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Behavioural Ecology, Reproductive Biology and Colour Change Physiology in the Stony Creek Frog (Litoria wilcoxii)

Author Kindermann, Christina

Published 2017

Thesis Type Thesis (PhD Doctorate)

School Griffith School of Environment

DOI https://doi.org/10.25904/1912/1098

Downloaded from http://hdl.handle.net/10072/367513

Griffith Research Online https://research-repository.griffith.edu.au

Behavioural ecology, reproductive biology and colour change physiology in the Stony Creek Frog (Litoria wilcoxii)

Christina Kindermann

B. Sc. (Hons)

Griffith University School of Environment

Environmental Futures Research Institute

Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy

July 2016

Abstract

Many animals possess the remarkable ability to change their skin colour. Colour change can have several potential functions, including communication, thermoregulation and camouflage. However, while the physiological mechanisms and functional significance of colour change in other vertebrates have been well studied, the role of colour change in amphibians is still relatively unknown and a disconnection between morphology, physiology and function exists in the literature (review presented in chapter 2).

In this thesis, I investigate these multidisciplinary components to understand the processes and functions of colour change in stony creek frogs (Litoria wilcoxii), which are known to turn bright yellow during the breeding season. By (1 – Chapter 3) examining the distribution and structure of dermal pigment cells, (2– Chapter 4) determining hormonal triggers of rapid colour change, (3– Chapter 5) investigating seasonal colour, hormone and disease relationships and (4– Chapter 6) determining the evolutionary functions of colour change, I provide a comprehensive explanation of this phenomenon in L. wilcoxii.

1) Dorsal skin colour in L. wilcoxii is determined by the arrangement of two types of chromatophore: melanophores and xanthophores. Rapid colour change is the result of pigment dispersion or aggregation in the melanophores which either exposes or covers the yellow xanthophores.

2) This pigment movement is triggered by the neuro-hormone adrenalin (typical of other species exhibiting rapid brightening). Male frogs turned a vivid yellow within 5 minutes following adrenalin injection and remained so for 3 to 5 hours before rapidly fading back to brown. This timing followed natural observations of amplexing males. Interestingly, adrenalin injections triggered colour change but not sperm release in male frogs, while Human chorionic gonadotropin (hCG) induced sperm release but not colour change.

3) At a seasonal level, reproductive hormone (testosterone) levels and dorsal colour score (yellowness) increased during breeding months whilst stress hormone (corticosterone) levels remained stable. Infection by Bd (Batrachochytrium dendrobatidis, the pathogen that induces chytridiomycosis) was associated with increased corticosterone and decreased testosterone, however did not appear to be influencing colour expression.

4) Behavioural experiments using model frogs ruled out female preference, and male-male competition was rarely observed, however male vocalisations and movement increased at the sight of a female and model female. Predation trials found no significant difference in attack rates between yellow and brown models exposed to natural field conditions.

Overall, this thesis demonstrates both seasonal and rapid dynamic colour changes in male Litoria wilcoxii. Rapid colour change is under neuro-hormonal control and functions in intersexual signal during amplexus. It is likely that seasonal increases in yellow colouration are related to reproductive hormone cycles. The physiological stress response associated with Bd infection could potentially suppress physiological aspects of reproduction, however more research is needed. Colour functions as an intrasexual signal following amplexus that could avert sperm competition and displacement by other males during amplexus, presenting a novel function for rapid dynamic colour change in amphibians. My research expands our understanding of the mechanisms, processes and potential functions of rapid colour change in dichromatic amphibians.

Declaration

This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

All research procedures reported in this thesis were undertaken under the Queensland Department of Environment and Heritage Protection (DEHP) research permit WISP13675913 and the Griffith University Animal Ethics Committee (AEC) permit #ENV/20/12/AEC.

______(Date) ______

Christina Kindermann

iii Acknowledgements

A number of people need to be thanked for helping me with the completion of my PhD. First and foremost, I would like to thank my family for always supporting me and believing that I can do anything I want. To my parents, Sonja and Andy, words cannot describe how much you have supported me, having a bed ready for me whenever I needed to escape city life made many parts of this journey easier. To my partner Basam, even though this doesn’t come close to what you mean to me all I can say is that I love you and I can’t wait for more adventures with you.

Thanks to my supervisor Jean-Marc Hero for sharing your fascination with amphibians and your passion for conservation. Most of all thanks for pushing me to publish my work, attend conferences, tutor field courses and assist in various other projects beyond the scope of my research. These experiences have been invaluable and have allowed me to develop as a scientist. Thanks also to Edward Narayan for taking the time to teach me in the lab and for your helpful comments on my papers. And thank you Guy Castley, for coming on board in the last minute and helping me get to the end.

There was a large amount of fieldwork involved in this project that would not have been possible without the help of numerous volunteers. I would especially like to thank Daniel Stellmacher, Kristian Owen, Corey Newell, Trish Hall, Sonia Marsonic, Tahlie Page, Billy Ross, Kat Lowe, Mariel Familer Lopez, Basam Tabet and many others who have up their free time to help me out. Thanks also to my wonderful office mate for the laughs, the support and sometimes needed distractions.

Much of this study was funded through a postgraduate research scholarship; Jean-Marc Hero provided the remaining funds. The histology component of this study would not have been possible without the facilities and training provided by The University of Queensland Histology Facility, thanks also to Andrew Weeks and Anthony Van Rooyen at Cesar for processing the Chytrid samples. I would also like to thank the journal editors and anonymous reviewers for providing constructive feedback on my submitted papers.

Finally, I would like to thank my frogs for giving me an insight into their fascinating world. It has been such a rewarding experience to be able to search for an answer to the questions no one really knew. The sleepless nights I spent along the rocky creek edges were definitely worth it.

Acknowledgement of co-authored papers included in this Thesis

Included in this thesis are four published papers (Chapters 3, 4, 5, and 6), one in review (Chapter 2) which are co-authored with my supervisors and one short communications (Appendix 1) of which I am the sole author. My contribution to each paper, how it relates to the overall thesis aim and bibliographic details are outlined at the front of the relevant chapter. All published papers are presented in their published format and papers in review are formatted according to journal specifications therefore spelling and formatting will vary depending on journal origins and specifications. The bibliographic details and status for these papers are:

 Kindermann C and Hero J-M (in review) Physiology, function and the ecological drivers of colour change in amphibians. Biological Journal of the Linnean Society.  Kindermann C, Hero J-M (2016) Pigment cell distribution in a rapid colour changing amphibian (Litoria wilcoxii). Zoomorphology 135 (2), 197-203, doi:10.1007/s00435- 016-0303-1  Kindermann C, Narayan E J, Hero J-M (2014) The Neuro-Hormonal Control of Rapid Dynamic Skin Colour Change in an Amphibian during Amplexus. PloS one 9 (12), e114120, doi: 10.1371/journal.pone.0114120  Kindermann C, Narayan E J, Hero J-M (2016) Does physiological response to disease incur cost to reproductive ecology in a sexually dichromatic amphibian species? Comparative Biochemistry and Physiology: Part A: Molecular & Integrative Physiology 203,220-226, doi: 10.1016/j.cbpa.2016.09.019  Kindermann C and Hero J-M (2016) Rapid dynamic colour change is an intrasexual signal in a lek breeding frog (Litoria wilcoxii). Behavioral Ecology and Sociobiology 70 (20), 1995-2003, doi: 10.1007/s00265-016-2220-1  Kindermann C. 2015. Litoria wilcoxii (Stony Creek Frog). Interspecific amplexus. Herpetological Review 46 (2): 235

The copyright status of the published papers is held by the relevant journals.

Co-authors contributed to these papers by providing (1) Guidance of experimental design; (2) laboratory and technical support; and (3) advice and comments of written material. Appropriate acknowledgements of those who contributed to the research but did not qualify as authors are included in the acknowledgments section of each paper.

v Supervisor: Jean-Marc Hero

vi “It seems to me that the natural world is the greatest source of excitement; the greatest source of visual beauty; the greatest source of intellectual interest. It is the greatest source of so much in life that makes life worth living.”

Sir David Attenborough

vii Table of Contents

Abstract ...... i Declaration ...... iii Acknowledgements ...... iv Acknowledgement of co-authored papers included in this Thesis ...... v Table of Contents ...... viii Chapter 1: General Introduction ...... 1 Chapter 2: Physiology, function and the ecological drivers of colour change in amphibians . 10 Chapter 3: Pigment cell distribution in a rapid colour changing amphibian (Litoria wilcoxii) ...... 36 Chapter 4: The Neuro-Hormonal Control of Rapid Dynamic Skin Colour Change in an Amphibian during amplexus ...... 44 Chapter 5: Does physiological response to disease incur cost to reproductive ecology in a sexually dichromatic amphibian species? ...... 56 Chapter 6: Rapid dynamic colour change is an intrasexual signal in a lek breeding frog (Litoria wilcoxii) ...... 64 Chapter 7: General conclusion ...... 74 Appendix 1: Litoria wilcoxii (Stony Creek Frog) Interspecific amplexus ...... 81 Appendix 2: Supporting information for chapter 2...... 83 Appendix 3: Supplementary data for chapter 4 ...... 110 Appendix 4: Chapter 6 supplementary information...... 121

viii Chapter 1: General Introduction

1.1 Thesis Background

Animal colouration plays a key role in communication and adaptation. And both natural and sexual selection have shaped its evolution (Chen et al., 2012). Skin colour may be controlled by genetics, endocrine and external environmental factors (Bagnara & Hadley, 1973) and is an important aspect of courtship, communication and displays of dominance in many species. Colour can be produced through pigments which absorb light of different wavelengths, through a structural system where colour is created through scattering of light, or through bioluminescence, whereby organisms produce light themselves (Booth, 1990).

In amphibians, skin colour is usually determined by three cell types (melanophores, xanthophores and iridophores) which are collectively called ‘the dermal chromatophore unit’ (Bagnara & Hadley, 1973; Bagnara et al., 1968). These cells can vary structurally depending on the location in the skin layer, age, and physiological state. Whilst differences exist among vertebrate groups and species, most exhibit some variation of this structural arrangement (Alibardi, 2012; Bagnara et al., 1968; Kuriyama et al., 2006). Colour change results from movement of pigment (physiological colour change) within these cells or a change in cell structure (morphological colour change) which alters the way light is reflected and therefore colour perceived (Booth, 1990).

The speed of colour change can vary greatly from slow unidirectional changes (ontogenetic colour change), most commonly from juvenile to adult life stages to rapid temporary changes that can occur in just a few minutes (Booth, 1990; Wente & Phillips, 2005). In some chameleons and fish, rapid changes occur in just seconds (Okelo, 1986). For example, the tropical flatfish (Bolthus ocellatus) can blend to a variety of backgrounds in less than eight seconds (Ramachandran et al., 1996). In amphibians colour adaptation for camouflage generally takes several hours (Hadley & Quevedo, 1967) and in some species (such as male moor frogs, Rana arvalis) it takes days or weeks for breeding colouration to develop (Ries et al., 2008). Faster changes during the breeding events have been documented in several amphibians including the stony creek frog (Litoria wilcoxii) and the yellow Neotropical toad (Amietophrynus lemairii) which can change colour within minutes (Bittencourt-Silva, 2014; Kindermann et al., 2013).

Colour change in amphibians is predominantly driven by different types of hormones. Melanocyte stimulating hormone (MSH) controls the darkening and lightening of skin colour through dispersing or aggregating melanin in the melanophores (Aspengren et al., 2009; Fernandez & Bagnara, 1991). The type of stimulus (e.g. hormones or neuro-hormones) and cell structure (presence and location of chromophore types) influence the pattern (ontogenetic or dynamic) and speed of colour change (Sköld et al., 2013). These systems are highly complex and species (particularly amphibians) can have differing responses (darkening or lightening) to certain hormones (Nielsen, 1978; Salim & Ali, 2011). External factors such as light and temperature also play a role in physiological colour change as they influence some of the hormones which trigger colour change. Again, the degree and type of effects vary among species (Filadelfi et al., 2005; King et al., 1994; Sköld et al., 2008; Tattersall et al., 2006). Light can also directly regulate colour change by stimulating receptors on chromophores, rather than stimulating the brain first (Oshima, 2001). This is important for ectothermic species as colour change is an important part of maintaining homeostasis (Norris, 1967).

The adaptive functions of colouration and colour change are complex. Natural and sexual selection for traits associated with three main functions appear to be most common. 1) predator avoidance through camouflage (Booth, 1990; Wente & Phillips, 2005), 2) communication (Stuart-Fox & Moussalli, 2009; Sztatecsny et al., 2012; Sztatecsny et al., 2010) and, 3) thermoregulation (King et al., 1994; Tattersall et al., 2006; Withers, 1995). Determining the basic function can be simple; e.g. if colour change is a function for camouflage an organism would match it background whereas colour change driven by sexual selection would see the organisms stand out more (at least in the breeding season) for mate selection. More specific reasons particularly in relation to dynamic colour change and communication are more complex and require detailed behavioural studies.

Studies of colour change can help give a greater understanding of evolutionary processes, especially those driving multimodal communication and animal behaviour. Studies have shown that colour polymorphisms in frogs have been linked to reduced extinction risks at species level (Forsman & Hagman, 2009) this highlights the importance of colour as an evolutionary driver. The study of physiological aspects of colour change also allows researchers to explore cell and whole organism interactions in a visual way. For example, amphibian and fish melanophores have been used as models for a general understanding of intracellular transport and organelle positioning (Svensson et al., 2005). This includes the

development of melanophores as biosensors and skin pigmentation in ecotoxicology studies (Sköld et al., 2013).

My study is motivated by the question: Why and how do frogs change colour? The work presented herein focuses on the stony creek frog, Litoria wilcoxii, that exhibit rapid reversible colour change during breeding (brown to yellow).

1. 2 Introduction to study species

The Stony Creek Frog (Litoria wilcoxii) is a common species of Hylidae commonly found along rocky creek sections across eastern Australia. Surrounding habitat is commonly rainforest or wet sclerophyll forest. This species (along with Litoria jungguy) was separated from Litoria lesueuri based on genetic evidence, thigh colour and distribution. The ranges of L. wilcoxii and L. jungguy overlap in north Queensland and they cannot be distinguished without DNA testing (Donnellan & Mahony, 2004).

Litoria wilcoxii are sexually dimorphic (males 45mm, females 85mm) and sexually dichromatic. Both sexes can be cream to dark brown above with white granular bellies, often with darker mottling; the inside of their thighs are light yellow (often brighter yellow in males) with black speckling (Vanderduys, 2012). Males display bright yellow dorsal colouration at night during the breeding season, especially when in amplexus. Males lack a vocal sac and therefore have a soft purr like call, lasting a few seconds (Vanderduys, 2012). Amplexus lasts several hours (Kindermann pers obs.) during which female’s lay over 1000 eggs that are attached to rocks or debris in still stream sections or pools (Anstis, 2013). This species is a prolonged breeder; males call from late winter to autumn and are most active on hot dry summer nights (Kindermann pers. obs.). During the breeding season (September to February) males form small leks and call from rocky or sandy stream banks close to the water (Anstis, 2013; Wells, 2007). Males greatly outnumber females in breeding aggregations; often females are only sighted along stream edges whilst in amplexus, but are commonly seen much further away from the stream in forested areas (Vanderduys, 2012). This lack of females in breeding aggregations may explain why males have often been observed in amplexus with different species similar in appearance to females, including Litoria nannotis (Anstis, 2013), Mixophyes iteratus and Rhinella marina (Kindermann pers. obs.).

Litoria wilcoxii are often said to be a reservoir host to the chytrid fungus Batrachochytrium dendrobatidis (Bd) as populations show no signs of decline despite having 28% prevalence of

chytrid infection (Kriger & Hero, 2007). Infections are highly seasonal with increases in infection rates occurring during spring (the start of the breeding season). Recently it has been suggested that L. lesueurii be just as susceptible to Bd infection but are not declining due to a higher fecundity then non-declining species (West et al., 2015). Previous studies on several species (including L. wilcoxii) have demonstrated that individuals infected with Bd have higher corticosterone (stress) levels compared to frogs that tested negative (Gabor et al., 2013; Kindermann et al., 2012; Peterson et al., 2013). These studies demonstrate negative sub-clinical effects of Bd on reservoir host species.

What is most unique about this species is their remarkable ability to rapidly change colour. This colour change, from brown to yellow (and back to brown) occurs in just a few minutes (Kindermann et al., 2012). Whilst dynamic colour change for social signalling (especially for breeding colouration) is common in amphibians, most changes seem to occur at a seasonal level. In addition, in many species that may exhibit rapid colour change, accurate details on timing are missing (Bell & Zamudio, 2012). Understanding the colour change mechanisms and processes in L. wilcoxii is therefore an important step in amphibian colour change research.

1. 3 Aims and structure of the thesis

The overall aim of this thesis was to determine the functional significance of colour change in L. wilcoxii by investigating the multidisciplinary components of dynamic colour change: morphology, physiology and function (evolutionary purpose). Specifically I will aim to investigate (1) the marked difference between ontogenetic, seasonal and dynamic colour change in amphibians and the hypotheses used to explain the evolutionary functions of these colour changes, (2) the cellular mechanisms and structure that enable L. wilcoxii to change colour, (3) how rapid dynamic colour change in L. wilcoxii is regulated (the hormones that trigger pigment movement), (4) seasonal colour and stress and reproductive hormone cycles and the possible influences of disease on reproductive physiology and secondary reproductive displays such as colour expression, and (5) the evolutionary function of rapid dynamic colour change in L. wilcoxii. Developing this conceptual framework for one species will allow future research to apply similar methods to investigate the phenomenon of dynamic colour change in other amphibians and ectotherms.

The first aim was achieved through a literature review (presented in chapter 2), which allowed me to identify key questions relating to colour change and sexual selection. I focused

on how colour change patterns are associated with amphibian breeding systems (Wells, 1977) to gain a greater understanding of the function of sexual dichromatism in amphibians. Aims 2-5 are presented in four studies which are reported in separate chapters (Chapters 3 to 6).

To understand fully how L. wilcoxii can rapidly change colour I investigated morphology (chapter 3) and physiology (chapter 4). The basic morphology of the dermal chromatophore unit is well known, however differences have been observed between species. In chapter 3 I used light microscopy to investigate differences in pigment cell structure between brown and yellow males and permanent and colour changing skin sections. This allowed an accurate comparison in basic morphology between L. wilcoxii and other colour changing amphibians.

To gain an understanding of the natural timing of colour change I observed pairs prior to and during amplexus, using digital photography to document colour change (chapter 4). Following this, I conducted manipulations where male frogs were treated with epinephrine and testosterone to determine if this rapid colour response was associated with neuro- hormone or reproductive hormone processes.

In chapter 5 I extended the physiology component of the study further and examined seasonal relationships among the stress hormone corticosterone, the reproductive hormone testosterone, reproductive colour displays (yellowness) and disease (Bd) in individual males. This allowed me to determine if Bd has an impact on hormone balance and reproductive colouration for L. wilcoxii. Determining if the physiological mechanisms that regulate breeding (reproductive hormones and dorsal colouration) are impacted by Bd infection will give a deeper insight to how species are being affected by disease on a physiological level. This is especially important to examine in species that are currently not showing declines as it provides insight into the sub-clinical effects of disease on reservoir host species.

Finally, in chapter 6 I investigate the evolutionary function of colour change in L. wilcoxii. In this chapter I investigate processes of sexual selection (female choice, male competition and intra-sexual signalling) to test the function of dynamic colour change. Furthermore I explore the hypothesis that colour change may simply be an accidental by-product of spermiation. And determine the costs (if any) of this dynamic colour change to male L. wilcoxii.

Following these chapters, the general conclusion (chapter 7) includes a summary of the findings reported in this thesis and describes future directions and implications of colour change research.

References

Alibardi, L. 2012. Cytology and localization of chromatophores in the skin of the Tuatara (Sphenodon punctaus). Acta Zoologica, 93(3), 330-337. Anstis, M. 2013. Tadpoles and frogs of Australia. New Holland publishers, Australia. Aspengren, S., Sköld, H., Wallin, M. 2009. Different strategies for color change. Cellular and Molecular Life Sciences, 66(2), 187-191. Bagnara, J.T., Hadley, M.E. 1973. Chromatophores and color change: the comparative physiology of animal pigmentation. Pearson Education, Limited, Univeristy of Michigan, USA. Bagnara, J.T., Taylor, J.D., Hadley, M.E. 1968. The Dermal Chromatophore Unit. The Journal of Cell Biology, 38(1), 67-79. Bell, R.C., Zamudio, K.R. 2012. Sexual dichromatism in frogs: natural selection, sexual selection and unexpected diversity. Proceedings of the Royal Society of London B: Biological Sciences, 283(1834), rspb20121609. Bittencourt-Silva, G. 2014. Notes on the reproductive behaviour of Amietophrynus lemairii (Boulenger, 1901)(Anura: Bufonidae). Herpetology Notes, 7, 611-614. Booth, C.L. 1990. Evolutionary significance of ontogenetic colour change in animals. Biological Journal of the Linnean Society, 40(2), 125-163. Chen, I., Stuart‐Fox, D., Hugall, A.F., Symonds, M.R. 2012. Sexual selection and the evolution of complex color patterns in dragon lizards. Evolution, 66(11), 3605-3614. Donnellan, S., Mahony, M. 2004. Allozyme, chromosomal and morphological variability in the Litoria lesueuri species group (Anura: Hylidae), including a description of a new species. Australian Journal of Zoology, 52(1), 1-28. Fernandez, P.J., Bagnara, J.T. 1991. Effect of background color and low temperature on skin color and circulating α-MSH in two species of leopard frog. General and Comparative Endocrinology, 83(1), 132-141. Filadelfi, A.M.C., Vieira, A., Louzada, F.M. 2005. Circadian rhythm of physiological color change in the amphibian Bufo ictericus under different photoperiods. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 142(3), 370-375. Forsman, A., Hagman, M. 2009. Association of coloration mode with population declines and endangerment in Australian frogs. Conservation Biology, 23(6), 1535-1543.

Gabor, C.R., Fisher, M.C., Bosch, J. 2013. A non-invasive stress assay shows that tadpole populations infected with Batrachochytrium dendrobatidis have elevated corticosterone levels. PloS one, 8(2), e56054. Hadley, M., Quevedo, W. 1967. The role of epidermal melanocytes in adaptive color changes in amphibians. Advances in biology of skin, 8, 337-359. Kindermann, C., Narayan, E.J., Hero, J.-M. 2014. The Neuro-Hormonal Control of Rapid Dynamic Skin Colour Change in an Amphibian during Amplexus. PloS one, 9(12), e114120. Kindermann, C., Narayan, E.J., Hero, J.-M. 2012. Urinary corticosterone metabolites and chytridiomycosis disease prevalence in a free-living population of male Stony Creek frogs (Litoria wilcoxii). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 162(3), 171-176. Kindermann, C., Narayan, E.J., Wild, F., Wild, C.H., Hero, J.-M. 2013. The effect of stress and stress hormones on dynamic colour-change in a sexually dichromatic Australian frog. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 165(2), 223-227. King, R.B., Hauff, S., Phillips, J.B. 1994. Physiological color change in the green treefrog: responses to background brightness and temperature. Copeia, 1994(2), 422-432. Kriger, K.M., Hero, J.M. 2007. Large‐scale seasonal variation in the prevalence and severity of chytridiomycosis. Journal of Zoology, 271(3), 352-359. Kuriyama, T., Miyaji, K., Sugimoto, M., Hasegawa, M. 2006. Ultrastructure of the dermal chromatophores in a lizard (Scincidae: Plestiodon latiscutatus) with conspicuous body and tail coloration. Zoological Science, 23(9), 793-799. Nielsen, H.I. 1978. The effect of stress and adrenaline on the color of Hyla cinerea and Hyla arborea. General and Comparative Endocrinology, 36(4), 543-552. Norris, K.S. 1967. Color Adaptation in Desert Reptiles and its Thermal Relationships. in: Lizard Ecology - a Symposium, (Ed.) W.W. Milstead, Vol. W. W. Milstead: . University of Missouri Press. Columbia, Missouri,USA, pp. 162-229. Okelo, O. 1986. Neuroendocrine control of physiological color change in Chameleo gracilis. General and Comparative Endocrinology, 64(2), 305-311. Oshima, N. 2001. Direct Reception of Light by Chromatophores of Lower Vertebrates. Pigment Cell Research, 14(5), 312-319. Peterson, J.D., Steffen, J.E., Reinert, L.K., Cobine, P.A., Appel, A., Rollins-Smith, L., Mendonça, M.T. 2013. Host stress response is important for the pathogenesis of the 7

deadly amphibian disease, chytridiomycosis, in Litoria caerulea. PloS One, 8(4), e62146. Ramachandran, V.S., Tyler, C.W., Gregory, R.L., Rogers-Ramachandran, D., Duensing, S., Pillsbury, C., Ramachandran, C. 1996. Rapid adaptive camouflage in tropical flounders. Nature, 379(6568), 815-8. Ries, C., Spaethe, J., Sztatecsny, M., Strondl, C., Hödl, W. 2008. Turning blue and ultraviolet: sex-specific colour change during the mating season in the Balkan moor frog. Journal of Zoology, 276(3), 229–236. . Salim, S., Ali, S. 2011. Vertebrate melanophores as potential model for drug discovery and development: a review. Cellular and Molecular Biology Letters, 16(1), 162-200. Sköld, H.N., Amundsen, T., Svensson, P.A., Mayer, I., Bjelvenmark, J., Forsgren, E. 2008. Hormonal regulation of female nuptial coloration in a fish. Hormones and Behavior, 54(4), 549-556. Sköld, H.N., Aspengren, S., Wallin, M. 2013. Rapid color change in fish and amphibians– function, regulation, and emerging applications. Pigment Cell & Melanoma Research, 26(1), 29-38. Stuart-Fox, D., Moussalli, A. 2009. Camouflage, communication and thermoregulation: lessons from colour changing organisms. Philosophical Transactions of the Royal Society B: Biological Sciences, 364(1516), 463-470. Svensson, P.A., Forsgren, E., Amundsen, T., Sköld, H.N. 2005. Chromatic interaction between egg pigmentation and skin chromatophores in the nuptial coloration of female two-spotted gobies. Journal of Experimental Biology, 208(23), 4391-4397. Sztatecsny, M., Preininger, D., Freudmann, A., Loretto, M.-C., Maier, F., Hödl, W. 2012. Don’t get the blues: conspicuous nuptial coloration of male moor frogs (Rana arvalis) supports visual mate recognition during scramble competition in large breeding aggregations. Behavioral Ecology and Sociobiology, 66(12), 1587-1593. Sztatecsny, M., Strondl, C., Baierl, A., Ries, C., Hödl, W. 2010. Chin up: are the bright throats of male common frogs a condition-independent visual cue? Animal Behaviour, 79(4), 779-786. Tattersall, G.J., Eterovick, P.C., de Andrade, D.V. 2006. Tribute to RG Boutilier: skin colour and body temperature changes in basking Bokermannohyla alvarengai (Bokermann 1956). Journal of Experimental Biology, 209(7), 1185-1196. Vanderduys, E. 2012. Field Guide to the Frogs of Queensland. CSIRO Publishing, Australia.

Wente, W.H., Phillips, J.B. 2005. Seasonal color change in a population of pacific treefrogs (Pseudacris regilla). Journal of Herpetology, 39(1), 161-165. West, M., Gillespie, G., McCarthy, M. 2015. Quantifying the demographic impact of Chytrid fungus on amphibian decline. The Australian Society of Herpetologists Annual meeting, Eilston, Victoria. Withers, P. 1995. Evaporative water loss and colour change in the Australian desert tree frog Litoria rubella (Amphibia: Hylidae). Records of the Western Australian Museum, 17, 277-282

Chapter 2: Physiology, function and the ecological drivers of colour change in amphibians

Forward: This chapter is a literature review examining current knowledge on the physiology and potential evolutionary functions of amphibian colour change. Relationships between colour change pattern and breeding system in dichromatic species are also examined to provide a deeper understanding of the role of sexual selection on this form of colour change and allow a comparison between other species and the focal species of this thesis (Litoria wilcoxii).

This chapter is a co-authored paper that has been submitted to Biological Journal of the Linnean Society and is under review. Supporting information (referred to within the article) is available for this manuscript in Appendix 2 of the thesis. My contribution to the paper involved: reviewing and analyzing the literature, contracting databases to compare information and writing the manuscript. The bibliographic details of the co-authored paper are:

Kindermann C, Hero J-M (manuscript in review) Physiology, function and the ecological drivers of colour change in amphibians. Biological Journal of the Linnean Society.

______(Date) ______

Student and corresponding author: Christina Kindermann

______(Date) ______

Supervisor and co-author: Jean-Marc Hero

10 Abstract

Amphibians have the incredible ability to change colour, yet the hormonal drivers and evolutionary functions are poorly known in most species. Herein we review the variations in timing, physiology and function of colour change in amphibians. Additionally, we investigate how sexual selection may drive colour change in anuran amphibians by examining breeding pattern and reproductive behaviour of sexually dichromatic species and relating these to function.

We identify three patterns of colour change based on speed, hormonal mechanisms and ecological function. 1. Ontogenetic colour change is a unidirectional, permanent change from a juvenile to an adult colour phase. The function of this change is mostly unknown but is driven by processes of natural and sexual selection. 2. Seasonal colour change is a temporary colour change that occurs over weeks or months. It commonly occurs in sexually dichromatic, explosive breeding species, where it functions as an intrasexual signal for sex recognition. 3. Rapid colour change occurs in just minutes or hours and is also reversible. It is commonly used as for camouflage or thermoregulation (78%). In some species (22%) only males change colour and this predominantly occurs in prolonged breeders where it functions as an intrasexual signal.

Future studies using a multi-disciplinary approach are required to understand the biological drivers and functions of colour change to provide a comprehensive understanding of this remarkable phenomenon.

Keywords: Colour change, sexual selection, natural selection, sexual dichromatism, amphibian reproduction, camouflage, thermoregulation, sexual signalling.

11 Introduction

Colour change is widespread in the animal kingdom. It occurs in many species from the well- known fascinating displays seen in chameleons and cephalopods to lesser known ectothermic animals including crustaceans, insects, fish, reptiles and amphibians (Abbott, 1973; Bagnara, Taylor & Hadley, 1968; Stuart-Fox & Moussalli, 2009). In many species colour change is an essential warning sign and camouflage mechanism, allows better capacity for thermoregulation and an integral part of mating and reproductive behaviour (Stuart-Fox & Moussalli, 2009). Colouration in lower vertebrates is produced by the absorption and reflection of light by specialized pigment containing cells called chromatophores (Bagnara, Fernandez & Fujii, 2007). In amphibians skin colour is predominantly based on the number and organisation of three types of chromatophore: melanophores (contain light absorbing pigments), xanthophores (contain bright coloured pigments such as pteridines or carotenoids) and iridophores (contain reflecting platelets). The arrangement, presence and location in the dermis can vary among species (Ali & Naaz, 2014; Bagnara et al., 1968) and the location on the body of the individual (Kindermann & Hero, 2016; Kuriyama, Miyaji, Sugimoto & Hasegawa, 2006).

Colour change is either achieved through a change in pigment cell organisation and number (morphological colour change), or by rapid pigment migration within chromatophores [physiological colour change] (Bagnara & Hadley, 1973). The lightening and darkening seen in many amphibians is a physiological colour change in which colour change is the result of pigment movement from the centre of the melanophore to the cell periphery where it covers the iridophores and/or xanthophores, increasing light absorption (Bagnara, 1964). A change in colour from a juvenile to adult colour phase is usually the product of morphological colour change as it involves the destruction and creation of new pigment or chromatophore types (Booth, 1990).

In amphibians colour change is mainly controlled by hormones or neuro-hormones in response to external triggers such as temperature, light intensity and behavioural cues. The types of hormones that control pigment movement have been well studied (Nery & Castrucci, 1997; Sköld, Aspengren & Wallin, 2013). Determining these physiological processes and causal mechanisms provides a conceptual framework for evolutionary studies, and therefore physiological processes were included where possible in this review. Physiological mechanisms are subject to selection just as much as the phenotypic expression and function,

and one cannot fully understand one without the other (Hofmann, Beery, Blumstein, Couzin, Earley, Hayes, Hurd, Lacey, Phelps, Solomon, Taborsky, Young & Rubenstein, 2014; Tinbergen, 1963). Although there is increasing knowledge in both physiology and function of colour change in amphibians a disconnection between them still exists, with studies often studying one component without relating it to the other (Bittencourt-Silva, 2014; Doucet & Mennill, 2010; Nielsen, 1978b).

Herein we examine the physiological structure and hormonal mechanisms of colour change, current knowledge on variations in colour change patterns (speed and duration), and review the ecological and evolutionary drivers of colour change in amphibians. Our literature search spanned across disciplines and includes past reviews, experimental studies, natural history notes, field guides and accounts from other researchers. We firstly compiled a list of all amphibians (anurans and urodela, excluding tadpoles) known to undergo colour change and where possible identified the duration, function and possible physiological process. We identified 282 species that matched our criteria; however, this number is likely to be an underestimate as many mentions of colour change may be in short natural history notes or anecdotal comments which are difficult to pick up in literature searches. For sexually dichromatic species (where colour is thought to be controlled by sexual selection) we examined the relationships between breeding system (according to Wells 1977) and colour change pattern to further elucidate the potential evolutionary function. Our study confirms the high diversity of physiological processes and function that exists in amphibians and highlights disconnection between physiological and evolutionary or behavioural studies; in particular those changes driven by sexual selection.

1. Patterns of colour change

The timing of colour change in amphibians can vary greatly from slow unidirectional changes which occur throughout the life of an individual, usually a juvenile to adult colour phase (Booth, 1990) to reversible changes that can take a few hours or minutes (Doucet & Mennill, 2010). In many descriptions of colour changing occurrences exact timings are left out, descriptions of ontogenetic colour changes usually just mention differences in juvenile and adult colouration (n=130 species) or in the case of reversible colour changes only seasonal (n=56 species) or diurnal (n=48 species) changes are reported. Other studies just use general observations such as ‘hours’ without including exact time measurements (n=13 species).

Specific timings from experimental studies have been documented in approximately 32 species (supplementary data tables 1 and 2).

Herein we distinguish between three forms of colour change, one unidirectional (ontogenetic) and two reversible changes (seasonal and rapid). It is important to note that individual species can exhibit more than one form of colour change driven by different functions. Ontogenetic colour change (OCC) is a unidirectional change in colouration usually from a juvenile colour phase to an adult colour phase. OCC is a form of morphological colour change, whereby the change in skin colour is a result of pigment synthesis and/or change in chromophore distribution through the dermis (Booth, 1990). OCC has been documented to occur in approximately 132 species (table 1) with roughly 50% displaying sexual dichromatism. OCC is common in species in the family Hyperoliidae where females and some males often develop different colours to juveniles (Richards, 1976). Seasonal colour change (SCC) has been recorded in at least 62 species (table 1). It is reversible and typically occurs during the breeding season where the male changes colour (82% brighten and 11% darken). In the remaining 7% seasonal variations in colour occur in both sexes. Due to the slower (days to months) development of pigment colouration, SCC is likely to be a form of morphological colour change (Rehberg-Besler, Mennill & Doucet, 2015). Rapid colour change (RCC) is another form of reversible colour change, lasting from minutes to several hours. Over 121 species (123 occurrences) have been recorded (table 1); due to the ephemeral nature of this form of colour change this number is likely to be an underestimate, in particular more subtle lightening or darkening of skin tone which most amphibians are assumed to be capable of (Bagnara & Hadley, 1973). Rapid colour change is a physiological change, where the movement of pigment within chromophores (predominantly the melanophores) creates colour change (Jørgensen & Larsen, 1960; Nielsen, 1978b).

The degree individual species change colour can range from differences in shade (like the light to dark green in Hyla crucifer or H. japonica) to vivid dichromatic changes in colour (like in the brown to yellow in Litoria wilcoxii) depending on the evolutionary function (Choi & Jang, 2014; Kats & Vandragt, 1986; Kindermann, Narayan & Hero, 2014). Males change colour in 22% of individuals (19% brighten and 3% darken). However, it is most common (78%) for both sexes to undergo colour change (both lighten and darken).

Table 1: Summary of colour changes associated for ontogenetic, seasonal and rapid colour change, showing the number of species documented to display each form of colour change and percentage of this for each pattern of colour change; note that some species exhibit more than one type of colour change or variation of change.

Both Both Both Male Male Female Female sexes sexes sexes Pattern of colour brightens darkens brightens darkens brighten darken change change #species #species #species #species #species #species #species (%) (%) (%) (%) (%) (%) (%)

Ontogenetic (n=132) 20 (15%) 3 (3%) 34 (26%) 16 (12%) 31 (23%) 28 (21%) 0 (0%)

Seasonal (n=62) 51 (82%) 7 (11%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 4 (7%)

Rapid (n=121) 23 (19%) 4 (3%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 96 (78%)

2. Physiology of colour change

Colour change in amphibians is predominantly driven by hormones and is regulated by the pars intermedia of the brain (Aspengren, Skold & Wallin, 2009). The hormonal drivers of colour change have been well researched in some species, mainly using in vitro studies such as skin bioassays (Nery & Castrucci, 1997) without much reference to the natural pattern or function of colour change. Serotonin (5HT) receptors have been found to regulate physiological colour change in the Indian bullfrog Hoplobatrachus tigerinus (Ali, Salim, Sahni, Peter & Ali, 2012), however whether this is related to the dynamic breeding colouration seen in males (Tabassum, Muhammad, Anwar, Mehmood, Hussain & Shahid, 2011) has not been determined. Experimental studies on whole organisms have primarily focused on background matching (Choi & Jang, 2014; Kats & van Dragt, 1986; King, Hauff & Phillips, 1994) and only more recently on other functions of colour change (Kindermann et al., 2014; Tang, Lue, Tsai, Yu, Thiyagarajan, Lee, Huang & Weng, 2014).

The most common and therefore most researched (n=9 species) hormone identified to trigger colour change is melanocyte stimulating hormone (MSH). MSH causes skin darkening by stimulating the dispersal of melanin into the fingers of the melanophores which cover the other cell types (Bagnara, 1964). Numerous other factors can inhibit or stimulate the release of melanin including other hormones, light and temperature (Roubos, Van Wijk, Kozicz, Scheenen & Jenks, 2010). Such factors are often associated with the speed and function of colour change. For example, the circadian hormone melatonin has been shown to regulate day/night colouration (Filadelfi, Vieira & Louzada, 2005) whilst catecholamine’s trigger more rapid changes (Novales & Davis, 1969). Direct effects of light and/or temperature on

colour change (without investigation of any associated hormonal triggers) have been recorded in at least 26 species (supplementary tables 1 and 2). We suspect that such responses are present in many other species where colour change is associated with camouflage and thermoregulation.

Steroid hormones (testosterone, oestrogen and progesterone) strongly influence aspects of reproduction such as breeding behaviour and the development of seasonal nuptial colouration (n=2 species) or ontogenetic sexual differences [n=2 species] (Sköld, Amundsen, Svensson, Mayer, Bjelvenmark & Forsgren, 2008; Sköld et al., 2013). In a study using Southern cricket frogs (Acris gryllus), Greenberg demonstrated that female and non-breeding males treated with testosterone pellets assumed breeding male colouration (darkening and development of yellow colouration on the vocal sac) whereas control males and females did not (Greenberg, 1942). Likewise, juvenile reed frogs Hyperolius argus and H. viridiflavus (which undergo ontogenetic changes) administered with testosterone and oestradiol assumed male or female colour patterns (Hayes & Menendez, 1999; Richards, 1982).

Although testosterone injections stimulated colour change from brown to yellow in male brown tree frogs (Buergeria robusta), the combination of testosterone, melatonin and prolactin also stimulated pigment migration. More specifically testosterone stimulated the dispersion of yellow pigment in the xanthophores whilst melatonin caused the dark melanophore pigment to aggregate, resulting in the brilliant yellow observed in breeding male (Tang et al., 2014). In this species colour change occurs over days or weeks during breeding, coinciding with this colour change were increases in high values of gonad indexes values and plasma testosterone (Tang 2014 pers. comm.). Early in vitro studies showed that steroid hormones (testosterone and progesterone) can cause melanosome expansion in Rana pipiens within 2 hours of treatment (Himes & Hadley, 1971) although this was not related to nuptial colouration.

A recent field study on the Australian hylid L. wilcoxii demonstrated that changes from brown to yellow after epinephrine (adrenalin) injection occurred within five minutes, the same time colour changes were observed in wild amplexing males (Kindermann et al., 2014). The results of this study provide further evidence of the role of neuro-hormones (epinephrine and nor-epinephrine) as rapid dispersal or aggregating agents demonstrated in early amphibian colour studies (Nielsen, 1978a) and that are well known in other colour changing

ectotherms including reptiles (Greenberg, 1990; Greenberg & Crews, 1990; Okelo, 1986) and fish (Fujii, 2000).

3. Evolutionary functions

Colour change is driven by processes of natural and sexual selection (Korzan, Robison, Zhao & Fernald, 2008; Stuart-Fox & Moussalli, 2009). In species where both sexes change colour (especially those undergoing RCC) processes of natural selection such as camouflage and thermoregulation appear to be the main drivers of colour change (King et al., 1994; Norris & Lowe, 1964; Stegen, Gienger & Sun, 2004). In sexually dichromatic species, where colour change occurs in one sex only (either rapidly or over a breeding season) it is most likely been driven by sexual selection (Bell & Zamudio, 2012; Rehberg-Besler et al., 2015; Sztatecsny, Preininger, Freudmann, Loretto, Maier & Hödl, 2012). Some specific ecological drivers or functions mentioned in the literature thus far include camouflage, thermoregulation (including UV protection), aposematism, mimicry, male competition, sex recognition and female choice (table 2).

3.1 Sexual selection and the association between dichromatism and breeding systems

The two breeding systems (prolonged and explosive) distinguished by Wells (1977) represent two ends of a scale, which ranges from one night breeding events, often seen in temperate environments to species where breeding has been observed throughout the year (Wells, 1977). We identified 145 species to display some form of sexual dichromatism and for these we determined the breeding system and other reproductive behaviours to investigate whether any relationships between colour change pattern (OCC, SCC and RCC) and breeding system (prolonged and explosive) were present. A chi‐squared test showed a significant result (X- squared = 12.4425, df = 2, p= 0.002) suggesting that there is a relationship between breeding system and colour change pattern and that these proportions are significantly different to chance expectation.

We found that sexually dichromatic species displaying ontogenetic and rapid colour change had a greater proportion of prolonged breeding species whereas in species undergoing SCC there was no difference between numbers of explosive or prolonged breeders (figure 1). Prolonged breeders spend a far greater investment into breeding, and female choice or male competition often play a role in mate selection (Bateson, 1983). For species displaying OCC relationships between breeding system and colour change are difficult to determine due to the

lack of data on current functions and breeding system (no breeding information was available for 40% of species). For species capable of RCC, the ability to switch colour on and off could allow signals to be utilised more effectively over a longer period (Wells, 1980). Bright colours could be effectively turned on if a prospective mate or conspecific was in sight and then turned off for predator avoidance or thermoregulatory requirements.

In explosive breeders, breeding is concentrated over a short period where all investment into reproduction takes place. Mate searching and scramble competition are behaviours associated with this breeding system (Wells, 1977), and temporary colour signals (SCC) can often act as a mate recognition tool (Sztatecsny et al., 2012). The advantages of this are likely to outweigh any costs associated (such as predation) due to the large number of individuals in breeding aggregations (Doucet & Mennill, 2010). More research into the function of seasonal colour change in prolonged breeders is needed.

Males developed brighter colouration in 15% of ontogenetic amphibians, which traditionally suggests sexual selection is the driver of colour change (Andersson & Iwasa, 1996). It is likely that female choice and aspects of sex recognition play a role for these types of colour change, but to date only a few studies have shown any conclusive results. A female preference for brighter males was shown in two species Oophaga pumilio and Scaphiopus couchi (Maan & Cummings, 2008; Vásquez & Pfennig, 2007). Because colouration is maintained throughout adulthood it is likely that colour is an indicator of male quality (Martín & López, 2010; Vásquez & Pfennig, 2007).

In many species, particularly those from the family Hyperoliidae, it is the female that most commonly undergoes colour change to become brighter (Schiøtz, 1999). Evidence in other species (especially birds) suggests that the function of this mode of colour change is sex role reversal where the males choose females or female-female competition is displayed (Hanssen, Folstad & Erikstad, 2006; Heinsohn, Legge & Endler, 2005; Murphy, Hernández- Muciño, Osorio-Beristain, Montgomerie & Omland, 2009) however this has not been investigated for amphibians. A more parsimonious explanation (which still requires more investigation) is sexual niche partitioning, where one sex is under greater predation risk (calling males or amplexing pairs); however this is mostly overlooked in amphibians. Difference in resource use or predation risk between life stages is common in amphibians (Lindquist & Hetherington, 1998) and it is therefore likely that sexual niche partitioning is more common than currently reported (Manthey & Grossmann, 1997).

Over 90% of seasonal colour changes occur during the breeding season and involve the male changing colour (see table 1). Numerous studies have shown that anurans have a limited capacity of mate recognition, and individuals (particularly explosive breeders from the family Bufonidae) have been observed in amplexus with numerous things ranging from other species to inanimate objects such as cans and even chunks of mud (Đorđević & Simović, 2014; Mollov, Popgeorgiev, Naumov, Tzankov & Stoyanov, 2010). In large breeding aggregations colour can function as a sex recognition tool which can prevent miss-pairing (a sometimes costly endeavour), whilst still allowing organisms to camouflage during non-breeding times. In a behavioural experiment in 2012 Sztatecsny et al. showed that male moor frogs (Rana arvalis) spent significantly more time amplexing a brown model frog (female colouration) then with a blue model (Sztatecsny et al., 2012). A similar study was repeated with the yellow neotropical toad Incilius luetkenii which showed similar results (Rehberg-Besler et al., 2015), suggesting this trait it appears to be a common function of SCC in sexually dichromatic species. Alternatively, in prolonged breeders undergoing SCC, male competition may be a driver of colour change. This was observed in Allobates caeruleodactylus where the blue toes in males (present during the breeding season) act as a male-male signal (Vitt & Caldwell, 2013). More research is needed to determine whether other processes of sexual selection play a role in this form of colour change.

Rapid colour change in males usually functions as a sexual signal or communication tool. Females showed a preference to the brightest throats in Guibemantis liber (Vences, M, 2015, pers. comm.), which stand out more due to rapid darkening or dorsal skin (exact timing not known). Whilst female preference for bright colours and visual signals has been observed in several species (Cornuau, Rat, Schmeller & Loyau, 2012; Hartmann, Hartmann & Haddad, 2004; Himstedt, 1979), the fitness component it signals (if at all) is often unknown. When colouration is the product of female choice it would seem that colour signals some kind of quality, however this can be difficult to quantify in rapid colour changing species. Most observations on female choice for colour or brightness are on species that exhibit permanent colours or ontogenetic colour change rather than rapid colour change (Gomez, Richardson, Lengagne, Plenet, Joly, Léna & Théry, 2009).

Male competition is common in amphibians and often occurs when females and or breeding sites are limited (Arak, 1983; Wells, 2007). Colour change during male-male aggression has been well documented in many animals in particular reptiles, often as a signal of dominance (Cooper & Greenberg, 1992; Greenberg, 2002; Ligon & McGraw, 2013). Colour change

during male-male aggression has also been observed in amphibians. Colostethus palmatas, Mannophryne collaris and Mannophryne trinitatis can change from brown to black within 10 minutes when calling, defending breeding territories or fighting another male, in the latter case and the loser will rapidly change back to its original brown colour while the winner stays black (Duellman & Trueb, 1994).

We identify a new form of sexual recognition, that is sexual recognition post amplexus, where colour change occurs after a female is secured (Kindermann et al., 2014) and functions as a signal to other males after amplexus has been initiated (Kindermann & Hero, 2016 pers. comm.). This form of colour change is likely to function as an intra-sexual signal to other males to avoid sperm competition. It is different than intra-sexual signalling (pre-amplexus) which is more of a sexual recognition tool in large breeding aggregations where both sexes are of a similar size (commonly seen in seasonal colour changing explosive breeding species). So far, this function has only been demonstrated in L. wilcoxii (although it likely to be the same for Litoria jungguy and Litoria lesueuri). More research is warranted on other species displaying similar forms (rapid brightening post amplexus) of colour change.

20 Legend: explosive 15 prolonged Numberofspecies 10

0 Ontogenetic Rapid Seasonal Colour change pattern

Figure 1: The distribution of breeding systems (from Wells 1977) for each pattern of colour change (ontogenetic, seasonal and rapid) in sexually dichromatic species. Bars represent explosive breeders (black bar), prolonged breeders (white bars). Note that some species exhibit more than one type of colour change.

3.2 Natural selection

Few studies (<10%) have investigated the ecological functions of OCC in sexually monochromatic species. Based on our analysis of the literature we have identified camouflage (5% of monochromatic species) or aposematism and mimicry (3% of monochromatic species) to be the main evolutionary function. Camouflage related to variations in habitat use and predation pressures between juveniles and adults is a likely explanation for many species displaying OCC however more studies are needed on this complex function. Many species in the genus Hyperolius exhibit this form of colour change, in this genus juveniles are usually plain in colouration (brown or green) and slowly develop brighter colours and patterns as adults, often in one sex only (Hoffmann & Blouin, 2000). The development of these changes coincides with sexual maturity and is driven by reproductive hormones (Hayes & Menendez, 1999; Richards, 1982). However, despite colour change being such a common occurrence in this family, the evolutionary specific mechanisms of why they change colour this have not been studied. This pattern of colour change may be important for juveniles as it is advantageous for them to delay the development of reproductive colours (to avoid predation) until they are ready to breed. Alternatively, bright colours in adults can be used as an aposematic signal which may not be as developed in juveniles. Atelopus zeteki are dark brown as juveniles and match the stream edge habitat they live in, over time they develop brilliant yellow colouration and become more toxic (Lindquist & Hetherington, 1998).

Ontogenetic colour changes from bright juveniles to dull adults may be driven by differing habitat or foraging requirements. In juvenile Eastern newts (Notophthalmus viridescens), the difference in light environments and predator visual systems between terrestrial and aquatic environments may drive the ontogenetic change from bright red to a dull brown-green (Huheey & Brandon, 1974; Pough, 1974). Another possible explanation is that the toxin (TTX) found in juvenile newts may not be as useful for aquatic adults because the toxin may disperse too quickly in water, making dull colouration more beneficial than aposematic colouration. There are also some species from the genus Pseudotriton which mimic the colouration of N. viridescens, the change in mimic species may be due to increased predation pressure (Pough, 1974). Juvenile Oreophryne ezra are black with golden spots and are active (and surprisingly conspicuous) during the day while the adults are light pink/peach and active at night, although there is currently no conclusive evidence of the function of this change, it may be related to the ambient light and vegetation type the individual uses or an aposematic function (Kraus & Allison, 2009).

Some species can change colour at seasonal levels, to cope with seasonal vegetation and/or temperature changes (Wente & Phillips, 2005). This form of seasonal colour change is rare and has only been recorded in 13% of SCC species. It is likely that this form of colour change has been interchanged with OCC or RCC as many studies don’t specify exact timings (Supplementary table 2). Seasonal changes corresponding to changing habitat have been observed in Rhinoderma darwinii (Bourke, Barrientos, Ortiz, Busse, Böhme & Bakker, 2011) and Hyla regilla (Wente & Phillips, 2003). Aspects of natural selection including camouflage (seasonal changes in habitat colouration), thermoregulation (changing colour to adapt to thermoregulatory constraints across seasons) are probably more common than reported here, however more detailed studies are required.

Most commonly RCC functions as a camouflage or thermoregulatory tool and to some degree is present in most amphibians (Hoffmann & Blouin, 2000). Evidence for background matching in cordata/urodela (Fernandez & Bagnara, 1991) and anurans (Kats & van Dragt, 1986) has been attributed to thermoregulation (over 30%) or ultraviolet protection (2%) as well as camouflage [20%] (Garcia, Straus & Sih, 2003; Garcia, Stacy & Sih, 2004). Rapid colour change responses vary from differences in day and night time colouration (Toledo & Haddad, 2009) to intermediate changes to intermediate changes to match background or respond to temperature changes (Kats & van Dragt, 1986). A surprising number of frogs spend a large amount of time basking in sunlight and colour change (lightening) can help reduce water loss, this explains why such a large number of species were identified to use colour as a thermoregulatory tool (table 2). Many species including Litoria rubella and Bokermannohyla alvarengai turn pale white when they bask regardless of their background colour. As lighter colours reflect more heat, it is assumed that colour change is a response to environmental conditions (i.e. heating by the sun) in species displaying this behaviour (Tattersall, Eterovick & de Andrade, 2006; Withers, 1995).

Numerous studies have shown that there is a strong effect of light and temperature on an organism’s capacity of colour change. The spring peeper (Hyla crucifer) matches its background (both light and dark) at a faster rate when temperatures are higher (Kats & van Dragt, 1986). And, both background appearance and temperature were shown to influence colour change in the American green tree frog (Hyla cinerea) (King et al., 1994). Such observations explain why so many studies and observational data hypothesise thermoregulation as an evolutionary function of colour change (table 2).

Table 2: Summary of the hypotheses that have been proposed to explain the function of ontogenetic, seasonal dynamic and rapid dynamic colour change; the number of species column represents records from peer-reviewed natural history notes, field guides or experimental reports. The confirmed studies column represents the number of species where the function has been experimentally confirmed or accurate data is available. It should be noted that some species exhibit more than one type of colour trait, and colour can have more than one function, for example some species change colour for thermoregulation and camouflage (not shown in the table).

Pattern of Number of Confirmed colour Function References species studies change

Aposematic signal (including mimicry) 4 3 1-4 Ontogenetic Camouflage (habitat/niche partitioning) 7 5 4-11 (n=132) Female choice 2 1 12,13

Camouflage/thermoregulation 8 13 14-20

Seasonal Male-male competition or signalling 3 5 21,23

(n=62) Sex recognition (pre-amplexus) 12 19 23-32

Female choice 1 2 33

Camouflage 22 18 18,23,29,34- 44

Thermoregulation 36 30 15, 29,38,45-56

Camouflage and thermoregulation 13 11 18,19, 29,38,57-65

Rapid UV protection 2 2 9-11

(n=121) Male-male competition 3 3 66,67

Sex recognition (pre-amplexus) 3 3 18,29

Sex recognition (post-amplexus) 3 3 18, 68, 69

Female choice 2 2 29

References: (1) Pough, 1974; (2) Huheey & Brandon, 1974; (3)Kraemer, Kissner & Adams, 2012 (4) Lindquist & Hetherington, 1998;(5)Hayes & Menendez, 1999; (6) Kramek & Stewart, 1980; (7) Manthey & Grossmann, 1997; (8) Larson, 1980; (9)Garcia et al., 2003; (10)Garcia & Sih, 2003; (11)Garcia et al., 2004; (12)Vásquez & Pfennig, 2007;(13)Maan & Cummings, 2008; (14) Gray, 1972; (15)Toledo & Haddad, 2009; (16)Bourke et al., 2011; (17) King & King, 1991;(18)Anstis, 2013;(19)Wente & Phillips, 2003; (20) Kingdon, 1989;(21) Vitt & Caldwell, 2013; (22) Hirschmann & Hödl, 2006; (23) Rödel, 2000;(24) Sztatecsny et al., 2012; (25) Sztatecsny, Strondl, Baierl, Ries & Hödl, 2010; (26) Doucet & Mennill, 2010; (27)Himstedt, 1979; (28) Rehberg-Besler et al., 2015; (29) Glaw & Vences, 2007; (30)Wogan, Win, Thin, Lwin, Shein, Kyi & Tun, 2003; (31)Tabassum et al., 2011;(32) Vanderduys, 2012;(33) Greenberg, 1942; (33) Biju, Bossuyt & Lannoo, 2005;(34) Bahir, Meegaskumbura, Manamendra-Arachchi, Schneider & Pethiyagoda, 2005;(34)Bagnara & Hadley, 1973; (35) Nielsen, 1979;(36)Machado, Menegucci, Mendes & Moroti, 2015(37)King et al., 1994; (38)Savage, 2002;(39) Fernandez Jr & Collins, 1988; (40)Wright, 2002;(41) Means & Karlin, 1989; (42)Parker, 2012; (43)Heinen, 1994; (44) Guyer, Donnelly & Donnelly, 2004 (45) Snyder & Hammerson, 1993; (46)Andreone, Rosa, Noël, Crottini, Vences & Raxworthy, 2010;(47) Conant & Collins, 1998; (48) Dehling & Grafe, 2008; (49) Schiøtz, 1999; (50)Tattersall et al., 2006; (51) Withers, 1995;(52) Kobelt & Linsenmair, 1986; (53) Pyburn, 1963;(54)Leenders, 2001; (55)Kenyon, Phillott & Alford, 2010; (56)Nielsen, 1980; (57) Choi & Jang, 2014; (58)Medina Barcenas, 2013; (59) Chester, 2010; (60) Kats & van Dragt, 1986;(61)Logier, 1952; (62)Roubos, 1997;(63)Escallón, 2008; (64)Mendelson III, Savage, Griffith, Ross, Kubicki & Gagliardo, 2008; (65) Vallan, Glaw, Andreone & Cadle, 1998;(66)Lüddecke, 1999; (67) Duellman & Trueb, 1994; (68) Kindermann et al., 2014; (69)Bittencourt-Silva, 2014.

3.3 Interactions between natural and sexual selection Although it easy to assume that there is only one selective pressure driving colour change evolution it is generally more complex (Norris, 1967; Stevens & Merilaita, 2011). The development of bright breeding colouration conflicts with an animal’s ability to avoid predation and rapid dynamic colour change can be a solution to this, as breeding colours can be turned on or off when needed (Sköld et al., 2013). In the neo-tropical toad Incilius luetkenii yellow males (which undergo a seasonal change from brown to yellow) faded rapidly (within hours) to brown after amplexus with a female, as well as after capture, indicating that although turning yellow is an important aspect to their courtship (possibly for sex recognition) it is also a costly signal (Doucet & Mennill, 2010; Rehberg-Besler et al., 2015). Similar observations occurred in the Madagascan Mantellid, Aglyptodactylus securifer where a captured individual displaying yellow breeding colouration had lost much of its brightness overnight in a collection bag (Vences, M, 2015, pers. comm.).

In contrast species, such as L. wilcoxii and Amietophrynus lemairii which both change colour in just a few minutes remain bright yellow during amplexus (Bittencourt-Silva, 2014; Kindermann et al., 2014). Bright yellow L. wilcoxii can also rapidly change back to brown if handled, suggesting that rapid colour change allows for camouflage when needed. There is little evidence of disadvantage of attaining bright breeding colouration in L. wilcoxii (Kindermann & Hero, 2016 pers. comm.); this suggests that either these species don’t have natural predators that rely on colour to detect species or the gain of this colour change over- ride the predation risks associated with bright colouration.

4. Conclusion and future directions

Colour change in amphibians is primarily the result of a change in pigment cell distribution and pigment movement within individual cell types (Bagnara & Hadley, 1973). The distribution of these cell types can determine the capacity for colour change in species (Szydłowski, Madej & Mazurkiewicz‐Kania, 2015). Pigment movement is mainly driven by hormones and the timing can vary from just minutes to permanent changes that occur over months or years (Booth, 1990; Kindermann et al., 2014). The type of hormone driving the colour change is often related to the speed of change, reproductive hormones trigger seasonal or ontogenetic colour changes that often occur in one sex only (Greenberg, 1942) whereas peptide and neuro-hormones stimulate rapid changes (Fernandez & Bagnara, 1991; Kindermann et al., 2014). The complexities and colours observed in amphibians are

astonishing. The use of these colours demonstrates the importance of vision as a communication tool and colour changing species are excellent models for evolutionary studies.

OCC is driven by natural and sexual selection and has a variety of functions, yet these have only been confirmed in approximately 10% of species. Despite the small amount of studies there appears to be a large variation in function including female choice, camouflage (niche partitioning) and aposematism. SCC is primarily driven by sexual selection, especially sex recognition as it is commonly observed in explosively breeding species. RCC is driven by natural and sexual selection. Colour changes for camouflage and thermoregulation are widespread and commonly reported and are mainly associated with diurnal / nocturnal changes, thermoregulatory requirements or in response to a predator attack. Sexual encounter colour change (intra sexual signalling, male-competition and female choice) has been reported in 22% of RCC studies, and may be more common than previously thought.

Our knowledge of the diversity of colour change in amphibians is growing however several limitations still exist. Many cases of colour change are brief explanations of the trait documented in field guides or anecdotal short notes (Bittencourt-Silva, 2014) or unpublished observations. Along with this there seems to be a trend that studies tend to look at one aspect of colour change, where the link between mechanism and function is unknown. This can leave out important gaps in the knowledge and therefore makes comparing processes between species more difficult.

Our understanding of colour production has been increased greatly by the understanding of morphology of pigment calls and the physiological processes of pigment and cell movement. However, this underlying physiology is often related to the evolutionary function and many studies miss this link. Future research should undertake an interdisciplinary approach combining morphology, physiology and function to make significant advances in the emerging field of colour change in amphibians.

Acknowledgements

This study was performed as part of the PhD research of Christina Kindermann at the Environmental Futures Research Institute, School of Environment, Griffith University. Thanks to all those who contributed species accounts for our database.

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Chapter 3: Pigment cell distribution in a rapid colour changing amphibian (Litoria wilcoxii)

Forward: This chapter examines the first component of colour change: the morphology (structure) of pigment cells. Determining the structure of pigment cells in Litoria wilcoxii provided a greater understanding of how this unique species can change colour.

This chapter is a co-authored paper that is published in Zoomorphology. My contribution to the paper involved: Designing and performing the research, analyzing the results and writing the manuscript. The bibliographic details of the co-authored paper are:

Kindermann C, Hero J-M (2016) Pigment cell distribution in a rapid colour changing amphibian (Litoria wilcoxii) Zoomorphology 135 (2), 197-203 doi:10.1007/s00435-016- 0303-1

______(Date) ______

Student and corresponding author: Christina Kindermann

______(Date) ______

Supervisor and co-author: Jean-Marc Hero

In order to comply with copyright this article has been removed

In order to comply with copyright this article has been removed In order to comply with copyright this article has been removed Chapter 7: General conclusion

Very few studies have taken a multidisciplinary approach to studying colour change in amphibians. Linking the structural and hormonal mechanisms of colour change with the adaptive function can bring about new discoveries to understanding the connections between physiological processes and behavioural ecology. The overall objective of this study was to better understand the mechanisms and functions of amphibian colour change using the stony creek frog Litoria wilcoxii as a model species.

A review of current literature on colour changing amphibians (presented in chapter 2) highlights the lack of knowledge about the details of colour change in individual species. Many descriptions of colour change were only found in field guides as unpublished observations. Or were presented as investigations that focussed solely on just one aspect of colour change (e.g. physiological or behavioural (Ries et al., 2008; Sztatecsny et al., 2012; Tang et al., 2014)). Ultimately this allowed me to test the hypotheses for dynamic colour change in L. wilcoxii.

Litoria wilcoxii is unique among amphibians not only due to the speed of change (which occurs in less than five minutes) but also that it mainly occurs after initiation of amplexus. The cellular structure of the skin determines capacity for colour change. The colour changing dorsal skin of Litoria wilcoxii has structures similar to the “dermal chromatophore unit” described earlier (Bagnara et al., 1968), however permanently coloured sections of thigh skin were markedly different. Rapid dorsal colour change occurs when dark pigment migrates from the centre of the melanophore into fingers that cover other cells (darkening), or vice versa (lightening) exposing the bright yellow chromatophores (chapter 3).

This rapid dispersal of pigment is achieved through stimulation by neuro-hormones, specifically adrenalin (Chapter 4) and predominantly occurs after initiation of amplexus. In addition, by examining seasonal cycles of stress and reproductive hormones and dorsal colour and the influence of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) it was determined that through increases in stress hormone levels and decreases in testosterone, physiological aspects of reproduction may be influenced by disease, however the capacity for colour change is not affected (Chapter 5). The parsimonious explanation for the function of this unusual phenomenon is an intrasexual signal following amplexus that could avert sperm competition by avoiding displacement by other males during amplexus (Chapter 6).

In chapter 2, I conducted a literature review to determine what is already known about amphibian colour change and secondly, to investigate the relationship with breeding system and colour change to elucidate the function of colour change in sexually dichromatic amphibians. Natural selection for camouflage and/or thermoregulatory requirements and sexual selection for signalling are the main evolutionary drivers of colour change. Many amphibian species exhibit more than one form of colour change (Stuart-Fox and Moussalli 2009). This suggests that the capacity for colour change may have evolved as a strategy to accommodate conflicting selective pressures (camouflage, signalling and thermoregulation). Alternatively, colour change may have initially evolved for just one function (i.e. camouflage), with an additional trait (i.e. sexual signalling) arising secondarily.

Ontogenetic colour change is unidirectional and driven by aspects of natural and sexual selection and about 50% of colour change occurs in one sex only. In dichromatic species this form of colour change is more prevalent in prolonged breeders. Seasonal colour change is a temporary colour change that most commonly occurs in dichromatic, explosive breeding species where it is likely to function as a sex recognition signal. Rapid colour change is also temporary and is commonly used for camouflage and thermoregulatory functions. In dichromatic species rapid colour change predominantly occurs in prolonged breeders where it functions as an intrasexual signal. These findings illustrate that there is a relationship between breeding system and colour change pattern. As more data on colour changing species becomes available, these relationships will be able to be interpreted in greater detail.

Whilst our knowledge of the diversity of colour change in amphibians is steadily increasing, most of the records/observations still come from brief descriptions field guides, anecdotal short notes (Bittencourt-Silva, 2014) or unpublished observations. And, although our understanding of colour production has been increased greatly by the many detailed physiological and morphological studies in the literature, there is a lack of evolutionary studies that can connect the physiological explanations. Detailed studies investigating only on trait can leave out important gaps in the knowledge and therefore makes comparing processes and functions between species more difficult. Future research should therefore take interdisciplinary approach combining morphology, physiology and function to make significant advances in the emerging field of colour change in amphibians.

Chapter 3 demonstrates the importance of understanding the way colour cells are structured to enable the physical response observed. Chromatophore morphology can elucidate the

pathway of pigment movement and type of colour change. It appears the dermal chromatophore structure is relatively conserved in colour changing skin sections for all vertebrates, however non-colour changing skin sections in L. wilcoxii are structurally simple, and do not have the mechanical structure required for colour change. This simple structure for skin sections that are permanently coloured has not been demonstrated previously. Future investigations comparing these structures in closely related species may shed light on the generality of this phenomenon. This study had its limitations as structures were only examined using light microscopy. The use of other microscope techniques such as TEM could show more detail and reveal the presence and location of iridophores (Ali and Naaz 2014).

The natural timing of dynamic colour change in L. wilcoxii is less than 5 minutes and largely occurs following initiation of amplexus, although males can display yellow colouration in the breeding season, even when females are not observed nearby. In chapter 4, this timing was replicated by injecting frogs with the neuro-hormone epinephrine (adrenalin). The reproductive hormone testosterone did not trigger colour change, although it may play a role in seasonal colour development. The hormonal processes of colour change are complex; this study was therefore very basic as only two hormones were investigated. Further investigation of natural fluctuations of these hormones and an investigation of other reproductive/colour change hormones will give a more detailed insight into this unique phenomenon.

Interactions among seasonal colour change, hormones and disease (chytridiomycosis) were investigated in chapter 5. Although L. wilcoxii populations appear to be stable, previous work showed that they too are affected (at least at a sub-clinical level) by Bd (Kindermann et al., 2012). A significant positive relationship between frogs that tested positive for Bd and corticosterone levels was observed and Bd infection status was associated with decreased testosterone levels. No significant relationship between Bd and dorsal colour was observed. This result shows that in addition to influencing the stress levels of individual frogs, the strongly seasonal dynamics of Bd are influencing physiological aspects of reproduction though decreased testosterone levels, however the capacity for colour change does not appear to be affected. This study has provided some insight into the effect of disease on reproduction; however the interactions between disease, stress and reproduction are complex. These relationships have been the subject of numerous studies (Blaustein et. al., 2012) and this growing body of research is revealing new differences and complexities between species (Brennelly et. al., 2015; West et. al., 2015). Further investigations into the important role of

life history (Zuk and Stoehr 2002) and other aspects of reproduction are needed as well as an integration of physiology and ecology (Seebacher and Franklin 2012).

The evolutionary function of colour change in L. wilcoxii is hypothesized to be an intrasexual signal following amplexus. This is based on three main results found in the study:1) the absence of female choice and increased male activity (calling and movement) when a female (or model female) is nearby suggests that this species displays a new form of colour change that’s acts as an intrasexual signal to avoid male displacement whilst in amplexus, 2) the hormonal pathways triggering colour change are different to those stimulating spermiation (adrenalin triggered colour change, hCG triggered sperm release), further emphasising that the role colour change plays in this species has a function and is not a by-product of hormonal stimulus, 3) we found no evidence of increased predation risk of yellow males suggesting that there is little or no cost in attaining yellow colouration during amplexus, a perceived “risky” period for any animal. Previous experiments have shown that anuran amphibians are able distinguish colour well in both dark and light environments (Aho et al., 1993; Gordon & Hood, 1976; Kicliter & Goytia, 1995). An addition, the primary predators of L. wilcoxii appear to be nocturnal snakes (such as the rough scaled snake Tropidechis carinatus) that rely on physical (movement) and chemical cues for detecting prey (Brischoux et al., 2010; Lillywhite, 2014; Shine & Charles, 1982), further suggesting that this strong visual signal is likely to be directed at conspecifics.

The breeding behaviour of L. wilcoxii remains relatively unknown, specifically in regards to egg laying (Anstis 2013) and therefore even simple observations such as this should be recorded and documented in future studies. Whilst it has been shown in chapter 6 that colour acts as a signal to other males, additional research on satellite males and male sneaking behaviour could shed some light on which male does get the female and how this is determined. Is it a case of first in best dressed or is there more at play? Future research investigating additional hormones that trigger colour change, in particular why epinephrine triggers colour change in L.wilcoxii. This could be achieved through the measurement of epinephrine in blood or urine samples in amplexing and non-amplexing males.

This thesis illustrates the complexity of colour change in animals, and colour change research. There are varying methods for quantifying colour (through photo analysis or spectrometry), administrating hormones (injection vs skin absorption), measuring hormones (urine or blood samples) and even microscope tequnique. Often the absence of a particular

method may be seen as a limitation. In this thesis the best available methods in relation to funding and ethics approval were used. For example digital image analysis rather than spectrometry was used as it is less invasive and therefore less likely to cause stress-induced skin colour changes (Greenberg, 2002). And, whilst measuring hormones in blood samples may have given a more accurate indication of hormonal processes, urinary hormone analysis was used as it is less invasive and less harmful to the animal (Narayan et al., 2010).

This thesis has provided a comprehensive understanding of colour change in Litoria wilcoxii and demonstrates novel mechanisms and functions. It is hypothesised that there are many more species which may exhibit similar physiological processes and even functions of colour change. Future research should focus on investigating the evolution of dynamic colour change in species with similar breeding systems.

References

Aho, A.-C., Donner, K., Helenius, S., Larsen, L.O., Reuter, T. 1993. Visual performance of the toad (Bufo bufo) at low light levels: retinal ganglion cell responses and prey- catching accuracy. Journal of Comparative Physiology A, 172(6), 671-682. Ali, S.A. and Naaz, I. 2014. Comparative light and electron microscopic studies of dorsal skin melanophores of Indian toad, Bufo melanostictus. Journal of Microscopy and Ultrastructure, 2(4), 230-235. Bagnara, J.T., Taylor, J.D., Hadley, M.E. 1968. The Dermal Chromatophore Unit. The Journal of Cell Biology, 38(1), 67-79. Blaustein, A. R., Gervasi, S. S., Johnson, P. T., Hoverman, J. T., Belden, L. K., Bradley, P. W., & Xie, G. Y. 2012. Ecophysiology meets conservation: understanding the role of disease in amphibian population declines. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 367(1596), 1688-1707. Brannelly, L. A., Hunter, D. A., Lenger, D., Scheele, B. C., Skerratt, L. F., & Berger, L. (2015). Dynamics of Chytridiomycosis during the Breeding Season in an Australian Alpine Amphibian. PloS one, 10(12), e0143629. Brischoux, F., Pizzatto, L., Shine, R. 2010. Insights into the adaptive significance of vertical pupil shape in snakes. Journal of Evolutionary Biology, 23(9), 1878-1885. Gordon, J., Hood, D. 1976. Anatomy and physiology of the frog retina. in: The Amphibian Visual System, pp. 29-86. Greenberg N. 2002. Ethological aspects of stress in a model lizard, Anolis carolinensis. Integrative and Comparative Biology 42: 526-540. Kicliter, E., Goytia, E.J. 1995. A comparison of spectral response functions of positive and negative phototaxis in two anuran amphibians, Rana pipiens and Leptodactylus pentadactylus. Neuroscience letters, 185(2), 144-146. Kindermann, C., Narayan, E.J., Hero, J.-M. 2012. Urinary corticosterone metabolites and chytridiomycosis disease prevalence in a free-living population of male Stony Creek frogs (Litoria wilcoxii). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 162(3), 171-176. Lillywhite, H.B. 2014. Percieving the snake's world stucture and function of snake organs. in: How Snakes Work: Structure, Function and Behavior of the World's Snakes, Oxford University Press. New York.

Narayan, E., Molinia, F., Christi, K., Morley, C., Cockrem, J., 2010. Urinary corticosterone metabolite responses to capture, and annual patterns of urinary corticosterone in wild and captive endangered Fijian ground frogs (Platymantis vitiana). Australian Journal of Zoology 58, 189-197. Ries, C., Spaethe, J., Sztatecsny, M., Strondl, C., Hödl, W. 2008. Turning blue and ultraviolet: sex-specific colour change during the mating season in the Balkan moor frog. Journal of Zoology, 276(3), 229–236. Seebacher F., Franklin C. E. 2012. Determining environmental causes of biological effects: the need for a mechanistic physiological dimension in conservation biology. Phil. Trans. R. Soc. B 367, 1607–1614. Shine, R., Charles, N. 1982. Ecology of the Australian Elapid Snake Tropidechis carinatus. Journal of Herpetology, 16(4), 383-387. Stuart-Fox D, Moussalli A. 2009. Camouflage, communication and thermoregulation: lessons from colour changing organisms. Philosophical Transactions of the Royal Society B: Biological Sciences 364: 463-470. Sztatecsny, M., Preininger, D., Freudmann, A., Loretto, M.-C., Maier, F., Hödl, W. 2012. Don’t get the blues: conspicuous nuptial colorationof male moor frogs (Rana arvalis) supports visual mate recognition during scramble competition in large breeding aggregations. Behavioral Ecology and Sociobiology, 66(12), 1587-1593. Tang, Z.-J., Lue, S.-I., Tsai, M.-J., Yu, T.-L., Thiyagarajan, V., Lee, C.-H., Huang, W.-T., Weng, C.-F. 2014. The hormonal regulation of color changes in the sexually dichromatic frog Buergeria robusta. Physiological and Biochemical Zoology, 87(3), 397-410. Zuk M., Stoehr A. M. 2002. Immune defense and host life history. Am. Nat. 160 (1), 9–22

Appendix 1: Litoria wilcoxii (Stony Creek Frog) Interspecific amplexus Article type: Natural History Note

Kindermann C. 2015. Litoria wilcoxii (Stony Creek Frog). Interspecific amplexus. Herpetological Review 46 (2): 235

Interspecific amplexus is most commonly associated with explosive breeding species or those with a strongly male-biased operational sex ratio such as bufonids (Wells 2007. The Ecology and Behavior of Amphibians. University of Chicago Press, Chicago, Illinois. 1148 pp.). During the breeding season (spring to autumn), male Litoria wilcoxii gather along creek edges and call for prospective females (Donnellan and Mahony 2004. Aust. J. Zool. 52:1–28). There is a strong male-biased sex ratio within these aggregations (pers. obs.). This species undergoes rapid color change during amplexus, whereby males turn from brown to bright yellow within minutes after initiation of amplexus (Kindermann et al. 2014. PloS one 9: e114120). Due to the strongly male biased sex ratio, access to females may be limited and intense male competition may occur.

Here I document two cases of interspecific amplexus involving male L. wilcoxii and one native and one introduced anuran. The first observation (Fig. 1) occurred at 2056 h on 21 October 2013, where I observed a male L. wilcoxii amplecting a juvenile Mixophyes iteratus (Giant Barred Frog) along a rocky section of the Nerang River in Numinbah Valley, Queensland, Australia (28.1770°S, 153.2280°E, WGS84; 155 m elev.). The M. iteratus did not attempt to move away from or dislodge the male and the pair remained in this way for several hours. Air temperature was 20.5°C and water temperature was 24.2°C.

The second case occurred in the same location at 2025 h on 4 November 2013, this time between a male L. wilcoxii and a Rhinella marina (Cane Toad). In this case a male L. wilcoxii was observed jumping towards the R. marina and immediately amplexing it; the R. marina made no attempt to dislodge the amplectant male. The pair remained in amplexus for several hours during which time the male L. wilcoxii underwent rapid color change from brown to yellow within 10 min. of amplexus initiation (Fig. 2).

Air temperature was 19.6°C and water temperature 23.3°C. It is worth noting that several more observations of interspecific amplexus between male L. wilcoxii and R. marina were made during the breeding season, showing that this may be a common occurrence, despite not being documented in the literature. In all cases the amplected frog was a similar size and

color to female L. wilcoxii, these observations indicate that males may be choosing females based on body size or color, the lack of females observed in the area might explain why males turn to other species.

Fig.1. Amplexus between a male Litoria wilcoxii and juvenile Mixophyes iteratus.

Fig. 2. Amplexus between a male Litoria wilcoxii and Rhinella marina. A) Color at first sighting. B) Color 10 minutes later.

Appendix 2: Supporting information for chapter 2

Table 1: Summary of colour change in dichromatic amphibians

Table 2: Summery of colour change in monochromatic amphibians

Table 1: Summary table of the physiological processes and functions of colour change in dichromatic amphibians with the addition of breeding system and behaviour descriptions. Adult (A), Juvenile (J), Male (M), Female (F), Ontogenetic colour change (OCC), Seasonal colour change (SCC), Rapid colour change (RCC), melanocyte - stimulating hormone (MSH), Epinephrine (Epi), Testosterone (T), oestrogen (E), progesterone (Pro), Adrenocorticotropic hormone (ACTH) temperature (temp), 5- hydroxytryptamine (5-HT). Colour changes occurring in both sexes, in addition to dichromatic species are present in some species and are included in this table.

Breeding Order Family Species Colour change description Pattern trigger (s) Behaviour Function Reference system Green (J) to brown (A)/blue OCC/ Anura Arthroleptidae Leptopelis notatus no data Unknown Schiøtz 1999 throats in calling males (M) RCC Green (J) to yellow dorsal pattern Harper et al. 2010; Toledo Anura Arthroleptidae Leptopelis parkeri OCC no data Unknown (M) and Haddad 2009 Leptopelis Hoffmann and Blouin Anura Arthroleptidae Green (J) to brown (F) OCC no data Unknown vermiculates 2000 Amietophrynus dense populations Anura Bufonidae Brown to yellow (M) OCC prolonged Unknown Baha El Din 1993 kassasii observed Amietophrynus Brown to yellow (M) breeding scramble Sex recognition Anura Bufonidae SCC explosive Rödel 2000 kisoloensis season competition (pre-amplexus) Amietophrynus RCC- scramble Sex recognition Anura Bufonidae Green to yellow (M) in minutes explosive Bittencourt-Silva 2014 lemairii SCC competition (pre-amplexus) Amietophrynus Brown to yellow (M) in breeding Sex recognition Anura Bufonidae SCC explosive stream breeding Rödel 2000 togoensis season (pre-amplexus) Brown pattern (J) to yellow brown Anura Bufonidae Amietophrynus xeros OCC explosive Unknown Rödel 2000 (M) scramble Hoffmann and Blouin Anura Bufonidae Anaxyrus canorus Brown (J) to tan (M) OCC explosive Unknown competition, 2000; Liang 2010 Atelopus male combat, Anura Bufonidae Brown (J) to yellow or blue (M) OCC prolonged Unknown Jaslow 1979; Savage 2002 chiriquiensis territory defence Blue grey to contrasting patterns Anura Bufonidae Atelopus senex OCC prolonged stream breeding Unknown Savage 2002 (M) Black and yellow to contrasting male combat and Anura Bufonidae Atelopus varius OCC prolonged Unknown Savage 2002 patterns (M) territory defence Brown to tan (M) in breeding scramble Anura Bufonidae Bufo bufo SCC explosive Unknown Reading and Clarke 1983 season competition Brown to yellow (M) in breeding scramble Possible sex Anura Bufonidae Bufo crocus SCC explosive Wogan et al. 2003 season competition recognition Brown to yellow (M) in breeding scramble Possible sex Anura Bufonidae Bufo japonicus SCC explosive Maeda and Matsui 1990 season competition recognition Brown to yellow (M) in breeding scramble Possible sex Anura Bufonidae Bufo macrotis SCC explosive Wogan et al. 2003 season competition recognition Duttaphrynus Brown to yellow throat (M) in some male Anura Bufonidae SCC explosive Unknown Khan 2001 melanostictus breeding season aggression Brown (J), black marks (F), Anura Bufonidae Incilius aurarius OCC prolonged stream breeding Unknown Mendelson et al. 2012 yellow brown (M)

Brown to yellow (M) in breeding RCC and scramble Sex recognition Anura Bufonidae Incilius luetkenii explosive Doucet and Mennill 2010 season, darken in a few hours SCC competition (pre-amplexus) Anura Bufonidae Incilius majordomus Tan brown (J) to tan yellow (M) OCC no data Unknown Savage et al. 2013

Brown, develop green mid dorsal Anura Bufonidae Incilius marmoreus OCC no data Unknown Duellman and Trueb 1994 mark (F) Incilius males call along Anura Bufonidae Brown to dark dorsal spots (F) OCC prolonged Unknown Savage 2002 melanochlorus streams Olive brown (J), red spots (F), scramble Anura Bufonidae Incilius periglenes OCC explosive Unknown Savage 1966 orange (M) competition Brown to yellow (M) in breeding Anura Bufonidae Peltophryne lemur SCC explosive Unknown Matos-Torres 2006 season OCC/ Filadelfi et al. 2005 Anura Bufonidae Rhinella icterica Brown green to yellow (M)/brown MSH explosive Thermoregulation RCC Toledo and Haddad 2009 explosive- scramble Anura Bufonidae Rhinella marina Brown to tan (M) OCC Unknown Zug and Zug 1979 prolonged competition Anura Bufonidae Rhinella veredas Brown green to yellow (M) OCC explosive Unknown Brandão et al. 2007

Dark brown green to tan brown Anura Bufonidae Rhinella yanachaga OCC no data Unknown Lehr et al. 2008 (F) Sclerophrys Brown to yellow (M) in breeding breeding Anura Bufonidae SCC prolonged Unknown Hillers and Rodel 2007 maculata season aggregations Black (J) black brown (F), white Calling males sit Anura Bufonidae Werneria preussi OCC prolonged Unknown Duellman and Trueb 1994 spots (M) next to water Pristimantis orange (J) to grey on thighs and direct Hoffmann and Blouin Anura Craugastoridae OCC prolonged Unknown polychrus venter (F) development 2000 Allobates male territory Anura Dendrobatidae Blue toes (M) in breeding season SCC prolonged Male competition Vitt and Caldwell 2013 caeruleodactylus defence Colostethus male defends Anura Dendrobatidae Brown to black (M) in minutes RCC prolonged Male competition Lüddecke 1999 palmatas territory Mannophryne Brown to black (M) in 1-10 male combat and Anura Dendrobatidae RCC prolonged Male competition Duellman and Trueb 1994 collaris minutes territory defence Mannophryne Brown to black (M) in 1-10 male combat and Anura Dendrobatidae RCC prolonged Male competition Duellman and Trueb 1994 trinitatis minutes territory defence male combat and Maan and Cummings Anura Dendrobatidae Oophaga pumilio Orange red to brighter colours (M) OCC prolonged Female choice territory defence 2009 Hoplobatrachus Brown to yellow (M) in breeding scramble Possible sex Ali et al. 2012; Tabassum Anura Dicroglossinae SCC 5-HT1,2, 3 explosive tigerinus season competition recognition et al. 2011 Gastrotheca direct Hoffmann and Blouin Anura Hemiphractidae green (J and F) to brown (M) OCC prolonged Unknown andaquiensis development 2000 Brown to yellow with dark throat RCC/ males defend Unknown/possible Anura Hylidae Acris gryllus T and temp prolonged Greenberg 1942 (M) in 1 month SCC territories F choice Brown pattern to yellow (M) in associated with Anura Hylidae Cyclorana brevipes SCC explosive Unknown Anstis 2013 breeding season heavy rain brown to yellow belly and dark associated with Anura Hylidae Cyclorana muculosa SCC explosive Unknown Anstis 2013 dorsum (M) in breeding season heavy rain Dendropsophus Anura Hylidae Brown to yellow (M) day to night RCC prolonged female choice Unknown Duellman 1969 carnifex

Dendropsophus Tan to yellow (M) to light Anura Hylidae RCC prolonged Female choice Unknown Halliday 2016 ebraccatus brown(F) day to night Dendropsophus Tan to yellow (M) to light male combat and Anura Hylidae RCC prolonged Unknown Motta et al. 2012 frosti brown(F) day to night territory defence Dendropsophus Anura Hylidae Brown to yellow (M) day to night RCC prolonged Unknown Motta et al. 2012 giesleri Dendropsophus yellow to tan day to night/Vocal RCC/ some female Anura Hylidae prolonged Unknown Duellman 1970 microcephalus sac yellow (M) in breeding season SCC choice Dendropsophus Anura Hylidae Brown to yellow (M) day to night RCC prolonged Unknown Motta et al. 2012 microps Dendropsophus Brown to yellow (M) in breeding defending of call Anura Hylidae SCC prolonged Unknown Haddad 1991 minutus season sites Dendropsophus Anura Hylidae Brown to yellow (M)day to night RCC prolonged Unknown Motta et al. 2012 parviceps Dendropsophus brown to yellow vocal sac (M) in Anura Hylidae SCC prolonged Unknown Duellman 2015 phlebodes breeding season Dendropsophus white to brown (F) to yellow (M) Thermoregulation/ Anura Hylidae RCC no data Halliday 2016 rhodopeplus day to night unknown Green/black throat (M) in 1-2 RCC/ Epi, T, light, King et al. 1994; Nielsen Anura Hylidae Hyla cinerea prolonged Camouflage hours SCC temp, MSH and Dyck 1978 males aggressive Anura Hylidae Hypsiboas boans Brown to orange brown (F) OCC prolonged Unknown Sumadh 2012 defend territory Brown to yellow (M) in breeding Anura Hylidae Hypsiboas fasciatus SCC prolonged Unknown Vitt and Caldwell 2013 season Hypsiboas Anura Hylidae Brown to yellow (M) day to night RCC prolonged Unknown Carvalho et al. 2010 multifasciatus Hypsiboas Thermoregulation Anura Hylidae Brown, lighten or darken in 1 hour RCC Light prolonged Medina Barcenas 2013 rosenbergi and camouflage Green grey mottling (J)to mottling males establish Anura Hylidae Isthmohyla calypsa OCC prolonged Unknown Lips et al. 2005 darkens (F) calling sites Isthmohyla Tan to yellow (M) in breeding scramble Anura Hylidae SCC explosive Unknown Savage 2002 pseudopuma season competition Litoria Brown to yellow (M) in breeding Anura Hylidae SCC prolonged Unknown Anstis 2013 booroolongensis season Brown to yellow (M) in breeding Anura Hylidae Litoria brevipalmata SCC prolonged Unknown Anstis 2013 season Cream to brown/ brown to yellow RCC- Camouflage/ Anura Hylida Litoria dentata prolonged Anstis 2013 (M) SCC unknown Brown stripes to yellow (M) Anura Hylidae Litoria jervisiensis RCC prolonged Unknown Payne 2014 During calling or amplexus Brown to yellow (M) in breeding Males form possible male Anura Hylidae Litoria jungguy RCC prolonged Anstis 2013 season aggregations signalling Brown to yellow (M) in breeding Males form possible male Anura Hylidae Litoria lesueuri RCC prolonged Anstis 2013 season aggregations signalling Brown to yellow throat (M) in Anura Hylidae Litoria nasuta RCC prolonged Unknown Anstis 2013 breeding season Anura Hylidae Litoria nigrofrenata Brown dorsum, yellow sides (M) RCC prolonged Unknown Anstis 2013

Green-gold to yellow, black throat Anura Hylidae Litoria raniformis SCC prolonged Unknown Anstis 2013 (M) in breeding season Brown to yellow (M) in breeding Anura Hylidae Litoria Revilata SCC prolonged Unknown Glaw and Vences 2007 season light brown in 1 hour/throat RCC/ Thermoregulation/ Anura Hylidae Litoria rubella Light prolonged Withers 1995 darkens in breeding season (M) SCC unknown Brown to yellow (M) in breeding RCC/ Anura Hylidae Litoria tyleri prolonged Unknown Anstis 2013 season /darken or lighten in hours SCC Litoria Brown to yellow (M) in breeding Anura Hylidae SCC prolonged Unknown Anstis 2013 watjulumensis season Males form Anura Hylidae Litoria wilcoxii Brown to yellow (M) in <5 mins RCC Epi prolonged Male signalling Kindermann et al. 2014 aggregations Osteocephalus Brown grey to yellow (M) in Scramble Anura Hylidae SCC explosive Unknown Jungfer and Hödl 2002 leprieurii breeding season competition beige brown to yellow orange (M) Anura Hylidae Scinax chiquitanus RCC no data Unknown Sturaro and Peloso 2014 day to night Brown tan to yellow (M) in male combat and Anura Hylidae Scinax elaechrous SCC explosive Unknown Savage 2002 breeding season territory defence Scinax Grey brown to yellow (M) day to Anura Hylidae RCC prolonged lek breeder Unknown Toledo and Haddad 2009 fuscomarginatus night Grey brown to yellow (M) in Anura Hylidae Scinax fuscovarius SCC explosive Unknown Toledo and Haddad 2009 breeding season Grey brown to yellow (M) day to Anura Hylidae Scinax hayii RCC prolonged Unknown Toledo and Haddad 2009 night Grey brown to yellow (M) in Anura Hylidae Scinax rizibilis SCC prolonged lek breeder Unknown Toledo and Haddad 2009 breeding season explosive- Anura Hylidae Scinax ruber Grey brown to yellow (M) OCC Unknown Wells 2007 prolonged Olive to tan (F)/yellow vocal sec OCC/ scramble Anura Hylidae Triprion petasatus explosive Unknown Duellman 2015 (M) in breeding season SCC competition Brown to green/grey vocal sac RCC/ Anura Hylidae Smilisca baudinii prolonged Camouflage Savage 2002 (M) in breeding season SCC Heterixalus Grey (J) to orange spots OCC/ Thermoregulation Anura Hyperoliidae no data Glaw and Vences 2007 alboguttatus (F)/lighten or darken RCC (?)/unknown Brown to olive with black spots OCC/ Thermoregulation Anura Hyperoliidae Heterixalus tricolor no data Schiøtz 1999 (F) /white to brown RCC (?) /unknown Heterixalus Beige (J) to black marking OCC/ Thermoregulation/ Anura Hyperoliidae Light no data Glaw and Vences 2007 variabilis (F)/White to beige RCC unknown Pale green (J) to brown with white Hayes and Menendez Anura Hyperoliidae Hyperolius argus OCC T and E no data Camouflage (M) spots (F) 1999 Hyperolius Yellow (J) to ventrum black with Anura Hyperoliidae OCC no data Unknown Schiøtz 1999 bolifambae white spots (F) Hyperolius Light brown (J) to dark with light Anura Hyperoliidae OCC no data Unknown Schiøtz 1999 brachiofasciatus spots (F) Hyperolius Green, white lines (J) to grey with Anura Hyperoliidae OCC no data Unknown Schiøtz 1999 camerunensis red spots (F) Anura Hyperoliidae Hyperolius Brown or green with stripe (J) to OCC prolonged Unknown Lötters et al. 2004

cinnamomeoventris green (F) Brownish green with light lines (J) Anura Hyperoliidae Hyperolius concolor OCC no data Unknown Schiøtz 1999 to green (F) Hyperolius Grey green, light green lines (J) to Anura Hyperoliidae OCC no data Unknown Schiøtz 1999 diaphanus green throat (F) Hyperolius Olive green pattern (J) black with Anura Hyperoliidae OCC no data Unknown Schiøtz 1999 dintelmanni green spots (F) Hyperolius Light green with stripe (J) to green Anura Hyperoliidae OCC prolonged Unknown Schiøtz 1999 fusciventris (F) Hyperolius Green with dark stripe (J) to Anura Hyperoliidae OCC prolonged Unknown Schiøtz 1999 guttulatus brown with spots (F) Brown green with stripes (J) to Anura Hyperoliidae Hyperolius kivuensis OCC prolonged Unknown Schiøtz 1999 green (F) Brown green with stripes (J) to Anura Hyperoliidae Hyperolius lateralis OCC prolonged Unknown Schiøtz 1999 green (F) Hyperolius Green to brown (J) to dark with Anura Hyperoliidae OCC prolonged Unknown Dyson et al. 1992 marmoratus coloured stripes (F) Grey brown (J) to greyish with Anura Hyperoliidae Hyperolius nimbae OCC no data Unknown Schiøtz 1999 black spots (F) Yellow with fine lines (J) to Anura Hyperoliidae Hyperolius pardalis OCC prolonged Unknown Schiøtz 1999 brown and spotted (F) Hyperolius Green (J) to fawn to orange brown Anura Hyperoliidae OCC no data Unknown Schiøtz 1999 phantasticus (F) Hyperolius Anura Hyperoliidae Brown (J) to green (F) OCC prolonged Unknown Schiøtz 1999 pickersgilli Hyperolius Brown with hourglass pattern (J) Anura Hyperoliidae OCC prolonged Unknown Schiøtz 1999 picturatus to brown (F) Hyperolius Brown green, light lines (J) to Anura Hyperoliidae OCC prolonged Unknown Schiøtz 1999 puncticulatus coloured stripes(F) Hyperolius Gold brown with stripes (J) to Anura Hyperoliidae OCC no data Unknown Schiøtz 1999 quinquevittatus green with stripes (F) Hyperolius Green (J) to dark with red, black, Anura Hyperoliidae OCC no data Unknown Schiøtz 1999 riggenbachi yellow pattern(F) Hyperolius Brown (J) to brown with red Anura Hyperoliidae OCC no data Unknown Schiøtz 1999 rubrovermiculatus vermiculation’s (F) Hyperolius Brown green, light lines (J) to Anura Hyperoliidae OCC prolonged Unknown Schiøtz 1999 sylvaticus very colourful (F) Hyperolius Brown with pattern (J) to grey Hoffmann and Blouin Anura Hyperoliidae OCC prolonged Unknown tuberculatus with black spots (F) 2000 Hyperolius Brown or mottled green (J) to Hoffmann and Blouin Anura Hyperoliidae OCC prolonged Unknown tuberilinguis green (F) 2000 Hyperolius v. Brown(J)to black & yellow OCC/ Thermoregulation Hoffmann and Blouin Anura Hyperoliidae prolonged glandicolor pattern(F)/light to dark RCC (?) /unknown 2000 Hyperolius v. Tan with dark stripes (J) to green OCC/ T, E, light and Thermoregulation/ Kobelt and Linsenmair Anura Hyperoliidae prolonged karissimbiensis with yellow spots (F)/light to dark RCC temp unknown 1986; Richards 1976 White (J) to light grey with black Hoffmann and Blouin Anura Hyperoliidae Hyperolius v. spatzi OCC prolonged Unknown spots (F) 2000 88

Hyperolius v. Tan, dark stripes (J) to green with OCC/ T, E, light, Thermoregulation/ Anura Hyperoliidae prolonged Richards 1976 viridiflavus yellow spots (F)/light to dark RCC and temp unknown Anura Hyperoliidae Hyperolius wermuthi Green (J) to red stripe (F) OCC no data Unknown Schiøtz 1999

Morerella Anura Hyperoliidae Orange to yellow (M) day-night RCC prolonged Unknown Rödel 2000 cyanophthalma Anura Limnodinastidae Notaden nichollsi grey throat (M) in breeding season SCC explosive Unknown Anstis 2013

Aglyptodactylus Brown to yellow (M) in breeding Anura Mantellidae SCC explosive form aggregations Unknown Glaw and Vences 2007 laticeps season Aglyptodactylus Brown to yellow (M) in breeding Sex recognition Anura Mantellidae SCC explosive form aggregations Glaw and Vences 2007 madagascariensis season (pre-amplexus) Aglyptodactylus Brown to yellow (M) in breeding RCC- possible sex Anura Mantellidae explosive form aggregations Glaw and Vences 2007 securifer season fade to brown in hours SCC recognition Blommersia Brown to yellow (M)/darken or OCC/ Thermoregulation/ Anura Mantellidae prolonged palm leaves Andreone et al. 2010 angolafa lighten RCC unknown Boophis Green to brown (J) to vivid green Anura Mantellidae OCC no data Unknown Glaw and Vences 2007 microtympanum (F) Anura Mantellidae Boophis pauliani Green (M) to light brown (F) OCC no data Unknown Glaw and Vences 2007

Green to black (white vocal sac large breeding Anura Mantellidae Guibemantis liber RCC explosive Female choice Glaw and Vences 2007 contrast) aggregations Scaphiophryne aggregate in small Anura Microhylidae Yellow, brown, black grey (M) OCC explosive Unknown Glaw and Vences 2007 gottlebei pools Neobatrachus Patchy brown to brown yellow in associated with Anura Myobatrachidae SCC explosive Unknown Anstis 2013 pelobatoides breeding season (M) heavy rain OCC/ Anura Pelodytidae Pelodytes caucasicus Brown to olive green (M) no data Unknown Arikan et al. 2007 SCC Phrynobatrachus Yellow vocal sac (M) in breeding vocal sac visual Anura Phrynobatrachidae SCC prolonged male signalling Rödel and Schiøtz 2004 alleni season signal Phrynobatrachus Yellow vocal sac (M) in breeding vocal sac visual Hirschmann and Hödl Anura Phrynobatrachidae SCC prolonged male signalling krefftii season signal 2006 Phrynobatrachus Brown-grey/brown to yellow RCC/ Camouflage/ Anura Phrynobatrachidae prolonged Rödel 2000 latifrons green (M) in breeding season SCC unknown Phrynobatrachus Light to dark brown to green Anura Phrynobatrachidae RCC no data Unknown Rödel 2000 natalensis dorsal patches(M) day to night Brown to green (M) in breeding Anura Pyxicephallinae Pyxicephalus edulis OCC explosive aggregates Unknown Rödel 2000 season Brown to yellow (M) in breeding aggregations, Anura Ranidae Clinotarsus curtipes SCC prolonged Unknown Tapley and Purushotham season some male combat Brown to yellow (M) in breeding Males call spread Anura Ranidae Hylarana aurata SCC prolonged Unknown Günther 2003 season out Brown to yellow (M) in breeding Anura Ranidae Hylarana volkerjane SCC no data Unknown Günther 2003 season Brown to yellow (M) in breeding Anura Ranidae Odorrana khalam SCC no data Unknown Jodi Rowley pers. comm season Brown to blue (M) in breeding scramble Anura Ranidae Rana arvalis SCC explosive Sex recognition Sztatecsny et al. 2012 season competition,

Green -brown hours / yellow RCC/ F choice, M Camouflage/ Anura Ranidae Rana clamitans MSH and Epi prolonged Parker 2012; Wells 1977 throat (M) in breeding season SCC defend territories unknown Brown to black brown (M) in Anura Ranidae Rana graeca SCC explosive Unknown Valakos 2008 breeding season Green to green with darker stripes sexual niche Manthey and Grossmann Anura Ranidae Rana hosii OCC no data (M) partitioning 1997 Anura Ranidae Rana longicrus Red brown to yellow brown (M) OCC prolonged Unknown Lue 1990

Brown in 5 days/brown to dark scramble Camouflage/ Anura Ranidae Rana sylvatica SCC explosive King and King 1991 brown (M) in breeding season competition unknown Brown in 45 min/dark with white RCC/ Light and scramble Camouflage/ Sztatecsny et al. 2010; Anura Ranidae Rana temporaria explosive throat (M) in breeding season SCC temperature competition Sex recognition Vences et al. 2002 Anura Ranidae Rana vaillanti Green to grey brown (F) OCC prolonged Unknown Ramirez et al. 1998

Sanguirana Green with orange spots to large Anura Ranidae OCC no data Unknown Fuiten et al. 2011 aurantipunctata colourful spots (F) Brown to yellow (M) in breeding T + melatonin Males form small Anura Rhacophoridae Buergeria robusta SCC prolonged Unknown Tang et al. 2014 season + prolactin aggregations Polypedates Yellow vocal sac (M) in breeding Anura Rhacophoridae SCC prolonged Unknown Kanamadi et al. 1993 maculatus season Large breeding Anura Scaphiopodidae Scaphiopus couchii Brown to yellow OCC explosive Female choice Vásquez and Pfennig 2007 aggregations Desmognathus brown with orange patches (J) to OCC/ Unknown/ Means and Karlin 1989; Chordata Plethodontidae no data apalachicolae brown (M) RCC Camouflage Verrell 1994 Ichthyosaura Grey and black to more patterned Chordata Salamandridae SCC no data Sex recognition Himstedt 1979 alpestris (M) in breeding season

Table 2: Summary table of the physiological process and functions of colour change in monochromatic amphibians (both sexes undergo colour change). Adult (A), Juvenile (J), Male (M), Female (F), Ontogenetic colour change (OCC), Seasonal colour change (SCC), Rapid colour change (RCC), melanocyte -stimulating hormone (MSH), Epinephrine (Epi), Testosterone (T), oestrogen (E), progesterone (Pro), Adrenocorticotropic hormone (ACTH) temperature (temp)

Order Family Species Colour change description Pattern Trigger (s) Function Reference Anura Arthroleptidae Leptopelis concolor Green (J) to brown (A) OCC Unknown Harper et al. 2010

Leptopelis Anura Arthroleptidae Green (J) to brown (A) OCC Unknown Schiøtz 1999 cynnamomeus Leptopelis Anura Arthroleptidae Green with yellow spots (J) to green (A) OCC Unknown Harper et al. 2010 flavomaculatus Leptopelis Anura Arthroleptidae Green (J) to brown (A) OCC Unknown Schiøtz 1999 mossambicus Leptopelis Anura Arthroleptidae Green (J) to brown (A) OCC Unknown Schiøtz 1999 occidentalis Anura Alytidae Alytes obstetricans Brown with olive spots-darkens and lightens RCC Camouflage Polo-Cavia et al. 2016 Emerald with gold stripes during day to dark Camouflage and Anura Batrachylidae Hylorina sylvatica RCC MSH Chester 2010 green at night thermoregulation Anaxyrus Anura Bufonidae Brown to dark brown in 15 min-2 hours RCC Temp, stress and, humidity Camouflage Heinen 1994 americanus Anura Bufonidae Anaxyrus boreas Brown with olive blotches RCC Temp Thermoregulation Feder 1992 Black (metamorphs) to yellow with black Camouflage- Anura Bufonidae Atelopus zeteki OCC Lindquist and Hetherington 1996 spots (J) to yellow (A) aposematism Anura Bufonidae Bufo houstonesis Beige (J) to grey white (A) OCC Unknown Hillis et al. 1984

Black, blue white lines or rings (J) to black or Anura Bufonidae Nectophryne afra OCC Unknown Blackburn and Droissart 2008 brown, yellow dorsolateral stripes (A) Black with silver bands and stripes (J) to Anura Bufonidae Nectophryne batesii OCC Unknown Blackburn and Droissart 2008 brown with dark blotches (A) Anura Bufonidae Rhinella arenarum Brown to light or dark in 2 hours RCC Camouflage Parker 2012

Anura Centrolenidae Cochranella mache Blue to green to deep blue RCC Unknown Cisneros-Heredia et al. 2008

Anura Craugastoridae Craugastor augusti Brown with white band to brown OCC Unknown Hoffmann and Blouin 2000

Strabomantis Camouflage and Anura Craugastoridae Brown to dark or light brown in 1 hour RCC Light Medina Barcenas 2013 bufoniformis thermoregulation Camouflage- Rhinoderma Anura Cycloramphidae Brown to green in a few months SCC match changing Bourke et al. 2011 darwinii environment Dendrobates Black with yellow stripes (J) to black spots Anura Dendrobatidae OCC Unknown Hoffmann and Blouin 2000 leucomelas on yellow stripes (A)

Anura Dendrobatidae Phyllobates bicolor Brown and yellow stripes (J) to yellow (A) OCC Unknown Myers et al. 1978

Phyllobates Black, gold dorsolateral stripes (J) to yellow

Anura Dendrobatidae OCC Unknown Myers et al. 1978 terribilis (A)

Eleutherodactylus After removal from Anura Eleutherodactylinae brown to tan in 30 mins RCC Camouflage Hedges et al. 1992 melacara bromeliad Gastrotheca Anura Hemiphractidae Green (J) to spotted brown olive (A) OCC Unknown Hoffmann and Blouin 2000 aureomaculata Camouflage and Anura Hemiphractidae Gastrotheca cornuta Dark brown to tan in an hour (day-night) RCC Light Medina Barcenas 2013; Savage 2002 thermoregulation Gastrotheca Anura Hemiphractidae Green (J) to brown grey (A) OCC Unknown Hoffmann and Blouin 2000 griswoldi Anura Hemiphractidae Gastrotheca helenae Tan (J) to brown (A) OCC Unknown Hoffmann and Blouin 2000

Grey stripe (J) to brown or green stripe (A) in Anura Hylidae Acris crepitans SCC Camouflage Gray 1972 2-4 months Anura Hylidae Afrixalus lacteus White to brown RCC Thermoregulation Schiøtz 1999

Anura Hylidae Agalychnis annae Green during day to blue purple at night RCC Thermoregulation Savage 2002

Agalychnis Green during day to blue purple or red brown Anura Hylidae RCC Thermoregulation Pyburn 1963 callidryas at night Green during day to blue purple or red brown Possible Anura Hylidae Agalychnis lemur RCC Savage 2002 at night thermoregulation Possible Anura Hylidae Agalychnis saltator Green during day to tan brown at night RCC Leenders 2001 thermoregulation Yellow green during day to dark green at Possible Anura Hylidae Agalychnis spurrelli RCC Savage 2002 night thermoregulation Bokermannohyla Anura Hylidae Light brown to dark brown in 30-60 min RCC Light and temperature Thermoregulation Tattersall et al. 2006 alvarengai Bokermannohyla Dark reddish brown to Black or white in 15 Anura Hylidae RCC Light Thermoregulation Toledo and Haddad 2009 circumdata min Anura Hylidae Cyclorana australis Green (JA) to brown (A) OCC Unknown Anstis 2013

Dendropsophus Anura Hylidae Red brown during day to tan at night RCC Unknown Moravec et al. 2006 juliani Dendropsophus Camouflage and Anura Hylidae Green to brown RCC Light Escallón 2008 labialis thermoregulation Anura Hylidae Dendropsophus salli brown during day to yellow at night RCC Unknown Jungfer et al. 2010

Duellmanohyla Anura Hylidae Brown to green RCC Thermoregulation Savage 1968 lythrodes Ecnomiohyla Camouflage and Anura Hylidae Mottled green to brown RCC Kubicki and Salazar 2015 bailarina thermoregulation Ecnomiohyla Anura Hylidae Brown-green (J) to lavender brown(A) OCC Unknown Hayes et al. 1986 fimbrimembra Ecnomiohyla Camouflage or Anura Hylidae Brown to brown and green RCC Mendelson III et al. 2008 rabborum thermoregulation Camouflage or Anura Hylidae Ecnomiohyla sukia Brown to brown and green RCC Savage and Kubicki 2010 thermoregulation Green to dark brown in hours/ develop RCC- Epi, light, temp, melatonin, α Thermoregulation Anura Hylidae Hyla arborea Gomez et al. 2009; Nielsen 1978 colourful vocal sac and stripe in days (M) SCC + β-MSH and ACTH /sexual signal

Bagnara et al. 1968; Snyder and Anura Hylidae Hyla arenicolor Brown to grey in a few hours RCC MSH Thermoregulation Hammerson 1993 Anura Hylidae Hyla chrysoscelis Green grey to brown in seconds RCC Light and temp Thermoregulation Conant and Collins 1998 Camouflage and Anura Hylidae Hyla crucifer Brown to light or dark in 45 min RCC Light and temp Kats and van Dragt 1986 thermoregulation Anura Hylidae Hyla gratiosa Bright green to olive green in 30 mins RCC Unknown Caldwell 1963

Green brown, dark lateral stripe to light stripe Anura Hylidae Hyla heinzsteinitzi RCC Temp Thermoregulation Grach et al. 2007 (day-night) Camouflage and Anura Hylidae Hyla japonica Green to brown in 1 hour RCC Choi and Jang 2014 thermoregulation Anura Hylidae Hyla meridionalis Green RCC Unknown Dufresnes et al. 2011; Stöck et al. 201

Camouflage and Stegen et al. 2004; Wente and Phillips Anura Hylidae Hyla regilla Brown to Green in hours0weeks RCC Light and temp thermoregulation 2003 and 2005 Anura Hylidae Hyla sarda Green to grey RCC Unknown Nöllert and Nöllert 1992

Anura Hylidae Hyla squirella Green to brown in 10 mins RCC Camouflage Wright 2002

Camouflage and Anura Hylidae Hyla versicolor Green grey to brown in seconds RCC Epi, Light and Temp Logier 1952; Parker 2012 thermoregulation Green (J) to tan or red brown (A)/tan or red OCC/ Thermoregulation Anura Hylidae Hypsiboas crepitans Murphy and Murphy 1997 brown to grey or green upon capture RCC /unknown Hypsiboas Tan with black legs and spots (J) to brown, Anura Hylidae OCC Unknown Toledo and Haddad 2009 geographicus green, tan, grey (A) Dark brown (J) to light brown with yellow Anura Hylidae Hypsiboas lundii OCC Unknown Toledo and Haddad 2009 pattern (A) Camouflage Anura Hylidae Hypsiboas prasinus Green to brown in a few months SCC Toledo and Haddad 2009 (habitat) Anura Hylidae Hypsiboas raniceps Green (J) to green, brown tan (A) OCC Unknown Toledo and Haddad 2009

Hypsiboas Dark brown (J) to light brown with yellow Anura Hylidae OCC Unknown Toledo and Haddad 2009 semilineatus pattern (A) White with brown spots (J) to brown, yellow, Anura Hylidae Hypsiboas pardalis OCC Unknown Toledo and Haddad 2009 green (A) bright green (J) to grey, tan to dark brown Anura Hylidae Litoria australis OCC Unknown Anstis 2013 (A) Litoria Anura Hylidae Brown (J) to green (A) OCC Unknown Anstis 2013 barringtonensis Anura Hylidae Litoria chloris Brown (J) to green (A) OCC Unknown Anstis 2013

Anura Hylidae Litoria citropa Brown (J) to green and red (A) OCC Unknown Anstis 2013

Anura Hylidae Litoria daviesae Golden brown (J) to green and brown stripes OCC Unknown Anstis 2013

Anura Hylidae Litoria genimaculata Green to brown in 1 week RCC Thermoregulation Kenyon et al. 2010

Anura Hylidae Litoria gracilenta Brown (J) to green (A) OCC Unknown Anstis 2013

Anura Hylidae Litoria infrafrentata yellow green to brown in 30 mins RCC Camouflage and Anstis 2013

thermoregulation Litoria Anura Hylidae Brown (J) to green or brown (A) OCC Unknown Anstis 2013 kroombitensis Anura Hylidae Litoria nudidigita Brown (J) to green (A) OCC Unknown Anstis 2013

Possible Anura Hylidae Litoria pearsoniana Green, beige or brown SCC Anstis 2013 camouflage Anura Hylidae Litoria phyllochroa Brown (J) to green (A) OCC Unknown Anstis 2013

Litoria Anura Hylidae Brown (J) to green or brown (A) OCC Unknown Anstis 2013 subglandulosa Anura Hylidae Litoria tornieri Beige during day to red brown at night RCC Camouflage Anstis 2013

Anura Hylidae Litoria xanthomera Brown (J) to green (A) OCC Unknown Anstis 2013

Osteocephalus Anura Hylidae Black-grey (J) to brown (F) or tan (M) OCC Unknown Jungfer and Hödl 2002 mimeticus Osteocephalus Anura Hylidae Grey (J) to brown (F) or tan (M) OCC Unknown Jungfer and Hödl 2002 taurinus Pachymedusa Anura Hylidae Brown to green RCC Light and temp Camouflage Bagnara and Hadley 1973 dacnicolor Camouflage or Anura Hylidae Phasmahyla guttata Green during day to purple brown at night RCC Machado et al. 2015 defence Possible Anura Hylidae Phasmahyla jandaia Green during day to purple brown at night RCC Toledo and Haddad 2009 thermoregulation Phasmahyla cochr Possible Anura Hylidae Green during day to purple brown at night RCC Toledo and Haddad 2009 anae thermoregulation Phyllomedusa Possible Anura Hylidae Green during day to purple brown at night RCC Toledo and Haddad 2009 azurea thermoregulation Phyllomedusa Possible Anura Hylidae Green during day to purple brown at night RCC Toledo and Haddad 2009 megacephala thermoregulation Phyllomedusa Possible Anura Hylidae Green during day to purple brown at night RCC Toledo and Haddad 2009 rohdei thermoregulation Anura Hylidae Smilisca phaeota Tan to green RCC Thermoregulation Leenders 2001

Yellow orange (J) to green (A)/green to black OCC/R Anura Hyperoliidae Acanthixalus sonjae Light Unknown Rödel 2000; Roedel et al. 2009 in a few mins CC Anura Hyperoliidae Afrixalus laevis White during day to yellow at night RCC Unknown Schiøtz 1999

Chrysobatrachus Green (J) to green and brown with black dots Anura Hyperoliidae OCC Unknown Schiøtz 1999 cupreonitens (A) Heterixalus Brown (J) to blue grey with yellow (A)/ OCC/R Thermoregulation Anura Hyperoliidae Light Glaw and Vences 2007 madagascariensis lighten to white CC /unknown Heterixalus Anura Hyperoliidae White during day to yellowish brown at night RCC Light Thermoregulation Glaw and Vences 2007 punctatus Hyperolius RCC- Anura Hyperoliidae Yellow green during day to white by night Camouflage Kingdon 1989 horstockii SCC Brown with darker spots (J) to lighter brown Anura Hyperoliidae Hyperolius mitchelli OCC Unknown Schiøtz 1999 with darker spots and white stripe (F)

Green or brown with light dorsolateral lines Anura Hyperoliidae Hyperolius molleri OCC Unknown (Schiøtz 1999 (J) green or brown (A) Anura Hyperoliidae Hyperolius nitidulus Bright colours during day to beige at night RCC Unknown Lampert 2001

Green (J) to silver to red to brown with dark OCC/ Anura Hyperoliidae Hyperolius ocellatus Unknown Schiøtz 1999 spots (F)/lighten or darken RCC Camouflage or Anura Mantellidae Boophis albilabris Greenish brown- darkens at night RCC Glaw and Vences 2007 thermoregulation Camouflage or Anura Mantellidae Boophis lichenoids Cream during day to mottled brown at night RCC Vallan et al. 1998 thermoregulation Boophis Light green with dark brown spots (J) to Anura Mantellidae OCC Unknown Glaw and Vences 2007 madagascariensi brown (A) Possible Anura Mantellidae Boophis viridis Green during day to reddish brown at night RCC Glaw and Vences 2007 thermoregulation Anura Microhylidae Oreophyrne ezra Brown with spots (J) to pink (A) OCC Unknown Kraus and Allison 2009

Brown bright blue spots (J)to beige brown Anura Microhylidae Stumpffia be OCC Unknown Köhler et al. 2010 (A) Anura Microhylidae Cophixalus kethuk Brown by day to orange brown T NIGHT RCC Unknown Kraus and Allison 2009

Anura Microhylidae Dyscophus antongilii Dull yellow to yellow (M) or red (F) OCC Unknown Tessa et al. 2007

Anura Microhylidae Dyscophus guineti Dull yellow to yellow (M) or red (F) OCC Unknown Glaw and Vences 2007

Red brown to grey brown, yellow to yellow Anura Microhylidae Otophryne pyburni RCC Unknown Campbell and Clarke 1998 to grey Heleioporus Anura Myobatrachidae Brown to brown with white spots OCC Unknown Anstis 2013 albpunctatus Camouflage and Filadelfi and Castrucci 1996; Roubos Anura Pipidae Xenopus laevis Grey brown-green patterns in 1 hour RCC Light, MSH, Epi and ACTH thermoregulation 1997 Ptychadena Anura Ptychadenidae Brown OCC Unknown Richards and Nace 1983 mascrareniensis Pelophylax Anura Ranidae Green to yellow RCC Camouflage Nielsen 1979 esculentus Green brown to dark brown in 5 hours (48 Possible Anura Ranidae Rana catesbeiana RCC MSH Camargo et al. 1999 hours to lighten) camouflage Olive brown patches darken or lighten in 10 Possible Anura Ranidae Rana chiricahuensis RCC MSH, light and temperature Fernandez and Bagnara 1991 mins camouflage MSH, T, Pro, Epi, light, Himes and Hadley 1971; Richards Anura Ranidae Rana pipiens Green brown patches RCC Unknown temp and Nace 1983 Green brown with black patches (J) to green Habitat difference between Anura Ranidae Rana septentrionalis OCC Camouflage Kramek and Stewart 1980 brown with brown patches (A) life stage Philautus spp Brown, darken or lighten to match leaf litter Anura Rhacophoridae RCC Camouflage Bahir et al. 2005 (ground nesting) during amplexes Raorchestes Anura Rhacophoridae Grey blue to dark grey <48 hours RCC Unknown Halley and Goel 2012 akroparallagi Raorchestes Brown or green to dark brown or <48 hours Anura Rhacophoridae RCC Unknown Halley and Goel 2012 chromasynchysi green

Raorchestes Anura Rhacophoridae Brown-green-darkens and lightens RCC Camouflage Biju et al. 2005 nerostagona Rhacophorus Anura Rhacophoridae Green during day to brown at night RCC Thermoregulation Dehling and Grafe 2008 penanorum Grey (J) to brown (A)/grey to brown in 4 OCC/ Camouflage/ Garcia et al. 2003; Garcia and Sih Chordata Ambystomatidae Ambystoma texanum Temp hours RCC UVR protection 2003; Garcia et al. 2004 Ambystoma Grey (J) to brown (A)/grey to brown in 4 OCC/ Camouflage/ Garcia et al. 2003; Garcia and Sih Chordata Ambystomatidae Temp barbouri hours RCC UVR protection 2003; Garcia et al. 2004 Ambystoma tigrinum darken or lighten (J), Pattern change (A) in 2- OCC/ Camouflage/ Chordata Ambystomatidae Light Fernandez Jr and Collins 1988 nebulosum 3 hours RCC Unknown Aneides Chordata Plethodontidae Green to grey (J), grey with white spots (A) OCC Camouflage Larson 1980 flavipunctatus Bolitoglossa Mottled dark to brown to salmon/tan in 15 Chordata Plethodontidae RCC Light Camouflage Guyer et al. 2004 colonnea mins Aposematism- Chordata Plethodontidae Plethodon cinereus Brown red (J) to green brown (A) OCC Kraemer et al. 2012; Tilley et al. 1982 mimicry Pseudotriton Aposematism- Huheey and Brandon 1974; Pough Chordata Plethodontidae Red (J) to brown (A) OCC montanus diastictus mimicry 1974 Pseudotriton Aposematism- Huheey and Brandon 1974; Pough Chordata Plethodontidae Red (J) to brown (A) OCC montanus mimicry 1974 Aposematism- Huheey and Brandon 1974; Pough Chordata Plethodontidae Pseudotriton ruber Red (J) to brown (A) OCC mimicry 1974 Notophthalmus Huheey and Brandon 1974; Pough Chordata Salamandridae Red (J) to green (A) OCC Aposematic signal viridescens 1974 Chordata Salamandridae Salamandra corsica Black and yellow (J) to more yellow (A) OCC Unknown Beukema 2011

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Savage, J. M., Ugarte, C. A., & Donnelly, M. A. (2013). A new species of earless toad (Bufonidae: Incilius) from western Panama. Copeia, 2013(1), 8-12. doi:http://dx.doi.org/10.1643/CH-12-095 Schiøtz, A. (1999). Treefrogs of Africa. Frankfurt, Germany: Chimaira. Snyder, G., & Hammerson, G. (1993). Interrelationships between water economy and thermo-regulation in the Canyon tree-frog Hyla arenicolor. Journal of arid environments, 25(3), 321-329. doi:10.1006/jare.1993.1065 Stegen, J. C., Gienger, C., & Sun, L. (2004). The control of color change in the Pacific tree frog, Hyla regilla. Canadian journal of zoology, 82(6), 889-896. doi:10.1139/z04-068 Stöck, M., Horn, A., Grossen, C., Lindtke, D., Sermier, R., Betto-Colliard, C., . . . Luquet, E. (2011). Ever-young sex chromosomes in European tree frogs. PLoS Biology, 9(5), e1001062. doi:10.1371/journal.pbio.1001062 Sturaro, M. J., & Peloso, P. L. V. (2014). A new species of Scinax Wagler, 1830 (Anura: Hylidae) from the middle Amazon river basin, Brazil. Papéis Avulsos de Zoologia (São Paulo), 54(2), 9-23. doi:10.1590/0031-1049.2014.54.02 Sumadh, K. (2012). Hypsiboas boans (Giant Tree Frog). Sztatecsny, M., Preininger, D., Freudmann, A., Loretto, M.-C., Maier, F., & Hödl, W. (2012). Don’t get the blues: conspicuous nuptial coloration of male moor frogs (Rana arvalis) supports visual mate recognition during scramble competition in large breeding aggregations. Behavioral Ecology and Sociobiology, 66(12), 1587-1593. doi:10.1007/s00265-012-1412-6 Sztatecsny, M., Strondl, C., Baierl, A., Ries, C., & Hödl, W. (2010). Chin up: are the bright throats of male common frogs a condition-independent visual cue? Animal Behaviour, 79(4), 779-786. doi:10.1016/j.anbehav.2010.01.003 Tabassum, F., Rais, M., Anwar, M., Mehmood, T., Hussain, I., & Khan, S. A. (2011). Abundance and breeding of the common skittering frog (Euphlyctis cyanophlyctis) and bull frog (Hoplobatrachus tigerinus) at Rawal Lake, Islamabad, Pakistan. Asian Herpetol Research, 2, 245-250. doi:10.3724/SP.J.1245.2011xxxxxx Tang, Z.-J., Lue, S.-I., Tsai, M.-J., Yu, T.-L., Thiyagarajan, V., Lee, C.-H.,Weng, C.-F. (2014). The hormonal regulation of color changes in the sexually dichromatic frog Buergeria robusta. Physiological and Biochemical Zoology, 87(3), 397-410. doi:10.1086/675678 Tapley, B., & Purushotham, C. B. (2011). Fighting behaviour in the Bicoloured frog Clinotarsus (Rana) curtipes Jerdon, 1854. Herpetology Notes, 4, 353-355 107

Tattersall, G. J., Eterovick, P. C., & de Andrade, D. V. (2006). Tribute to RG Boutilier: skin colour and body temperature changes in basking Bokermannohyla alvarengai (Bokermann 1956). Journal of Experimental Biology, 209(7), 1185-1196. doi:10.1242/jeb.02038 Tessa, G., Guarino, F. M., Giacoma, C., Mattioli, F., & Andreone, F. (2007). Longevity and body size in three populations of Dyscophus antongilii (Microhylidae, Dyscophinae), the tomato frog from north-eastern Madagascar. Acta Herpetologica, 2(2), 139-146. doi:http://dx.doi.org/10.13128/Acta_Herpetol-2218 Tilley, S. G., Lundrigan, B. L., & Brower, L. P. (1982). Erythrism and mimicry in the salamander Plethodon cinereus. Herpetologica, 38(3), 409-417. Toledo, L. F., & Haddad, C. F. (2009). Colors and some morphological traits as defensive mechanisms in anurans. International Journal of Zoology, 2009. doi:10.1155/2009/910892 Valakos, E. D., Pafilis, P., Sotiropoulos, K., Lymberakis, P., Maragou, P., & Foufpoulos, J. (2008). The amphibians and reptiles of Greece. Frankfurt, Germany: Chimaira/Serpent’s Tale. Vallan, D., Glaw, F., Andreone, F., & Cadle, J. E. (1998). A new treefrog species of the genus Boophis (Anura: Ranidae: Rhacophorinae) with dermal fringes from Madagascar. Amphibia-Reptilia, 19(4), 357-368. Vásquez, T., & Pfennig, K. S. (2007). Looking on the bright side: females prefer coloration indicative of male size and condition in the sexually dichromatic spadefoot toad, Scaphiopus couchii. Behavioral Ecology and Sociobiology, 62(1), 127-135. doi:10.1007/s00265-012-1412-6 Vences, M., Galán, P., Vieites, D. R., Puente, M., Oetter, K., & Wanke, S. (2002). Field body temperatures and heating rates in a montane frog population: the importance of black dorsal pattern for thermoregulation. Annales Zoologici Fennici, 39, 209-220. Verrell, P. A. (1994). The courtship behaviour of the Apalachicola dusky salamander, Desmognathus apalachicolae Means & Karlin (Amphibia Caudata Plethodontidae). Ethology ecology & evolution, 6(4), 497-506. doi:10.1080/08927014.1994.9522974 Vitt, L. J., & Caldwell, J. P. (2013). Herpetology: an introductory biology of amphibians and reptiles (4th ed.). USA: Academic Press, Elsevier Inc. Wells, K. D. (1977). Territoriality and male mating success in the green frog (Rana clamitans). Ecology, 58(4), 750-762. doi:10.2307/1936211

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Wells, K. D. (2007). The Ecology and Behavior of Amphibians. Chicago, Illinois, USA: University of Chicargo Press. Wente, W. H., & Phillips, J. B. (2003). Fixed green and brown color morphs and a novel color‐changing morph of the Pacific tree frog Hyla regilla. The american naturalist, 162(4), 461-473. doi:10.1086/378253 Wente, W. H., & Phillips, J. B. (2005). Seasonal color change in a population of pacific treefrogs (Pseudacris regilla). Journal of Herpetology, 39(1), 161-165. doi:10.1670/0022-1511 Withers, P. (1995). Evaporative water loss and colour change in the Australian desert tree frog Litoria rubella (Amphibia: Hylidae). Records of the Western Australian Museum, 17, 277-282. Wogan, G. O., Win, H., Thin, T., Lwin, K. S., Shein, A. K., Kyi, S. W., & Tun, H. (2003). A new species of Bufo (Anura: Bufonidae) from Myanmar (Burma), and redescription of the little known species Bufo stuarti Smith 1929. Proceedings of the California Academy of Sciences, 54(7), 141-153. Wright, A. H. (2002). Life-histories of the Frogs of Okefinokee Swamp, Georgia: North American Salientia (Anura) (Vol. 2). Cornell University, USA: Cornell University Press. Zug, G. R., & Zug, P. B. (1979). The marine toad, Bufo marinus: a natural history resume of native populations: Smithsonian Institution Press Washington, DC

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Appendix 3: Supplementary data for chapter 4

Table S1: Individual frog colour score values at for each time point and treatment.

Frog time colour Treatment 1 120 3.156 epinephrine injection 1 60 3.218 epinephrine injection 1 20 3.179 epinephrine injection 1 10 3.175 epinephrine injection 1 30 3.102 epinephrine injection 1 5 3.046 epinephrine injection 1 0 -1.455 epinephrine injection 2 5 -0.9 saline injection 2 120 -0.9 saline injection 2 0 -0.97 saline injection 2 10 -1.05 saline injection 2 20 -1.43 saline injection 2 60 -1.72 saline injection 2 30 -1.77 saline injection 3 30 3.188 epinephrine injection 3 60 3.17 epinephrine injection 3 120 3.139 epinephrine injection 3 20 3.114 epinephrine injection 3 10 2.951 epinephrine injection 3 5 2.862 epinephrine injection 3 0 -2.941 epinephrine injection 4 5 2.18 saline injection 4 0 2.16 saline injection 4 10 2.15 saline injection 4 20 2.09 saline injection 4 120 2.08 saline injection 4 60 2.06 saline injection 4 30 2.04 saline injection 5 60 3.212 epinephrine injection 5 120 3.145 epinephrine injection 5 5 2.922 epinephrine injection 5 10 2.917 epinephrine injection 5 30 2.842 epinephrine injection 5 20 2.735 epinephrine injection 5 0 -1.846 epinephrine injection 6 0 0.4 saline injection 6 5 -0.41 saline injection 6 30 -0.42 saline injection 6 10 -0.52 saline injection 6 60 -0.67 saline injection 6 20 -0.77 saline injection 6 120 -0.79 saline injection

7 30 3.119 epinephrine injection 7 20 3.095 epinephrine injection 7 120 3.079 epinephrine injection 7 60 3.066 epinephrine injection 7 5 2.949 epinephrine injection 7 10 2.946 epinephrine injection 7 0 0.287 epinephrine injection 8 30 1.69 saline injection 8 120 1.48 saline injection 8 60 1.42 saline injection 8 10 1.37 saline injection 8 20 1.31 saline injection 8 5 1.29 saline injection 8 0 1.12 saline injection 9 120 3.098 epinephrine injection 9 60 3.053 epinephrine injection 9 30 3.011 epinephrine injection 9 10 2.958 epinephrine injection 9 20 2.838 epinephrine injection 9 5 2.774 epinephrine injection 9 0 0.852 epinephrine injection 10 30 0.86 saline injection 10 120 0.59 saline injection 10 60 0.43 saline injection 10 0 0.42 saline injection 10 20 -0.15 saline injection 10 5 -0.19 saline injection 10 10 -0.24 saline injection 11 120 2.491 epinephrine drops 11 60 2.471 epinephrine drops 11 30 2.441 epinephrine drops 11 20 2.314 epinephrine drops 11 10 2.106 epinephrine drops 11 5 0.385 epinephrine drops 11 0 -1.246 epinephrine drops 12 120 2.001 epinephrine drops 12 60 1.999 epinephrine drops 12 30 1.994 epinephrine drops 12 20 1.594 epinephrine drops 12 10 0.659 epinephrine drops 12 5 0.551 epinephrine drops 12 0 0.128 epinephrine drops 13 120 2.611 epinephrine drops 13 20 2.56 epinephrine drops 13 10 2.368 epinephrine drops 13 60 2.241 epinephrine drops 13 30 2.174 epinephrine drops 13 5 2.02 epinephrine drops

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13 0 1.091 epinephrine drops 14 120 2.636 epinephrine drops 14 60 2.606 epinephrine drops 14 20 2.573 epinephrine drops 14 30 2.48 epinephrine drops 14 10 0.834 epinephrine drops 14 5 -1.015 epinephrine drops 14 0 -1.254 epinephrine drops 15 120 1.936 epinephrine drops 15 60 1.352 epinephrine drops 15 20 1.187 epinephrine drops 15 10 1.067 epinephrine drops 15 30 0.929 epinephrine drops 15 5 0.535 epinephrine drops 15 0 -0.154 epinephrine drops 16 0 -0.05 testosterone drops 16 5 0.05 testosterone drops 16 10 0.62 testosterone drops 16 20 0.5 testosterone drops 16 30 0.87 testosterone drops 16 60 0.85 testosterone drops 16 120 1.07 testosterone drops 17 0 0.11 testosterone drops 17 5 -0.96 testosterone drops 17 10 -0.4 testosterone drops 17 20 -0.2 testosterone drops 17 30 0.07 testosterone drops 17 60 0.09 testosterone drops 17 120 0.19 testosterone drops 18 0 -0.54 testosterone drops 18 5 -0.96 testosterone drops 18 10 -0.09 testosterone drops 18 20 -0.19 testosterone drops 18 30 -0.13 testosterone drops 18 60 -0.22 testosterone drops 18 120 -0.22 testosterone drops 19 0 0.55 testosterone drops 19 5 0.56 testosterone drops 19 10 0.49 testosterone drops 19 20 0.55 testosterone drops 19 30 -0.13 testosterone drops 19 60 -0.35 testosterone drops 19 120 -0.35 testosterone drops 20 0 -0.03 testosterone drops 20 5 -1.12 testosterone drops 20 10 -1.26 testosterone drops 20 20 -0.1 testosterone drops 20 30 0.1 testosterone drops

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20 60 -0.15 testosterone drops 20 120 -0.09 testosterone drops 21 0 0.85 testosterone injection 21 5 0.82 testosterone injection 21 10 0.83 testosterone injection 21 20 0.86 testosterone injection 21 30 0.87 testosterone injection 21 60 0.84 testosterone injection 21 120 0.91 testosterone injection 22 0 -0.44 oil injection 22 5 -0.39 oil injection 22 10 -0.38 oil injection 22 20 -0.43 oil injection 22 30 -0.46 oil injection 22 60 -0.43 oil injection 22 120 -0.31 oil injection 23 0 1.79 testosterone injection 23 5 1.81 testosterone injection 23 10 1.85 testosterone injection 23 20 1.82 testosterone injection 23 30 1.83 testosterone injection 23 60 1.87 testosterone injection 23 120 1.91 testosterone injection 24 0 1.89 oil injection 24 5 1.95 oil injection 24 10 1.73 oil injection 24 20 1.68 oil injection 24 30 1.63 oil injection 24 60 1.61 oil injection 24 120 1.68 oil injection 25 0 0.69 testosterone injection 25 5 0.71 testosterone injection 25 10 0.68 testosterone injection 25 20 0.66 testosterone injection 25 30 0.77 testosterone injection 25 60 0.81 testosterone injection 25 120 0.92 testosterone injection 26 0 0.96 oil drops 26 5 1.05 oil drops 26 10 0.87 oil drops 26 20 0.22 oil drops 26 30 0.73 oil drops 26 60 0.73 oil drops 26 120 0.73 oil drops 27 0 -0.16 oil drops 27 5 0.35 oil drops 27 10 0.08 oil drops 27 20 0.13 oil drops

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27 30 0.36 oil drops 27 60 0.58 oil drops 27 120 0.81 oil drops 28 0 -0.34 oil drops 28 5 0.16 oil drops 28 10 0.14 oil drops 28 20 -0.08 oil drops 28 30 0.14 oil drops 28 60 -0.08 oil drops 28 120 -0.29 oil drops 29 60 0.7 saline drops 29 20 0.67 saline drops 29 30 0.66 saline drops 29 5 0.65 saline drops 29 0 0.63 saline drops 29 120 0.62 saline drops 29 10 0.6 saline drops 30 60 -0.32 saline drops 30 120 -0.33 saline drops 30 20 -0.38 saline drops 30 30 -0.41 saline drops 30 5 -0.49 saline drops 30 10 -0.51 saline drops 30 0 -0.53 saline drops 31 0 -0.31 oil injection 31 5 -0.42 oil injection 31 10 -0.36 oil injection 31 20 -0.19 oil injection 31 30 -0.22 oil injection 31 60 -0.16 oil injection 31 120 -0.15 oil injection 32 0 1.245 oil injection 32 5 1.28 oil injection 32 10 1.26 oil injection 32 20 1.31 oil injection 32 30 1.24 oil injection 32 60 1.16 oil injection 32 120 1.14 oil injection 33 0 -0.04 oil injection 33 5 -0.12 oil injection 33 10 -0.18 oil injection 33 20 -0.25 oil injection 33 30 0.22 oil injection 33 60 -0.28 oil injection 33 120 -0.25 oil injection 34 0 2.43 oil drops 34 5 2.44 oil drops 34 10 2.47 oil drops

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34 20 1.71 oil drops 34 30 1.9 oil drops 34 60 1.71 oil drops 34 120 1.9 oil drops 35 0 -0.55 oil drops 35 5 -0.44 oil drops 35 10 -0.38 oil drops 35 20 -0.26 oil drops 35 30 0.02 oil drops 35 60 -0.16 oil drops 35 120 0.02 oil drops 36 0 1.47 testosterone injection 36 5 1.53 testosterone injection 36 10 1.65 testosterone injection 36 20 1.61 testosterone injection 36 30 1.64 testosterone injection 36 60 1.651 testosterone injection 36 120 1.895 testosterone injection 37 0 -0.74 testosterone injection 37 5 -0.75 testosterone injection 37 10 -0.73 testosterone injection 37 20 -0.68 testosterone injection 37 30 -0.67 testosterone injection 37 60 -0.63 testosterone injection 37 120 -0.2 testosterone injection 38 0 1.68 saline drops 38 10 1.61 saline drops 38 5 1.58 saline drops 38 20 1.38 saline drops 38 120 1.23 saline drops 38 60 1.21 saline drops 38 30 1.19 saline drops 39 30 1.136 saline drops 39 0 0.14 saline drops 39 20 0.137 saline drops 39 10 0.136 saline drops 39 60 0.132 saline drops 39 120 0.131 saline drops 39 5 0.13 saline drops 40 10 0.21 saline drops 40 60 0.208 saline drops 40 120 0.197 saline drops 40 5 0.195 saline drops 40 20 0.192 saline drops 40 0 0.19 saline drops 40 30 0.19 saline drops 41 0 -1.279 natural 42 0 -0.128 natural

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43 0 2.85 natural 44 0 -2.239 natural 45 0 -2.011 female 46 0 -1.19 natural 47 0 -1.104 natural 48 0 1.404 natural 49 0 2.264 natural 50 0 1.917 natural 51 0 1.917 natural 52 0 1.917 natural 53 0 -1.074 natural 54 0 1.343 natural 55 0 2.204 natural 56 0 2.064 natural 57 0 -1.53 natural 58 0 -1.917 natural 59 0 -1.582 natural 41 5 2.665 natural 42 5 2.67 natural 43 5 2.926 natural 44 5 2.397 natural 45 5 -2.204 female 46 5 2.378 natural 47 5 2.746 natural 48 5 2.404 natural 49 5 3.17 natural 50 5 2.917 natural 51 5 2.4 natural 52 5 2.27 natural 53 5 2.22 natural 54 5 2.546 natural 55 5 2.204 natural 56 5 2.33 natural 57 5 2.148 natural 58 5 1.986 natural 59 5 2.057 natural 41 10 2.498 natural 42 10 3.538 natural 43 10 2.924 natural 44 10 2.454 natural 45 10 -2.235 female 46 10 2.378 natural 47 10 2.746 natural 48 10 2.404 natural 49 10 3.073 natural 50 10 2.917 natural 51 10 2.4 natural 52 10 2.217 natural

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53 10 2.474 natural 54 10 2.548 natural 55 10 2.304 natural 56 10 2.33 natural 57 10 2.169 natural 58 10 1.682 natural 59 10 2.218 natural 41 20 2.641 natural 42 20 3.239 natural 43 20 2.948 natural 44 20 2.454 natural 45 20 -2.228 female 46 20 2.378 natural 47 20 2.746 natural 48 20 2.345 natural 49 20 3.103 natural 50 20 2.917 natural 51 20 2.4 natural 52 20 2.417 natural 53 20 2.615 natural 54 20 2.486 natural 55 20 2.204 natural 56 20 2.33 natural 57 20 2.327 natural 58 20 1.986 natural 59 20 2.416 natural 41 30 2.653 natural 42 30 3.533 natural 43 30 2.763 natural 44 30 2.852 natural 45 30 -2.203 female 46 30 2.383 natural 47 30 2.746 natural 48 30 2.315 natural 49 30 3.103 natural 50 30 2.8 natural 51 30 2.4 natural 52 30 2.3 natural 53 30 2.663 natural 54 30 2.346 natural 55 30 3.015 natural 56 30 2.23 natural 57 30 2.863 natural 58 30 2.393 natural 59 30 2.414 natural 42 60 2.77 natural 43 60 2.82 natural 44 60 2.847 natural

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45 60 -2.235 female 46 60 2.478 natural 47 60 2.746 natural 48 60 2.284 natural 49 60 3.106 natural 50 60 2.83 natural 51 60 2.4 natural 52 60 2.4 natural 53 60 2.4 natural 54 60 2.546 natural 55 60 2.769 natural 56 60 2.23 natural 57 60 2.863 natural 58 60 2.4 natural 59 60 2.391 natural 42 90 2.789 natural 43 90 2.47 natural 44 90 2.84 natural 45 90 -2.086 female 46 90 2.395 natural 47 90 2.746 natural 48 90 2.284 natural 49 90 3.103 natural 50 90 2.8 natural 51 90 2.4 natural 52 90 2.563 natural 53 90 2.4 natural 54 90 2.476 natural 55 90 2.769 natural 56 90 2.33 natural 57 90 2.863 natural 58 90 2.447 natural 59 90 2.37 natural 42 120 2.695 natural 43 120 2.462 natural 44 120 2.818 natural 45 120 -2.207 female 46 120 2.578 natural 47 120 2.746 natural 48 120 2.284 natural 49 120 3.103 natural 50 120 2.8 natural 51 120 2.4 natural 52 120 2.4 natural 53 120 2.563 natural 54 120 2.483 natural 55 120 2.769 natural 56 120 2.33 natural

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57 120 2.563 natural 58 120 2.391 natural 59 120 2.379 natural 42 150 2.732 natural 43 150 2.423 natural 44 150 2.735 natural 45 150 -2.011 female 46 150 2.478 natural 47 150 2.746 natural 48 150 2.284 natural 49 150 3.103 natural 50 150 2.8 natural 51 150 2.4 natural 52 150 2.4 natural 53 150 2.4 natural 54 150 2.646 natural 55 150 2.729 natural 56 150 2.33 natural 57 150 2.709 natural 58 150 2.4 natural 59 150 2.415 natural 42 180 3.301 natural 43 180 2.456 natural 44 180 3.008 natural 45 180 -2.104 female 46 180 2.595 natural 47 180 2.746 natural 48 180 2.284 natural 49 180 3.103 natural 50 180 2.3 natural 51 180 2.4 natural 52 180 2.3 natural 53 180 2.506 natural 54 180 2.746 natural 55 180 2.769 natural 56 180 2.33 natural 57 180 2.473 natural 58 180 2.37 natural 59 180 2.403 natural 44 210 2.734 natural 45 210 -2.135 female 46 210 -0.648 natural 47 210 2.746 natural 48 210 2.276 natural 49 210 3.103 natural 50 210 2.2 natural 51 210 2.4 natural 52 210 2.4 natural

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53 210 2.4 natural 54 210 2.546 natural 55 210 2.061 natural 56 210 -1.873 natural 57 210 2.6 natural 58 210 2.363 natural 59 210 2.4 natural 42 240 -1.066 natural 44 240 -1.233 natural 45 240 -2.128 female 46 240 -1.096 natural 47 240 2.746 natural 48 240 1.023 natural 49 240 2.073 natural 50 240 1.936 natural 51 240 1.936 natural 52 240 1.936 natural 53 240 1.936 natural 54 240 2.533 natural 55 240 0.823 natural 56 240 -2.217 natural 57 240 1.836 natural 58 240 1.936 natural 59 240 1.936 natural 45 270 -2.111 female 47 270 2.746 natural 48 270 0.986 natural 50 270 -0.778 natural 51 270 -1.078 natural 52 270 -1.078 natural 53 270 -2.078 natural 54 270 2.046 natural 55 270 -2.786 natural 57 270 -1.064 natural 58 270 -1.157 natural 59 270 -1.078 natural 45 300 -2.094 female 47 300 2.746 natural 50 300 -1.078 natural 51 300 -1.078 natural 52 300 -1.078 natural 53 300 -2.585 natural 54 300 -0.606 natural 57 300 -1.379 natural 58 300 -1.276 natural 59 300 -1.078 natural 47 330 -0.878 natural 54 330 -1.178 natural

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Appendix 4: Chapter 6 supplementary information

Paint, Dye and Plasticine details and RGB:

 Amplexing male: (R: 254, G:243, B: 48)

 Brown male: (R: 94, G: 64, B: 29)

 Yellow plasticine: CO126775 (R: 255, G: 255, B: 0)

 Rit Dye Tinte: Lemon Yellow (R: 255, G: 255, B: 90)

 Rit Dye Tinte: Cocoa Brown (R: 11, G: 84, B: 52)

 Reeves Acrylic Paint: Lemon Yellow and Medium Yellow blend (R: 254, G: 251,

B:28)

 Reeves Acrylic Paint: Burnt Umber and Burnt Sienna blend(R: 115, G: 73, B: 26)

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