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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
i
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
ii
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
iii
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
iv
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
v
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
vi
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
1
CHAPTER 1
General Introduction
2
Chapter 1
3
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
4
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
5
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
6
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
7
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
8
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
9
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
10
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
11
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
12
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
13
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
14
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
15
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
16
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
17
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
18
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
19
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
20
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
21
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
22
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
23
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
24
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.).
Chapter 1
25
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).
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.
a
)
b c
d e f
g h i
Chapter 1
26
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
27
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
28
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
29
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
30
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
31
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
32
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53
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
54
Chapter 2
55
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
56
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
57
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
58
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
59
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
60
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
61
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 - Floating - Submerged
% cover % cover % 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
62
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
63
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
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64
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
65
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
66
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
67
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
68
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
69
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
70
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
71
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
72
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
73
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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77
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)
78
Chapter 3
79
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
80
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
81
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).
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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
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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).
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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
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(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
Species # Clutches collected
Collection site Coordinates Date of
collection
Start of experiment
t
End of experiment
Pelophylax perezi
11 Grândola (stream) 38º07’N, 8º34’W 16-Jun 02-Jul 09-Nov
Hyla meridionalis
9 Grândola (temporary
ponds) 38º06’N, 8º34’W 13-Mar 08-Apr 15-Aug
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
Bufo calamita 9 Grândola (temporary/
ephemeral ponds) 38º05’N, 8º35’W 22-Apr 19-May 09-Aug
Pelodytes ibericus
10 Grândola (temporary
ponds) 38º06’N, 8º33’W 26-Dec 23-Jan 22-Jun
Pelobates cultripes
8 Grândola (temporary
ponds) 38º09’N, 8º31’W 28-Nov 15-Dec 05-Sep
Discoglossus galganoi
9 Grândola (ephemeral
ponds) 38º06’N, 8º34’W 08-Jan 24-Jan 13-May
Alytes cisternasii
- Grândola (stream) 38º05’N, 8º33’W 04-Dec 15-Dec 28-Jul
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
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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.
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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
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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
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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
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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).
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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,
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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
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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 χ
2 P Wald χ
2 P Wald χ
2 P Wald χ
2 P Wald χ
2 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
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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 * * *
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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
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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
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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
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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
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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
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100
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).
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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
Chapter 3
102
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
Chapter 3
103
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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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
Chapter 4
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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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117
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
Species Nr. clutches
collected Date of
collection Start of
experiment Morphology registered
Gosner stage
End of 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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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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119
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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120
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).
Chapter 4
121
-0.04
-0.02
0.00
0.02
0.04
No predator Crayfish Dragonfly
RW
1 =
39.5
5%
Pelobates cultripes
-0.04
-0.02
0.00
0.02
0.04
No predator Crayfish Dragonfly
RW
1 =
62.5
7%
Pelodytes ibericus
-0.04
-0.02
0.00
0.02
0.04
0.06
No predator Crayfish Dragonfly
RW
1 =
80.1
1%
Alytes cisternasii
-0.08
-0.04
0.00
0.04
0.08
0.12
No predator Crayfish Dragonfly
RW
1 =
88.8
0%
Discoglossus galganoi
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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122
-0.02
-0.01
0.00
0.01
0.02
0.03
No predator Crayfish Dragonfly
RW
1 =
42.7
6%
Hyla meridionalis
-0.04
-0.02
0.00
0.02
0.04
No predator Crayfish Dragonfly
RW
1 =
42.5
7%
Pelophylax perezi
-0.02
-0.01
0.00
0.01
0.02
0.03
No predator Crayfish Dragonfly
RW
1 =
37.9
4%
Hyla arborea
-0.03
-0.02
-0.01
0.01
0.02
No predator Crayfish Dragonfly
RW
1 =
46.1
6%
Bufo bufo
-0.02
-0.01
0.00
0.01
0.02
No predator Crayfish Dragonfly
RW
1 =
44.5
8%
Bufo calamita
Fig. 1 Extended
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123
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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124
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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125
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 - -
Chapter 4
126
a)
b)
c)
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.
Chapter 4
127
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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128
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
A. cisternasii N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. r = -0.687 P = 0.028
N.S. N.S.
D. galganoi r = 0.882 P = 0.001
N.S. N.S. N.S. r = -0.787 P = 0.007
N.S. r = -0.639 P = 0.047
N.S. N.S. N.S. N.S. N.S.
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.
P. ibericus N.S. N.S. r = 0.741 P = 0.014
N.S. r = 0.853 P = 0.002
N.S. N.S. N.S. r = -0.820 P = 0.004
N.S. r = -0.889 P = 0.001
N.S.
B. bufo r = -0.907 P < 0.001
r = -0.765 P = 0.010
r = -0.861 P = 0.001
N.S. N.S. N.S. r = 0.849 P = 0.002
r = 0.708 P = 0.022
r = 0.871 P = 0.001
r = 0.728 P = 0.017
N.S. N.S.
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.
H. arborea N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. r = -0.830 P = 0.011
N.S.
H. meridionalis N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. r = -0.700 P = 0.024
N.S. N.S. N.S.
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
Chapter 4
129
Table 4. Extended.
Time met-Mass met Morphol-Behav
Dragonfly Crayfish Dragonfly Crayfish
N.S. r = -0.827 P = 0.003
r = -0.819 P = 0.004
N.S.
N.S. N.S. r = -0.789 P = 0.007
N.S.
N.S. N.S. N.S. N.S.
N.S. N.S. r = -0.637 P = 0.047
N.S.
r = 0.875 P = 0.001
r = 0.803 P = 0.005
r = -0.907 P < 0.001
r = -0.635 P = 0.048
N.S. N.S. N.S. N.S.
N.S. N.S. N.S. N.S.
N.S. N.S. r = -0.887 P = 0.001
N.S.
N.S. N.S. N.S. N.S.
Chapter 4
130
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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131
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
Chapter 4
132
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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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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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
Chapter 4
135
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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136
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
Chapter 4
137
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
12
34
5
6
7
8
9
10
11
12
13
14
15
16
17
18
1920
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
144
145
CHAPTER 5
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
146
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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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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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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
Type of water body
Rice field on the bank of the
Caia river Caia river
Melides stream and rice fields
Monchique river
Affluent of the Seixe river
Coordinates 38º52’N 7º03’W
39º07’N 7º17’W
38º08’N 8º44’W
37º17’N 8º33’W
37º22’N 8º38’W
Elevation a.s.l 170 m 270 m 25 m 200 m 100 m
Approximate coexistence time
32 years 32 years 21 years 0 years 0 years
Date of collection
10-June 16-June 08-July 18-June 18-June
Number of clutches collected
8 10 10 9 12
Start of experiment
02-July 02-July 17-August 04-August 05-August
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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
Chapter 5
155
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
Chapter 5
156
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 χ
2 P Wald χ
2 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).
Chapter 5
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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).
-0.03
-0.02
-0.01
0.00
0.01
0.02
0.03
RW
1 =
39.7
4%
a)
No predator Crayfish
LC1 LC2 SC NC1 NC2
-0.02
-0.01
0.00
0.01
0.02
0.03
RW
2 =
15.0
4%
b)
LC1 LC2 SC NC1 NC2
Population
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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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
Chapter 5
162
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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163
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
Chapter 5
164
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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CHAPTER 6
General Discussion
174
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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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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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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
↑ cage avoidance
↓ activity
↑ cage avoidance -
↑ headbody depth
↑ tail fin depth
↑ tail muscle depth
↑ tail fin insertion
↓ time to metamorphosis
↑ mass at metamorphosis
↑ growth rate -
Discoglossus galganoi ↓ activity
↑ cage avoidance
↓ activity
↑ cage avoidance -
↑ headbody depth
↑ tail fin depth
↑ tail muscle depth
↑ tail fin insertion
-
↑ time to metamorphosis
↓ growth rate
Pelobates cultripes ↓ activity
↑ cage avoidance
↓ activity
-
↑ tail fin depth
↑ tail muscle depth
↑ tail fin insertion
↓ time to metamorphosis
-
Pelodytes ibericus -
↓ activity
↑ cage avoidance -
↑ headbody depth
↑ tail fin depth
↑ tail fin insertion
-
↑ mass at metamorphosis
↑ growth rate
Bufo bufo ↓ activity
↑ cage avoidance
↓ activity
↑ cage avoidance
↑ headbody length
↑ headbody length
↓ time to metamorphosis
↓ mass at metamorphosis
↓ time to metamorphosis
↓ mass at metamorphosis
↓ growth rate
Bufo calamita - - - - - -
Hyla arborea ↓ activity ↓ activity
↑ cage avoidance - ↑ tail fin depth
↑ tail fin insertion
↓ time to metamorphosis
↑ growth rate
↑ growth rate
Hyla meridionalis ↓ activity
↓ activity
↑ cage avoidance -
↑ headbody depth
↑ tail fin depth
↑ tail fin insertion
-
↑ mass at metamorphosis
Pelophylax perezi ↓ activity*
↓ activity
↓ headbody length**
↑ tail muscle depth**
↑ tail fin insertion**
↑ headbody depth
↑ tail fin depth
↑ 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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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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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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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 Defences Survival
Probability of reproducing in a crayfish invaded
habitat
Population resilience
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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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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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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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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191
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