Seleção Sexual E Modelagem Visual Em Ameivula Ocellifera

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE BIOCIÊNCIAS PROGRAMA DE PÓS-GRADUAÇÃO EM ECOLOGIA

CAROLINA MARIA CARDOSO AIRES LISBOA

SELEÇÃO SEXUAL E MODELAGEM VISUAL EM

AMEIVULA OCELLIFERA

NATAL – RN - BRASIL

2017

CAROLINA MARI A CARDOSO AIRES LISBOA

SELEÇÃO SEXUAL E MODELAGEM VISUAL EM AMEIVULA OCELLIFERA

Tese apresentada ao Programa de Pós-Graduação em Ecologia da Universidade Federal do Rio Grande do Norte, como parte das exigências para a obtenção do título de Doutora em Ecologia

Orientador: Dr. Gabriel Corrêa Costa

Coorientador: Dr. Daniel Marques de Almeida Pessoa

Orientador no período sanduíche: Dr. Barry Sinervo - University of California Santa Cruz

NATAL – RN - BRASIL

2017

Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Prof. Leopoldo Nelson - •Centro de Biociências - CB

Lisboa, Carolina Maria Cardoso Aires. Seleção sexual e modelagem visual em Ameivula ocellifera / Carolina Maria Cardoso Aires Lisboa. - Natal, 2017. 128 f.: il.

Tese (Doutorado) - Universidade Federal do Rio Grande do Norte. Centro de Biociências. Programa de Pós-Graduação em Ecologia. Orientador: Prof. Dr. Gabriel Corrêa Costa. Coorientador: Prof. Dr. Daniel Marques de Almeida Pessoa. Coorientador: Prof. Dr. Barry Sinervo.

1. Preferência de fêmeas - Tese. 2. Interações entre machos - Tese. 3. Sinalização UV - Tese. 4. Performance - Tese. 5. Qualidade de machos - Tese. 6. Modelagem visual - Tese. I. Costa, Gabriel Corrêa. II. Almeida Pessoa, Daniel Marques de. III. Sinervo, Barry. IV. Universidade Federal do Rio Grande do Norte. V. Título.

RN/UF/BSE-CB CDU 392.6

AGRADECIMENTOS

Agradecer é reconhecer que não somos autossuficientes e que recebemos da vida o presente de contarmos uns com os outros. Agradeço carinhosamente aos que tornaram esta tese possível.

Ao Programa de Pós-graduação em Ecologia-PPGEco da Universidade Federal do Rio

Grande do Norte, em especial aos dedicados professores, funcionários e colegas, por proporcionarem tanto crescimento com suas ideias e conhecimentos enriquecedores.

À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES e ao

Programa Doutorado Sanduíche no Exterior-PDSE pelas bolsas concedidas e ao Conselho

Nacional de Desenvolvimento Científico e Tecnológico-CNPq pelo financiamento do projeto.

Ao orientador Gabriel Corrêa Costa, pela credibilidade, confiança e constante suporte durante a realização deste projeto. Agradeço imensamente pela oportunidade.

Ao coorientador Daniel Marques de Almeida Pessoa, pelo apoio logístico e intelectual e pelas inúmeras reuniões bastante produtivas para discussão das metodologias.

Ao orientador no período sanduíche, Barry Sinervo, por prontamente me acolher em seu laboratório e por compartilhar um pouco da sua experiência ao longo da estadia na UCSC.

Às colegas de pesquisa e amigas Katalin Bajer e Orsolya Molnár pelas orientações, pela companhia no campo e nos experimentos e por todos os agradáveis momentos. Vocês foram fundamentais para a realização desta pesquisa e me ensinaram mais do que podem imaginar.

Aos amigos e colegas de laboratório Marc Huber, Cleto Freire, Bruno Maggi, Gustavo

Carvalho, André Bruinje, Juan Pablo, Brunno Freire, Eliana Faria, Vinícius São Pedro e

Caterina Penone, pelo auxílio nas coletas, pela convivência e pela agradável companhia.

Aos colegas da UCSC Gabriel Caetano, Pauline Blaimont, Carla Sette e Joseph Stewart pela acolhida e pelos bons momentos de convívio.

À minha família, em especial aos meus pais e aos meus queridos José Petronilo e Ravi, por serem meu alicerce e minha dose diária de amor. À vocês dedico esta tese.

“But the bright colours with which so many lizards are ornamented, as well as their various curious appendages, were probably acquired by the males as an attraction, and then transmitted either to their male offspring alone, or to both sexes. Sexual selection, indeed, seems to have played almost as important a part with reptiles as with birds; and the less conspicuous colours of the females in comparison with the males cannot be accounted for, as Mr. Wallace believes to be the case with birds, by the greater exposure of the females to danger during incubation”.

Charles Darwin The Descent of Man, and Selection in Relation to Sex

RESUMO

A seleção sexual é responsável pela evolução de diversos sinais visuais conspícuos utilizados na comunicação intra e interespecífica de inúmeras espécies. Os lagartos têm sistema visual com fotorreceptores que são sensíveis aos comprimentos de onda UV, e algumas espécies utilizam ornamentos de cor UV na comunicação. Neste estudo, utilizamos espectrofotometria para obter evidências de ornamentação UV em lagartos Ameivula ocellifera. Utilizando um aparato experimental em formato de arena, obtivemos evidências do papel da sinalização UV na seleção sexual (preferência de fêmeas e competição entre machos). Nossos resultados revelaram que a sinalização UV é importante na preferência de fêmeas, uma vez que as mesmas exibem preferência por machos com maior reflectância UV em relação aos machos com reflectância experimentalmente reduzida. Também descobrimos que os machos com UV reduzido não foram mais propensos a perder disputas do que os controle, embora quanto maior a diferença de reflectância UV entre os pares, menor o tempo de avaliação entre os rivais antes do combate. Para avaliar se os sinais de cor são informativos da qualidade, testamos dois ornamentos de machos de A. ocellifera contra traços morfológicos e desempenho fisiológico.

Descobrimos que machos maiores apresentaram comprimentos de onda UV e médio mais intensos nos ocelos dorsolaterais e, em contraste, os machos de cabeça menor tiveram o croma

UV mais intenso nas escamas ventrais exteriores (EVEs). Concluímos que a mesma característica de cor transmite diferentes mensagens dependendo da posição do sinal no corpo dos lagartos, sendo um indicativo de estratégias alternativas de sinalização. Além disso, um maior brilho nas EVEs foi associado a maior força de mordida, sendo este um sinal confiável de capacidade de luta do macho. Esses resultados sugerem que existe um sistema de sinalização múltipla na espécie. Por fim, modelamos os sistemas visuais de A. ocellifera e de dois tipos de predadores (ave de rapina e serpente) para descobrir como as manchas de coloração são percebidas e explorar as consequências da coloração conspícua em termos de pressões

seletivas. Encontramos dicromatismo entre os sexos, com a reflectância UV de machos mais visíveis e altamente distinguíveis dos das fêmeas a partir do sistema visual de A. ocellifera. Os sinais UV foram altamente perceptíveis quando em contraste com a coloração do corpo e do ambiente natural para A. ocellifera e, menos mas ainda perceptíveis, para os predadores, concordando com a hipótese da condução sensorial. Esta tese esclarece o papel dos sinais sexuais e sua importância nas comunicações intra e interespecíficas em lagartos. Nossas descobertas baseiam futuros estudos sobre evolução e comportamento e expandem o conhecimento acerca das seleções natural e sexual propostas por Darwin.

PALAVRAS-CHAVE: Preferência de fêmeas, Interações entre machos, Sinalização UV,

Performance, Qualidade de machos, Modelagem visual

ABSTRACT

Sexual selection is responsible for the evolution of many conspicuous visual signals used in intra and interspecific communication of innumerous species. Lizards have acute visual systems with retinal photoreceptors that are sensitive to UV wavelengths, and some species use UV colour ornaments for communication. In this study, we used UV full-spectrum reflectance spectrophotometry to collect data from Ameivula ocellifera UV structural colouration. Using an arena-form experimental set, we obtained evidence for the role of UV signaling in sexual selection (mate choice and male-male interactions). Our results showed that

UV chroma is important in female association preference, as females exhibit spatial preference for males of higher UV reflectance over males with experimentally reduced UV reflectance.

We also found that A. ocellifera males with experimentally reduced UV reflectance were not more likely to lose contests than control males, although bigger the difference of UV reflectance between pairs, smaller the evaluation time between rivals before the contest. We also tested two male ornaments in A. ocellifera against morphological traits and physiological performance to assess whether colour signals are informative for male quality traits. We found that larger males had more intense short (UV) and medium wavelength chroma on dorsolateral eyespots and, in contrast, smaller-headed males had more intense UV chroma on outer ventral scales (OVS). We concluded that the same colour trait convey different messages depending on the body position of the signal, perhaps indicative of alternative signalling strategies.

Moreover, higher brightness on OVS signals were associated with stronger bite force, being a reliable signal of fighting ability. These results suggest that there is a multiple signalling system in our model species. Finally, we modeled the visual system of A. ocellifera, snake and avian predators to access how colour patches appear to the receivers. We found that there are dichromatism between sexes, with UV signals of males more conspicuous in reflectance and highly distinguishable from females to conspecifics visual system. UV signals were highly

perceptible from body colouration and from natural background to conspecifics and less but still perceptible to predators, agreeing with sensory drive hypothesis. This thesis enlighten the role of sexual signals and their importance on intra and interspecific communications in lizards.

Our findings support further studies on evolution and behavior and expand the knowledge on natural and sexual selections initiated by Darwin.

KEYWORDS: Female preference, Male-male interactions, UV Signaling, Male quality traits,

Performance, Visual modeling

SUMÁRIO

INTRODUÇÃO GERAL ...... 1 Sinalização visual ...... 2 Sinalização UV ...... 4 Estrutura da tese ...... 5 REFERÊNCIAS ...... 6 CAPÍTULO I: Female Brazilian whiptail lizards (Ameivula ocellifera) prefer males with high ultraviolet ornament reflectance ...... 10 ABSTRACT...... 11 INTRODUCTION...... 12 MATERIAL AND METHODS ...... 14 RESULTS AND DISCUSSION ...... 19 REFERENCES ...... 24 CAPÍTULO II: The role of UV colour signals on male competition in the Brazilian whiptail lizard (Ameivula ocellifera) ...... 33 ABSTRACT...... 34 INTRODUCTION...... 35 METHODS ...... 37 RESULTS ...... 43 DISCUSSION ...... 45 REFERENCES ...... 47 CAPÍTULO III: Similar ornaments, distinct messages: sexual signals in male Brazilian whiptail lizard (Ameivula ocellifera) ...... 55 ABSTRACT...... 56 INTRODUCTION...... 58 METHODS ...... 62 RESULTS ...... 66 DISCUSSION ...... 67 REFERENCES ...... 73 CAPÍTULO IV: Colour signals conspicuousness of Brazilian whiptail lizard (Ameivula ocellifera) through conspecifics and predators visual systems ...... 82 ABSTRACT...... 82 INTRODUCTION...... 83 MATERIAL AND METHODS ...... 87 RESULTS ...... 90 DISCUSSION ...... 92 REFERENCES ...... 98 CONSIDERAÇÕES FINAIS ...... 117 ANEXOS ...... 119

INTRODUÇÃO GERAL

A seleção sexual é um conceito introduzido por Darwin no livro A Origem das Espécies (1859), elaborando a ideia em A Descendência do Homem e Seleção em Relação ao Sexo (1874). Pode ser definida como um caso especial de seleção natural que atua na variação entre membros de um sexo na habilidade de obter parceiros, cópulas ou fertilizações (Savalli, 2001). Darwin identificou que a seleção sexual pode operar de duas formas: através da competição para atrair membros do sexo oposto, referindo-a como seleção de parceiros ou seleção intersexual; ou através da disputa entre membros de um mesmo sexo ao acesso a parceiros ou a recursos que irão atrair parceiros, conhecida como seleção intrasexual ou, por usualmente envolver machos, competição entre machos.

Seleção intersexual de parceiros e competição intrasexual são responsáveis pela evolução de diversos tipos de ornamentos ou sinais visuais conspícuos (revisados por

Andersson, 1994). Nesse contexto, interessantes experimentos têm sido conduzidos com aves, como por exemplo o alongamento da cauda em machos de andorinha-das-chaminés, Hirundo rustica, os levando a obter parceiras mais rapidamente que os machos com caudas mais curtas, aumentando assim seu sucesso reprodutivo (Møller, 1998); ou com fêmeas silvestres do tentilhão Carpodacus mexicanus que, podendo escolher dentre quatro machos, mostravam significativa preferência pelo mais colorido (Hill, 1990).

Embora esses sinais sejam frequentemente atribuídos à seleção de parceiros, eles podem funcionar como “aviso” da presença de um indivíduo em um habitat complexo (Fleishman,

1988), para mediar disputas (Murphy et al., 2009), sinalizar dominância (Zucker, 1989) ou habilidade de combate para intimidar rivais em competições intrasexuais (Thompson e Moore,

1991). As disputas podem variar consideravelmente no grau de escalada dos rituais de exibição para combates. Contudo, se os adversários podem avaliar a probabilidade relativa de ganhar a competição, então o resultado pode ser resolvido sem a necessidade de disputas escaladas 2

(Maynard Smith e Parker, 1973, 1976). Experimentos de disputas pareadas sugerem isso, como os com a codorna-de-Gambel, Callipepla gambelii, nos quais o aprimoramento das plumas da cabeça dos machos tornava-os vencedores mais prováveis, enquanto a remoção os tornava mais propensos a perder disputas (Hagelin, 2002).

Sinalização visual

Sistemas visuais fotossensíveis ao ultravioleta foram estudados em alguns grupos de lagartos, como em Sphaerodactylidae (Ellingson et al., 1995), Gekkonidae (Loew, 1994; Roth e Kelber, 2004), duas espécies de Chamaeleonidae (Bowmaker et al., 2005), um Cordylidae

(Fleishman et al. 2011), em muitos Lacertidae (Pérez i de Lanuza e Font 2014, Martin et al.

2015) e em várias espécies de lagartos do gênero Anolis (Fleishman et al., 1993; Loew et al.,

2002). Por outro lado, uma ampla variedade de espécies de lagartos possuem ornamentos com coloração UV, que podem ser utilizados como sinais visuais (e.g. LeBas e Marshall 2000,

Stuart-Fox et al. 2007, Font et al. 2009), mas em poucos casos foi provado que a reflectância

UV realmente influencia a escolha de parceiros (Thorpe e Murielle 2001, Bajer et al.2010).

Apesar de os sinais de cor poderem exibir uma ampla gama de funções, eles são inúteis se receptor do sinal não puder detectá-lo. Portanto, a detectabilidade é uma parte crítica do desenho de um sinal visual (Dawkins e Guilford 1997; Fleishman 2000, Leal e Fleishman

2004).

Estudos sugerem que a visão UV é importante na comunicação intraespecífica em animais com ornamentos reflexivos, como na corte (Silberglied, 1978) e preferência de fêmeas em borboletas (Kemp, 2007; Robertson, 2005); comportamento territorial em peixes de recifes de corais (Siebeck, 2004); escolha de parceiros em aves (Andersson et al., 1998; Bennett et al.,

1996; Hunt et al., 1997; Hunt et al., 1999); e preferências de machos e fêmeas em répteis

(LeBas e Marshall, 2000; Bajer et al., 2010), com interesse crescente nesses tipos de estudos. 3

Contudo, a avaliação de cores na comunicação animal deve ser realizada de forma independente do sistema visual humano (Endler, 1990; Bennett et al., 1994), pois temos apenas três classes espectrais de células cone da retina, percebendo a luz somente nos comprimentos de onda entre 400 e 700 nm. Portanto, não estamos aptos a percebê-la na faixa ultravioleta

(300-400 nm), dependendo de equipamentos (Endler, 1990) para medir fisicamente a faixa de comprimento de onda visível das espécies estudadas.

Entre os lagartos, estudos de reflectância UV na coloração corporal foram realizados em espécies de Agamidae (Le Bas e Marshall, 2000), Chamaeleonidae (Gehring e Witte, 2007),

Scincidae (Blomberg et al., 2001), Lacertidae (Molina-Borja et al., 2006; Pérez I de Lanuza e

Font, 2007; Font et al., 2009), e especialmente em Polychrotidae do gênero Anolis (Macedonia,

2001; Stoehr e McGraw, 2001; Thorpe, 2002; Macedonia et al., 2003; Thorpe e Stenson, 2003).

A coloração corporal de algumas espécies de lagartos possuem ocelos, manchas aproximadamente circulares de escamas nos flancos de indivíduos adultos, contrastando com as cores laterais dos corpos, como observados no lagarto ocelado Lacerta (Timon) lepida do sudoeste da Europa (Bischoff et al., 1984; Hall, 2008; Font, 2009).

Apesar da grande variedade de ornamentos coloridos e dos complexos comportamentos de exibição, sabe-se pouco sobre a seleção sexual ou escolha de parceiros em lagartos (e.g.

Tokarz, 1995; Olsson e Madsen, 1998; LeBas e Marshall, 2000; LeBas e Marshall, 2001; López et al., 2002; López e Martín, 2005; Martín e López 2009). A escolha de parceiros depende frequentemente da coloração, pois ela pode sinalizar a qualidade do macho e é, portanto, adequada para prever com confiança o resultado da escolha de parceiros pelas fêmeas

(Andersson, 1994). Apesar de algumas conclusões de que fêmeas de lagartos geralmente não escolhem seus parceiros (Olsson e Madsen 1995; Tokarz 1995), foram registradas algumas evidências desse comportamento (Censky, 1997; Bajer et al., 2010). 4

Há evidências de que o tamanho do corpo e da cabeça dos machos possui um importante papel na preferência das fêmeas de lagartos (e.g. Stamps 1983; Cooper e Vitt 1993), mas poucos estudos têm abordado a escolha de parceiros baseada na coloração (ex. Hamilton e

Sullivan, 2005; LeBas e Marshall, 2000; LeBas e Marshall, 2001; Bajer et al., 2010). Antes de

LeBas e Marshall (2000, 2001), os estudos utilizavam definições de cor subjetivas, sem explorar o papel dos espectros de cor na escolha de parceiros.

Sinalização UV

O papel da sinalização ultravioleta na comunicação intraespecífica tem sido estudado recentemente em lagartos (Stapley e Whiting, 2006; Whiting et al., 2006; Font et al., 2009;

Bajer et al., 2010). Em relação à escolha de parceiros, os únicos estudos que envolvem o papel do UV em répteis são os de LeBas e Marshall (2000) com o agamídeo Ctenophorus ornatus, no qual o macho prefere fêmeas com alta reflectância de UV, e o recente Bajer et al. (2010) com o lagarto verde europeu (Lacerta viridis), no qual as fêmeas preferem machos com alta reflectância ultravioleta na região gular. O papel dos sinais UV nas disputas entre machos tem sido explorado em alguns estudos (Alonso-Alvarez et al., 2004; Siebeck, 2004; Siefferman e

Hill, 2005). Em lagartos, somente os estudos de Stapley e Whiting (2006) e Whiting et al.

(2006) são conhecidos.

Baseados nesses aspectos, podemos sugerir a hipótese de que a reflectância UV nos padrões de coloração corporal é utilizada como sinalização em outras famílias de lagartos que vivem em ambientes tropicais ricos em UV, como os Teiidae. Podemos também inferir que esses padrões são importantes sinais de comunicação intra e interespecífica.

Consequentemente, o presente estudo visa explorar experimentalmente esses aspectos, utilizando como sistema de estudo o lagarto Ameivula ocellifera.

Estrutura da tese

Num primeiro momento, nossos resultados revelaram que machos e fêmeas de Ameivula ocellifera são mutualmente ornamentados (exibem manchas com coloridos similares) e possuem dicromatismo sexual críptico (machos refletem a cor ultravioleta com mais intensidade do que as fêmeas). Estas características fizeram da espécie um modelo ideal para testar o papel dos ornamentos sexualmente selecionados em relações intra e intersexuais, e levantaram questões sobre quais aspectos qualitativos tais ornamentos poderiam sinalizar durante a comunicação intraespecífica e como se dá a percepção desses sinais. Assim, os quatro capítulos dessa tese, aqui brevemente resumidos, foram estruturados de acordo com tais questões, conforme se segue.

No primeiro capítulo, realizamos experimentos de seleção sexual para verificar como os ornamentos com coloração ultravioleta (UV), especificamente os ocelos e escamas ventrais exteriores (EVEs), influenciam na escolha do parceiro sexual pelas fêmeas em A. ocellifera. O resultados desde estudo foram publicados na revista Behavioural Processes (Anexo I). No segundo capítulo, realizamos experimentos de competição entre machos para observar o papel da coloração UV sobre tais interações. No terceiro capítulo, buscamos a relação entre a coloração dos ornamentos e os aspectos qualitativos dos machos, testados através de medições de força de mordida, performance locomotora e morfometria, para determinar quais características tais ornamentos sinalizam e observar a honestidade dos sinais. No quarto capítulo, realizamos um estudo de modelagem visual em A. ocellifera em parceria com o laboratório do Dr. Barry Sinervo, em Santa Cruz, CA, EUA, durante o doutorado sanduíche oferecido pelo Programa Doutorado Sanduíche no Exterior (PDSE) da CAPES. Os sistemas visuais de A. ocellifera e de dois tipos de predadores foram modelados para desvendar como a coloração conspícua de A. ocellifera é percebida.

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CAPÍTULO I: FEMALE BRAZILIAN WHIPTAIL LIZARDS (AMEIVULA OCELLIFERA)

PREFER MALES WITH HIGH ULTRAVIOLET ORNAMENT REFLECTANCE

Carolina M. C. A. Lisboa1*, Katalin Bajer,1,2 Daniel M. A. Pessoa3 Marc Huber1 and Gabriel

C. Costa4

1Laboratory of Biogeography and Macroecology, Department of Ecology, Federal University of Rio Grande do Norte, Campus Universitário, Lagoa Nova, Natal, RN, 59078-900, Brazil.

2Behavioural Ecology Group, Department of Systematic Zoology and Ecology, Eötvös Loránd

University, Pázmány Péter sétány 1/c, H-1117, Budapest, Hungary

3Laboratory of Sensory Ecology, Department of Physiology, Federal University of Rio Grande do Norte, Campus Universitário, Lagoa Nova, Natal, RN, 59078-900, Brazil.

4Department of Biology, Auburn University at Montgomery, Montgomery, AL 36124, USA.

Running title: UV preference in lizard sexual selection

Manuscript type: Original article

*Corresponding author. Laboratory of Biogeography and Macroecology, Department of

Ecology, Federal University of Rio Grande do Norte, Campus Universitário, Lagoa Nova,

Natal, RN, 59078-900. Phone/Fax: 55-84-3342-2334. Email: [email protected]

ABSTRACT

Conspicuous colouration is an important way of social communication in many taxa. The role of ultraviolet (UV) signals in intraspecific communication has only recently been studied in lizards, and there is not a general understanding of the adaptive role of UV colouration. Colour ornaments can signal male quality in mate choice and are therefore suitable for reliably predicting the outcome of female preference. Here, we tested the potential role of UV colouration in female spatial preference in a non-territorial teiid lizard, Ameivula ocellifera.

We experimentally manipulated the UV reflectance of size-matched male pairs and tested the effects of our treatment on females’ spatial distribution. We found that females associated with males of higher UV reflectance, suggesting that UV colour can be an important clue during mate preference decisions. Our results provide the first empirical evidence for the importance of UV colouration in female preference in a mutually ornamented lizard species.

Keywords: female preference, mate choice, sexual selection, signalling, ultraviolet colouration 12

INTRODUCTION

Conspicuous colour patches are important elements of social communication (Bradbury and

Vehrencamp 2011). The adaptive significance of colour signals has been shown in numerous studies, including sex recognition (Schultz and Fincke 2009), mate acquisition (Bennett et al.

1997; MacDougall and Montgomerie 2003) and rival deterring (Pryke et al. 2001). Studies on sexual colour signals commonly use model systems showing substantial sexually dimorphic ornamentation, often even define sexually selected traits as being sexually dimorphic (e.g.

Andersson 1994). However, there are many species in which both males and females are similarly ornamented (mutually ornamented species, Nordeide et al. 2013). To understand how sex-specific evolutionary mechanisms are responsible for maintaining sexually monomorphic ornamentation, it is important to explore the function of the ornament as a social or sexual signal in mutually ornamented species.

Reliability of information emitted by signaller arises from costs linked to signal elaboration, maintenance or behavioural displays (Grafen 1990; Zahavi 1975). For instance, carotenoid-based colouration is costly to produce (reviewed in: Hill 2006; Svensson and Wong

2011), as carotenoid pigments need to be incorporated by a special diet and there is trade-off between their utilization as ornament components versus physiologically important molecules

(Blount and McGraw 2008; McGraw et al. 2006). Hence, carotenoid colours were thoroughly investigated in a range of model taxa including fishes (Amundsen and Forsgren 2001; Endler

1980), birds (for review, see McGraw et al. 2006) and lizards (Hamilton et al. 2013). In contrast to pigment-based colours, a range of light scattering, light reflecting or light grating nanostructures produce structural colours throughout the whole spectra (including ultraviolet -

UV or blue). Structural colours were at first regarded as cheap to produce and thus having no importance in individual quality signalling (Kemp et al. 2011; Prum and Torres 2004).

However, recent studies on UV ornaments found that UV colour is related to various aspects 13 of individual quality such as feeding history (Lim and Li 2007), body condition (i de Lanuza et al. 2014; Kemp and Rutowski 2007) and social dominance (Bajer et al. 2010; Whiting et al.

2006), suggesting that UV colouration can have costs similar to those of pigment based colours and thereby act as a quality signal (Prum 2006). In lizards, signalling via UV is largely assumed

(Fleishman et al. 2011), since UV ornaments are widespread in the taxa (Pérez i de Lanuza et al. 2013b) and UV perception is a conservative trait in the group (Fleishman et al. 1993; i de

Lanuza and Font 2014; Loew et al. 2002). Both correlative and experimental studies found that attributes of UV colouration act as a signal in intrasexual competition for mates, such as male dominance (Bajer et al. 2011; Martín and López 2009), fighting ability (i de Lanuza et al. 2014;

Stapley and Whiting 2006) and aggression (Whiting et al. 2006). Furthermore, although controversial, there is also supporting information for the role of UV colouration in intersexual selection in lizards. Field studies showed that elaborate UV ornamentation influenced mate acquisition of males (Martín and López 2009; Olsson et al. 2011). Moreover, manipulative laboratory experiments showed a role for UV colouration in both female and male mate preference (Bajer et al. 2010; Lebas and Marshall 2001; but see Tokarz 1995). These studies suggest that UV signals can play a pivotal role in mate selection.

Lizards frequently exhibit colourful eyespots and outer ventral scales - OVS (Arnold

1989) on their flanks, forming two rows of conspicuous patches on the lateral side of animals.

Only a few studies investigated the colour attributes of these patterns. Molina-Borja et al.

(2006) found that males of a lacertid lizard Gallotia galloti express more intense UV colouration on their eyespots than females. Similarly, Font et al. (2009) showed that colouration of Lacerta lepida is more shifted to short wavelengths in males than females, and also raised the possibility that UV eyespots may function as sexual or social signals. Lateral colour patches are often thought to have a signalling function because they are commonly 14 displayed during social interactions, and form a distinctive pattern contrasting to visual background (Stuart–Fox and Ord 2004).

The aim of our study was to investigate the role of UV colour of eyespots and OVS ornaments in female choice. We tested mate preferential decisions of females in Ameivula ocellifera, a small, mutually ornamented teiid lizard. We conducted experimental tests controlling for body size as a cue during mate assessment, enabling females to choose from males based exclusively on colouration. We tested the hypothesis that UV colouration acts as a signal of mate quality and therefore influences receptive females’ spatial distribution. As females’ spatial preference, and consequently, mate acquisition of males was shown to be strongly correlated with paternity in lizards (Olsson et al. 2011), we considered female association to males as indicative of mate preference.

MATERIAL AND METHODS

Study system

The Brazilian whiptail lizard, A. ocellifera Spix 1825, is distributed throughout Northeast of

Brazil (Harvey et al. 2012; Oliveira et al. 2015). Some populations exhibit sexual dimorphism in body size and relative head size, with sexually mature males being larger and having bigger relative head size than females (Mesquita and Colli 2003; Vitt 1983). Both males and females exhibit bright blue-turquoise eyespots and outer ventral scales (OVS) on their flanks. Both eyespots and OVS show strong UV reflectance. Sexual activity of A. ocellifera seems to be associated with different climate cycles the species complex faces throughout its distribution area, ranging from clear cyclic reproductive periods to continuous reproduction with multiple clutches per year (Cruz 1996; Mesquita and Colli 2003; Vitt 1983). As there is no available information about the sexual cycle of A. ocellifera from our population, we decided to time our experiment based on reproductive status data of 175 dissected specimens captured between 15

March 2013 and March 2014 (unpublished data). Females with vitellogenic ovarian follicles and males with enlarged testes and convolvulated epididymides were considered as reproductively active. Frequency of sexually active adult females and males suggested that reproductive period in this region occurs during the dry season, peaking in November-January.

Collection and husbandry

We collected 38 adult males and 19 adult females in December 2013 in an Atlantic Forest fragment at Centro de Lançamento Barreira do Inferno (CLBI), an area managed by Brazilian military in the municipality of Parnamirim, RN, Brazil (5º55’2’’S; 35º10’4’’W). We collected lizards using a 1 km long transect of 20 pitfall trap units. A unit consisted of four plastic buckets of 60 L each, arranged in a Y shape formation and connected by 5 m long plastic foil drift fences. A total of 80 buckets were kept open during 15 consecutive days, and were checked regularly during the day in order to avoid stress by natural factors such as heat or predation, and to prevent capturing two or more specimens in the same trap at the same time. Only individuals with intact or fully regenerated tails were used in the experiment. We checked receptivity of collected females by presence/absence of mating scars (Fitze et al. 2005).

Captured animals were transported to the laboratory at Universidade Federal do Rio Grande do

Norte.

We weighed specimens with a digital scale and measured their snout-vent length

(SVL), head length, head height, and head width with a digital calliper (Mitutoyo Inc., Japan).

We housed lizards individually in plastic boxes (size: 41 cm X 28 cm X 12 cm; length, width, height, respectively). Illumination was provided by Repti Glo 5.0 Tropical Full Spectrum

Terrarium Lamps (Exo Terra, Rolf C. Hagen Inc., Holm, Germany), and photoperiod was held natural (12 L: 12 D). Temperatures were set to match range of natural habitat of the study population, from 22–25 ºC at night to 27–30 ºC during the day. We placed folded cardboard 16 pieces into boxes for shelters. Lizards were fed daily with Tenebrio molitor larvae and termites

(Isoptera), and water was provided ad libitum.

Colour measurements

We measured colouration of eyespots and OVS with an Ocean Optics USB4000-UV-VIS type spectrometer, equipped with a DH-2000-BAL light source and a QR450-7-XSR bifurcated fibber-optic probe (Ocean Optics Inc., Dunedin, Florida). A custom-made probe holder made of anodized aluminium held the single ending of the probe. The probe holder kept the probe in a constant 3 mm distance and 90° angle to surface. We measured colouration of patches with three largest diameters for both eyespots and OVS. We used the three readings to calculate a mean reflectance for both eyespots and OVS and used them as separate variables during analyses. Reflectance was calculated relative to a diffusive white reference (WS-1-SL

Spectralon Reflectance Standard, Ocean Optics, Inc.) and dark reference (= no incoming light) using SpectraSuite software (Ocean Optics, Inc.). We took measurements across the spectrum of 300–700 nm wavelengths. There is no available information about the visual system and spectral sensitivity of A. ocellifera, therefore we decided to measure colouration within the broadest range of wavelengths known to be visible to lizards (Fleishman et al. 2011). White and black reference was re-measured regularly to avoid spectrometer ‘drift’ (Endler and

Mielke-Jr. 2005).

To characterize colouration of eyespots and OVS, we derived five variables from spectral data: (1) Total brightness - average reflectance over the range of 300 and 700 nm, (2)

UV brightness - average reflectance over the range of 300 and 400 nm (3) UV chroma - percentage of average reflectance measured over the range of 300 and 400 nm compared to total brightness (R300–400/R300–700), (4) Visible brightness [VIS] - average reflectance over the range of 400 and 700 nm, (5) VIS chroma - percentage of average reflectance measured 17 over the range of 400 and 700 nm compared to total reflectance (R400–700/R300–700). We took colour measurements one day before the experiments and right after applying treatments

(UV-deprived or control, see below).

Assemblage of male pairs

We assigned males (N=39) into pairs based on their snout-vent length (SVL), allowing a maximum difference of 2 mm. We summarized variables describing head size with a Principal

Components Analysis (PCA) on head length, head width and head height (Table 1). We used scores as a separate variable (“Head size”) in subsequent analyses. We ran paired t-tests to examine whether members of male pairs (assigned to UV-deprived and control treatment) differed in their morphological variables (SVL, Body weight, Head size).

Colour manipulation

To disentangle the effect of UV colouration from other traits that might possibly be connected to UV, we experimentally manipulated UV colouration of 19 male pairs (2 pairs were later excluded from analysis see details below). We applied a UV blocking sunscreen (Vichy Capital

Soleil SPF 50, Vichy Laboratoires, Vichy, France) (Olsson et al. 2011) mixed with common

Vaseline® (as carrier substance) on UV-deprived males’ (N=19) eyespots and OVS, while control males received pure Vaseline® (control males, N=19). Members of a male pair were randomly assigned to either UV-deprived or control treatments. The sunscreen used to block

UV is perfume-free and there is no record of any detrimental effects it may have on lizards, even in long-term use (Olsson et al. 2011). We did not detect any change in our sunscreen- bearing experimental animals’ behaviour, physiology or colouration (no apparent swelling, fading or discolouring was observed). 18

We used paired t-tests and Wilcoxon signed rank tests (where assumptions of normality were violated) to examine whether eyespot and OVS colouration of males assigned to control and UV deprived groups differed before treatment.

Female association preference experiment

In order to test the effect of UV colour on female spatial preference, we ran mate choice 19 trials (males N=38, females N= 19). Females were randomly assigned to male pairs. Our experimental arenas were modified after (Bajer et al. 2010) and consisted of a large terrarium of 80cm in length, 60cm in width and 50cm height. In short, we divided the arenas (N=5) into three compartments for the two stimuli males (25x30 cm each) and the female (40x50 cm).

Females were randomly assigned to arenas. Control and UV-deprived males were randomly assigned to sides of the arena (to avoid biased choices due to potential side preference of females) and could not see each other during the trials. Within the female compartment, a neutral and two preference areas were defined that allowed the female to stay close to either one of the males while being able to stay out of sight of the other male (i.e preference towards male A or B) or seeing both males at the same time (i.e. no preference). We used plexi glass dividers with high UV transmittance (UV transmittance at 315-380 min 80 %, at 380-400 min

90 %; Plexiglas GS4012, Evonik Ind. AG, Darmstadt, Germany) between compartments of the males and the female. As a result, females were only able to estimate visual signals for male assessment (i.e. chemical stimuli were excluded from female compartment). Trials were conducted at the Laboratory of Sensory Ecology at UFRN from 14th to 18th December, during the activity period of our study population (between 08.00 and 16.00). The experimental arenas were illuminated with Repti Glo 5.0 Tropical Full Spectrum Terrarium Lamps (Exo Terra, Rolf

C. Hagen Inc., Holm, Germany). Air temperature ranged from 28 and 32°C. We allowed acclimatization to the compartments for 15 min before starting trials. Observation was made 19 from behind a blind. We did not observe any signs of stress during trials. We recorded female location every 10 min for 6 h, resulting in a total of 36 records per trial. Experimental animals could not see the observer. After every trial, we thoroughly washed the arena with alcohol

(70%) in order to remove any chemical stimuli of individuals of previous trials. We excluded trials where females were observed in the neutral area for more than 50% of total number of observations or did not visit all areas at least once. Based on these criteria, three trials were excluded and further statistical analyses were run on data from valid experiments (N=17).

Every individual was used only once during the trials. After experiments, all males and females were released at the site of capture.

Statistical analysis

To determine whether we can collapse colour data gathered for eyespots and OVS and use it as a single variable, we analysed spectral data in two ways. First, we analysed a saturated model

(separate eyespot and OVS), followed by a model with pooled body regions. We found higher support for the first model (results not shown), and therefore we used separate colour variables for eyespots and OVS in the subsequent analysis. We tested the efficiency of our treatment by testing Total brightness, UV brightness, UV chroma, VIS brightness and VIS chroma between

UV-deprived and control males using Wilcoxon signed rank tests. The number of female sightings was entered as a response variable and male treatment (UV-deprived vs. control) as a factorial predictor. Statistical analyses were performed with SPSS v19 (SPSS Inc., Chicago,

IL, USA) and R v3.2.1 (R Core Team, Vienna, Austria).

RESULTS AND DISCUSSION

The first PC of males head size explained 96.54% of total variance (eigenvalue =2.896, all factor loadings >0.981). We did not find any difference between SVL (t18=0.987, p=0.337), 20

Body weight (t18=1.021, p=0.321) or Head size (t18=0.885, p=0.388) when comparing UV- deprived and control males. A visual inspection of reflectance curves of intact eyespot and

OVS colouration showed that both ornaments reflect light in UV range (300-400 nm; Fig.1a- b). There was no significant difference in the colour variables between UV deprived and control males prior to manipulation (all p>0.080). After treatment, control males’ colouration did not change significantly compared to their intact colouration (all p>0.078), while reflectance in

UV range dropped significantly in UV-deprived males (eyespot UV brightness: Z=-3.179, p=0.001; UV chroma: Z=4.281, p>0.001; OVS UV brightness: Z=3.849, p=0.001, UV chroma: t18 =3.676, p=0.001).

UV brightness and UV chroma from eyespots and OVS were significantly lower in

UV-deprived males than in control males (Table 2). We did not find significant difference between UV-deprived and control males in VIS brightness on both ornaments, or VIS chroma of eyespots, while VIS chroma from OVS of UV-deprived males was higher than that of control males (Table 2). UV-deprived and control males did not differ in their total brightness (Table

2). Nonetheless, UV reflection of both control and UV-deprived males was within the natural range when compared to a larger dataset of intact males caught in 2013 (Fig. 2a-d). As control males not only had higher UV reflectance, but also were also less chromatic in VIS range on their OVS, we cannot completely rule out the possibility that ‘visible’ colouration of this ornament also affected male assessment. However, a visual inspection of reflectance curves shows that treatment hardly affected the visible range in OVS (Fig. 1a-b). Hence, the difference between VIS chroma of UV-deprived and control groups is a result of intensity decrease in short wavelengths, and consequently a change in contribution to the rest of the spectra, rather than a brightness change in visible wavelengths.

Although we did not test the role of VIS chroma in affecting female spatial preference, we found a positive correlation between medium wavelength chroma with snout-vent length 21 and body condition in both sexes (unpublished data), suggesting that non-UV colours can also play a role in mate selection in A. ocellifera. Medium and short wavelength adjacent colour patches can enhances chromatic contrast within each other and with the visual background because they are complementary, which means that each one reflects most in the region of the spectrum where the other does not (Pérez i de Lanuza and Font 2015). This is a well-known strategy to maximize conspicuousness (Endler 2012), especially in many lacertid lizard species

(Pérez i de Lanuza and Font 2016), because contrasting colours stimulate retinal cones in opposite ways (Endler 1992). Complementary colour patches within-body colouration may enhances conspicuousness of sexual signals in A. ocellifera and are probably advantageous to fitness, thus selection may favor the evolution of these contrasting spectral combinations in our study species. However, further experiments are necessary to confirm this statement.

Female sightings were significantly higher on control males’ side than on UV-deprived males’ side (Wilcoxon signed rank test: Z=-2.979, p=0.003, Fig. 3), with GLZ models also providing results (Z=0.110, p<0.001). Therefore, female spatial distribution was positively associated with males with higher UV reflectance on their eyespots and OVS. Our experiment provides evidence that sexually receptive A. ocellifera females show preferential spatial distribution towards males with higher UV reflectance over males with experimentally reduced

UV reflectance. As males did not differ systematically in traits other than colouration, we assume that their preference shows the communicative function of eyespot and OVS colouration during male assessment. Mutual ornamentation is rarely investigated in terms of mate selection (for review, see Kraaijeveld et al. 2007). To our knowledge, this is the first study experimentally investigating the role of UV colouration in female preference in a mutually ornamented lizard species. Our results support the hypothesis that females use UV reflective eyespots and/or OVS of males as a clue during mate assessment, suggesting that eyespot and

OVS colouration is a sexual signal in males. Previous studies on female association preference 22 for UV-manipulated male signals in a lacertid species (Lacerta viridis) showed similar results, as females preferred males with nuptial throat colouration with higher UV reflectance (Bajer et al. 2010). Teiids and lacertids are close-related groups (Pyron et al. 2013) and both perform female accompaniment (Bajer et al. 2010, Ribeiro et al. 2011) that allows females to be choosy by assessing their mates. Additionally, multidisciplinary approaches and behavioral tests confirmed that many lacertids are capable of UV vision (Pérez i de Lanuza and Font 2014).

Thus, UV colour seems to be an important signal in mate acquision in Lacertoidea. Regarding other lizard groups, UV colour preference was found in male (LeBas and Marshall 2000) but not in female choice (LeBas and Marshall 2001) in the agamid lizard Ctenophorus ornatus.

Colour-based female mate preference was also found in a multivariate analysis in ornate tree lizard (Urosaurus ornatus; Hamilton and Sullivan 2005) using human visual perception, showing that colour assessment can be a widespread mate choice strategy in many lizards groups.

There are several scenarios as to why females prefer to associate with males of more intense UV colouration. UV reflective ornaments can function as amplifiers in the studied signalling system. Signal theory predicts that visual signals that are more contrasting to their background are more conspicuous (Endler 1990), and selection for effective signalling favours highly conspicuous signals (Dawkins 1993). Many lizard species bear colourful eyespots and

OVS patterns that stretch along the lateral side of the animals, making them easier to detect when, for example, males pose during mating behaviour (Stuart–Fox and Ord 2004). Moreover,

UV or blue signals are particularly salient in light conditions of woodland shade, where ambient light is rich in UV and green leaves reflect very low in the shorter wavelengths (Théry

2001). Thus, although we have no information on visual sensitivity of A. ocellifera, it is possible that in the habitat used by individuals of our study population, male assessment is easier when based on highly conspicuous (UV reflective) ornaments rather than an otherwise 23 cryptically coloured body silhouette. Body size, for example, can be easier to assess during courtship of males, and female choosiness for larger males is frequently found in lizards

(Hamilton and Sullivan 2005; Hofmann and Henle 2006; Richard et al. 2005). Considering that females chose males with greater UV reflectance even with the elimination of body size differences in our experiment, UV reflective ornaments may signal individual quality irrespective of body size. Costliness ensures honesty of individual quality signals (Grafen

1990; Zahavi 1975). Structural colours have been shown to be both condition dependent and costly in several taxa, signalling male quality directly (Bajer et al. 2012; Molnár et al. 2012;

Siefferman and Hill 2005; Simmons and Bailey 1993). Moreover, environmental factors like food availability and temperature (Bajer et al. 2012; Figuerola and Senar 2005; Penteriani et al. 2006) can affect expression of structural colouration, making UV signals especially costly to produce in ectotherms (Bajer et al. 2012). UV colour signals were also found to advertise dominance and health status (Martín and López 2009; Molnár et al. 2013; Molnár et al. 2012) and to be condition dependent (Martin et al. 2013; Molnár et al. 2012) in lizard species. As genes resulting higher condition, dominance or better parasite resistance can be inherited by offspring (Drews 1993; Roulin et al. 2001; Rowe and Houle 1996), it can be adaptive for females to choose males signalling better individual quality in these aspects. Finally, as characteristics of UV signals can also be influenced by environmental factors, they are suitable to signal home range quality, either in terms of available food or heterogeneous microhabitats needed for effective thermoregulation; both important aspects of individual quality in lizards

(Adolph 1990; Bauwens et al. 1996; Huey et al. 2009). Thus, choosy females can also gain direct benefits by assessing mate quality through their structural colouration.

Taken together, we raise the possibility that UV ornaments of males act either as amplifiers of other intrinsic individual quality traits, as quality signals per se or are parts of a multiple signalling system, facilitating the reliability of signals in a social context (Hamilton 24 and Sullivan 2005; Møller and Pomiankowski 1993; Rowe 1999). However, information conveyed by eyespot and OVS colouration and the selective pressures acting on these ornaments are still to be investigated in both sexes. Thus, future work must be conducted to establish information on (i) the proximate factors affecting UV colour development, (ii) whether UV colouration also has a function in sexual or social context in females and (iii) whether male and female A. ocellifera colouration provides a base for assortative mating, as it was shown in other mutually ornamented species (MacDougall and Montgomerie 2003; Pérez i de Lanuza et al. 2013a).

Ethical Approval

None of the experimental animals suffered any injury. Animal Ethics Committee of UFRN

(protocol #040/2013) approved our study. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Collection permits were granted by ICMBio (nº 23164-1) and CLBI. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Funding

This project was funded by a CAPES Science without borders young talent research grant BJT

# 043/2012 and CNPq Universal grant # 474392/2013-9. GCC thanks CNPq Grants

#302776/2012-5 and 201413/2014-0

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Table 1 – Factor loadings of Principal Components Analysis (PCA) on head measurements.

Head measurements PC1

Head Width 0.939

Head Height 0.909

Head Length 0.937

% explained variance 86.201

Table 2 – Comparison of colour traits of eyespots and OVS between control and UV-deprived

A. ocellifera. Mean values for control and UV-deprived followed by statistics and p values.

UV brightness (eyespot and OVS), UV Chroma (eyespots), and Total brightness (OVS) were tested with Wilcoxon signed ranked test, all other comparison were made with paired t-tests.

Eyespot OVS

Colour measure Mean t/Z p Mean t/Z p

UV brightness 6.07/3.84 -2.722 0.006 27.84/21.48 -2.107 0.037

UV Chroma 0.51/0.38 -3.258 0.001 0.72/0.55 4.295 0.001

VIS brightness 12.71/12.53 -0.087 0.932 43.63/47.48 -0.987 0.338

VIS Chroma 1.02/1.07 -1.436 0.170 1.19/1.22 -3.377 0.004

Total brightness 12.26/11.50 0.432 0.671 39.18/39.25 -0.260 0.795

Figures

Fig1 – Spectral reflectance of A. ocellifera eyespots (a) and OVS (b): before treatment

(diamond line), control (circle line) and UV-deprived (square line). Mean reflectance, closed dots and 95% confidence intervals – error bars are shown for every 20 nm.

Fig2 – UV brightness (a, c) and UV chroma (b, d) of UV-reduced (N=17) and control male

(N=17) A. ocellifera eyespots (a,b) and OVS (c, d) compared to intact ranges measured in 2013

(N=175) (boxes denote 95% confidence intervals and whiskers minimum–maximum ranges).

Fig3 – Number of female sightings between control and UV-deprived males. Means and 95% confidence intervals are shown.

CAPÍTULO II: THE ROLE OF UV COLOUR SIGNALS ON MALE COMPETITION IN

THE BRAZILIAN WHIPTAIL LIZARD (AMEIVULA OCELLIFERA)

Carolina M. C. A. Lisboa1*, Katalin Bajer,1,2 Orsolya R. Molnár3, Daniel M. A. Pessoa4 and Gabriel C.

Costa5

1 Laboratory of Biogeography and Macroecology, Department of Ecology, Federal University of Rio

Grande do Norte, Campus Universitário, Lagoa Nova, Natal, RN, 59078-900, Brazil.

2 Behavioural Ecology Group, Department of Systematic Zoology and Ecology, Eötvös Loránd

University, Pázmány Péter sétány 1/c, H-1117, Budapest, Hungary.

3 Department of Biological Sciences, Life Sciences Center, Dartmouth College, 78 College Street,

Hanover, NH 03755, USA.

4 Laboratory of Sensory Ecology, Department of Physiology, Federal University of Rio Grande do

Norte, Campus Universitário, Lagoa Nova, Natal, RN, 59078-900, Brazil.

5 Department of Biology, Auburn University at Montgomery, Montgomery, AL 36124, USA.

*Corresponding author. Email: [email protected]

ABSTRACT

Colouration can signal individual quality traits and may influence mate choice and intrasexual competition outcomes. Previous work demonstrated, for example, that intense male UV colour of Brazilian whiptail lizards (Ameivula ocellifera) was preferred by females in experimental setups. Conspicuous ornaments used by females in mate choice are usually signals of male quality, and thus can be used by males to evaluate rivals in male contests and avoid costly fights. Here we aimed to test whether UV colour influences male-male competition in A. ocellifera, by experimentally decreasing the intensity of UV colouration of randomly chosen male members from otherwise similar male pairs and testing whether difference in UV colour can affect contest outcome. We confirmed colour treatment effectiveness by modelling lizard’s visual system and searching for differences in UV perception between treatments. Our results showed that bigger the difference on rival’s UV coloration, faster they get into a fight, suggesting that colour dissimilarities can influence duration of contests. Our study supports predictions of ‘war of attrition’ game, in which contests with higher asymmetries are resolved faster. We concluded that UV colour traits that affects outcome of female choice also influences male contests and works as a sexual signal in both interactions in A. ocellifera.

Keywords: animal communication; contests; visual modelling; sexual selection; ultraviolet 35

INTRODUCTION

For many animal species with good visual acuity, body colouration is essential in social communication, being a source of signals for interspecific recognition (Schultz and Fincke

2009), intrasexual communication, such as male competition (Bajer et al. 2011), and intersexual communication, such as courtship (Stuart-Fox and Goode 2014) or mate choice

(Pearn et al. 2001). These apparently simple colour traits are, in fact, a multilayered and three- dimensional structurally complex that may contain different types of pigments, structural colours or an association of both (Grether et al. 2004). Each colour element may raise independently, influenced by a variety of selective pressures such as social interactions or predation (Macedonia et al. 2002).

Structural coloration is produced by light reflective or light scattering microstructures, for example, by the nanometer-scale structure of the integument in lizards, usually conferring blue and ultraviolet (UV) colouration (Lanuza and Font 2010). This type of colouration plays a role in intraspecific communication in many taxa, as in the courtship (Silberglied and Jr.

1978) and female preference in butterflies (Kemp 2007); aggressiveness during territorial behaviour in coral reef fishes (Siebeck 2004); male attractiveness in newts (Secondi et al.

2012); mate choice (Siefferman and Hill 2005) and male-male interactions (Rémy et al. 2010) in birds; male (LeBas and Marshall 2000) or female preference (Bajer et al. 2010) and male- male competition in reptiles (Bajer et al. 2011); scent marks recognition in mammals (Melin et al. 2012).

In male-male contests, the outcome may be influenced by structural colours of male presumed visual badges, being a signal for advertising territorial status (Schultz and Fincke

2009), indicating aggressiveness (Korsten et al. 2007) or fighting ability (Alonso-Alvarez et al. 2004). In lizards, studies on Augrabies flat lizards (Platysaurus broadleyi) (Stapley and

Whiting 2006, Whiting et al. 2006) have shown that UV reflective colour expression can be 36 related with a variety of male traits in male-male competition, such as aggressiveness and fighting ability, and may honestly advertise status to rivals. On European green lizard (Lacerta viridis), artificially UV-decreased males lost most of the paired contests, which indicates that structural UV colour signals male dominance (Bajer et al. 2011). Although these studies successfully related colour badges and male individual quality, confirmation of visual perception of ornaments from target species is an essential but absent issue that would determine if colours are indeed visible and how do they act on sexual selection.

Colour ornaments can be honest signals that predict male quality. For example, in male sand lizards (Lacerta agilis), size and saturation of the green lateral badges are strongly correlated with mass and body condition (Olsson 1994). Phenotypic traits such as body size and colour patches were also able to influence contest dynamics and outcomes in L. agilis, as specimens with more conspicuous patches were more likely to be winners. The general method to test if a male trait functions as a signal in male-male competition is to search for a connection between the potential signal trait variability (e.g. shape, size, and colour) and successfulness of the bearer during aggressive interaction (Censky 1995, Salvador and Veiga 2001, Stapley and

Whiting 2006, Bajer et al. 2011).

Contests may considerably vary in degree of escalation, from ritualized displays to fighting, and competitors are assumed to minimize potential costs by mutually assessing each other’s potential qualities (Smith and Parker 1976). According to predictions of ‘war of attrition’ game (Maynard Smith and Parker 1973, Maynard Smith 1974), contests with higher asymmetries are resolved faster, and mutual evaluation time decreases with the traits difference. Structural colouration asymmetries are important in resolving conflicts and avoid fights by signaling relative individual quality (Siefferman and Hill 2005, Bajer et al. 2011).

Our previous experiments showed that UV chroma is important in female association preference, as they exhibit spatial preference for males with higher UV reflectance in 37 matched male pairs (Lisboa et al. 2017). In this work, we aim to test whether UV reflectance influences the outcome of male-male competition in our model species, the Brazilian whiptail lizard (Ameivula ocellifera). By artificially reducing UV reflectance in a member of a size- matched male pair, we expect that (1) manipulated males are more likely to lose contests than control males, similarly to European green lizard (Lacerta viridis) (Bajer et al. 2011); (2) higher the dissimilarity on UV colour between opponents, lower the time of assessment before engage to a fight, as predicted by ‘war of attrition’ game (Maynard Smith and Parker

1973, Maynard Smith 1974). By that, we intent to have an insight on how colour signals may influence sexual selection in our model species.

METHODS

Study system

The Brazilian whiptail or Spix’s whiptail lizard (Ameivula ocellifera Spix 1825) is a small- sized (<100 mm in body size) lizard, distributed throughout Northeast Brazil (Oliveira et al.

2015). Many populations exhibit sexual dimorphism in body size and relative head size, with sexually mature males being larger and having longer, wider, and higher heads than those of the females, which have relatively longer bodies (Vitt 1983, Mesquita and Colli 2003). On

Brazilian coastal regions, females reproduce continuously throughout the year, with a peak at the end of the rainy season (Zanchi-Silva et al. 2014) and most clutches are composed of two eggs, with little variation among population (Menezes and Rocha 2014).

Adult Brazilian whiptail lizards have bright blue (to the human eye) eyespots (ocelli) on their ﬂanks. Eyespots are relatively round-shaped colour patches on the lizards’ body side that usually show contrasting colouration to the surrounding body surface. Like many other teiids, A. ocellifera has an additional row of blue coloured patches on their outer ventral scales

- OVS (Arnold 1989). These patches are irregular shaped, they include a few coloured scales, 38 rather than being regular shaped motifs, but usually cover a higher proportion of the body surface then the eyespots. Both the eyespots and the OVS reflect in the UV range, and that relative UV intensity is body size dependent in both sexes.

Collection and husbandry

During two reproductive seasons, we collected 88 adult A. ocellifera males, 40 animals in

December of 2013 and 48 in October-November of 2014. We performed collections in an

Atlantic Forest fragment in Centro de Lançamento Barreira do Inferno (CLBI), an area managed by the Brazilian military in the municipality of Parnamirim, RN, Brazil (5°55'24 .92

"S 35 ° 10'4 .93).

We caught lizards through active search and manual noosing, using a loop made of fishing line, and using a 1 km long transect of 20 pitfall trap units. A unit consisted of four plastic buckets of 60 L each, arranged in a Y shape formation and connected by 5 m long plastic foil drift fences. We kept a total of 80 buckets opened during 15 consecutive days in each reproductive season, and checked regularly during the day in order to avoid stress by natural factors as heat or predation, and to prevent having two or more specimens in the same trap at the same time. In case we captured two or more lizards in the same bucket, we released them far from the collecting area. We used only individuals with intact or fully regenerated tails in the experiment. Captured animals were placed in cloth bags and sent to the laboratory of

Universidade Federal do Rio Grande do Norte (UFRN; Natal, RN, Brazil) morphological and colour measurements, as well as male-male contest experimental trials.

After capture, we measured lizards’ morphological variables. We weighed them with a digital scale (Scientech SA 210, Scientech Inc. Boulder, Colorado) and measured their snout- vent length (SVL) and head length (from the tip of the snout to the commissure of the mouth; to the nearest 0.01 mm), head height (at its highest point; to the nearest 0.01 mm) and head 39 width (at its broadest point; to the nearest 0.01 mm) with a digital caliper (Paquimeter Absolute,

Mitutoyo Inc., Japan, 0.01mm precision). Lizards were housed individually in plastic boxes

(size: 41 cm x 28 cm x 12 cm; length, width, height, respectively). Illumination was provided by Repti Glo 5.0 Tropical Full Spectrum Terrarium Lamps (Exo Terra, Rolf C. Hagen Inc.,

Holm, Germany), and the photoperiod was held natural (12 L: 12 D). Temperatures ranged according to the natural habitat of the study population, from 22–25 ºC at night to 27–30 ºC during the day. We placed folded cardboard pieces into boxes for shelters. The lizards were fed daily with Tenebrio molitor larvae and water was provided ad libitum.

Colour measurements

We measured colouration of the lizards’ eyespots and outer ventral scales (OVS) with a spectrometer type Ocean Optics USB4000, used with a DH-2000-BAL light source and a

QR450-7-XSR bifurcated fiber-optic probe (Ocean Optics Inc., Dunedin, Florida). The single ending of the probe was held by a custom-made probe holder made of anodized aluminum that allowed us to measure the colouration of small-sized patches (as eyespots and OVS) without their surrounding areas, by illuminating an area of 3 mm in diameter. The probe holder kept the probe in a constant 3mm distance and 90° angle to the surface. As within-individual variance in the UV colouration of the eyespots and OVS was not tested previously, we created a representative sample of the potentially uneven colouration (Endler and Mielke-Jr. 2005) by taking three consecutive readings of three randomly chosen patches in the case of eyespots and three randomly chosen patches in the case of OVS. We calculated a mean reflectance for both the eyespots and the OVS and used them as separate variables during the analyses. Reflectance was calculated relative to a diffusive white reference (WS-1-SL Spectralon Reflectance

Standard, Ocean Optics, Inc.) and dark reference (= no incoming light) using the SpectraSuite software (Ocean Optics, Inc.) (Whiting et al. 2006). Measurements were taken across the 40 spectrum of 320 –700 nm. There is no available information about the visual system and the spectral sensitivity of A. ocellifera, therefore we decided to measure the colouration within the broadest range of wavelengths known to be visible to lizards (Fleishman et al. 2011). We re- measured white and black references regularly to avoid spectrometer ‘drift’.

A visual inspection of the spectra of eyespots and OVS of A. ocellifera (N=77) collected in 2014 revealed that there is a distinctive reflectance peak in the UV range, around 355 nm, and another one in the human visible range, around 550 nm. To characterize the colouration of the eyespots and OVS, we calculated five variables: (1) brightness, the total reflectance over the range of 320 to 700 nm; (2) UV chroma, the percent of reflectance measured over the range of 320 to 400 nm compared to total reflectance (R320–400/R320–700); (3) UV hue, that was calculated as the wavelength with the maximum slope of the curve in the UV range (Andersson et al. 1998); (4) ‘green’ chroma, the percent of reflectance measured over the range of 400 to

700 nm compared to total reflectance (R400–700/R320–700); (5) ‘green’ hue, that was calculated as the wavelength with the maximum slope of the curve in the human visible range

(Andersson et al. 1998).

Visual modelling

To test the experimental colour treatment effectiveness from A. ocellifera visual perspective, we modeled lizard’s visual system to access chromatic contrasts between intact and UV- treated males. We also modeled chromatic and achromatic contrasts between experimental groups (control and UV-reduced) to search for differences in UV perception between rivals.

Due to lack of information, and in order to model how A. ocellifera visual system would detect differences in spectra of eyespots and OVS from conspecifics, we used the photoreceptors’ sensitivity curves from the common lizard Zootoca vivipara (Martin et al.

2015), the phylogenetically closest taxon (Pyron et al. 2013) that had been studied with respect 41 to its visual sensitivity. To build the model, we quantified the quantum catch for each photoreceptor (Qi default) using a relative cone abundance of 1:2:5:9 (empirical model). We measured the illuminant from inside the experimental arenas (in µmol m-2 s-1) and we used a default ideal background in the model. We used the receptor noise model to estimate the chromatic distance before (intact) and after (control and manipulated) experimental treatments and between the two experimental groups (control and manipulated spectra; see section 2.6) in

Just Noticeable Difference (JND) perceptual units, in which chromatic contrast can be perceptible by the given visual system if exceeds the threshold of 1 JND. Here, we considered chromatic distances between 1-4 JND as ‘poorly perceptible’ and above 4 JND as ‘highly perceptible’ (Martin et al. 2015). Analyses were performed on Pavo Package (Maia et al. 2013) of R 3.2.1 (R 2015).

Male pairs and colour manipulation

We experimentally manipulated the UV colouration of 40 males from 2013 season paired according to their snout-vent length, allowing a maximum difference of 2 mm. We measured

OVS and ocelli UV chroma and brightness prior and after colour manipulation, and provided the matched members of the pairs one to another. Experimental males had UV reduced using a mixture of Vichy Capital Soleil 50 sun protection factor sunscreen (Olsson et al. 2011) and common vaseline moisturizer. Control animals received only vaseline. We paired males through Excel size sorted dataset.

We summarized the variables describing the head shape with a Principal Components

Analysis (PCA) on head length, head width and head height. The PCA resulted in one PC with an eigenvalue >1 (2.904), describing head size (all factor loadings <−0.983). We used that variable (“Head size” - HS) in subsequent analyses. We used paired t tests to examine whether

UV-deprived and control males within a pair differed in their morphological [snout-vent length 42

(SVL), body weight (BW), head size (HS)] variables before the colour manipulation. We did not find any difference between the SVL (t16=1.196, p=0.249), BW (t16=1.049, p=0.310) or HS

(t16=0.912, p=0.375) of UV-deprived and control males.

A visual inspection of the reflectance curves of the intact eyespot and OVS colouration showed that there is a distinctive peak in the UV range in both cases (320-400 nm; Fig. 1a, b).

After treatment, reflectance in the UV range dropped significantly both in UV-deprived and control males compared to intact males (see Fig. 2a, b and statistical results), but still stayed within the natural range of male colouration found in the study population (Fig. 2a, b). We used paired t tests and Wilcoxon signed rank tests (where assumptions of normality were violated) to examine whether eyespot and OVS colouration differed accidentally before the treatment.

There was no significant difference between total brightness (eyespot: t16=0.938, p=0.362;

OVS: t16=-1.552, p=0.140), UV brightness (eyespot: Z=-0.828, p=0.407; OVS: t16=0.180, p=0.859) UV chroma (eyespot: t16=-0.183, p=0.857; OVS: t16=2.007, p=0.062), ‘green’ brightness (eyespot: Z=-0.876, p=0.381; OVS: t16=-1.472, p=0.161) or ‘green’ chroma

(eyespot: t16=-0.086, p=0.933; OVS: t16=0.424, p=0.677). Hence, male pairs were randomly assembled in terms of both morphological and colour variables, and differed systematically only in their UV colouration.

Male-male interactions experiment

Males within the 20 pairs of 2013 season were assigned randomly to the control or UV- manipulated groups. Initial morphological and colour differences between treatment groups were tested with paired t-tests. To see whether our UV-reducing treatment was successful, we used paired t-tests to compare colour variables between treatment groups. Fight success between manipulated and control males was analyzed with a Chi-squared test based on a 2 x 2 contingency table with treatment and success entered. 43

We provided the matched members of the pairs one to another, in a two-compartmented arena divided by a paper card. Experimental animals could not see the observer and every individual was used only once during the trials. After every trial, we thoroughly washed the arena with alcohol (70%) in order to remove any chemical stimuli of individuals of previous trials. After a 15 min adaptation period, we raise the paper card, exposing them to their respective pairs. If lizards did not display aggressive behavior (approaching contestant with supplanting, chasing, and biting, forcing the contestant either to respond aggressively or escape) after raised the middle wall of glass terraria within 20 min, we considered the trial unsuccessful. Based on these criteria, three trials were excluded and further statistical analyses were run on data from valid experiments (N=16). The trials terminated when a male first escaped the other. The escaping male was assigned as loser, while the other as winner. If control males win significantly more contests, it would suggest that UV colours can determine competition outcome by being a signal of male quality. The time of pair’s mutual evaluation, between the raise of paper card and the first escape of the loser, was recorded in seconds. We performed a Generalized Linear Model (GLZ) on evaluation time and differences between members of the pairs on manipulated UV chroma in order to search for dissimilarities that would confirm predictions that contests with higher asymmetries are resolved faster. All analyses were performed on R v3.2.1 (R Core Team, Vienna, Austria). After experiments, all males were released at the site of capture. Permits for collecting were granted by ICMBio (nº

23164-1) and CLBI. None of the experimental animals suffered any injury. Animal Ethics

Committee of UFRN (protocol #040/2013) approved our study.

RESULTS

Neither morphology (SVL, BW and HS) nor colour (total brightness, ‘green’ chroma and UV chroma) differed between the UV-deprived versus control males prior to manipulation (all 44 p>0.161). After the treatment, both the eyespots’ and OVS’s UV brightness and UV chroma were significantly lower in UV-deprived males than in control males (UV brightness, eyespots:

Z=-2.722, p=0.006; OVS: Z=-2.107, p=0.037; UV chroma, eyespots: Z=-3.258, p=0.001;

OVS: t16=4.295, p=0.001). We did not find significant differences between UV-deprived and control males with respect to their ‘green’ brightness (eyespots: t16=-0.087, p=0.932; Fig. 2c;

OVS: t16=-0.987, p=0.338) or ‘green’ chroma of eyespots (t16=-1.436, p=0.170; Fig. 2d).

However, OVS’s ‘green’ chroma of UV-deprived males were higher than that of control males

(t16=-3.377, p=0.004). Hence, our treatments were effective in reducing relative UV reflectance

(and were strong enough to affect the overall reflectance).

The visual modelling results corroborated colour treatment effectiveness, as chromatic contrasts between intact and control males were poorly or non-discernible (eyespots: 1.36 JND;

OVS: 0.76 JND) whereas intact and UV-reduced contrasts were highly discernible (eyespots:

7.17 JND; OVS: 4.57 JND). Chromatic contrasts were also highly discernible (> 4 JND) from each other between control and UV-reduced animals in both eyespots and OVS ornaments

(Table 1), which means that, from lizards’ visual perspective, the experimental groups differed in terms of colour. Additionally, based on achromatic contrast, the experimental groups differed in brightness of the eyespots but not in brightness of the OVS. UV quanta catches differed in approximately 50% on UVS cones between control and UV-reduced patches, while for the other three cone types the values were considerably equivalents (Table 1).

The manipulation of UV reflectance did not determined fight success (X2= 0.54, p =

0.46), and males with reduced UV lost fights in approximately 50% of the trials. However, UV relative intensity was negatively correlated with evaluation time (Z15= -3.314, p<0.001), which means that the bigger the difference between control and manipulated colouration, the faster they get into a fight.

DISCUSSION

Spectra and visual perception analyses confirmed that we successfully decreased UV reflectance of eyespots and OVS in our experiments, hence we could test the role of this colour component in isolation. We found that A. ocellifera males with experimentally reduced UV reflectance were not more likely to lose contests than control males, but UV affected evaluation time on initial stages of contests. Despite methodological differences, our study corroborates what was found in field experiments on Platysaurus broadleyi, which contest outcome was not influenced by throat UV-reduction, but UV-reduced males were more likely to be challenged than control males, indicating that UV was important during the initial stage of opponent assessment (Stapley and Whiting 2006, Whiting et al. 2006). However, our result differs from a similar experiment on Lacerta viridis, in which reduced UV throat males were more likely to lose fights than control males, indicating that UV spots influences fight success on male competition (Bajer et al. 2011). Studies with different groups also corroborate the role of UV in male contest, such as with eastern bluebirds, Sialia sialis, which competition experiments showed that structural plumage colour is a condition-dependent trait that may be used to accurately assess fighting ability (Siefferman and Hill 2005). On Gallotia galloti lizard, a species which also displays lateral and ventrolateral blue patches and is sexually dichromatic in the UV, male contest experiments found that competitors did not differ in the number of lateral patches, but the first lateral patch of winners was lighter than that of loser (Molina-Borja et al. 1998), although the influence of UV was not taken into account. In our study species, other traits may be responsible for signaling dominance, as head size for example (because we controlled SVL, BW and HS on male pairs, we could not evaluate their influence), that is known to be an important trait in antagonistic interactions during their defense of foraging territories, and males with larger heads tend to have an advantage over males with smaller heads (Vitt 1983). Chemical cues might also influence male interactions, as observed in 46

Podarcis hispanica (López and Martín 2002). In addition, there might be innumerous others selective forces acting on colour ornamentation in A. ocellifera, as environmental or predation pressures.

Males equally-matched in size are generally slower to escalate to a fight. With a lack of a clear discrepancy in size between opponents, males must acquire information regarding their opponents’ strengths or weaknesses. In this context, some asymmetric feature will be taken as a 'cue' by which a contest can be settled conventionally. Colour signals play, at this moment, an important role as a signal of opponent’s quality. In A. ocellifera contests, difference in rivals’ relative UV intensity affected evaluation time, as it decreases with the increase of coloration asymmetries, suggesting that UV colour signals may play a role in faster resolving conflicts and avoiding fights by signaling relative individual quality. Our data agreed with a basic prediction from the ‘war of attrition’ game (Maynard Smith and Parker 1973, Maynard

Smith 1974), an 'evolutionarily stable strategy' (ESS) which says that contests between equally matched opponents should lead to longer battles than contests between opponents that are unequally matched or, in other words, higher the asymmetries, faster the contest is solved.

In sum, here, in a robust study, we established through empirical behavioral evidence, supported by spectrometry and visual modelling, that UV colour influence male competition through the time of assessment during contests. These conclusions augments the body of evidences that male colour badges plays a role in sexual selection by providing information to conspecifics in competition and mate choice. Furthermore, as A. ocellifera males and females display the same colour traits, other questions on how these sexually selected ornaments influences intra and intersexual interactions in this model system can be raised. Surprisingly, the role of colour ornaments in females remains almost unknown, as female signaling generally has been neglected in studies of ornamentation, although females often compete for limited resources. Recent studies on birds suggested that female ornaments might signal quality 47

(Siefferman and Hill 2005), dominance (LeBas 2006) or social status (Murphy et al. 2009).

Thus, we should expect that females A. ocellifera also have evolved these signals to reduce risks on agonistic intrasexual interactions, by allowing rivals to assess their fighting ability.

Moreover, additional studies are needed to highlight the complexity of this cryptic dichromatic mating and social system.

ACKNOWLEDGEMENTS

We thank to Mélissa Martin for providing the spectral sensitivity data. We thank Marc Huber,

Bruno Maggi, Gustavo Carvalho, Juan Pablo Zurano, André Bruinje and Cleto Freire for their help in collecting specimens and performing measurements. This project was funded by a

CAPES Science without borders young talent research grant BJT # 043/2012 and CNPq

Universal grant # 474392/2013-9. GCC thanks CNPq Grants #302776/2012-5, 201413/2014-

0 and 302297/2015-4.

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TABLE

Table 1. Results of visual perception of control and UV-reduced eyespots and OVS ornaments of males Brazilian whiptail lizards (Ameivula ocellifera) in quantum catch (Qi) to each of the four types of photoreceptor cells (UVS, SWS, MWS, LWS) modelled from Zootoca vivipara. Lum refers to achromatic stimuli (white). Chromatic distances in JND (just-noticeable difference) between reflectances of control and UV-reduced eyespots and OVS, given in chromatic contrast (ΔS) and achromatic contrast (ΔL). Distances between 1-4 JND are considered poorly discernible, while distances above 4 JND are highly discernible, under the conditions considered, within the given visual system.

Quantum catch (Qi) JND

Patch Mean Mean Mean Mean Mean UVS SWS MWS LWS Lum Δ S Δ L (Range) (Range) (Range) (Range) (Range)

Control eyespot 2.913 2.170 1.159 1.247 4.513 4.721 3.775 3.996 4.227 4.468 7.996 3.242 UV-reduced eyespot 1.426 (1.426-2.913) 1.334 (1.160-1.335) 4.928 (4.513-4.928) 4.216 (3.775-4.216) 4.709 (4.227-4.709)

Control OVS 9.955 7.892 3.985 3.902 1.502 1.472 1.168 1.154 1.318 1.302 4.900 0.775 UV-reduced OVS 5.828 (5.828-9.955) 3.819 (3.819-3.985) 1.441 (1.441-1.502) 1.140 (1.141-1.168) 1.285 (1.285-1.318) 52

FIGURES

Figure 1. Spectral reflectance (+95% CI) measured in the 320–700 nm range on the eyespot (a) and OVS patch (b) of male Ameivula ocellifera prior to manipulation (dotted line; n=40) and after the UV-reducing (solid line; n=20) and control (dashed line; n=20) treatments. UV range = 320–400 nm, blue range = 400–490 nm.

Figure 2. Comparison of eyespots’ UV brightness (a) and chroma (b), VIS brightness (c) and chroma (d), and total brightness (e) after the UV-depriving and control treatments were applied. Means+95% CI are shown. Asterisks (*) denote significant differences.

Figure 3. Comparison of outer ventral scales (OVS) UV brightness (a) and chroma (b),

VIS brightness (c) and chroma (d), and total brightness (e) after the UV-depriving and control treatments were applied. Means+95% CI are shown. Asterisks (*) denote significant differences.

CAPÍTULO III: SIMILAR ORNAMENTS, DISTINCT MESSAGES: SEXUAL

SIGNALS IN MALE BRAZILIAN WHIPTAIL LIZARD (AMEIVULA OCELLIFERA)

Carolina Lisboa1*, Katalin Bajer,1,2 Orsolya Molnár3, Barry Sinervo4 and Gabriel C.

Costa5

1 Laboratory of Biogeography and Macroecology, Department of Ecology, Federal

University of Rio Grande do Norte, Campus Universitário, Lagoa Nova, Natal, RN,

59078-900, Brazil.

2 Behavioural Ecology Group, Department of Systematic Zoology and Ecology, Eötvös

Loránd University, Pázmány Péter sétány 1/c, H-1117, Budapest, Hungary

3 Department of Biological Sciences, Life Sciences Center, Dartmouth College, 78

College Street, Hanover, NH 03755, USA

4 Department of Ecology and Evolutionary Biology, Earth and Marine Science Building,

University of California Santa Cruz, Santa Cruz, CA 95064, USA

5 Department of Biology, Auburn University at Montgomery, Montgomery, AL 36124,

USA

*Corresponding author. Email: [email protected]

ABSTRACT

Sexual selection acts on mating success resulting in conspicuous sexual colour signals in lizard species. These signals can reliably advertise individual quality through their elaboration and maintenance costs. However, as selection affects a suite of whole- organism traits (i.e. physiology, metabolism, morphology, etc.), it is important to consider whole-animal performance as a direct target of sexual selection because it contributes directly to outcomes of male-male competition or can be the traits used in mate choice.

Here, we investigated male characteristics that are closely related but rarely tested together, despite their importance in understanding the evolution of signal design. By testing colour components of two ornaments in Brazilian whiptail lizard Ameivula ocellifera against morphological quality traits and male’s physiological performance, we were able to gain insight into traits that can be directly affected by selection. We found that larger males had more intense short (UV) and medium wavelength chroma on dorsolateral eyespots, indicating that individuals with better survival and/or foraging ability can afford the costs of intense coloration. In contrast, smaller-headed males had more intense UV chroma on outer ventral scales (OVS), suggesting that this badge might signal quality to conspecifics. We concluded that the same colour trait conveys different messages depending on the body position of the signal, perhaps indicative of alternative signalling strategies. Moreover, higher brightness on OVS signals were associated with stronger bite force, which indicates that this colour component can reliably signal fighting ability in males. These results suggest that colour signals are informative for male quality traits that are important in social or sexual selection. Additionally, we provide evidence of distinct colour ornaments signalling different aspects of the quality on the same individual as parts of a multiple signalling system in our model species.

Keywords: Multiple signal system; bite force; sprint speed, sexual selection; ultraviolet colouration 58

INTRODUCTION

Sexual ornaments in many animal species may function as signals in conspecific recognition (Wallace 1889) and communication (Darwin 1871), evolved through processes of sexual selection during mate choice and/or intrasexual competition

(Andersson 1994, Berglund et al. 1996). Modified properties of these traits transmit information about the bearer or its environment to conspecifics, incipient species, or species. There are two types of signals, those that can act as honest signal of individual quality to receivers because of their elaboration and maintenance costs (Zahavi 1975,

Zahavi 1977, Grafen 1990), and those that do not currently signal quality of the bearer, but may be a dishonest signal (e.g. Kokko 1997, Candolin 1999). The reliability of the signal is usually measured by the correlation between the signal variable properties (e.g. size of the colour patches, colour brightness, chroma, hue, etc.) and the bearer’s attribute of interest (e.g. Molnár et al. 2012) or the benefits obtained by the receiver from knowing the signal content (e.g. Kekalainen et al. 2010, Giraudeau et al. 2011). The correlation is in turn consistent of an honest signal if it is “honest on average” (Johnstone and Grafen

1993, Kokko 1997), that is, contains sufficient information to benefit the receiver and for the signalling system to be stable.

The variety of signals is more diverse than the species that bearers them. There are many cases in which one species display multiple signals, each one carrying messages intended for different receivers (i.e. male or female conspecifics, predators) with distinct interests (Møller and Pomiankowski 1993, Pryke et al. 2001), complicating selection on a multiple signalling system (Lancaster et al. 2009, Lancaster et al. 2014). Colouration is an ideal example of a multiple signalling system (Grether et al. 2004) often found in birds and lizards (e.g. Senar et al. 2003, Martín and López 2009, Molnár et al. 2012), which the 59 system itself can be a set of colour ornaments and/or multiple colour components of single patches (e.g. Plasman et al. 2015).

Animals express their colouration through four fundamental mechanisms: (i) light absorbing molecules with wavelength-selective sensitivity (pigment-based colours as yellow, red and brown) carried by chromatophor cells; (ii) light scattering and reflecting microstructures of the integument (structural colours as ultraviolet [UV] and blue) (Senar et al. 2003, Grether et al. 2004); (iii) light production by bioluminescent processes or; (iv) by fluorescence, which biochemical compounds absorbs part of the light and reemits in a different region of the spectrum (Kemp et al. 2011). The two former mechanisms have primarily differing physiological pathways, however, several processes can affect elaboration of both types of colouration (Calisi and Hews 2007, Peters et al. 2007), and both can be costly as well (Prum 2006). The most well-known example is the carotenoid pigment-based colouration (yellow, orange and red colours). Carotenoids cannot be synthetized de novo, and animals have to take them through their diet, consequently it may be costly to acquire and sequester in signals and thus it can be an honest signal of individual quality (Kodric-Brown 1989, Locatello et al. 2006, Pryke et al. 2007).

Structural colours can also indicate individual quality, as they require precise development of reflective structures in iridophores, thus might be indicate of good genes

(Shawkey et al. 2003). As colour traits are often linked to individual quality, they will vary in response to the environment of the bearer or the ability to keep resources that are needed to maintain intense colours.

The development and maintenance of colour signals usually depends on an individual’s physiological ability (i.e. individual performance) to survive and reproduce

(Guilford and Dawkins 1991), which is directly affected by sexual selection pressures

(Sinervo et al. 2000, Miles et al. 2001, Lailvaux and Irschick 2006), that in turn drives 60 the evolution of signal design. Whole-organism performance can be assessed by quantifying the maximal ability of the animal in accomplishing an ecologically relevant task through total or partial body dynamics (Lailvaux and Irschick 2006), such as sprint speed (Irschick and Garland 2001) or bite force (Anderson et al. 2008), typically under controlled laboratory conditions. Sprint speed is the most commonly studied and understood aspect of performance (Garland and Losos 1994), as it can be a predictor of animal's health and body condition (Garrido and Pérez-Mellado 2014), resource-holding potential (Peterson and Husak 2006), foraging efficiency (Huey and Pianka 1981), dominance (Garland et al. 1990, Robson and Miles 2000), reproductive success (Husak et al. 2007), survivorship (Miles 2004) and survival under predatory pressure (Ekner-

Grzyb et al. 2013). Bite force capacity, in turn, is also an important proxy of dominance

(Lailvaux et al. 2004, Huyghe et al. 2005, Husak et al. 2006) and territorial quality

(Lappin and Husak 2005) as well as head size (Molina-Borja et al. 1998, Gvozdik and

Van Damme 2003, Perry et al. 2004) due to their direct correlation between these two traits (Herrel et al. 1999). All these aspects influenced by performance are important determinants of male fitness, thus providing support for the hypothesis that sexual selection operate on whole-animal performance traits.

Despite of the large range of colourful ornamentation lizards display, few studies have investigated the relationship between colour sexual signals and whole-animal performance (e.g. Meyers et al. 2006), especially in Neotropical lizard species (e.g.

Plasman et al. 2015). In these species with continuously varying colouration, studies of the potential signal role of colour traits regarding whole-animal performance may lead to a better understanding of the evolution of different reproductive strategies and signal design. The role of male lizard colour ornaments has been widely proved to be important in intraspecific relationships (reviewed in Cooper and Greenberg 1992), as in determining 61 the outcome of male contests (e.g. Alonso-Alvarez et al. 2004, Stapley and Whiting 2006,

Whiting et al. 2006, Korsten et al. 2007, Schultz and Fincke 2009, Bajer et al. 2011) and mate acquisition (e.g. Martín and López 2009, Bajer et al. 2010, Olsson et al. 2011, Fitze et al. 2014). Thus, Neotropical lizards are ideal model systems for studying colour signals containing quality information measured through performance, in order to obtain a broader view of signals’ roles in sexual selection.

In previous experimental studies, we found that female Brazilian whiptail lizards

(Ameivula ocellifera Spix, 1825) preferred males with more conspicuous UV ornaments

(Lisboa et al. 2017). This colour component also influenced male contest assessment, but did not predict the outcome of competition (Lisboa et al. unpublished data). Based on these results, we can infer that UV colour ornaments on A. ocellifera are under sexual selection and hold information of male quality. Thus, here we tested the hypotheses that

(1) UV colour in male A. ocellifera is an honest signal, and (2) different components of the colour ornaments signal different aspects of male quality. Specifically, we investigated the relationships between (1) short wavelength (UV) chroma, as it affects mate choice and male competition in our species (Lisboa et al. 2017); (2) medium wavelength (green) chroma, because it correlates with male quality and predicts competition outcome in other species (Olsson 1994a, b); (3) total brightness, as it signals quality in other species (LeBas and Marshall 2000, Martín and López 2009, Molnár et al.

2012) and body size, relative head size and body condition, because these traits are known to affect fitness of lizards (Hamilton and Sullivan 2005, Hofmann and Henle 2006,

Molnár et al. 2012). Both traits also comprise two key aspects of male performance capacity (maximum bite force and maximum sprint speed), as whole-animal performance has a major influence on evolutionary fitness and is a target of selection pressures (Husak and Fox 2008, Irschick et al. 2008) in species bearing continuously varying colour traits 62

(Vanhooydonck et al. 2005, Meyers et al. 2006). Additionally, we expected that our results might support assumptions about the evolution of a multiple signalling system, by testing the role of each colour component and each colour signal and their relation with male quality.

METHODS

Study system

The Brazilian whiptail lizard (A. ocellifera) is a diurnal and active foraging species that occur in Northeast Brazil (Oliveira et al. 2015). The reproductive season lasts up to four months with females presumably mating with multiple males (Vitt 1983). Sexes show differences in head size (Mesquita and Colli 2003) and colouration, with cryptic sexual dichromatism in multiple colour attributes (Molnár et al. unpublished data). Both sexes are mutually ornamented (i.e. with similar elaborate ornamentation), exhibiting two lines of bright UV-blue patches: eyespots on their dorsolateral body region and outer ventral scales (OVS) on flank. In terms of eyespots and OVS, they display cryptic dichromatism to human eyes, as the males’ ornaments reflect UV light more strongly than the corresponding patches in females.

Collection and husbandry

We collected animals in October-November of 2014. We captured 48 adult males A. ocellifera in an Atlantic Forest fragment in Pium district, in the municipality of

Parnamirim, RN, Brazil (5° 54′ 22″ S, 35° 15′ 37″ W), through active search and manual noosing, using a loop made of fishing line. Only individuals with intact or fully regenerated tails were used in the study. Captured animals were placed in cloth bags and transported to the laboratory at Universidade Federal do Rio Grande do Norte (UFRN; 63

Natal, RN, Brazil), where animals were weighed with a analytical balance (AUW220D,

Shimadzu Corp., Kyoto, Japan, 0.01mg precision) and had their snout-vent length (SVL or body size, distance between the tip of the snout and the posterior edge of the cloaca, to the nearest 0.01 mm), head length (from the tip of the snout to the commissure of the mouth; to the nearest 0.01 mm), head height (at its highest point; to the nearest 0.01 mm) and head width (at its broadest point; to the nearest 0.01 mm) measured with a digital caliper (Paquimeter Absolute, Mitutoyo Inc., Kanagawa, Japan, 0.01mm precision). We calculated a standard condition index (residuals of mass/body size) for each individual.

After measurements, males were placed in individual plastic boxes (size: 41 cm X 28 cm

X 12 cm; length, width, height, respectively) with folded cardboard pieces for shelters, water ad libitum and Tenebrio molitor larvae as food provided once a day. The boxes were under artificial light provided by Repti Glo 5.0 Tropical Full Spectrum Terrarium

Lamps (Exo Terra, Rolf C. Hagen Inc., Holm, Germany). Photoperiod (12 L: 12 D) and temperatures (22–25 °C at night and 27–30 °C during the day) were held natural according to the natural habitat of the study population. Animals spent no more than one week in captivity and were released at the site of capture. None of the experimental animals suffered any injury. Collection permits were granted by ICMBio (nº 23164-1) and CLBI. Animal Ethics Committee of UFRN (protocol #040/2013) approved our study.

Colour measurements

The colouration of the eyespots and OVS were measured with a spectrometer (Ocean

Optics USB4000-UV-VIS), used with a light source (DH-2000-BAL) and a bifurcated fiber-optic probe (QR450-7-XSR) (Ocean Optics Inc., Dunedin, Florida) with a customized probe holder on the single ending, made from the same material as the RPH-

1 probe holder (Ocean Optics Inc.). Reflection measurements with and without the probe 64 holder were similar, taken in a constant 3mm distance and 90° angle to the surface, with an illuminated diameter of 3mm. We measured the colouration of patches with the three largest diameters for both eyespots and OVS. We used the three readings to calculate a mean reflectance for both the eyespots and the OVS and used them as separate variables during the analyses. Reflectance was calculated relative to a diffusive white reference

(WS-1-SL Spectralon Reflectance Standard, Ocean Optics, Inc.) and dark reference (= no incoming light) using the SpectraSuite software (Ocean Optics, Inc.) (Whiting et al.

2006). White and black reference were re-measured regularly to avoid spectrometer

‘drift’ (Endler and Mielke-Jr. 2005). There is no available information about the visual system and the spectral sensitivity of A. ocellifera, therefore we decided to measure the colouration within the broadest range of wavelengths known to be visible to lizards

(Fleishman et al. 2011), across the spectrum of 320–700 nm wavelengths.

Colouration of the eyespots and OVS were characterized by three variables: (1) brightness, the total reflectance over the range of 320 and 700 nm; (2) short wavelength

(UV) chroma, the percentage of reflectance measured over the range of 320 and 400 nm compared to total reflectance (R320–400/R320–700) and; (3) medium wavelength chroma, the percentage of reflectance measured over the range of 400 and 700 nm compared to total reflectance (R400–700/R320–700).

Bite force measurements

We measured bite force using an isometric force transducer (type 9203, Kistler Inc.,

Winterthur, Switzerland) connected to a charge amplifier (Kistler Inc.) following Herrel et al. (1999). We stimulated lizards to open their jaws and grab the bite plates of the force transducer by softly tapping the side of the snout, as it usually triggers aggressive responses on lizards. As body temperature affects performance capacity in lizards 65

(Angilletta 2009), we conducted measurements at the same ambient temperature of 27–

30 °C and every male was kept in the experiment room for 30 minutes to let them to acclimatize. We scored bite performance trials as “accepted” if individuals kept biting on the plates at least for three seconds. For every male, we made five repetitions, and only the highest bite force out of the five was used during the subsequent analysis.

Locomotor performance measurements

We measured sprint speed of male lizards with a 2m long racetrack composed by eight segments of 25 cm covered with sandpaper as a substrate, to provide adequate traction and stable substrate for running, in a randomly assigned order. Prior to every trial, individuals were held for 30 minutes to allow them to acclimatize to the ambient temperature (27–30 °C) of the trial. Lizards were chased down the racetrack at maximum speed by tapping them with a soft brush. We did three repetitions for each individual along three consecutive days, one per day, to allow them sufficient time to recover between trials. We filmed the running across the tracks in dorsal view with a Canon 60D camcorder set at 60 frames s–1, mounted 150 cm above the surface and used Adobe

Premiere Pro CS6 version 6.0.5 (Adobe Systems Inc., San Jose, California, United States) to analyze image data. To get the maximum sprint speed of each individual we transformed the measurement units in meters s–1, chose the fastest run in a 25 cm length of each individual as a maximum sprint speed and compared best performance results to colour traits and morphology. By that, we expect to find that better performers signals quality by displaying more conspicuous colour components, as well as having larger body and head sizes and better body condition.

Statistical analysis 66

We run a Principal Components Analysis (PCA) on the three measured head variables

(head length, height and width) in order to reduce dimensions. The first principal component (Head PC) described 81% of the total variation (eigenvalue=2.43), and showed positive correlation with all original variables (factor loadings: head height =

0.88; head length = 0.92; head width = 0.90).

We corrected morphological and performance variables for body size (SVL) to avoid collinearity by conducting Linear Regressions with Head PC, Body weight (BW) or Maximum bite force as response variables and SVL as explanatory variable. We extracted unstandardized residuals from analyses and used them as variables in subsequent analyses as Relative head size (head size vs. SVL), Condition (BW vs. SVL) and Maximum bite force residuals (maximum bite force vs. SVL).

We performed General Linear Models (GLMs) on eyespot and OVS separately to search for possible correlations between raw colour variables (Total brightness, Short wavelength chroma and Medium wavelength chroma) versus morphology and performance variables, with colour variables as dependent factors and SVL, Relative head size, Condition, Maximum bite force residuals and Maximum sprint speed as independent variables predictors. Colour components of each ornament are assumed to signal some aspect of male quality, thus we expect to detect positive relations between some variables in order to find the role of each component on intraspecific communication. All analyses were performed in R v3.2.1 (R 2015).

RESULTS

For dorsolateral eyespots, we found a positive relationship between UV chroma and SVL

(t47=2.24; p=0.03; Fig. 1a; Table 1). We also found a positive relationship between

Medium wavelenght chroma and SVL (t47=2.79; p=0.01; Fig. 1b; Table 1). These results 67 suggest that males with higher relative UV and “green” intensity of eyespots were larger in size. For outer ventral scales (OVS) on flank, we found that Short wavelenght chroma showed a negative correlation with relative head size (t47=-2.44; p=0.02; Fig. 1c; Table

1) and total brightness correlated positively with maximum bite force (t47=2.73; p=0.01;

Fig. 1d; Table 1). Sprint speed and body condition did not significantly correlate with any variables.

DISCUSSION

We found evidences that ultraviolet (UV) colour is indeed an honest signal of individual quality as reflected in performance and size, corroborating with previous studies showing that UV is affected, for example, by body condition (Molnár et al. 2012), food intake

(Lim and Li 2007), immune response (Martín and López 2009, Griggio et al. 2010) or territorial and quality of behavioral thermoregulation (Bajer et al. 2012). It is also a hunting cue to predators (Honkavaara et al. 2002) and a intraspecific signal, working as a sexual signal in mate choice (Bajer et al. 2010) and male competition (Stapley and

Whiting 2006, Whiting et al. 2006, Bajer et al. 2011, Olsson et al. 2011).

Our results indicated that larger A. ocellifera males carry more conspicuous UV dorsolateral eyespots. On the dorsum, coloration was supposedly cryptic to avoid predation costs, but as predicted by Zahavi’s Handicap Hypothesis (Zahavi 1975), these ornaments may also work as handicaps attracting both predators and the attention of females, being a reliable signal of male quality due to viability costs. Thus, males with higher UV reflective eyespots and greater speed are probably more able to avoid the potential costs of predation risks due to attracting predators, being therefore better quality individuals for females. Male body size is known as the main trait that influences reproductive success in lizards, as larger males usually sire more offspring (Calsbeek and 68

Sinervo 2002, Stapley and Keogh 2006), as well as it is a significant predictor of fighting ability (Whiting et al. 2006). Additionally, body size is a signal of male quality which some female lizard species prefer (Cooper Jr and Vitt 1993, Censky 1997), and it can also be considered a condition-dependent trait (Schulte-Hostedde et al. 2005). When the expression of the ornament is related to condition or has evolved condition-dependent expression because the ornament is a handicap, it will reliably reflect male’s good genes, as predicted by good genes hypothesis (Kirkpatrick and Ryan 1991, Andersson 1994,

Kokko 1998, Kokko et al. 2003, Kotiaho and Puurtinen, 2007). These males will be preferred by females due to relation between male quality and superior genes (Andersson

1994) or to avoid harassment from other males, which would allow females to increase foraging time (Censky 1997). Therefore, UV on eyespots are honest signals of good male quality.

However, UV intensity on the outer ventral scales (OVS) ornament was negatively related to relative head size. Similarly, UV was related to quality traits in male European green lizards, as UV throat colour signals femoral pores asymmetry and low body condition (Molnár et al. 2012). In Augrabies flat lizards, territorial males had higher throat

UV chroma but lower body condition than floater males (Whiting et al. 2006). These results suggest that high UV chroma on intraspecific ornaments might be a signal of quality in males or that there are social costs because more active and territorial males might lose their quality through social interactions. In lizards, higher dominance status can be usually predicted by larger head size (Molina-Borja et al. 1998, Gvozdik and Van

Damme 2003) and higher sprint speed (Garland et al. 1990, Robson and Miles 2000). We found that UV chroma was negatively related to head size and marginally negatively correlated with sprint speed in males. As females A. ocellifera exhibit preference for males with higher UV-reflective ornaments (Lisboa et al. 2017), female preference may 69 be based on other male traits not related to dominance (e.g. López et al. 2002, Lailvaux and Irschick 2006, Stapley 2008). Many previous studies suggested that male dominance and dominance signals are not attractive to females, so dominance may not be a reliable predictor of better genetic quality or paternal care for females (Qvarnström and Forsgren

1998, López et al. 2002).

These results corroborate our hypothesis that eyespots and OVS ornaments and their respective colour components seems to be maintained by different selective forces in A. ocellifera, signaling different aspects of male quality. This result can be reconciled with reference to the multiple message hypothesis suggested by Moller and

Pomiankowski (1993) to explain the evolution of multiple secondary sexual characters, which states that different components of ornaments produced through distinct metabolic pathways can signal different aspects of individual quality. Interestingly, we also found that the same UV colour component on different ornaments can signals different aspects of male quality. The apparently contradictory results on eyespot and OVS UV chroma may be explained by mating success, which results from the balance between two opposite selective forces: dominance status and female preference (Andersson 1994).

This result also indicates that the UV component of coloration has dual messages and costs, depending on the body position of the signal. Body location of the signal is an important target of selection, as evidences suggest that the evolution of ornaments exposed to visual predators are mainly constrained by natural selection (Lancaster et al.

2009, Lancaster et al. 2014) while unexposed ornaments are driven by sexual selection

(Stuart–Fox and Ord 2004). As an example, males of the lizard Iberolacerta monticola compensate for the increased visual conspicuousness of exposed UV-blue lateral ocelli by being shyer (an antipredatory behavior), while they do not compensate for relatively unexposed UV-blue ventral spots (Cabido et al. 2009). On the other hand, male agonistic 70 displays as lateral compression in Gallotia galloti maximally exposes dorso- and ventrolateral UV-blue patches, suggesting a role in intraspecific signaling (Bohórquez-Alonso and Molina-Borja 2014). Because of its ventrolateral (flank) location, OVS UV chroma ornaments in A. ocellifera might be better noticed by conspecifics, while being less conspicuous to predators looking from above, such as birds of prey with UV visual acuity

(Cuthill et al. 2000) (Stuart-Fox et al. 2003). The potential social costs on OVS or predation costs on eyespots of conspicuous UV colouration may be offset by benefits of female mating preference in A. ocellifera (Lisboa et al. 2017).

We observed that A. ocellifera males with higher competitive ability, expressed here as bite force, displayed brighter OVS, a ventrolateral badge visible for rivals. Total brightness comes from the thickness and spacing of reflecting nanometric platelets in iridophore cells of skin layer, which determines the wavelength composition and proportion of incident light reflected (Grether et al. 2004). Therefore, total brightness has the potential to function as quality indicator, being a predictor of male’s dominance

(Martín and López 2009), age, relative head size, health status (Molnár et al. 2012), as well as female’s receptivity (LeBas and Marshall 2000). Bright structural coloration also has been shown to be costly (Prum 2006, Bajer et al. 2012); it is therefore possible that only individuals with higher performance can bear the survival costs to maturity, when females might choose them as mates. Brightness can also work as a head size amplifier

(Martín and López 2009, Molnár et al. 2012) but in A. ocellifera, higher brightness on

OVS could work as a body size amplifier as well, being a good predictor of individual fitness to conspecifics.

Medium wavelength (green) chroma in A. ocellifera is a cryptic dorsolateral colour component resulted from overlap of skin layers of structural blue and pigment- based yellow reflectance (Grether et al. 2004). The latter might be produced by 71 carotenoids synthetized by intake of food containing such pigments (McGraw et al. 2006).

Intense carotenoid coloration has been shown to signal better health state (Hidalgo-Garcia

2006), foraging ability (Karino and Shinjo 2007) or survival (Mateos-Gonzalez et al.

2014), therefore healthier A. ocellifera individuals may have these qualities, achieving a larger body size and expressing more intense green coloration on the dorsolateral eyespots. Green badges are also an important signal for sand lizards (Lacerta agilis) males, as they strongly correlate with mass, body condition and fighting ability (Olsson

1994a), predict male genetic quality (Olsson et al. 2005) and affect male contests (Olsson

1994b), mating strategies (Olsson et al. 2000) and mate acquisition (Anderholm et al.

2004).

Sprint speed did not significantly correlate with any variables. However, it marginally correlated positively with Medium wavelength chroma on eyespots and negatively with UV chroma on OVS. Both relations were expected, as sprint speed usually increases with body size (Garland and Losos 1994), which was also related with

Medium wavelength chroma on eyespots, as well as it is a significant predictor of dominance (Garland et al. 1990, Robson and Miles 2000), thus it may be important in determining the Darwinian fitness of male lizards (Sinervo and Lively 1996). On OVS, higher UV reflectance was also related to smaller head sizes and can again be related to low-quality or younger males, with lower sprint speeds.

Although theoretical and empirical work supports the notion that body condition is linked to male quality and, consequently, is a predictor of the expression of sexually selected male traits (Zahavi 1977, Iwasa and Pomiankowski 1991, Kemp and Rutowski

2007) and of individual performance (Schluter et al. 1991, Clobert et al. 2000, Jennions et al. 2001), we found no correlation between these traits. Similar studies also could not establish that male ornaments and performance are condition-dependent traits (e.g. Husak 72 et al. 2006), suggesting that male quality may comprise other aspects than body condition as, for example, access to good quality territories and/or resources for females (Kotiaho

2001). Another possibility is that the relation mass/body size does not accurately predicts condition by itself, because many other factors related to somatic state, genotype and epigenetic state can influence condition as well (Hill 2011). The availability of body resources is one aspect of somatic state that is a key determinant of individual condition, but it does not completely describes condition in a way that we can fully understand the relation with ornament expression.

In this study, we tested three colour components from two distinct ornaments against physiological (i.e. locomotor and bite performance) and morphological variables in A. ocellifera males, thereby revealing a multiple signaling system that signals distinct aspects of individual quality in A. ocellifera. Here we also provide evidence of the multiple message hypothesis suggested by Moller and Pomiankowski (1993) to explain the evolution of multiple secondary sexual characters. Moreover, we were able to predict the costs of elaborate colour, particularly those related to UV colour, but the exact costs of structural colours are still poorly known. Specifically, larger and older males had more intense short and medium wavelength chroma on eyespots, indicating that only individuals with better survival and/or foraging ability can afford to express intense coloration. Hence, both colour components may be acting as sexual signals. On the other hand, smaller head males had more intense UV chroma on OVS, suggesting that UV on

OVS might signals low quality on males. Interestingly, the same colour trait conveys different information on each ornament, as high-reflective UV component can signal larger body size on eyespots and smaller head and lower sprint speed on OVS, suggesting that the role of colour trait depends on the body position of the signal. Additionally, on

OVS, higher brightness signals stronger bite force, which is an honest predictor of 73 fighting ability to conspecifics. Taken together, these results confirms our hypotheses that

UV is an honest signal and that colour components can signal distinct aspects of male quality. Future studies are necessary to evaluate how each colour component may affects intra and interspecific signal receivers’ behavior, to assess a more accurate role of colour traits. Furthermore, colour perception depends on visual acuity of the receiver, and yet many studies (e.g. Thorpe 2002, Lanuza and Font 2007) had called for visual sensitivity as a fundamental issue to take into account in future behavioural studies.

ACKNOWLEDGEMENTS

We thank Bruno Maggi, Juan Pablo Zurano, André Bruinje, Marc Huber and Cleto Freire for their help in collecting specimens and performing measurements. This project was funded by a CAPES Science without borders young talent research grant BJT #043/2012 and CNPq Universal grant # 474392/2013-9. GCC thanks CNPq Grants #302776/2012-5 and 201413/2014-0.

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FIGURE

Figure 1. Results of General Linear Models regarding correlations between morphological and performance variables and coloration of male Brazilian whiptail lizards (Ameivula ocellifera; N=48). (a) Eyespot UV chroma showed a positive connection with SVL. (b) Eyespot medium wavelength chroma was positively associated with SVL. (c) OVS UV chroma showed negative correlation with relative head size. (d)

OVS total brightness showed a positive correlation with maximum bite force. Solid line represents linear fit to data.

TABLE

Table 1. Results of General Linear Models on eyespot and OVS colour variables vs. morphological and performance variables of Brazilian whiptail lizards (Ameivula ocellifera; N=48). Non-significant effects are shown as seen at time of removal during backward stepwise model selection. R², F values, parameter estimates (B), standard errors

(SE) and t values are provided for each variable. Asterisks (*) show significant final model P values.

Colour variable Effect R² F B SE t value P Eyespot UV chroma SVL 0.098 5.015 0.011 0.005 2.240 0.030* Rel. head size 0.145 3.825 -0.080 0.051 -1.573 0.123 Condition 0.175 1.778 -0.019 0.042 -0.443 0.660 Max. bite force res. 0.165 2.908 0.003 0.003 1.031 0.308 Max. sprint speed 0.171 2.215 -0.041 0.077 -0.528 0.600 Eyespot MW chroma SVL 0.143 7.656 0.008 0.003 2.767 0.008* Rel. head size 0.238 2.619 -0.005 0.035 -0.141 0.889 Condition 0.237 3.345 -0.019 0.020 -0.940 0.352 Max. bite force res. 0.221 4.177 -0.002 0.002 -1.180 0.244 Max. sprint speed 0.197 5.521 0.073 0.042 1.745 0.088 Eyespot brightness SVL 0.036 0.318 0.041 0.155 0.267 0.791 Rel. head size 0.035 0.387 -0.802 1.896 -0.423 0.674 Condition 0.031 0.466 0.565 1.111 0.508 0.614 Max. bite force res. 0.015 0.719 0.087 0.102 0.848 0.401 Max. sprint speed 0.025 0.579 1.517 2.267 0.669 0.507 OVS UV chroma SVL 0.165 2.890 0.004 0.003 1.186 0.242 Rel. head size 0.082 4.107 -0.067 0.033 -2.027 0.048* Condition 0.207 2.800 0.039 0.026 1.509 0.139 Max. bite force res. 0.219 2.351 0.002 0.002 0.804 0.426 Max. sprint speed 0.138 3.600 -0.083 0.049 -1.709 0.094 OVS MW chroma SVL 0.023 1.106 0.002 0.002 1.052 0.298 Rel. head size 0.051 0.794 -0.018 0.025 -0.716 0.478 Condition 0.057 0.504 -0.006 0.020 -0.297 0.768 Max. bite force res. 0.055 0.622 -0.001 0.002 -0.389 0.699 Max. sprint speed 0.040 0.944 -0.031 0.035 -0.888 0.379 OVS brightness SVL 0.199 5.597 -0.611 0.316 -1.932 0.060 Rel. head size 0.222 3.073 -3.233 3.831 -0.844 0.403 Condition 0.209 3.885 1.737 2.303 0.754 0.455 Max. bite force res. 0.133 7.041 0.589 0.222 2.654 0.011* Max. sprint speed 0.223 2.412 -1.029 5.016 -0.205 0.838

CAPÍTULO IV: COLOUR SIGNALS CONSPICUOUSNESS OF BRAZILIAN

WHIPTAIL LIZARD (AMEIVULA OCELLIFERA) THROUGH CONSPECIFICS AND

PREDATORS VISUAL SYSTEMS

Carolina M. C. A. Lisboa1*, Barry Sinervo1, Daniel M. A. Pessoa2 and Gabriel C. Costa3

1Department of Ecology and Evolutionary Biology, Earth and Marine Sciences Building,

University of California, Santa Cruz, CA, USA

2Laboratory of Sensory Ecology, Department of Physiology, Federal University of Rio

Grande do Norte, Campus Universitário, Lagoa Nova, Natal, RN, 59078-900, Brazil

3Department of Biology, Auburn University at Montgomery, Montgomery, AL 36124,

USA

ABSTRACT

Sexual selection is the mechanism behind the evolution of many conspicuous visual signals in lizards, and the interaction with natural selection may result in complex colour ornamentation which perception may vary depending on the audience. Here, we aim to evaluate conspicuousness of Brazilian whiptail lizard (Ameivula ocellifera) colouration from the perspective of the visual system of conspecifics and avian and snake predators, which are rarely considered together, by modeling visual perception using the receptor noise model. Our results showed dichromatism between sexes, with UV signals of males more conspicuous in reflectance and highly distinguishable from females to conspecifics visual system. UV signals were in general highly perceptible from body colouration and from natural background to both conspecifics and predators. Results agreed with sensory drive hypothesis prediction that colour signals are shaped by evolution to be more conspicuous to conspecifics than to predators. Interaction of complementary colour 83 patches promotes conspicuousness, which is modeled by selective forces of natural and sexual selections. By modeling visual perception, we accessed how patches appear to the receivers and explored the consequences of conspicuous colouration in terms of the selective pressures in our study population.

Keywords: visual modeling, UV signaling, sexual dimorphism, sensory drive hypothesis, complementary colouration

INTRODUCTION

According to Darwin’s evolutionary studies (Darwin 1859, 1871), sexual selection is the mechanism behind the evolution of many ornaments including conspicuous visual signals

(reviewed by Andersson 1994), being the most important elements of social communication (Bradbury and Vehrencamp 2011). Colour signals are mainly used to advertise individual quality, but their adaptive significance has been shown by numerous studies regarding sex recognition (Schultz and Fincke 2009), mate acquisition (Bennett et al. 1997, MacDougall and Montgomerie 2003) and the deterrence of rivals (Pryke et al. 2001). These signals also have costs of increasing detection of the bearer to predators

(Endler 1992, Stuart–Fox and Ord 2004), being this way influenced by natural selection.

Thus, depending on the relative importance of each selective pressures, sexual and natural selections interact to influence colour ornamentation.

Recent models of signal evolution usually take into account many aspects, such as the type of receiver of the signal (conspecifics, predators) and their respective purposes, psychology and sensory system, the signal properties and function and/or the properties of the background environment (Endler 1992, Rowe 1999, Stuart–Fox and Ord 2004).

The way a receiver perceives colour signals and how detectable these signals are against 84 a given background are a current research topic (Bennett and Théry 2007, Kemp et al.

2009) and can be estimated through visual modeling, by analysis of reflectance data

(Vorobyev and Osorio 1998, Vorobyev et al. 1998, Maia et al. 2013).

One of the main model systems used for studies on interaction of colouration and colour vision are diurnal lizards, which have a diverse colorful ornaments and acute tetrachromatic visual systems (Pérez i de Lanuza and Font 2014). Detection and perception of colour signals depends on complex visual systems with photopigments that are usually specified by the wavelength of peak absorbance, and include long, medium, short and very short wavelength sensitive classes (LWS, MWS, SWS and VS/UVS classes, respectively; Kelber et al. 2003). Lizard vision includes a small percentage of ultraviolet (UV) cone photoreceptors in the total cone population, which are associated with colourless oil droplets that are transparent down to short wavelengths (Fleishman et al. 1993, Loew et al. 2002).

UV-photosensitive vision has been studied in some lizard groups, as sphaerodactylids (Ellingson et al. 1995), geckos (Loew 1994, Roth and Kelber 2004), two species of chamaeleonids (Bowmaker et al. 2005), a cordylid (Fleishman et al. 2011), many lacertids (Pérez i de Lanuza and Font 2014, Martin et al. 2015) and species of anoline lizards (Fleishman et al. 1993, Loew et al. 2002). Furthermore, a number of lizard species have been shown to exhibit contrasting UV colour ornaments, which may be used as signals (e.g. LeBas and Marshall 2000, Stuart-Fox et al. 2007, Font et al. 2009), and in a few cases, it has indeed been shown that UV reflectance influences mate preference

(Thorpe and Murielle 2001, Bajer et al. 2010, Lisboa et al. 2017).

While colour signals may serve a variety of functions, a signal is useless if the intended receiver does not detect it. Thus, detectability is a critical feature of a visual signal design (Leal and Fleishman 2004), and conspicuous signals over a contrasting body 85 background colouration are better detectable by the intended receiver. Eyespots can be very conspicuous and high-contrasting body marks, as observed in Lacerta (T.) lepida and other ocellated lizards, in which part of the colour pattern resembles the background environment, whereas the blue eyespots contrast strongly with the background and may function as intraspecific signals (Font et al. 2009) or as disruptive patterns to avoid avian predators (Stevens and Cuthill 2006). Environmental characteristics, such as substrate type, habitat openness or vegetation cover, affects light conditions and colour contrasts and may shape signals attributes that are most readily perceived by the sensory system of the receiver.

In our previous studies with the Brazilian whiptail lizard (Ameivula ocellifera), we experimentally demonstrated that UV reflectance influence female preference for males with higher UV reflectance (Lisboa et al. 2017). The ability of this species in discriminating between different colour reflectances is limited by the spectral sensitivity and the relative density of each class of photoreceptor in its retina, for which there are no studies available. We are therefore interested in determining whether the detection and discrimination of colour differences works in A. ocellifera, by predicting the relative effectiveness with which signal draws the visual attention of conspecific lizards, which we refer to as the signal’s detectability.

Ameivula ocellifera is an interesting species to study for two reasons. First, their colouration shows eyespots or ocelli, approximately circular motifs of scales on the ﬂanks of adult individuals, with contrasting colors on the sides of their bodies, similar to some lacertid taxa (Font et al. 2009). They also have conspicuously colored scales at the boundary between the lateral and ventral body surfaces, termed outer ventral scales

(OVS), also found in lacertids (Font et al. 2009, Marshall and Stevens 2014). In A. ocellifera, both the eyespots and the colored OVS, which appears blue to a human 86 observer, are UV reflective and play a role as a mate quality signal (Lisboa et al. 2017).

Additionally, other traits of interest are the presence of orange patches on their throat and a green dorsum, both especially conspicuous during reproductive season. Second, the species inhabits open areas in UV-rich habitats, which provides an interesting background for conspecific visual detection. The population under study settles on a tropical region in which the incidence of ambient UV is extremely high, due to geographical position and scarce cloud cover. Therefore, both the contrasting body colour signal and the UV-rich background provides a good opportunity to study signal’s detectability in our model species. This will enlighten questions about the roles of spectral sensitivities and visual backgrounds in the evolution of sexual ornamentation and the antagonism between sexual and natural selections, as the first acts on visual signals to maximise conspicuousness

(Endler and Théry 1996, Andersson 2000), while the former often favours cryptic coloration (Andersson 1994).

The aim of the current study was to evaluate the conspicuousness of A. ocellifera colour patches from the perspective of the visual system of conspecifics, by estimating:

(1) reflectance and contrast between patches of males and females, to search for sexual dimorphism as an indicative of sexual selection; (2) contrasts between spectral reflectances of body patches, to specify the role and conspicuousness of each patch or sexual signal on body colouration; and (3) contrast between body patches and natural backgrounds, to evaluate differences in conspicuousness under distinct light conditions and backgrounds. Additionally, we modeled the visual system of two potential predators to: (1) search for potential differences in perceived conspicuousness of signals and body patches of A. ocellifera to distinct receivers; and (2) test the predictions of the sensory drive hypothesis (Endler 1992) that colour signals are shaped by evolution to be more conspicuous to conspecifics than to other receivers. By modeling visual perception, we 87 can access how patches appear to the receivers and explore the consequences of conspicuous colouration in terms of the selective pressures in our study population.

MATERIAL AND METHODS

Study system and collection

The Brazilian whiptail lizard, Ameivula ocellifera Spix 1825, is a small-sized lizard that is distributed throughout Northeast of Brazil (Harvey et al. 2012, Oliveira 2014, Oliveira et al. 2015). Some populations of Ameivula ocellifera exhibit sexual dimorphism in body size and relative head size, with sexually mature males being larger and having a larger relative head size than those of the females (Vitt 1983, Mesquita and Colli 2003).

We collected 35 adult males and 20 adult females of A. ocellifera lizards in an

Atlantic Forest fragment in Centro de Lançamento Barreira do Inferno (CLBI), an area managed by the Brazilian military in the municipality of Parnamirim, RN, Brazil

(5°55'24.92"S 35°10'4.93). We sampled using a 1 km long transect of 20 pitfall trap units.

A unit consisted of four plastic buckets of 60 L each, arranged in a Y shape formation and connected by 5 m long plastic foil drift fences. We kept a total of 80 buckets opened during 15 consecutive days, and checked regularly during the day to avoid stress by natural hazards such as heat or predation. Captured animals were transported to the laboratory of Universidade Federal do Rio Grande do Norte (UFRN; Natal, RN, Brazil) for colour measurements. Animals spent no more than one week in captivity and were released at the site of capture. None of the experimental animals suffered any injury.

Lizard reflectance measurements

We measured ten body regions of each lizard: head, throat patch, dorsolateral eyespots, dorsum, OVS, belly, dorsal tight, ventral tight, dorsal tail base and ventral tail base. We 88 measured each body region three times on three different locations and the used the mean as the representative spectrum. We took all spectral reflectance measurements within two days of the lizards being in captivity.

We measured body colouration with a spectrometer type Ocean Optics USB4000-

UV-VIS, with a DH-2000-BAL light source and a QR450-7-XSR bifurcated fiber-optic probe (Ocean Optics Inc., Dunedin, Florida). The probe was modified with a custom- made probe holder, made of anodized aluminium in order to restrict the illuminated area to 3 mm in diameter, allowing us to measure the colouration of small-sized patches (such as eyespots and OVS), while excluding their surrounding areas. The probe holder maintained the probe in a constant 3mm distance and 90° angle to the surface.

Measurements taken by the RPH-1 probe holder (Ocean Optics Inc.) and our customized probe holder gave similar results. Reflectance was calculated relative to a diffusive white reference (WS-1-SL Spectralon Reflectance Standard, Ocean Optics, Inc.) and dark reference (= no incoming light) using the SpectraSuite software (Ocean Optics, Inc.)

(Whiting et al. 2006). No information is available for the visual system and the spectral sensitivity of A. ocellifera, although these are conservative traits in lizard group.

Therefore we decided to measure the colouration within the broadest range of wavelengths known to be visible to diurnal lizards (Fleishman et al. 2011), from 300 to

700 nm. White and black references were re-measured regularly to avoid spectrometer

‘drift’ (Endler and Mielke-Jr. 2005).

Background and irradiance measurements

We obtained background spectral reflectance measurements from sand and leaf litter under full sun and shrub shade, grass and bush leaves near or upon which lizards were first sighted or captured, five times for each site. Measurements of natural background 89 spectral reflectance followed the same protocol as that used on lizard’s reflectance spectra. We averaged reflectance spectra in each locality to have a mean background reflectance (Fig. 1).

To measure irradiance (µmol m-2 s-1 nm-1; Fig. 2) we used an Ocean Optics S2000 spectrophotometer and an Ocean Optics cosine adaptor head, held overhead and oriented upward, taken under a clear and cloudless sky, between 10:00 and 12:00h.

Visual Modeling

A widely used method to model visual perception is to use the receptor noise model, which estimates the distance between two spectra in the chromatic and achromatic spaces in Just Noticeable Difference (JND) units (Maia et al. 2013). JND is a perceptual unit in which chromatic contrast can be either perceptible (≥ 1 JND) or not (< 1 JND). When the color contrast between two objects produces a value that exceeds the threshold of 1 JND, the target is to be considered detectable against the background (Osorio et al. 2004). Here we assumed that colour distances lower than 1 JND were ‘not distinguishable’, those between 1-4 JND were ‘poorly distinguishable’, and those higher than 4 JND were

‘highly distinguishable’ (Martin et al. 2015). Using this method, we will be able to detect sexual dimorphism by distinguishable chromatic contrasts between males and females, to evaluate conspicuousness of each body patch and colour signals and from patches against natural environment and to assess potential lower perception of colour signals from predators vision. Although the differences in JNDs of lizard colours against backgrounds are unlikely to change, we incorporated lighting conditions (full sun, shadow) into the calculations. The visual model estimates chromatic (colour) distance (ΔS) based on the four single sensitive cones (ultraviolet, short wavelength, medium wavelength and long wavelength) and achromatic (bright) distance (ΔL) based on the double cone, which is 90 responsible for detecting differences in signal luminance for lizards (Osorio and

Vorobyev 2005). We conducted visual modeling using the R v3.2.1 (R 2015) package

“Pavo” (Maia et al. 2013).

To evaluate A. ocellifera conspecifics visual discrimination, we used photoreceptors’ peak absorbance from Zootoca vivipara (Fig. 3A), a species from the family Lacertidae, which is a close phylogenetic group. Lacertids and teiids are closely related groups both at morphological (Caldwell 1999, Abdala and Moro 2003) and molecular phylogenies (Pyron et al. 2013). In addition, Z. vivipara is similar to A. ocellifera in their habit and habitat (both species are active foragers that lives in grassy open habitats), as well as the higher abundance of UV cones that improves the detection of small variations in UV reflectance (Martin et al. 2015).

Regarding discrimination by visual predators, we applied our model to the spectral sensitivity from two types of potential predators (Fig. 3B, C). The diurnal snake

Philodryas patagoniensis (Fig. 3B), which is known to prey on Ameivula spp. (Peloso and Pavan 2007) and is common on our study site. We also used avian raptor sensitivity peaks, because birds of prey are notorious predators of Ameivula spp (Koski et al. 2015), and some species of Accipitridae such as the Roadside Hawk Rupornis magnirostris (Fig.

3C) are frequent in our study site. Einat Hauzman (2016 pers. comm.) and Lind et al.

(2013) provided snake and avian raptor sensitivity peaks data, respectively.

RESULTS

Spectral analyses of body patches from males and females showed that all patches but throat and ventral tail base were more conspicuous in males, particularly eyespots and 91

OVS (Figure 4). Eyespots, OVS and throat colours were highly distinguishable between males and females (Table 1).

Our visual modeling analyses on body patches showed that, from the perspective of conspecifics, the contrasts (chromatic distance) of eyespots and OVS colour sexual signals against dorsum and belly colour background are highly perceptible (≥ 4 JND), with OVS slightly more than eyespots, but poorly distinguishable (1-4 JND) between each other. Throat colour against belly background was also poorly distinguishable (Table

2).

From the perspective of predators, results were similar between each type, but chromatic distances were slightly more contrasting for avian raptors (Table 2). Eyespots and OVS were both highly distinguishable against dorsum and, consequently, very conspicuous to predators, but poorly or not distinguishable against each other. OVS against belly chromatic distance was highly perceptible for conspecifics but poorly distinguishable for both visual predators, suggesting that OVS is an intraspecific signal.

Achromatic distances of all variables were highly detectable from all visual systems.

From a lizard’s perspective, chromatic contrasts of colour patches against background environment were highly distinguishable in general, except for throat x sand under shrub shade (Table 3). All body patches but the dorsum were more perceptible under full sun than under shaded environment.

The snake P. patagoniensis better detects sexual signals (eyespots, OVS and throat) chromatic contrasts under full sun than under shaded environments or against bush or grass. Avian raptors can barely detect chromatic contrasts of sexual signals unless these are presented against leaf litter under full sun. Dorsum is poorly detectable only against grass by both predators. In general, conspecifics visual discrimination was better in detecting contrasts than by the visual system of both predators. 92

DISCUSSION

Through a lizards’ visual system perception, A. ocellifera sexual signals (eyespots and OVS) were proven to be highly distinguishable against both body and environmental backgrounds. This result has implications for the success of signals transmission, since highly detectable colour patterns facilitate the perception, discrimination and memorization by conspecifics (Rowe 1999, Candolin 2003, Bradbury and Vehrencamp

2011).

Chromatic distances of male UV patches were highly distinguishable from those of the female, and spectral reflectances of UV patches were higher in males. These findings complement each other and confirm that UV sexual signals are more conspicuous in males, corroborating with Darwin’s theory (Darwin 1871) of sexual selection that explains the presence of exaggerated male secondary sexual traits, which should be opposed by natural selection.

Our visual modeling results show that UV-blue signals eyespots and OVS were both highly detectable against brown-green dorsum background from conspecifics perspective, but OVS more than eyespots, and they were poorly but still distinguishable between each other in chromatic distance. Achromatic distance reveals better the difference between UV-blue signals, as OVS are bright and smooth laminar scales which luminance may be more detectable for lizards than the granular scales of eyespots. In the same way, our previous studies on A. ocellifera colouration indicates that the different body locations of these similar signals may result in them conveying different information

(Lisboa et al. unpublished data), as OVS on flanks region are more visible to conspecifics and, as expected, more reflective and discernible against dorsum colouration than are eyespots. 93

The abundance of UV cones is important for lizard chromatic resolution, and the presence of this cone type in our model strongly improves visual performance for detecting variations in colour signals of A. ocellifera, which are essential for sex recognition and intra and intersexual competition in our study species. Superabundance of UVS cones in the retina of Platysaurus broadleyi also enhances discrimination of small variations in UV on the throats of conspecifics (Fleishman et al. 2011), and multidisciplinary approaches and behavioral tests confirmed that many lacertids are capable of discrimination with their UV vision (Pérez i de Lanuza and Font 2014).

These UV signals are less conspicuous to both snake and avian predators with different visual systems, agreeing with previous findings on other lizard species and their predators (Marshall and Stevens 2014, Pérez i de Lanuza and Font 2015). This results is in agreement with the predictions of the sensory drive hypothesis in that if predators and prey exhibit different spectral sensitivities, colour signals will evolve in ways that make them more conspicuous to conspecifics than to predators (Endler 1992). Our snake predator is trichromatic, lacking oil droplets, so it has poor colour discrimination in chromatic distance of lizard colour patches. Macedonia (2009) found similar results, with conspicuousness of Crotaphytus dickersonae greatest for the lizard and avian visual models than for the snake visual model. Regarding avian visual sensitivity, Lind et al.

(2013) showed that raptor eyes filter out much UV light, so relatively long wavelengths predominantly arrives at the retina. Thus, UV signals emanating from the lizards are better tuned to the visual sensitivities of lizards than to the avian raptors. Marshall and Stevens

(2014) found that the UV-sensitive Aegean wall lizards (Podarcis erhardii) appeared twice as conspicuous to conspecifics than to avian predators against the same visual background. 94

Chromatic contrast between OVS and the continuum rows of white belly scales was poorly or non-detectable by predators but highly distinguishable by conspecifics.

OVS always have higher UV peaks than eyespots in both sexes and are located in a vetrolateral region that is poorly visible to predators. Evidence from other species supports the hypothesis that UV-blue OVS convey information about male quality, as body condition and/or fighting ability (Pérez i de Lanuza and Font 2014). Although more research is needed, these findings may indicate that OVS signal can be used as a “private” conspecific communication signal by A. ocellifera that is imperceptible to predators, as seen in fish species that use UV wavelength band as a private communication channel invisible to predators (Cummings et al. 2003).

Dorsolateral eyespots, on the other hand, are highly distinguishable by predators when contrasting against dorsum. In previous studies, we found that larger males had more intense short (UV) and medium wavelength chroma on dorsolateral eyespots, indicating that individuals with better survival and/or foraging ability can afford the costs of intense coloration (Lisboa et al. unpublished data). We also found female preference for males with higher UV chroma on eyespots and OVS (Lisboa et al. submitted). Thus, eyespots may function as a “handicap” or honest signal of individual quality to receivers because of their predation costs (Zahavi 1975, Zahavi 1977, Grafen 1990). Alternatively, eyespots may have a camouflage function that conveys protection against predators.

According to Thayer’s Hypothesis (Thayer 1909), eyespots and other high-contrast markings create false edges that distract predator away from the body outline, or they can also generate a dazzle coloration on the moving prey, making the estimates of speed and trajectory difficult for the predator to evaluate. If so, the handicap hypothesis could not account for female preference of more reflective UV eyespots on males. Although the firsts empirical tests rejected Thayer’s Hypothesis (Stevens et al. 2008, Stobbe and 95

Schaefer 2008), further studies may be required for A. ocellifera to elucidate the role of eyespots on cryptic colouration and female preference.

UV-orange throat patches have a step in reflectance in the 500-600 nm interval

(Figure 4), and females had slightly more conspicuous patches in medium and long wavelengths, which may be due to a carotenoid-based colouration of gravid females, as found in Crotaphytus dickersonae (Macedonia et al. 2009). However, the orange spots on throats are visible especially during reproductive season in both sexes, and may have a signaling function in short UV and long wavelength spectra. A white belly provides the strongest dorsoventral countershading, and the robusts chromatic and achromatic contrasts of belly versus dorsum found in A. ocellifera may provide protection against predators. On the other hand, ventral non-white colouration such as an orange throat may reduce the efficiency of countershading (Thayer 1909) and turn the lizards more conspicuous to predators and conspecifics. As shown in Table 2, chromatic and achromatic contrasts of throat against dorsum were less contrasting than belly against dorsum in A. ocellifera. Because of these colour association with the lateral patches, these long wavelength bands can function as amplifiers, by contributing in maximize the overall pattern conspicuousness and improving the receiver’s ability to detect or assess the signal (Pérez i de Lanuza and Font 2015, 2016). However, to successfully determine whether throat UV-orange patches have amplifier or signaling function it would be necessary to determine its effects on the response of receivers through behavioural experiments.

Long- (orange throat) or medium- (dorsolateral brown-green) and short- (eyespots and OVS) wavelength colour patches are basically complementary, which means that each one reflects most in the region of the spectrum where the other does not (Pérez i de

Lanuza and Font 2015). This enhances chromatic contrast between each signal element 96

(especially on adjacent patches) and with the visual background, which is a well-known strategy to maximize conspicuousness (Endler 2012) because contrasting colours stimulate retinal cones in opposite ways (Endler 1992). This is a common strategy in many lacertid lizard species (Pérez i de Lanuza and Font 2016), which are closely related to teiids such as A. ocellifera (Pyron et al. 2013). However, in all lacertid species studied,

UV-reflecting colour patches are located either laterally or ventrally, but not in both locations (Pérez i de Lanuza and Font 2016). In contrast to lacertids, A. ocellifera showed a secondary ventral UV peak and UV-reflecting eyespots and OVS, which may be characteristics that make A. ocellifera complex colour pattern and signaling system more challenging to study.

Although inconspicuous colouration should prevail because it is hard to be detected by predators, benefits provided through sexual selection are thought to outweigh the costs imposed by natural selection. Complementary colour patches within-body colouration enhances conspicuousness of sexual signals in A. ocellifera and may be advantageous to fitness, thus selection may favor the evolution of these contrasting spectral combinations in our study species. Colour association was shown to be non- random in lacertid lizards phylogeny, with long- and short-wavelength colours selected for increased conspicuousness (Pérez i de Lanuza and Font 2016). Additionally, these contrasting colour patterns probably evolved in a correlated fashion because they promote conspicuousness and consequently improves signal efficiency. As UV in lizards is a structural component produced by interactions between incident light and nanostructures in the skin while long wavelength signals are mainly pigment-based (Grether et al. 2004,

Haisten et al. 2015), it is unlike that this association of two distinct mechanisms is driven by genetic correlation, in which traits are partially determined by the same or linked loci.

This colour pattern is probably driven instead by correlated selection (Sinervo and 97

Svensson 2002), in which the same environmental pressure acts on seemingly unrelated traits. Correlational selection is often a feature of signals that serve anti-predator function

(e.g., alternative escape behavior) and sexually selected functions in the context of choice for phenotype integration (Lancaster et al. 2009, 2010).

Target-background analysis reveals that, from a lizard’s perspective, chromatic contrasts of colour patches against background environment were highly distinguishable, except for UV-orange throat in sand under shrub shade. Shaded environments do not favor long wavelength colouration as orange patches because there is no direct sunlight strong in long wavelengths to be reflected. Chromatic contrast of conspecifics were large, but not consistently more distinguishable for predators, in all visual backgrounds. As the number of visual backgrounds and variables increase, it is expected that the probability of selected colour patches that are more detectable to conspecifics than to predators become increasingly unlikely (Kemp et al. 2008). Our study population inhabits UV-rich open habitats, with sparsely distributed shrubs or small trees providing shade under predominantly blue skies (Figure 1). These characteristics bias the ambient light towards short wavelengths that enhances conspicuousness of UV-reflecting body patches (Endler and Théry 1996) and consequently improves signalling (Molina-Borja et al. 2005). This contrast between UV patches and natural environment suggests the influence of selective pressures (Lanuza and Font 2010), as visual signals can be exploited by both conspecifics and predators, and habitat complexity influences the conspicuousness of signallers

(Stuart–Fox and Ord 2004).

In summary, our results quantified colour patches and signal conspicuousness in relation to each other and to natural backgrounds, considering the visual system properties of both predators and conspecifics, which are rarely considered together in studies of signal evolution. We have shown that phenotypic colouration in our study species reveals 98 contrasting adjacent colour patches that play a role in sexual signaling and camouflage.

Interaction of complementary long-, medium- and short-wavelengths colour patches promotes conspicuousness, which is modeled by selective forces of natural and sexual selection. These conspicuous signals are better tuned to the visual systems of conspecifics than to that of their snake and avian predators, indicating that the conflicting selective forces can affect detectability of signals by different receivers and signal location on the body. Further work should focus on behavioural studies that reveal the role of long- and medium- wavelength conspicuous colour patches in our study species by evaluating receiver’s responses and experiments to evaluate risk of detection by predators.

ACKNOWLEDGEMENTS

We thank to Mélissa Martin and Einat Hauzman for providing the spectral sensitivity data from Z. vivipara and P. patagoniensis, respectively, and to Gustavo Carvalho, Marc

Huber, Orsolya Molnár, Bruno Maggi, André Bruinje and Cleto Freire for their help in collecting specimens and performing measurements. Collection permits were granted by

ICMBio (nº 23164-1) and CLBI. Animal Ethics Committee of UFRN (protocol

#040/2013) approved our study. This project was funded by CAPES/PDSE BEX6383/15-

7 scholarship to CMCAL, CAPES Science Without Borders young talent research grant to BJT # 043/2012 and CNPq Universal grant # 474392/2013-9. GCC thanks CNPq grants

#302776/2012-5 and 201413/2014-0.

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Figure 1. Spectral reflectance of natural background under different light conditions and microhabitats used by Ameivula ocellifera. Means (N=5) are represented by solid lines ±

SE (grey surrounds).

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Figure 2. Habitat irradiance sampled from the study site.

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Figure 3. Relative sensitivity for each cone class in (A) the lizard species used to model

Ameivula ocellifera visual system, the common lizard Zootoca vivipara (Martin et al.

2015); (B) a snake predator of A. ocellifera, Philodryas patagoniensis (Einat Hauzman

2016 pers. comm.); and (C) a general avian raptor spectral sensitivity (Lind et al. 2013).

Numbers above each curve indicate peak wavelength absorbance for violet-sensitive 112

(VS), short- (SWS), middle- (MWS), and long- (LWS) wavelength-sensitive visual pigments.

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Figure 4. Spectral reflectance of ten Ameivula ocellifera body regions. Males (N=35) are represented by solid lines ± SE (grey surrounds) and females (N=20) by dashed lines ±

SE (grey surrounds).

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Table 1. Chromatic (ΔS) and achromatic (ΔL) contrasts of body colour patches of

Ameivula ocellifera males and females from the perspective of conspecifics (Zootoca vivipara visual system), in units of just noticeable differences (JND). *Highly distinguishable (≥ 4 JND) contrasts.

JND Males x Females ΔS ΔL Belly 0.79 3.72 Dorsum 3.49 4.51 Eyespots 5.40* 3.03 Dorsal thigh 1.38 4.17 OVS 6.76* 6.23 Head 0.55 4.77 Throat 5.47* 4.66 Tail base 2.47 4.80 Ventral thigh 1.12 5.54 Ventral tail base 0.84 1.88

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Table 2. Chromatic (ΔS) and achromatic (ΔL) contrasts between body colour patches of

Ameivula ocellifera from the perspectives of conspecifics (Zootoca vivipara visual system), of a snake predator (Philodryas patagoniensis) and of an avian raptor, in units of just noticeable differences (JND).

JND Patch x Patch A. ocellifera P. patagoniensis Avian raptor ΔS ΔL ΔS ΔL ΔS ΔL Dorsum x Eyespots 9.41 13.31 4.70 22.78 7.42 18.69 Dorsum x OVS 10.91 36.28 5.60 46.97 8.55 42.34 OVS x Belly 4.11 16.91 1.73 13.43 2.22 14.42 Belly x Throat 3.48 15.72 1.99 19.78 2.73 17.90 OVS x Eyespots 1.64 22.61 0.90 23.86 1.18 23.32 Throat x Eyespots 7.32 25.05 1.13 17.68 4.12 19.99 Throat x Dorsum 7.63 37.79 4.49 40.84 5.18 39.13 Belly x Dorsum 8.26 52.95 4.33 60.11 6.70 56.49

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Table 3. Chromatic (ΔS) and achromatic (ΔL) contrasts between body colour patches of Ameivula ocellifera and background environment from the perspectives of conspecifics (Zootoca vivipara visual system), of a snake predator (Philodryas patagoniensis) and of an avian raptor, in units of just noticeable differences (JND). JND Patch x Background A. ocellifera P. patagoniensis Avian raptor ΔS ΔL ΔS ΔL ΔS ΔL Eyespots x Sand under full sun 10.17 56.65 9.55 55.87 2.27 55.26 Eyespots x Sand under shrub shade 6.78 14.91 3.40 9.85 3.69 11.76 Eyespots x Leaf litter under full sun 15.41 11.72 13.19 7.63 8.29 9.42 Eyespots x Leaf litter under shrub shade 8.09 8.55 6.07 13.16 3.44 11.50 Eyespots x Bush leaves 4.76 28.77 2.85 23.63 3.43 25.89 Eyespots x Grass 5.09 6.41 3.30 2.04 3.96 3.83 OVS x Sand under full sun 11.06 34.03 10.25 32.01 3.03 31.93 OVS x Sand under shrub shade 6.70 7.70 2.83 14.01 4.14 11.56 OVS x Leaf litter under full sun 16.89 10.89 14.01 16.22 9.43 13.90 OVS x Leaf litter under shrub shade 7.68 31.17 5.37 37.02 3.59 34.82 OVS x Bush leaves 5.64 6.16 2.68 0.23 4.43 2.57 OVS x Grass 6.57 16.20 4.19 21.82 5.09 19.49 Dorsum x Sand under full sun 9.65 70.37 7.61 78.92 6.78 74.26 Dorsum x Sand under shrub shade 10.14 28.63 7.21 32.90 6.06 30.76 Dorsum x Leaf litter under full sun 9.64 25.44 9.83 30.68 4.68 28.43 Dorsum x Leaf litter under shrub shade 12.38 5.16 9.87 9.89 7.10 7.50 Dorsum x Bush leaves 7.43 42.49 5.85 46.68 4.21 44.90 Dorsum x Grass 4.64 20.13 2.00 25.09 3.53 22.83 Throat x Sand under full sun 10.61 32.99 10.22 38.44 4.46 35.52 Throat x Sand under shrub shade 3.38 8.75 2.87 7.58 0.92 7.98 Throat x Leaf litter under full sun 15.79 11.93 13.60 9.79 8.24 10.31 Throat x Leaf litter under shrub shade 6.31 32.21 5.60 30.58 2.05 31.24 Throat x Bush leaves 5.02 5.11 1.91 6.20 2.04 6.16 Throat x Grass 5.53 17.24 3.54 16.38 3.24 15.91 117

CONSIDERAÇÕES FINAIS

Os experimentos de seleção sexual conduzidos aqui mostraram que os ornamentos com coloração ultravioleta (UV), os ocelos e escamas ventrais exteriores (EVEs), influenciam na escolha do parceiro sexual pelas fêmeas. A coloração ultravioleta (UV) foi importante nos experimentos de associação espacial de fêmeas, já que as mesmas exibiram preferência por machos com maior reflectância UV em seus ornamentos, em relação aos machos com coloração UV experimentalmente reduzida. Com isso, vimos que os ornamentos desempenham um papel importante na seleção de parceiros pelas fêmeas da espécie, pois sinalizam a qualidade dos machos.

Apesar de não observarmos importância da coloração sobre as interações entre machos nos experimentos de competição, visto que os machos com reflectância UV experimentalmente reduzida não foram mais propensos a perder o combate do que os machos controle, a reflectâcia UV foi correlacionada negativamente com o tempo de avaliação. Portanto, concluímos que a coloração UV não é um sinal de dominância entre os machos da espécie, embora influencie o tempo de avaliação durante as disputas.

A relação entre a coloração dos ornamentos e os aspectos qualitativos dos machos foram testados através de medições de força de mordida, performance locomotora e morfometria. Com isso, determinamos quais características tais ornamentos sinalizam e pudemos definir a honestidade dos sinais. Nas EVEs encontramos ligações entre as variáveis brilho e força de mordida, entre cor UV e tamanho relativo da cabeça, e uma correlação marginal entre UV e performance locomotora. Já nos ocelos, encontramos um correlação marginal entre a velocidade da corrida e a cor verde, além da associação entre intensidade do verde e do UV com o comprimento rostro-cloacal dos animais. Tais resultados sugeriram que os caracteres de cor são informativos sobre performance e tamanho nos machos e, consequentemente, revelam atributos do parceiro sexual para as 118 fêmeas, sendo assim sinais honestos de qualidade. Também observamos que ornamentos distintos sinalizam diferentes aspectos qualitativos dos machos, em um sistema de sinalização múltiplo que pode estar sob influência de diferentes pressões seletivas para que sejam mantidas suas características.

Por fim, realizamos um estudo de modelagem visual no qual os sistemas visuais de A. ocellifera e de dois tipos de predadores foram modelados para desvendar como a coloração conspícua de A. ocellifera é percebida. Vimos que a reflectância dos sinais UV de machos é altamente distinguível da de fêmeas a partir do sistema visual de A. ocellifera e que também foram, em geral, altamente perceptíveis quando em contraste com a coloração do corpo e do fundo natural. A partir da visão dos predadores, os sinais UV foram menos mas ainda perceptíveis, concordando com a hipótese de condução sensorial.

Vimos ainda que cores complementares promovem maior visibilidade e são moldadas por pressões seletivas na população estudada.

As contribuições ao conhecimento sobre seleção natural e, mais especificamente, sobre seleção sexual contidas aqui são de grande relevância para o entendimento da evolução dos sinais sexuais em lagartos e para o esclarecimento das formas de comunicação intra e interespecíficas nesses animais, além de suprirem uma lacuna sobre este tipo de estudo em lagartos neotropicais.

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ANEXOS