roqueras (Podarcis muralis) evitan el uso de refugios que contienen señales químicas de las culebras lisas europeas (Coronella austriaca), y si este comportamiento de evitación se mantiene a lo largo del tiempo o las lagartijas lo modifican. Los resultados sugieren que las lagartijas roqueras detectaron las señales químicas de las culebras lisas dentro de los refugios y, a corto plazo, disminuyeron el uso de refugios que contenían olor de depredador e incrementaron sus movimientos de escape. Sin embargo, esta respuesta de evitación pareció disminuir a largo plazo. Las lagartijas, al investigar en ocasiones sucesivas a lo largo del tiempo, pudieron valorar de nuevo que realmente la culebra no estaba presente y modificaron el uso del refugio, disminuyendo su respuesta de evitación. Por lo tanto, las lagartijas parecen ser capaces de valorar las variaciones temporales en el riesgo de depredación por culebras dentro de los refugios. Capítulo 5 Efectos del ecoturismo: múltiples depredadores 291 Chemical assessment of predation risk in the wall lizard, Podarcis muralis, is influenced by time exposed to chemical cues of ambush snakes Abstract Lizards often respond to predator presence by increasing refuge use. However, this behaviour may expose lizards to saurophagous snakes, which inhabit the same refuges to ambush their lizard prey. Snakes, which are not always visible, deposit chemical trails that can be detected by lizards. Even though there are obvious advantages of using chemical cues, chemical detection of predators might lead to very conservative estimates of risk. This is because chemical cues might indicate that an area was risky in the recent past, but not necessarily at the current time. We examined experimentally whether wall lizards (Podarcis muralis) avoid using refuges that contain chemical cues of smooth snakes (Coronella austriaca), and whether this avoidance response is maintained long term or whether it can be modified. Results suggest that wall lizards detected the chemical cues of smooth snakes inside refuges, and, in the short term, decreased the use of predator-scented refuges and increased their escape movements. However, this avoidance response seemed to decrease in the long term. By investigating the refuge again over subsequent time periods, lizards reassessed whether the snake was actually present, modified their refuge use and decreased their avoidance response. Therefore, wall lizards seem able to assess temporal variations in predation risk by snakes inside refuges and to respond accordingly. Introduction Predation is a major selective force. However, since animals must accomplish more in their lifetime than simply avoiding predation, natural selection favours individuals that minimise their individual risk of mortality while attending to other demands (Lima and Dill 1990). Chemosensory cues may reliably reveal Capítulo 5 Efectos del ecoturismo: múltiples depredadores 292 the presence of predators and they may also provide information on predator activity level and diet (Kats and Dill 1998). Snakes deposit chemical trails that can be detected by lizards with their highly developed vomeronasal system (Cooper 1990; Van Damme et al. 1995; Downes and Shine 1998a; Van Damme and Quick 2001; Downes and Bauwens 2002). Because snakes are not always visible, their chemical stimuli may be particularly important for lizards that share the same refuges (Downes and Shine 1998a). For example, some geckos used their chemosensory ability to avoid entering rock crevices with snake scent (Downes and Shine 1998a,b). Prey, such as lizards, often respond to predator presence by increasing refuge use (Greene 1988; Sih et al. 1992). However, refuges may have some costs that should be minimised, such as the loss of time available for other activities, or physiological costs (Dill and Fraser 1997; Sih 1997; Martín and López 1999a,b). In addition, some types of refuges may only be useful against some particular type of predators, or may expose prey to other types of predators (Sih et al. 1998). For example, lizards may face saurophagous, ambush-hunting snakes that share the same refuges (Downes and Shine 1998a). The ability to detect the chemical cues of a snake may help lizards to survive an encounter with a predator (Downes 2002). Even though there are obvious advantages of using chemical cues, especially when other cues are unavailable, chemical detection of predators may lead to very conservative estimates of risk because they indicate that a given area was risky at a certain point in time but not necessarily a current risk (Kats and Dill 1998; Turner and Montgomery 2003). Thus, according to the threat sensitivity hypothesis (Helfman 1989), natural selection should favour individuals that take action appropriate to the magnitude of threat, rather than avoiding the use of refuges in response to all kinds of predator chemical cues. The wall lizard, Podarcis muralis, offers an excellent opportunity to study the patterns of avoidance of hazardous refuges. Wall lizards respond to predator presence in the open by increasing refuge use (Martín and López 1999b). However, by doing this, they may expose themselves to increased predation risk inside refuges by ambush- hunting smooth snakes (Coronella Capítulo 5 Efectos del ecoturismo: múltiples depredadores 293 austriaca). This is a lizard specialist that hunts by ambush foraging, hidden in rock crevices (Rugiero et al. 1995; Galán 1998), and has a geographic distribution and habitat preferences that overlap frequently with those of wall lizards. Previous studies have shown that P. muralis is able to detect and discriminate the chemical cues of smooth snakes (Amo et al. 2004). In this paper, we examined experimentally whether wall lizards avoid using refuges that contain chemical cues of smooth snakes, and whether this avoidance response is maintained long term or whether it can be modified. Material and Methods During May 2001, we captured adult P. muralis by noosing (9 males and 10 females; snout-vent length, SVL, ± SE = 66 ± 2 mm) at a rock wall (120 m long, 5 m high) near Cercedilla (Madrid Province, Spain). We also captured in the same wall two adult smooth snakes to be used as source of scent of potential predators. Lizards were individually housed at .El Ventorrillo. Field Station 5 km from the capture site, in outdoor 60 × 40 cm PVC terraria containing sand substratum and rocks for cover. Every day, they were fed mealworm larvae (Tenebrio molitor) dusted with multivitamin powder for reptiles, and water was provided ad libitum. The photoperiod and ambient temperature was that of the surrounding region. Lizards were held in captivity at least one month before testing to allow acclimation to laboratory conditions. To prevent lizards from having contact with the scent stimuli before they were tested, the snakes were housed separately in glass terraria (60 × 30 × 20 cm) with sawdust on the substrate to obtain their scent. Due to its absorbent properties, the odourless sawdust is an excellent method for obtaining snake scent without disturbing the animal. All the animals were healthy during the trials and were returned to their exact capture sites at the end of experiments. The experiment was performed under licence from the Madrid Environmental Agency (‘Consejería del Medio Ambiente de la Comunidad de Madrid’). To compare the behaviour of lizards when they found a potentially unsafe refuge (i.e. with snake chemical cues) or an unfamiliar but predator-free refuge, we used two terraria (60 × 40 × 30 cm). Terraria were divided into two halves, Capítulo 5 Efectos del ecoturismo: múltiples depredadores 294 and had two refuges placed symmetrically on either side, one in front of the other, with a distance of 15 cm between them. The refuges were flat rocks (10 × 7 cm) placed 2 cm above the substrate, allowing lizards to hide under them. In the ‘predator’ treatment, the terrarium had a refuge containing chemical cues of a smooth snake and an odourless refuge. In the ‘control’ treatment both refuges were odourless. To add the predator scent to the refuge, we used sawdust that had been in the terrarium of the snakes for at least three days, moistened with deionized water. In the odourless refuges, we applied some deionized water to a similar quantity of odourless sawdust. In both cases, sawdust was placed on the ground, inside the refuge. We did not include a pungent control (e.g. perfume) in the experimental design because results of previous experiments showed that P. muralis cannot distinguish it from water and from other biologically irrelevant odours, but can distinguish it from snake scent (Amo et al. 2004). Moreover, P. muralis does not modify the use of refuges containing a pungent odour, compared to an odourless control (Amo, López and Martín, unpublished data). Every lizard was tested in each treatment once in a randomised block design, and order of trials was counterbalanced. One trial was conducted per day for each animal. Trials were conducted under outdoor conditions during July 2001 between 1200-1700 hrs when lizards were fully active. Lizards were allowed to bask in their home terraria for at least two hours before trials. After each trial the cages and the refuges were cleaned thoroughly with water and detergent for 20 min and dried at the outdoor temperature. We used new stimuli in each trial to avoid the mixture of odours. Experiments were recorded on videotape (Hi-8 format, 25 frames s-1) using a Sony CCD-TR810E video camera aligned perpendicularly over the terrarium. Lizards were filmed as they moved spontaneously along the terrarium during 25 min. The experimenter was not present during filming to avoid disturbing lizards. After this, we noted the location of each lizard in the terrarium every 30 min over the subsequent five hours. Later, we analysed the tapes and noted lizard behaviour in the experimental half of the terrarium (i.e. the half that contained the snake-scented refuge in the ‘predator’ Capítulo 5 Efectos del ecoturismo: múltiples depredadores 295 treatment, or one of the odourless refuges in the ‘control’ treatment). We noted the total time spent in the experimental area, time spent in movement, motionless, or standing up trying to escape (i.e. the lizard stands in a upright position against the wall of the terrarium and performs scratching movements with the forelegs), and total time spent inside each refuge. To determine possible changes through time in the responses, we divided each 25 min period into five consecutive periods of 5 min each. We chose this interval of time because previous results of Thoen et al. (1986) showed that the responses of common lizards, Lacerta vivipara, to the scent of smooth snakes was different in the first 5 min of the trial than afterwards. We used two-way repeated measures analysis of variance (ANOVA) to test for differences between treatments (‘control’ vs. ‘predator’) and among the five time sequences of each individual (within-subjects factor). Data of total time spent in the experimental half of the terrarium were log-transformed, which successfully normalize the data. We used the time spent in movement, motionless, and in standing up acts in the experimental area, and the time spent in each refuge, in relation to the total time spent in the corresponding area. Angular transformations of all percentages were made to normalize the data. Differences in the location of lizards during the subsequent five hours between treatments were analysed with one way ANOVA. We calculated the number of times that lizards were observed outside of refuges, and the number of times the lizards were seen hidden in the experimental refuge in relation to the number of times that the lizards were inside any refuge. Data were log- and arcsin- transformed, respectively, to normalize data. Tests of homogeneity of variances (Levene.s test) showed that in all cases variances were not significantly heterogeneous after transformation. Pairwise comparisons of means were planned using the Tukey.s honestly significant difference (HSD) test (Sokal and Rohlf 1995). Capítulo 5 Efectos del ecoturismo: múltiples depredadores 296 Results Total time that lizards spent in the experimental area did not differ significantly between treatments (repeated measures two way ANOVA; F1,18 = 0.52, P = 0.48), although there were significant differences among sequences (F4,72 = 2.47, P < 0.05). The interaction was not significant (F4,72 = 1.12, P = 0.35). Lizards decreased the time that they spent in the experimental half of the terrarium over time, although there were only significant post-hoc differences between the first and the third sequence (Tukey.s test, P = 0.04). Time spent moving did not differ significantly between treatments (F1,18 = 0.10, P = 0.75), but there were significant differences among sequences (F4,72 = 11.92, P < 0.0001) (Fig. 1a). The interaction was not significant (F4,72 = 1.50, P = 0.21). Lizards decreased their movement rate across time, especially after the first 5 min. Thus, there were significant differences between the first sequence and the subsequent four (P < 0.001 in all cases), but not between the rest of sequences (P > 0.21 in all cases). Time spent motionless did not differ significantly either between treatments (F1,18 = 0.58, P = 0.46), or among sequences (F4,72 = 0.31, P = 0.87), but the interaction was significant (F4,72 = 2.51, P < 0.05) (Fig. 1b). Lizards increased the time spent motionless in the ‘predator’ treatment whereas they decreased it in the ‘control’ treatment in the course of time, although post hoc comparisons did not show significant differences (Tukey`s test, P > 0.10 in all cases). Duration of standing up acts did not differ significantly between treatments (repeated measures two way ANOVA; F1,18 = 2.14, P = 0.16), although there were significant differences among sequences (F4,72 = 6.07, P = 0.0003) and the interaction was significant (F4,72 = 4.34, P = 0.003) (Fig. 1c). During the first 5 min the time spent by lizards in standing up acts was significantly higher in the ‘predator’ treatment than in the ‘control’ one (P = 0.006). Whereas, later, there were no significant differences either between sequences, when considering each treatment alone, or between treatments in each sequence (P > 0.57 in all cases). Capítulo 5 Efectos del ecoturismo: múltiples depredadores 297 Figure 1. Percent time (mean + SE) spent (a) in movement, (b) motionless, and (c) in standing up acts, in relation to the total time spent in the experimental area, in the ‘control’ (open boxes) and ‘predator’ (black boxes) treatments. Time spent inside the refuge did not differ significantly either between treatments (repeated measures two way ANOVA; F1,18 = 0.001, P = 0.98) or among sequences (F4,72 = 2.06, P = 0.09), but the interaction was significant (F4,72 = 3.94, P = 0.006) (Fig. 2). During the first 5 min there were no significant differences between treatments in time spent in refuges (P = 0.30). However, in the course of time lizards increased the time they spent in the control refuge (differences between the first and the other five sequences, P = 0.02), but they did not increase it in the refuge containing chemical signals of a snake (P > 0.97 in all cases). Nevertheless, in the long term (i.e. in the subsequent five hours), the number of times that lizards were observed out of a refuge did not Capítulo 5 Efectos del ecoturismo: múltiples depredadores 298 significantly differ between treatments (control: 3 ± 1 times; predator: 4 ± 1 times; one way ANOVA, F1,18 = 1.02, P = 0.33). Also, there was no significant difference between treatments in the use of the experimental refuge (number of times in the experimental refuge/number of times in any of the two refuges, control: 56 ± 7 %; predator: 51 ± 6 %; F1,18 = 1.26, P = 0.28). Thus, in the long term lizards did not avoid to hide in the refuge soiled with snake scent. Fig. 2 Percent time (mean + SE) spent inside the experimental refuge in relation to the total time spent in the experimental area, in the ‘control’ (open boxes) and ‘predator’ (black boxes) treatments. Discussion Results of this study suggest that wall lizards were able to detect the chemical cues of smooth snakes, and to use them in the short term to assess the potential risk of predation inside a refuge, but that after some time lizards were able to reassess whether the snake was actually present and modified their response. To avoid the risk of predation by ambush snakes, lizards initially modified their behaviour and their use of potentially hazardous refuges. During the first few minutes, lizards spent the same time in both types of refuges. However, later on, lizards decreased their use of the predator-scented refuge, whereas they increased the use of the odourless refuge. This could be explained if lizards approached refuges and spent some time investigating the source of the odour, but after discriminating the snake scent, they decided to avoid using the unsafe refuge. Our results agree with previous studies that have shown that other lizard species avoid using retreats that were soiled with snake.s scent (Downes and Shine 1998a; Downes and Bauwens 2002). Lizards also modified their locomotor patterns in the predator treatment. Previous studies have shown that prey exposed to a potential predator odour often show behavioural changes such as reduced activity (Van Damme et al. 1990), increased refuge use (Kiesecker et al. 1996) or reduced use of the potential risky area (Downes and Shine 1998a). Our results suggest that Capítulo 5 Efectos del ecoturismo: múltiples depredadores 299 lizards increased their escape behaviour (i.e. standing up acts) when they found chemical cues of a snake inside a refuge. Podarcis sicula lizards also increase the time spent in standing up acts when they found chemical cues of a snake on the ground (Downes and Bauwens 2002). Also, wall lizards showed a similar behaviour when they found chemical cues of a snake on the open ground of a terrarium (Amo, López and Martín, unpublished data). These results suggest that lizards perceived an increase in the risk of remaining near a potentially unsafe area and that they responded by trying to escape from the terrarium. A similar response to predator chemicals was observed in larval Ambystoma salamanders, which decreased movement only in the absence of a refuge; otherwise, increased movement in an effort to reach a refuge (Sih and Kats 1991). Also, increased movement in larval toads in response to an alarm substance may represent refuge-seeking behaviour (Hews 1988). Wall lizards also tended to maintain the time spent motionless in the risky area while they decreased it in the control area across the time. By standing still, lizards may try to visually detect the snake in a potentially unsafe area (Avery 1991, 1993; McAdam and Kramer 1998). However, this avoidance response seemed to decrease in the long term. Chemical detection of a snake may indicate that a refuge was risky at a certain point in time but it does not necessarily indicate a current risk. Thus, chemical assessment might lead to excessively conservative estimates of risk if prey continue avoiding the refuge despite the absence of the predator. By investigating the refuge again over the subsequent minutes, lizards may assess the absence of the snake. Thus, wall lizards responded to the temporal decrease in the risk of predation inside the refuge by decreasing their avoidance response and increasing the use of such refuge. Similarly, the avoidance response to predator chemical cues diminished with time in Physa snails (Turner and Montgomery 2003) and Plethodon cinereus salamanders (Sullivan et al. 2002). In contrast, garden skinks, Lampropholis guichenoti, avoided the use of predatorscented areas during six months (Downes 2001). However, in this case, the predator odour was replaced weekly. Thus, skinks probably continued avoiding the risky Capítulo 5 Efectos del ecoturismo: múltiples depredadores 300 area because they could perceive a fresh stimulus every week. An explanation for the lack of avoidance behaviour across the time may be that wall lizards are able to assess the age of the chemical cues they found. However, our experiment did not test this effect and, thus, further research is needed to examine whether wall lizards have this ability. Acknowledgements We thank two anonymous reviewers for helpful comments, Kevin Pilz for checking the english, and ‘El Ventorrillo’ MNCN Field Station for use of their facilities. Financial support was provided by the MCYT projects BOS 2002-00598 and BOS 2002-00547, and and ‘El Ventorrillo’ CSIC grant to L. Amo. References Amo L, López P, Martín J (2004) Wall lizards combine chemical and visual cues of ambush snake predators to avoid overestimating risk inside refuges. Anim Behav 67: 647-653 Avery RA (1991) Temporal dynamics of a vigilance posture in the ruin lizard Podarcis sicula. Amphibia-Reptilia 12: 352-356 Avery RA (1993) Experimental analysis of lizard pause-travel movement: pauses increase probability of prey capture. Amphibia-Reptilia 14: 423-427 Cooper WE Jr (1990) Chemical detection of predators by a lizard, the broad-headed skink (Eumeces laticeps). J Exp Zool 256: 162-167 Dill LM, Fraser AHG (1997) The worm re- turns: hiding behaviour of a tube- dwelling marine polychaete, Serpula vermicularis. Behav Ecol 8: 186-193 Downes S (2001) Trading heat and food for safety: costs of predator avoidance in a lizard. Ecology 82: 2870- 2881 Downes S (2002) Does responsiveness to predator scents affect lizard survivorship? Behav Ecol Sociobiol 52: 38-42 Downes S, Bauwens D (2002) Does reproductive state affect a lizard.s behavior toward predator chemical cues? Behav Ecol Sociobiol 52: 444- 450 Downes S, Shine R (1998a) Sedentary snakes and gullible geckos: predator- prey coevolution in nocturnal rock- dwelling reptiles. Anim Behav 55: 1373- 1385 Downes S, Shine R (1998b). Heat, safety or solitude? Using habitat selection experiments to identify lizard’s priorities. Anim Behav 55: 1387-1396 Galán P (1998). Coronella austriaca - Laurenti 1768. In: Ramos MA (ed) Fauna Ibérica. Vol. 10. Museo Nacional de Ciencias Naturales, CSIC, Madrid, pp 364-375 Greene HW (1988) Antipredator mechanisms in reptiles. In: Gans C, Billet F, Maderson PFA (eds) Biology of the reptilian, vol 16. Wiley, New York, pp 1-152 Gans C, Huey RB (Eds). New York: John Wiley and Sons. Helfman GS (1989) Threat-sensitive predator avoidance in damselfish- trumpetfish interactions. Behav Ecol Sociobiol 24: 47-58 Hews DK (1988) Alarm response in larval western toads, Bufo boreas: release of larval chemicals by a natural predator and its effects on predator capture efficiency. Anim Behav 36: 125-133 Capítulo 5 Efectos del ecoturismo: múltiples depredadores 301 Kats LB, Dill LM (1998) The scent of death: chemosensory assessment of predation risk by prey animals. Ecoscience 5: 361- 394 Kiesecker JM, Chivers DP, Blaustein AR (1996) The use of chemical cues in predatory recognition by western toad tadpoles. Anim Behav 52: 1237-1245 Lima SL, Dill LM (1990) Behavioral decisions made under the risk of predation: a review and prospectus. Can J Zool 68: 619-640 Martín J, López P (1999a). When to come out from a refuge: risk-sensitive and state-dependent decisions in an alpine lizard. Behav Ecol 10: 487-492 Martín J, López P (1999b) An experimental test of the costs of antipredatory refuge use in the wall lizard, Podarcis muralis. Oikos 84: 499-505 McAdam AG, Kramer DL (1998) Vigilance as a benefit of intermittent locomotion in small mammals. Anim Behav 55: 109- 117 Rugiero L, Capula M, Filippi E, Luiselli L (1995) Food habits of the Mediterranean populations of the smooth snake (Coronella austriaca). Herpetol J 5: 316-318 Sih A (1997) To hide or not to hide? Refuge use in a fluctuating environment. Trends Ecol Evol 12: 375-376 Sih A, Kats LB (1991) Effects of refuge availability on the responses of salamander larvae to chemical cues from predatory green sunfish. Anim Behav 42: 330-332 Sih A, Kats LB, Moore RD (1992) Effects of predatory sunfish on the density, drift and refuge use of the stream salamander larvae. Ecology 73: 1418-1430 Sih A, Englund G, Wooster D (1998) Emergent impact of multiple predators on prey. Trends Ecol Evol 13: 350-355 Sokal RR, Rohlf FJ (1995) Biometry, 3rd. ed. New York: W. H. Freeman and Co. Sullivan AM, Maerz JC, Madison DM (2002) Anti-predator response of red- backed salamanders (Plethodon cinereus) to chemical cues from garter snakes (Thamnophis sirtalis): laboratory and field experiments. Behav Ecol Sociobiol 51: 227-233 Thoen C, Bauwens D, Verheyen R (1986) Chemoreceptive and behavioural responses of the common lizard Lacerta vivipara to snake chemical deposits. Anim Behav 34: 1805-1813 Turner AM, Montgomery SL (2003) Spatial and temporal scales of predator avoidance experiments with fish and snails. Ecology 84: 616-622 Van Damme R, Quick K (2001) Use of predator chemical cues by three species of lacertid lizards (Lacerta bedriagae, Podarcis tiliguerta, and Podarcis sicula). J Herpetol 35: 27-36 Van Damme R, Bauwens D, Vanderstighelen D, Verheyen RF (1990) Responses of the lizard Lacerta vivipara to predator chemical cues: the effects of temperature. Anim Behav 40: 298-305 Van Damme R, Bauwens D, Thoen C, Vanderstighelen D, Verheyen RF (1995) Responses of naive lizards to predator chemical cues. J Herpetol 29: 38-43 303 Capítulo 6 Capítulo 6 Conclusiones y perspectivas 305 CONCLUSIONES Y PERSPECTIVAS Conclusiones • Todas las especies de lacértidos que se han estudiado en esta tesis, Lacerta monticola, L. lepida, Podarcis muralis, y Psammodromus algirus, presentan infecciones por parásitos sanguíneos (hemogregarinas), que, en algunos casos, parecen tener un papel importante en la condición corporal y estado de salud de los individuos (capítulo 2). • El efecto deletéreo de los parásitos parece ser más evidente cuando otro factor, como un incremento en el riesgo de depredación debido por ejemplo a la degradación del hábitat (capítulo 3) o al ecoturismo (capítulo 4), afecta a la condición física de los individuos. • La modificación del medio natural supone una pérdida de hábitat óptimo para algunas especies, por lo que cambios drásticos del hábitat, como las repoblaciones con pinos en lugares originalmente ocupados por robledales, conllevan la desaparición de unas especies y la colonización de otras (capítulo 3). • Los cambios en la estructura de la vegetación también implican un incremento en el riesgo de depredación percibido por las lagartijas (capítulo 3), ya que, por ejemplo, pueden ser más fácilmente detectables en áreas degradadas. Las lagartijas responden a este incremento en el riesgo con estrategias antidepredatorias. Presentan estrategias preventivas como la selección de microhábitats seguros, y son capaces de modificar sus patrones de locomoción para minimizar el riesgo al desplazarse por zonas inseguras. Además, ante el ataque de un depredador, presentan estrategias de escape acordes al nivel de riesgo (capítulo 3). • El ecoturismo supone un incremento en el riesgo de depredación para las lagartijas, ya que responden a las personas como a depredadores (capítulo 4). Ante el ataque simulado de un depredador, las lagartijas responden igual en zonas con alto o bajo nivel de ecoturismo. Por ello, en zonas con alto Capítulo 6 Conclusiones y perspectivas 306 nivel de ecoturismo, las lagartijas deben realizar frecuentemente estrategias antidepredatorias (capítulo 4). • Los comportamientos antidepredatorios son energéticamente costosos, por lo que un incremento en la frecuencia de estos comportamientos debido al incremento del riesgo en medios degradados o con una alta afluencia de turistas, conlleva una pérdida de condición corporal (capítulos 3 y 4). Por ejemplo, el aumento de la velocidad de carrera como estrategia preventiva (capítulo 3), las mayores distancias de aproximación permitidas a los depredadores (capítulo 3), o el incremento en el uso de refugios, con la consiguiente pérdida de oportunidades de alimentación o los costes fisiológicos debido al tiempo pasado a bajas temperaturas en el interior de éstos (capítulo 4), suponen una disminución de la condición corporal. • La pérdida de condición corporal parece afectar en algunos casos a la capacidad de las lagartijas para desarrollar una respuesta inmune adecuada para la defensa frente a parásitos (capítulos 3 y 4), lo que puede incrementar los efectos negativos de los parásitos y afectar a la eficacia biológica de los individuos. • El uso de refugios, como estrategia antidepredatoria, puede incrementar el riesgo de depredación por culebras que cazan al acecho en el interior de los refugios (capítulo 5), por lo que el ecoturismo podría causar un incremento en la depredación debido al efecto de múltiples depredadores. Esto es debido a que dos depredadores actuando simultáneamente, y que requieren respuestas conflictivas, pueden causar una facilitación de la depredación. Por tanto, ambos depredadores obtendrían ventajas y la presa sufriría un incremento en el riesgo de depredación. Sin embargo, las lagartijas parecen valorar adecuadamente el riesgo de depredación por culebras utilizando para ello señales químicas y visuales, y abandonan antes los refugios cuando encuentran varias señales de la presencia de una culebra en el interior del refugio. Así mismo, son capaces de estimar el tiempo que las señales químicas llevan depositadas y la presencia de la culebra para no sobreestimar el riesgo. También, cuando son atacadas presentan estrategias de escape flexibles de forma que evitan el Capítulo 6 Conclusiones y perspectivas 307 uso de refugios cuando pueden eludir el ataque de otra forma (capítulo 5). • La flexibilidad en las estrategias antidepredatorias también permite a las lagartijas hacer frente a un incremento en el riesgo de depredación sin incurrir en costes excesivos de estas estrategias, no sólo en términos de posible mortalidad debido a culebras sino también en relación a los costes fisiológicos del uso de refugios (capítulo 4). De esta forma, las lagartijas que viven en zonas degradadas no permanecen tanto tiempo escondidas en el interior de los refugios como las lagartijas que viven en zonas naturales, con menor riesgo de depredación, y que no tienen que realizar esta estrategia tan frecuentemente. Además, las lagartijas con peor condición corporal reducen el tiempo pasado en los refugios después de un ataque, mientras que las lagartijas en mejor condición, que pueden asumir más costes, permanecen más tiempo escondidas (capítulo 4). • Tanto los cambios antropogénicos del medio, como la deforestación y degradación de la vegetación o el ecoturismo son dos factores que implican un incremento en el riesgo de depredación para las lagartijas, al que responden incrementando el uso de estrategias antidepredatorias. Los costes de estas estrategias afectan a la condición física de los individuos, y por tanto, pueden incrementar los efectos negativos de los parásitos. Esta pérdida de condición corporal puede repercutir en su eficacia biológica a largo plazo. Por tanto, estos dos factores podrían afectar al mantenimiento de las poblaciones de lacértidos de la Sierra de Guadarrama. Perspectivas Las principales conclusiones de esta tesis doctoral son apenás una primera aproximación al estudio del efecto que los cambios antropogénicos en el medio pueden causar en las poblaciones de lagartijas. Este estudio se ha centrado exclusivamente en el incremento en el riesgo de depredación asociado a la influencia humana y a las estrategias antidepredatorias de las lagartijas para hacer frente este riesgo y a sus costes fisiológicos asociados. Sin embargo, a lo largo de este estudio se han ido sugiriendo otros factores que podrían afectar al mantenimiento de las poblaciones. Por ejemplo, la realización Capítulo 6 Conclusiones y perspectivas 308 de estrategias antidepredatorias tiene unos costes que no han sido examinados en esta tesis, como la pérdida de oportunidades de reproducción o defensa del territorio que pueden estar afectando a las interacciones sociales de los individuos y que podrían afectar por tanto a la selección sexual. Por otro lado, la modificación del medio natural puede afectar también a las poblaciones de presas de las lagartijas. Una limitación o simplemente un cambio en la disponibilidad de presas podría afectar no sólo a la eficacia de la alimentación, sino también a la búsqueda de alimento, el tiempo de manipulación de las presas y un largo etcétera. La degradación de la vegetación también podría causar cambios en el ambiente térmico, incrementando los costes de termorregulación para los individuos, que tendrían menos tiempo para realizar otras actividades. Este cambio afectaría de manera especial a las hembras gestantes, lo que podría provocar una disminución de la eficacia biológica de los individuos. Estos son sólo unos pocos ejemplos de los factores que, además del incremento en el riesgo, podrían afectar al mantenimiento de las poblaciones de lagartijas, lo que muestra que hay mucho por hacer en este tema. Los resultados de este estudio sugieren correlaciones entre las estrategias antidepredatorias y la condición corporal de los individuos. En algunos casos se han realizado estudios de laboratorio para tratar de aislar el efecto observado, o bien las conclusiones se han basado en estudios previos que aislaban ese efecto. Por ejemplo, en el estudio del efecto del ecoturismo en la lagartija roquera, un experimento previo demostraba que una alta frecuencia de comportamientos antidepredatorios como el uso de refugios afectaba a la condición de los individuos. Sin embargo, en otros casos sería necesario realizar experimentos controlando otras posibles variables. Por ejemplo, sería necesario examinar en el caso de las hembras de P. algirus que un incremento en la magnitud de las estrategias antidepredatorias conlleva una disminución en la condición corporal. Además sería muy interesante conocer los efectos a medio y largo plazo de esta disminución en la condición corporal. Por ejemplo, se podrían realizar experimentos para ver este efecto no sólo durante la gestación Capítulo 6 Conclusiones y perspectivas 309 sino también en la puesta y supervivencia de la descendencia. Así mismo, en algunos de estos estudios se ha examinado la respuesta inmune mediada por linfocitos T de los individuos. Sin embargo los resultados no han sido concluyentes en muchos experimentos. El bajo número muestral y el desconocimiento de otros factores que pueden afectar a la respuesta inmune, como podría ser la temperatura corporal de los individuos pueden haber enmascarado posibles resultados de esta medida. Por tanto, son necesarios estudios más detallados para examinar el efecto de la degradación del medio en el estado de salud de los individuos. Los resultados de este estudio muestran que, en muchos casos, el efecto de la degradación del medio o bien del ecoturismo sobre la condición física de los individuos es notable a lo largo del período de actividad de las lagartijas, ya que a lo largo de todo este tiempo los individuos han tenido que realizar frecuentemente estrategias antidepredatorias y han ido sufriendo sus costes. Por lo tanto, sería recomendable realizar todos los experimentos durante todo el período de actividad de las distintas especies. De la misma forma, estudios a largo plazo serían no sólo interesantes sino necesarios para una valoración mejor de los efectos de los cambios en el medio sobre las poblaciones. En resumen, esta tesis doctoral aporta unos conocimientos preliminares sobre las consecuencias de la influencia humana en el comportamiento antidepredatorio y la condición física y estado de salud de los individuos de varias poblaciones de lagartijas. Los resultados que se han obtenido no hacen sino resaltar el escaso conocimiento que se tiene de estos efectos, sugiriendo la necesidad de examinar en profundidad estos y otros factores que podrían estar afectando al mantenimiento de las poblaciones de lacértidos de la Sierra de Guadarrama. Tesis Luisa Amo de Paz Portada Índice Contenidos Agradecimientos Capítulo 1 Capítulo 2 Capítulo 3 Capítulo 4 Capítulo 5 Capítulo 6