Predator-Prey Interactions Between an Invasive Crayfish Predator and Native Anurans: Ecological and Evolutionary Outcomes

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA ANIMAL

PREDATOR-PREY INTERACTIONS BETWEEN AN INVASIVE CRAYFISH PREDATOR AND NATIVE ANURANS: ECOLOGICAL AND EVOLUTIONARY OUTCOMES

ANA LUÍSA SUMARES DA CRUZ NUNES

DOUTORAMENTO EM BIOLOGIA ESPECIALIDADE DE ECOLOGIA

2011

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA ANIMAL

PREDATOR-PREY INTERACTIONS BETWEEN AN INVASIVE CRAYFISH PREDATOR AND NATIVE ANURANS: ECOLOGICAL AND EVOLUTIONARY OUTCOMES

ANA LUÍSA SUMARES DA CRUZ NUNES

TESE ORIENTADA POR:

DR. RUI MIGUEL REBELO

DR. ANSSI LAURILA

DOUTORAMENTO EM BIOLOGIA ESPECIALIDADE DE ECOLOGIA

2011

This study was funded by Fundação para a Ciência e a Tecnologia (FCT) through a PhD grant (SFRH/BD/29068/2006) and by the research project POCI/BIA-BDE/56100/2004.

This dissertation should be cited as: Nunes AL (2011) Predator-prey interactions between an invasive crayfish predator and native anurans: ecological and evolutionary outcomes. PhD Thesis, Universidade de Lisboa, Portugal.

Nota Prévia A presente tese apresenta resultados de trabalhos já publicados ou em preparação para publicação (capítulos 2 a 5), de acordo com o previsto no nº 1 do artigo 41º do Regulamento de Estudos Pós-Graduados da Universidade de Lisboa, publicado no Diário da República II série nº 209 de 30 de Outubro de 2006. Tendo os trabalhos sido realizados em colaboração, a candidata esclarece que participou integralmente na concepção dos trabalhos, obtenção dos dados, análise e discussão dos resultados, bem como na redacção dos manuscritos.

Lisboa, Novembro de 2011

Ana Luísa Sumares da Cruz Nunes

Aos papás e à mana

ACKNOWLEDGMENTS

After almost five years of hard work, finally this thesis comes to an end. I guess one of the things that it made me realise is my unbelievable capacity of never being able to achieve deadlines. However, it also allowed me to become a mini-scientist and to get to experience things that will forever be in my mind and that make me who I am today.

I start by thanking my supervisors, Rui Rebelo and Anssi Laurila. To Rui, for convincing me to apply for a PhD grant, even though I was very reluctant to start a life as a researcher at the time. I turned out to be right, science is way too scientific for me, but I cannot be happier for having done this PhD. Thank you for teaching me so many things about the natural world, for going with me to the field every time I needed, for your constant good mood but especially for, other than a supervisor, being a true good friend. To Anssi, the nicest Finnish person I have ever met, thank you for promptly accepting me as your half PhD student and for allowing me to work in Uppsala for two years. Thank you for all the chats in your office about work and many other things, from wolves and bears to the horrible Swedish weather. And for always being so patient about me handing in work late.

Thank you both so much for helping me make this thesis a nice scientific work, but especially for being always present, available and extremely funny supervisors. Throughout the discussion of the thesis I use the personal pronoun “I”, but this work is really “ours”.

To Fundação para a Ciência e Tecnologia I thank for my PhD grant (SFRH/BD/29068/2006) and for funding through the Project POCI/BIA-BDE/56100/2004. I also thank Stiftelsen för Zoologisk Forskning for funding.

To Dr. Miguel Tejedo for having received us so well in Seville and having allowed us to perform fieldwork in Doñana.

To my master students, Erika, Susana and Cátia, for making the huge experiment in the Herdade a doable thing and for going to Grândola, even at weekends, to change waters and feed the predators. Thank you for all your help in the field and in the lab.

To Pedro Andrade, the best field assistant ever, for keeping me company in Grândola so many times, for being so good in fixing problems and in building weird necessary things and for talking about crazy nonsense sometimes. You made my fieldwork extremely pleasant and easy.

To Sandra Amaral for making me go to Rui’s office many years ago and help trace a way into studying amphibians. For going with me through the hard process of learning about amphibian larvae (“Is this an Alytes or a Pelobates?”). To Maria João, for teaching me almost everything I know about amphibians and for calling me to Sweden when I needed help with statistics. Both you and Mário have been really nice friends.

To Helder, an amphibian companion, for the help with the phylogenetic trees, but mostly for sharing the love for amphibians and always being ready to talk about them. Thank you for always being ready to help and for hosting me in Seville during the congress.

To all the friends I made in Sweden, thank you so much for having made life in the north a, not only bearable, but extremely nice thing. You have allowed me to experience many incredibly nice and different situations and to live the real grad student life.

To everyone in the department, thank you for making work a very nice place, filled with friends. Thank you for the thursday pancakes, sushi lunches, friday fikas and Christmas dinners.

To Marianne, for all the help in solving ‘money issues’, you were great!

To Jan L, for always asking me about the thesis and for giving me nice hints on how a paper should be written.

To the frog group, Emma, Björn, Magnus, Rike, Sandra H, Katja, German and Alex, thanks for sharing your knowledge with me and for being nice friends. To Rike, thanks for being such a lovely office mate and for letting me feed your snails.

To the people from the Iberian (not Spanish) office, Violeta, Maria, Germán and Alex, for always speaking in english with me and for all the healthy gossip. To Germán and Alex, thank you for all the help in my papers and for showing me the wonders of doing fieldwork with frogs in Sweden. To Alex, for all the chocolates in the fika room (no more oranges for you!), for the company in the office when no one else was there and, especially, for having become a very good friend.

To the Costa Rica crew, Biao, Jonathan, Jenny and Caro, for an amazing trip to the other side of the world. Thank you, Biao, for being such an easy going person and for always being in a good mood. Thank you, Jonathan, for being my friend, despite I though you didn’t like me in the beginning and for showing me so many birds in Costa Rica. You are lovely! Jenny, the atypical Swedish girl, thank you for teaching me how to do kannelbulle and for all the shared lunches outside. To Caro, thank you for all the time spent together, during and after work, for always liking the things I cooked, for letting me get to see a black grouse (or many) and, especially, for the very nice friendship that we have.

To other people from EBC, Jürg, Maria, Omar, Yoshi, Christina, Kasia, Anna, Richard and Andreas for all the dinners, parties and beers in the Nations. To Kasia, the fortunately more PopBio than AnimEcol girl, thanks for being so nice and caring and for making me understand how much a real scientist can love her job.

To my Portuguese family in Uppsala, Tiago and Vanda, for being very close and good friends and for allowing me to not forget my mother tongue while I was away.

To my ‘Colombian’ family, Daniel C, Daniel O, Tobias, Angie, Caro, Jessica, Ricardo, Manda, Tanai, Sahar, Tobi, Anna, Ola, Claire and Sara for all the friendship and for making my stay in Uppsala much better than I could wish for. Thanks for all the shared moments, especially in our amazing trips to Sälen and Colombia. Muitossshhh obrigadossshhh, from radiosssshh portuguessshhhh. To Caro, Angie and Tobias, for always being there for me.

To my dearest friends from the “Sala dos terminais”, Ana Leal, Nuno, Tatá, Mafalda, Joana, Miguel and Luís, thank you for making it really nice to go to work, even at the final stages of this thesis (especially when it was a cake day). Thank you for giving me all the strength I needed to finish this, you were the ones who made it possible for me to hand this in now. I especially thank you for constantly asking if I needed help and for the last minute reviewing of the thesis.

To all my korfball people, for often making me think of things other than work, for all the holidays spent together and for, after all these years, still not understanding what my job was. Ralha and David, now you can finally see what ‘your’ money was used for. To my twins, Sara and Ana, thank you for coming to Grândola to help me in the field. Xiquinho, thank you for, even when I was far, always being close through the internet.

To Joana Vinagre, Henrique, Natércia and Sónia for coming all the way from Portugal to experience the ‘warm’ Swedish summer, making my first year in Uppsala much nicer, at a time I wanted to come back to Portugal. To Natércia, for always asking me about the thesis and for being there, even if through a phone call, every time I needed. To Sónia, thank you for your help changing waters and feeding tadpoles and for all the times you stayed at home keeping me company while I had to work.

To Joaninha and Ricardo, the ever present university friends, for the solid friendship and for always encouraging me to finish this fast. To Lena for, even far, still asking how the thesis was going and for chatting with me about phenotypic plasticity.

To Joana Vinagre, our long time friendship, with its highs and lows, says it all. Thank you for your constant presence in my life.

Aos meus padrinhos, Augusta e Rufino, por todos os fantásticos almoços e por sempre estarem presentes na minha vida.

Aos meus pais e à minha irmã, por serem uma fonte de inspiração de vida e por me terem feito quem sou hoje. Por me terem deixado invadir a cave deles com milhares de girinos e lagostins e ainda me terem ajudado no trabalho. Por sempre me terem apoiado em todas as fases deste doutoramento, e por nunca me terem pressionado para o terminar. Mamã, obrigada por tudo teres feito para estar aqui hoje e não te preocupes que vou poder repetir a mesma palavra duas vezes quando defender a tese.

To all the people I did not mention, but that I met through this journey, be it in congresses, in the field, in trips or in the university, thank you for being a part of this work.

TABLE OF CONTENTS

Abstract i Resumo ii

CHAPTER 1. General Introduction 1

1.1. Predator-prey interactions 3

Defensive antipredator strategies 3

- Cues used to assess predation risk 8

1.1.2. Unsuccessful predation attempts and nonlethal prey injury 10

1.2. Invasive alien species 12

1.3. Inducible responses in the presence of invasive alien predators 16

1.4. Species in focus 20

1.4.1. The invasive predator: red swamp crayfish (Procambarus clarkii) 21

1.4.2. The native prey: anurans from the Southwest of the Iberian Peninsula 24

1.5. P. clarkii and the anurans from Southwestern Iberian Peninsula 27

1.6. Goals and outline of the thesis 28

1.7. Papers presented 31

1.8. References 32

CHAPTER 2. 53 Nunes AL, Cruz MJ, Tejedo M, Laurila A, Rebelo R Nonlethal injury caused by an invasive alien predator and its consequences for an anuran tadpole.

CHAPTER 3. 77 Nunes AL, Richter-Boix A, Laurila A, Rebelo R Do anuran larvae respond behaviourally to chemical cues from an invasive crayfish predator? A community-wide study.

CHAPTER 4. 109 Nunes AL, Orizaola G, Laurila A, Rebelo R Morphological and life-history responses and integration of defensive traits in an anuran community invaded by an exotic predator.

CHAPTER 5. 145 Nunes AL, Orizaola G, Laurila A, Rebelo R Population divergence in antipredator defences against an invasive predator: does coexistence time promote the evolution of adaptive responses?

CHAPTER 6. General Discussion 173

6.1. Crayfish as an agent of tail injury in anurans 175

6.2. Phenotypic variation in Iberian tadpoles 176

6.3. Plastic responses of Iberian anurans to a native and an exotic predator 178

6.4. Implications of P. clarkii invasion for anuran communities 184

6.5. Control and management of P. clarkii and conservation of native amphibians 187

6.6. Final considerations 189

6.7. References 191

ABSTRACT

The introduction of exotic species, particularly invasive aquatic predators, is a pervasive phenomenon worldwide, largely implicated in amphibian declines. As native prey do not share a co-evolutionary history with exotic predators, they often lack effective antipredator defences, which may endanger their populations. Alternatively, due to their strong predation pressure, invaders may induce rapid evolutionary changes in invaded communities.

The red swamp crayfish Procambarus clarkii is a widespread invasive predator abundant in freshwater habitats in Southwestern Iberian Peninsula. It is an efficient predator of Iberian amphibian larvae and it can exert strong impact upon them. The main goal of this thesis was to assess the impacts of P. clarkii in fitness components of larval Iberian anurans in order to better understand variation in vulnerability of native prey to novel predators. This was done by performing a series of laboratory experiments.

P. clarkii incurred tail injury in tadpoles at very high rates, which affected tadpole morphology and survival, and may have delayed fitness costs. Seven out of nine anuran species showed predator-induced behavioural plasticity in the presence of P. clarkii, but only two showed morphological plasticity. Life-history responses, elicited by four species, were very variable and species-specific, contrarily to behavioural defences that were similar across species. More studies are needed to settle the adaptive value of these responses. Populations of Pelophylax perezi differed in antipredator defences according to coexistence time with crayfish and it is suggested that P. clarkii may have selected for rapid evolution of defences in a long-term invaded population.

Since there are no easy solutions to cope with this invasion, understanding interactions between P. clarkii and native species is critical for predicting how this invasion may alter the dynamics of natural communities and for directing conservation efforts towards the most vulnerable species, as is the case of Pelodytes ibericus, an Iberian endemic.

Keywords: Biological invasions, aquatic invasive predator, inducible defences, nonlethal injury, amphibians, Iberian Peninsula

RESUMO

A introdução de espécies exóticas, fenómeno que actualmente ocorre a um ritmo muito elevado, é considerada uma das principais ameaças para a biodiversidade global. Quando novas espécies de predadores são introduzidas fora da sua área de distribuição original podem causar impactos negativos na estrutura e funcionamento dos ecossistemas invadidos. Em particular, a invasão de ecossistemas aquáticos por grandes predadores tem sido apontada como uma das causas que mais tem contribuído para o declínio de populações de anfíbios, fenómeno que está a acontecer a nível global. Muitas vezes isto pode dever-se ao facto de as espécies de anfíbios não reconhecerem o predador introduzido e, como tal, não terem a capacidade de responder com os seus mecanismos antipredatórios naturais, que podem consistir em alterações comportamentais, morfológicas ou de parâmetros da história vital.

Estas defesas naturais aumentam a probabilidade de sobrevivência das espécies-presa perante predadores nativos e o facto de muitas vezes não serem utilizadas na presença de predadores exóticos aumenta grandemente a sua vulnerabilidade.

Grande parte dos estudos que documentam impactos negativos para espécies de anfíbios decorrentes da introdução de predadores exóticos diz respeito à introdução de peixes não nativos. No entanto, já foram também documentados os impactos negativos da introdução de lagostins exóticos. O lagostim-vermelho-americano, Procambarus clarkii, é uma espécie nativa do Centro-Sul dos Estados Unidos da América e do Nordeste do México que, com fins comerciais, foi introduzida em vários países de todo o mundo, estando actualmente estabelecida em todos os continentes, excepto na Austrália e na Antártida. Esta espécie foi introduzida pela primeira vez em território europeu em 1973, na área de Badajoz (Espanha), e no ano seguinte na zona de Sevilha, nas marismas do rio Guadalquivir. Através das bacias hidrográficas do Guadiana e do Tejo a espécie rapidamente se expandiu para Portugal, sendo actualmente muito comum e abundante em praticamente toda a Península Ibérica, sobretudo no Sul. No Sudoeste da Península Ibérica, uma região sem espécies de lagostim de água doce

ii nativas, o P. clarkii pode ser encontrado em habitats permanentes, temporários ou mesmo efémeros, muitas vezes coexistindo com várias espécies de anfíbios. Nesta área existem catorze espécies de anfíbios, dez das quais são anuros. São estes a rã verde (Pelophylax perezi), a rela meridional (Hyla meridionalis), a rela comum (Hyla arborea), o sapo comum (Bufo bufo), o sapo corredor (Bufo calamita), o sapinho de verrugas verdes ibérico (Pelodytes ibericus), o sapinho de verrugas verdes (Pelodytes punctatus; não estudado aqui), o sapo de unha negra

(Pelobates cultripes), a rã de focinho pontiagudo (Discoglossus galganoi) e o sapo parteiro ibérico (Alytes cisternasii). Sabe-se que o P. clarkii é um predador eficaz de ovos e larvas de todas as espécies de anfíbios existentes nesta área, podendo também infligir-lhes danos sub- letais, como cortes nas caudas. A sua presença parece ter fortes consequências negativas para estas comunidades de anfíbios, tendo sido registada uma diminuição da riqueza específica de anfíbios em diversas áreas invadidas.

Embora existam estudos anteriores que já demonstraram alguns dos efeitos negativos que a introdução de P. clarkii acarreta para as comunidades de anfíbios do Sudoeste da Península, nenhum deles examinou os possíveis impactos indirectos que a presença de P. clarkii pode ter sobre larvas de anfíbios desta área. Assim, esta tese teve como principal objectivo avaliar que tipo de efeitos não letais pode o lagostim exercer nas formas larvares de nove das dez espécies de anuros existentes no Sudoeste da Península Ibérica e que consequências podem os mesmos ter para a fitness dos indivíduos. Os resultados permitirão entender melhor que espécies de anuros são mais vulneráveis a invasões deste predador.

Foi avaliado o papel do P. clarkii como agente causador de elevadas frequências de cortes na cauda em girinos de Pelobates cultripes e investigado em laboratório que tipo de consequências podem esses danos ter para um girino nas suas taxas de mortalidade, crescimento e desenvolvimento. Foi também realizada uma experiência em laboratório na qual larvas de todas as espécies de anuros do Sudoeste da Península foram sujeitas à presença de pistas químicas de P. clarkii, associadas ou não a cheiros de conspecíficos predados, com o

iii intuito de investigar se os girinos apresentam ou não respostas antipredatórias

(comportamentais, morfológicas ou na história vital) a este predador exótico. Estas respostas foram comparadas com as apresentadas face a um predador nativo, larvas de libélula (Género

Aeshna). Foi ainda efectuada uma outra experiência, na qual girinos de cinco populações de

Pelophylax perezi com diferentes histórias de coexistência com o P. clarkii foram expostos à presença deste predador, de modo a testar se em populações com uma mais longa história de contacto com o P. clarkii são induzidas respostas antipredatórias diferentes das induzidas em populações com menor ou nenhum tempo de coexistência com este predador.

No Parque Natural de Doñana, a frequência média de girinos de P. cultripes capturados com cortes na cauda foi significativamente maior em locais onde o P. clarkii estava presente. A perda de porções de cauda causou uma ligeira diminuição na sobrevivência dos girinos, mas não afectou as suas taxas de crescimento e desenvolvimento, embora possa haver custos tardios que não foram aqui detectados. As lesões na cauda causaram uma alteração na morfologia caudal dos girinos após a regeneração, com os girinos lesionados a desenvolverem músculos caudais mais altos e barbatanas caudais mais baixas. Estes efeitos variaram com a frequência de corte e disponibilidade de alimento, tendo tido maior expressão nos indivíduos mais alimentados.

A presença de lagostim, sobretudo quando associada a pistas químicas de conspecíficos predados, induziu alterações no nível de actividade e na distância ao predador em girinos de sete das nove espécies estudadas. Quatro espécies alteraram parâmetros da sua história vital, enquanto apenas duas apresentaram alterações morfológicas em resposta ao predador exótico. Enquanto as respostas comportamentais apresentadas pelas várias espécies foram muito semelhantes, as alterações observadas na história vital variaram muito de espécie para espécie, indicando respostas específicas a este predador consoante a espécie-presa em causa.

Por outro lado, quando expostas ao predador nativo, oito das nove espécies mostraram plasticidade fenotípica em vários dos parâmetros estudados. Foi também encontrada uma

iv maior integração nas respostas apresentadas quando as espécies se encontravam na presença do predador nativo do que do exótico.

As populações de P. perezi com diferentes histórias de contacto com o P. clarkii diferiram nas respostas antipredatórias apresentadas. Enquanto os girinos de populações que nunca coexistiram com este predador diminuíram os níveis de actividade na sua presença, os girinos de populações com mais de trinta anos de coexistência não o fizeram. Em vez disso, alteraram a sua morfologia para uma forma que melhora a sua capacidade de fuga à predação por P. clarkii, o que parece indicar que houve, neste caso, evolução rápida de respostas ao lagostim e adaptação local à sua presença.

Este trabalho permitiu concluir que em sete das nove espécies de anuros do Sudoeste da

Península Ibérica aqui estudadas foram induzidas respostas antipredatórias na presença não letal de um lagostim exótico. Assumindo que as respostas são efectivas na redução da vulnerabilidade ao lagostim, estas sete espécies parecem estar mais aptas a sobreviver e resistir à invasão dos habitats aquáticos por P. clarkii. De qualquer forma, é essencial a realização de estudos futuros que permitam entender se as respostas aqui apresentadas conferem, de facto, vantagens para a sobrevivência a este predador. Por outro lado, as espécies que não responderam a P. clarkii serão potencialmente mais vulneráveis à predação por este lagostim e estarão, a longo prazo, sujeitas a declínios populacionais ou mesmo extinções locais. Pelodytes ibericus é um destes casos, o que a torna numa espécie cujas populações deveriam ser monitorizadas, tendo em conta o seu estatuto endémico no

Sudoeste da Península Ibérica e a sua distribuição relativamente limitada.

Uma vez que P. clarkii se encontra actualmente largamente distribuído pela Península Ibérica e que a sua erradicação é impossível com os meios actuais, a implementação de medidas que permitam a coexistência entre este predador exótico e as comunidades nativas de anfíbios é uma das poucas opções que resta. Neste sentido, estudar a dinâmica das interacções entre

v predadores exóticos e presas nativas, assim como identificar as espécies mais vulneráveis a invasões e com diferentes potenciais para evolução rápida de defesas antipredatórias podem ser ferramentas fundamentais para que as populações-presa persistam em áreas invadidas.

Palavras-chave: Invasões biológicas, predador aquático introduzido, plasticidade fenotípica, predação subletal, anfíbios, Península Ibérica

CHAPTER 1

General Introduction

Chapter 1

1. GENERAL INTRODUCTION

1.1. Predator-prey interactions

Predation is a major selective force influencing the dynamics, structure and evolutionary processes of prey communities (Lima and Dill 1990, Johnson and Agrawal 2003). Predators affect dynamics of prey populations not only by direct predation, but also by imposing direct or indirect nonlethal effects upon them (Lima 1998, Preisser et al. 2005, Sih et al. 2010). Direct nonlethal effects result from prey employing costly defensive antipredator strategies (Lima

1998, Preisser et al. 2005) or from consequences of injury caused by incomplete or unsuccessful predation attempts (Morin 1985, Harris 1989). Predators may also impact prey communities via indirect effects propagated through the trophic web that end up impacting the focal prey species (Wilbur 1997, Peacor and Werner 2001, Werner and Peacor 2003). In fact, nonlethal predatory effects have been suggested to exert as strong or stronger effects than direct consumption in the dynamics of prey populations, and this seems to be particularly evident in aquatic ecosystems (Preisser et al. 2005, Werner and Peacor 2006).

Defensive antipredator strategies

In order to evade predation risk, prey have developed a variety of predator avoidance defences. These defences can be constitutive and expressed at all times, or inducible and only expressed when predators are present. Constitutive defences, which are permanently expressed, are elicited when predation risk is stable over time (Clark and Harvell 1992, Edgell et al. 2009). On the contrary, inducible defences are expressed in response to spatially or temporally variable predation risk and depend on the existence of cues that allow prey to accurately detect the risk of predation. Further, inducible defences often incur fitness costs in

Chapter 1 the absence of predators, which prevents them from becoming constitutive (Harvell 1990,

Clark and Harvell 1992, Tollrian and Harvell 1998).

Inducible defences are a widespread form of phenotypic plasticity, the capacity of an individual to alter its phenotype in response to alterations in environmental biotic or abiotic factors

(Pigliucci 2001, DeWitt and Scheiner 2004, Whitman and Agrawal 2009). Phenotypic plasticity is ubiquitous and has been documented in many organisms (reviewed in Tollrian and Harvell

1998, Sultan 2000, Pigliucci 2001, DeWitt and Scheiner 2004, Whitman and Agrawal 2009).

Through predator-induced plasticity prey express phenotypic alterations in behaviour, morphology, life-history or physiology which, by reducing vulnerability to predators, often act as adaptive antipredator defences (Tollrian and Harvell 1998, Agrawal 2001). Inducible antipredator defences are very variable and occur in a diversity of taxa. Some of the best known examples include the production of defensive chemicals in plants (Berenbaum and

Zangerl 1998), diel vertical migration in cladocerans (e.g. Bollens et al. 1992, Boersma et al.

1998), formation of defensive structures such as helmets and neck teeth in cladocerans (e.g.

Spitze 1992, Boersma et al. 1998), changes in shell shape and thickness in molluscs (e.g.

Trussell and Smith 2000, Hoverman and Relyea 2007), alterations in body shape in fish (e.g.

Brönmark and Pettersson 1994) and alterations in behaviour and morphology in anuran tadpoles (e.g. McCollum and Van Buskirk 1996).

Many experimental studies show that predator-induced phenotypic plasticity is widespread in larval amphibians (reviewed in Lima and Dill 1990, Kats and Dill 1998, Benard 2004). In response to strong predation pressure, many amphibian larvae alter their behaviour, morphology, physiology or life-history. While little attention has been devoted to the study of physiological plasticity (but see Steiner and Van Buskirk 2008), plasticity in behaviour, morphology and life-history in response to predator cues has been thoroughly explored in this group.

Chapter 1

Behavioural defensive strategies in tadpoles may consist in aggregation and formation of schools (e.g. Watt et al. 1997, DeVito 2003, Spieler 2003), a shift to safer microhabitats (e.g.

Cruz and Rebelo 2005), spatial avoidance of the predator (e.g. Laurila et al. 1997, Relyea 2001,

Nicieza et al. 2006) or a reduction in activity level (e.g. Skelly and Werner 1990, Skelly 1992,

Griffiths et al. 1998, Anholt et al. 2000, Relyea 2001, 2004, Richardson 2001, Van Buskirk 2002,

Richter-Boix et al. 2007, Laurila et al. 2006, 2008). All these behavioural alterations benefit tadpoles, as they reduce the frequency of predator-prey encounter rates or of predator attacks, which increases their odds of survival (Lima and Dill 1990, Kats and Dill 1998, McPeek

2004). Specifically, a decrease in activity level is one of the most commonly reported behavioural defences against predators and seems to be an important mechanism decreasing predation rates (Lima and Dill 1990, Van Buskirk and McCollum 2000a). This defence may be so widespread because movement is generally used by predators to detect prey, usually causing a positive correlation between activity level and mortality risk (Werner and Anholt 1993, Skelly

1994). As such, there should be strong selection against tadpoles having high activity levels in the presence of predators, in order to minimize the chances of prey detectability and of predators incurring fatal attacks.

Plastic alterations in morphology are also pervasive responses to predation risk in larval amphibians (e.g. McCollum and Van Buskirk 1996, McCollum and Leimberger 1997, Van

Buskirk et al. 1997, Van Buskirk and McCollum 2000a, Lardner 2000, Relyea and Werner 2000,

Relyea 2001, 2004, Van Buskirk 2002, LaFiandra and Babbitt 2004, Laurila et al. 2006, 2008,

Richter-Boix et al. 2007, Van Buskirk 2009, Hettyey et al. 2010). These usually consist in an increase in tadpole’s tail depth, an enhancement of distal tail coloration, and the development of smaller headbodies. Although the functionalities of these traits are not completely understood, smaller headbodies and deeper tails may enhance swimming ability, enable tadpoles to generate faster swimming bursts and improve manoeuvrability (McCollum and

Leimberger 1997, Dayton et al. 2005; but see Van Buskirk and McCollum 2000b, Johnson et al.

Chapter 1

2008). Deeper and more brightly coloured tails may lure predator strikes away from the headbody into the more regenerable tail and may also facilitate membranous tail ripping when a predator attack takes place (Doherty et al. 1998, Van Buskirk et al. 2003). These predator- induced alterations in morphology increase the ability to escape from attacking predators and enhance the probability of survival under predation risk (McCollum and Van Buskirk 1996, Van

Buskirk et al. 1997, Van Buskirk and McCollum 2000a, Dayton et al. 2005, Álvarez and Nicieza

2006, Johnson et al. 2008, Hettyey et al. 2010).

Frequently, the expression of behavioural and morphological defences entail growth and development costs for prey, due to either low foraging rates or a high investment in production and maintenance of defensive structures (e.g. Skelly 1992, McCollum and Van

Buskirk 1996, Van Buskirk 2000, Relyea 2002, LaFiandra and Babbitt 2004, Steiner 2007a). This leads to the growth/predation risk trade-off hypothesis, which states that increasing defences in order to avoid predation risk often comes at the expense of reduced growth (e.g. Werner and Anholt 1993, Werner 1986, Lima and Dill 1990, Lima 1998, McPeek 2004, Stoks et al.

2005). Further, this often results in reduced body size and/or delayed metamorphosis which can decrease future fitness by reducing viability during harsh conditions, especially in seasonal environments, delaying maturity and reducing size at first reproduction and reproductive success (Smith 1987, Semlitsch et al. 1988, Berven 1990).

In the presence of predators, phenotypic plasticity in prey life-history may either derive from predator-induced alterations in behaviour or morphology or may result from direct responses to predation risk (Beckerman et al. 2007, Relyea 2007, Steiner 2007b, Higginson and Ruxton

2009). In organisms with complex life histories, such as amphibians, predation risk can alter the timing of major life-history transitions, by shaping both hatching and metamorphic decisions. Amphibians have been shown to alter the timing of hatching in order to reduce the risk of predation, either by accelerating or delaying it (e.g. Johnson et al. 2003, Warkentin

2011). Both time to and size at metamorphosis can also experience plastic adjustments in

Chapter 1 response to predation pressure (reviewed in Benard 2004, Relyea 2007, Tejedo et al. 2010).

These traits are critical life-history attributes that greatly determine post-metamorphic fitness in amphibians (Smith 1987, Semlitsch et al. 1988, Berven 1990). In response to predatory cues, amphibians can shorten their time to metamorphosis and, by leaving the risky predator environment earlier, increase their chances of survival (Babbitt and Tanner 1998, Benard 2004,

Moore et al. 2004, Higginson and Ruxton 2009). Nevertheless, a reduced time to metamorphosis seems to be a quite rare response, maybe because shorter larval periods may induce smaller metamorphic sizes which, in turn, can lead to reduced post-metamorphic fitness (Relyea 2007). Increasing larval growth rate and size at metamorphosis can also be a direct and effective response to predation, since large size can provide a safe refuge from gape or size-limited predators (Urban 2007). This can also be advantageous because larger sizes at metamorphosis are associated with larger sizes at maturity, increased fecundity and/or reproductive success (Smith 1987, Semlitsch et al. 1988, Berven 1990).

A number of theoretical studies have predicted how predation risk should alter the time and size at which prey metamorphose (e.g. Wilbur and Collins 1973, Werner 1986, Rowe and

Ludwig 1991, Abrams and Rowe 1996, Higginson and Ruxton 2010) but, except for the most recent study, their predictions (metamorphosis earlier and at a smaller size) rarely match with empirical results (Benard 2004, Relyea 2007). This indicates that predator-induced alterations in life-history traits are complex and highly variable, which probably reflects the fact that these alterations are often costs of plastic behavioural or morphological defences (Relyea 2007,

Steiner 2007b).

Often, behavioural and morphological defences, as well as life-history trait alterations, are not just direct and independent responses to predation, but occur simultaneously and largely influence each other (Lima 1998, Van Buskirk and McCollum 2000a, Steiner 2007b). These traits are often functionally correlated and express complex patterns of association, indicating phenotypic trait integration (Relyea 2001, Steiner 2007b). The degree of phenotypic

Chapter 1 integration will then depend on the magnitude of the functional relationships among traits

(Relyea 2001). Many different and complex associations between activity, tail depth, growth rate, body size and larval period are possible and these relationships are frequently not linear

(Relyea 2001, Benard 2004, Steiner 2007b, Laurila et al. 2008, Higginson and Ruxton 2009). For example, behavioural and morphological defences may complement each other, but they may also augment each other, since a reduction in activity may save energy, which can then be used to develop morphological defensive structures (Johansson and Andersson 2009). Further, although inducible defences may incur fitness costs, other studies have, in contrast, shown that either reducing activity or inducing morphological defences in the presence of predators may also result in increased growth rates (Spitze 1992, Peacor 2002, Johansson and Andersson

2009). As a result, relationships between different traits are very variable and several different outcomes may result from predator-induced plastic trait alterations.

In amphibians, antipredator defences induced by prey can change through ontogeny (e.g. Kats and Dill 1998, Eklöv and Werner 2000, Relyea 2003, Laurila et al. 2004, Hettyey et al. 2010).

During development, larvae experience periods of different vulnerability to predation - due, for instance, to ontogenetic changes in body size - which may require different types and magnitudes of antipredator defenses (Werner and Anholt 1993, Relyea 2003). Generally, prey vulnerability and intensity of responses to predators decrease as prey size increases, the strongest responses being elicited early in tadpole development (Eklöv and Werner 2000,

Relyea 2003, Laurila et al. 2004).

Cues used to assess predation risk

In order for prey to accurately assess predation risk and to elicit adequate antipredator defences, reliable cues providing information on the potential predator have to be available. In aquatic systems, prey use a variety of visual, tactile or chemical cues separately or in different

Chapter 1 combinations to evaluate and adjust to predation risk (Lima and Dill 1990, Smith 1992, Kats and Dill 1998). In these environments, chemical cues acquire special relevance as a reliable source of information, because they allow prey to detect predators from a safe distance and because they are effective in low-visibility conditions, such as at night, in turbid waters, or in densely vegetated habitats (Kats and Dill 1998, Petranka and Hayes 1998, Ferrari et al. 2010).

Chemically-mediated predator detection is widespread among aquatic animals, such as invertebrates, fish and amphibians (reviewed in Chivers and Smith 1998, Kats and Dill 1998,

Wisenden 2003, Ferrari et al. 2010). The chemical cues to which prey respond may arise from predator-specific odours (predator kairomones), may be cues actively or passively released by disturbed or injured prey (disturbance or alarm cues) or may be digestive by-products released after predators consume prey (diet cues) (Chivers and Smith 1998, Wisenden 2003,

Schoeppner and Relyea 2005, 2009, Fraker et al 2009, Ferrari et al. 2010).

Predator kairomones are chemical signals resulting from the predator’s scent itself, which are received by and favour prey, but do not benefit the predator (Kats and Dill 1998, Ferrari et al.

2010). They are important for prey to obtain information about predation risk and often allow for antipredator responses to be elicited in order to evade predators (e.g. Petranka and Hayes

1998, Pettersson et al. 2000, Bryer et al. 2001, Gallie et al. 2001, Van Buskirk and Arioli 2002,

Vilhunen and Hirvonen 2003). Disturbance cues consist in chemical signals released by disturbed or distressed prey prior to being captured, and seem to be quite widespread among aquatic taxa (reviewed in Ferrari et al. 2010). When these cues are detected by other prey, they usually induce weaker antipredator responses than alarm cues do, because they indicate less risk (Chivers and Smith 1998, Gonzalo et al. 2010). Alarm cues are chemicals released by prey upon mechanical damage caused by a predator during a predation event (Chivers and

Smith 1998, Ferrari et al. 2010). They are produced by specialised skin cells and/or released through a regulated secretory process, inform other (conspecific or not) prey about the presence of an active and dangerous predator in the environment and invoke antipredator

Chapter 1 responses (Chivers and Smith 1998, Fraker et al. 2009, Ferrari et al. 2010). Several aquatic organisms have been shown to respond to damage-released alarm cues, probably because they provide an early warning of a predator’s presence and facilitate responses to a wide array of predators (reviewed in Chivers and Smith 1998, Wisenden 2003, Ferrari et al. 2010). Diet cues are post-digestion chemicals released by the predator’s digestive system during defecation, most likely resultant from prey tissues or prey alarm cues modified by the predator’s digestive enzymes (Wisenden 2003, Schoeppner and Relyea 2009, Ferrari et al.

2010). Diet cues also induce plastic alterations in prey and several studies have shown that many prey species only elicit antipredator responses if the predator has been fed conspecifics of the focal prey species (e.g. Stirling 1995, McCollum and Leimberger 1997, Slusarczk 1999,

Schoeppner and Relyea 2005, 2009). Nevertheless, many of these studies cannot distinguish if prey are responding to either damage-released alarm cues, to a combination of alarm cues and predator-specific odours or to diet-released cues; they can only assure that cues from predation events mediate the responses (but see e.g. LaFiandra and Babbitt 2004, Schoeppner and Relyea 2009). Still, cues resulting from digestion seem to be very important for some prey species to induce complete and effective antipredator defences (Schoeppner and Relyea

2009). Further, by using cues that provide information about both the identity of the predator and of the consumed prey species, prey will more finely assess predation risk, which can be beneficial, since overresponding to non dangerous threats would divert energy away from other fitness-related activities.

Unsuccessful predation attempts and nonlethal prey injury

Predators are often unsuccessful in their attempts to capture prey (Morin 1985, Abrams 1989).

Most often these failed or incomplete predation attempts result in sublethal damage being inflicted to the attacked prey. Nonlethal injury is conspicuous in nature and can, in the long

Chapter 1 term, depress prey survival (Harris 1989). A particular and common form of predator-inflicted injury consists in autotomy, the self-induced release of a body part as an escape strategy to predator attacks, a phenomenon observed in animals such as damselflies, salamanders and lizards (Maginnis 2006).

In freshwater habitats, it is common to find amphibian prey injured due to ineffective predation attempts (Wilbur and Semlitsch 1990, Bowerman et al. 2010). Specifically, anuran larvae are often found with heavily damaged tails and the pattern and severity of damage seems to vary from species to species (Wilbur and Semlitsch 1990, Tejedo 1993, Axelsson et al.

1997, McCollum and Leimberger 1997, Blair and Wassersug 2000, Nyström et al. 2001). In fact, tail loss in larval anurans may be an important mechanism in reducing the effects of predation, because the high fragility of their tail fin allows it to be easily torn by predators, helping tadpoles survive otherwise lethal attacks (Wilbur and Semlitsch 1990, Doherty et al. 1998, Van

Buskirk et al. 2003).

Still, caudal injury may incur substantial short and long term costs to amphibians. Short term costs consist in a decreased tail length and consequent reduced swimming ability, since tail length affects tadpole swimming speed (Wassersug and Sperry 1977, Figiel and Semlitsch

1991, Teplitsky et al. 2005; but see Van Buskirk and McCollum 2000b). The diminished swimming capacity may affect escape abilities and increase vulnerability to predation and the odds of tadpoles suffering further damage (Semlitsch 1990, Figiel and Semlitsch 1991, Teplitsky et al. 2005). Tail loss may also significantly reduce fitness in the short term, because the process of tail regeneration that follows increases energetic expenditure and diverts energy away from growth and development into the repair of the damaged tissues (Wassersug and

Sperry 1977, Semlitsch and Reichling 1989, Van Buskirk and McCollum 2000b). Nonlethal caudal injury may also potentially incur long term costs in terms of reduced larval growth, development and survival (Morin 1985, Semlitsch and Reichling 1989, Semlitsch 1990, Wilbur and Semlitsch 1990, Parichy and Kaplan 1992). As the tadpole tail is resorbed and its materials

Chapter 1 reinvested during metamorphosis, delayed costs of the loss of tail tissue may only arise at metamorphosis. Tadpoles with injured tails may emerge later and at a smaller size, which can have severe consequences for post-metamorphic fitness (Smith 1987, Semlitsch et al. 1988,

Berven 1990). Still, consequences of tail injury seem to be quite variable and depend on both resource availability and injury frequency and severity (Parichy and Kaplan 1992).

1.2. Invasive alien species

Invasive alien species are species introduced outside their natural range, which populations grow successfully and get established in the new habitat and that often become a nuisance and a threat to native biota (Parker et al. 1999, Mack et al. 2000, Richardson et al. 2000,

Lockwood et al. 2007). The rate at which novel species are introduced into new environments has reached an unprecedented rate and is continuously increasing (Vitousek et al. 1997,

Thompson 1998, Strauss et al. 2006). While spread of invasive species may be due to natural processes, most dispersion across biogeographical barriers is a direct consequence of human activities such as agriculture, aquaculture, recreation and global commerce (Lodge 1993,

Vitousek et al. 1997, Mack et al. 2000, Sakai et al. 2001, Lockwood et al. 2007).

The introduction and spread of non-native species into new regions is of global concern, because it poses a threat to natural ecological communities and is nowadays one of the major challenges facing global biodiversity (Lodge 1993, Vitousek et al. 1997, Mack et al. 2000, Sala et al. 2000). Invasive species may alter native communities in many ways, acting as novel predators, vectors of pathogens, parasites and competitors of natives or by promoting hybridization with indigenous species (Mack et al. 2000, Sakai et al. 2001, Mitchell et al. 2006).

By creating novel ecological contexts, introduced species alter the structure and functioning of invaded ecosystems, often causing irreversible environmental damage which results in the displacement or extirpation of native species (Vitousek et al. 1997, Parker et al. 1999, Mack et

Chapter 1 al. 2000, Sakai et al. 2001, Clavero and García-Berthou 2005, Schlaepfer et al. 2005, Mitchell et al. 2006, Salo et al. 2007). Examples of invasions causing decline or extinction of local native species have been documented worldwide. Among the most ecologically destructive are the introduction of the Asian chestnut blight fungus (Cryphonectria parasitica) in eastern United

States, water hyacinth (Eichornia crassipes) in tropical lakes and rivers all around the world, crayfish plague (Aphanomyces astaci) in Europe and cane toad (Rhinella marina) in Australia, all considered by the IUCN Invasive Species Specialist Group to be among the 100 worst invaders in the world (http://www.issg.org/database/welcome).

The introduction of alien predators, in particular, can have significant negative impacts on native prey, not only at an individual or population levels, but often at community and ecosystem levels (e.g. Townsend 1996, Mack et al. 2000, Kats and Ferrer 2003, Salo et al.

2007). Predator introductions are most dramatic on island ecosystems, where prey often lack historical coexistence with certain predator archetypes (sensu Cox and Lima 2006). The major declines and extinctions of native mammals in Australia caused by the introduction of red foxes (Vulpes vulpes) and feral cats (Felis catus) illustrate this well (Dickman 1996). Also, the introduction of the exotic brown tree snake (Boiga irregularis) on the island of Guam has had an enormous negative impact on native reptiles, birds and mammals (Rodda et al. 1997).

Freshwater ecosystems seem to be especially vulnerable to introduced predators, which may reflect multiscale widespread prey naiveté towards invasive predators in these habitats (Sala et al. 2000, Cox and Lima 2006). These systems have been particularly affected by predator invasions, one reason for that being the stocking of freshwater habitats throughout the world with non-native fish predators for recreational purposes or elimination of pests and which often result in serious negative impacts for native biota (Mooney and Cleland 2001, Cox and

Lima 2006, Gherardi 2007a).

Chapter 1

Crayfish have also been reported as high-impact freshwater invaders which, due to their omnivorous diet (on detritus, algae, plants, invertebrates, amphibians and fish), act as keystone species, often having severe negative effects at multiple trophic levels in freshwater food webs (Lodge et al. 1994, Nyström et al. 1996, Nyström et al. 2001, Dorn and Wojdak

2004, Rodríguez et al. 2005, Gherardi and Acquistapace 2007). In Europe, there are only five native crayfish species, all from the family Astacidae; however, several other crayfish species, mostly indigenous from North America (mainly family Cambaridae), have been introduced into

European inland waters (Lodge et al. 2000, Holdich and Pöckl 2007, Holdich et al. 2009). These introductions were often deliberate and due to commercial reasons (stocking or aquaculture) or pet trade (Lodge et al. 2000, Gherardi 2006, Holdich et al. 2009). Invasive crayfish have greatly contributed to marked declines in European crayfish species because they have facilitated the spread of the crayfish plague in Europe, a disease caused by an oomycete

(Aphanomyces astaci), which does not affect North American species, but causes severe kill offs in native species and also because of their superior competitive abilities (Lodge et al. 2000,

Holdich et al. 2009). Nowadays the invasive crayfish species with the widest distribution and of most concern in Europe are Orconectes limosus (spiny-cheek crayfish), Pacifastacus leniusculus

(signal crayfish) and Procambarus clarkii (red swamp crayfish) (Holdich et al. 2009).

Amphibians are very vulnerable to aquatic invasions and the introduction of exotic predators has been implicated in many amphibian declines (reviewed by Kats and Ferrer 2003). In fact, alongside habitat destruction, pollution and the emergence of infectious diseases, this is one of the main factors contributing to the worldwide amphibian declines and extinctions

(reviewed in Alford and Richards 1999, Blaustein and Kiesecker 2002, Collins and Storfer 2003).

The introduction of non-native amphibians and reptiles outside their native range, even though often overlooked, has been shown to have negative consequences for native amphibian populations (Scalera 2007, Polo-Cavia et al. 2010, Shine 2010). American bullfrogs

(Lithobates catesbeianus) are of special interest because, despite originating from the United

Chapter 1

States, they are now widespread all over the world and, where introduced, have been implicated in declines or displacement of indigenous amphibian species (Kiesecker and

Blaustein 1997, 1998, Kats and Ferrer 2003, Pearl et al. 2004, Adams and Pearl 2007).

In most cases, however, impacts have been caused by fish predators, recognized as major contributors to drastic reductions and, at times, extirpation of native amphibian populations

(reviewed by Kats and Ferrer 2003; e.g. Finlay and Vredenburg 2007). For example, the introduction of mosquitofish has contributed to the strong reduction of several amphibian species around the world: Taricha torosa in Southern California (Gamradt and Kats 1996),

Lissotriton helveticus and Mesotriton alpestris in Europe (Denoël et al. 2005) and Litoria aurea in Australia (White and Pyke 1996, Hamer et al. 2002). Amphibians from mountainous areas seem to be particularly affected by fish invasions, since many studies have shown that the introduction of exotic fish into montane lakes for recreational purposes has caused a decrease in amphibian abundance and diversity in several regions (e.g. Spain: Braña et al. 1996; North

America: Pilliod and Peterson 2001, Knapp 2005, Pearson and Goater 2008, Pope 2008, Pilliod et al. 2010).

Crayfish have also been reported as important contributors to declines of native amphibians

(Kats and Ferrer 2003). The introduced signal crayfish (Pacifastacus leniusculus) predates on embryonic and larval amphibians and its presence seems to have caused negative impacts in invaded amphibian populations in southern Sweden (Axelsson et al. 1997, Nyström and

Abjörnsson 2000, Nyström et al. 2001, Nyström et al. 2002). The introduction and spread of the exotic red swamp crayfish (Procambarus clarkii) in mountain streams in California has been indicated as the most likely cause for the declines in populations of the California newt Taricha torosa. These declines were probably due to efficient predation by crayfish on newt eggs and larvae, increased rates of tail injury and decreased amphibian breeding activity in sites colonized by the crayfish (Gamradt and Kats 1996, Gamradt et al. 1997). Laboratory experiments have shown P. clarkii to consume eggs and larvae of several native European

Chapter 1 amphibians (Gherardi et al. 2001, Renai and Gherardi 2004, Cruz and Rebelo 2005, Cruz et al.

2006b). Further, many field studies have documented a negative association between the presence of some amphibian species and this exotic crayfish in Europe (Beja and Alcazar 2003,

Rodríguez et al. 2005, Cruz et al. 2006a,b, 2008, Ficetola et al. 2011).

1.3. Inducible responses in the presence of invasive alien predators

For prey to detect and recognise predators and elicit adequate antipredator mechanisms there needs to be a shared evolutionary history between predator and prey (Strauss et al. 2006, Sih et al. 2010). When invasive predators colonise areas outside their historical geographic range, new predator-prey interactions are established and the lack of evolutionary experience with the invader may render native prey unable to recognise the novel predator as a dangerous threat. Prey evolutionary naïveté may then either cause a failure to respond to novel predators or result in inappropriate antipredator defences, compromising their ability to avoid predation by exotic predators (Cox and Lima 2006, Strauss et al. 2006, Sih et al. 2010). The degree of such naïveté can be higher if the phylogenetic and ecological similarity between the invader and native predators is low, which may increase the impact that the introduction has in the invaded ecosystem (Cox and Lima 2006, Strauss et al. 2006, Sih et al. 2010). Hence, a novel predator very distantly or not at all related to native predators is very likely to cause a high impact in native prey communities (Ricciardi and Atkinson 2004, Strauss et al. 2006, Sih et al.

2010).

Several studies have reported an inability of native prey to elicit antipredator responses when exposed to cues from introduced predators (e.g. Gamradt and Kats 1996, Hutson et al. 2005,

McLean et al. 2007, McEvoy et al. 2008, Polo-Cavia et al. 2010, Gomez-Mestre and Diaz-

Paniagua 2011). This lack of responses is especially likely to occur when exotic predators invade systems that have historically lacked predators, such as some oceanic islands or alpine

Chapter 1 lakes (Braña et al. 1996, Rodda et al. 1997, Blackburn et al. 2004, Knapp 2005). At times, animals also show ineffective or inappropriate responses towards exotic predators (Sih et al.

2010). This is the case of the native European water vole Arvicola terrestris, which responds to the introduced American mink Neovison vison by taking refuge in a burrow, which results as an ineffective response against the exotic predator (Macdonald and Harrington 2003). Surplus killing events of native mammals and birds by foxes (Vulpes vulpes) in Australia have been also suggested to be largely the result of ineffective antipredator defences (Short et al. 2002). This lack of or maladaptive responses to invaders may be a major contributor to heavy native prey mortality in the face of non-native predators and to the dramatic impacts often observed following invasions (Cox and Lima 2006, Sih et al. 2010).

In contrast, some prey species are able to identify alien predators as potential threats and elicit effective antipredator avoidance responses when exposed to their cues. This ability of responding with defences to unnatural predators probably plays a key role in enabling native prey and invasive predators to successfully coexist in the same environment. Alterations in prey behaviour, morphology and life-history in response to exotic predators have been documented in a wide variety of organisms, such as cladocerans (e.g. Cousyn et al. 2001, Fisk et al. 2007), bivalves (e.g. Freeman and Byers 2006, Whitlow et al. 2003, Whitlow 2010), snails

(e.g. Trussel and Smith 2000, Edgell et al. 2009), fish (e.g. Nannini and Belk 2006, McLean et al.

2007, Stuart-Smith et al. 2008, Rehage et al. 2009), amphibians (e.g. Kiesecker and Blaustein

1997, 1998, Griffiths et al. 1998, Pearl et al. 2003, Marquis et al. 2004, Moore et al. 2004,

Bosch et al. 2006, Epp and Gabor 2008, Gall and Mathis 2010), birds (e.g. Peluc et al. 2008) and mammals (e.g. Russel and Banks 2007, Sündermann et al. 2008). Although many of these studies do not clearly address which process allows for prey to induce phenotypic alterations in the presence of novel predators, this can be achieved through innate, learned or evolved mechanisms.

Chapter 1

Prey may innately detect unnatural threats and elicit suitable avoidance mechanisms against introduced predators in the absence of a common evolutionary history when the exotic predator is sufficiently similar to a native predator, if prey show generalized antipredator responses or if they rely on general predation cues to evaluate and respond to predation risk

(Rehage et al. 2009). An invasive alien predator which is closely phylogenetically or phenotypically similar to native predators, may also be ecologically similar (e.g. in terms of cues or predation technique), which promotes prey responses to the novel predator (Ferrari et al. 2007, Epp and Gabor 2008, Rehage et al. 2009, Sih et al. 2010, 2011). Innate responses to non-native predators may also occur if prey exhibit generalized antipredator responses, whereby exposure to different types of predation risk induce the development of the same defences. Examples of this include avoidance responses to any large moving animal or to a general fish odour (Magurran 1990, Gherardi et al. 2011). Probably many times this is verified because some prey rely on general predation cues to evaluate and respond to predation risk.

In aquatic systems, general cues usually consist in disturbance or damage alarm cues released by stressed or captured prey or in chemicals resulting from the predator’s digestion (reviewed in Chivers and Smith 1998, Ferrari et al. 2010). If, in the face of predation events, the presence of an invasive predator is associated with these general cues, predators may become chemically ‘labelled’ by their diet, facilitating detection of and responses to the novel potential threat (Marquis et al. 2004, Sündermann et al. 2008, Sih et al. 2010, 2011).

Even if animals do not innately respond to a novel predator they may, through time and due to experience, learn the identity of the invader. Learning may play a key role in allowing native prey species to evoke appropriate antipredator defences against unknown predators

(reviewed in Ferrari et al. 2010). In aquatic systems learning occurs through the coupling of conspecific alarm cues with specific-predator odours and often only requires a single conditioning event (Mirza et al. 2006). After this conditioning period, prey learn to elicit antipredator responses to the predator odour alone, allowing for subsequent recognition of

Chapter 1 the novel predator stimuli. Learned recognition of predator odours has been widely reported among freshwater organisms, especially in fish (e.g. Korpi and Wisenden 2001, Kelley and

Magurran 2003, Kristensen and Closs 2004, Ferrari et al. 2005, Ferrari and Chivers 2006) and amphibians (e.g.Mandrillon and Saglio 2005, Mirza et al. 2006, Gonzalo et al. 2007, Ferrari et al. 2008, Shah et al. 2010), but also in other taxa (e.g. crustaceans: Bool et al. 2011).

Nevertheless, it is to notice that, even though learning can be highly beneficial for recognising novel predators, it does not necessarily mean that the responses elicited will be effective in evading that specific predator (Sih et al. 2010, 2011).

Since invasive predators are agents of novel selection, native species may evolve in response to biological invasions. Given enough time, the strong selection pressure imposed by invaders may induce evolutionary changes in native prey, promoting the evolution of predator recognition and adaptive antipredator defences (Schlaepfer et al. 2005, Strauss et al. 2006, Sih et al. 2010). This evolutionary process frequently occurs in contemporary timescales, often within decades, being called as rapid (Reznick and Ghalambor 2001). For rapid evolutionary changes to occur a particularly strong and consistent selection pressure has to be imposed by the novel predator and there has to be a differential genetic susceptibility of individuals to the predator. Further, native prey populations have to be large enough to withstand the negative ecological effects incurred by the invader in the short term (Reznick and Ghalambor 2001,

Strauss et al. 2006). Several studies have documented rapid directional adaptive evolution in native organisms following invasions (reviewed in Thompson 1998, Reznick and Ghalambor

2001, Strauss et al. 2006; e.g. Shine 2011), and introduced predators are often the selective agent involved. Rapid evolution of behavioural, morphological or life-history adaptations in response to exotic predators is pervasive and has been reported in prey such as bivalves (e.g.

Freeman and Byers 2006), snails (e.g. Trussel and Smith 2000, Edgell et al. 2009), crustaceans

(e.g. Cousyn et al. 2001, Fisk et al. 2007), fish (e.g. O’Steen et al. 2002) and amphibians (e.g.

Kiesecker and Blaustein 1997). Specifically in amphibians, animals with a generation time much

Chapter 1 longer than the other abovementioned taxa, tadpoles of the native red-legged frog (Rana aurora) have evolved the ability to detect and respond with adaptive avoidance behaviours to chemical cues of the introduced bullfrog (Rana catesbeiana) in a period of time as short as 70 years (Kiesecker and Blaustein 1997). The evolution of adaptive responses in native prey populations is the most effective mechanism in enhancing the probability of long term prey population persistence.

Finally, within the same community, species may greatly vary in their ability to perceive and elicit responses to invasive predators. For instance, Pearl et al. (2003) found different responses in two native anurans to cues from two introduced predators, the bluegill sunfish

(Lepomis macrochirus) and the red swamp crayfish (P. clarkii): no evidence of predator recognition was found in the Pacific treefrog (Pseudacris regilla), while the northern red-legged frog (Rana aurora aurora) responded behaviourally to both introduced predators. As such, it is not easy to predict how a certain prey species will react to the presence of an exotic predator.

Some features that may help explain variation in the ability of prey to respond to novel predators may be the type of cues that prey use to gauge risk (use of broad, general cues as opposed to specific predator cues), the existence of natural traits that facilitate escape to predators and the extent of phenotypic plasticity that species present (Sih et al. 2011). Many more factors will surely affect this and, in the end, all of them will influence a species ability to survive and, in the long term, thrive in invasion altered environments.

1.4. Species in focus

The Iberian Peninsula currently maintains over 70 non-indigenous freshwater species. These species include a wide variety of invertebrate and vertebrate taxa, flatworms, molluscs, crustaceans and fish being the most common ones (García-Berthou et al. 2007, Cobo et al.

2010). In this area, the most abundantly spread invasive decapod crustacean is the red swamp

Chapter 1 crayfish, Procambarus clarkii (Gherardi 2006, García-Berthou et al. 2007). At the same time, there is only one native species of crayfish (Austropotamobius pallipes), which is currently thought extinct in Portugal, and which has recently been proposed as being non-native to the

Iberian Peninsula (Holdich et al. 2009). The introduction of invasive aquatic species, especially large predators, into the Iberian Peninsula acquires special relevance, since this area is a hotspot of freshwater biodiversity comprising, among others, several endemic and nearly- endemic amphibian species (Loureiro et al. 2008).

The invasive predator: red swamp crayfish (Procambarus clarkii Girard, 1852)

P. clarkii is a crayfish species originally endemic to northeastern Mexico and southcentral USA that, due to its commercial value, has been largely introduced outside its natural range. It has proven a successful invader, being now present in all continents except Australia and

Antarctica, making it the most cosmopolitan crayfish species in the world (Hobbs et al. 1989,

Gherardi 2006, Holdich et al. 2009). P. clarkii was first introduced into Spain (and Europe) in

1973 near Badajoz and one year later near Seville, for aquaculture purposes and with the aim of supplementing crayfish stocks, after many native crayfish populations had been devastated by the crayfish plague (Habsburgo-Lorena 1983, Gherardi 2006, Holdich et al. 2009). P. clarkii easily managed to escape the sites of release, soon colonising and expanding into new areas, which was aided by the spread of live specimens by fishermen (Geiger et al. 2005, Gherardi

2006). Both this, and the fact that environmental characteristics in the Iberian Peninsula seem to be highly suitable for the establishment of this invader, allowed for its subsequent expansion all through the Peninsula, where it is currently extremely widespread and abundant

(Correia 2002, Gil-Sánchez and Alba-Tercedor 2002, Gherardi 2006, Holdich et al. 2009,

Capinha and Anastácio 2011). Probably through common hydrographic basins and with the help of fisherman it invaded Portugal, where its presence was first registered in 1979, in the

Chapter 1

Caia River, located quite close to Badajoz (Ramos and Pereira 1981). By 1984 it had arrived to the Mondego region and, by 1990, it was already abundant in the Sado River basin (Southwest

Portugal) (Anastácio et al. 1995, R. Rebelo, pers. comm.).

Numerous biological and ecological characteristics of P. clarkii allow this crayfish to be such a successful invader (reviewed in Geiger et al. 2005). P. clarkii exhibits rapid growth, high fecundity, high reproductive potential, early maturity and it may, sometimes, have multiple generations in a single year (Hobbs et al. 1989, Anastácio and Marques 1995, Gutiérrez-Yurrita and Montes 1999). It is capable of plastically adjusting its reproductive life cycle to the specific habitat conditions, allowing it to adapt to a diversity of environments (Gutiérrez-Yurrita and

Montes 1999). Further, it has a generalist and opportunistic diet, feeding on detritus, as well as on diverse plant and animal resources, such as algae, macrophytes, macroinvertebrates, amphibians and fish, according to their availability (e.g. Gherardi et al. 2001, Correia 2002,

2003, Renai and Gherardi 2004, Correia et al. 2005, Gherardi and Acquistapace 2007, Correia and Anastácio 2008). P. clarkii is also capable of learning the identity of new unknown predators, which may contribute to its adaptation to novel environments (Gherardi et al.

2011). This crayfish inhabits a wide variety of freshwater habitats including rivers, lakes, ponds, canals and marshes and it has a high tolerance to adverse and extreme environmental conditions, namely drought, high temperatures, low oxygen and pollution (Gutiérrez-Yurrita and Montes 1999, Ilhéu et al. 2007, Capinha and Anastácio 2011). Finally, it has a high dispersal ability, since overland dispersal over long distances seems to be a frequent phenomenon (Gherardi and Barbaresi 2000, Barbaresi et al. 2004, Aquiloni et al. 2005, Kerby et al. 2005, Cruz and Rebelo 2007). All these traits allow crayfish to quickly build extremely large populations and promote its high invasive potential.

P. clarkii exerts wide environmental impacts in invaded communities, and often affects the structure and functioning of aquatic ecosystems, being considered an ‘ecosystem engineer’

(Gherardi 2007a). By consuming and destroying aquatic macrophytes and decreasing

Chapter 1 macroinvertebrate abundance, it causes a reduction in food resources and in habitat complexity, such as refuges and spawning sites for fish and amphibians (Gutiérrez-Yurrita and

Montes 1999, Correia 2002, Rodríguez et al. 2003, 2005, Gherardi and Acquistapace 2007).

This, together with its bioturbation activity (promoted by burrowing, ingestion of sediment particles and alterations in dissolved inorganic nutrients), may lead to an increase in water turbidity due to blooms of surface microalgae and to particle resuspension (Rodríguez et al.

2003, 2005, Geiger et al. 2005, Gherardi 2007b). In its turn, this turbid state will reduce light penetration and plant primary production, which can be followed by losses in biodiversity

(Rodríguez et al. 2003, 2005, Geiger et al. 2005). All these effects, alongside with crayfish consuming rice seedlings, are known to negatively impact agricultural areas in several regions, including the Iberian Peninsula (reviewed in Geiger et al. 2005). Both this and the fact that, due to burrowing, P. clarkii may cause the collapse of banks in dams or dykes, indicate that this crayfish may have serious economic impacts in areas where it has been introduced (Geiger et al. 2005).

As a predator, P. clarkii relies more on chemical rather than visual cues to detect prey, being particularly active at night (even though activity may also be high during the day) (Gherardi and Barbaresi 2000, Aquiloni et al. 2005). At times, it adopts a sit-and-wait strategy to capture prey, but it often acts as a moderately active predator, especially during the juvenile stage

(Gherardi et al. 2001, Renai and Gherardi 2004, Correia et al. 2005, Gomez-Mestre and Diaz-

Panigua 2011, A.L. Nunes, pers. obs.). Through ontogeny, its diet shifts from being more carnivorous as juveniles to more herbivorous in adult stages (Correia 2003, Correia et al. 2005).

It seems to be a more efficient predator than native European crayfishes and to be immune to toxic chemical compounds that protect some prey species from other predators (Gamradt and

Kats 1996, Gherardi et al. 2001, Renai and Gherardi 2004, Cruz and Rebelo 2005).

P. clarkii is an efficient predator of a wide array of aquatic organisms. It has been reported to feed on a variety of insects, crustaceans, snails and both eggs and larvae of fish and

Chapter 1 amphibians (e.g. Gamradt and Kats 1996, Gherardi et al. 2001, Correia 2002, 2003, Renai and

Gherardi 2004, Correia et al. 2005, Cruz and Rebelo 2005, Ilhéu et al. 2007). Other than predating them, P. clarkii may affect amphibians through different processes. It is capable of inflicting serious injury to amphibian prey (Gamradt et al. 1997, Leite 2003) and, similarly to other invasive crayfishes (e.g. Nyström and Abjörnsson 2000, Nyström et al. 2001), it induces alterations in behaviour, morphology and life-history of larval amphibians (Pearl et al. 2003,

Cruz and Rebelo 2005, Ortiz-Santaliestra et al. 2010, this study). Finally, it can cause the displacement of amphibian populations from their natural breeding habitats and affect their reproductive success (Gamradt and Kats 1996, Gamradt et al. 1997, Cruz et al. 2006a,b, 2008,

Ficetola et al. 2011).

The native prey: anurans from the Southwest of the Iberian Peninsula

In the Southwest of the Iberian Peninsula there are 14 amphibian species, ten anurans and four urodeles. In this study only the anuran community is in focus, which is comprised by the

Iberian water frog (Pelophylax perezi Seoane 1885), the Mediterranean tree frog (Hyla meridionalis Boettger 1874), the European tree frog (Hyla arborea Linnaeus 1758), the common toad (Bufo bufo Linnaeus 1758)1, the natterjack toad (Bufo calamita Laurenti 1768), the Iberian parsley frog (Pelodytes ibericus Sánchez-Herráiz, Barbadillo, Machordom and

Sanchiz 2000)2, the common parsley frog (Pelodytes punctatus Daudin 1802; not in focus here), the Western spadefoot toad (Pelobates cultripes Cuvier 1829), the Iberian painted frog

(Discoglossus galganoi Capula, Nascetti, Lanza, Bullini and Crespo 1985) and the Iberian midwife toad (Alytes cisternasii Boscá 1879) (Fig. 1). This area holds a remarkable endemic

1 A very recent study suggests that Iberian populations of B. bufo belong to a clade including North African and French populations, denominated as Bufo spinosus (Recuero et al. 2011).

2 Pelodytes ibericus and Pelodytes punctatus were until recently considered the same species and their distribution limits in the Iberian Peninsula are still not well established (Sánchez-Herráiz et al. 2000). Recent genetic data suggests that our study populations likely belong to P. ibericus (J. Díaz-Rodríguez, pers. com.). 24

Chapter 1 anuran community: out of ten species, two are endemic for the Southwest region (P. ibericus and A. cisternasii), one is endemic from the Iberian Peninsula (D. galganoi) (Loureiro et al.

2008) and five others have a fairly restricted distribution outside Portugal and Spain

(Pelophylax perezi, Hyla meridionalis, Pelodytes punctatus and Pelobates cultripes). Moreover, three of these species have the status of ‘near threatened’ either internationally (H. arborea and A. cisternasii) or nationally (D. galganoi) (Cabral et al. 2005).

a b c )

d e f

g h i

Fig. 1. Photographs of the larval and adult stages of the nine Iberian anurans studied: a) Alytes cisternasii, b) Pelodytes ibericus, c) Pelobates cultripes, d) Hyla arborea, e) Hyla meridionalis, f) Pelophylax perezi, g) Bufo bufo, h) Bufo calamita and i) Discoglossus galganoi. Photos of adult animals of pictures a), b), d) and g) taken by Erika Almeida.

Chapter 1

The nine studied anuran species are distributed along different breeding habitats, largely defined by an hydroperiod gradient. A. cisternasii, B. bufo, H. arborea and P. perezi mainly breed in permanent and extensive water bodies, such as streams, rivers and dams, although they may also occur in temporary ponds. P. cultripes, P. ibericus and H. meridionalis generally occur in temporary ponds, while B. calamita is typically found in small ephemeral ponds. D. galganoi usually inhabits these latter habitats, but at times can be found in temporary water bodies (Diaz-Paniagua 1990, Van Buskirk 2002, Richter-Boix et al. 2007, Loureiro et al. 2008).

Larvae of these species also have a different use of space in the water bodies, occupying different microhabitats. Species like B. calamita, D. galganoi, P. perezi and A. cisternasii are typical bottom-dwellers, spending most of the time at the floor of the ponds or streams (Diaz-

Paniagua 1985, Cruz and Rebelo 2005). On the contrary, P. cultripes, P. ibericus and the two hylids are mainly found in the water column. P. ibericus also uses the shallow margins and Hyla spp. are highly associated with vegetated areas of the ponds (Diaz-Paniagua 1985, Cruz and

Rebelo 2005). Tadpoles of B. bufo are continually active, but most of their time is spent in open water areas (Cruz and Rebelo 2005).

Previous studies have tested the existence of predator-induced behavioural, morphological or life-history plasticity in most of these species. In the presence of predators (usually Dytiscid

(Insecta: Coleoptera) or Aeshnid (Insecta: Odonata) larvae), most of these anuran species alter their phenotypes in order to avoid or be defended from predation. Although results often vary from study to study, all the nine anuran species seem to be able to develop behavioural antipredator responses (B. bufo: Semlitsch and Gavasso 1992, Laurila et al. 1998; B. calamita:

Tejedo 1993; A. cisternasii: Cruz and Rebelo 2005; D. galganoi: Nicieza et al. 2006; P. perezi:

Gonzalo et al. 2007, Richter-Boix et al. 2007, Polo-Cavia et al. 2010, Gomez-Mestre and Díaz-

Paniagua 2011; P. ibericus: Cruz and Rebelo 2005; P. cultripes: Polo-Cavia et al. 2010; H. meridionalis: Richter-Boix et al. 2007; H. arborea: Van Buskirk 2002). Morphological and life- history plasticity in the presence of predators has also been reported in B. bufo (Álvarez and

Chapter 1

Nicieza 2006, Richter-Boix et al. 2007, Van Buskirk 2009), D. galganoi (Nicieza et al. 2006), P. perezi (Richter-Boix et al. 2007, Gomez-Mestre and Díaz-Paniagua 2011) and the two Hyla species (Lardner 2000, Van Buskirk 2002, 2009, Richter-Boix et al. 2007, Polo-Cavia et al. 2010).

B. calamita consistently shows no alterations in morphology, while it has the capacity of altering life-history traits (Lardner 2000, Richter-Boix et al. 2007). In A. cisternasii and P. ibericus, predator-induced alterations in morphology and life-history have not been studied before the present study, but A. obstetricans and P. punctatus, species closely related to these, have been shown to alter these traits. P. fuscus, a species close to P. cultripes, did not show alterations in growth or morphology when exposed to an invertebrate predator (Lardner

2000).

1.5. P. clarkii and the anurans from Southwestern Iberian Peninsula

In the Southwest of the Iberian Peninsula, the area of introduction of P. clarkii, this crayfish is capable of successfully colonising all types of aquatic habitats where amphibians are found, including small and shallow ponds (Cruz and Rebelo 2007). In this region, no native crayfish species ever existed. Eggs and larvae of all the anuran species present in this area are readily preyed by the crayfish (Cruz and Rebelo 2005, Ortiz-Santaliestra et al. 2010). Even bufonids, known to rely on chemical toxic deterrents as defences against predators are promptly consumed by P. clarkii (Gherardi et al. 2001, Renai and Gherardi 2004, Cruz and Rebelo 2005).

There have also been reports of this crayfish inflicting nonlethal tail injury in species such as P. cultripes, H. meridionalis and P. perezi (Leite 2003).

Cruz and Rebelo (2005) found that all but two (B. bufo and P. cultripes) of these nine anuran species reduced activity level and/or shifted microhabitat use in the presence of P. clarkii.

However, another study has shown that crayfish cues induce alterations in larval swimming activity in tadpoles of P. cultripes (Ortiz-Santaliestra et al. 2010). On the other hand, Gomez-

Chapter 1

Mestre and Díaz-Paniagua (2011) reported a failure of P. perezi larvae to express inducible defences against this crayfish.

Several field studies have documented strong negative impacts of this crayfish on Iberian amphibians. In the Sado River basin, Cruz et al. (2006a) found that the presence of P. clarkii in a water body was a negative predictor of the use of that water body by P. cultripes, B. bufo and possibly P. ibericus for reproduction. In addition, amphibian species richness was lower in places colonised by the crayfish. The presence of P. clarkii also seems to greatly affect the breeding habitat use and reproductive success of B. calamita in Doñana Natural Park (Cruz et al. 2006b). Further, Beja and Alcazar (2003) have documented a negative correlation between the distribution of this exotic crayfish and of some amphibian species in a Nature reserve in

Southwestern Portugal. A study performed in Italy observed that larval abundance of all studied amphibian species, including B. bufo, was negatively associated with the presence of P. clarkii (Ficetola et al. 2011). Finally, invasion by P. clarkii has caused the disappearance of certain amphibian species from specific areas. In a lagoon in Spain, five of the six amphibian species present became extremely rare and two of them (B. calamita and P. cultripes) disappeared after crayfish arrival (Rodríguez et al. 2005). P. clarkii also seems to have caused strong reductions or the local extinction of H. arborea, P. perezi and P. punctatus in a Nature reserve in Portugal (Cruz et al. 2008).

1.6. Goals and outline of the thesis

One of the ways to evaluate the impacts of exotic predator introductions on prey communities is by experimentally assessing the effects of the predator on different components of prey fitness and survival. This may help clarifying some of the mechanisms that make prey species more or less affected by invasions.

Chapter 1

Larval anurans provide excellent study models for investigating inducible defences in the presence of exotic predators, since they most often show predator-induced phenotypic plasticity. Since predation during aquatic stages is one of the dominant forces regulating amphibian populations (Smith 1983, Azevedo-Ramos et al. 1999), lacking appropriate defences towards an invasive predator may render the invaded communities at serious risk. Therefore, the ongoing invasion of P. clarkii in the Iberian Peninsula presents a very good opportunity to study early responses of native prey to the presence of novel predators.

The main goal of this thesis was to study the outcomes of recently-established predator-prey interactions following invasion by the aquatic predator P. clarkii on amphibian communities of the Southwest of the Iberian Peninsula. In particular, more specific objectives were:

1. To evaluate the importance of P. clarkii as an agent of tail injury on free-living tadpoles and to assess fitness and survival costs of tail damage for tadpoles;

2. To assess if larvae of the nine anuran species from the Southwest of the Iberian Peninsula are able to induce behavioural, morphological or life-history responses in the presence of P. clarkii and how integrated these responses are;

3. To investigate if tadpoles from populations differing in historical exposure to P. clarkii showed dissimilar antipredator responses to this crayfish.

This thesis is structured as a compilation of four scientific papers (one published, one in review and two manuscripts), each corresponding to one chapter, that focus on these specific goals.

This is preceded and followed by two more general sections (Chapters 1 and 6) which present, respectively, a general introduction to the themes focused in the following chapters and a general discussion in which the most relevant results of the preceding chapters are discussed and integrated.

Chapter 1

The first goal of this thesis was addressed in Chapter 2, where results from a field study and a laboratory experiment were integrated. First, the role of P. clarkii as an important inducer of tail damage in tadpoles of the Western spadefoot toad, P. cultripes, was tested by comparing frequencies of tadpole caudal injury in temporary ponds, with and without crayfish, in

Southwestern Spain. Secondly, to investigate if tail damage can have important consequences for tadpole morphology, life-history and survival, and how these consequences may differ according to available energetic resources, frequency of tail injury and food availability were manipulated in the laboratory in a factorial experiment.

The second objective was examined in Chapters 3 and 4, in which a laboratory experiment was performed to evaluate if the nine anurans studied here were able to elicit behavioural

(alterations in activity level and spatial avoidance), morphological (alterations in tadpole headbody and tail morphology) and life-history (alterations in growth rate, time to and mass at metamorphosis) responses when exposed to P. clarkii chemical cues. These antipredator responses were compared with those elicited towards a common native predator, the larval dragonfly Aeshna sp. For this, tadpoles were raised in the laboratory and exposed to scents of a caged crayfish, a dragonfly larva or no predator scents. The role of tadpole ontogeny and of chemical cues from predation events in shaping behavioural responses was also investigated, as well as the existence of integration among all phenotypic responses.

Lastly, to fulfil the third goal, P. perezi tadpoles from populations with longer (ca. 30 yr), shorter (ca. 20 yr) or no coexistence time with P. clarkii were reared in the laboratory in the nonlethal presence of this predator and their behavioural and morphological responses were analysed (Chapter 5). This also allowed testing for the existence of evolved adaptive responses of a native amphibian to this invasive predator.

Chapter 1

1.7. Papers presented

Chapter 2

Nunes AL, Cruz MJ, Tejedo M, Laurila A, Rebelo R (2010) Nonlethal injury caused by an invasive alien predator and its consequences for an anuran tadpole. Basic and Applied Ecology 11: 645- 654.

Chapter 3

Nunes AL, Richter-Boix A, Laurila A, Rebelo R (in review) Do anuran larvae respond behaviourally to chemical cues from an invasive crayfish predator? A community-wide study. Oecologia.

Chapter 4

Nunes AL, Orizaola G, Laurila A, Rebelo R. Morphological and life-history responses and integration of defensive traits in an anuran community invaded by an exotic predator (manuscript).

Chapter 5

Nunes AL, Orizaola G, Laurila A, Rebelo R. Population divergence in antipredator defences against an invasive predator: does coexistence time promote the evolution of adaptive responses? (manuscript).

Chapter 1

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CHAPTER 2

Nonlethal injury caused by an invasive alien predator and its consequences for an anuran tadpole

Nunes AL, Cruz MJ, Tejedo M, Laurila A, Rebelo R

Basic and Applied Ecology 11: 645-654

Chapter 2

Nonlethal injury caused by an invasive alien predator and its consequences for an anuran tadpole

Abstract

Nonlethal tail injury resulting from unsuccessful predation attempts is common in anuran larvae and can potentially induce significant fitness costs in terms of survival and growth. We tested the hypotheses that the alien red swamp crayfish, Procambarus clarkii, is an important inducer of tail injury in tadpoles of the Iberian spadefoot toad, Pelobates cultripes, and that tail damage can have important consequences for the tadpoles’ life history and morphology. This was investigated by first estimating frequencies of caudal injury in P. cultripes tadpoles in temporary ponds, with and without crayfish. Secondly, we performed a laboratory experiment in which four levels of tail injury frequency were combined with two levels of food availability.

The frequency of tadpoles with damaged tails was higher in ponds with crayfish and the presence of this predator was the strongest predictor of tail injury frequency in a pond.

Induced tail loss decreased larval survivorship and affected tail morphology, with injured tadpoles developing deeper tail muscles and shallower tail fins. The magnitude of these effects depended on injury frequency, as well as on food availability. The results suggest that P. clarkii is inflicting tail injuries at much higher levels than those occurring before its introduction; these injuries affect tadpole morphology and may induce delayed fitness costs.

Keywords: Unsuccessful predation, alien crayfish, tail injury, tadpoles, costs

Chapter 2

Introduction

Invasions of exotic species are one of the major factors contributing to worldwide amphibian decline (Kats & Ferrer 2003). Freshwater ecosystems are particularly vulnerable to nonindigenous predators, such as crayfish (Hobbs, Jass & Huner 1989). Crayfish, often successful invaders, can impact native communities across multiple trophic levels and readily prey upon amphibians (Nyström, Svensson, Lardner, Brönmark & Granéli 2001). Crayfish introductions can then have important ecological consequences for amphibians, since predation is one of the major biotic factors structuring amphibian larval communities (Wilbur

1980).

Amphibian larvae are often found with damaged tails resulting from unsuccessful predation attempts, which can be caused by a wide variety of vertebrate (turtles, salamanders, newts) and invertebrate (dragonfly and beetle larvae, crayfish) predators (e.g. Morin 1985; Wilbur &

Semlitsch 1990; Tejedo 1993; Nyström et al. 2001). Tadpoles continuously exposed to predators are likely to accumulate several injuries. Injury frequencies in natural populations are influenced by predator densities and often also by factors such as the diversity and complexity of microhabitats (Morin 1985; Figiel & Semlitsch 1991).

The highly fragile tail fin which is easily torn by predators, can help tadpoles escape and survive otherwise lethal attacks (Doherty, Wassersug & Lee 1998; Van Buskirk, Anderwald,

Lupold, Reinhardt & Schuler 2003). In this aspect, predator-induced tail loss in amphibian larvae resembles autotomy, a widespread antipredator strategy involving self-induced release of a body part, which helps animals escape predator attacks (see Maginnis 2006 for a review).

Similarly to autotomy, tail loss has an obvious immediate survival benefit, but its potential costs are substantial and include reduced swimming performance and increased energetic expenditure for regenerating damaged tissue (Wassersug & Sperry 1977; Van Buskirk &

McCollum 2000). Swimming performance is not only affected by tail length, but also by

Chapter 2 tadpole morphology, implying an important role of tadpole shape in predator escape abilities.

In the presence of predators, many species develop deeper tails, which increase their survival under predation risk, either due to enhanced swimming performance caused by larger tail muscles (McCollum & Leimberger 1997; Teplitsky, Plenet, Léna, Mermet, Malet et al.2005;

Wilson, Kraft & Van Damme 2005) or by enabling nonlethal ripping of the deep tail fin

(Doherty et al. 1998; Van Buskirk et al. 2003). Regeneration following tail loss may also impose a cost because energy previously used for growth and development is then diverted to repair the damaged tissue (Semlitsch & Reichling 1989). However, the consequences of tail loss seem to depend on injury frequency and severity, as well as on the amount of food available to tadpoles. Injury effects become more pronounced when energy is limited (Parichy & Kaplan

1992).

In the Southwest of the Iberian Peninsula, the Iberian spadefoot toad (Pelobates cultripes) often coexists with the invasive red swamp crayfish (Procambarus clarkii). This invasive crayfish was introduced in this area, previously devoid of freshwater crayfish, in the early

1970’s (Gutiérrez-Yurrita & Montes 1999). It is an active predator of P. cultripes tadpoles (Cruz

& Rebelo 2005), which can inflict tail injury in mesocosms (A. L. Nunes, pers. obs.).

Furthermore, it typically consumes and damages aquatic macrophytes, which might reduce periphyton biomass, an important food source for tadpoles (Gutiérrez-Yurrita & Montes 1999;

Nyström et al. 2001). P. cultripes breeds mostly in temporary ponds that lack fish and other large aquatic predators, but which P. clarkii is able to colonize (Cruz & Rebelo 2007). Cruz,

Rebelo & Crespo (2006) showed that, nowadays, the probability of a water body being used as a breeding site by P. cultripes is significantly reduced by P. clarkii’s presence.

The aim of this study was two-fold. First, we assessed the importance of P. clarkii as a tail injury agent on free-living tadpoles, by comparing frequencies of tadpoles with tail damage in ponds with and without P. clarkii in southwestern Spain. Secondly, we experimentally manipulated frequency of tail injury and food availability in the laboratory, in order to assess

Chapter 2 survival, growth and development costs of tail damage for P. cultripes tadpoles. This integrated approach allowed us to consider a large number of factors that should be addressed in a field study and, at the same time, look into interactions that are often difficult to establish in complex systems but that can easily arise in a controlled laboratory setting.

Methods

Field study

The field work was conducted in Parque Natural del Entorno de Doñana, southwestern Spain.

P. clarkii was introduced in the Doñana marshes in 1974 and probably colonized this area through La Rocina Creek, the main permanent tributary to the Guadalquivir marshlands and the most important source of P. clarkii colonizers in the park (Gutiérrez-Yurrita & Montes

1999). The park contains a series of ecologically similar temporary ponds, free of fish due to their hydrological cycle, but where crayfish can attain very high densities. Only ponds far from a crayfish source remain uncolonized (Cruz & Rebelo 2007).

Sampling methods

In February-April 2004, we sampled 54 temporary ponds in the study area but, for this study, we selected the 31 ponds in which P. cultripes tadpoles were found (Fig. 1). To investigate the presence and abundance of P. clarkii individuals and P. cultripes tadpoles we used 5-6 baited funnel traps (1.5 and 5 L) in each pond. These traps were left for one night and checked the following morning. Five dip-net sweeps (30 cm diameter, 2 mm mesh size) were also performed in each pond. Ponds having no crayfish were repeatedly trap-sampled - up to 3 times -, in order to confirm crayfish absence. Cruz et al. (2006) showed these methods

Chapter 2 adequate to assess the presence of P. clarkii. All animals were released back in the ponds after sampling.

Fig. 1. Map of the study area (surroundings of Doñana Natural Park) and its location in the Iberian Peninsula, showing the 31 temporary ponds selected for this study. The star indicates the location where the clutches for the laboratory experiment were collected.

The frequency of injured tadpoles in each pond was estimated only from dip-net sweeps, because tadpoles captured in the traps might have become injured while being confined with predators. Tadpoles were considered as having tail injury if a part of their tail had been ripped off and not yet regenerated. In order to assess if the frequency of injured tadpoles in a pond was influenced by the presence of crayfish, we had to account for variables such as the

Chapter 2 presence of other potential aquatic predators, vegetation cover and pond dimensions. Aquatic insects from the families Dytiscidae (adults and larvae), Aeshnidae, Cordulegasteridae,

Notonectidae, Corixidae, Coenagrionidae and vertebrate urodeles (Pleurodeles waltl, Triturus marmoratus, Lissotriton boscai), were sampled using dip-net sweeps and funnel traps as described above. Emergent, floating and submerged vegetation cover were estimated using six

1-m transects, in which the vegetation composition was determined every 10 cm (ten points per transect). Vegetation cover was the percentage of points covered by each type of vegetation. Pond area (m2) and maximum depth (cm) were also measured.

Statistical analyses

As ponds with and without crayfish may differ in their predator communities, we estimated and compared relative abundances of invertebrate and vertebrate predators present in these two types of ponds. Abundances were calculated considering the most effective sampling method for each predator and based on catch per sampling effort. Trap captures were thus used to estimate abundances of P. clarkii and Dytiscidae; for all other predators dip-net sweeps proved more efficient. We used t-tests to compare the abundances of different predators in ponds with and without crayfish. We compared frequencies of tail injury in ponds with and without crayfish using a Mann-Whitney U test. We also performed Spearman correlations between frequency of tail injury and abundances of all predators sampled. Effect size estimates were determined by Cohen’s d.

We used Generalized Linear Models to investigate which factors influenced the frequency of injured tadpoles. We used a binomial function in which the number of tadpoles having tail damage in each pond was considered the dependent variable and the total number of tadpoles caught in that pond treated as a trials variable. As we did not find differences in the abundance of predators in ponds with and without crayfish (see results), we used predator

Chapter 2 presence/absence data for the biotic variables, whereas the abiotic variables were treated as continuous (Table 1). We performed a series of univariate analyses and the variables with P- values < 0.25 in the Wald test were selected for the multivariate analysis. We also checked for collinearity between abiotic variables using a Pearson correlation coefficient. Variables were excluded from the multivariate model using a backward stepwise elimination procedure, until all the remaining variables were significant predictors (P< 0.05). GLMs were performed using the software package SPSS (version 15.0).

Table 1. Abiotic and biotic variables (and correspondent measurement units) recorded in each pond and used to perform the Generalized Linear Model. For the biotic variables 0 means absence and 1 means presence of a predator group in a pond

Variables Measurement units Abiotic

Aquatic vegetation

- Emergent % cover - Floating % cover - Submerged % cover Pond area m2

Pond maximum depth cm

Biotic

Crayfish 0/1

Dytiscidae (adults) 0/1

Dytiscidae (larvae) 0/1

Aeshnidae 0/1

Cordulegasteridae 0/1

Notonectidae 0/1

Corixidae 0/1

Coenagrionidae 0/1

Vertebrate predators 0/1

Chapter 2

Laboratory experiment

Experimental procedure

On November 30th 2005 we collected four clutches of P. cultripes in a pond in Serra de

Grândola, southwestern Portugal (38º04’N, 8º34’W) (Fig. 1), colonized by P. clarkii. The eggs were taken to the Faculty of Sciences, University of Lisbon, where the experiment took place.

Tadpoles were kept in clutch-specific 20-L aquaria filled with aged tap water changed regularly and fed commercial fish food ad libitum. When they reached Gosner developmental stage 25

(operculum closure over gills; Gosner 1960) we randomly selected 120 tadpoles from each clutch and kept them individually in 0.4-L plastic cups filled with aged water, changed every two days. Throughout the experiment, water temperature was kept between 19.5 and 22.5 ºC and the photoperiod was 12L: 12D.

We used a factorial experimental design with frequency of injury (four levels), food availability

(high/low) and different clutches (four levels) as factors, with each treatment combination replicated 15 times, resulting in a total of 480 tadpoles as experimental units. Frequency of caudal injury ranged from no injury (control) to three injuries per tadpole, inflicted in events separated by 20 days. The first injury event, in which tadpoles from injury levels 1, 2 and 3 suffered tail damage, took place January 16th (day 0 of the experiment) and the last one (injury only at level 3) March 13th 2006. Tail damage was inflicted so that the final tail length equalled

0.5 × headbody length, the amount of tail left being always proportional to the tadpole size.

Immediately after the damage, inflicted with a disinfected scalpel, tadpoles were placed individually in aged water and none showed signs of infection. Tadpoles regenerated their tails in 10-15 days.

The tadpoles were fed 0.05 g of commercial fish food (Tetra Min) and boiled lettuce, according to two different treatments of food availability. Tadpoles from the high-food treatment were fed every two days, while tadpoles from the low-food treatment were only fed every fifth day.

Chapter 2

The lower food treatment was known to limit growth and developmental rates of P. cultripes tadpoles in the laboratory (A. L. Nunes, unpublished data).

At days 20, 40 and 60 (20 days after each injury event and immediately before the next one), all the individuals were photographed in side view against a grid background, in order to obtain data on their morphology. Gosner development stage was also determined and survival was registered at day 60. All the photographs were analysed using software Image J 1.38x

(Image Processing and Analysis in Java) and body length, maximum body depth, maximum tail fin depth and maximum tail muscle depth were recorded to the nearest 0.01 mm. Tail traits were always measured in the undamaged part of the tail.

Statistical analyses

All analyses on morphology were conducted on log transformed data in order to fulfil the assumptions of analysis of variance. Prior to analyzing morphological data, trait values were corrected for variation in body size because morphological measurements are usually highly correlated. In order to do this, body size was defined as the first component in a principal component analysis performed using a correlation matrix. All the traits loaded highly positively on the first axis, and it always explained more than 80% of the total variation. Body size (PC1) was then used as a covariate in two-way fixed effect ANCOVAs (factors: food availability and injury treatment) to compare tadpole morphology in the different treatments (García-Berthou

2001). Clutch identity was included in the initial analyses as a random factor, but as it did not affect the results qualitatively in any of the analyses (in all cases, P> 0.1), it was not included in the final models.

In the analyses of morphology at days 20, 40 and 60, we compared treatments according to the injury events that had already been inflicted. For example, in day 20 we compared the control tadpoles with tadpoles that tail damage induced once, whereas in day 60, after

Chapter 2 tadpoles had been injured three times, all injury treatments were compared. The initial models included all possible interactions with the covariate PC1, but all non-significant interactions were removed from the final model. Post-hoc pairwise comparisons were performed using

Bonferroni corrections. Effect sizes for the analysis of variance were calculated using partial eta squared. Developmental rates were calculated as the difference between final and initial

Gosner development stage divided by the number of days of the experiment and analyzed with two way ANOVAs. Tadpole survival was analyzed as a binomial variable, using a factorial logistic regression. In this case, odds ratios were used to compute effect sizes. All values in the results are expressed as mean ± 1 S.E.

Results

Field study

P. clarkii was found in 16 of the 31 ponds with P. cultripes. In all ponds with P. clarkii, we found evidence of crayfish reproduction (juveniles or brooding females). All the other predators were found both in ponds with and without crayfish. The abundance of P. cultripes tadpoles did not differ between ponds with and without crayfish (11.83 ± 4.18 individuals/trap and 12.51 ± 9.51 individuals/trap, respectively; t29 = 0.07, P = 0.947, ES = 0.025).

Abundance of invertebrate and vertebrate predators was also similar in ponds with and without P. clarkii (t29 = -0.06, P = 0.952, ES = 0.022 and t29= 1.16, P = 0.254, ES = 0.031, respectively). Likewise, there were no significant differences in relative abundances of each group of invertebrate and vertebrate predators between ponds with and without crayfish (t- tests, P > 0.05; Fig. 2). In ponds with crayfish, P. clarkii was usually the most abundant predator

(Fig. 2).

Chapter 2

Fig. 2. Relative abundance (mean and standard error) of the invertebrate and vertebrate predators present in temporary ponds, with and without crayfish. See methods for abbreviations of predator group names.

The frequency of tail injury in P. cultripes tadpoles was higher in ponds with than without crayfish (Mann-Whitney U test, Z = -3.59, P < 0.001, ES = 0.901; Fig. 3). Frequency of tail injury was significantly correlated with the abundance of crayfish (rs = 0.63, N = 31, P < 0.001), but not with abundance of any other predator (all P > 0.05). When testing for which factors predicted the frequency of tail damage found in different ponds, the final model retained only four factors, two biotic and two abiotic. The frequency of injured tadpoles was higher in ponds with adult Dytiscidae and P. clarkii than in the ponds where these predators were absent

(Table 2). Crayfish proved to be the strongest positive predictor of the frequency of injured tadpoles in a water body (β = 3.62 ± 0.45). In ponds with P. clarkii the probability of finding a tadpole with tail damage was 37.4 times higher (95% C.I.: 15.49 – 90.28) than finding it in a pond without P. clarkii. An increase in pond area increased the frequency of tail injury, whereas tail injuries decreased with increasing vegetation cover (Table 2).

Chapter 2

Fig. 3. Mean frequency of tadpoles (+ S.E.) having tail injury in ponds with and without crayfish.

Laboratory experiment

In the beginning of the experiment (day 0) there were no differences in the morphology of

tadpoles assigned to different treatments (for all morphological traits, P > 0.05). At days 20, 40

and 60, only food availability had an influence on body size (PC1) and developmental stage of

the tadpoles, with animals from the high-food treatment being larger and more developed

(F1,161 = 479.46, P < 0.001, ES = 0.749, F1,306 = 1770.21, P < 0.001, ES = 0.853 and F1,418 = 2311.66,

P < 0.001, ES = 0.847 respectively, for body size; F1,212 = 285.69, P< 0.001, ES = 0.574, F1,324 =

710.13 P < 0.001, ES = 0.687 and F1,418 = 550.19, P < 0.001, ES = 0.568 respectively, for

developmental stage).

Table 2. Estimated coefficients (β) with standard errors (S.E.), estimated odds ratio (exp(β)) and P-values obtained via the Wald χ2 test for the variables retained in the final Generalized Linear Model as being significant predictors of the frequency of tail injury in P. cultripes Variables Coefficient (β) S.E. Wald d.f. P Exp (β) Constant -7.871 1.428 30.396 1 <0.001 0 Dytiscidae presence (adults) 3.274 0.546 36.014 1 <0.001 26.418 Crayfish presence 3.622 0.450 64.858 1 <0.001 37.396 Emergent vegetation -1.571 0.461 11.621 1 0.001 0.208 Pond area 0.928 0.275 11.381 1 0.001 2.529 For the categorical variables the reference category is 0 (= absence of predator).

Chapter 2

Twenty days after the first injury event and after complete tail regeneration, injured tadpoles had shallower tail fins and deeper tail muscles than uninjured tadpoles (Table 3; Fig. 4).

Individuals from the high-food treatment had deeper tail muscles than the ones from the low-food treatment, but there were no differences between food treatment groups for the tail fin depth (Table 3; Fig. 4). At day 40, animals injured a second time had again shallower tail fins than uninjured animals and animals injured only once; food availability did not have a significant effect (Table 3; Fig. 4). For tail muscle depth, a significant interaction between food availability and frequency of injury showed that injured tadpoles from the high-food treatment had deeper tail muscles than uninjured tadpoles, whereas there were no differences at the low-food level (Table 3; Fig. 4). After all the injury levels were induced (day 60), tadpoles at high-food had deeper tail muscles and shallower tail fins than tadpoles at low-food (Table 3;

Fig. 4). Variation in tail fin depth due to tail damage depended on food availability, with injured tadpoles having slightly deeper tail fins in the low-food treatment and shallower tail fins in the high-food treatment (Table 3; Fig. 4).

Fig. 4. Tail morphology (tail fin and tail muscle depth) of tadpoles subjected to high (black squares) and low food availability (open circles) at days 20, 40 and 60 (20 days after each injury event). Frequency of injury varied in accordance with the injury events that were successively inflicted. Data are least-square means ± S.E.

Chapter 2

The PC1 x Food/Injury interaction was significant for tail muscle depth at day 40 and for tail fin depth at day 60. However, one-way ANCOVAs performed separately for high and low-food treatments showed exactly the same trends as the two-way ANCOVAs.

Table 3. Analysis of covariance (ANCOVA) on the effects of food availability and frequency of injury on tadpoles tail morphology (tail fin and tail muscle depth) 20, 40 and 60 days after the beginning of the experiment. PC1 is the first component from a principal component analysis and represents body size (used as a covariate). ES stands for effect size

Day 20 Tail Fin Depth Tail Muscle Depth Source d.f. F P ES d.f. F P ES Food(F) 1 0.000 0.985 0 1 6.752 0.010 0.040 Injury(I) 1 7.710 0.006 0.046 1 36.294 <0.001 0.185 FxI 1 0.050 0.823 0 1 0.290 0.591 0.002 PC1 1 211.183 <0.001 0.569 1 261.834 <0.001 0.621 PC1xF PC1xI Error 160 160

Day 40 Tail Fin Depth Tail Muscle Depth Source d.f. F P ES d.f. F P ES Food(F) 1 3.025 0.083 0.010 1 45.916 <0.001 0.132 Injury(I) 2 15.116 <0.001 0.090 2 25.119 <0.001 0.143 FxI 2 2.766 0.065 0.018 2 5.515 0.004 0.035 PC1 1 346.266 <0.001 0.532 1 242.842 <0.001 0.446 PC1xF 1 25.118 <0.001 0.077 PC1xI 2 4.678 0.010 0.030 Error 305 302

Day 60 Tail Fin Depth Tail Muscle Depth Source d.f. F P ES d.f. F P ES Food(F) 1 20.133 <0.001 0.046 1 221.709 <0.001 0.348 Injury(I) 3 1.939 0.123 0.014 3 2.532 0.057 0.018 FxI 3 3.027 0.029 0.022 3 0.116 0.951 0.001 PC1 1 547.693 <0.001 0.570 1 162.022 <0.001 0.280 PC1xF 1 23.186 <0.001 0.053 1 15.483 <0.001 0.036 PC1xI 3 2.897 0.035 0.021 Error 413 416

Chapter 2

Survival after 60 days was marginally significantly affected by injury treatments (logistic regression, Wald χ2 = 7.20, d.f. = 3, P = 0.066), with animals injured twice (0.842 ± 0.033 (mean

± S.E.); Wald χ2 = 5.76, d.f. = 1, P = 0.016, ES = 0.329) or thrice (0.858 ± 0.032; Wald χ2 = 4.36, d.f. = 1, P = 0.037, ES = 0.375) having lower survival than animals with no tail injuries (0.942 ±

0.021). Neither food availability, nor the interaction between the two factors influenced tadpole survival (Wald χ2 = 0.15, d.f. = 1, P = 0.698, ES = 1.357 and Wald χ2 = 4.74, d.f. = 3, P =

0.192, respectively).

Discussion

We found that the presence of the invasive species P. clarkii was associated with a greatly increased rate of tail injury in P. cultripes tadpoles. The positive correlation found between the frequency of tail injury and crayfish abundance emphasizes the serious consequences that the high P. clarkii densities can have for P. cultripes tadpoles, especially if we consider injury frequency as an indicator of predator pressure (Morin 1985). Furthermore, P. cultripes tadpoles seem to lack behavioural antipredator defenses against P. clarkii, such as decreased activity under predation risk (Cruz & Rebelo 2005), making them more prone to both direct predation and nonlethal attacks. In addition, high fecundity, rapid growth and ability to adjust its reproductive cycle to the hydrological conditions allow P. clarkii to quickly build extremely large populations in these fish-free habitats, which can become a serious risk to amphibian populations (Hobbs et al. 1989; Gutiérrez-Yurrita & Montes 1999).

Dytiscid beetles also proved important agents of injury for P. cultripes tadpoles, reflecting the high predation pressure they can impose (Tejedo 1993). Tejedo (1993) suggested that the large body size of P. cultripes tadpoles could be an important antipredator mechanism against

Dytiscus pisanus larvae. This can make them hard to capture not only because of their large size, but also because larger tadpoles are faster swimmers (Wassersug & Sperry 1977; Van

Chapter 2

Buskirk & McCollum 2000). This may allow tadpoles to frequently escape only at the cost of a ripped tail.

Tadpole tail injuries decreased with increasing emergent vegetation cover, probably because vegetation provides shelter for prey by decreasing predator encounter rates (Babbitt & Tanner

1998). On the other hand, frequency of injured tadpoles increased with pond area. Pond size can influence important biotic and abiotic factors in a community. For instance, Pearman

(1995) found that increasing pond area led to a greater incidence of Dytiscid beetles. In fact, in the present study there was a positive correlation between pond area and adult Dytiscid abundance, which may explain the increase in tail injuries with increased pond size.

In our experimental study, we found a tendency for larval survivorship being affected by increased frequency of caudal injury. In the laboratory, even under high-food conditions, repeated injuries seem to impose a cost in terms of reduced survival to P. cultripes tadpoles.

Parichy and Kaplan (1992) also observed reduced survivorship of injured tadpoles of Bombina orientalis, but only under low-food conditions. Similarly, tail loss in the salamander

Ambystoma talpoideum was negatively correlated with survival (Semlitsch & Reichling 1989).

This mortality effect can be caused by physiological stress responses or infection due to wounds (Langkilde & Shine 2006). However, in our field study, although tail injury frequency was much higher in ponds with than without crayfish, tadpole abundances were similar in the two types of ponds. Assuming there are no confounding factors, this suggests that, in our study area, there is no apparent survival cost caused by tail damage. Alternatively, if there is a survival cost, tadpole abundances in ponds with and without crayfish might not have differed due to crayfish indirect effects in other potential tadpole predators, such as macroinvertebrates or urodeles. If predation risk by these predators is reduced by crayfish- induced antipredator behaviours (Gamradt, Kats & Anzalone 1997; Nyström et al. 2001;

Werner & Peacor 2003), predation on tadpoles will decrease and population abundances may become similar to those in crayfish-free ponds.

Chapter 2

Also, contrary to our expectations, we did not find a perceptible cost of tail injury on the development and growth of P. cultripes tadpoles, even under relatively poor food conditions.

Other studies have also found weak or no effects of tail damage on developmental and growth rates of tadpoles, suggesting that rapid regeneration could reduce the ecological costs of tail damage (Wilbur & Semlitsch 1990; McCollum & Leimberger 1997). Following injury, all our tadpoles regenerated their tails in less than 15 days. However, if tadpoles compensate for tail injury by adopting antipredator behaviours like hiding, decreasing activity or moving to areas less suitable for predators, this may reduce their foraging success and ultimately growth under more natural conditions (Semlitsch 1990; Figiel & Semlitsch 1991).

Parichy and Kaplan (1992) found that B. orientalis tadpoles with injured tails had decreased growth and development at metamorphosis, but only under limited food conditions. This led to a later emergence and a smaller size at metamorphosis. Hence, even though in the laboratory there were no evident growth costs twenty days after the injury events, it is quite likely that there are delayed costs of tail damage for tadpoles. If tadpoles suffer repeated injuries during the larval stage, several regeneration episodes will take place, implying major shifts in resource allocation to support the rebuilt structures. The potential costs of transferring energy away from processes supporting growth into the reconstruction of the tail might only arise at metamorphosis, a period of profound physiological and morphological changes (Werner 1986). Injured animals may metamorphose at smaller sizes, which often leads to reduced postmetamorphic survival, delayed maturity and decreased size at first reproduction (Smith 1987; Semlitsch, Scott & Pechmann 1988). Indeed, in our study area, a sample of newly-metamorphosed individuals from ponds with crayfish had significantly smaller weights and body sizes than those from ponds without crayfish (M. Tejedo, A.L. Nunes, M. J.

Cruz, R. Rebelo, unpublished data). However, we can only speculate that this might arise from differences in frequencies of tail damage, because many other factors may influence it.

Chapter 2

The nonlethal injuries inflicted in our experiment induced changes in tail morphology: damaged animals had, in general, shallower tail fins, with deeper tail muscles. As measurements of tail traits were performed in the undamaged part of the tail, this indicates plastic changes in the original tail muscle and fin. This suggests that regeneration is not only producing new structures, but also influencing growth of the existing ones. To our knowledge, this is the first record of morphological plasticity in P. cultripes tadpoles. The increase in tail muscle depth as a response to caudal injury is in accordance with morphological responses of several tadpole species exposed to predators (e.g. Relyea 2001; Teplitsky et al. 2005; Wilson et al. 2005). In Scaphiopus holbrookii, a species from the same family of P. cultripes, tadpoles having predator-induced deeper tails exhibited faster burst swimming speeds, which probably promote a higher promptness to escape an attack (Dayton et al. 2005; Teplitsky et al. 2005).

The larger muscle, apart from allowing tadpoles to outswim predators may also enable them to survive attacks by facilitating tail ripping via enhanced thrust (Dayton et al. 2005). We can only speculate how well this morphological response would suit P.clarkii’s predation attempts, but the ability of a tadpole to generate bursts of speed or to facilitate membranous tail ripping is probably a useful feature for avoiding unpredictable attacks.

Food availability proved to be an important factor in determining changes in tail depth. In the beginning of the experiment all injured tadpoles increased their muscle depth but, after the second injury, low-food tadpoles did not increase it anymore. This was probably due to energy limitation, suggesting that food levels may shape the consequences of tail injury. The response of altering muscle depth was mainly found at high resource availability and is probably absent in macrophyte impoverished wetlands invaded by P. clarkii. If habitat productivity is dampened by crayfish presence, the effects of tail injury might be more severe for tadpoles, as they lack the necessary energy to recover from them.

To summarize, in areas where P. clarkii is established, it is probably imposing a high predation pressure on P. cultripes, as reflected by the high frequencies of tadpoles with tail injuries.

Chapter 2

However, tail damage is probably not critical for tadpole survival, because animals often survive attacks to the tail. In fact, in our experiment, tail injury only imposed a marginal cost in tadpole survival. It also stimulated the growth of the tail muscles, which did not exact an immediate cost on growth and development, probably reflecting an adaptation to repeated tail injuries. Nevertheless, costs may arise from shifts in resource allocation that have little immediate impact, but that alternatively compromise future fitness components. Moreover, as true fitness costs may be underestimated in laboratory conditions, studying these mechanisms under more natural conditions is essential to understand potential impacts for amphibians in systems invaded by this crayfish.

Acknowledgments

We thank Sandra Amaral for her help in the field and Nídia Fernandes, Ricardo Rocha, Sofia

Viegas, Pedro Andrade and Vasco Furtado for their help in the experiment. We also thank

Pedro Segurado for drawing the map. We thank the Portuguese Instituto da Conservação da

Natureza e da Biodiversidade (ICNB) for the permits. This research was supported by the FCT

Project POCI/BIA-BDE/56100/2004 and by the European Community-Access to Research

Infrastructure Action of the Improving Human Potential Programme in Doñana Biological

Station (ECODOCA).

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Chapter 2

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Chapter 2

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Chapter 2

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CHAPTER 3

Do anuran larvae respond behaviourally to chemical cues from an invasive crayfish predator?

A community-wide study

Nunes AL, Richter-Boix A, Laurila A, Rebelo R

Oecologia (in review)

Chapter 3

Do anuran larvae respond behaviourally to chemical cues from an invasive crayfish predator? A community-wide study

Abstract

Antipredator behaviour is an important fitness component in most animals. A co-evolutionary history between predator and prey is important for prey to respond adaptively to predation threats. When non-native predator species invade new areas, native prey may not recognise them and lack effective antipredator defences. However, responses to novel predators can be facilitated by chemical cues from the predators’ diet. The red swamp crayfish Procambarus clarkii is a widespread invasive predator in the Southwest of the Iberian Peninsula, where it preys upon native anuran tadpoles. In a laboratory experiment we studied behavioural antipredator defences (alterations in activity level and spatial avoidance of predator) of nine anurans in response to P. clarkii chemical cues and compared them with the defences towards a native predator, the larval dragonfly Aeshna sp. To investigate how chemical cues from consumed conspecifics shape the responses, we raised tadpoles with either a tadpole-fed or starved crayfish or dragonfly larva, or in the absence of a predator. Six species significantly altered behaviour in the presence of crayfish, and this was largely mediated by chemical cues from consumed conspecifics. In the presence of dragonfly most species exhibited behavioural defences and often these did not require the presence of cues from predation events.

Responding to cues from consumed conspecifics seems to be a critical factor in facilitating certain behavioural responses to novel exotic predators; this finding can be useful for predicting antipredator responses to invasive predators and help directing conservation efforts to the species at highest risk.

Keywords: Tadpole, activity level, spatial avoidance, behavioural plasticity, exotic predator

Chapter 3

Introduction

Behaviour plays a decisive role in shaping the outcome of predator-prey interactions (Lima and

Dill 1990, Lima 1998, Ferrari et al. 2010). Predation is an important selective force acting on the behaviour of prey species and, in order to minimise predation risk, many organisms have evolved a variety of predator-avoidance behavioural defences (reviews in Lima and Dill 1990,

Kats and Dill 1998). A decrease in activity level is one of the most common and effective behavioural antipredator responses and decreases vulnerability to predation (Lima and Dill

1990, Kats and Dill 1998). Spatial avoidance of predators also acts as an antipredator defence by reducing the rate of predator-prey encounters and, consequently, predation risk (Laurila et al. 1997, Relyea 2001, Nicieza et al. 2006). However, these behavioural shifts often incur costs to animals due to reduced resource acquisition, which can alter growth, development and life- history patterns (Werner and Anholt 1996, Lima 1998, but see Steiner 2007). Therefore, there should be strong selection on prey to recognise dangerous predators, to accurately determine predation risk and to adjust antipredator responses accordingly.

Invasive non-native predators are a worldwide threat to biodiversity (Blackburn et al. 2010).

Freshwater ecosystems are amongst the most invaded and particularly vulnerable to introduced predators (Lodge et al. 1998, Cox and Lima 2006). For instance, many amphibian population declines have been associated with the introduction of exotic aquatic predators

(review in Kats and Ferrer 2003). When predators invade areas outside their historical geographic range, the lack of a common evolutionary history with native prey species often renders the latter more prone to suffering heavy predation (Cox and Lima 2006, Gall and

Mathis 2010). This is because the evolutionary naïveté of prey may either cause a failure to recognise and respond to novel predation threats, or result in inappropriate or ineffective antipredator defences (Cox and Lima 2006, Strauss et al. 2006, Sih et al. 2010). The degree of naïveté can depend on the similarity of the invader to native predators, since phylogenetic

Chapter 3 relatedness is often associated with ecological similarity (Cox and Lima 2006, Sih et al. 2010).

For instance, more distantly related aquatic predator species are likely to have more dissimilar chemical signatures. Hence, a novel predator with no related species in the native community may pose a higher threat, as it is more unlikely to be recognised as dangerous by native prey

(Strauss et al. 2006, Gall and Mathis 2010, Sih et al. 2010). Several studies have reported a lack of native prey responses to aquatic invasive predators, which may cause profound changes in the invaded ecosystems. A classic example is the introduction of the Nile perch (Lates niloticus) in Lake Victoria, which caused a severe decline in native cichlids, probably due to lack of predator recognition (Witte et al. 1992).

In aquatic ecosystems, predator chemical cues are particularly important for prey in assessing predation risk (Kats and Dill 1998). The chemicals to which prey respond may be predator- specific odours, cues that are actively or passively released by injured or consumed conspecifics or, more frequently, a combination of both (Chivers and Smith 1998, Schoeppner and Relyea 2009, Fraker et al. 2009, Hettyey et al. 2010). Several studies have shown that fed predators commonly elicit strong antipredator defences while starved predators often do not

(Stirling 1995, Slusarczk 1999, Schoeppner and Relyea 2005, 2009, but see Petranka and Hayes

1998, Van Buskirk and Arioli 2002). Furthermore, prey responses often depend on the phylogenetic distance between consumed and responding prey, consumed conspecifics and individuals of closely related species eliciting a stronger response (e.g. Kats and Dill 1998,

Laurila et al. 1998, Schoeppner and Relyea 2005, 2009). As a predator may become chemically

‘labelled’ by its diet, recognition of a novel predator can be facilitated if diet chemical cues are associated with it (reviewed in Ferrari et al. 2010). However, studies on prey responses to invasive predators generally do not take into account the role of chemical cues originating from consumed conspecifics and their potential importance on enabling predator recognition

(but see Marquis et al. 2004).

Chapter 3

Freshwater crayfishes, which have been widely introduced outside their native range, can have major impacts in native prey species and cause severe population declines (Hobbs et al. 1989,

Lodge et al. 1998, Cox and Lima 2006, Larson and Olden 2010). In the southwest of the Iberian

Peninsula, most freshwater habitats have been invaded by Procambarus clarkii (red swamp crayfish), an exotic crayfish endemic to northeastern Mexico and southcentral USA (Hobbs et al. 1989, Gherardi 2006). This crayfish was introduced in Spain in 1973 and by the 1990s it was already abundant in southwestern Portugal, an area previously devoid of freshwater crayfishes or functionally similar species (Habsburgo-Lorena 1983, Almaça 1991). This area holds a remarkable endemic anuran community: out of nine species three are endemic to the Iberian

Peninsula and three others have a restricted distribution outside Portugal and Spain (Gasc et al. 1997). P. clarkii readily preys upon eggs and larvae of all these anurans (Cruz and Rebelo

2005). Due to its high fecundity and rapid growth, it can quickly build extremely large populations, especially in fish-free habitats, and become a serious threat to amphibian populations (Gherardi 2006, Larson and Olden 2010).

The ability of anuran tadpoles to detect and respond to P. clarkii is largely unknown, despite the importance of this information for understanding the extent to which native amphibians are able to cope with this invasive predator. In this study, our main goal was to understand if anuran tadpoles from a community in southwest Iberian Peninsula invaded by P. clarkii ca. 25 years ago are able to exhibit behavioural defences in the presence of chemical cues from this crayfish. For this, we performed a laboratory experiment in which we assessed changes in tadpole activity level and spatial avoidance of the predator in the presence of chemical cues from the invasive crayfish and compared them with those elicited in the presence of a common native predator, the larval dragonfly Aeshna sp. We also investigated the role of chemical cues from consumed conspecifics by comparing behavioural responses in the presence of starved and conspecific-fed predators. A previous study has shown that many of our study species may change their behaviour under direct predation risk by P. clarkii (Cruz

Chapter 3 and Rebelo 2005), but no study has compared these responses to those elicited in the presence of a native predator, nor investigated the role of predator chemical cues or cues from predated conspecifics in it. Finally, we evaluated how these behavioural responses changed during tadpoles’ ontogeny.

The anuran community in Southwestern Portugal consists of nine species: Iberian water frog

(Pelophylax perezi), Mediterranean tree frog (Hyla meridionalis), European tree frog (Hyla arborea), common toad (Bufo bufo), natterjack toad (Bufo calamita), Iberian parsley frog

(Pelodytes ibericus), Western spadefoot toad (Pelobates cultripes), Iberian painted frog

(Discoglossus galganoi) and Iberian midwife toad (Alytes cisternasii). Except the two Bufo species, these species (or very closely related species) show behavioural plasticity in presence of caged Aeshnid dragonflies (Van Buskirk 2002, Nicieza et al. 2006, Richter-Boix et al. 2007,

Gomez-Mestre and Díaz-Paniagua 2011), and we expect them to show behavioural responses in the presence of this native predator. In P. cultripes behavioural responses to Aeshnids have not been studied before; we predict that behavioural defences may be unnecessary, because the large body size of these tadpoles acts as an important antipredator mechanism (Tejedo

1993). Concerning responses to the exotic crayfish, we predict most of the species to not detect and respond to this novel predator; however, chemical cues from consumed conspecifics may ease these responses. Cruz and Rebelo (2005) have shown that these anuran species differ in vulnerability to predation by P. clarkii, the two Bufo species, P. cultripes and D. galganoi being the most susceptible species. This may indicate that these species lack appropriate defences towards this crayfish. Finally, we predict antipredator defences to be strongest in the beginning of tadpole development because prey vulnerability and responses to predators decrease as prey size increases (e.g. Eklöv and Werner 2000; Hettyey et al. 2010).

Chapter 3

Materials and methods

Study species and their maintenance

This study was conducted in the surroundings of the Sado River basin (SW Portugal), an area that was colonised by P. clarkii in the 1980s (R. Rebelo pers. obs.). In this area, P. clarkii can be found in all types of water bodies (e.g. streams, temporary ponds), including small and shallow ones (Cruz and Rebelo 2007). Aeshna larvae are voracious native predators of tadpoles, widely abundant in water bodies throughout this area. All anuran species included in this study co- occur with both these predators.

The experiment took place in a laboratory of the field station of Centro de Biologia Ambiental

(Grândola, southwest Portugal, 38º06.482’N, 8º34.140’W), from 15th December 2007 to 15th

November 2008. This extended experimental period was due to differences in breeding phenology of the study species. Several egg masses of each species were collected from ponds located in Alentejo region, southwest Portugal (Table 1). In A. cisternasii, males show parental care until tadpoles become free-swimming, and the tadpoles used in the experiment had been very recently released in the ponds. All the ponds had established crayfish populations, with evidence of crayfish reproduction (juveniles or brooding females). Clutches were kept in several species specific 5L aquaria filled with spring water from the field station, until tadpoles reached Gosner stage 25 (operculum closure over gills; Gosner 1960). Larvae were fed commercial fish food and boiled lettuce ad libitum every two days. Throughout the experiment water was changed every 5 days. Water temperature was 18.0 ± 0.17 ºC (mean ± SE) and the photoperiod was 12L: 12D.

Predators were collected from local streams or ponds. Adult crayfish were captured using baited funnel traps and late instar dragonfly larvae using dip-nets. Predators were transferred to the laboratory and kept either in 40L aquaria (crayfish) or individually in 1.2L plastic boxes

Chapter 3

(dragonflies). We fed crayfish with commercial fish food and small invertebrates, and

dragonflies with tadpoles and Ephemeroptera larvae.

Table 1. Number of clutches collected, collection site (type of water body and coordinates), date of their collection and dates of start and end of the experiment for each of the nine studied species

# Clutches Date of Start of End of Species Collection site Coordinates collected collection experiment experiment t Pelophylax 11 Grândola (stream) 38º07’N, 8º34’W 16-Jun 02-Jul 09-Nov perezi

Hyla Grândola (temporary 9 38º06’N, 8º34’W 13-Mar 08-Apr 15-Aug meridionalis ponds)

Hyla arborea 10 Melides (rice field) 38º08’N, 8º44’W 05-Jun 17-Jun 25-Sep

Bufo bufo 6 Grândola (stream) 38º09’N, 8º39’W 07-Feb 19-Feb 04-Jun

Grândola (temporary/ Bufo calamita 9 38º05’N, 8º35’W 22-Apr 19-May 09-Aug ephemeral ponds)

Pelodytes Grândola (temporary 10 38º06’N, 8º33’W 26-Dec 23-Jan 22-Jun ibericus ponds)

Pelobates Grândola (temporary 8 38º09’N, 8º31’W 28-Nov 15-Dec 05-Sep cultripes ponds)

Discoglossus Grândola (ephemeral 9 38º06’N, 8º34’W 08-Jan 24-Jan 13-May galganoi ponds)

Alytes - Grândola (stream) 38º05’N, 8º33’W 04-Dec 15-Dec 28-Jul cisternasii

Experimental setup

We performed a factorial experiment using the nine anuran species and five predator

treatments, each combination being replicated five times. This resulted in a total of 225

experimental units, each consisting of a plastic tank (39 × 28 × 28cm) filled with 10L of water.

At the start of the experiment (day 0), ten tadpoles at developmental stage 25 and a predator

cage were added to each tank. The five predator treatments were: fed crayfish, fed dragonfly,

starved crayfish, starved dragonfly and control (no predator). The predators were placed in the

cages one day after the tadpoles. A single predator was placed in a cylindrical opaque floating

Chapter 3 cage, which sides were covered with fine mesh netting allowing chemical cues to flow out of the cage. Only chemical predator cues (no visual or tactile cues) were available for the tadpoles. Crayfish and dragonfly cages had a diameter of 85 and 62 mm, respectively. In the control treatments, we randomly used empty predator cages from one of the two sizes. While inside the predator cage in each experimental container, predators from the ‘fed’ treatments were fed three focal species tadpoles every other day. Starved predators were not fed during the experiment. Prior to entering the experiment all predators were starved for, at least, 48 hours. All the predators were replaced every two weeks so that the starved predators could be fed.

Response variables and statistical analyses

We recorded tadpole behaviour (activity levels and spatial avoidance) in the beginning (time period 1), middle (time period 2) and close to the end (time period 3) of larval development.

As the length of larval period differs among different species, the dates of behavioural observations - as well as headbody size and Gosner developmental stage (Gosner 1960) at each date - also varied (Table 2). On each observation day, tadpole activity and spatial avoidance were estimated by counting the number of active tadpoles and the number of tadpoles close to the predator cage in each tank at five repeated occasions between 8 and 12 a.m., separated by at least 30 minutes. A tadpole was considered active when it was actively swimming (either slowly or with fast bursts of speed), feeding (even if not substantially altering position) or simply undulating its tail (without actively swimming). Tadpoles were considered to be close to the predator cage if they were either in physical contact with the cage or not more than 1cm away from it.

Chapter 3

Table 2. Days of the experiment, Gosner developmental stage (according to Gosner 1960) and headbody size when behaviour was sampled in the three time periods (early, middle and late in development) for each species

Species Day of the experiment Median Gosner stage Average headbody size Time 1 Time 2 Time 3 Time 1 Time 2 Time 3 Time 1 Time 2 Time 3 Pelophylax perezi 11 41 63 25 33 40 5.26 12.56 15.90 Hyla meridionalis 8 42 57 25 32 40 5.53 10.14 11.35 Hyla arborea 8 36 50 25 30 41 5.41 8.06 11.11 Bufo bufo 8 31 51 25 34 41 5.82 9.11 10.71 Bufo calamita 9 29 37 26 34 42 4.03 6.99 8.73 Pelodytes ibericus 14 27 49 25 32 39 5.61 11.11 13.01 Pelobates cultripes 13 123 199 25 30 41 5.63 14.77 23.66 Discoglossus galganoi 14 34 40 27 35 42 4.40 9.35 10.01 Alytes cisternasii 13 59 89 25 30 39 6.83 12.49 13.91

When making comparisons across taxa, there is a need to examine whether the phenotypic

traits in focus are correlated with the phylogenetic history of the focal species. Since nine

different anuran species were used in this study, we followed Abouheif’s (1999)

recommendation of testing for phylogenetic trait independence prior to analysing our data. If

no significant phylogenetic autocorrelation is detected among species (failure to reject the null

hypothesis of phylogenetic independence), phylogenetically comparative methods do not have

to be used and conventional statistical analyses can be applied (Abouheif 1999). To test for the

assumption of phylogenetic independence within our data set we first calculated, for each

species and predator treatment separately, the extent of predator-induced behavioural

plasticity. This was estimated as the proportional change in either activity level or spatial

avoidance in each of the four predator environments relative to the no predator environment

([behaviour in predator presence – behaviour in predator absence]/ behaviour in predator

absence; Richardson 2001, Van Buskirk 2002). Using these values, a Test For Serial

Independence (TFSI) was performed for each treatment, using the program “Phylogenetic

Independence, Version 2.0” (Abouheif 1999, Reeve and Abouheif 2003). The phylogenetic tree

used was constructed based on Duarte et al. (2011) (Fig. 1). Phylogenetic autocorrelation was

Chapter 3 calculated in the form of a C‐statistics and topology of the original data was randomised

10.000 times to estimate the null hypothesis.

Fig. 1 Assumed phylogenetic relationships (based on Duarte et al. 2011) for the nine anuran species used in this experiment: Pelophylax perezi, Hyla meridionalis, Hyla arborea, Bufo calamita, Bufo bufo, Pelodytes ibericus, Pelobates cultripes, Discoglossus galganoi and Alytes cisternasii

Since for all predator treatments we accepted the null hypothesis of phylogenetic independence in behavioural plasticity in both traits (see results), subsequent statistical analyses did not need to be phylogenetically corrected. To examine differences in species behavioural responses to the exotic and the native predators over their development, we used

Generalized Linear Models with a Binomial error distribution and a logit link function. As behaviour was recorded in three different time periods, a repeated measures approach was used to assess the effect of time on species responses. The total number of active tadpoles or tadpoles close to the predator cage per container pooled over the five observations in each time period was used as the dependent variable and the total number of tadpoles inside a container pooled over the five observations in each time period was treated as a trials

Chapter 3 variable. We started by performing an analysis at the community level, comparing responses of all the studied species in the different predator treatments. Subsequently we looked at a species-specific level to help identify more precisely antipredator responses of each species.

Since most individual species showed a significant interaction between time period and predator treatment (see results), we also performed separate analyses for species in each time period in order to investigate responses over time in more detail. Multiple planned comparisons between predator treatments were performed using Bonferroni corrections.

These comparisons were chosen in order to answer the questions which species are responding to the native or the invasive predators (control treatment vs. all others), do cues from consumed conspecifics play a role in this (fed vs. starved treatments) and how similar is the response to the two predators (fed dragonfly vs. fed crayfish and starved dragonfly vs. starved crayfish). Generalized Linear Mixed models using block as a random factor were initially performed, but as this factor was never significant for the models, it was discarded from the definitive analyses.

In order to understand if species responding more to the native predator also respond more to the exotic predator and if these responses are mediated by cues from consumed conspecifics,

Pearson correlations between predator-induced behavioural plasticity in the two starved and in the two fed predator treatments were calculated. We used data from time period 1 as this was the period when the strongest responses were observed. If species antipredator responses are mainly mediated by consumed conspecifics cues, we expect to find a positive correlation between the two fed predator treatments and no correlation between the two starved treatments. If, alternatively, species require the presence of predator-specific cues to respond, we expect a strong response to the dragonfly and a weak or no response to the crayfish (due to the lack of recognition of crayfish kairomones), resulting in either a negative or no correlation between the two fed and the two starved treatments. To investigate which amount of predator-induced behavioural plasticity in the fed treatments was due to the

Chapter 3 presence of cues from consumed conspecifics (hereafter plasticity due to CC cues) we estimated, for each predator and species, the proportional change in activity level or spatial avoidance in the fed predator treatment relative to the starved predator treatment

([behaviour in presence of fed predator – behaviour in presence of starved predator]/ behaviour in presence of starved predator). We then performed a correlation between the plasticity due to the CC cues in the presence of dragonfly and crayfish. If all plasticity is attributed to the CC cues, plasticity should be the same in presence of the two predators and a close to perfect correlation should be observed, since the number of tadpoles fed to the two different predators was equal.

Results

We did not find a significant phylogenetic autocorrelation for behavioural plasticity in activity level (C = 0.177, P = 0.22 for the fed dragonfly treatment, C = 0.125, P = 0.27 for the fed crayfish treatment, C = 0.040, P = 0.41 for the starved dragonfly treatment, C = 0.077, P = 0.32 for the starved crayfish treatment) and spatial avoidance (C = 0.114, P = 0.27 for the fed dragonfly treatment, C = 0.353, P = 0.09 for the fed crayfish treatment, C = 0.141, P = 0.24 for the starved dragonfly treatment, C = 0.015, P = 0.52 for the starved crayfish treatment) of the nine species in any of the four different predator treatments, suggesting no association between behavioural plasticity and species phylogenetic history.

When comparing behaviour among the nine anuran species, both activity level and spatial avoidance varied across time periods (Wald χ2 = 106.28, df = 2, 538, P < 0.001 for activity level;

Wald χ2 = 51.05, df = 2, 538, P < 0.001 for spatial avoidance). Further, a significant interaction between time period and predator treatment indicates complex behavioural responses of tadpoles to different predator regimes throughout their development (Wald χ2 = 28.91, df = 8,

538, P < 0.001 for activity level; Wald χ2 = 143.73, df = 8, 538, P < 0.001 for spatial avoidance).

Chapter 3

When analysing behavioural responses to predators, we always refer to comparisons between each predator and the no-predator (control) treatments, unless otherwise noted.

When looking at species separately, time period was an important factor in determining activity level in most of the species. B. bufo, H. meridionalis, P. cultripes, H. arborea, B. calamita and P. perezi increased their overall activity with time, whereas D. galganoi decreased activity (in all analyses, P < 0.001 for the factor ‘time period’; Fig. 2). Only A. cisternasii and P. ibericus did not show an alteration of activity with time (P > 0.06; Fig. 2). A significant interaction between time period and predator treatment was found for all species except B. calamita and P. ibericus, indicating that alterations in behavioural responses to predators throughout development were common (for all analyses, P < 0.05; Fig. 2). For B. calamita no differences were detected between predator treatments (Wald χ2 = 8.924, df = 4,

60, P = 0.063), while P. ibericus reduced activity in the presence of dragonfly, with a stronger response to the fed than to the starved predator (Wald χ2 = 51.179, df = 4, 60, P < 0.001; Fig.

2).

In species which behavioural responses varied over time, dragonfly presence (together with predated conspecifics) strongly affected tadpole activity level, with reduced activity in all time periods in all species except P. cultripes and P. perezi (Tables 3, 4, Fig. 2). B. bufo was the only species showing a continuous and similar in magnitude response to both dragonfly (fed and unfed) and crayfish (fed) predators, this response becoming stronger with developmental time

(Tables 3, 4, Fig. 2). An initial significant response to the fed crayfish and a later response to the starved dragonfly arose in A. cisternasii, while in D. galganoi these responses appeared later in development and were stronger in the fed treatments. Both hylid species were less active in the presence of the crayfish predator late in their development, either when the predator was fed (H. meridionalis) or unfed (H. arborea) (Tables 3, 4, Fig. 2). For P. cultripes we found an initial response of reduced activity to both fed predators, the response to the crayfish being weaker than to the dragonfly, which later disappeared. For P. perezi, early in ontogeny,

Chapter 3 tadpoles from the fed dragonfly treatment reduced activity, while the ones from the starved dragonfly treatment showed a significantly higher proportion of active tadpoles (Tables 3, 4,

Fig. 2).

Fig. 2 Proportion of active tadpoles (mean ± SE) of the nine anuran species, in presence of five predator treatments, in three different time periods

Chapter 3

Table 3. Generalized Linear Model for the activity level and spatial avoidance of the nine anuran species when subjected to different predator treatments, in each of the three time periods. The analyses were not performed for P. ibericus because it did not show a significant interaction between predator treatment and time period for any of the traits. P-values < 0.05 are marked in boldface.

Activity level Spatial avoidance Time 1 Time 2 Time 3 Time 1 Time 2 Time 3 Wald χ2 P Wald χ2 P Wald χ2 P Wald χ2 P Wald χ2 P Wald χ2 P P. perezi 32.086 <0.001 11.515 0.021 14.014 0.007 7.096 0.131 1.854 0.763 10.647 0.031 H. meridionalis 31.937 <0.001 18.023 0.001 38.538 <0.001 42.314 <0.001 35.606 <0.001 8.021 0.091 H. arborea 19.614 0.001 21.778 <0.001 13.538 0.009 ------B. bufo 51.938 <0.001 75.177 <0.001 134.372 <0.001 45.998 <0.001 11.381 0.023 27.548 <0.001 B. calamita ------0.001 1.000 7.082 0.132 8.601 0.072 P. cultripes 31.154 <0.001 22.096 <0.001 2.213 0.697 8.367 0.079 13.724 0.008 13.678 0.008 D. galganoi 59.337 <0.001 65.994 <0.001 50.534 <0.001 19.707 0.001 21.666 <0.001 14.307 0.006 A. cisternasii 34.349 <0.001 33.652 <0.001 23.999 <0.001 24.084 <0.001 46.338 <0.001 51.320 <0.001

Chapter 3

Table 4. Results of planned comparisons between predator treatments for tadpole activity level and spatial avoidance after analysis shown in Table 3. Results are not shown for P. ibericus because an analysis per period was not performed (see Results) and for B. calamita because no significant differences were found. Significant values after Bonferroni correction (alpha level set to 0.006) are marked with an asterisk. ‘Drag’ stands for dragonfly and ‘Crayf’ for crayfish.

P. perezi H. meridionalis H. arborea B. bufo P. cultripes D. galganoi A. cisternasii Time period 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Activity level Control-Fed Drag * * * * * * * * * * * * * * * * Control-Starved Drag * * * * * * * * Control-Fed Crayf * * * * * * * * Control-Starved Crayf * * Fed Drag-Fed Crayfish * * * * * * * * * * * Starved Drag-Starved Crayf * * * * * * * * Fed Drag-Starved Drag * * * * * * * * * * * Fed Crayf-Starved Crayf * * * * * * Spatial avoidance Control-Fed Drag * * * * * * * * * * * Control-Starved Drag * * * * * * * * Control-Fed Crayf * * * * * * * Control-Starved Crayf * * Fed Drag-Fed Crayfish * * * * * * Starved Drag-Starved Crayf * * * * * Fed Drag-Starved Drag * * Fed Crayf-Starved Crayf * * *

Chapter 3

When looking at species-specific spatial avoidance, A. cisternasii, P. ibericus, B. bufo, H. arborea and P. perezi did not alter the degree of predator spatial avoidance with time (in all analyses, P > 0.2 for the factor ‘time period’; Fig. 3). Once again, a significant interaction between time period and predator treatment was found for all species except H. arborea and

P. ibericus, indicating alterations in antipredator responses through ontogeny (for all analyses,

P < 0.05; Fig. 3). Both H. arborea (Wald χ2 = 12.916, df = 4, 60, P = 0.012; Fig. 3) and P. ibericus

(Wald χ2 = 31.907, df = 4, 60, P < 0.001; Fig. 3) avoided the predator cage in the presence of fed dragonfly.

Fig. 3 Proportion of tadpoles near the predator cage (mean ± SE) of the nine anuran species, in presence of five predator treatments, in three different time periods

Chapter 3

In each time period, all significant responses of spatial avoidance consisted of moving away from the predator cage. Tadpoles of A. cisternasii showed continuous and strong avoidance behaviour throughout development to both predators (both fed and starved). This was the only species avoiding the predator cage in the starved crayfish treatment (Tables 3, 4, Fig. 3).

D. galganoi, B. bufo and H. meridionalis strongly avoided the cage with the dragonfly predator

(fed and unfed), and both D. galganoi (time periods 1 and 3) and B. bufo (period 1) also avoided the fed crayfish predator as strongly as they avoided the native predator. Tadpoles of

P. cultripes avoided the cage with fed crayfish in time period 2 (Tables 3, 4, Fig. 3).

No correlation was detected between species-level plasticity in activity level in the starved dragonfly and starved crayfish treatments (r = 0.269, N = 9, P = 0.484; Fig. 4a). However, there was a significant positive correlation between species plasticity in the fed dragonfly and fed crayfish treatments (r = 0.717, N = 9, P = 0.03; Fig. 4b). A positive correlation was also found between the two predator treatments for plasticity in activity attributed to the CC cues (r =

0.699, N = 9, P = 0.036; Fig. 4c), suggesting that the correlation between plasticity in the two fed treatments was due to among-species variation in plasticity to CC cues. Concerning behavioural plasticity in spatial avoidance of the predator, no significant correlations were found between fed (r = 0.317, N = 9, P = 0.406) or starved treatments (r = 0.09, N = 9, P =

0.889), or for plasticity to the CC cues (r = 0.039, N = 9, P = 0.920).

Discussion

This study provides the first community-wide assessment of antipredator behaviour of anuran larvae in response to chemical cues of the invasive crayfish P. clarkii, the most cosmopolitan crayfish species in the world (Gherardi 2006). Even though P. clarkii was relatively recently introduced in our study area, we found that six out of nine prey species responded

Chapter 3 behaviourally to this predator. As expected, these responses were greatly mediated by chemical cues resulting from predation events.

Fig. 4 Relationship between behavioural plasticity in activity level of the nine anuran species in the presence of dragonfly and crayfish, in time period 1: a) plasticity in presence of the starved predators: for each predator axis, ([activity with starved predator present – activity with predator absent]/activity with predator absent); b) plasticity in presence of the fed predators: for each predator axis, ([activity with fed predator present – activity with predator absent]/activity with predator absent); c) plasticity attributed to the consumed conspecifics chemical cues: for each predator axis, ([activity with fed predator present – activity with starved predator present]/activity with starved predator present). The grey line in graph c) represents the expected relationship if species respond to diet cues exactly in the same way in the dragonfly and crayfish treatments. See figure 1 for species names abbreviations. Plasticity values below zero indicate a decrease in activity in the presence of predators and positive plasticity values reflect an increase in activity with predators present

Behavioural responses to the native and introduced predators

In the presence of dragonfly, six species had a strong behavioural response (A. cisternasii, D. galganoi, B. bufo, H. meridionalis, H. arborea and P. ibericus), two had a moderate response (P. cultripes and P. perezi), while one species had no significant response (B. calamita). In general, activity levels were greatly reduced and strong spatial avoidance of the predator was observed in the presence of the native predator. These are common and usually effective antipredator responses (reviews in Lima and Dill 1990, Kats and Dill 1998), often reported for larval

Chapter 3 amphibians exposed to odonate predators (e.g. Werner and Anholt 1996, Relyea 2001,

Richardson 2001, Nicieza et al. 2006). The strong responses observed here indicate that, as expected, most of these anuran species do have the potential of exhibiting behavioural strategies to avoid predation. The significant behavioural response of B. bufo to the dragonfly was somewhat unexpected, since most studies report a lack of behavioural antipredator responses in this species (e.g. Laurila et al. 1997, Richter-Boix et al. 2007). However, although bufonids are known to rely on chemical toxic deterrents as defences against predators, several invertebrate predators (including P. clarkii) are resistant to them, probably inducing tadpoles to use alternative defences (Semlitsch and Gavasso 1992, Cruz and Rebelo 2005). Laurila et al.

(1998) also reported a weak decrease in B. bufo activity in the presence of a dragonfly.

We found that six tadpole species (A. cisternasii, B. bufo, D. galganoi, H. meridionalis, H. arborea and P. cultripes) significantly altered activity level and/or showed spatial avoidance in the presence of P. clarkii. Although these responses were generally weaker than the ones elicited towards the native predator, this indicates that the ability to respond to the invasive crayfish is quite pervasive in this amphibian community. These results are in line with previous studies that have shown amphibians to alter behaviour as a response to chemical cues of non- native predators, such as P. clarkii (e.g. Pearl et al. 2003, Marquis et al. 2004, Gall and Mathis

2010). Pearl et al. (2003) found different responses in two native anurans to cues from an introduced predator which, together with our study, show that species from the same community may commonly respond differently to exotic predators.

Differences in the ability of prey species to respond behaviourally to the crayfish could have arisen due to several aspects of species ecology, such as their habitat use or body size (and associated swimming abilities). Anuran species face different selective environments along the pond permanency gradient, ranging from permanent lakes and ponds to ephemeral pools

(Wellborn et al. 1996). Since permanent water bodies have a higher predator abundance and diversity than ephemeral ones, species using the former habitats (P. perezi, H. arborea, B. bufo

Chapter 3 and A. cisternasii in our case) are likely to have more generalized (as opposed to predator- specific) antipredator defences that allow responses to a wide array of predators, likely facilitating responses to novel invasive predators (Richter-Boix et al. 2007, Smith and Awan

2009, Gómez and Kehr 2011). Indeed, in this study, three of the four species typically inhabiting permanent water bodies responded to the exotic predator (A. cisternasii, B. bufo and H. arborea). Nonetheless, P. perezi, also a permanent pond species, did not respond to the crayfish, indicating that the hydroperiod gradient does not explain all the differences in species antipredator responses. Alternatively, prey that have small body size and relatively weak swimming ability, such as the bufonids and D. galganoi, should elicit antipredator defences in the presence of many different predators. Two of these species (the exception being B. calamita) indeed responded to the crayfish predator. However, species with larger body size and good swimming ability, such as the hylids, also elicited behavioural responses to the exotic crayfish. The evolutionary history of a species can also be important in defining differences in species antipredator responses (Richardson 2001); however, we did not find any phylogenetic autocorrelations in the behavioural responses of these species, indicating that evolutionary history does not seem to play an important role here (see also Richter-Boix et al. 2007). On the whole, since none of the previously mentioned factors entirely explains differences in behavioural responses to the exotic predator, it is possible that all these factors interact to define the final species-specific responses.

The display of behavioural responses by B. bufo, D. galganoi and P. cultripes towards P. clarkii is surprising, since an earlier study found that these species were the most vulnerable to P. clarkii predation (Cruz and Rebelo 2005). This might indicate that in Cruz and Rebelo’s study tadpoles did not have enough time to develop behavioural defences against the crayfish (it only lasted 48 hours) or, alternatively, that the behavioural responses shown here may be a non-effective response towards this exotic predator. For example, because crayfish rely much more on chemical rather than on visual cues to detect prey, decreased activity in response to

Chapter 3 crayfish may render inactive tadpoles to an equal risk to that experienced by active ones

(Aquiloni et al. 2005).

The importance of chemical cues from consumed conspecifics

In this study, most species significantly changed behaviour in the presence of fed predators, suggesting that cues from consumed conspecifics play a very important role in inducing anuran behavioural responses. However, six out of nine species also altered behaviour when exposed to chemical cues from the starved dragonfly indicating that predator-specific odours from a native predator are, in many cases, sufficient to launch behavioural defences.

In the three species that responded strongly to the exotic predator (A. cisternasii, B. bufo and

D. galganoi), behavioural plasticity (considering activity level) to the fed crayfish was mostly explained by plasticity to the CC cues (cues from consumed conspecifics), indicating that the response to the exotic predator was mediated by cues resulting from predation events. These results are in accordance with those by Marquis et al. (2004), who suggested the presence of conspecific alarm cues to be crucial in chemical detection of predators by B. bufo. Injured tadpoles of this species are known to release alarm cues - chemicals released by specialised cells in the skin - that induce antipredator responses, such as a reduction in activity, in conspecifics (Chivers and Smith 1998, Hagman 2008, Fraker et al. 2009). Responding to cues from injured conspecifics is frequent among anuran species (Chivers and Smith 1998, Hagman

2008, Ferrari et al. 2010) and may also be the case for A. cisternasii and D. galganoi (S. Amaral,

M.J. Cruz and R. Rebelo, unpublished data). Responding to broad and general alarm cues seems to be a critical factor in allowing tadpoles to elicit behavioural defences to novel invasive predators and makes common evolutionary history between predator and prey unnecessary for responses to be elicited (Sih et al. 2010).

100

Chapter 3

Behavioural responses across ontogeny

As predicted, tadpoles of all species (except P. ibericus) showed different antipredator responses to predator chemical cues over ontogeny. All the species responding to the native predator elicited behavioural defences in the first developmental period, indicating an early development of antipredator behaviour, which is in accordance with other studies (e.g.

Petranka and Hayes 1998, Laurila et al. 2004). However, not all these species elicited defences in time period three, at times the antipredator response disappearing as the tadpoles grew larger. This indicates that, as tadpole size increased, vulnerability to predation probably decreased, reducing the importance of behavioural defences (e.g. Eklöv and Werner 2000,

Hettyey et al. 2010). In the case of P. cultripes, overall activity level greatly increased at the same time as the antipredator response disappeared, suggesting that in this case large body size and high activity levels are linked with decreased behavioural responses towards predators. On the other hand, since predation risk did not increase with time in this experiment, we cannot exclude the possibility that, in some of the species, tadpoles might have stopped responding due to habituation (Magurran 1990).

Behavioural plasticity correlations

We found a positive correlation between predator-induced behavioural plasticity in activity level in the presence of the fed native and exotic predators. Since there was no relationship between plasticity in the two starved treatments, the former correlation was probably a result of the correlation between plasticity to the CC cues (cues from consumed conspecifics) in the two predator treatments. This reinforces the idea that the antipredator responses were greatly mediated by chemical signals resulting from predation events upon conspecifics. Further, it suggests that the magnitude of antipredator responses to native predators, when greatly

101

Chapter 3 mediated by CC cues, can be a good proxy for predicting the potential antipredator responses to novel predators in amphibians and possibly even in other species. Still, the correlation between plasticity to the CC cues in the two predator treatments was not close to perfect, as expected if all the observed plasticity was due to the CC cues. Instead, species plasticity to the

CC cues was stronger in the presence of dragonfly than in the presence of crayfish, indicating that additional information from the predator contributed to an increased plastic response when the dragonfly was present. Once again, there seems to be a difference in species responses depending on whether they co-evolved for a long time with the predator or not.

The fact that we found no significant correlations between predator treatments for plasticity in predator spatial avoidance was probably due to many species not altering this trait in predator presence and to the wide variation in responses often observed (large standard errors). This suggests that plasticity in this trait is less widespread among species from this community than plasticity in activity level. Alternatively, it may also indicate that, for some species, chemical cues are not enough to induce alterations in this behavioural trait, maybe because it implies more risk or is more costly.

Conclusions

Since P. clarkii arrived to the southwest of Portugal in the 1980s, larval anurans have been exposed to high predation pressure imposed by this exotic predator (Cruz and Rebelo 2005).

Our experiment showed that six out of nine anuran species present in this area showed behavioural responses to this novel predator. We suggest that these species, provided the behavioural responses are effective, will be better able to persist with the continued expansion and establishment of this introduced predator. However, since failure to recognise an exotic species as a predator is probably one of the most damaging forms of prey naiveté

(Cox and Lima 2006), the remaining three species - unless eliciting alternative defences - will

102

Chapter 3 be highly vulnerable, which may lead to population declines or even local extinctions. Since one of these species, P. ibericus, is an Iberian endemic with a very limited distribution area

(Loureiro et al. 2008), serious conservation problems may arise in the near future. Evaluating which species rely on chemical cues from predation events in their responses to predators may be a useful tool for understanding the potential of native prey species to respond to invasive alien predators and help direct conservation efforts to the most susceptible species.

Acknowledgments

We thank Pedro Andrade, Erika Almeida, Susana Alves and Cátia Guerreiro for their invaluable help in the field and experimental work, Hélder Duarte for providing data on species phylogeny and Jesús Díaz-Rodríguez for providing an insight on the Pelodytes taxonomy. Permits were provided by the Portuguese Instituto da Conservação da Natureza e da Biodiversidade (ICNB).

This research was funded by the FCT Project POCI/BIA-BDE/56100/2004, FCT grant

SFRH/BD/29068/2006 and by Stiftelsen för Zoologisk Forskning.

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CHAPTER 4

Morphological and life-history responses and integration of defensive traits in an anuran community invaded by an exotic predator

Nunes AL, Orizaola G, Laurila A, Rebelo R

Manuscript

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Morphological and life-history responses and integration of defensive traits in an anuran community invaded by an exotic predator

Abstract

Predator-induced phenotypic plasticity has been widely documented in response to native predators, but studies examining the extent to which prey can respond to exotic predators are scarce. As native prey do not share a long evolutionary history with exotic predators, they often lack or develop ineffective defences against them. This may lead to population declines and even extinctions, making exotic predators a serious threat to biodiversity. Here we studied if tadpoles of an anuran community in the Southwest of the Iberian Peninsula developed morphological or life-history responses when exposed to chemical cues of the exotic red swamp crayfish, Procambarus clarkii, a predator recently introduced in this area. We reared tadpoles of nine species until metamorphosis and examined predator-induced responses in terms of larval morphology, growth and development. These responses were compared with the ones developed in the presence of a native predator, the larval dragonfly Aeshna sp.. Eight of the nine species altered their morphology or life-history in response to the native predator, but only four responded to the exotic predator, indicating among-species variation in the ability to respond to novel predators. While morphological defences were generally similar across species (deeper tail fins) and almost exclusively elicited in the presence of the native predator, life-history responses were very variable and commonly elicited in the presence of the exotic predator. Phenotypic trait correlations were more common and the defences more integrated in the presence of dragonfly than crayfish. Five species, of which Pelodytes ibericus is an Iberian endemic, did not respond to crayfish. The lack of response may increase the risk of local extinctions and ultimately reduce diversity of the invaded amphibian communities.

Understanding how native prey species vary in their responses to invasive predators is

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Chapter 4 important in predicting the negative impacts caused by altered predator-prey interactions following biological invasions.

Keywords: Tadpole, exotic crayfish, morphology, life-history, phenotypic integration

Introduction

Invasive species are currently one of the most serious ecological threats to biodiversity. They can modify ecosystems through habitat alteration, introduction of new diseases, genetic introgression, competition and predation (Lockwood et al. 2007). By creating new interactions with native species, invasives may drive native populations to extinction and profoundly alter ecosystems (Lockwood et al. 2007). Invasive predators have drastically reduced or extirpated populations of native species, largely due to a lack of co-evolutionary history between predator and prey (reviews in Kats and Ferrer 2003, Cox and Lima 2006). When a predator invades a new area, native prey may not be able to detect or identify the novel predator as a dangerous threat, resulting in no or inappropriate antipredator responses (Cox and Lima 2006,

Sih et al. 2010). This lack of native prey responses to invasive predators is likely to increase prey mortality, and severely impact invaded populations and communities (e.g. Witte et al.

1992, Polo-Cavia et al. 2010).

On the other hand, naturally co-occurring predators and prey share a common evolutionary history, and prey are usually able to detect and evaluate predation risk and use adequate defensive mechanisms that enhance their survival (Lima and Dill 1990, Sih et al. 2010).

Predator-induced defences are a widespread form of phenotypic plasticity and involve alterations in behaviour, morphology, physiology and life-history (e.g. Lima and Dill 1990,

Benard 2004). Morphological defences typically consist of developing defensive structures, such as spines and helmets in crustaceans, alterations of body shape in fish and amphibian

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Chapter 4 larvae (e.g. deeper body and tail) and increased shell thickness in snails, all of which improve survival under predation risk (e.g. Spitze 1992, McCollum and Van Buskirk 1996, Hoverman and

Relyea 2007, Johansson and Andersson 2009). For example, in amphibians, a deeper tail lures predator strikes away from the vulnerable headbody into the regenerable tail (Van Buskirk et al. 2003) and may enable tadpoles to generate faster swimming bursts and improve manoeuvrability (McCollum and Leimberger 1997, Dayton et al. 2005).

Predators can also induce alterations in the life-history of their prey (Benard 2004, Beckerman et al. 2007, Relyea 2007). Predation risk can affect key life-history decisions such as time to and size at metamorphosis, which can strongly influence individual fitness (reviews in Benard

2004, Relyea 2007). For instance, a reduction in time to metamorphosis can be a direct response to predation risk which allows prey to leave earlier the risky predacious environments, thereby reducing mortality risk (Benard 2004, Nicieza et al. 2006, Higginson and

Ruxton 2009). As large size can provide a refuge from predation, increasing growth rate and size at metamorphosis can also be a direct and adaptive response to predation by gape or size- limited predators (Urban 2007b). Importantly, however, the growth/predation risk trade-off is a common constraint documented for many organisms: higher growth rate often comes at the expense of increased vulnerability to predators (e.g. Werner 1986, Lima and Dill 1990, McPeek

2004).

An integrated view of inducible defences states that selection acts in favour of expressing simultaneously multiple integrated defensive traits (Steiner and Pfeiffer 2007). Defensive traits are often correlated and the degree of phenotypic trait integration depends on the magnitude of the functional relationships among traits (Relyea 2001). Behavioural and morphological defences, as well as life-history trait alterations, are not just direct and independent responses to predation, but occur together and often depend on each other, promoting phenotypic integration (Steiner 2007). Some of these relations indicate trade-offs among traits and reflect either fitness costs or benefits associated with the development of inducible defences (e.g.

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Van Buskirk 2000, Relyea 2001, Higginson and Ruxton 2009). For example, behaviourally and morphologically defended prey may allocate fewer resources into growth and development, often resulting in reduced body size or delayed metamorphosis (Spitze 1992, Van Buskirk 2000,

Higginson and Ruxton 2010; but see Steiner 2007). However, these defences may also result in increased growth rates, indicating that the link between behavioural and morphological defences and growth rate is not necessarily straightforward (Spitze 1992, Peacor 2002,

McPeek 2004, Johansson and Andersson 2009). Relationships between different traits, as well as trait integration, vary across species and populations and can be altered by environmental conditions (Relyea 2001, Benard 2004, Laurila et al. 2008, Higginson and Ruxton 2009,

Johansson and Andersson 2009). For instance, if exposed to an exotic predator, prey may show a more weakly integrated defensive response than in the presence of a native predator, due to the lack of co-evolutionary history selecting for an integrated response.

Many amphibians are negatively impacted by the introduction of invasive species (Kats and

Ferrer 2003), and their larval stages provide a very good study model for examining inducible defences in the presence of exotic predators. In amphibians, larval predation is one of the major sources of mortality (Werner 1991) and lacking appropriate defences towards an exotic predator may render the invaded communities at serious risk. Even so, within a community, species may greatly vary in their ability to detect and respond to novel predators. For instance, prey species that use more general - as opposed to predator-specific - chemical cues to assess predation risk, are more likely to respond to novel exotic predators (Sih et al. 2011, Chapter 3).

In amphibians, examples of these cues are chemical alarm cues released by injured or consumed conspecifics (Sih et al. 2011). To date, several studies have examined behavioural responses to exotic predators in amphibians (e.g. Pearl et al. 2003, Polo-Cavia et al. 2010,

Chapter 3). However, only few have investigated morphological and life-history responses (e.g.

Moore et al. 2004, Gomez-Mestre and Díaz-Paniagua 2011), and none of these have used a community-wide approach.

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The red swamp crayfish Procambarus clarkii, endemic to northeastern Mexico and southern

USA, is a successful invader worldwide (Holdich et al. 2009). It was introduced in Spain in 1973 and has now established populations all over the Iberian Peninsula (Holdich et al. 2009).

Several studies have documented strong negative impacts of this crayfish on Iberian amphibians (e.g. Cruz et al. 2006, 2008). A recent community-wide study in southwestern

Portugal showed that six out of nine anuran species elicited behavioural defences when exposed to P. clarkii, while eight species responded to a common native predator (dragonfly larva (Aeshna sp.); Chapter 3). Species not responding behaviourally to the crayfish may be highly vulnerable to this predator, unless they are able to use alternative defences against it. In this paper, we aimed to understand if anuran species from this invaded community show predator-induced plasticity in morphology and life-history characters in the presence of this exotic crayfish and compared these defences to the responses to a native dragonfly larva.

Further, we explored if integration of defences differed in the presence of the native and exotic predators. We predict that most anuran species will respond to the native predator, and that species using more general chemical cues to respond to predators (such as Alytes cisternasii, Bufo bufo and D. galganoi in this community; Chapter 3) are more likely to show morphological or life-history alterations in the presence of the novel predator. Finally, we predict patterns of trait integration to vary across species and expect to find more trait correlations and a stronger pattern of integration in the presence of the native than in the presence of the exotic predator.

Methods

Study species and experimental procedure

This study was conducted in an area located in the Sado River basin (SW Portugal), where a variety of freshwater habitats ranging from streams to rice fields can be found (Cruz et al.

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2006). This area, which naturally lacks any other freshwater crayfishes, was invaded by P. clarkii ca. 25 years ago (R. Rebelo, pers. obs.). Nine anuran species occur in this area: Iberian midwife toad (Alytes cisternasii), Iberian painted frog (Discoglossus galganoi), Western spadefoot toad (Pelobates cultripes), Iberian parsley frog (Pelodytes ibericus), common toad

(Bufo bufo), natterjack toad (Bufo calamita), European tree frog (Hyla arborea), Mediterranean tree frog (Hyla meridionalis) and Iberian water frog (Pelophylax perezi). Three of these species are Iberian endemics (A. cisternasii, D. galganoi and P. ibericus) and three others (P. cultripes,

H. meridionalis and P. perezi) have a restricted distribution outside Iberia (Loureiro et al. 2008).

P. clarkii is an efficient predator of larvae of all these species and is now abundant throughout southwestern Portugal (Cruz and Rebelo 2005). The native predator used in this study, late- instar dragonfly larvae (Aeshna sp.), are voracious tadpole predators, widespread in the study area. All the anuran species included in this study presently co-occur with both these predators.

Because of differences in breeding phenology among the species, experiments were conducted in different times throughout the year. From November 2007 to June 2008, according to species phenology, several clutches of eight of the nine anuran species were collected in streams or ponds in the study area (Table 1). For A. cisternasii, a species in which males carry the eggs until hatching, larvae were collected very shortly after being released in the water. A reproducing P. clarkii population was present in all the water bodies from where egg masses were collected. The egg clutches were transported to the field station of the

Centre for Environmental Biology in Grândola (38º06’N, 8º34’W), where the experiment took place. Embryos and larvae were kept in several species-specific 5L containers filled with spring water that was changed every three days, and were fed commercial fish food and boiled lettuce ad libitum. After reaching Gosner stage 25 (operculum closure over gills; Gosner 1960) they were randomly assigned to different treatments and the experiment was initiated. Adult crayfish and late-instar larval dragonflies were caught in ponds close to the field station (< 30

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km). While not in the experiment, crayfishes were kept in several 40L plastic tanks and fed

commercial fish food and small invertebrates, whereas dragonfly larvae were kept individually

in 1.2 L plastic boxes and fed Ephemeroptera larvae. Prior to entering the experiment

predators were starved for 48-72 hours.

Table 1. Number of clutches collected, dates of collection, of the start of the experiment, of photography (median Gosner stage at this date) and of the end of the experiment for each of the nine studied species

Nr. clutches Date of Start of Morphology Gosner End of Species collected collection experiment registered stage experiment

Alytes cisternasii - 04-Dec 15-Dec 12-Fev 30 28-Jul Discoglossus galganoi 9 08-Jan 24-Jan 26-Fev 35 13-May Pelobates cultripes 8 28-Nov 15-Dec 16-Apr 30 05-Sep Pelodytes ibericus 10 26-Dec 23-Jan 20-Fev 32 22-Jun Bufo bufo 6 07-Feb 19-Feb 20-Mar 34 04-Jun Bufo calamita 9 22-Apr 19-May 17-Jun 34 09-Aug Hyla arborea 10 05-Jun 17-Jun 23-Jul 30 25-Sep Hyla meridionalis 9 13-Mar 08-Apr 20-Mai 32 15-Aug

Pelophylax perezi 11 16-Jun 02-Jul 03-Sep 33 09-Nov

We performed a factorial experiment using nine anuran species and three predator

treatments. At the beginning of the experiment, groups of ten tadpoles were allocated to

plastic containers (39 × 28 × 28 cm) filled with 10 L of water. A cylindrical predator cage, either

containing one crayfish (cage diameter 85 mm), one dragonfly larva (cage diameter 62 mm) or

no predator (randomly one of the two diameters) was added to each container. Cages were

opaque and covered with fine mesh netting on both sides, so that chemical cues could flow

out, but visual or tactile cues were not available. Predators were fed three tadpoles

conspecifics of the experimental individuals every second day. The three different predator

treatments were “crayfish”, “dragonfly”, and “control” (empty predator cage). For each of the

nine species all treatments were replicated once in each of five randomized blocks, resulting in

a total of 135 experimental units. Once tadpoles approached metamorphosis (Gosner stage 42:

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Chapter 4 emergence of at least one forelimb), we checked the containers daily for metamorphosed individuals, which were then removed. Throughout the experiment, the tadpoles were fed fish flakes and boiled lettuce ad libitum three times per week and water was changed every five days. Water temperature was 18.0 ± 0.17 ºC (mean ± SE) and the photoperiod 12L:12D.

Response variables

In order to examine tadpole morphology each larva was photographed with a digital camera in side view against a grid background. Photographs were taken, depending on species, when tadpoles were between Gosner developmental stages 30 and 35 (see Table 1). Photographs of five random tadpoles per container were loaded into MakeFan7 (Sheets 2009) to create a standardized template for digitizing the different landmarks. The body shape of tadpoles was captured by digitizing 20 landmarks on each individual (Appendix 1), using tpsDig2 software

(Rohlf 2008). Some of the landmarks were chosen according to specific and easily identifiable anatomical points in the tadpole body (e.g. the centre of the eye, the tip of the tail muscle and fin), while others were defined by using proportional distances within a structure (Appendix 1).

We conducted landmark-based geometric morphometrics analyses on these digitized landmarks; this method is a powerful tool for analyzing and visualizing morphological variation between individuals (Rohlf and Marcus 1993, Zelditch et al. 2004). Landmark coordinates were imported into tpsRelw (Rohlf 2007) to create relative warps which, in a multivariate geometric morphometrics analysis, are the equivalent to the principal component scores from Principal

Component Analysis. Relative warp scores on tadpole morphology were estimated using the consensus (i.e. average) morphology for each experimental container in order to avoid pseudoreplication. Consensus conformations were estimated using tpsRelw (Rohlf 2007).

At metamorphosis, tadpoles were blotted dry and weighed with a digital balance to the nearest 0.01 g (mass at metamorphosis). Time to metamorphosis was defined as the number

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Chapter 4 of days elapsed between the beginning of the experiment (day of Gosner stage 25) and the onset of metamorphosis (day of Gosner stage 42). Growth rate was estimated as the quotient between mass at and time to metamorphosis.

Statistical analyses

All morphological analyses were conducted on relative warp scores and independently for each of the nine studied species. The body shape of tadpoles was examined using the first relative warp yielded by the geometric morphometrics analyses (RW1) which accounted, on average, for over 50% of the total morphological variance (Fig. 1; see Orizaola et al. (2011) for a similar approach). The predator effects on the other RWs were negligible in all species and are not presented here. We analyzed the effects of the different predator treatments on tadpole morphology by using univariate analyses of variance (ANOVAs) with type III sum of squares, where RW1 scores were the dependent variable describing tadpole morphology.

Post-hoc analyses were performed using Tukey HSD tests.

To investigate for the effects of predator treatments in larval life-history traits (time to and mass at metamorphosis, and growth rate), we first performed a multivariate analysis of variance (MANOVA) for each species, followed by univariate ANOVAs for each trait. Tank means were always used as the unit of analysis, and values were log transformed in order to fulfil the assumptions of analysis of variance. Block effects were not significant for any of the traits and were excluded from the analyses. Post-hoc comparisons were performed using

Tukey HSD tests. An analysis comparing all the studied species was not performed, since species responses were expected to differ, and we were mostly interested in examining if a specific species was responding or not to the exotic predator.

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To determine whether predator-induced changes in morphology were associated with induced changes in life-history characters, we performed Pearson correlations between traits, within each species and across predator environments. However, in order to examine if the presence of an invasive predator induced relationships different than the ones induced by a native predator, correlations were performed on tank means (n = 10 per species), separately for crayfish and dragonfly treatments (plus the control treatment). In order to obtain a more integrative approach, we also used behavioural data from a companion paper (Chapter 3) to investigate relationships between alterations in behaviour (the average proportion of active tadpoles across ontogeny for each species), morphology and life-history traits. Across-species correlations were not performed because we were interested in looking at trade-offs and integration within each species.

Results

Morphological responses

All species except B. calamita modified their morphology when exposed to the native predator

(Table 2; Fig. 1). Of these eight species, all except B. bufo responded by increased tail fin depth, and all but B. bufo and P. perezi had a more anterior insertion of the tail fin to the headbody

(Fig. 1). Induced tadpoles of A. cisternasii, D. galganoi, P. perezi and P. cultripes also had deeper tail muscles. The three former species together with P. ibericus and H. meridionalis also increased their headbody depth (Fig. 1). B. bufo altered its morphology by increasing headbody length (Fig. 1). On the contrary, when exposed to crayfish, only B. bufo changed their morphology, the response being similar to that found in the presence of the dragonfly (Table

2; Fig. 1).

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0.06 Alytes cisternasii 0.04

0.02

0.00

RW1 = 80.11% -0.02

-0.04 No predator Crayfish Dragonfly

0.12 Discoglossus galganoi 0.08

0.04

0.00

-0.04 RW1 = RW1= 88.80% -0.08 No predator Crayfish Dragonfly

0.04 Pelobates cultripes

0.02

0.00

-0.02 RW1 = RW1= 39.55%

-0.04 No predator Crayfish Dragonfly

0.04 Pelodytes ibericus

0.02

0.00

-0.02 RW1 = 62.57%

-0.04 No predator Crayfish Dragonfly

Fig. 1 Mean ± S.E. values of Relative Warp 1 (RW1) representing tadpoles shape of nine anuran species (A. cisternasii, D. galganoi, P. cultripes, P. ibericus, B. bufo, B. calamita, H. arborea, H. meridionalis and P. perezi) in the presence of the different predator treatments. Drawings placed on the right hand side of each graph show the shape of larvae representing the extreme positive (black) and negative (grey) scores of RW1.

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0.02 Bufo bufo

0.01

-0.01

-0.02 RW1 = RW1= 46.16%

-0.03 No predator Crayfish Dragonfly

0.02 Bufo calamita

0.01

0.00

-0.01 RW1 = RW1= 44.58%

-0.02 No predator Crayfish Dragonfly

Hyla arborea 0.03 0.02 0.01 0.00

RW 1 = RW= 1 37.94% -0.01 -0.02 No predator Crayfish Dragonfly

0.03 Hyla meridionalis 0.02 0.01 0.00

-0.01 RW1 = RW1= 42.76% -0.02 No predator Crayfish Dragonfly

0.04 Pelophylax perezi

0.02

0.00

-0.02 RW 1 = RW= 1 42.57% -0.04 No predator Crayfish Dragonfly

Fig. 1 Extended

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Table 2. Univariate ANOVAs on the morphology (first relative warp, RW1) of the nine anuran species in presence of the different predator treatments (left hand panel). P-values for the Tukey post-hoc tests referring to comparisons between the control and each predator treatment (right hand panel). P-values < 0.05 are marked in boldface

Morphology Dragonfly Crayfish

df F P P P A. cisternasii 2, 15 165.786 <0.001 <0.001 0.745 D. galganoi 2, 15 79.434 <0.001 <0.001 0.419 P. cultripes 2, 15 7.207 0.009 0.012 0.907 P. ibericus 2, 15 42.452 <0.001 <0.001 0.610 B. bufo 2, 15 28.615 <0.001 <0.001 0.005 B. calamita 2, 15 0.121 0.887 - - H. arborea 2, 15 13.654 0.001 0.003 0.910 H. meridionalis 2, 15 8.047 0.006 0.035 0.589 P. perezi 2, 13 14.796 0.001 0.001 0.164

Life-history responses

Species differed substantially in life-history traits. B. calamita and D. galganoi had the shortest larval periods (ca 50 days; Fig. 2), roughly 25% of the larval period of A. cisternasii and P. cultripes, which had the slowest development. Excluding P. cultripes, a species that grew much faster and attained a much higher mass than all the others, larvae of A. cisternasii and P. perezi had the highest masses at metamorphosis and P. perezi the highest growth rates. In contrast,

B. calamita had the lowest growth rate and was smallest at metamorphosis (Fig. 2).

MANOVAs detected treatment effects in life-history traits for all the anuran species (P < 0.038) except for P. perezi (P = 0.489). In B. calamita none of the univariate ANOVAs was significant

(Table 3). The univariate ANOVAs followed by Tukey tests showed that five species - B. bufo, D. galganoi, H. arborea, H. meridionalis and P. ibericus - significantly changed life-history traits as a response to the native predator (Table 3). Induced tadpoles of P. ibericus increased growth rate and metamorphosed at larger mass (Table 3; Fig. 2). Tadpoles of H. meridionalis also

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Chapter 4 attained a larger mass at metamorphosis, while H. arborea tadpoles increased their growth rates. On the contrary, B. bufo tadpoles decreased both growth rate and time to metamorphosis, which resulted in a smaller mass at metamorphosis (Table 3; Fig. 2). Tadpoles of D. galganoi also reduced their growth rates, but they took longer to metamorphose; this was the only species to increase time to metamorphosis in response to predator presence

(Table 3; Fig. 2).

Four species altered their life-history traits in the presence of crayfish (A. cisternasii, B. bufo, H. arborea and P. cultripes; Table 3). From these species, only B. bufo and H. arborea also responded to the native predator, the responses to the two predators being very similar, but with some small differences. In response to crayfish, B. bufo did not alter growth rate (while reducing time to and mass at metamorphosis) and H. arborea, in addition to increasing growth rate, also reduced time to metamorphosis (Table 3; Fig. 2). Tadpoles of both A. cisternasii and

P. cultripes also reduced time to metamorphosis when exposed to crayfish. Finally, tadpoles of

A. cisternasii grew faster and attained higher masses (Table 3; Fig. 2).

Trait correlations

None of the paired trait correlations was statistically significant for B. calamita, P. cultripes and

P. perezi (Table 4). When exposed to the dragonfly larvae six species showed significant correlations between different traits, while only two species had significant correlations in the presence of crayfish. Although there were generally relatively few significant trait correlations per species, many more significant relationships were found in the presence of the native than in the presence of the exotic predator (19 vs. 7, Table 4).

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Table 3. Analyses of larval life-history traits (time to metamorphosis, mass at metamorphosis and growth rate) for each of the nine anuran species. Results (P- values) of the univariate ANOVAs performed for each trait (df = 2,15) are shown on the left hand side of each column. Results (P-values) of the Tukey post-hoc tests are shown on the right hand side and values refer to comparisons of each predator treatment with the control treatment. P-values < 0.05 are marked in boldface

Time to metamorphosis Mass at metamorphosis Growth rate

P Dragonfly Crayfish P Dragonfly Crayfish P Dragonfly Crayfish

A. cisternasii <0.001 0.834 <0.001 0.022 0.177 0.018 <0.001 0.322 <0.001 D. galganoi <0.001 0.001 0.727 0.766 - - 0.004 0.014 0.886 P. cultripes 0.012 0.621 0.012 0.838 - - 0.640 - - P. ibericus 0.429 - - <0.001 0.002 0.374 0.001 0.002 0.950 B. bufo 0.001 0.001 0.006 <0.001 <0.001 0.003 0.019 0.031 0.995

B. calamita 0.053 - - 0.110 - - 0.254 - - H. arborea 0.044 0.737 0.043 0.067 - - 0.002 0.031 0.002 H. meridionalis 0.118 - - 0.007 0.030 0.746 0.120 - - P. perezi 0.957 - - 0.054 - - 0.067 - -

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Fig. 2 a) Time to metamorphosis, b) Mass at metamorphosis and c) Growth rate (mean ± SE) of nine anuran species, in presence of the different predator treatments. The three treatments are control (No pred), crayfish (Crayf) and dragonfly (Dragonf). Note different scales in the graphs in the end of each row, for species having very high values of a specific trait.

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In the presence of dragonfly larvae, D. galganoi had a negative correlation between activity level (behaviour) and morphology - low activity was correlated with deeper headbody, tail fin and tail muscle in this species. Further, induced morphology was correlated with longer larval period (as was also activity) and reduced growth rate. In fact, D. galganoi was the only species for which there was a negative correlation between morphology and growth rate (Table 4). On the contrary, for P. ibericus there was a positive correlation between these two traits; tadpoles that grew faster (and had larger mass at metamorphosis) also had deeper headbody and tail fin. In both this species and H. meridionalis, induced morphology was associated with low activity level which, in turn, was associated with higher mass (and in the case of P. ibericus with higher growth rate) (Table 4). Activity level was also negatively correlated with growth rate in H. arborea.

For species showing trait correlations when exposed to crayfish, A. cisternasii exhibited a different relationship than the one shown in the presence of the dragonfly. When exposed to dragonfly, there was a significant negative correlation between behaviour and morphology, lower activity being correlated with deeper headbody, tail fin and tail muscle (Table 4). On the other hand, in the presence of crayfish, A. cisternasii activity was negatively correlated with mass. There was also a negative correlation between time to and mass at metamorphosis

(Table 4). B. bufo was the only species showing very similar correlations in the presence of the native and the exotic predators. Induced morphology was negatively correlated with both time to and mass at metamorphosis (the latter only in dragonfly presence), while activity was positively correlated with these two metamorphic traits. There was also a positive correlation between time to and mass at metamorphosis and a negative correlation between activity and morphology. This indicates that lower activity was associated with longer headbody and a faster metamorphosis at lower mass (Table 4).

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Table 4. Phenotypic Pearson correlations (r and P values) performed between morphological, behavioural and life-history traits for the nine studied species. N = 10 for all the correlations. Correlations for which P > 0.05 are marked as non significant (N.S.).

Time met-Morphol Mass met-Morphol Growth rate-Morphol Time met-Behav Mass met-Behav Growth rate-Behav

Dragonfly Crayfish Dragonfly Crayfish Dragonfly Crayfish Dragonfly Crayfish Dragonfly Crayfish Dragonfly Crayfish r = -0.687 A. cisternasii N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. P = 0.028 r = 0.882 r = -0.787 r = -0.639 D. galganoi N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. P = 0.001 P = 0.007 P = 0.047

P. cultripes N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.

r = 0.741 r = 0.853 r = -0.820 r = -0.889 P. ibericus N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. P = 0.014 P = 0.002 P = 0.004 P = 0.001 r = -0.907 r = -0.765 r = -0.861 r = 0.849 r = 0.708 r = 0.871 r = 0.728 B. bufo N.S. N.S. N.S. N.S. N.S. P < 0.001 P = 0.010 P = 0.001 P = 0.002 P = 0.022 P = 0.001 P = 0.017

B. calamita N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.

r = -0.830 H. arborea N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. P = 0.011 r = -0.700 H. meridionalis N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. P = 0.024

P. perezi N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. Time met - Time to metamorphosis; Mass met - Mass at metamorphosis; Morphol - Morphology; Behav - Behaviour

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Table 4. Extended.

Time met-Mass met Morphol-Behav

Dragonfly Crayfish Dragonfly Crayfish r = -0.827 r = -0.819 N.S. N.S. P = 0.003 P = 0.004 r = -0.789 N.S. N.S. N.S. P = 0.007

N.S. N.S. N.S. N.S.

r = -0.637 N.S. N.S. N.S. P = 0.047 r = 0.875 r = 0.803 r = -0.907 r = -0.635 P = 0.001 P = 0.005 P < 0.001 P = 0.048

N.S. N.S. N.S. N.S.

r = -0.887 N.S. N.S. N.S. P = 0.001

N.S. N.S. N.S. N.S.

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Discussion

We found that both morphological and life-history plasticity in response to predator chemical cues were widespread in a community of larval anurans. Plastic responses were more common in response to the native than to the exotic predator and, responses to the exotic predator, when present, were often qualitatively different from the responses to the native predator.

Furthermore, phenotypic integration was overall much higher in the presence of the native than in the presence of the exotic predator.

Eight of our study species showed antipredator responses in the presence of the native dragonfly (all but B. calamita), while only four showed plasticity in the presence of the exotic crayfish (A. cisternasii, B. bufo, H. arborea and P. cultripes). In larval anurans, previous studies have reported morphological and life-history responses to chemical cues from both caged dragonfly (e.g. Laurila et al. 1998, Relyea 2001, Richter-Boix et al. 2007) and crayfish predators

(e.g. Nyström and Abjörnsson 2000, Saglio and Madrillon 2006). In this study, a higher proportion of the species responding to a specific predator altered morphology in the presence of dragonfly (eight in eight for dragonfly against one in four for crayfish), whereas in the presence of crayfish a higher proportion altered life-history traits (four in four for crayfish against five in eight for dragonfly). This may suggest that life-history characters are more evolutionary labile and more readily altered in response to new selective pressures than morphological characters (Blomberg et al. 2003). In addition, if life-history alterations are more costly than morphological ones (Higginson and Ruxton 2010), they may be selected against when co-evolutionary history between predator and prey is longer. Alternatively, responses to the native predator may not be efficient against the invasive predator, suggesting predator- specific defences (e.g. Relyea 2001, 2002). In fact, among the species that responded to the two predators, only B. bufo exhibited the same type of defences, while both A. cisternasii and

P. cultripes had completely dissimilar responses, altering only morphology in the presence of

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Chapter 4 dragonfly and only life-history traits in the presence of crayfish. Further studies testing the efficiency of these inducible responses against the two different predators would be valuable

(e.g. Gomez-Mestre and Diaz-Paniagua 2011).

Morphological responses

Dragonfly presence strongly affected tadpole morphology, with tadpoles of most species showing quite similar morphological plastic responses. These consisted of developing a deeper headbody, tail fin and tail muscle and a more anterior insertion of the tail fin to the headbody.

The development of deep tails when exposed to chemical cues from predatory dragonflies is a widespread adaptive response in larval anurans (e.g. Relyea 2001, Van Buskirk 2009), which enhances the probability of a tadpole escaping a predator attack (e.g. McCollum and Van

Buskirk 1996, Van Buskirk et al. 2003, Dayton et al. 2005, Johnson et al. 2008). A more anterior insertion of the tail fin into the upper part of the headbody has also been previously reported as a response to predation risk (Van Buskirk 2009). The headbody region of five species (A. cisternasii, D. galganoi, H. meridionalis, P. ibericus and P. perezi) also changed with dragonfly predation cues, with induced tadpoles having deeper headbodies. This may not have a direct defensive function, but may enable tadpoles to have a greater gut surface area for food digestion, which would facilitate accumulating energy reserves, maybe allowing for less foraging activity (Van Buskirk 2009).

The only species for which we detected morphological plasticity in response to crayfish was B. bufo. Previous studies have generally failed to detect predator-induced morphological plasticity in this species (e.g. Lardner 2000, Richter-Boix et al. 2007). However, also using geometric morphometrics tools, Van Buskirk (2009) recently reported the same morphological alteration that we found for this species in predator presence (increased headbody size). This may indicate that morphological alterations in B. bufo have not been previously detected due

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Chapter 4 to technical constraints. Further, when faced with crayfish, responses of B. bufo were the same as in the presence of dragonfly indicating that the same type of defences are used against the native and invasive predators. This reinforces the idea that, in this species, responses to the exotic predator are mainly mediated by the presence of general alarm cues, rather than to predator specific cues (Semlitsch and Gavasso 1992, Chapter 3). B. calamita was the only species lacking morphological plasticity, which is in accordance with previous studies (Lardner

2000, Richter-Boix et al. 2007).

Life-history responses

Contrary to induced morphological responses, life-history alterations were extremely variable among the anuran species and the two predators. The large variation in life-history responses supports the view that these changes can either be a direct response to predation risk or a consequence of the development of behavioural and/or morphological inducible defences

(Steiner 2007). The most frequent alterations were found in time to metamorphosis and the least frequent in mass at metamorphosis. This suggests less plasticity in the latter trait, possibly because mass at metamorphosis is a very important life-history attribute in determining adult fitness in amphibians, and because a threshold size has to be attained before metamorphosis can occur (Wilbur and Collins 1973, Relyea 2007, Higginson and Ruxton

2009). P. perezi and B. calamita were the only species showing no significant predator-induced plasticity in life-history traits.

B. bufo tadpoles reduced both time to and mass at metamorphosis in the presence of dragonfly and crayfish (as well as growth rate in dragonfly presence) and the responses to the two predators were very similar. This again suggests a generalized response towards predators in B. bufo (probably due to a response to alarm cues), which may facilitate responses to novel invasive predators (Sih et al. 2011, Chapter 3). Laurila et al. (1998) found a similar response to

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Chapter 4 a dragonfly predator. Metamorphosing earlier is a rare, but potentially advantageous response to predators which has been reported also in other bufonids (Relyea 2007). Contrary to B. bufo, tadpoles of D. galganoi exposed to a dragonfly predator significantly prolonged time to metamorphosis, most likely due to strong morphological and behavioural defences, as has been previously shown for this species (Nicieza et al. 2006). A longer larval period can be detrimental for ephemeral pond species like D. galganoi because it is likely to increase mortality when the pond dries. Morphological defences were negatively correlated with growth, suggesting that they were more costly than behavioural defences (Van Buskirk 2000).

Three of our study species significantly increased growth rates in the presence of either dragonfly (P. ibericus and H. arborea) or crayfish (A. cisternasii and H. arborea). Increased growth is not a common prey response to caged predators, but has been previously reported in amphibians (Werner 1991, McCollum and Leimberger 1997, Van Buskirk and Relyea 1998,

Peacor 2002, Teplitsky et al. 2003, Urban 2007a) and other taxa (e.g. Crowl and Covich 1990,

Spitze 1992, Johansson and Andersson 2009). Increased growth rates may be due to physiological changes in metabolism, such as increased food assimilation or food conversion efficiency (McCollum and Van Buskirk 1996, McPeek 2004, Stoks et al. 2005, Steiner 2007,

Dmitriew 2011). If tadpoles reach larger size through these mechanisms, this may act as a defence against size-limited predators (Urban 2007b, Dmitriew 2011), and Beckerman et al.

(2007) proposed that size-selective predation may induce direct physiologically mediated alterations in life-history traits of prey. Although dragonflies and crayfish are not strictly gape- limited predators, dragonflies are less able to capture and handle prey above a certain size

(Werner and McPeek 1994). Increased growth rates may also be achieved by increased foraging effort and activity (McPeek 2004, Urban 2007a), which was not the case here (see

Table 4). On the contrary, in A. cisternasii and P. ibericus we found a negative correlation between behaviour and mass at metamorphosis and/or growth rate, suggesting that less active animals grew faster and became larger. While this contrasts with the classical

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Chapter 4 behaviourally-mediated growth/predation risk trade-off, other studies have also found an association between reduced activity and higher growth rates in predator presence. Johansson and Andersson (2009) proposed that the reduced activity of crucian carp in the presence of pike allowed more energy to be saved and then allocated to growth. Our finding that exposure to predators can have a net positive effect on growth, even when behavioural defences are elicited, reinforces the idea that a decoupling between behaviour and growth is common (see also McPeek 2004, Stoks et al. 2005). It should be noticed that, in spite of its benefits, accelerated growth may compromise post-metamorphic fitness by preventing allocation to other important traits or functions, such as immune defence and resistance to oxidative stress

(Dmitriew 2011).

Two of the species that grew faster in predator presence also took a shorter time to reach metamorphosis (A. cisternasii and H. arborea). This was only observed in response to crayfish and may indicate the perception of a higher mortality risk in the aquatic habitat. While these concurrent alterations in time to and mass at metamorphosis are not predicted to occur in nature by recent dynamic state-dependent models (Higginson and Ruxton 2010, but see

Teplitsky et al. 2003), they can be beneficial for tadpoles avoiding crayfish predation.

Trait correlations

The patterns of trait integration varied widely across species and predator environment. In general, more species showed trait correlations when exposed to the native than to the exotic predator, and within species traits were generally more integrated (higher number of trait correlations) in the presence of the native predator. D. galganoi, B. bufo and P. ibericus showed a highly integrated response in the presence of the dragonfly larvae. Interestingly, B. bufo showed similar integration in response to both crayfish and dragonfly, again suggesting that a response to alarm chemicals rather than to predator cues may have homogenized the

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Chapter 4 responses in this species. A. cisternasii showed a higher level of phenotypic integration in crayfish than dragonfly presence, which might suggest an adaptive response to this novel predator. In this case, trait correlations found in the presence of dragonfly and crayfish were dissimilar because this species responded to the crayfish by altering different suites of traits than when responding to the dragonfly. H. arborea and P. cultripes, although eliciting defences to the crayfish predator showed no trait integration.

In the presence of the same predator, correlations were very variable from species to species, suggesting species-specific defensive strategies. Further, the same traits were sometimes related in opposite manners in the same predator environment. For instance, in A. cisternasii, time to and mass at metamorphosis were negatively correlated, whereas in B. bufo these traits were positively related. This shows that traits are not linearly related across species and that outcomes are specific for each prey species. Nevertheless, the correlation between behaviour and morphology was consistently negative across species, indicating that a decrease in activity did not reduce the energy necessary for eliciting morphological defences. On the contrary, alterations in morphology could have been indirect effects of reduced tadpole activity, as has been observed before (e.g. Johansson and Andersson 2009). This suggests that these two types of defences may frequently complement or augment each other, which is predicted to maximize fitness (Steiner and Pfeiffer 2007).

Conclusions

In conclusion, our work highlights the importance of studying whole communities to evaluate responses to predators, adding to the growing evidence that different species use specific suites of defences against the same predator. Our results reinforce that extant interspecific differences in behavioural, morphological and physiological characteristics as well as in species ecology, select for different adaptive antipredator responses in different species (Lardner

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2000). We found that, from the nine anurans present in the study area, four species reacted to crayfish cues in the examined traits. In three of these species, crayfish presence produced a different suite of defences than in the presence of dragonfly, indicating predator-specific responses and the ability to detect dissimilar predator signals. This suggests detection of the exotic crayfish as a threat, either by recognition of this novel invasive predator, or by a mere reaction to an unknown chemical stimulus possibly resembling kairomones emitted by another predator in the community. P. ibericus and P. perezi, while responding to the dragonfly, did not respond to the exotic predator (a similar result was also found in behaviour; Chapter 3), apparently not recognizing the crayfish as a threat. As such, these species seem to be undefended against P. clarkii, which may lead to population declines and compromise their persistence in habitats invaded by the crayfish. This acquires special relevance in the case of P. ibericus, a species endemic to the Iberian Peninsula (Loureiro et al. 2008). Since most inland aquatic habitats in the Iberian Peninsula are already invaded by P. clarkii, and most of the areas not invaded seem to be suitable for its establishment (Capinha and Anastácio 2011), special efforts are needed to determine more precisely the level of threat that this invasive crayfish poses to these native anurans. Examining how prey species vary in their responses to introduced predators is essential for understanding the dynamics of predator-prey interactions following biological invasions and, ultimately, for preventing extinctions in invaded communities.

Acknowledgements

We thank Pedro Andrade, Erika Almeida, Susana Alves and Cátia Guerreiro for their invaluable help in the field and experimental work. We also thank Jesús Díaz-Rodríguez for helping with the Pelodytes taxonomy. Comments from Frank Johansson greatly improved the manuscript.

Permits were provided by the Portuguese Instituto da Conservação da Natureza e da

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Biodiversidade (ICNB). This research was funded by the FCT Project POCI/BIA-

BDE/56100/2004, FCT grant SFRH/BD/29068/2006 and Stiftelsen för Zoologisk Forskning.

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Appendix 1. Location of the 20 landmarks digitized for estimating body shape of each tadpole and detailed explanation of their position

11 13 4 3 17 7 14 5 2 8 20 1 19 9 15 18 6 10 12 16

1 - Most anterior point of the headbody 2 - Centre of the eye 3 - Point of intersection between the headbody and the upper edge of the tail fin 4 - Point of the tail fin at two thirds of the distance between landmarks 1 and 9 5 - Point of the headbody (dorsal side) at two thirds of the distance between landmarks 1 and 9 6 - Point of the headbody (ventral side) at two thirds of the distance between landmarks 1 and 9 7 - Point of intersection between the headbody and the upper edge of the tail muscle 8 - Point of intersection between the headbody and the central line of the tail muscle 9 - Point of intersection between the headbody and the lower edge of the tail muscle 10 - Point of intersection between the anus and the lower edge of the tail fin 11 - Point of the tail fin (dorsal side) at one quarter of the distance between landmarks 8 and 20 12 - Point of the tail fin (ventral side) at one quarter of the distance between landmarks 8 and 20 13 - Point of the tail fin (dorsal side) at half distance between landmarks 8 and 20 14 - Point of the tail muscle (dorsal side) at half distance between landmarks 8 and 20 15 - Point of the tail muscle (ventral side) at half distance between landmarks 8 and 20 16 - Point of the tail fin (ventral side) at half distance between landmarks 8 and 20 17 - Point of the tail fin (dorsal side) at three quarters of the distance between landmarks 8 and 20 18 - Point of the tail fin (ventral side) at three quarters of the distance between landmarks 8 and 20 19 - Tip of the tail muscle 20 - Tip of the tail fin

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Population divergence in antipredator defences against an invasive predator: does coexistence time promote the evolution of adaptive responses?

Nunes AL, Orizaola G, Laurila A, Rebelo R

Manuscript

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Population divergence in antipredator defences against an invasive predator: does coexistence time promote the evolution of adaptive responses?

Abstract

Invasive alien predators can impose strong selection on native prey populations and induce rapid evolutionary change in the invaded communities. The red swamp crayfish Procambarus clarkii has invaded most freshwater habitats in the Iberian Peninsula, where it preys on native amphibian larvae. We raised Iberian waterfrog (Pelophylax perezi) tadpoles from populations differing in historical exposure to P. clarkii (30 years, 20 years or no coexistence) and examined how coexistence time shaped antipredator responses in different P. perezi populations. We show that tadpoles from non-invaded populations reduced activity level in the presence of P. clarkii, whereas long-term invaded populations did not. Further, tadpoles from one of the long- term invaded populations responded to the crayfish with inducible morphological defences

(shorter headbody and deeper tail). These results suggest that not reducing activity level and inducing morphological defences are likely adaptive responses to the predation pressure imposed by this crayfish. Although quite recent (ca. 32 years), it is possible that the strong predation pressure imposed by P. clarkii has selected for rapid evolution of morphological responses in populations of P. perezi and accounts for the divergence in antipredator defences found between different populations. The considerable among-population variation found in prey responses to an invasive predator stresses that integrative approaches may be especially relevant to understand the outcomes of predator-prey interactions in biological invasions.

Keywords: Historical coexistence, invasive predator, antipredator defences, population divergence, rapid evolution

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Introduction

Predation is an important selective force acting on the fitness of prey organisms, and there is strong selection on prey populations to evolve traits that enhance their ability to escape or deter predation (Lima and Dill 1990). Prey develop a wide variety of inducible antipredator strategies which involve modifications in behaviour, morphology and life-history that reduce their vulnerability to predators (Lima and Dill 1990, Benard 2004). Antipredator behaviours can consist of changing microhabitat use, aggregating, using refuges, spatially avoiding the predator and, most commonly, reducing activity levels; these alterations are adaptive as they reduce predator-prey encounter rates and prey detectability (Lima and Dill 1990, Kats and Dill

1998). Morphological defences typically consist of defensive structures, such as spines, changes in body shape or shell thickness, which increase the probability of escaping an attack and enhance the chances of survival (e.g. Spitze 1992, Trussel and Smith 2000, Relyea 2001).

Successful predator recognition and effective avoidance strategies typically occur when predator and prey have a common evolutionary history (Strauss et al. 2006, Sih et al. 2010).

Nowadays, invasive alien predators are introduced into new environments at an unprecedented rate, resulting in new predator-prey interactions between organisms sharing no evolutionary history (Thompson 1998, Strauss et al. 2006). Invaded prey populations often suffer dramatic declines or even extinctions due to their inability to respond with appropriate antipredator defences (Cox and Lima 2006, Strauss et al. 2006, Sih et al. 2010). However, by imposing strong selection pressure upon native organisms, invasive predators may induce rapid evolutionary changes in native species, through which prey evolve the ability to recognise and respond to them (Schlaepfer et al. 2005, Strauss et al. 2006, Sih et al. 2010). For rapid adaptation to occur, selection pressure imposed by the exotic predator has to be sufficiently strong and consistent, and native populations have to be genetically variable in susceptibility to the predator (Strauss et al. 2006). Natural selection will favour prey individuals

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Chapter 5 that can detect and exhibit predator avoidance in response to the novel predator. Many examples of rapid directional adaptive evolution in native prey species in response to introduced predators have been reported in several taxa including molluscs, crustaceans, fish, amphibians and reptiles (Kiesecker and Blaustein 1997, Losos et al. 2004, O’Steen et al. 2002,

Freeman and Byers 2006, Fisk et al. 2007).

However, some prey species are able to ‘innately’ detect and elicit adaptive responses to stimuli of novel predators in the absence of a common evolutionary history (Epp and Gabor

2008, Peluc et al. 2008, Sündermann et al. 2008, Rehage et al. 2009). This ability may be due to the latter’s phylogenetic or phenotypic similarity to native predators co-occurring with the prey (Epp and Gabor 2008, Rehage et al. 2009). Prey may also exhibit generalized antipredator responses, whereby exposure to predation risk induces the development of defences, regardless of previous direct experience with a particular predator (e.g. response to a general fish odour or to a large moving object; Magurran 1990, Gherardi et al. 2011). ‘Innate’ responses of native prey to invasive predators can also be facilitated if detection of predators relies on general predation cues, such as cues associated with the predator’s diet or disturbance/damage alarm cues released by startled or attacked prey (Sündermann et al.

2008, Ferrari et al. 2010). Alternatively to ‘innate’ responses, prey may learn to identify and respond to alien predators through experience by pairing novel predator scents with cues released by attacked or consumed conspecifics. Hence, prey may learn to ‘label’ exotic predators as a threat respond to them (review in Ferrari et al. 2010).

Freshwater environments, which have been widely invaded by exotic species, are particularly vulnerable to introduced predators (Cox and Lima 2006). One of the most conspicuous non- native predator species introduced worldwide is the red swamp crayfish (Procambarus clarkii), originally endemic from southcentral USA and northeastern Mexico (Hobbs et al. 1989,

Gherardi 2006). This invasive species, which was first recorded in the Iberian Peninsula in 1973, has rapidly spread throughout the area and is now an abundant species in most freshwater

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Chapter 5 habitats all over the Peninsula (Holdich et al. 2009). P. clarkii is an extraordinarily successful invader, often altering the dynamics of the ecosystems it colonises (e.g. Geiger et al. 2005). It is an efficient predator of amphibian larvae known to have strong negative impacts on Iberian amphibians (Cruz and Rebelo 2005, Cruz et al. 2008).

Tadpoles of the Iberian waterfrog, Pelophylax perezi, exhibit typical behavioural (reduced activity level) and morphological (shorter and deeper headbodies and deeper tails) plastic antipredator responses when exposed to chemical stimuli of native aquatic predators (Gonzalo et al. 2007, Richter-Boix et al. 2007, Polo-Cavia et al. 2010, Gomez-Mestre and Diaz-Paniagua

2011, Chapters 3 and 4). These behavioural and morphological alterations are common responses of larval anurans to native caged predators, which are known to increase the odds of survival in predator presence (Skelly 1994, Van Buskirk and Relyea 1998, Teplitsky et al.

2005). In the presence of P. clarkii, however, three studies did not detect behavioural or morphological responses of P. perezi to the predator (Gomez-Mestre and Diaz-Paniagua 2011,

Chapters 3 and 4). Nevertheless, Gomez-Mestre and Diaz-Paniagua (2011) found that induced morphological defences elicited by P. perezi in the presence of a native larval dragonfly predator (a deeper and more pigmented tail) increased tadpole survival in the presence of P. clarkii.

In this study, using five populations of P. perezi that differ in coexistence time with P. clarkii, we investigated if coexistence with the introduced predator affects the ability to respond to this invasive crayfish. We compared behavioural and morphological responses of five populations from three areas characterized by different exposure times to P. clarkii: longer (ca.

30 yr), shorter (ca. 20 yr) or no coexistence time with this predator. Assuming that P. clarkii imposes strong selection on P. perezi populations, we expect that tadpoles from invaded populations will show antipredator defences to the crayfish (such as adaptive alterations in morphology), while individuals from non-invaded areas will show no responses to this predator.

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Methods

Study populations

P. perezi is the most abundant and widely distributed anuran in Portugal (Loureiro et al. 2008).

It inhabits a wide variety of aquatic habitats, but prefers permanent water bodies (Almeida et al. 2001). It is commonly found in both P. clarkii invaded and non-invaded areas.

We selected populations of P. perezi from southern Portugal, the region where P. clarkii was first recorded in Portugal and where no native crayfishes exist (Ramos and Pereira 1979,

Almaça 1991). P. clarkii is currently widespread over large areas throughout Portugal, and we were not able to find a region where the three different types of populations could be found.

Hence, we collected populations with different coexistence times with crayfish from three geographically distinct areas. We first chose P. perezi populations from the area of the Caia

River, an affluent of the Guadiana River (Table 1, Fig.1). This is the area where P. clarkii was first recorded in Portugal in 1979 (Ramos and Pereira 1979), and populations from Caia are probably the ones in Portugal which have been exposed to P. clarkii for the longest time

(Longer Coexistence time – LC). Two study populations 34 km apart from each other were collected in this region (Fig.1). Secondly, we collected one population in Melides, an area which was invaded by the crayfish around 1990 (R. Rebelo pers. obs.; Table 1, Fig.1). P. perezi populations from this area have coexisted with crayfish for a shorter time than those from the

Caia region (Shorter Coexistence time – SC). This region is characterized by extensive areas of rice culture, where the crayfish can attain very high densities. Finally, we collected P. perezi populations that have never been in contact with P. clarkii (No Coexistence – NC) in the

Monchique region (Table 1, Fig.1). This is a mountain range located in the far South of

Portugal, where strong water flow and topography have probably so far prevented invasion by

P. clarkii (Gil-Sánchez and Alba-Tercedor 2002, Kerby et al. 2005, Cruz and Rebelo 2007).

Repeated surveys using baited traps during 2009 proved this area still devoid of crayfish (A.L.

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Nunes and R. Rebelo, unpubl.). In this region, we collected two different study populations situated 12 km apart and located in different hydrographic basins, separated topographically by a marked ridge (Table 1, Fig.1).

All study populations were collected from slow-flowing sections of streams harbouring a rich fauna of native predators (insects, native fish, and snakes). Trapping and visual observations confirmed that dense P. clarkii populations coexisted with LC and SC populations.

Table 1. Area, coordinates, elevation above sea level of collection sites, type of water body, approximate coexistence time between Procambarus clarkii and Pelophylax perezi and dates of collection and of the start of the experiment for each of the five studied populations of P. perezi

Population LC1 LC2 SC NC1 NC2 Area Caia Caia Melides Monchique Monchique Rice field on Melides Type of water Monchique Affluent of the the bank of the Caia river stream and body river Seixe river Caia river rice fields 38º52’N 39º07’N 38º08’N 37º17’N 37º22’N Coordinates 7º03’W 7º17’W 8º44’W 8º33’W 8º38’W Elevation a.s.l 170 m 270 m 25 m 200 m 100 m Approximate coexistence 32 years 32 years 21 years 0 years 0 years time Date of 10-June 16-June 08-July 18-June 18-June collection Number of clutches 8 10 10 9 12 collected Start of 02-July 02-July 17-August 04-August 05-August experiment

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Fig. 1. Map of Portugal with the areas of origin of each study population (LC – Longer Coexistence populations, SC – Shorter Coexistence population and NC – No Coexistence populations). The black triangle indicates the location where P. clarkii was first introduced in Spain and the white triangle indicates the site where crayfish presence was first registered in Portugal. Grey areas indicate regions 700 m a.s.l.

Experimental procedure

During June and July 2009, we collected several egg masses of Iberian waterfrog from each of the abovementioned populations (Table 1). The eggs were taken to the laboratory in the field station of Centro de Biologia Ambiental in Grândola (38º06’N, 8º34’W), where the experiments took place. Clutches were kept in different 1L containers filled with well water until tadpoles reached Gosner developmental stage 25 (Gosner 1960), when the experiment was initiated.

Adult crayfish were caught in streams close to the field station using baited funnel traps. While not in the experiment, the crayfish were kept in 28 × 20 × 14cm containers and fed commercial fish food ad libitum.

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To examine the effects of crayfish chemical cues on P. perezi antipredator defences, we used a factorial design crossing population origin with two predator treatments. Each combination was replicated once in each of five randomized blocks. Populations represented different coexistence times with the exotic crayfish: Longer Coexistence time (LC1 and LC2), Shorter

Coexistence time (SC) and No Coexistence (NC1 and NC2) (Table 1). The two predator treatments were the presence or absence of a single caged crayfish predator. Each experimental unit consisted of a 39 × 28 × 28cm container filled with 10L of well water, ten tadpoles and a suspended predator cage. Predator cages were opaque plastic cylinders

(diameter 85 mm, length 16 cm) with top and bottom covered with fine mesh netting (1.5 mm), allowing the tadpoles to receive chemical, but not visual or tactile cues from the crayfish.

Containers in the no-predator treatment had an empty cage. Crayfish were fed three P. perezi tadpoles every other day, and the tadpoles were fed commercial fish food and boiled lettuce ad libitum every three days. Water was changed weekly. Photoperiod was kept 12D:12N throughout the experiment and the water temperature was 21.0 ± 0.08 ºC (mean ± SE).

Response variables

We recorded tadpole activity at the beginning of the experiment (day 10) and in the middle of the larval development (day 60). The number of active tadpoles in each tank was recorded between 9 and 12 am in five sequential moments, each observation separated by at least 30 minutes. Tadpoles were considered active if they were swimming, foraging or moving their tails.

Tadpole morphology was recorded at day 60 of the experiment by placing each tadpole in a small Plexiglas box with a grid background and taking a side-view digital photograph. We used geometric morphometrics tools to examine variation in tadpole morphology (Rohlf and Marcus

1993, Zelditch et al. 2004). Photographs of five random individuals per container were loaded

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Chapter 5 into MakeFan7 (Sheets 2009) to produce deformation grids describing tadpole body shape. In each tadpole 19 landmarks representing an adequate coverage of the tadpole body shape were marked using the image analysis software tpsDig2 (Rohlf 2008; Fig. 2). These landmarks were chosen based on previous studies (Orizaola et al. 2011, Chapter 4). In order to extract tadpole shape variables, photographs with landmark coordinates were imported into tpsRelw

(Rohlf 2007). This software allowed us to generate a mean (consensus) landmark configuration for each experimental container, described by several components (relative warps), which were later used as shape variables in the statistical analysis. The two first relative warps (RW1 and RW2) explained 54.78% of the initial morphological variance (Fig.4) and were used as dependent variables in the analyses.

Fig. 2. The 19 landmarks digitized for estimating body-shape in each tadpole.

Statistical analysis

Variation in antipredator responses among populations was analysed for activity level and morphology. For activity, we performed a Generalized Linear Model (GLM), using a Binomial error distribution and a logit link function. The total number of active tadpoles per container summed for the five observations in each time period was used as the dependent variable and the total number of tadpoles pooled over the five observations in each container as the trials variable. As activity was registered in two different time periods, a repeated measures approach was used. Pairwise comparisons were performed to inspect differences among populations. To examine differences in morphology of different populations, we used

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Chapter 5 univariate analyses of variance (ANOVAs) followed by Tukey HSD post-hoc tests. Population was always used as a fixed factor because study populations were selected based on the time of coexistence with P. clarkii. Since we found differences between populations and a significant (or nearly significant) difference for the population × treatment effect (see Results),

GLMs (activity) or univariate ANOVAs (morphology) were performed for each population separately. Initially, block was used as a random factor and mixed models were performed.

However, this factor was always non-significant (P >0.1) and it was removed from the final models. All analyses were performed using the software package SPSS (version 19.0).

Results

Activity level increased as tadpole development progressed (Table 2, Fig.3). A significant interaction between time period and population was detected, indicating that activity varied differently among populations throughout tadpole development (Table 2, Fig.3). Further, we found significant differences in activity level among different populations. Populations LC1 and

LC2 had lower activity than the other populations (pairwise comparisons, P< 0.05; Table 2,

Fig.3). Populations NC1 and NC2 had similar activity levels and population SC had higher activity than all the other populations (pairwise comparisons, P> 0.05; Table 2, Fig.3). A significant interaction between population and predator treatment indicated that populations differed in their responses to the crayfish (Table 2). When looking at each population separately, early in larval development, only population SC reduced activity in the presence of crayfish cues. Later in ontogeny, the two NC populations strongly reduced their activity levels in predator presence, while LC and SC populations showed no alterations in activity (Table 2,

Fig.3).

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Table 2. Generalized Linear Models for the activity level of P. perezi populations when exposed to control or crayfish treatments, in the two time periods. Left-hand panel shows analysis for all populations and right-hand panel analysis for each population separately. df= 4,77 for all the factors involving the ‘population’ term and df= 1,77 for all the other factors. P-values < 0.05 are marked in boldface

Activity level Period 1 Period 2

Wald χ2 P Wald χ2 P Wald χ2 P Period 29.486 < 0.001 Period × Population 20.891 < 0.001 LC1 0.150 0.698 0.419 0.517 Period × Predator Treatment 4.182 0.041 LC2 0.001 0.969 0.430 0.512 Period × Pop × Pred Treat 7.397 0.116 SC 8.673 0.003 1.739 0.187 Population 105.418 < 0.001 NC1 0.394 0.530 19.926 < 0.001 Predator Treatment 13.689 < 0.001 NC2 2.967 0.085 23.002 < 0.001 Pop × Pred Treat 9.842 0.043

Fig. 3. Proportion of active tadpoles (mean ± SE) of the five studied populations when exposed to control (white square) or crayfish (black square) treatments, in the two time periods (LCs – Long Coexistence populations, NCs – No Coexistence populations and SC – Short Coexistence population).

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We found significant differences between populations in morphological traits, (F4,36 = 20.185,

P< 0.001 for RW1 and F4,36 = 8.240, P< 0.001 for RW2). Population LC2 did not differ from

populations LC1 and NC2 and population NC1 had a similar morphology to population NC2.

Population SC was different from all the other populations except LC1, having longer and wider

headbodies, wider tails and a more anterior insertion of the tail fin to the headbody (post-hoc

tests, P< 0.05; Fig.4).

No predator Crayfish a) 0.03

0.02

0.01

0.00 RW1 = RW1= 39.74%

-0.01

-0.02

-0.03 LC1 LC2 SC NC1 NC2 Population b) 0.03

0.02

0.01

RW2 = RW2= 15.04% 0.00

-0.01

-0.02 LC1 LC2 SC NC1 NC2 Population

Fig. 4. Mean ± SE values of a) Relative Warp 1 (RW1) and b) Relative Warp 2 (RW2) representing tadpoles shape of the five populations in the presence of control (white square) or crayfish (black square) treatments (LCs – Long Coexistence populations, NCs – No Coexistence populations and SC – Short Coexistence population). Drawings on the right hand side of each graph show the shape of larvae representing the extreme positive (upper panel) and negative (lower panel) scores of RW1 or RW2.

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There was a non significant tendency towards an interaction between the factors ‘population’ and ‘predator treatment’ in RW2 (F4,36 = 2.129, P= 0.097), while this did not happen for RW1

(F4,36 = 0.813, P= 0.525). When looking at populations separately, population LC1 showed significant alterations in RW2, with tadpoles developing smaller headbodies, deeper tail muscles and a more anterior insertion of the tail fin into the headbody in the presence of crayfish (F1,6 = 10.489, P= 0.018; Fig.4); no evidence for alterations in morphology was found in other populations or in RW1.

Discussion

We found that tadpoles of P. perezi respond to chemical cues of P. clarkii. Specifically, tadpoles from one of the populations with a long history of contact with P. clarkii (henceforth LC) showed morphological plasticity in crayfish presence, while there was a reduction in activity in response to the crayfish in no coexistence time populations (NC). Our results suggest that the length of coexistence time with the invasive predator has an important role in defining the type of responses elicited. Further, previous studies have failed to detect responses of P. perezi to P. clarkii (Gomez-Mestre and Diaz-Paniagua 2011, Chapter 3), indicating that there is considerable variation among local populations in the ability to induce plastic responses towards this exotic predator.

The plastic responses to P. clarkii differed between populations coexisting with the crayfish and populations that have never been in contact with it. NC populations greatly reduced their activity levels (in time period 2) in the presence of crayfish; this is a common adaptive defence against predators (Lima and Dill 1990, Kats and Dill 1998). However, coexistence populations generally did not alter their activity level in the presence of the crayfish. The hypothesis that baseline activity level in LC populations is so low that a reduction in its level would not be possible is unlikely, as both this (Polo-Cavia et al. 2010, Gomez-Mestre and Diaz-Paniagua

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2011, Chapter 3) and other anuran species with low baseline activity (e.g. Richardson 2001,

Laurila et al. 2006) are able to reduce their activity in response to chemical cues from other predators. We suggest two different hypotheses to explain the lack of behavioural responses in populations coexisting with crayfish.

First, given that the LC populations had quite low baseline activity levels, this lack of response could suggest the existence of a constitutive behavioural defence in these populations, which may evolve under uniform and strong predation risk (Moran 1992, Edgell et al. 2009). In this case, strong selection by P. clarkii would have promoted the loss of behavioural plasticity in these populations, leading to permanently well defended inactive tadpoles, irrespective of the presence of crayfish in the environment. However, we do not think this is the most likely hypothesis because the population with a short coexistence time with crayfish (SC) had high baseline activity level, yet it only reduced activity in crayfish presence early in ontogeny.

Alternatively, we suggest that reducing activity is not the most effective defence against this predator and that not altering activity may be an evolved adaptive response to the predator’s presence. Although some previous studies have documented a reduction in prey activity in crayfish presence (Bryan et al. 2002, Marquis et al. 2004, Mandrillon and Saglio 2005, Chapter

3), many others have reported no alterations or an increase in prey activity level in the presence of crayfish (Rahel and Stein 1988, Lefcort 1996, Åbjörnsson et al. 2000, Nyström et al.

2001, Correia et al. 2005, Leite et al. 2005, Hoverman and Relyea 2008). In fact, this defence may not be very effective against P. clarkii which is a moderately active predator (Gomez-

Mestre and Diaz-Panigua 2011) and relies more on chemical rather than visual cues to detect prey (Aquiloni et al. 2005). This may put inactive tadpoles at the same risk as active ones and make swimming ability more important than inactivity in avoiding crayfish predators (Lefcort

1996, Nyström et al. 1999). Even though in their case tadpole activity was recorded in the presence of a free-ranging crayfish, Cruz and Rebelo (2005) did not find any relationship between activity rates or plasticity in activity and survival to P. clarkii. Future studies

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Chapter 5 investigating more thoroughly if induced behavioural alterations are adaptive under direct predation by the crayfish are clearly needed.

When exposed to crayfish, tadpoles from one of the longer coexistence populations (LC1) altered morphology by developing a smaller headbody, a deeper tail muscle and a more anterior insertion of the tail fin into the upper part of the headbody, all of which are common adaptive morphological alterations of larval anurans to predators (McCollum and Van Buskirk

1996, Relyea 2001, Teplitsky et al. 2005, Van Buskirk 2009). Gomez-Mestre and Diaz-Paniagua

(2011) showed that similar morphological alterations - induced by dragonflies - are efficient in decreasing P. perezi mortality imposed by P. clarkii. This suggests that the morphological response to P. clarkii in LC1 is adaptive (as is the lack of response in behaviour) and that this population has adapted locally by evolving morphological defences against the invasive crayfish. This result agrees with the idea that invasive species can be a strong selective force generating local adaptation and rapid evolution (Strauss et al. 2006, Sih et al. 2010). While our study reports the fastest evolution of predator defences documented for an amphibian (ca. 32 years, 10-15 P. perezi generations), there are several studies reporting rapid evolution in response to selection from invading predators (Schlaepfer et al. 2005, Freeman and Byers

2006, Strauss et al. 2006), including one case in amphibians (Kiesecker and Blaustein 1997).

Considering this, we find surprising the fact that LC2 did not respond with morphological defences to crayfish chemical stimuli (despite a tendency for that). We hypothesize this may be because LC1 is located at a rice field in a bank of the Caia River, very close to where it meets the Guadiana River (see Fig.1), the river through which P. clarkii likely expanded into Portugal, before colonizing the Caia river (Ramos and Pereira 1981). Further, rice fields are one of the preferred habitats of the crayfish in the Iberian Peninsula, where they attain very high densities (Anastácio et al. 1995, Fidalgo et al. 2001). This suggests that LC1 may have coexisted for a slightly longer time with more stable populations of P. clarkii than LC2. Alternatively, this

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Chapter 5 may indicate differences between the populations in the intensity of selection imposed by the crayfish or differences in the evolutionary potential of the populations.

Interestingly, Gomez-Mestre and Díaz-Paniagua (2011) did not find morphological responses of

P. perezi to P. clarkii in a population from Doñana National Park where P. clarkii was found already in 1974. While we cannot exclude population differences in intensity of selection or in

P. perezi’s potential to evolve responses to P. clarkii, the discrepancy between those and the present results may be explained by the more robust approach used to examine tadpole morphology in the present study.

The fact that NC populations altered behaviour in crayfish presence indicates the detection of a threat by these populations and the ability of tadpoles to elicit defences towards a predator with which they have had no previous evolutionary contact. This suggests an ‘innate’ response of these populations to the introduced crayfish predator. Many species innately recognise and respond to chemical stimuli from native predators (review in Wisenden 2003), but examples of

‘innate’ recognition of exotic predators in amphibians are not common (but see Epp and Gabor

2008, Rehage et al. 2009). Naive populations responding to unknown predators usually share an evolutionary history with native predators closely related to the novel predator (Epp and

Gabor 2008, Rehage et al. 2009). Austropotamobius pallipes, the only native crayfish species in

Portugal, is now extinct across the Portuguese territory. Its original geographical range did not reach southern Portugal (Almaça 1991), suggesting that the responses of NC populations are not likely due to the phylogenetic similarity to a native predator in the community (Strauss et al. 2006). Instead, the responses may have been facilitated by the detection of cues from predation events, resulting from injured or digested conspecifics (Ferrari et al. 2010) and/or by an innate antipredator response towards generalized predator signals (Gherardi et al. 2011).

The fact that crayfish presence induced a similarly strong behavioural response in the two NC populations reinforces the idea of a general response towards predators or diet cues, independent of the specific predatory threat.

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The differences between coexistence and no-coexistence populations may be related to a differential perception of different populations in the available predation cues present in our experimental containers. Tadpoles may require more complete information about both the predator species and the predator diet (not only the predator diet alone) before investing in longer-term and less reversible morphological defences (Relyea 2003, Schoeppner and Relyea

2005, Orizaola et al. 2011). By having locally adapted to P. clarkii, coexistence populations (or at least LC1) may be able to detect and respond to specific kairomones from this predator. In contrast, NC populations probably lack this ability, having only detected alarm or diet cues resulting from predation events and eliciting short-term and more reversible behavioural defences.

We found significant among-population variation according to populations’ geographical area of origin. There was a clear geographical pattern of phenotypic divergence in larval behaviour and morphology between populations from Caia (LC) and Monchique (NC), the former being less active and usually having deeper headbodies and tails than the latter, suggesting either genetic population differentiation or maternal effects. By influencing embryo size and energy, maternal investment may greatly affect an individual’s phenotype, especially through alterations in growth rate (Laugen et al. 2002). Genetic differences may result from adaptation to local habitat features such as temperature, canopy cover, native predators, competitors or food availability (Relyea 2002, Van Buskirk and Arioli 2005, Richter-Boix et al. 2010). However, we note that the populations from the two regions experience quite similar native predator regimes (diverse and dense populations of predators), and large population differences in native predation pressure are not likely to be important here. Studies using multiple populations from several different geographical areas can definitely determine the role of rapid evolution in favouring local adaptation in populations invaded by P. clarkii.

This study highlights the importance of studying population variation in antipredator defences towards introduced predators and suggests that invasive alien predators may drive rapid

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Chapter 5 evolutionary change in invaded communities. Taking into account its enormous invasion potential in the Iberian Peninsula (Capinha and Anastácio 2011), P. clarkii is likely to colonize

Monchique and other crayfish-free regions in the future. This study shows that some invaded prey populations do have the potential to locally adapt to the crayfish presence. However, given the strong selection pressure that this predator imposes (Cruz and Rebelo 2005, Cruz et al. 2008, Nunes et al. 2010, Ficetola et al. 2011), other invaded populations or populations not yet invaded may not be able to evolve defensive adaptations rapidly enough to hinder strong declines or even extinction. As amphibian species appear to differ in their ability to respond to

P. clarkii (Chapters 3 and 4), future studies should span populations of several species in order to identify the ones at most risk.

Acknowledgments

We thank Bruno Carreira, Rosário Sumares, João Nunes and Sónia Longueira for their help in the laboratory. We also thank Rui Braz for designing the map of P. perezi populations. Permits were provided by the Portuguese Instituto da Conservação da Natureza e da Biodiversidade

(Licence n.º228/2009/CAPT/TRANS). This research was funded by the FCT Project POCI/BIA-

BDE/56100/2004, FCT grant SFRH/BD/29068/2006 and by Stiftelsen för Zoologisk Forskning.

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General Discussion

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6. GENERAL DISCUSSION

In this thesis, I conducted a thorough analysis of the antipredator strategies used by larval anurans from the Southwestern Iberian Peninsula to respond to the exotic crayfish P. clarkii.

More specifically, I assessed the importance of P. clarkii as a tail injury agent on anuran tadpoles and the consequences of tail injury to prey. I examined the behavioural, morphological and life-history antipredator responses to P. clarkii in the whole community of larval anurans and analysed how strongly these responses were phenotypically integrated.

Finally, I compared crayfish-induced phenotypic plasticity in invaded and non-invaded anuran populations.

The work presented here helps to understand how anuran prey populations deal with invasion by a novel introduced predator and sets the scene for future research in this area. The aim of this chapter is to synthesise and integrate the main findings exposed in the previous chapters in the light of current knowledge on biological invasions, to set the results in an evolutionary framework and to make recommendations on how to promote persistence of vulnerable prey species.

6.1. Crayfish as an agent of tail injury in anurans

This work showed that P. clarkii incurs nonlethal tail injury on P. cultripes tadpoles at a much higher rate than native predators do, indicating that this invasive predator is a strong agent of injury in invaded anuran populations. This may severely impact native amphibians, since I found a tendency for decreased larval survival as the frequency of caudal injury increased, even when resource availability was high (Chapter 2).

Nonlethal injury induced alterations in tadpole tail morphology, with injured animals having deeper tail muscles. Since a deeper tail improves the odds of survival in the presence of P.

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Chapter 6 clarkii (Gomez-Mestre and Diaz-Paniagua 2011) and this seems to be an adaptive response evolved by populations with a long coexistence time with crayfish (Chapter 5), the induced morphological alterations following tail injury may increase the survival probability of the tadpoles.

Tail regeneration and alterations in tail morphology did not incur costs in terms of tadpole growth and development, even though these may only arise later in life. In future studies, animals surviving injury should be followed until metamorphosis and at post-metamorphic stages, to examine if repeated tail loss has effects on traits such as body mass, jumping ability or resistance to desiccation. It is also important to note that P. cultripes has a very long larval period, which may allow it to better recover from tail injury than other species with shorter larval periods and for which consequences of tail loss may be more severe.

6.2. Phenotypic variation in Iberian tadpoles

My experiments allowed documentation of species differences in phenotypic traits. In fact, the phenotypic variation of some larval anurans in focus in this thesis has never been studied before, as is the case of A. cisternasii, P. cultripes and P. ibericus (but see Diaz-Paniagua 1985 for P. cultripes).

Out of the nine study species, these three species were the ones having the longest larval periods: 112 days for P. ibericus, 200 days for A. cisternasii and 222 days for P. cultripes

(Chapter 4). Contrary to A. cisternasii, which typically inhabits permanent water bodies, P. cultripes reproduces in temporary ponds. The long larval period of P. cultripes suggests that there is either great larval mortality due to pond desiccation, or that tadpoles accelerate their development when cues from a decreasing water level are present. In comparison with the other species, P. cultripes had a very high growth rate and mass at metamorphosis (Chapter 4).

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This suggests that the tadpoles have to maintain very high activity level (and high foraging rates), which was indeed observed late in their development (Chapter 3).

In general, species inhabiting ephemeral and temporary habitats are expected to be very active and maintain high feeding rates in order to develop fast and reach metamorphosis before the water body dries (Wellborn et al. 1996, Laurila and Kujasalo 1999, Anholt et al.

2000, Richter-Boix et al. 2007). On the contrary, permanent ponds species do not need to cope with periodic drying, and are expected to be less constrained in terms of behaviour and development. However, when looking at baseline activity levels of the nine anuran species

(Chapter 3), I found that B. calamita and D. galganoi, both species from ephemeral habitats, had quite low baseline activity levels and that B. bufo, a permanent pond species, had a moderately high activity level. This indicates that the relationship between hydroperiod gradient and baseline levels of activity does not hold in this community (see also Richardson

2001, Van Buskirk 2002). Species phylogeny did not explain this pattern since I did not find a phylogenetic autocorrelation in baseline activity level of the study species and since species closely related to these do not have similar baseline activity levels to the ones found here

(Richter-Boix et al. 2007).

Further, species with the highest activity level were not the ones with the shortest time to metamorphosis, suggesting that, contrary to results of some other studies (e.g. Relyea 2001,

Richter-Boix et al. 2007), activity was not linearly related to developmental rate (Chapters 3 and 4). In some of these species behavioural changes appeared to have little impact on developmental rates, suggesting a decoupling between activity level and food consumption rate (McPeek 2004, Steiner 2007b). Hence, as behaviour seems to be uncoupled from development, it is not surprising that activity of different species does not reflect differences in pond hydroperiod.

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Different species have different phenotypic traits and, in fact, even the same species in different studies often differs in phenotype, which may reflect experimental or methodological differences, but most likely reflects population genetic divergence (e.g. for P. perezi: Richter-

Boix et al. 2007, Polo-Cavia et al. 2010, Egea-Serrano et al. 2011, Gomez-Mestre and Diaz-

Paniagua 2011). As seen in Chapter 5, intraspecific phenotypic variation is often high in larval amphibians (e.g. Relyea 2002b, Van Buskirk and Arioli 2005, Laurila et al. 2006, 2008). More studies examining among-population phenotypic variation are essential, especially considering that some anuran species from the Iberian Peninsula show significant patterns of population subdivision (e.g. Rosa 1995, Gonçalves et al. 2009) and that the taxonomy of many of these species is constantly under revision (e.g. P. ibericus, Sánchez-Herráiz et al. 2000; B. spinosus,

Recuero et al. 2011).

6.3. Plastic responses of Iberian anurans to a native and an exotic predator

Although species differed in their responses to predators, I found that, with the exception of B. calamita, all anurans from Southwestern Iberian Peninsula showed predator-induced phenotypic plasticity. Surprisingly, only one of these species did not show any alteration in phenotype in the presence of the exotic predator, indicating that native prey species are not completely defenceless against invasive predators.

All species responded to the presence of dragonfly with alterations in behaviour and morphology. However, while all but P. ibericus responded to the crayfish behaviourally, only B. bufo and P. perezi altered morphology in its presence (Chapters 3 and 4, Table 1). Even though

P. perezi did not show any change in behaviour, morphology or life-history in Chapters 3 and 4, in Chapter 5 I found that some P. perezi populations do have the ability to respond to P. clarkii.

In this case, time of coexistence with the invasive predator was the factor behind different types of responses. As discussed in Chapter 5, this suggests genetic variation in antipredator

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Chapter 6 responses, evolved through natural selection imposed by the invasive predator. I found that only two out of nine species altered morphology in the presence of crayfish, while eight species did so in the presence of dragonfly (Table 1). This cannot be due to morphological defences not being effective against the crayfish predator, as Gomez-Mestre and Diaz-

Paniagua (2011) showed that tadpoles with increased tail depth are better in surviving predation by P. clarkii (see also Chapter 5). The lack of morphological defences may then be a consequence of these defences being less reversible than behavioural ones (Relyea 2001,

2003, Orizaola et al. 2011) and, as a result, not being as easily elicited in the presence of an unknown chemical stimulus, even if associated with distress cues from predated conspecifics

(Schoeppner and Relyea 2009). An alternative hypothesis relates to the fact that morphological characters are considered less plastic than behavioural traits and so less readily altered in response to new selective pressures (Relyea 2001, Blomberg et al. 2003, Blumstein 2006). At the same time, it suggests that alterations in behaviour are likely more prompt responses of prey when a chemical cue is detected in the environment, being it a predator odour or simply cues from consumed conspecifics. In the three species that were most responsive to the crayfish behaviourally (A. cisternasii, B. bufo and D. galganoi), cues resulting from predation events largely mediated behavioural plasticity (Chapter 3), reinforcing the idea that behavioural antipredator responses often derive from the presence of cues from consumed conspecifics (Chapter 5, Schoeppner and Relyea 2005). In light of these results, the fact that A. cisternasii also reduced activity level when exposed to cues from starved crayfish is intriguing and deserves further study.

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Table 1. Antipredator responses elicited by the nine anuran species in the presence of the invasive (P. clarkii) or native (Aeshna spp.) predator.

Behaviour Morphology Life-history

Crayfish Dragonfly Crayfish Dragonfly Crayfish Dragonfly Alytes cisternasii ↓ activity ↓ activity ↑ headbody depth ↓ time to metamorphosis ↑ cage avoidance ↑ cage avoidance ↑ tail fin depth ↑ mass at metamorphosis - - ↑ tail muscle depth ↑ growth rate ↑ tail fin insertion Discoglossus galganoi ↓ activity ↓ activity ↑ headbody depth ↑ time to metamorphosis ↑ cage avoidance ↑ cage avoidance ↑ tail fin depth ↓ growth rate - - ↑ tail muscle depth ↑ tail fin insertion Pelobates cultripes ↓ activity ↓ activity ↑ tail fin depth ↓ time to metamorphosis ↑ cage avoidance - ↑ tail muscle depth - ↑ tail fin insertion Pelodytes ibericus ↓ activity ↑ headbody depth ↑ mass at metamorphosis - ↑ cage avoidance - ↑ tail fin depth - ↑ growth rate ↑ tail fin insertion Bufo bufo ↓ activity ↓ activity ↑ headbody length ↑ headbody length ↓ time to metamorphosis ↓ time to metamorphosis ↑ cage avoidance ↑ cage avoidance ↓ mass at metamorphosis ↓ mass at metamorphosis ↓ growth rate

Bufo calamita ------Hyla arborea ↓ activity ↓ activity ↑ tail fin depth ↓ time to metamorphosis ↑ growth rate ↑ cage avoidance - ↑ tail fin insertion ↑ growth rate Hyla meridionalis ↓ activity ↓ activity ↑ headbody depth ↑ mass at metamorphosis ↑ cage avoidance - ↑ tail fin depth - ↑ tail fin insertion Pelophylax perezi ↓ activity* ↓ activity ↓ headbody length** ↑ headbody depth ↑ tail muscle depth** ↑ tail fin depth - - ↑ tail fin insertion** ↑ tail muscle depth

* only crayfish-free populations ** only a long-term crayfish invaded population

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However, behavioural responses elicited in the presence of fed crayfish may not necessarily be the most effective defenses against this predator. This seems to be the case for P. perezi, a species for which a reduction in activity level likely represents a poorly effective defence towards P. clarkii (Chapter 5). This may also hold true for A. cisternasii, a species that, similarly to P. perezi, uses the bottom of ponds or streams as a preferred microhabitat (Díaz-Paniagua

1985, Cruz and Rebelo 2005). On the contrary, for species mainly found in shallow margins of ponds or in open water (B. bufo, Hyla spp. and P. cultripes) (Díaz-Paniagua 1985, Cruz and

Rebelo 2005), a decrease in activity, together with a safe distance to the predator, will surely be adaptive. A. cisternasii has been reported to change microhabitat use in P. clarkii’s presence: it avoids pond bottom and moves to the shallow margins (Cruz and Rebelo 2005), making low activity an effective response. Predation experiments assessing the survival value of different behavioural responses against P. clarkii would help settling their adaptive value and should clearly follow.

The lack of antipredator responses towards both predators in B. calamita can be explained by the fact that this species reproduces in extremely ephemeral ponds, where no large aquatic predators usually exist (Diaz-Paniagua 1990, Richter-Boix et al. 2007), making it highly vulnerable to P. clarkii predation (Cruz and Rebelo 2005, Cruz et al. 2006b).

While predator-induced responses to the native dragonfly were usually strong and highly integrated, responses to the invasive crayfish were often weak and generally not integrated

(Chapters 3 and 4). The exceptions were B. bufo, which showed similar integration to both predators, and A. cisternasii, for which a more integrated response was found in the presence of crayfish than dragonfly (Chapter 4). These two species, together with H. arborea and P. perezi (only one long-term coexistence population), were the most responsive to the novel predator. Further, A. cisternasii and H. arborea altered different suites of traits in the presence of the exotic and the native predator, and responded behaviourally when exposed to chemical cues from the starved crayfish (Chapters 3 and 4). This suggests that these species are capable

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Chapter 6 of detecting crayfish kairomones in the environment and associate them with a signal of danger, different to that caused by the native dragonfly predator. This may indicate that the study populations of A. cisternasii and H. arborea have already evolved the ability to recognise and respond to cues from the novel predator, although I cannot be as sure as in the case of long-term coexistence populations of P. perezi (Chapter 5). Studies assessing rapid adaptive evolution on these two species would be very interesting, for example testing antipredator responses in the F1 generation of several populations with different histories of contact with P. clarkii, in order to understand the genetic basis of the observed changes (Reznick and

Ghalambor 2001).

The four abovementioned most responsive species to the crayfish all breed in permanent water bodies. Even though the type of habitat where species reproduce is not the only factor determining responses to this invasive predator (Chapters 3 and 4), this trend may be due to these habitats being the main crayfish sources, where more stable and long lasting crayfish populations have likely been established.

Costs of inducible defences were quite rare in this anuran community, at least under the experimental conditions used in this study. Although the expression of behavioural and morphological defences commonly incurs proximate costs in terms of growth and development (reviewed by DeWitt et al. 1998, Van Buskirk 2000, Relyea 2002a, Steiner 2007a, b, Callahan et al. 2008, Auld et al. 2010), other studies have either found weak or no costs

(Scheiner and Berrigan 1998, Van Buskirk and Saxer 2001, Teplitsky et al. 2003, Van Buskirk and Steiner 2009). Here, potential costs of defence were only detected in two out of nine anuran species, D. galganoi and B. bufo (Chapter 4). This may be related to these two species having the smallest body size of all species (excluding B. calamita; Chapter 3), making any alteration in their energy reserves due to induced defences (more energy expenditure or less energy gain) much more detrimental than for larger species. This reinforces the idea of the existence of prey-specific costs (Callahan et al. 2008). Yet, the possibility that more costs were

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Chapter 6 not detected under our experimental conditions due to food conditions being unrestricted, probably allowing tadpoles to attain high energy levels, cannot be excluded.

I also found that inducible defences were concurrent with increased growth and developmental rates in some of the species. Both A. cisternasii and H. arborea reduced their activity level while they increased growth rate and attained metamorphosis faster in the presence of crayfish (and at a larger size, in the case of A. cisternasii; Chapter 4). Similarly,

Teplitsky et al. (2003) found that, when exposed to fish chemical cues, tadpoles of H. arborea grew and developed faster (although they did not detect any behavioural alterations).

Attaining metamorphosis earlier and at an equal or larger mass has not been predicted by recent dynamic state-dependent models (Higginson and Ruxton 2010). This may be related to limitations in the models. For instance, the models assume that when prey are inactive there is no food consumption which, in the light of present results, is unlikely to be true. The models also do not allow for a negative association between activity/foraging rate and growth or mass at metamorphosis, contrary to what was observed in this (Chapter 4) and other recent studies

(Johansson and Andersson 2009). It is clear that future models should incorporate this information.

The general lack of costs observed in this community and the decoupling observed between behaviour and growth/development may help explain why species from temporary habitats, contrary to what is predicted, did not invest less in behavioural defences than species from permanent habitats (Chapters 3 and 4). It is commonly stated that species inhabiting temporary water bodies, due to having a limited time span to reach metamorphosis, cannot afford to reduce activity level as a behavioural defence (Wellborn et al. 1996, Laurila and

Kujasalo 1999, Richardson 2001, Richter-Boix et al. 2007). However, if a reduction in activity level does not cause reduced growth or development in temporary pond species (as was observed here; see the section ‘Species phenotypic traits’ and Chapter 4), individuals with behavioural defences will still grow fast enough and reach metamorphosis before the pond

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Chapter 6 dries. In fact, other authors have also reported as strong behavioural defences in temporary as in permanent habitats species (Van Buskirk 2002).

Overall, the capacity to elicit antipredator responses towards an invasive crayfish predator and the type of responses elicited varied a lot among the study species. Some of the species did not respond to the crayfish at all (B. calamita and P. ibericus), some responded with only few trait alterations (H. meridionalis and D. galganoi), one responded using a generalized response promoted by alarm cues (B. bufo), three responded by changing a suite of traits in a likely efficient manner (A. cisternasii, H. arborea and P. cultripes) and one seems to have evolved adaptive morphological responses to this predator (P. perezi). According to our results, P. ibericus, which responds strongly to the native but not to the invasive predator, is one of the most vulnerable species to crayfish introduction in this community. In fact, previous studies have shown that P. clarkii is a negative predictor of the presence of this species in a water body (Beja and Alcazar 2003, Cruz et al. 2006a), and that the crayfish has caused the local extinction of a closely related species (P. punctatus) in a Nature Reserve (Cruz et al. 2008).

6.4. Implications of P. clarkii invasion for anuran communities

The species showing the strongest antipredator responses to the crayfish (A. cisternasii, B. bufo, H. arborea and P. cultripes) can be predicted to be the ones with the highest survivorship to direct predation by P. clarkii. However, this does not seem to be the case if we compare the results obtained here with those obtained by Cruz and Rebelo (2005) (Table 2). In their study,

B. calamita, B. bufo and P. cultripes were the most vulnerable species to P. clarkii which, excluding B. calamita, were species defended against P. clarkii in our study (Chapters 3 and 4,

Table 2). Conversely, for other species, results of the two studies overlapped, as in the case of

A. cisternasii and H. arborea, both well defended species, apparently less susceptible to predation (Chapters 3 and 4, Table 2) (Cruz and Rebelo 2005).

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While the discrepancy between these studies may be partially caused by methodological constraints, the results suggest that predator-induced defences may not be enough to explain tadpole vulnerability to P. clarkii. Still, some of the antipredator responses assumed here as defences against the crayfish, such as a reduced time to metamorphosis, although beneficial, do not influence vulnerability to a predator before or when an attack takes place. This type of defences should, in turn, be reflected in the susceptibility of natural anuran populations to invasion by P. clarkii. Several aspects of tadpole ecology probably act together in explaining vulnerability to this novel predator (e.g. antipredator defences, swimming ability, microhabitat use), and studies aiming to understand which factors are the most important and in which way they interact are an essential next step to understand which species are more vulnerable to invasions.

Neither anuran antipredator defences (Chapters 3, 4 and 5, Table 2) nor survival as measured by Cruz and Rebelo (2005) seem to provide a general mechanistic explanation for the patterns of anuran displacement and declines observed in crayfish-invaded areas (Table 2) (Cruz et al.

2006a, 2008). For example, for B. bufo and P. cultripes, both defended species (Chapters 3 and

4, Table 2), crayfish presence was a negative predictor of their breeding probability (Cruz et al.

2006a). However, a consistent trend was found for Pelodytes spp., a species poorly defended against P. clarkii (although with quite high survival), to suffer strongly from crayfish invasion

(Chapters 3, 4 and 5, Table 2) (Cruz et al. 2006a, 2008). Further, the distribution of some species associated with permanent water bodies (A. cisternasii and H. arborea) was not affected by crayfish presence in the Sado region (Cruz et al. 2006a), which may be related to these species being efficiently defended against this predator (Chapters 3 and 4). Yet, field data from another region in Portugal (Paúl do Boquilobo Nature Reserve) showed that populations of H. arborea, apparently resistant to crayfish in Sado, were excluded or strongly reduced after crayfish arrival (Cruz et al. 2008).

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Table 2. Approximate ranking of species vulnerability to P. clarkii inferred by different studies and considering anuran antipredator defences (Chapters 3, 4 and 5), survival (Cruz and Rebelo 2005), probability of reproducing in a crayfish invaded habitat (Cruz et al. 2006a) and population resilience (Cruz et al. 2008). Antipredator Probability of reproducing Population Defences Survival in a crayfish invaded resilience - habitat B. calamita B. calamita P. cultripes Pelodytes punctatus P. ibericus B. bufo B. bufo H. arborea H. meridionalis P. cultripes P. ibericus P. perezi D. galganoi D. galganoi H. arborea P. cultripes P. perezi A. cisternasii P. perezi* A. cisternasii B. calamita B. bufo P. ibericus P. perezi H. arborea H. arborea H. meridionalis + A. cisternasii H. meridionalis D. galganoi

* only a long-term crayfish invaded population

These lines of evidence suggest that the impacts that P. clarkii exerts in amphibian populations

do not derive solely from direct predation and that the dynamics of declining amphibian

communities is not only influenced by species’ responses or survival to crayfish predation. P.

clarkii must affect amphibian populations through other indirect ways and, in fact, invaders

such as crayfish can impact organisms at different ecological and multitrophic levels (Nyström

et al. 2001, Rodríguez et al. 2005). By greatly increasing the rate at which tadpoles suffer tail

injury, crayfish may affect anuran survival and metamorphic recruitment (Chapter 2). By

grazing and nonconsumptive destruction of macrophytes, crayfish reduce the area available

for the growth of periphyton, affecting the quantity of food resources for tadpoles (Lodge et

al. 1994, Nyström et al. 2001, Rodríguez et al. 2005). Macrophyte consumption also reduces

structural complexity of the aquatic habitat, decreasing shelters and destroying egg-laying sites

of some amphibian species (Gutiérrez-Yurrita and Montes 1999, Rodríguez et al. 2005). Dense

crayfish populations can also affect water quality by increasing turbidity and nutrient release,

which is associated to its intense foraging and burrowing activity (Rodríguez et al. 2003, 2005).

The type of resources exploited by P. clarkii coupled with their action as bioturbators, may lead

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Chapter 6 to habitat degradation and dramatic direct and indirect effects on amphibian populations

(Geiger et al. 2005, Rodríguez et al. 2005).

6.5. Control and management of P. clarkii and conservation of native amphibians

As introduced species become established and abundant in their new habitats, eradication and management options become more limited (Sakai et al. 2001, Lockwood et al. 2007). In the

Iberian Peninsula, P. clarkii has already passed the early stages of invasion - establishment and spread - when total eradication should be attempted, being now at the ‘ecological impact’ stage, which demands for intensive control (Kolar and Lodge 2001, Sakai et al. 2001, Simberloff

2003, Lockwood et al. 2007). Although there are examples of successful control of established invasive species ( see examples in Simberloff 2003), for aggressive and very successful invaders, such as P. clarkii, eradication and direct control are very unlikely or even impossible

(Barbaresi and Gherardi 2000, Lodge et al. 2000, Kerby et al. 2005, Gherardi 2007). Among the techniques that can be used to control or eradicate invasive crayfish are mechanical (trapping and electrofishing), physical (draining ponds or creating barriers), chemical (use of biocides) or biological (microbial insecticides and introduction of fish predators) approaches, some of which have been proven unfruitful in the elimination of P. clarkii (Anastácio et al. 2000, Kerby et al. 2005, Freeman et al. 2010). Additionally, given P. clarkii’s ability to quickly expand and its enormous invasion potential in the Iberian Peninsula there is likely to be only very few crayfish-free aquatic habitats in the near future (Capinha and Anastácio 2011, Liu et al. 2011).

Considering this scenario, only a few options are left to sustain persistence of amphibian populations in the Southwest of the Iberian Peninsula. First of all, given enough resources, control or eradication of crayfish in small ephemeral or temporary ponds with the highest suitability for amphibians could be an important measure, since anuran species from these

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Chapter 6 habitats seem to be the most undefended against the crayfish (Chapters 3, 4 and 5; see also

Cruz et al. 2006a). This is especially relevant for endemic species, such as P. ibericus and D. galganoi (Loureiro et al. 2008).

Another sustainable solution consists of finding strategies that allow coexistence between P. clarkii and vulnerable native prey species, for which the results of the present study may help setting a head start (Carroll 2011). Since anuran populations seem to be able to, through time, evolve efficient morphological defences against this invasive crayfish (Chapter 5), management plans could promote survival of native prey populations long enough to allow adaptive evolution to occur (Schlaepfer et al. 2005, Lankau et al. 2011). This could be promoted by, for instance, increasing structural habitat complexity in order to provide more shelters and refuge areas for amphibian larvae to hide from crayfish attacks (Axelsson et al. 1997, Babbitt and

Tanner 1998). Yet, caution has to be taken because some other predators, such as dragonflies, often use vegetation cover as a preferential place to launch their attacks. In fact, the directional selection imposed by P. clarkii should be combined with prey population growth, which may be a key factor hindering extinction and allowing for rapid evolution to occur

(Reznick and Ghalambor 2001). Hence, population size of P. clarkii should be controlled so as to not cause extirpation of the native prey populations and allow population growth.

Another approach consists of stocking naïve or recently invaded populations with individuals from longer-term invaded populations, with already evolved adequate morphological defences towards P. clarkii to, via hybridization, increase the evolutionary potential of the former prey populations (Schlaepfer et al. 2005, Lankau et al. 2011). However, this option may be quite risky, because it may promote loss of intraspecific adaptive phenotypic trait variation, pervasive in several anuran species from the Iberian Peninsula (e.g. Chapter 5, Rosa 1995,

Gonçalves et al. 2009, Egea-Serrano et al. 2009). Finally, the capacity that some amphibians have of, through association with alarm cues, learning to recognise and respond to the scent of novel predators (Ferrari et al. 2010), may be used as a tool by conservation biologists

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(Schlaepfer et al. 2005). By ‘teaching’ individuals that a certain predator is dangerous, prey will avoid it by adopting antipredator defences, hopefully adequate to the specific predator (Sih et al. 2011).

It is important to note that rapid evolution of morphological responses to P. clarkii was only detected in populations of P. perezi (Chapter 5), and the evolution-based management techniques proposed above are dependent on the assumption that a similar pattern will occur in populations of the other Southwestern Iberian Peninsula anuran species. Species with a similarly short generation time as P. perezi (2-3 years), high reproductive output and short larval period, such as both Hyla spp. and B. bufo (Almeida et al. 2001), may have high potential to locally adapt to the presence of P. clarkii. Further, this may be enhanced for H. arborea, a species that, along with P. perezi, reproduces in late spring/early summer, when crayfish also reproduces, making selection predation pressure stronger and maybe promoting local adaptation (provided selection is not too strong) (Gutiérrez-Yurrita and Montes 1999, Almeida et al. 2001).

6.6. Final considerations

Invasive species are altering the biotic structure of many ecosystems and have become a major cause of extinction. The high rate of anthropogenic-mediated species introductions will continue to promote faunal homogenization, making the number of biological invasions inevitably destined to increase. Further, global climate change will most likely allow for exotic species to expand their ranges even more (e.g. Liu et al. 2011).

This work adds to the growing evidence that the invasion of the Southwest of the Iberian

Peninsula by the invasive crayfish P. clarkii impacts native anuran prey populations. This is reflected by the high rates of tadpole tail injury found in crayfish-invaded populations, the

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Chapter 6 alterations in tadpole behaviour, morphology and life-history caused by crayfish presence and the evolutionary changes observed in populations with a long coexistence time with P. clarkii.

The results found here extend the taxonomic occurrence of predator-induced plasticity, here reported for the first time for some Iberian anurans. Antipredator responses towards the invasive predator varied greatly within the same community, which can make the impact of P. clarkii on amphibian populations uneven and hard to predict. More studies are clearly needed and they should focus on the most vulnerable species to this invasion, such as P. ibericus, an endemic Iberian species (Loureiro et al. 2008). P. clarkii proved to be a strong selective agent, promoting adaptive evolution in populations of P. perezi in a quite short period of time. As such, and since there are no easy solutions to cope with P. clarkii’s invasions, employing strategies that allow native communities to coexist with this predator are probably the best option left. Still, there is no doubt that P. clarkii will continue to induce deep alterations in amphibian communities in Southwestern Iberian Peninsula in the future.

Understanding the dynamics of the interactions between exotic predators and native prey and what role the former may have in structuring the future evolutionary pathway of invaded systems is now essential and requires the convergence in the fields of ecology and evolution.

Given the more ecological approach of this thesis, I hope that these results can be combined with more evolutionary-oriented studies to improve the understanding of the essentially irreversible outcomes resulting from the invasion of P. clarkii.

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