unesp UNIVERSIDADE ESTADUAL PAULISTA Instituto de Biociências de Rio Claro Departamento de Zoologia

Transcrição

1 unesp UNIVERSIDADE ESTADUAL PAULISTA Instituto de Biociências de Rio Claro Departamento de Zoologia BIOLOGIA REPRODUTIVA DE Scinax fuscomarginatus EM UM FRAGMENTO DE CERRADO NO SUDESTE DO BRASIL LUÍS FELIPE DE TOLEDO RAMOS PEREIRA Dissertação apresentada ao Instituto de Biociências da Universidade Estadual Paulista Julio de Mesquita Filho, Campus de Rio Claro, para a obtenção do título de Mestre em Ciências Biológicas (Área de Zoologia). Rio Claro Estado de São Paulo Brasil Abril de 2004

2 unesp UNIVERSIDADE ESTADUAL PAULISTA Instituto de Biociências de Rio Claro Departamento de Zoologia BIOLOGIA REPRODUTIVA DE Scinax fuscomarginatus EM UM FRAGMENTO DE CERRADO NO SUDESTE DO BRASIL LUÍS FELIPE DE TOLEDO RAMOS PEREIRA Orientador: Prof. Dr. DENIS OTÁVIO VIEIRA DE ANDRADE Co-orientador: Prof. Dr. CÉLIO FERNANDO BAPTISTA HADDAD Dissertação apresentada ao Instituto de Biociências da Universidade Estadual Paulista Julio de Mesquita Filho, Campus de Rio Claro, para a obtenção do título de Mestre em Ciências Biológicas (Área de Zoologia). Rio Claro Estado de São Paulo Brasil Abril de 2004

3 Enfunando os papos, Saem da penumbra, Aos pulos, os sapos. A luz os deslumbra. Em ronco que aterra, Berra o sapo-boi: "Meu pai foi à guerra!" "Não foi!" "Foi!" "Não foi!". O sapo-tanoeiro, Parnasiano aguado, Diz: "Meu cancioneiro É bem martelado. Vede como primo Em comer os hiatos! Que arte! E nunca rimo Os termos cognatos! O meu verso é bom Frumento sem joio Faço rimas com Consoantes de apoio. Vai por cinqüenta anos Que lhes dei a norma: Reduzi sem danos A formas a forma. Clame a saparia Em críticas céticas: Não há mais poesia, Mas há artes poéticas..." OS SAPOS Urra o sapo-boi: "Meu pai foi rei" "Foi!" "Não foi!" "Foi!" "Não foi!" Brada em um assomo O sapo-tanoeiro: "A grande arte é como Lavor de joalheiro. Ou bem de estatuário. Tudo quanto é belo, Tudo quanto é vário, Canta no martelo." Outros, sapos-pipas (Um mal em si cabe), Falam pelas tripas: "Sei!" "Não sabe!" "Sabe!". Longe dessa grita, Lá onde mais densa A noite infinita Verte a sombra imensa; Lá, fugindo ao mundo, Sem glória, sem fé, No perau profundo E solitário, é Que soluças tu, Transido de frio, Sapo-cururu Da beira do rio Manuel Bandeira, 1918

4 ii AGRADECIMENTOS Ao prof. Célio Fernando Baptista Haddad a quem devo em muito pelo que hoje sei sobre anfíbios, pela orientação e amizade, principalmente durante os cafezinhos, momentos nos quais mais aprendi, tanto sobre Biologia, como sobre política, universidades, ética, churrasco, fotografia, etc... Ao prof. Denis Otávio Vieira de Andrade pela orientação, mesmo que tangencial, pela continuidade dos trabalhos antigos, mas sobretudo pelo companheirismo e resistência, afinal já se foram mais de seis anos de orientação! Aos Zoólogos que também contribuíram para minha formação, principalmente os professores Augusto Abe, Rogério Bastos, Márcio Martins, Ivan Sazima, Ariovaldo Cruz Neto e Francisco Braga. Ao pessoal dos laboratórios de Herpetologia de Rio Claro: Juliana, Cynthia, Luís, Moisés, Anne e Flávio, Luciana Lugli, Paulo e Marília, Luciana Bolsoni, Paulo Perereca, Marcos, Olívia e João pelo companheirismo e pela ajuda durante toda a realização da dissertação, desde o auxílio na elaboração do projeto, coleta de dados, análises multivariadas, discussões científico-herpetológicas e até a redação final dos capítulos. À galera de Itirapina que foi fundamental para animar aquele Cerrado, especialmente Elaine e Hilton, Tozetti e Victor, Beto, Cínthia e Ricardo, Jota, Spina e Cia; eventualmente Márcio Martins, Kelly, Jeanne e Chan também contribuíram para azeitonar minhas madrugadas em Itirapina. À administração da Estação Ecológica de Itirapina, principalmente a Denise Zancheta e os funcionários do Instituto Florestal, especialmente Gilson, que por repetidas vezes me deu uma forcinha nos atoleiros do Cerrado. É claro, não podia deixar de mencionar a marcante onipresença e simpatia de Dna. Izabel, quase sempre disposta a nos divertir.

5 iii Ao pessoal extracurricular de Rio Claro, os quais sempre me acompanharam nas horas vagas: Milho Xangrilá, Fábio Longarina e TC, Prima e Zé, Akio, Cauré, Ju Zina, Yara e Bira, Sabrina, Renata Udulutsch, Renata Campanhã e Lye, Sarahinha, João Ennser, Elis Regina e Talento. Ao povo de sampa, principalmente Quito, Gus, Raf s, Amaral, Ivan, Evandro e Bel, os quais eternamente farão parte de minhas conquistas. À Débora pela paciência, companheirismo e amor, os quais parecem ser infinitos. À toda minha família, mas essencialmente aqueles sem os quais tudo ficaria muito mais difícil: Regina, Cris, Neide, Iza, Jair, Eliza e Raphael. Por fim, ao Conselho Nacional de Desenvolvimento Científico e Técnico (CNPq) pela bolsa de estudos concedida (processo no /2003-3) e a Idea Wild pelos equipamentos doados.

6 iv ÍNDICE Resumo... Abstract... Introdução Geral... Conhecimento atual sobre Scinax fuscomarginatus... Objetivos O Cerrado e a Área de Estudo... Capítulo 1: ACOUSTIC REPERTOIRE AND CALLING SITE OF SCINAX FUSCOMARGINATUS (ANURA, HYLIDAE) DURING REPRODUCTION.. Abstract... Introduction... Methods... Results... Discussion... Acknowledgments... Literature Cited... Capítulo 2: REPRODUCTIVE BIOLOGY AND CHORUS ORGANIZATION OF SCINAX FUSCOMARGINATUS (ANURA, HYLIDAE)... Abstract... Introduction... Methods... Results... Discussion... Acknowledgments... References... Considerações Finais

7 1 RESUMO O estudo da biologia reprodutiva de populações de anfíbios anuros compreende diversos aspectos de história natural, comportamento e ecologia, tais como: I) a caracterização do modo reprodutivo; II) a caracterização das diferentes vocalizações emitidas e de seus contextos de emissão; III) a verificação da ocorrência de seleção sexual e de segregação de recursos limitados; IV) a descrição de comportamentos sociais e de características morfológicas dos adultos, formas larvárias, desovas e ovos da espécie. O presente estudo investigou a biologia reprodutiva de Scinax fuscomarginatus sob todos os enfoques enumerados acima. As observações foram realizadas no período vespertino e noturno, entre setembro de 2002 e março de 2004 em um fragmento conservado de Cerrado na Estação Ecológica de Itirapina, localizada nos Municípios de Itirapina e Brotas, interior do Estado de São Paulo. A espécie reproduziu em ambientes lênticos e temporários, sendo o padrão reprodutivo prolongado e a organização dos agregados reprodutivos em forma de leks. Os machos defenderam territórios através de interações acústicas, visuais e físicas, sendo que os machos vencedores foram sempre os residentes. Foi observado comportamento de macho satélite, embora interceptação de fêmeas ou de casais amplectados por machos satélites não tenha sido observada. Nos ambientes estudados o número de machos foi sempre maior que o de fêmeas, gerando baixas razões sexuais operacionais. Foram descritos em detalhe quatro tipos de vocalizações (canto de anúncio, canto esporádico, canto territorial e canto de briga) e seus respectivos contextos de emissão. Com exceção do canto de anúncio, as demais vocalizações apresentadas são inéditas para a espécie. O amplexo foi axilar e os ovos foram depositados diretamente na água, no fundo de poças temporárias. Os ovos possuíam duas cápsulas gelatinosas, as quais conferem maior espaçamento entre os embriões. A população estudada de S. fuscomarginatus apresentou uma gama de comportamentos sociais elaborados, incluindo organização espacial entre machos, interações agonísticas (acústicas, visuais e físicas) e um provável comportamento de corte (canto de corte), características comuns à espécies de padrão reprodutivo prolongado.

8 2 ABSTRACT Studies on the reproductive biology of anuran populations combine diverse aspects of natural history, behavior, and ecology, such as: I) characterization of the reproductive mode; II) characterization of the vocalizations produced by the species and the contexts of emission; III) verification of the occurrence of sexual selection and segregation of limited resources; IV) description of the social behaviors and morphologic characteristics of the adults, tadpoles, clutches, and eggs. The present study investigated the reproductive biology of Scinax fuscomarginatus considering all features presented above. Observations occurred during the evening and night between September 2002 and March 2004 in a fragment of Cerrado at the Estação Ecológica de Itirapina, located at the counties of Itirapina and Brotas, interior of the state of São Paulo, southeastern Brazil. The species reproduced in lentic and temporary ponds. The reproductive pattern was prolonged and the mating system was characterized as lek. Males defended territories by acoustic, visual, and physical interactions. Winner males were always the residents. Satellite behavior was observed, although none female or amplected couple interception was observed. At the studied ponds the number of males (calling or not) was always higher than the number of females, generating low operational sex ratios. Four different vocalizations (advertisement, sporadic, territorial and fighting calls) were described in detail. With the exception of the advertisement call, the remaining calls are novel for the species. The amplex was axillary and the eggs were laid directly on the water, in the bottom of temporary ponds. The eggs had two gelatinous layers, which conferred spacing among the embryos. The studied population of S. fuscomarginatus showed an elaborated range of social behaviors, including spatial organization among males at the chorus, agonistic interactions (acoustic, visual, and physical), and a probable courtship behavior (courtship call). These characteristics are common in prolonged breeding species.

9 3 INTRODUÇÃO GERAL Os estudos com anfíbios anuros na região neotropical e em ambiente natural têm se dedicado principalmente aos aspectos da biologia reprodutiva das espécies (Martins, 1988; 1993; Haddad & Cardoso, 1992; Freitas et al., 2001; Wogel et al., 2002). Estudos como estes procuram descrever a temporada de atividade reprodutiva das populações, geralmente baseada na atividade de vocalização dos machos adultos (Pombal Jr., 1997; Bernarde & Machado, 2001; Toledo et al., 2003), ou juvenis (Lampert & Linsenmair, 2002) e inferem sobre a influência de fatores abióticos sobre a atividade reprodutiva (Aichinger, 1987). Alguns desses estudos investigam também os fatores responsáveis pela seleção sexual, verificando aspectos reprodutivos quantitativos e de relações de fecundidade (Prado & Uetanabaro, 2000; Bastos & Haddad, 2001). Ademais, como os anfíbios anuros se destacam por sua alta capacidade de emissão sonora, muitos estudos em campo já foram realizados sobre esse tema (ver Ryan, 2001). Com o passar dos anos, diversas funções sociais e diferentes contextos de emissão dos cantos foram identificados (revisões em Wells, 1988; Haddad, 1995). Por exemplo, a comunicação sonora entre os anfíbios anuros pode ser utilizada para a atração de parceiros sexuais (Haddad & Cardoso, 1992; Schwartz, 1994; Brenowitz & Rose, 1999) ou pode estar relacionada à defesa e delimitação de territórios, podendo evitar combates físicos entre machos que disputam recursos limitados, como os sítios de vocalização (Wells, 1988; Bastos & Haddad, 2001) e/ou desova (Martins et al., 1998). A vocalização ainda pode funcionar como um eficiente meio de segregação reprodutiva pré-zigótica (Oldham & Gerhardt, 1975; Etges, 1987; Cardoso & Viellard, 1990; Bourne & York, 2001). A vocalização é, portanto, um eficiente mecanismo de comunicação entre os anfíbios anuros (Ryan, 2001). Apesar disso, o conhecimento

10 4 sobre os diversos aspectos relativos à comunicação acústica, incluindo as características estruturais do repertório acústico das espécies de anuros neotropicais, é ainda escasso. Outro parâmetro freqüentemente investigado durante a atividade reprodutiva dos anfíbios anuros é a territorialidade dos machos, incluindo diversos estágios de interações agressivas, gerada pela disputa de recursos limitados, como sítios de vocalização, sítios de desova, e/ou parceiras sexuais (Wells, 1977a; 1978; Cardoso & Haddad, 1984; Martins et al., 1998; Bastos & Haddad, 1995; 2002; Guimarães & Bastos, 2003). Em conjunto, os estudos sobre biologia reprodutiva dos anuros neotropicais indicam que os acasalamentos não ocorrem ao acaso, podendo ser definidos por seleção de machos pelas fêmeas, ou como resultado das interações acústicas e agressivas entre machos (Arak, 1983, Ryan, 1985; Olson et al., 1986; Wagner & Sullivan, 1992; Brenowitz & Rose, 1999). Assim, processos de seleção sexual podem estar atuando no sentido de favorecer o dimorfismo sexual na maioria das espécies de anuros (Shine, 1979). O dimorfismo sexual é causado pela diferença entre a soma de todas as pressões seletivas que afetam características dos machos e a soma de todas as pressões que afetam características das fêmeas (Ralls, 1976). Em relação ao tamanho e massa corpórea, as principais pressões para o dimorfismo sexual mencionadas na literatura são: I) pressão para incremento de fecundidade das fêmeas em virtude do aumento do tamanho corpóreo (Crump, 1974; Shine, 1979); II) pressão para aumento do tamanho corpóreo dos machos em virtude de interações agressivas (Davies & Halliday, 1977; Shine, 1979; Katsikaros & Shine, 1997); III) maior taxa de mortalidade dos machos, não permitindo que atinjam a mesma idade e, conseqüentemente, o mesmo tamanho das fêmeas (Shine, 1979; Monnet & Cherry, 2002); ou IV) diferentes taxas de crescimento entre machos e fêmeas (Halliday & Verrell, 1988).

11 5 Muitas espécies do gênero Scinax Wagler 1830 possuem dimorfismo sexual e possuem padrões reprodutivos variados (Lutz, 1973; Haddad et al., 1990; Heyer et al., 1990; Bourne, 1992; Bevier, 1997; Toledo et al., 2003). Quanto ao sítio de desova, algumas espécies ovipõem em ambientes lóticos (e.g., S. albicans e S. trapicheiroi: Carvalho e Silva & Carvalho e Silva, 1994) ou em ninhos de espuma (e.g., S. rizibilis: Haddad et al., 1990). Entretanto, na maioria das espécies as fêmeas depositam os ovos em ambientes aquáticos lênticos, nos quais os girinos se desenvolvem (Lutz, 1973; Bastos & Haddad, 1999). Este modo reprodutivo foi considerado como sendo o modo basal no cenário evolutivo proposto por Duellman (1985). Quanto à temporada reprodutiva (sensu Wells, 1977b), são conhecidas tanto espécies de padrão reprodutivo explosivo [e.g., S. fuscovarius: Bertoluci, 1998; Toledo et al., 2003; S. ruber: Bourne, 1992; Bevier, 1997; Scinax sp. (aff. similis): Toledo et al., 2003], quanto de padrão reprodutivo prolongado (e.g., S. albicans: Carvalho e Silva & Carvalho e Silva, 1994; S. boulengeri: Bevier, 1997; S. rizibilis: Bastos & Haddad, 1999; S. centralis e S. fuscomarginatus: Bernarde & Kokubum, 1999; Bastos et al., 2003a). Conhecimento Atual Sobre Scinax fuscomarginatus Scinax fuscomarginatus é um hilídeo de pequeno porte, com pouco mais de 2 cm de comprimento rostro-cloacal (Lutz, 1973). É uma espécie típica de áreas abertas, como o Cerrado e Pantanal (Araujo & Colli, 1998), distribuindo-se pelo leste da Bolívia, por todo território paraguaio e nordeste da Argentina. No Brasil a espécie pode ser encontrada desde a região sul ao planalto central e do leste da região sudeste ao extremo oeste da região pantaneira (Frost, 2002) (Figura 1).

12 6 Figura 1. Mapa da América do Sul indicando (em cinza) a distribuição geográfica aproximada de Scinax fuscomarginatus (segundo Frost, 2002). Dentre as espécies do gênero, S. fuscomarginatus caracteriza-se por machos que se agregam em torno de poças permanentes ou temporárias para a reprodução, utilizando a vegetação marginal como sítio de vocalização (Lutz, 1973; Rossa-Feres & Jim, 2001; Bastos et al., 2003a). Quanto ao repertorio acústico, apenas o canto de anuncio é conhecido (Pombal Jr. et al., 1995; Bastos et al., 2003a; 2003b), sendo que variações na sua estrutura foram reconhecidas quando diferentes populações foram estudadas (Bueno, 2001; Bueno et al., 2002). Relações de esforço reprodutivo dos machos já foram brevemente discutidas (Prado & Haddad, 2003). Sabe-se que a desova é depositada diretamente em corpos d água lênticos (Bastos et al., 2003a). Foram descritos o desenvolvimento larval e os girinos desta espécie, outrora tratada como Hyla parkeri (Vizoto, 1967) e Ololygon

13 7 fuscomarginata (Altig & Johnston, 1986). Ademais, são encontrados na literatura curtos relatos sobre predação de adultos por sanguessugas (Brandão & Garda, 2000) e sobre a gama de comportamentos defensivos apresentados por machos adultos (Toledo, no prelo). Objetivos Esta dissertação é apresentada em dois capítulos, redigidos em inglês e editados no formato de periódicos especializados. Os capítulos possuem objetivos distintos, sendo que o primeiro deles visou: (I) descrever o repertório acústico de Scinax fuscomarginatus; (II) identificar os possíveis contextos de emissão de cada uma das vocalizações reconhecidas; (III) determinar a temporada de vocalização; (IV) identificar e descrever os sítios de vocalização utilizados; e (V) realizar comparações com outras populações a fim de propiciar uma melhor compreensão sobre os padrões de vocalização desta espécie. O segundo capítulo é dedicado a: (I) descrição da organização espacial e temporal dos indivíduos presentes nos agregados reprodutivos; (II) caracterização dos comportamentos associados à biologia reprodutiva, como comportamento de formação de casais, interações agressivas entre machos e comportamento de macho satélite; (III) verificação de aspectos quantitativos tais como relações de fecundidade, esforço reprodutivo e dimorfismo sexual; e (IV) caracterização da desova e sua caracterização em ambiente natural.

14 8 O Cerrado e a Área de Estudo O Cerrado é um importante ecossistema que cobre cerca de 23 % (aproximadamente 2 milhões de km 2 ) do território brasileiro, estendendo-se das margens da Floresta Amazônica até áreas fragmentadas nos Estados de São Paulo e Paraná (Ratter et al., 1997). Apesar do Cerrado ser bastante extenso em território nacional, a herpetofauna deste bioma ainda é muito pouco conhecida, tanto do ponto de vista taxonômico, quanto ecológico (Araujo & Colli, 1998; Colli et al., 2003). Além de pouco conhecida, o Cerrado está sendo continuamente ameaçado, principalmente por atividades agropecuárias. Estas atividades afetaram severamente o Cerrado nos últimos 30 anos, restando atualmente menos de 60 % da sua cobertura original (Ratter et al., 1997). No Estado de São Paulo, por exemplo, o cerrado já ocupou cerca de 14 % do território, mas atualmente ocupa apenas 1 % (248,8 mil km 2 ), e apenas 18 % da área remanescente está protegida por 32 unidades de conservação e de reserva legal (Fiori & Fioravanti, 2001). Essas reservas, todavia, constituem-se basicamente de fragmentos do ecossistema original. Sendo assim, estudos biológicos nos fragmentos restantes são de extrema urgência, principalmente nas áreas mais afetadas como as do Estado de São Paulo (Martins, 2001). A Estação Ecológica de Itirapina pertence ao Instituto Florestal do Estado São Paulo e localiza-se a aproximadamente 230 km do Município de São Paulo, nos municípios de Itirapina e Brotas (Figura 2) e compreende uma das últimas áreas preservadas de Cerrado do Estado. A área da reserva é de cerca de ha de vegetação natural de cerrado pouco perturbada, incluindo várias fisionomias, como campo limpo, campo sujo, campos cerrados, áreas brejosas e matas de galeria (Martins,

15 9 2001) (Figura 3). O clima da região caracteriza-se por um inverno frio e seco e um verão quente e chuvoso. Figura 2. Estação Ecológica de Itirapina (EEc), localizada entre áreas de plantio de Eucaliptus spp. e Pinus spp. (Ripasa e Eex = Estação Experimental de Itirapina), próxima à cidade de Itirapina, interior do Estado de São Paulo, sudeste do Brasil.

16 10 C C C C M CL CL M CL CL Figura 3. Foto aérea da Estação Ecológica de Itirapina e suas principais fisionomias: CL = campo limpo; CS = campo sujo; CC = campo cerrado; MG = mata de galeria.

17 11 REFERÊNCIAS Aichinger, M Annual activity patterns of anurans in a seasonal Neotropical environment. Oecologia, 71: Altig, R. & Johnston, G. F Major characteristics of free-living anuran tadpoles. Smithsonian Herpetological Information Service, 67: Arak, A Male-male competition and mate choice in anuran amphibians. In: Male Choice, p Bateson, P., Ed., Cambridge, Cambridge University Press. Araujo, A. F. B. & Colli, G. R Biodiversidade do Cerrado: herpetofauna. In: workshop "ações prioritárias para a conservação da biodiversidade do Cerrado e Pantanal". Disponível em: < Bastos, R. P. & Haddad, C. F. B Vocalizações e interações acústicas em Hyla elegans (Anura, Hylidae) durante atividade reprodutiva. Naturalia, 20: Atividade reprodutiva de Scinax rizibilis (Bokermann) (Anura, Hylidae) na floresta Atlântica, sudeste do Brasil. Revista Brasileira de Zoologia, 16(2): Larvas de Scinax rizibilis (Bokerman) (Anura, Hylidae): avaliando o efeito parental. Revista Brasileira de Zoologia, 18(4): Acoustic and aggressive interactions in Scinax rixibilis (Anura: Hylidae) during the reproductive activity in southeastern Brazil. Amphibia Reptilia, 23: Bastos, R. P., Motta, J. A. O., L. P. Lima & Guimarães, L. D. 2003a. Anfíbios da Floresta Nacional de Silvânia, Estado de Goiás. Stylo gráfica e editora, Goiás, 82p.

18 12 Bastos, R. P., Bueno, M. A. F., Dutra, S. L. & Lima, L. P. 2003b. Padrões de vocalização de anúncio em cinco espécies de Hylidae (Amphibia: Anura) do Brasil central. Comunicações do Museu de Ciências e Tecnologia da PUCRS, Série Zoológica, 16(1): Bernarde, P. S. & Kokubum, M. N. C Anurofauna do Município de Guararapes, Estado de São Paulo, Brasil (Amphibia: Anura). Acta Biologica Leopoldensia, 21(1): Bernarde, P. S. & Machado, R. A Riqueza de espécies, ambientes de reprodução e temporada de vocalização da anurofauna em Três Barras do Paraná, Brasil (Amphibia: Anura). Cuadernos de Herpetolgía, 14(2): Bertoluci, J Annual patterns of breeding activity in Atlantic rainforest anurans. Journal of Herpetology, 32(4): Bevier, C. R Breeding activity and chorus tenure of two neotropical hylid frogs. Herpertologica, 53(3): Bourne, G. R Lekking behavior in the Neotropical frog Ololygon rubra. Behavioral Ecology and Sociobiology, 31: Bourne, G. R. & York, H Vocal behaviors are related to nonrandom structure of anuran breeding assemblages in Guyana. Ethology, Ecology and Evolution, 13: Brandão, R. A. & Garda, A. A Scinax fuscomarginatus (NCN). Predation. Herpetological Review, 31(3): 171. Brenowitz, E. A. & Rose, G. J Female choice and plasticity of male calling behaviour in the pacific treefrog. Animal Behaviour, 57:

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21 15 Katsikaros, K. & Shine, R Sexual dimorphism in the tusked frog, Adelotus brevis (Anura: Myobatrachidae): the roles of natural and sexual selection. Biological Journal of the Linnean Society, 60: Lampert, K. P. & Linsenmair, K. E Alternative life cycle strategies in the west African reed frog Hyperolius nitidulus: the answer to an unpredictable environment? Oecologia, 130: Lutz, B Brazilian Species of Hyla. Austin, University of Texas Press. Martins, M Biologia reprodutiva de Leptodactylus fuscus em Boa Vista, Roraima (Amphibia: Anura). Revista Brasileira de Biologia, 48: Observations on the reproductive behaviour of the smith frog, Hyla faber. Herpetological Journal, 3: Ecologia dos Cerrados de Itirapina. Disponível em: < Martins, M., Pombal Jr., J. P. & Haddad, C. F. B Escalated aggressive behaviour and facultative parental care in the nest building gladiator frog, Hyla faber. Amphibia-Reptilia, 19: Monnet, J. M. & Cherry, M. I Sexual size dimorphism in anurans. Proceedings of the Royal Society of London, B 269: Oldham, R. S., & Gerhardt, H. C Behavioral isolating mechanisms of the treefrogs Hyla cinerea and H. gratiosa. Copeia, 2: Olson, D. H., Blaustein, A. R. & O Hara, R. K Matting patterns vatiability among western toad (Bufo boreas) populations. Oecologia, 70:

22 16 Pombal Jr., J. P Distribuição espacial e temporal de anuros (Amphibia) em uma poça permanente na Serra de Paranapiacaba, sudeste do Brasil. Revista Brasileira de Biologia, 57(4): Pombal Jr., J. P., Bastos, R. P. & Haddad, C. F. B Vocalizações de algumas espécies do gênero Scinax (Anura, Hylidae) do sudeste do Brasil e comentários taxonômicos. Naturalia, 20: Prado, C. P. A. & Haddad, C. F. B Testes size in Leptodactylid frogs and occurence of multimale spawning in the genus Leptodactylus in Brazil. Journal of Herpetology, 37(2): Prado, C. P. A. & Uetanabaro, M Reproductive strategies of Leptodactylus chaquensis and L. podicipinus in the Pantanal. Brazilian Journal of Herpetology, 34(1): Ralls, K Mammals in which females are larger than males. In: Monnet, J. M. and Cherry, M. I Sexual size dimorphism in anurans. Proceedings of the Royal Society of London, B 269: Ratter, J. A., Ribeiro, J. F. & Bridgewater, S The Brazilian cerrado vegetation and threats to its biodiversity. Annals of Botany, 80: Rossa-Feres, D. C. & Jim, J Similaridade do sítio de vocalização em uma comunidade de anfíbios anuros na região de noroeste do Estado de São Paulo, Brasil. Revista Brasileira de Zoologia, 18(2): Ryan, M. J The Túngara Frog: a study in sexual selection and communication. Chicago, University of Chicago Press Anuran Communication. Smithsonian Institution Press, Washington, London, 252p.

23 17 Shine, R Sexual selection and sexual dimorphism in the Amphibia. Copeia, 1979(2): Schwartz, J. J Male advertisement and female choice in frogs: recent findings and new approaches to the study of communication in a dynamic acoustic environment. American Zoologist, 34: Toledo, L. F. Scinax fuscomarginatus (NCN). Defensive behavior. Herpetological Review, in press. Toledo, L. F., Zina, J. & Haddad, C. F. B Distribuição espacial e temporal de uma comunidade de anfíbios anuros do Município de Rio Claro, São Paulo, Brasil Holos Environment, 3(2): Vizoto, L. D Desenvolvimento de anuros da região norte-ocidental do Estado de São Paulo. Faculdade de Filosofia, Ciências e Letras de São José do Rio Preto, editora Rio Preto, p Wagner, W. E. & Sullivan, B Chorus organization in the gulf coast toad (Bufo valliceps): male and female behavior and the opportunity for sexual selection. Copeia, 1992(3): Wells, K. D. 1977a. The social behaviour of anuran amphibians. Animal Behavior, 25: b. Territoriality and male mating success in the green frog (Rana clamitans). Ecology, 58(4): Territoriality in the green frog (Rana clamitans): vocalizations and acoustic behavior. Animal Behavior, 26: The effect of social interactions on anuran vocal behavior, p In: B. Fritzsch, M. J. Ryan, W. Wilczynski, T. E. Hetherington and W. Walkowiak

24 18 (Eds.). The evolution of the amphibian auditory system. John Wiley and sons, New York. Wogel, H., Abrunhosa, P. A. & Pombal Jr., J. P Atividade reprodutiva de Physalaemus signifer (Anura, Leptodactylidae) em ambiente temporário. Iheringea, Série Zoológica, 92(2):

25 19 Chapter 1 ACOUSTIC REPERTOIRE AND CALLING SITE OF SCINAX FUSCOMARGINATUS (ANURA, HYLIDAE) DURING REPRODUCTION LUÍS FELIPE TOLEDO and CÉLIO FERNANDO BAPTISTA HADDAD

26 20 JOURNAL OF HERPETOLOGY ACOUSTIC REPERTOIRE AND CALLING SITE OF SCINAX FUSCOMARGINATUS (ANURA, HYLIDAE) DURING REPRODUCTION Luís Felipe Toledo 1 and Célio Fernando Baptista Haddad 1 1 Departamento de Zoologia, Instituto de Biociências, Universidade Estadual Paulista, Caixa Postal 199, CEP , Rio Claro, São Paulo State, Brasil. toledolf@hotmail.com Corresponding author: LFT: toledolf@hotmail.com Running title: acoustic repertoire and calling site of S. fuscomarginatus

27 21 ABSTRACT. Vocalizations produced by anuran amphibians are considered an interespecific pre-zygotic segregation mechanism fundamental for intraspecific communication. Calling helps on the spatial organization of males at breeding sites, promotes the attraction of new individuals to the chorus, and attracts mature females for reproduction. In the present study, we describe four distinct vocalizations (advertisement, sporadic, territorial, and fighting calls) emitted by males of Scinax fuscomarginatus. We also characterize their contexts of emission for each of these calls. With the exception of the advertisement call, the remaining descriptions are novel. We also described the calling sites, the calling season, and the extension of calling activity in single nights. Field work was made in a fragment of Cerrado biome (Estação Ecológica de Itirapina), Municipalities of Itirapina and Brotas, state of São Paulo, southeastern Brazil. At this ecological station, S. fuscomarginatus exhibited a complex acoustic repertoire, social interactions, and prolonged reproductive pattern. Males made use of three different strategies to avoid acoustic overlap, which may hinder disadvantageous female attraction. The advertisement call presented structural differences when compared to another population of state of São Paulo, but not to with populations from central Brazil.

28 22 INTRODUCTION Anuran amphibians are distinguished by their capacity of sound production, generally by air flow throughout its vocal cords (Duellman and Trueb, 1994, but see exception in Souza and Haddad, 2003). Vocalizations have many social functions and are produced in different contexts (reviews in Wells, 1988; Haddad, 1995). Acoustic communication between anurans can be used to attract mates (e.g., Haddad and Cardoso, 1992; Schwartz, 1994; Brenowitz and Rose, 1999). Also, vocalizations may be related to defense of territory, avoiding physical encounters between males that compete for a limited resource, such as calling sites (e.g., Wells, 1988; Bastos and Haddad, 2002) and/or egg laying sites (e.g., Martins et al., 1998). Calling however, can also attract (Ryan et al., 1982) or prevent potential predators (Sazima, 1975), and works as an efficient inhibitor of pre-zygotic segregation (Oldham and Gerhardt, 1975; Etges, 1987; Cardoso and Viellard, 1990; Bourne and York, 2001). Calling activity is, therefore, a primary communication mechanism for anuran amphibians (Ryan, 2001). The structural characteristics of the acoustic repertoire of Neotropical anuran species remain poorly investigated. For Scinax fuscomarginatus, only the description of the advertisement call is available (see Pombal Jr. et al., 1995; Bastos et al., 2003a; 2003b). This call, however, may vary among different populations (Bueno, 2001; Bueno et al., 2002). Regarding the calling season, there is one study that males vocalized for a short period (Pombal Jr., 1997) and another that males called for over three months (Bernarde and Kokubum, 1999). The present study describes the acoustic repertoire and the calling sites of Scinax fuscomarginatus, and tries to identify the possible contexts in which different vocalizations were emitted, and determines the calling season for this species.

29 23 MATERIALS AND METHODS Study area. Field work was done at the Estação Ecológica de Itirapina (EEI) located at the Municipalities of Itirapina and Brotas, state of São Paulo, Brazil. The EEI is under the administration of the Instituto Florestal de São Paulo and it is one of the last areas of pristine Cerrado at this state. The reserve has 2,300 ha of area including typical phytophysiognomies such as campo limpo, campo sujo, campo cerrado, swamps, and gallery forests (Martins, 2001). The climate of EEI is characterized by a cold and dry winter, and a hot and rainy summer, with the highest temperatures, days of rain, and rainfall in January (Fig. 1). Its sandy soil needs a large water volume prior to the formation of temporary ponds (person. obs.). Rainfall (mm) A Oct Nov Dec Jan Feb Mar Apr Days of rain Relative humidity (%) B Oct Nov Dec Jan Feb Mar Apr Fig. 1. (A) Total monthly rainfall (bars) and total monthly days of rain (line); and (B) mean monthly relative humidity (bars) and mean monthly temperature (line) at the Estação Ecológica de Itirapina, state of São Paulo, Brazil, between October 2002 and April Temperature (ºC) Observations were conducted in two temporary ponds, named Lagoa do Trem and Lagoa da Ponte. The first is rectangular and with approximately 250 m 2 in the portion studied (Fig. 2). The Lagoa da Ponte has a rounded shape, with approximately 500 m 2 of area and 40 m in diameter. The vegetation of both ponds is similar, with

30 24 predominance of small herbaceous shrubby vegetation, which the most common is Andropogon lateralis (Poaceae). These plants grow both in the central part and on the edges of the ponds, forming small islands of vegetation on the interior of the ponds (Fig. 2). Fig. 2. Studied portion of Lagoa do Trem, Estação Ecológica de Itirapina, state of São Paulo, Brazil. The arrows indicate the islands of vegetation formed by clumps of Andropogon lateralis. Data collection and analysis. Observations were made between September 2002 and March During the rainy season field expeditions were made with duration of 4 to 10 consecutive nights. The observation periods begun just before sunset and ended when the calling activity of the species had decreased considerably, or when stopped. We did not considered the time adjustments established by the daylight saving time. For the observations were used white-light flashlights, avoiding that the central focus of the flashlight illuminated the animals directly. For the observation of individual behavior and intraspecific interactions we used the sampling methods of ad libitum, focal-animal, all occurrences of some behaviors, and sequences (see Altmann, 1974; Lehner, 1996).

31 25 The individuals observed in the field were weighted with field weighting scales with g of precision, and measured (SVL = snout-vent length) with a 0.05 mm precision caliper. We also registered the hour of the observation, the individual activity, sex, presence or absence of mature ovules (when females), and nearest distance between conspecifics. For individual recognition, the animals were toe-clipped following the table presented by Powell and Knesel (1992). After that, they were released at the same place where they were collected. Acoustic census of males in calling activity were made hourly from the beginning to the end of the calling activity. Vocalizations were recorded with a Marantz cassette recorder, model PMD222, with an external directional microphone Audiotecnica, model AT835b, positioned ca. 50 cm from the calling male. We used BASF cassette tapes model CEII Chrome Extra, at 4.75 cm/s of speed. The sound analyses were made in a Macintosh computer, using the Canary software, configured with 16 bits of resolution, 22 khz of frequency sampling, FFT and frame length of 256 samples. The terminologies used for the vocalization analyses follow those adopted by Duellman and Trueb (1994): vocalization was assumed to be any sort of sound emission made by the vocal apparatus of the animal, where calls and notes are categories of vocalizations. Notes were treated as sound emissions characterized by discrete temporal unities that compose the call. The call was considered to be the sound emission formed by one (simple call) or more (compost call) stereotyped notes, which can be multipulsed or not. Pulses were considered as sub unities of notes, not always distinguishable. Abiotic factors that could interfere in the activity of the anurans were registered (time, air and water temperatures, measured with a 0.5 ºC precision mercury thermometer, and rain occurrence). Additional climatic data (relative humidity, temperature, rainfall, and days of rain per month) were obtained at the meteorological

32 26 station of the Fazenda Seriema, located ca. 15 km from the study site. Meteorological factors were correlated with the number of calling males which attended the chorus in the same day or with the mean monthly activity of the species at EEI. The calling sites were characterized by measuring height and distance of the calling site from the nearest pond margin; perimeter, height, and form of the occupied vegetation clump; and its distance to the nearest vegetation clump. For the analysis of statistical correlations and influences, after verified the homogeneity of variance premises and data normality, we used the r coefficient of Pearson and linear regression analysis, respectively. Significant values were considered when P < 0.05 (Zar, 1999).

33 27 RESULTS The calling season of Scinax fuscomarginatus at the EEI was extended from December to March for two consecutive reproductive seasons ( and ). The calling season began after the formation of lentic and temporary aquatic environments. The Lagoa do Trem was formed before the Lagoa da Ponte, hence the presence of calling males on these habitats occurred in distinct periods (Fig. 3). The number of calling males remained relatively high for two to three weeks after the start of the calling activity, and decreased rapidly after that (Fig. 3). The number of calling males was positively correlated with the monthly rainfall (r = 0.755; P = 0.05), days of rain per month (r = 0.868; P = 0.033), and mean monthly air temperature (r = 0.763; P = 0.046), but not with the relative humidity (r = ; P = 0.958). Males were recaptured always at the same pond where they were captured and marked, generally in successive nights, using exactly the same calling site. The individuals remained in the chorus for three consecutive nights in average (SD = 2.27; range = 1 8 consecutive nights; N = 22). One calling male was recaptured 15 days after its first capture.

34 /16/2002 1/4/2003 1/6/2003 1/14/2003 2/6/2003 2/8/2003 2/10/2003 2/12/2003 2/14/2003 3/8/2003 3/15/2003 Number of calling males Lagoa do Trem Lagoa da Ponte Field trips Fig. 3. Scinax fuscomarginatus calling season in two temporary ponds (Lagoa do Trem and Lagoa da Ponte) at the Estação Ecológica de Itirapina, state of São Paulo, Brazil, between December 2002 and March We observed males calling between 0 and 150 cm above the ground or from the water surface (Fig. 4A), mainly above the border vegetation of the temporary ponds. However, some calling males were also found as far as 2.3 m from the pond (Fig. 4B). During calling, males perched on branches of the emerging vegetation, with their heads usually oriented vertically and upwards (Fig. 5), but also adopting different positions (Table 1). Males could be oriented to the central part of the pond, to its margins, or even oriented parallel to the nearest margin (Table 1).

35 29 Males observed (%) A) B) Males observed (%) 0 0 to to to to to to to to 80 Calling site height (cm) 81 to to 100 > to to to -1 0 to to to 150 Margin distance (cm) 151 to 200 > Fig. 4. Characterization of the calling sites explored by males of Scinax fuscomarginatus at the Estação Ecológica de Itirapina. A) Height of the calling site, and B) distance of the calling site in relation to the nearest pond margin. The negative values represents measurements toward the central part of the pond; N = 58 in both cases. Fig. 5. Adult male of Scinax fuscomarginatus during calling activity on emerging vegetation at the Lagoa do Trem, Estação Ecológica de Itirapina, state of São Paulo, southeastern Brazil.

36 30 Table 1. Position of males of Scinax fuscomarginatus during calling activity at Estação Ecológica de Itirapina, state of São Paulo, Brazil. Numbers in parenthesis indicate the total number of observations. Calling male position Observations (%) In relation to the margins (57) Oriented to the center of the pond 33.9 Oriented to the margin 38.7 Parallel to the margin 27.4 In relation to the ground (61) Parallel 21.2 Perpendicular Head upwards 27.3 Head downwards 21.2 Total 48.5 Diagonal Head upwards 16.7 Head downwards 13.6 Total 30.3 Males also called from small gramineous clumps in the middle of the ponds. In 94.7% of the observations (total N = 62), only one male was observed occupying a single clump in the middle of the pond. On the remaining clumps (N = 5) satellite males were observed. There was no significant correlation between the physical characteristics of the anurans and any of the four analyzed physical characteristics of the clumps (Table 2). Table 2. Pearson coefficient (r) and significance (P) for the correlation between physical characteristics of adult calling males of Scinax fuscomarginatus and four structural parameters of their calling site at the Estação Ecológica de Itirapina, state of São Paulo, southeastern Brazil (N = 17). Calling male SVL r P Mass P r Clump perimeter Clump height Clump volume Distance to the nearest clump

37 31 Four distinct vocalizations emitted by males of Scinax fuscomarginatus were recognized. All vocalizations were simple and formed by one multipulsed note, but showed distinct physical structures (Table 3) and were emitted in different social contexts. Advertisement call (Fig. 6A). The single note that forms this call has the mean dominant frequency (3.82 ± 0.22 khz) lower than the remaining calls described for this species. It is emitted with variable intervals, between 0.58 and s, frequently higher than the duration of the note (540.1 ± ms). Advertisement calls in antiphony were also registered (Fig. 7). The intervals between pulses of the same call did not showed great variation (see Table 3), generating a significant and positive regression between the number of pulses per call and the duration of the call (r 2 = 0.58; P < ; Fig. 8). Sporadic call (Fig. 6B). Formed by one note that has frequency amplitude similar to those of the advertisement and territorial calls. However, the sporadic call has the highest interval between pulses (4.3 ± 4.9 ms), the lowest number of pulses per call (from 21 to 48), and the shortest duration of the call (304.5 ± 55.4 ms). There are two bands of dominant frequency in this vocalization; the first band has mean frequency of 4.0 khz and the second of 4.6 khz. This call was emitted most frequently by males during the beginning and during the end of the calling activity, when the density of calling males was lower (Fig. 9). Territorial call (Fig. 6C). Formed by a single note with dominant frequency amplitude similar to the amplitudes of the other calls (between 2.09 and 5.32 khz). This was the longest call (718.9 ± 71.5 ms) recorded and the one with the highest number of pulses (from 111 to 150 pulses per call). It shows temporal variation across its emission. It is possible to analyze territorial call notes in two different parts; the initial and central

38 32 portion of the note have the duration of the pulses and the interval of pulses higher than the final portion of the note (Table 3). Fighting call (Fig. 6D). Emitted with a lower intensity than the calls described above. Among them, it is the call that presents the lowest duration (85.0 ± 8.0 ms) and the lowest duration of the pulses (always less than 1 ms). It is the call that has the highest pulse repetition rate, which were always higher than 200 per call, without distinguishable intervals among them. The fighting call has the minimum frequency (1.8 khz) lower than the other calls (territorial: 2.09 khz; advertisement: 2.4 khz; sporadic: 2.8 khz) and the mean frequency amplitude (4.1 khz) higher than the other calls (sporadic: 2.4 khz; advertisement: 3.16 khz; territorial: 3.2 khz). The fighting calls were emitted by males during physical interactions (see Chapter 2). Fighting calls were repeatedly emitted, but without regular repetition rate over the whole combat by both males (see Chapter 2).

39 Table 3. 33

40 34 Frequency (khz) A) B) Frequency (khz) Frequency (khz) C) Time (ms) Frequency (khz) D) Time (ms) Time (ms) Time (ms) Fig. 6. Sonogram (above) and ocilogram (below) of (A) advertisement call (air temperature = 19.5 ºC), (B) sporadic call (air temperature = 23 ºC), (C) territorial call (air temperature = 24 ºC), and (D) fighting call (air temperature = 24 ºC) emitted by males of Scinax fuscomarginatus. Recordings made between February 2003 and January 2004, at the Estação Ecológica de Itirapina, state of São Paulo, Brazil.

41 35 Frequency (khz) A B A B A B A B A B 0,5 1 1,5 2 2,5 3 3,5 4 4,5 Time (s) Fig. 7. Antiphony of the advertisement call of Scinax fuscomarginatus. Letters A and B represents calls emitted by two males during vocal interaction. Recordings were made in January 2004 (air temperature = 21 ºC), at the Estação Ecológica de Itirapina, state of São Paulo, Brazil. 140 Number of pulses/note Duration of the note (ms) Fig. 8. Linear regression between number of pulses per note/call and duration of the note/call (N = 102) of the Scinax fuscomarginatus advertisement call at the Estação Ecológica de Itirapina, state of São Paulo, Brazil.

42 36 We found a negative correlation between body mass and SVL in relation to the advertisement call dominant frequency (r = -0.83; r 2 = 0.68; P = ; and r = -0.82; r 2 = 0.57; P = , respectively) (Fig. 8). SVL and body mass were not correlated to the number of pulses per note, duration of the call, and calling rate (P > 0.05 for all cases). There was also no correlation between air temperature and the dominant frequency, number of pulses per call, duration of the call, or vocalization rate (P > 0.05 for all cases) A) B) Dominant frequency (khz) Dominant frequency (khz) Mass (g) SVL (mm) Fig. 8. Linear regression (lines) between mass and dominant frequency (A), and between SVL and dominant frequency (B) of the advertisement call of Scinax fuscomarginatus at the Estação Ecológica de Itirapina, municipalities of Itirapina and Brotas, state of São Paulo, Brazil; N = 13. The calling activity of S. fuscomarginatus began one or two hours before the sunset and continued for up to eight hours after the sunset. The number of calling males emitting advertisement calls increased for about one hour after the sunset, when the peak of calling activity was reached. After that, the number of calling individuals decreased gradually. The territorial call was emitted at higher rates in the beginning of the calling activity, and it was emitted again only during male interactions (see chapter

43 37 2) (Fig. 9). The calling rate (number of calls per minute) varied across the night, and it was positively correlated with the number of calling males present at the chorus (r = 0.82; P = 0.047) (Table 4). In four occasions males were heard calling between 1100 and 1300 h, calling rarely and generally emitting sporadic calls rather than advertisement calls. Number of males Advertisement Territorial Sporadic Hours in relation to the sunset Fig. 9. Calling activity pattern of Scinax fuscomarginatus at the Estação Ecológica de Itirapina, Municipalities of Itirapina and Brotas, state of São Paulo, Brazil. Mean values (bars) and standard deviation (lines) of seven nights for the advertisement call, and results for one night for the remaining vocalizations. The number of males refers to calling individuals observed in an area of approximately 250 m 2. Table 4. Number of calling males of Scinax fuscomarginatus in an area of approximately 250 m 2, mean number of advertisement, territorial, and sporadic calls emitted by six males heard from a steady position over one night (January 10, 2004) at the Estação Ecológica de Itirapina. The sampling interval was two minutes, and the data were corrected for one minute sampling. The values are expressed as mean ± standard deviation (range). Hours in relation to the sunset Advertisement Vocalization rate (calls/minute) Territorial Sporadic Number of calling males ± 1.76 (6 to 9) 3.3 ± 1.2 (2 to 4.3) 0.16 ± 0.29 (0 to 1.5) ± 2.47 (15 to 19) ± 0.5 (13.5 to 14.5) ± 2.08 (8 to 12) ± 1.76 (6 to 9.5) ± 3.77 (3.5 to 11) 0.78 ± 0.51 (0.3 to 1.3) 0.83 ± 1.44 (0 to 2.5) 03

44 38 DISCUSSION Although the correlation between temperature, days of rain, and rainfall with the number of calling males, the presence of ponds was the limiting factor for the beginning of the calling season of Scinax fuscomarginatus at EEI. Males that called at Lagoa da Ponte only began the calling activity when the pond was present, approximately one month after the beginning of the calling activity at Lagoa do Trem (Fig. 3). Scinax fuscomarginatus presented a prolonged breeding pattern (sensu Wells, 1977) at EEI (see also Chapter 2). The fact that individuals were not recaptured out of the pond where they were first captured, indicate reproductive site phylopatry for S. fuscomarginatus, at least during the calling season. The fidelity to the calling site and the permanence of males at the breeding site for consecutive days can increase the chances of mating by the males (Murphy, 1994) and decrease the number of agonistic interactions, since the calling territories do not need to be established again over the nights. The calling site of S. fuscomarginatus at EEI was similar to sites observed in other studies. This species uses calling perches at the margins of temporary lentic ponds (Rossa-Feres and Jim, 2001; Bastos et al., 2003) or permanent ponds (Pombal Jr. et al., 1995; Bernarde and Kokubum, 1999; Bastos et al., 2003). Males prefer calling sites between 0 and 50 cm high in dense or sparse shrubby vegetation (Pombal Jr. et al., 1995; Rossa-Feres and Jim, 2001). The males positioning at the chorus during vocalizations seems to be random (Rossa-Feres and Jim, 2001), and may depend on the vegetation architecture used as perch. Likewise, the lack of correlation between the analyzed structural characteristics of the vegetation and the physical characteristics of

45 39 the calling males suggests that the calling site choice is influenced by other factors. It is possible that factors, such as male density of the same or of different species, interfere in the calling site choice (see Schwartz and Wells, 1984; Etges, 1987). Scinax fuscomarginatus presented a complex acoustic repertoire, with at least four different vocalizations. This is characteristic of species with prolonged breeding pattern (Wells, 1977; Bastos and Haddad, 1995; Guimarães and Bastos, 2003) and with complex social behavior (Martins and Haddad, 1988; Bastos and Haddad, 1999). The advertisement call can have several functions: (I) attraction of other males to the chorus; (II) female attraction; (III) and/or regulation of the spatial distribution among males present at the core (Haddad, 1995; Ryan, 2001). The dominant frequency and call duration of S. fuscomarginatus of EEI were slightly smaller when compared to another population from the state of São Paulo, southeastern Brazil (see Pombal Jr. et al., 1995). However, it was not different from one population from the state of Goiás, central Brazil (see Bastos et al., 2003a). The description presented by Bokermann (1967) for the advertisement call of one species in the Municipality of Rio de Janeiro, treated as Hyla fuscomarginata, refers to the advertisement call of Scinax alter (see Izeckshon and Carvalho e Silva, 2001 for the occurrence of S. alter and the absence of S. fuscomarginatus in the municipality of Rio de Janeiro). Therefore, besides taxonomic misunderstandings, the differences observed among the different studies may reflect populational divergences, already described for S. fuscomarginatus (see Bueno, 2001; Bueno et al., 2002) and other anuran species (e.g., Cardoso and Haddad, 1984; Heyer and Reid, 2003). However, the acoustic differences observed could be an artifact generated, or attenuated if the differences really exists, due to different calling analysis methodology (e.g., different softwares), or due to different recording equipment used (e.g., different microphones, recording tapes, or recorders)

46 40 (Balmires et al., 1997; Bastos et al., 2003a). Moreover, in our study always more than 10 individuals were present at the chorus during the recordings, while on the recordings made by Pombal Jr. et al. (1995) only two individuals were calling. Hence, there are differences in the social circumstances on which the recordings were made, which may result in acoustic variation (see Wells, 1988). The territorial call seemed to maintain the spatial segregation among males present at the breeding site, as reported for other anuran species (e.g., Brenowitz and Rose, 1999). This call was emitted in the beginning of the calling activity, and could indicate the delimitation of a territory to be occupied and defended throughout the night. Further emissions of territorial calls were heard only when males invaded other territories, generally finishing by the intruder departure (see Chapter 2). These observations reveal the efficiency of this kind of vocalization, which can prevent fight during agonistic interactions (Wells, 1988; Chapter 2). Fighting may cause serious injuries to anurans including death and have elevated energy costs (Kluge, 1981; Martins and Haddad, 1988). Escalated aggressive behavior, also observed in S. fuscomarginatus (Chapter 2), is a mechanism widespread among anuran amphibians, especially in hylids (e.g., Brenowitz, 1989; Bastos and Haddad, 1995; Martins et al., 1998). We were unable to identify any social context associated to the emission of the sporadic call. However, since these calls were always emitted at the beginning of the calling activity, at the end of it, and during the day, allied to the fact that its physical structure is similar to the advertisement call, we suggest that the sporadic call is am attempt of the males to produce advertisement calls, which is hindered by an adequate state of the vocal chords (Haddad, 1987). The sporadic calls produced in the beginning of the night would be a consequence of the quiescent state of the vocal chords after

47 41 many hours of inactivity. At the end of the calling activity the sporadic calls were emitted only when few individuals were still calling. Therefore, the production of sporadic calls at that moment could be done by males that were not calling for an hour or a few hours, time enough to make the vocal chords quiescent. Similar callings were reported to Hyla minuta (Haddad, 1987) and H. faber (Martins and Haddad, 1988), but in the second case they were named as initial call, because they were heard just in the beginning of the calling period. Although the social context of the emission of fighting calls is clear (see also Chapter 2), it was not possible to demonstrate its function. There are reports that this call is associated to the escalated aggressive behavior observed during agonistic interactions (e.g., Bastos and Haddad, 1995; Martins et al., 1998), however there are no demonstrations for such speculation and its function remain unclear. The correlation observed between SVL and body mass vs. advertisement call dominant frequency reinforces the indication that there is a negative correlation between size and mass vs. calling frequency in anurans (Robertson, 1986; Given, 1987; Sullivan, 1982; 1992; Guimarães and Bastos, 2003). This correlation is originated by the difference of the laryngeal components of individuals of different sizes (Martin, 1972). Hence, it is possible that many of the calling site intruders evaluate the opponent s condition by its advertisement call frequency (Robertson, 1986) and leave before any physical or even acoustic interaction (Martins et al., 1998; Chapter 2). Vocalizations in anurans, especially advertisement calls, are related to the reproductive success of the calling individual, since they attract mature females and repel competitive males in the vicinity (Haddad, 1995; Bastos and Haddad, 2002). In this context, three strategies were used by males of Scinax fuscomarginatus at EEI: (I) calling males divided the physical environment, showing preference for distances much

48 42 larger than the distance in which agonistic interactions were observed (see Chapter 2); (II) nearby males emitted advertisement calls in antiphony; and (III) males increased their calling rate when a higher number of males were present at the chorus. The first strategy avoids interception of attracted females by opportunistic males (e.g., Haddad, 1991). The first and second strategies reduce the probability of signal overlap among calling males (Dyson and Passmore, 1992; Bastos and Haddad, 1999). The third strategy increases the advertisement call overlaps most of the time; however, it also increases the amount of acoustic information available for females (Bastos and Haddad, 1995) and increases the chances of emitting calls during some short silent periods of the chorus. Hence, these three strategies increase the chances of calling males of attracting females (Wells and Schwartz, 1984; Wells, 1988), and at least the first and the second strategies reduce the competition among conspecifics (Wollerman, 1999). ACKNOWLEDGMENTS. We are grateful to Denis O. V. Andrade, Marcos G. Papp, and Luís O. M. Giasson for the discussion and suggestions on early versions of the manuscript; Débora Y. Campos, Bruna Franco, and João Giovanelli for helping on field activities; Denise Zancheta for the access permission to the Estação Ecológica de Itirapina; IBAMA and IF for the licenses conceded (license n. 026/02 RAN IBAMA, proc. n /02-61 and autos n /02 COTEC letter 471/2002, respectively); IDEA WILD for equipment donation; CNPq (proc. n /2003-3) for the Masters scholarship and FAPESP (proc. n. 01/ ) and CNPq for granting the Herpetology lab.

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55 49 Chapter 2 REPRODUCTIVE BIOLOGY AND CHORUS ORGANIZATION IN SCINAX FUSCOMARGINATUS (ANURA, HYLIDAE) LUÍS FELIPE TOLEDO and CÉLIO FERNANDO BAPTISTA HADDAD

56 50 AMPHIBIA REPTILIA REPRODUCTIVE BIOLOGY AND CHORUS ORGANIZATION IN Scinax fuscomarginatus (ANURA, HYLIDAE) Luís Felipe Toledo 1 and Célio Fernando Baptista Haddad 1 1 Departamento de Zoologia, Instituto de Biociências, Universidade Estadual Paulista, Caixa Postal 199, CEP , Rio Claro, Brasil. toledolf@hotmail.com Corresponding author: LFT: toledolf@hotmail.com Running title: Reproductive biology of S. fuscomarginatus

57 51 Abastract Reproduction is one of the most studied aspects of the natural history of Neotropical anurans. Likewise, the present study investigated the reproductive biology of Scinax fuscomarginatus in one of the last Cerrado fragments of the state of São Paulo. Naturalistic observations were made between September 2002 and March 2004 at Estação Ecológica de Itirapina (EEI), state of São Paulo, southeastern Brazil. Reproduction occurred in lentic and temporary water bodies. The species exhibited a prolonged breeding pattern and a lek mating system. Males were smaller than females and defended territories by means of acoustic, visual, and physical interactions. Winner males were residents, but they did not differ in size and mass from the intruders males. Satellite behavior was observed, but none female or amplected couple interception was registered. The number of males at the chorus was always higher than the number of females, generating low operational sex ratios. Scinax fuscomarginatus presented the generalized reproductive mode in which the eggs are laid in the water and the tadpoles are aquatic. The amplex was axillary and the eggs were deposited at the bottom of temporary ponds. Oocytes, egg-masses, and eggs were described. Egg size, pigmentation, and shape seems to be associated to the biotic and abiotic characteristics of the microhabitat of deposition and are thought to afford protection against ultraviolet radiation, predation, and permitting egg respiration in hypoxic environments.

58 52 Introduction Studies with anurans in natural environment in the Neotropics have focused mainly on the reproductive biology of the species (e.g., Martins, 1988; 1993; Haddad and Cardoso, 1992; Freitas et al., 2001; Wogel et al., 2002; Guimarães and Bastos, 2003). These studies usually describe the reproductive season of the populations, generally based on the calling activity of adult males (e.g., Pombal Jr., 1997; Bernarde and Machado, 2001) and juveniles (Lampert and Linsenmair, 2002), and comment about the abiotic factors that might influence the patterns of reproductive activity (Aichinger, 1987). Some detailed studies investigated factors that might be involved in sexual selection, verifying quantitative reproductive aspects and size-fecundity relationships (e.g., Prado et al., 2000; Bastos and Haddad, 2001). Territoriality of males is another commonly studied aspect of the reproductive biology of anurans, including agonistic interactions for limited resources, such as calling sites, egg laying sites, and/or females (Wells, 1977a; 1978; Cardoso and Haddad, 1984; Martins et al., 1998; Bastos and Haddad, 1995; 2002). Studies on reproductive biology conducted on Neotropical anurans show that mates are not formed by chance, being defined through female selection, or as a result of the aggressive interactions among males (Arak, 1983, Ryan, 1985; Olson et al., 1986; Wagner and Sullivan, 1992; Brenowitz and Rose, 1999). Therefore, sexual selection may be a causal factor to the size sexual dimorphism found in the majority of the anuran species (Shine, 1979). Sexual dimorphism is caused by the difference between the sum of all selective pressures that affects males characteristics and the sum of all selective pressures that affects females characteristics (Ralls, 1976). In relation to length and mass, the main explanations leading to sexual dimorphism are: (I) selective pressure for the fecundity

59 53 increment of the female, that results in body size enlargement (Crump, 1974; Shine, 1979); (II) selective pressure for the increase of male body size as a consequence of aggressive interactions (Davies and Halliday, 1977; Shine, 1979; Katsikaros and Shine, 1997); (III) higher mortality rate of males, not permitting them to reach the same age and, consequently, the same size of females (Shine, 1979; Monnet and Cherry, 2002); and (IV) different growing rates between the sexes (Halliday and Verrell, 1988). Most species in the genus Scinax Wagler 1830 exhibit sexual dimorphism in size, where females are generally larger than males (Lutz, 1973; Heyer et al., 1990). The genus is also characterized by having diverse reproductive patterns (Haddad et al., 1990; Bourne, 1992; Bevier, 1997; Toledo et al., 2003). Scinax rizibilis lays eggs on foam nests (Haddad et al., 1990), and few other species of Scinax lay eggs in lotic water bodies (e.g., S. albicans and S. trapicheiroi: Carvalho e Silva and Carvalho e Silva, 1994). However, in the majority of the species, females lay their eggs in lentic aquatic environments where the tadpoles grow (Lutz, 1973: Haddad et al., 1990; Bourne, 1992; Bevier, 1997; Toledo et al., 2003), the basal reproductive mode proposed by Duellman (1985). In regard to the reproductive pattern (sensu Wells, 1977a), there are Scinax species with the explosive reproductive pattern [e.g., S. fuscovarius: Bertoluci, 1998; Toledo et al., 2003; S. ruber: Bourne, 1992; Bevier, 1997; Scinax sp. (aff. similis): Toledo et al., 2003] and species that exhibit the prolonged reproductive pattern (e.g., S. albicans: Carvalho e Silva & Carvalho e Silva, 1994; S. boulengeri: Bevier, 1997; S. rizibilis: Bastos and Haddad, 1999; S. centralis and S. fuscomarginatus: Bernarde and Kokubum, 1999; Bastos et al., 2003). Scinax fuscomarginatus is characterized by males that join around permanent or temporary ponds for reproduction, using the boundary vegetation as calling sites (see Chapter 1; Lutz, 1973; Bernarde and Kokubum, 1999; Rossa-Feres and Jim, 2001;

60 54 Bastos et al., 2003). Only the advertisement call of this species is currently known (references in Chapter 1) and its reproductive effort relationships have been discussed before (Prado and Haddad, 2003). It is also known that S. fuscomarginatus lay their eggs directly on lentic water bodies (Bastos et al., 2003), and that its larval development has been described previously (as Hyla parkeri in Vizoto, 1967; and as Ololygon fuscomarginata in Altig and Johnston, 1986). Finally, there are anecdotal reports about adult predation by leeches (Brandão and Garda, 2000) and about the variety of defensive behaviors presented by adult males (Toledo, in press). Herein, we studied the reproductive biology of S. fuscomarginatus through the: (I) description of the spatial and temporal organization of the individuals at the breeding site; (II) characterization of the behaviors associated to reproductive biology, such as: pair formation; aggressive interactions among males; and satellite males behavior; (III) verification of quantitative aspects of reproduction, such as: fecundity, reproductive effort of females, and sexual dimorphism; and (IV) characterization of the egg-masses and its arrangements.

61 55 Methods Study area The present study was carried out at the Estação Ecológica de Itirapina (EEI), located on the municipalities of Itirapina and Brotas, central São Paulo State, southeastern Brazil. The EEI has approximately 230 km 2 of natural vegetation, including at least five distinct Cerrado physiognomies (Martins, 2001). On the physiognomies of campo sujo and campo limpo it is common the formation of lentic and temporary ponds, with the water temperature varying from 20 to 45 ºC (pers. obs.). These water bodies are formed essentially during the summer (between November and March) and make possible the reproduction of several anuran species, including Scinax fuscomarginatus (Toledo, 2003). Data collection Field trips, lasting from four to 10 consecutive days, were made between September 2002 and March Observations began one hour before the sunset and finished when the calling activity decreased considerably. During the observations, head flashlights were used, avoiding that the central focus of illumination reached directly the animals. The following sampling methods were used for individual and intraspecific behavior observations: ad libitum, focal-animal, all occurrences of some behaviors, and sequences (Altmann, 1974; Lehner, 1996). Individuals were weighted with 0.05 g of precision and the snout-vent length (SVL) was measured with a caliper to the nearest 0.05 mm. The following parameters were registered: air temperature, observation time, distance to the nearest conspecific

62 56 individual, distance and height of each individual from the pond, sex, and presence or absence of ovules in the females. For individual recognition, the animals were toeclipped following Powell and Knesel (1992), and released exactly at the same place from where they were captured. The operational sex ratios followed Emlen and Oring (1977): OSR = (number of females at the pond) / (number of males at the pond + number of females at the pond). The eggs were collected from clutches deposited by pairs maintained in plastic bags; eggs were conserved in 5 % formalin between 48 and 72 hours after spawning. The eggs were counted, measured, and classified according to the Gosner s table (Gosner, 1960). Statistical analysis The homogeneity of variances and normality of the data set were verified. For comparisons between mass and SVL of winner vs. loser males involved in agonistic interactions, or between calling/dominant vs. silent/satellite males, we used a paired t test. We used a t-student test to compare distances among males present at the reproductive aggregation. The comparison among male mass and size vs. female mass and size was made with an U-test of Mann-Whitney. In all cases, significant values were considered when P < 0.05 (Zar, 1999).

63 57 Results The reproductive season of Scinax fuscomarginatus at Estação Ecológica de Itirapina, based on the calling activity of males, lasted approximately three and a half months, from December 2002 to March 2003, and from December 2003 to March These periods coincided with the rainy and hot season of the year, as well as with the presence of temporary lentic ponds (see Chapter 1). In the beginning of the reproductive season, only males were observed at the reproductive aggregation. Females were found only approximately 20 days after the beginning of the calling season. The first aquatic larvae were found ca. 30 days after the beginning of the calling season in the same ponds where the naturalistic observations took place. Metamorphic individuals were found 40 and 50 days after the beginning of the calling season, holding on branches of vegetation above the water or swimming in the ponds. Adult females after oviposition were significantly larger, but not heavier, than adult males (table 1). Table 1. Comparison of snout-vent length (SVL) and body mass between adult males and females of Scinax fuscomarginatus at Estação Ecológica de Itirapina, municipalities of Itirapina and Brotas, São Paulo State. Values expressed as mean ± standard deviation (range; N). Males Females P U (after oviposition) SVL (mm) ± ± ( ; 52) Mass (g) 0.56 ± 0.07 ( ; 51) ( ; 5) 0.58 ± 0.06 ( ; 5) Males of S. fuscomarginatus used the edge vegetation of temporary ponds as calling sites (see Chapter 1). Despite the apparently erratic distribution of the calling sites around the temporary ponds (see fig. 2 in Chapter 1), 52% of the calling males were found separated by 100 to 300 cm from each other. For distances equal or inferior

64 58 to 30 cm, direct interactions between males were observed (fig. 1). Hence, The calling territory has approximately 60 cm in diameter. Inside the territory of a resident male two sorts of interactions were identified: (I) while the dominant male was calling, one or two other males assumed a submission posture, remaining silently as satellite males; or (II) the resident male defended its calling site by means of aggressive behaviors, and if the intruder did not respect the aggressive signs the interaction could end in physical combats (fig. 2). 25 Observations (%) >400 Distance between males (cm) Figure 1. Distance between Scinax fuscomarginatus nearest males at Estação Ecológica de Itirapina during the calling activity. White bars represent distances where satellite males or agonistic interactions were observed; grey bars represent the distances when solitary males were observed calling without occurrence of agonistic interactions (N = 56).

65 Silent intruder male invades resident s territory (n = 8) 59 Resident male emitting advertisement calls Resident male emit territorial calls (n = 8) Intruder assumes submission posture (satellite behavior) with its vocal sac deflated and with its belly touching the substrate (n = 5) Loser male moves away (n = 5) Both males call, advertisement and/or territorial calls (n = 4) Resident male emit advertisement call (n = 7) Males interact physically emitting fighting calls (n = 2) Figure 2. Schematic summary of the agonistic interactions between adult males of Scinax fuscomarginatus at Estação Ecológica de Itirapina, São Paulo state, southeastern Brazil. When its territory was invaded, the resident male turned towards the intruder and started to emit territorial calls (calls described in Chapter 1). If the intruder did not assume a submission posture, as satellite male, both males initiated an acoustic confront. First, the intruder could emit few advertisement calls (described in Chapter 1), but soon began to emit territorial calls. Males called intensively, although silent periods of up to 30 seconds occurred, the acoustic confront restarted when one of the males called again. During the calling contest, both resident (N = 1) and intruder (N = 1), could perform rapid extension and contraction of arms and legs.

66 60 If the intruder did not move away, the resident male approached the intruder giving short distance hops, starting the physical combat. During the combat, males gave chest-to-chest hugs, with the heads oriented to the same direction, and both males emitted fighting calls (see chapter 1). In two fights observed the loser male moved away just after the combat. These combats lasted for 15 and 70 seconds. Winner males were always the residents. These males were not significantly larger (t = 1.23; df = 5; P = 0.273) nor heavier (t = 1.17; df = 5; P = 0.296) than intruders or satellite males (table 2). Females were not observed defending territories or involved in agonistic interactions. Table 2. Mass and snout-vent length (SVL) of Scinax fuscomarginatus males (winners or dominants and losers or satellites) of the agonistic encounters observed at the Estação Ecológica de Itirapina, state of São Paulo, Brazil. Values of SVL and mass are expressed as mean ± standard deviation (range; N). In only one combat the males were captured, measured, and weighted. Encounters without combat Encounters with combat SVL (mm) Mass (g) SVL (mm) Mass (g) Winners or 22.2 ± ± dominants Losers or satellites ( ; 5) 22.4 ± 1.48 ( ; 7) ( ; 5) 0.55 ± 0.08 ( ; 7) Satellite male behavior was observed in few occasions, on which one male (dominant male) emitted advertisement calls, and one (N = 5) or two other males (N = 1) remained in submission posture, with the head and belly touching the substrate, and with the vocal sac deflated (satellite males). On three occasions, the dominant males were removed by the observer and after two to four minutes after the removal of the dominant male, the satellite males began to emit advertisement calls. Satellite males were observed independently of chorus density and approximation of females to groups of dominant and satellite males was not observed.

67 61 Throughout the study, only nine females were observed, five of which were captured. The operational sex ratio obtained for nights with presence of females were in average (SD = 0.053; range = ; N = 6 nights). Two females with ovules in different stages of development were captured near to the temporary ponds in January 2003; only females with mature ovules were found in the breeding site moving toward calling males (fig. 3A). Gravid females were also observed motionless for up to four minutes in front of calling males, head oriented toward them (fig. 3B). After that, females were observed moving toward the nearest male, or in the direction of other males in the vicinity. When the male perceived the presence of a female, he moved to her, emitting courtship calls with short duration and low energy intensity (not recorded) (N = 1). When the male was less than 10 cm from the female, he jumped on the female s dorsum, and amplected her axillary (fig. 3C). During the amplexus the male remained with his ventral region totally in contact with the dorsal surface of the female. With his arms, the male performed arrhythmic contractions on the female s flanks, in a rate of six to eight per minute. Pair formation occurred after 0159 h, in average, after the sunset (range = 0105 h 0225 h; N = 4). Between 0230 h and 4h after the amplexus, the female moved down from the vegetation branches to the pond s water level (mean = 0315 h; N = 4). Afterward, the female with the amplected male jumped into the water and remained fluctuating on the water surface or resting on the emerging branches of the aquatic vegetation for approximately five minutes (fig. 3D). Subsequently, the pair dived and remained submerged for 20 seconds at most, before return to water surface. These dives were repeated from four to six times, when oviposition occurred, 0530 h, in average, after the sunset (range = 0445 h 0605 h; N = 4). Then, still inside the water, the male released the female and the

68 62 individuals rapidly moved to the edge of the ponds, hiding on the closest vegetation clump (N = 3). A C B D Figure 3. Pair formation and egg-laying sequence of Scinax fuscomarginatus at Estação Ecológica de Itirapina, São Paulo state, southeastern Brazil. A) Calling male; B) female oriented toward a calling male; C) axillary amplected pair; D) amplected pair resting at the vegetation on the water surface of a temporary pond (indicated by an arrow). Gravid females had a mean of 346 ovules in their ovaries (SD = 33.5; range = ; N = 4), with approximately 0.82 mm in diameter (SD = 0.07; range = mm; N = 12). Ovules were black on the animal pole and beige on the vegetative pole (fig. 4A). The egg-mass is formed by a single and circular layer of eggs, whose coloration and diameter was similar to those of the ovules. Eggs had two concentric gelatinous capsules (absent in the ovules). Eggs at stage 13 had a mean diameter of 0.98 mm (SD = 0.07; range = mm; N = 18). The inner gelatinous capsule had a mean diameter of 1.17 mm (SD = 0.04; range = mm; N = 6) while the outer

69 63 capsule had a mean diameter of 2.65 mm (SD = 0.34; range = mm; N = 8) (fig. 4B). When submerged in water, the eggs remained with the animal pole upwards. A B I O Figure 4. A) Mature ovules in the ovaries; and B) fertilized egg of Scinax fuscomarginatus of the Estação Ecológica de Itirapina, São Paulo state, Brazil. The arrows show the external limits of the inner (I) and outer (O) gelatinous capsules. Four amplected pairs were measured and weighted. Male body mass corresponded to 65.17% of the female body mass after egg-laying (SD = 4.25; range = 62.5 to 71.43%). Male SVL corresponded to 95.76% of the female SVL (SD = 2.45; range = %). The mass of ovules represented 37.13% of the mass of the females after oviposition (SD = 9.72; range = %; N = 4). A mean of 86.22% of the eggs were fertilized (SD = 11.19; range = %; N = 3).