Seleção sexual e modelagem visual em Ameivula ocellifera

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(1)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.

(2) 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.

(3) 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.

(4) 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..

(5) “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.

(6) 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.

(7) 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.

(8) 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.

(9) 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.

(10) 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.

(11) 1. 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.

(12) 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..

(13) 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)..

(14) 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..

(15) 5 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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(20) 10 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. 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. Laboratory of Sensory Ecology, Department of Physiology, Federal University of Rio Grande. do Norte, Campus Universitário, Lagoa Nova, Natal, RN, 59078-900, Brazil. 4. Department 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: carolisboabio@yahoo.com.br.

(21) 11 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.

(22) 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.

(23) 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.

(24) 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.

(25) 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.

(26) 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.

(27) 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 sunscreenbearing experimental animals’ behaviour, physiology or colouration (no apparent swelling, fading or discolouring was observed)..

(28) 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.

(29) 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 (t 18=0.987, p=0.337),.

(30) 20 Body weight (t 18=1.021, p=0.321) or Head size (t 18=0.885, p=0.388) when comparing UVdeprived 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.1ab). 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.

(31) 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.

(32) 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.

(33) 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.

(34) 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. REFERENCES Adolph SC (1990) Influence of behavioral thermoregulation on microhabitat use by two Sceloporus lizards Ecology:315-327.

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