UNIVERSIDADE ESTADUAL DE CAMPINAS INSTITUTO DE BIOLOGIA
IZADORA COSTA VIDIGAL DE FREITAS
CARACTERIZAÇÃO MORFOLÓGICA E ACÚSTICA DE POPULAÇÕES DE CROSSODACTYLUS DUMÉRIL & BIBRON, 1841 (ANURA, HYLODIDAE): IMPLICAÇÕES TAXONÔMICAS E DISTRIBUIÇÃO
MORPHOLOGICAL AND ACOUSTIC DESCRIPTION OF CROSSODACTYLUS DUMÉRIL & BIBRON, 1841 (ANURA, HYLODIDAE) POPULATIONS: TAXONOMIC STATUS AND DISTRIBUTION
CAMPINAS
(2017)
IZADORA COSTA VIDIGAL DE FREITAS
CARACTERIZAÇÃO MORFOLÓGICA E ACÚSTICA DE POPULAÇÕES DE CROSSODACTYLUS DUMÉRIL & BIBRON, 1841 (ANURA, HYLODIDAE): IMPLICAÇÕES TAXONÔMICAS E DISTRIBUIÇÃO
MORPHOLOGICAL AND ACOUSTIC DESCRIPTION OF CROSSODACTYLUS DUMÉRIL & BIBRON, 1841 (ANURA, HYLODIDAE) POPULATIONS: TAXONOMIC STATUS AND DISTRIBUTION
Dissertação apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do Título de Mestra em Biologia Animal, na área de Biodiversidade Animal.
Dissertation presented to the Institute of Biology of the University of Campinas in partial fulfillment of the requirements for the degree of Master in Animal Biology, in Biodiversity area.
Orientador: DR. ARIOVALDO ANTONIO GIARETTA
ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELA ALUNA IZADORA COSTA VIDIGAL DE FREITAS E ORIENTADA PELO DR. ARIOVALDO ANTONIO GIARETTA.
CAMPINAS
(2017)
Campinas, 17 de Novembro de 2017
COMISSÃO EXAMINADORA
Prof.(a) Dr. Ariovado Antonio Giaretta (orientador)
Prof.(a). Dr.(a) Rachel Montesinos Martins Pereira
Prof.(a) Dr(a). Thais Helena Condez
Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se encontra no processo de vida acadêmica do aluno.
A Ata da defesa com as respectivas assinaturas dos membros encontra-se no SIGA/Sistema de Fluxo de Dissertação/Tese e na Secretaria do Programa (Programa de Pós-Graduação em Biologia Animal) da Unidade (Instituto de Biologia).
Á minha família...
AGRADECIMENTOS
Agradeço meu orientador, o Dr. Ariovaldo A. Giaretta, por contribuir na minha formação acadêmica nos últimos três anos. Muito obrigada pela oportunidade e por todo aprendizado que obtive até agora.
Aos colegas de laboratório Dr. Lucas Borges Martins, MSc. Felipe Andrade e MSc. Isabelle Haga pelo apoio nos trabalhos de campo. Um agradecimento em especial, ao Dr. Thiago Ribeiro de Carvalho por colaborar extensivamente no desenvolvimento do primeiro capítulo e por todas as discussões sobre bioacústica. Elas foram valiosas. Muito obrigada!
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-446935/2014-0) e a Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG-PPM 00510-15) pelo apoio financeiro do nosso laboratório.
O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001.
À minha família, que mesmo com dificuldades em vários momentos, me deu suporte em todos os momentos que precisei.
RESUMO O gênero Crossodactylus Duméril & Bibron, 1841 abriga 14 espécies e se distribui desde o Alagoas, passando pelo sudeste e sul brasileiro, até o sul do Paraguai e norte da Argentina. Crossodactylus, um dos gêneros mais problemáticos sob perspectiva taxonômica e sistemática, dentro da família Hylodidae, frequentemente está associado a riachos dentro de matas caracterizadas pela formação da Floresta Atlântica e campos montanhosos. Além disso, se trata de um gênero com muitas espécies crípticas, onde estudos taxonômicos integrativos que utilizam caracteres morfológicos, bioacústicos e moleculares são cada vez mais frequentes. O presente estudo obteve em campo espécimes vivos, dados acústicos e tecidos de duas populações de Crossodactylus, uma atribuída à C. werneri, e outra à C. caramaschii com o objetivo de verificar suas respectivas posições taxonômicas adequadas e caracterizá-los acusticamente. Com base em uma abordagem integrativa, descrevemos o canto inédito para a primeira população citada, além de abordar dados de variação de morfologia do adulto, descrição da larva, informações inéditas relacionadas à história natural para o gênero, aumento do limite de distribuição até então conhecido, e discutimos sua posição em status específico com base em distância genética entre sequências 16s. Avaliamos ainda uma população de Crossodactylus, considerando além de morfologia e morfometria, dados acústicos e genéticos, levantando evidências para uma nova espécie dentro do gênero.
ABSTRACT Crossodactylus Duméril & Bibron, 1841 comprises 14 species and occurs from Alagoas (brazilian northeastern), through southeastern and southern in Brazil, to southern Paraguay and northern Argentina. Crossodactylus, the most problematic gender under taxonomic and systematic perspectives within the family Hylodidae, is often found in rivulets and montane fields. Besides, it’s a gender with many cryptic species, and studies under integrative approach, which use characters such as morphology, bioacoustics and genetics, are becoming more frequent. Through field works, we get live specimens, acoustic data and tissues from two populations of Crossodactylus, one attributed to C. werneri, and the other to C. caramaschii,. So, the aim of this study was to verify the suitability between these populations and their respective taxonomic status. In an integrative approach, we describe at first time the advertisement call of the first cited population. Besides, we share data on variable morphology, tadpole description, natural history, distribution and genetics. We also evaluated a population of Crossodactylus, considering in addition to morphology and morphometry, acoustic and genetic data, raising evidences for a new species within the genus.
Sumário
1 INTRODUÇÃO 10 1.1 Introdução geral 10 1.2 Taxonomia e o conceito de espécie 10 1.3 Tipos de dados usados no reconhecimento de espécies de Anura 11
2 OBJETIVOS 16
3 REFERÊNCIAS BIBLIOGRÁFICAS 17
4 RESULTADOS 25 4.1 Manuscrito I (aceito no periódico Zootaxa) 25 4.1.1 Abstract 25 4.1.2 Introduction 26 4.1.3 Material And Methods 28 4.1.4 Results 31 4.1.5 Discussion 33 4.1.6 Acknowledgements 37 4.1.7 References 38 4.1.8 Figure captions 45 4.1.9 Appendices 47 4.1.9.1 Tables 51 4.1.9.2 Figures 55 4.2 Manuscrito II (Formatado nos moldes do periódico Zootaxa). 60 Abstract 60 4.2.2 Introduction 60 4.2.3 Materials and Methods 61 4.2.4 Results and Discussion 64 Conclusions 70 4.2.5 Acknowledgements 71 4.2.6 References 72 4.2.7 Figure captions 77 4.2.8 Appendices 78 4.2.9 Tables 82 4.2.9.1 Figures 86
5 CONCLUSÕES GERAIS 95
6. REFERÊNCIAS BIBLIOGRÁFICAS GERAIS 96
7. ANEXOS 103 7.1. Termos de sigilo 103 7.2. Declaração referente à bioética e Biossegurança 100 7.3 Declaração de não violação de direitos autorais 101
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1 INTRODUÇÃO
1.1 Introdução geral
Desvendar a biodiversidade exige não somente conhecimento de todas as espécies, mas também sobre como elas se distribuem e como se relacionam evolutivamente (Diniz-Filho et al. 2013). No entanto, mesmo em regiões bem estudadas, o montante de informações básicas sobre a biologia das espécies ainda é escasso, além de não receber a devida importância (Matthews 2015). Este quadro é agravado ainda pelo declínio de muitas populações, resultante do desmatamento, contaminação do ambiente, comércio ilegal, ou mesmo por surgimento de patógenos, como infecções causadas por fungos, no caso dos anfíbios (Collins & Crump 2009).
A riqueza mundial dos anuros atualmente compreende 6806 espécies (Frost 2017), estando a maior parte concentrada nos trópicos (Duellman & Trueb 1986). No último levantamento, foram contabilizadas para o Brasil 1039 espécies (Segalla et al. 2016), o que representa 15,5% da diversidade mundial do grupo. Esta riqueza se deve às grandes dimensões do país, e a integração de amplas zonas climáticas (MMA 2013), as quais formam zonas biogeográficas como a Amazônia, o Cerrado e a Mata Atlântica. Estes dois últimos merecem atenção especial, pois estão entre os 34 centros de alta de diversidade biológica e estão sujeitos a fortes ameaças pela ação antropogênica, além de já ter sido constatada perda de grande proporção da extensão geográfica original destes biomas (Myers et al. 2000). Estima-se que desde meados dos anos 80, 34 espécies de anfíbios foram extinguidas, quase 2000 estão ameaçadas e cerca de 3000 estão sofrendo declínio populacional (Stuart et al. 2004).
1.2 Taxonomia e o conceito de espécie
A taxonomia é o ramo científico responsável por trazer a tona o conhecimento da diversidade, ou seja, ela identifica, descreve, nomeia e a classifica todos os grupos de organismos, viventes e extintos (Wilson 2004; Padial et al. 2010; Schlick-Steiner et al. 2010; Kaiser et al. 2013), servindo de base para estudos nas mais diversas áreas (Wilson 2004; Dayrat 2005; Schlick-Steiner et al. 2010).
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Apesar de sua grande relevância, esta vertente vinha sofrendo crises em virtude da falta de interação entre as diferentes linhas de pesquisa envolvidas na delimitação das espécies (Dayrat 2005). No entanto, o desenvolvimento e a universalização de tecnologias, atualmente propicia o surgimento de uma nova abordagem: a Taxonomia Integrativa (sensu Dayrat 2005; Padial et al. 2010). Esta abordagem é caracterizada pelo uso de variadas bases de dados, que são complementares entre si, e que potencializam o rigor e a confiabilidade científica dos resultados (Schlick-Steiner et al. 2010).
O estudo da taxonomia se dá em diferentes níveis. Enquanto que a beta-taxonomia lida com análises mais inclusivas em relação aos organismos, a alfa-taxonomia tem como modelo de estudo as espécies (Bickford et al. 2007; Schlick-Steiner et al. 2010), e como unidade fundamental nesta área, é imprescindível delimitá-las a fim de entender verdadeiramente se tais organismos objetos de estudo, pertencem ou não a mesma entidade biológica (Dayrat 2005).
A discussão relacionada ao que considerar espécie, é extensa (e.g. de Queiroz 2007; Vences & Wake 2007; Wiens 2007). O problematização maior gira em torno da confusão entre os parâmetros de delimitação com o próprio conceito teórico (de Queiroz 2007). São conhecidos 24 conceitos de espécie, destacando-se o ecológico, genético, evolutivo, filogenético, e o biológico propriamente (Mayden 1997).
A fim de sanar tal problema, de Queiroz (2007) propõe o conceito unificado de espécie, baseado no elemento comum dentre todas as conceituações existentes, denominando- o ―unidade conceitual subjacente‖. Desta forma, espécies são linhagens metapopulacionais (subpopulações conectadas) com evolução independente, tornando-se feneticamente diferentes e diagnosticáveis em termos de estados de caracteres fixos, ligados ao isolamento reprodutivo destas entidades (de Queiroz 2007).
1.3 Tipos de dados usados no reconhecimento de espécies de Anura
A taxonomia clássica utiliza padrões de morfologia e morfometria para o reconhecimento das espécies. Atualmente, para os Anura, além dessa linha, cada vez mais se complementam os dados a partir da bioacústica e genética. Neste trabalho, estas três linhas de
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evidência foram utilizadas na avaliação taxonômica das populações, e foram comparadas tanto com dados disponíveis na literatura e Genbank, quanto originais.
A comunicação acústica surgiu diversas vezes durante a história evolutiva de diferentes grupos, frequentemente em contexto reprodutivo e de defesa de território (Duellman & Trueb 1986; Searcy & Anderson 1986; Gerhardt & Ruber 2002). Esta é uma característica que exerce diferentes funções (Duellman & Trueb 1986; Wells 2007; Toledo et al. 2015), e para os Anura, são classificados em três tipos, anúncio, territorial e de defesa, sendo o primeiro tipo a principal forma de comunicação, pois permite o macho atrair fêmeas e sinalizar a ocupação do território (Duellman & Trueb 1986; Gerhardt 1991; Gerhardt & Ruber 2002; Wells 2007). Variações neste tipo de canto podem indicar importantes características do macho, como tamanho corporal, o que influencia diretamente na escolha da fêmea e na territorialidade (Davies & Halliday 1978; Asquith & Altig 1990; Gerhardt 1991; Wagner 1992; Gerhardt & Ruber 2002), funcionando assim, como importante fator de seleção sexual e consequente limitador de fluxo gênico (Ryan & Rand 1993), os quais são relevantes em processos de especiação (Blair 1958; Martof 1961; Duellman & Trueb 1986; Searcy & Anderson 1986; Ryan et al. 1990; Ryan & Rand 1993).
Por sua natureza espécie-específica, os cantos de anúncio são amplamente utilizados no reconhecimento e diagnose de espécies de anuros (e. g. Padial & De la Riva 2009; Köhler et al. 2015). Além disso, representam uma importante base de dados para estudos em escala macroevolutiva, que se relacionam desde a avaliação de presença de sinal filogenético (sensu Blomberg et al. 2003) e de processos que produzem os padrões observados em espécies viventes (e. g. Ryan & Rand 1993; Cocroft & Ryan 1995), até a reconstruções filogenéticas com base em evidência total (Cocroft 1994; Cannatella et al., 1998; Heyer 1998) ou mesmo de forma isolada, a fim de se comparar com outros tipos de dados (Cannatella et al. 1998; Wollenberg et al. 2007; Goicoechea et al. 2010).
Atualmente os métodos moleculares têm agregado grande valor no conhecimento da diversidade mundial (Funk et al. 2011). Cada vez mais nos deparamos com o uso de genes mitocondriais e nucleares, além do "barcoding", em diagnoses (Vieites et al., 2009; Padial et al. 2010) principalmente naqueles grupos considerados crípticos (Faivovich et al., 2010).
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1.4. Caracterização do táxon-modelo: Crossodactylus (Anura, Hylodidae)
A família Hylodidae Günther, 1859 é composta por anuros do gênero Crossodactylus, Hylodes e Megaelosia, no inglês, popularmente conhecidos como ―spinythumb frogs‖, ―torrent frogs‖ e ―big-tooth frogs‖, respectivamente. Atualmente, a distribuição conhecida para esta família vai desde o nordeste brasileiro até o sul do Paraguai e norte da Argentina (Frost 2017).
A relação de Hylodidae com outras famílias de anuros é extensivamente discutida na literatura e as hipóteses filogenéticas são diversas. Tal discrepância pode ser resultado do baixo número de táxons ao representar a família nestas análises, além do uso de alguns tipos de parâmetros morfológicos que dão margem para uma interpretação incorreta (e.g. uso de caracteres homoplásicos ao invés de sinapomorfias, Pinna 1996). Embora existam tais discrepâncias nestas relações, a monofilia do grupo foi testada e corroborada muitas vezes como parte de amplos estudos (e.g., Lynch 1971; Haas 2003; Frost et al. 2006; Grant et al. 2006; Pyron & Wiens 2011).
Crossodactylus Duméril & Bibron (1841), o gênero mais problemático sob perspectiva taxonômica e sistemática (Heyer et al. 1990; Izecksohn & Carvalho-e-Silva 2001; Haddad et al. 2003; Ribeiro et al. 2005; Pimenta et al. 2008, 2014), atualmente compreende 14 espécies (Frost 2017), frequentemente associadas a pequenos riachos no interior de matas caracterizadas pelas formações de Floresta Atlântica e campos montanhosos (Nascimento et al. 2005; Giaretta & Facure 2008; Pimenta et al. 2015). Tanto na literatura quanto em coleções científicas, ainda são encontrados diversos espécimes identificados erroneamente ou mesmo sem identificação (Pimenta et al. 2008; 2014). A falta de rigor na identidade de todo este material de referência que temos até hoje está relacionada à falta de dados que tangem a variação tanto inter quanto intraespecífica e distribuição geográfica do grupo. Esta situação ainda é enfatizada pela morfologia e acústica conservada, o que torna estas espécies crípticas e consequentemente de difícil distinção sem análise detalhada (Caldart et al. 2011; Pimenta et al. 2008; 2014; 2015).
Caramaschi & Sazima (1985) ao descrever C. bokermanni, dividiu as espécies até então conhecidas em três grupos fenéticos com base em dois caracteres morfológicos: comprimento do focinho e formato do canthus rostralis. Estes grupos eram: (1) Grupo C.
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gaudichaudii, caracterizado pelo focinho acuminado e canthus rostralis bem definido, e representado até então por C. aeneus, C. bokermanni e C. gaudichaudii; (2) Grupo C. trachystomus, definido por focinho curto/arredondado e canthus rostralis pouco definido, no qual estavam compreendidos C. dispar, C. grandis e C. trachystomus; e (3) C. schmidti (monotípico), com focinho muito curto, canthus rostralis sem definição (arredondado) e grande espaço interorbital. Todas as espécies descritas a posteriori (e.g. C. cyclospinus, C. dantei e C. lutzorum) foram alocadas dentro do grupo C. gaudichaudii. No entanto, Pimenta et al. (2008) ao acessar variação morfológica e acústica de C. bokermanni verificou que tais caracteres não se aplicavam a espécie, além do que, tal espécie se tornou sinônimo júnior de C. trachystomus (Pimenta et al. 2015), até então alocada em um grupo com características diferentes.
Muitas destas espécies, ainda hoje, são tidas como de ampla distribuição, além do que muitas destas sobrepõem-se, como é o caso de C. gaudichaudii e C. aeneus (Frost 2017). Este é um fator que pode dificultar o reconhecimento da diversidade real dentro grupo, como visto por Pimenta et al. (2014) para C. dispar, o qual era relacionado a diversas populações, distribuídas desde o sudeste até o sul brasileiro e ainda Misiones, na Argentina, até que foram reconhecidas três espécies diferentes dentro deste complexo: C. boulengeri, C. timbuhy e C. werneri (Pimenta et al. 2014). Para C. caramaschii, também são relacionadas várias populações até o sul do Brasil, mas pouco se sabe sobre a variação entre estas (obs. Pessoal; Pimenta et al. 2014).
Estas espécies por último citadas foram descritas com base em morfologia externa de espécimes coletados a mais de quatro décadas, assim como C. franciscanus, a última espécie do gênero descrita até hoje. Além disso, não são vistos em suas localidades-tipo deste então (Pimenta et al. 2014; 2015). Desta forma, pouco é conhecido além de parâmetros morfológicos e morfométricos destas espécies, como canto, genética, larva, dentre outros. Isto dificulta também a avaliação do status de conservação atual destes animais.
Nesta dissertação, o objetivo geral foi avaliar a posição taxonômica de duas populações de Crossodactylus: uma atribuída à C. werneri, com registro de distribuição inédita, e outra população de perto relacionada à C. caramaschii. Para tal objetivo, foram
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acessadas outras linhas de evidência além de morfologia externa e morfometria: o repertório acústico, larva e distância genética (16S- mitocondrial), em uma abordagem integrativa.
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2 OBJETIVOS
Avaliar a posição taxonômica de duas populações de Crossodactylus, caracterizando morfométrica e acusticamente, comparando-as com outras populações de espécies proximamente relacionadas e integrar esses dados com os moleculares, sendo uma destas populações, uma potencial espécie inédita para a Ciência.
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4 RESULTADOS
4.1 Manuscrito I (aceito no periódico Zootaxa)
Vocalizations, tadpole, and natural history of Crossodactylus werneri Pimeta, Cruz &
Caramaschi, 2014 (Anura: Hylodidae), with comments on distribution and intraspecific
variation
IZADORA VIDIGAL1,2*, THIAGO R. DE CARVALHO3, RUTE B. G. CLEMENTE-
CARVALHO4,5 & ARIOVALDO A. GIARETTA1
1 Laboratório de Taxonomia, Sistemática e Evolução de Anuros Neotropicais, Universidade
Federal de Uberlândia (UFU), campus Ituiutaba, Ituiutaba, MG, Brazil.
2 Programa de Pós-Graduação em Biologia Animal, Instituto de Biologia, Universidade
Estadual de Campinas (Unicamp), Campinas, SP, Brazil.
3 Laboratório de Herpetologia, Instituto de Biociências/I.B., Departamento de Zoologia,
Universidade Estadual Paulista (UNESP), campus Rio Claro, SP, Brazil.
4 Programa de Pós-Graduação em Ecologia de Ecossistemas, Universidade de Vila Velha
(UVV), Vila Velha, ES, Brazil.
5 Present address: Department of Biology, Queen’s University, K7K4W8, Kingston, ON,
Canada.
* Corresponding author. E-mail: [email protected]
4.1.1 Abstract Crossodactylus werneri was described based on specimens collected in the 1970’s at Parque
Nacional do Itatiaia, being also reported for nearby localities. We collected specimens that we assigned to C. werneri, and recorded calls of the species during fieldworks at Serra das
26
Cabras, (Campinas, state of São Paulo). In this paper, we describe for the first time the vocalizations, tadpole, coloration in life, and comment on aspects of the natural history of C. werneri. Besides, the examination of specimens in zoological collections allowed us to extend the geographic range for this species. We also make remarks on morphological/chromatic variation and provide 16S rDNA sequences for the species. Adults were found along a slow- flowing streamlet with sandy/muddy bottom within a small fragment of secondary forest.
Males called between sunset and first hours of the night. Advertisement call consisted of series of pulsed notes. Call duration lasted around 3 s, emitted at the highest rate of 17 calls per minute and six notes per second. Note duration lasted around 18 ms. Notes had poorly defined pulses (irregular and/or weak amplitude modulations along the note). The dominant frequency was about 3380 Hz. Territorial call had a long, well-defined pulsed portion followed by a higher-amplitude ―squeak‖. The dominant frequency was around 3400 Hz.
Tadpoles were essentially similar to those of other Crossodactylus species, except by not having nostril ornamentation. Our record of C. werneri in Serra das Cabras might be regarded a rediscovery of this species since C. werneri had not been recorded for more than 30 years until our first record of C. werneri in the field from 2011 and subsequent years. Our record is approximately 100 km west, and Mococa 200 km northwest, from Santo Antônio do Pinhal, the westernmost previous record for C. werneri up to date. Gene sequences (16S rRNA) give insights into the genetic divergence between C. werneri and some congeners.
Keywords: Advertisement call, Atlantic Forest, geographical distribution, territorial call.
4.1.2 Introduction Crossodactylus Duméril & Bibron currently comprises 14 species distributed from
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northeastern to southern Brazil (Caldart et al. 2013), and southern Paraguay to northeastern
Argentina (Frost 2016). These species are often associated with the Atlantic Forest in habitats of montane rivulets (Nascimento et al. 2005; Giaretta & Facure 2008). This genus has been considered a taxonomically problematic group, in which associations between populations and nominal species may be challenging (Heyer et al. 1990; Izecksohn & Carvalho-e-Silva
2001; Haddad et al. 2003; Ribeiro et al. 2005; Pimenta et al. 2008, 2014). Such taxonomic difficulties partly reflect intraspecific variation in morphology and color patterns, and the scarcity of information for topotypes, which are in many cases rare or have not been registered since their original descriptions.
Advertisement calls have been described only for six species (Weygoldt & Carvalho- e-Silva 1992; Bastos & Pombal 1995; Nascimento et al. 2005; Pimenta et al. 2008, 2015;
Caldart et al. 2011), so have been tadpoles (Bokermann 1963; Caramaschi & Sazima 1985;
Caramaschi & Kisteumacher 1989; Francioni & Carcerelli 1993; Faivovich 1998; Nascimento et al. 2005; Laia & Rocha 2012; Silva-Soares et al. 2015).
Pimenta et al. (2014) reviewed the taxonomy of the species complex of
Crossodactylus dispar and recognized three different species under the name C. dispar Lutz,
1925. One of them, Crossodactylus werneri [as Crossodactylus dispar in Cochran 1955
―1954‖ and Bastos & Pombal 1995], was described based on specimens collected in the
1970’s at Parque Nacional do Itatiaia (Mantiqueira mountain range), being also reported for nearby localities (Pimenta et al. 2014).
During fieldwork at Serra das Cabras (Campinas, state of São Paulo), we collected specimens that we assigned to Crossodactylus werneri. Here, we describe for the first time the vocalizations, tadpole, color in life, and provide data on the natural history of C. werneri.
Additionally, we extend the geographic range of this species and provide further information
28
on morphological/chromatic variation and genetic divergence inferred from 16S rDNA.
4.1.3 Material And Methods Fieldwork was carried out at Serra das Cabras (22°54′43′′S; 46°49′56′′W, 950 m above sea level), District of Joaquim Egídio, Municipality of Campinas, state of São Paulo, southeastern
Brazil. Based on morphological traits, specimens housed in zoological collections from
Valinhos (23°00′18′′S; 46°57′50′′W, 860 a.s.l.) and Mococa (21°44′33′′S; 47°04′03′′W, 670 m a.s.l.), both localities also in the state of São Paulo, were also identified as belonging to C. werneri. However, these specimens were not included in the morphometric analysis because of small sample sizes (two males from Mococa and no male from Valinhos). We compared these specimens to data from the literature (Pimenta et al. 2014) and topotypes housed in zoological collections. Specimens examined (Appendix I) are housed in the following
Brazilian collections: Collection of Amphibians of the Museu de Biodiversidade do Cerrado at Universidade Federal de Uberlândia (AAG-UFU), Uberlândia, state of Minas Gerais;
Museu de Zoologia da Universidade Estadual de Campinas (ZUEC-AMP) at Universidade
Estadual de Campinas, Campinas, São Paulo; and the Collection Célio Fernando Baptista
Haddad (CFBH) at Universidade Estadual Paulista, Rio Claro, São Paulo.
Advertisement calls were recorded at Serra das Cabras using a Sennheiser K6/ME67 directional microphone and a Marantz PMD 671 digital recorder set at a sampling rate of 48.0 kHz and a sample size of 16 bits. We applied a high band pass filter (cutoff frequency = 150
Hz) to recordings with background noise. Acoustic variables were analyzed in Raven Pro 1.4 software, 64-bit version (Bioacoustics Research Program 2012) using the following settings: window type = Hann, window size = 256 samples, 3dB filter bandwidth = 270 Hz, overlap =
85%, hop size = 0.79 ms, DFT size = 1024 samples, grid spacing = 46.9 Hz. Sound figures
29
were generated using Seewave package version 1.6 (Sueur et al. 2008), in R version 2.13.0 (R
Development Core Team 2015), with Hann window, FFT size of 256 points, and 85% overlap. Acoustic terminology (Appendix II) mostly follows those proposed by Duellman &
Trueb (1994) and Cocroft & Ryan (1995). Call rise time was measured through Peak Time function; minimum frequency and dominant frequency were measured through Low
Frequency and Peak Frequency functions, respectively. When present, isolated notes emitted before the advertisement call (see Results) were not taken into account to determine call duration and note rate. We referred to territorial calls (sensu Toledo et al. 2015) for those emitted during male-male interactions (field observations). Call vouchers and sound files analyzed are housed in the acoustic database of A. A. Giaretta laboratory (Appendix III).
Morphometric measurements (variables listed in Appendix IV) were taken with digital calipers or under a stereomicroscope coupled to an ocular piece, following Duellman (1970), except for foot length, which is in accordance with Pimenta et al. (2014). Morphometric data were tested for intraspecific variability (topotypes x other populations) using both the
Discriminant Analysis of Principal Components [DAPC (Jombart et al. 2010); package adegenet 1.4-2 (Jombart 2008)] and the non-parametric Random Forests model [(Breiman
2001); package randomForest 4.6-10 (Liaw & Wiener 2002)]; both were run in R. For those variables indicated as important by the discriminant methods, the significance of the differences was evaluated according to the ―wilcox_test‖ function in the package coin 1.1-2
(Hothorn et al. 2008) of R.
Data on tadpoles and natural history were gathered at Serra das Cabras during six field trips between 06 November 2011 and 18 September 2015, which comprised both summer
(wet/rainy) and winter (dry) seasons. Tadpoles were collected in September 2015 with a sieve at sites where males were calling. The identification of the tadpoles was based on previous
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descriptions available for congeners (e.g., Crossodactylus aeneus Müller, 1924 in Silva-
Soares et al. 2015). Besides, syntopic tadpoles of two other species that have reproductive activity during the winter [Aplastodiscus leucopigyus (Cruz & Peixoto, 1985) and Scinax hiemalis (Haddad & Pombal, 1987)] were collected in late developmental stages so that identification was possible. Differentiation from tadpoles of the syntopic hylodid Hylodes sazimai Haddad & Pombal, 1995 was based on following features: body shape (ovoid in C. werneri; elliptical in H. sazimai), the absence of a ventral depression, and coloration (dorsum light gray in C. werneri; brown with dark brown spots in H. sazimai) (Haddad & Pombal
1995). Tadpoles were measured for 17 variables (Appendix IV) according to Altig &
McDiarmid (1999). Tadpoles (Appendix I) were at stages 31 and 34 (Gosner 1960) and are also housed in AAG-UFU collection (AAG-UFU 5236: a lot containing 18 tadpoles). Some portions of the pictures from preserved adults and tadpoles were slightly edited in their contour to remove flash shadowing.
The genomic DNA was isolated from muscle preserved in ethanol 100%. Tissue samples were digested with proteinase K, following a modified salt-extraction protocol
(Aljanabi & Martinez 1997). Molecular variation was assessed for 16S rRNA mitochondrial gene by PCR amplification using the primers 16Sar-L and 16Sbr-H (Palumbi et al. 1991) according to Goebel et al. (1999). The PCR products were purified using Qiagen PCR
Purification Kit (Qiagen) and were sequenced by Macrogen Inc. (Seoul, South Korea).
Sequences obtained were subjected to comparisons in GenBank 16S rRNA sequences available for Crossodactylus species (C. aeneus – KM390791; C. caramaschii Bastos &
Pombal, 1995 – AY263235, KJ961569; C. schmidti Gallardo, 1961 – AY843579,
HQ290948). Molecular distances between Crossodactylus species were computed as uncorrected p-distances (Yang 2006) using Mega 6 software (Tamura et al. 2013). Sequences
31
obtained in this study were deposited in GenBank under the accession numbers
KU215900−02.
4.1.4 Results Natural History
Adults were found along a slow-flowing streamlet (30 cm wide, 1−5 cm deep) with sandy/muddy bottom within a small secondary forest patch surrounded by Eucalyptus plantations. Males were calling along the streamlet, even though they were more abundant in a lentic portion of the streamlet with muddy bottom/substrate. Males (N > 20; N = 6 visits) called sat in shallow water, often hidden underneath fallen leaves. Calling activity started around sunset and extended to the first half of the night (between 16:00 to 00:00 h). Males called year around, including winter (dry season), when tadpoles were collected in late developmental stages (31−34). A female bearing mature eggs was found among calling males during dry season (September/2015). During summer (rainy season), syntopic anuran species in calling activity were: Boana faber (Wied-Neuwied), Boana prasina (Burmeister), Hylodes sazimai Haddad & Pombal, Ischnocnema juipoca (Sazima & Cardoso), Phyllomedusa burmeisteri Boulenger, and Proceratophrys boiei Miranda-Ribeiro.
Vocalizations (N = 10 males recorded, 79 calls, 1074 notes; Table 1, Fig. 1)
Comparative acoustic data for C. werneri and other Crossodactylus species are in Table 1.
Advertisement call consisted of a train of about 20 notes, resembling trills (Fig. 1A). Call duration lasted around 3 s, emitted at the highest rate of 17 calls per minute and six notes per second. Note duration lasted around 18 ms, note interval around 157 ms. Pulses within notes were poorly defined, i.e., exhibiting weak and/or irregular amplitude modulations along the
32
note. The call dominant frequency was around 3380 Hz, minimum frequency were around
1800 Hz. The call rise time was around 70%. Four out of 10 males occasionally issued 1−10 isolated notes (mean: 4.8; SD: 2.6) in-between advertisement calls, which had the same temporal and spectral structure of advertisement notes (Fig. 1A).
We recorded a second call type that we interpreted as territorial since it was released concurrently with other nearby calling males (N = 3 males). Territorial calls (N = 49) (Fig.
1B) had a long, well-defined pulsed portion, emitted at a rate of around 260 pulses/s, and ending with a higher-amplitude ―squeak‖ (call rise time > 80%). Call duration was around
260 ms, emitted at a rate of about 65 calls per minute. The dominant frequency was around
3400 Hz.
Morphology and color patterns
Adults (Table 2, Fig. 2)
An adult male from Serra das Cabras and a topotype Crossodactylus werneri are depicted in Fig. 2. Snout-vent length (SVL) was similar between topotypes (male SVL
19.9−23.0 mm; N = 6) and specimens from Serra das Cabras (male SVL 20.9−25.4 mm; N =
13) (W = 0.84, p = 0.39); the other features showed no significant difference either. The
Random Forests model (38% error rate) and DAPC (Fig. 3) showed a weak discrimination between topotypes and specimens from Serra das Cabras. Morphometric features for specimens from Valinhos and Mococa are also provided in Table 2. Some of the topotypes
(ZUEC-AMP 8310−3 and 10127) and most specimens from the other populations have rounded rather than truncate toe tips (Pimenta et al. 2014). All specimens analyzed possess a white stripe on the upper lip, light-colored lateral stripes (Fig. 2B), and belly with dark reticulations on a whitish cream background color (Fig. 2C). Males from Serra das Cabras (N
33
= 13) have fringes on tarsi and toes more developed than in females (N = 10), in addition to hypertrophied forearms.
Tadpoles (Table 3, Fig. 4)
Tadpoles have an ovoid body in dorsal and lateral views (Fig. 4). Snout rounded in dorsal and lateral views. Eyes are dorsally positioned. Nostrils dorsally positioned, rounded, without ornamentation, at mid-distance from the snout tip to the eyes. Spiracle sinistral, short, with its inner margin attached to the body. Tail fins higher than the body. Dorsal fin originates at ¾ of body length, slightly deeper than ventral fin. Oral disc ventrally positioned. Marginal papillae uniseriate, interrupted anteriorly; few scattered submarginal papillae present. Tooth row formula 2(2)/3(1), jaw sheaths wide and serrated; upper jaw sheath slightly arched and with a pointed, medial projection, and lower jaw sheath V-shaped. Vent tube dextral, originated at ventral fin, short, widely open. In preservative, body dark gray and tail light gray with scattered dark gray blotches.
Molecular data
Sequences of the 16S rRNA mitochondrial gene of Crossodactylus werneri from Serra das Cabras had no intra-group variation (0.0%). In contrast, interspecific comparisons resulted in 8%, 4.5%, and 5% of divergence from C. aeneus, C. caramaschii, and C. schmidti, respectively (Table 4).
4.1.5 Discussion Crossodactylus werneri has a particular behavior among Hylodidae by the fact that males call during the night and such feature appears to represent a reversion to the ancestral state for
34
Anura (Duellman & Trueb 1994). Besides, this species uses muddy portions of slow-flowing watercourse while other congeneric species, as well as other Hylodidae, are expected to call along rapid flowing streams with sandy-rocky bottom (Heyer et al. 1990; Giaretta & Facure
2008).
The advertisement call of Crossodactylus werneri is distinct from the calls of its congeners by having fewer notes per call. Similarly to C. cyclospinus, C. schmidti, and C. trachystomus, C. werneri has long note duration and note interval. As described by Pimenta et al. (2008) and Pimenta et al. (2015) for C. trachystomus and C. franciscanus Pimenta,
Caramaschi & Cruz, the advertisement call of C. werneri gradually increases in amplitude, reaching maximum amplitude about midway of call duration. Besides, as previously reported for other species of Crossodactylus (Pimenta et al. 2015), C. werneri may also emit isolated notes in-between advertisement calls. Detailed comparisons with the call of C. timbuhy
(Pimenta et al. 2015; as C. cf. dispar in Weygoldt 1986) were not possible due to its poor characterization.
Despite the advertisement calls of hylodid species have often been described as exhibiting tonal (well-marked harmonics) components (Hartmann et al. 2006; Caldart et al.
2011; Pimenta et al. 2015), we did not detect this pattern in the call of C. werneri; rather, notes had energy distributed over a broad-bandwidth frequency (Fig. 1). Also, the call of C. werneri has the lowest dominant frequency in comparison with those of congeners (Table 1).
On the other hand, advertisement calls of Hylodes and other Crossodactylus species have their dominant frequency usually coincident with the second or third harmonics (Caldart et al.
2011; Lingnau & Bastos 2007; Lingnau et al. 2008). This can be understood in an ecological context, given that these frogs almost always call in environments with stream noise and high- frequency signals could minimize masking interference in low-frequency noisy environments
35
(Hödl & Amézquita 2001). With that said, the lower dominant frequency of C. werneri may supposedly be related to the low environmental interference by stream noise (slow-flowing water) at the habitat where this species occurs at Serra das Cabras. It is worth highlighting that the congener C. schmidti was shown to perform short-term adjustments in acoustic traits in response to stream-generated noise (Caldart et al. 2016).
Only Crossodactylus gaudichaudii, C. schmidti, and C. cyclospinus had calls other than the advertisement call described (Caldart et al. 2011; Nascimento et al. 2005; Weygoldt
& Carvalho-e-Silva 1992, respectively). The call resembling the most the territorial call of C. werneri with respect to its temporal structure is that of C. cyclospinus, but they vary in note duration (C. werneri: 261 ms, C. cyclospinus: 11 ms; Nascimento et al. 2005) and dominant frequency (C. werneri: 4641 Hz, C. cyclospinus: 3454 Hz; Nascimento et al. 2005).
Despite the slight difference found in toe tip shapes (rounded rather than truncate as described by Pimenta et al. 2014), we observed that this trait varied among topotypes of
Crossodactylus werneri as well. Differences in color, such as patterns between snout and shoulder (upper lip), presence of lateral stripe, and belly (Pimenta et al. 2014) were regarded as variable and related to the preservation conditions of the types, which were collected more than 40 years ago.
Up to date, six Crossodactylus species have their tadpoles described: C. aeneus (Silva-
Soares et al. 2015); C. cyclospinus (Nascimento et al. 2005); C. dispar [Bokermann 1963
(this tadpole is ambiguously assigned to this species, as pointed by Pimenta et al. 2014)]; C. gaudichaudii (Francioni & Carcerelli 1993), C. schmidti (Faivovich 1998), and C. trachystomus [Caramaschi & Sazima 1985 (as C. bokermanni); Caramaschi & Kisteumacher
1989]. The differentiation between the tadpoles of C. werneri and those of the other six species was based on the combination nostril ornamentation, and upper jaw shape. In contrast
36
to some other species, C. werneri tadpoles do not show any nostril ornamentation [discrete ornamentation in C. aeneus, and well-ornamented in C. trachystomus and C. dispar (Silva-
Soares et al. 2015; no mention to the other species)]. Oral morphology is similar among congeners, described as having a medial pointed projection on upper jaw, whose degree of development varies among species, being pointed in C. werneri (present study) and still more protruding in C. aeneus (Silva-Soares et al. 2015). However, this structure is not described for
C. trachystomus, C. gaudichaudii, and C. schmidti (Nascimento et al. 2005) and poorly characterized for other species.
Our record at Serra das Cabras is located approximately 100 km west, and Mococa
200 km northwest, from Santo Antônio do Pinhal, the westernmost record for Crossodactylus werneri up to date (Fig. 5). Unfortunately, we were unable to obtain tissue samples from topotypes or from the other localities (Mococa and Valinhos), neither were we to obtain gene sequences from collections or from GenBank database. It is noteworthy that C. werneri had not been recorded since 1978 at the type locality (Pimenta et al. 2014), which corresponds to an interval of 33 years without any record for the species until 2011, when we collected the first specimens of C. werneri in the field, even though there are non-topotypical specimens housed in zoological collections that need to be reidentified (including our specimens of C. werneri examined in Appendix I) since the species description by Pimenta et al. in 2014.
Interestingly, this phenomenon of declines and disappearance are also valid for other congeners, such as C. boulengeri, C. dispar, and C. grandis, which have not been reported since the early or late 1970’s (Pimenta et al. 2014). The gene sequences (16S rRNA) now available for C. werneri shed light on the divergence rates (4.5−8.0%) among C. werneri and a few congeners (C. aeneus, C. caramaschii, and C. schmidti). Even so, molecular data for other Crossodactylus species are required so we could generate a better estimate of the
37
genetic diversity associated with the nominal species of Crossodactylus, which might be of great help in future studies addressing taxonomic and systematic issues in this group of
Neotropical frogs.
4.1.6 Acknowledgements We thank F.S. Andrade and I.A. Haga for their help in the field and for making available sound recordings. Lucas B. Martins critically read an early version of the manuscript. Leo R.
Malagoli provided helpful insights. Financial support by Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do
Estado de Minas Gerais (FAPEMIG). A research grant to AAG by CNPq. A doctoral fellowship by Fundação de Amparo à Pesquisa no Estado de São Paulo (FAPESP; TRC:
#2012/15763‒7) and a Master’s fellowship by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; IV). Collection permit was granted by Instituto Chico Mendes
(ICMBio/SISBIO #02015.008064/02–51).
38
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4.1.8 Figure captions Figure 1. (A) From top to bottom: oscillogram of a 29-note advertisement call of
Crossodactylus werneri preceded by five isolated notes (second note identified by a red arrow); spectrogram of three median notes and respective oscillogram. Sound file:
Crossod_werneriSousasSP1aAAGm671. (B) Spectrogram and respective oscillogram of the territorial call of Crossodactylus werneri. Sound file:
Crossod_werneriSousasSP1eAAGm671. Further information on these recordings is provided in Appendix III.
Figure 2. (A) Adult male specimens of Crossodactylus werneri in life from Serra das Cabras,
Campinas, São Paulo: above—AAG-UFU 0981, SVL 21.7 mm; below—AAG-UFU 0982,
SVL 21.6 mm. (B) Adult males of Crossodactylus werneri in dorsal view. Left—Serra das
Cabras, Campinas, São Paulo (AAG-UFU 1880; SVL 23.0 mm); right—Parque Nacional do
Itatiaia, Rio de Janeiro (topotype ZUEC-AMP 7981; SVL 22.1 mm). (C) Adult males of
Crossodactylus werneri from Serra das Cabras (Campinas, São Paulo), depicting variation in size and in the degree of reticulation on the belly (right—AAG-UFU 1875, SVL 21.6 mm; left—AAG-UFU 1878, SVL 25.1 mm).
Figure 3. A density plot of the first discriminant axis (DAPC) on morphological traits from adult males of Crossodactylus werneri. Note the low discrimination among topotypes (blue) and specimens from Serra das Cabras (red). Six principal component axes were retained and explained 95% of total variance.
Figure 4. The tadpole (stage 31) of Crossodactylus werneri. From top to bottom: lateral view; dorsal and ventral views of body; oral disc (bottom right). Specimen from Serra das Cabras,
Campinas, São Paulo (lot AAG-UFU 5236).
Figure 5. Geographic distribution of Crossodactylus werneri in southeastern Brazil. Red circle: type locality (Itatiaia, between the limits of the Brazilian states of Rio de Janeiro, São
46
Paulo, and Minas Gerais); white circles: localities reported by Pimenta et al. (2014); white triangles: new records (present study) from Mococa (above), and Serra das Cabras
(Campinas) and Valinhos (below).
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4.1.9 Appendices Appendix I. Additional specimens examined (adults and tadpoles).
Crossodactylus aeneus.—BRAZIL: RIO DE JANEIRO: Guapimirim: ZUEC-AMP
20459, 20974; Duque de Caxias: ZUEC-AMP 17578–88.
Crossodactylus boulengeri.—BRAZIL: SÃO PAULO: Capão Bonito: CFBH 35180.
Crossodactylus caramaschii.—BRAZIL: SÃO PAULO: Iporanga: ZUEC-AMP 8255–8
(paratypes).
Crossodactylus gaudichaudii.—BRAZIL: RIO DE JANEIRO: Rio de Janeiro: ZUEC-
AMP 13552–4, 17569–77.
Crossodactylus grandis.—BRAZIL: RIO DE JANEIRO: Lagoa Azul (Parque Nacional do Itatiaia): ZUEC-AMP 10; SÃO PAULO: Campos do Jordão: ZUEC-AMP 991.
Crossodactylus schmidti.—ARGENTINA: MISIONES: San Vicente: CFBH 9495–8.
Crossodactylus trachystomus.—BRAZIL: MINAS GERAIS: Santana do Riacho: ZUEC-
AMP 2287; Jaboticatubas: ZUEC-AMP 2531–2, ZUEC-AMP 2540–1.
Crossodactylus werneri.—BRAZIL: SÃO PAULO: Campinas (Serra das Cabras): AAG-
UFU (males): 0848, 0982, 1876, 1879–80, 1882–85, 3912; (females) 0981, 1875, 1877–78,
1881 3913, 4612; 5236 (lot containing 18 tadpoles), ZUEC-AMP (males): 8923–24; (females)
3188, 8922, 8925; Mococa: ZUEC-AMP (males) 8189, 8198; (females) 8187, 8191–5;
Valinhos: ZUEC-AMP 10867–9, 10875; RIO DE JANEIRO: Itatiaia, Parque Nacional do Itatiaia
(topotypes): ZUEC-AMP (male) 7981; (female) 7982; Itatiaia (near Parque Nacional do
Itatiaia, BR 354, km 9): ZUEC-AMP (males) 8310–3, 10126; (female) 10127, CFBH 9411–6.
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Appendix II. Acoustic terminology modified from Duellman & Trueb (1994) and Cocroft & Ryan (1995). Acoustic analysis was conducted in Raven Pro 1.4.
Call trait Definition Reference Notes per call Number of discrete acoustic units produced in a series (= call) Duellman & Trueb (1994) Call duration (s) Duration of note series Duellman & Trueb (1994) Call rate (Total number of calls – 1)/time from beginning of first call to beginning of last call. Cocroft & Ryan (1995) Given as calls/min. Intercall interval (s) Duration between the end of a call and the beginning of the next Present study Note duration (ms) Duration of discrete acoustic units, emitted either isolated or comprising note series Duellman & Trueb (1994) Internote interval (s) Duration between the end of one note and the beginning of the next Present study Note rate (Total number of notes – 1)/ time from beginning of first note to beginning of last note in Cocroft & Ryan (1995) a series. Given as notes/s Pulse rate (Total number of pulses – 1)/time from beginning of first pulse to beginning of last pulse Cocroft & Ryan (1995) of a note. Given as pulses/s Minimum frequency (Hz) Minor frequency detected for the notes on spectrogram window, measured manually Present study using the Low Frequency function. Given as the average from all notes analyzed. Dominant frequency (Hz) Frequency containing the greatest sound energy of the whole call (note series). Measured Duellman & Trueb (1994) using the Peak frequency function. Call Rise time (%) Time from beginning to peak amplitude of the call. Measured using the Peak time Cocroft & Ryan (1995) function.
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Appendix III. Sound files analyzed for Crossodactylus werneri from Serra das Cabras, (Campinas, São Paulo, Brazil). For each male recorded, the recording date, water and air temperatures, and voucher males (AAG-UFU accession numbers) are provided.
Sound files:
Crossod_werneriSousasSP1a-fAAGm671 [six recordings (―a-f‖) for male 1]; Crossod_werneriSousasSP2AAGm671 (one recording for male 2);
Crossod_werneriSousasSP3AAGm671 (one recording for male 3); Crossod_werneriSousasSP4a-dAAGm671 [four recordings (―a-d‖) for male
4]; Crossod_werneriSousasSP5a-dAAGm671 [four recordings (―a-d‖) for male 5]; Crossod_werneriSousasSP6AAGm671 (one recording for male 6); Crossod_werneriSousasSP7a-cAAGm671 [three recordings (―a-c‖) for male 7]; Crossod_werneriSousasSP8aAAGm671 [one recording for male 8]; Crossod_werneriSousasSP9aAAGm671 [one recording for male 9]; Crossod_werneriSousasSP10a-eAAGm671 [five recordings (―a- e‖) for male 10].
Date Voucher male Male recorded Water (°C) Air (°C) 06/Nov/2011 AAG-UFU 0848 Male 1 (six recordings) 18 18 06/Nov/2011 - Male 2 (one recording) 18 18 06/Nov/2011 - Male 3 (one recording) 18 18 05/Jan/2012 - Male 4 (four recordings) 19 19 05/Jan/2012 AAG-UFU 0981 Male 5 (four recordings) 19 19 02/Nov/2013 AAG-UFU 1875 Male 6 (one recording) 18 19 07/Set/2014 - Male 7 (three recordings) 19 20 07/Set/2014 - Male 8 (one recording) 19 20 07/Set/2014 AAG-UFU 3912 Male 9 (one recording) 19 20 07/Set/2014 AAG-UFU 3913 Male 10 (five recordings) 19 20
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Appendix IV. Morphometric terminology for adults and tadpoles.
Abbreviations Terminology Adults SVL Snout-vent length HL Head length HW Head width TBL Tibia length THL Thigh length FL Foot length TD Tympanum diameter ED Eye diameter END Eye-nostril distance NSD Nostril-snout distance IND Internarial distance IOD Interocular distance Tadpoles TL Total length BL Body length BW Body width BW Body height TL Tail length TH Tail height DFH Dorsal fin height VFH Ventral fin height IND Internostril distance IOD Interorbital distance ED Eye diameter ND Nostril diameter END Eye-nostril distance NSD Nostril-snout distance ESD Eye-snout distance SSD Snout-spiracle distance ODW Oral disc width
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4.1.9.1 Tables Table 1. Acoustic traits of the advertisement call of Crossodactylus werneri and those of the other six congeners with acoustic data available. Data provided as mean ± SD (range) [sample size for the variable]. Hyphens denote unavailable data. C. werneri C. caramaschii C. cyclospinus C. franciscanus C. gaudichaudii C. schmidti C. trachystomus Serra das Bastos & Pombal Nascimento et al. Pimenta et al. (2015) Weygoldt & Caldart et al. Pimenta et al. Call traits Cabras, SP (1995) (2005) Carvalho-e-Silva (2011) (2008) N = 10 males (1992) Notes/call 19.9 ± 4.3 56.8± 5.4 63.3 ± 11.9 48.0 ± 18.2 - 35.0 ± 6.64 58.8 ± 26.2 (12‒25) (49‒69) (35‒98) (36‒84) (25‒130) (13‒45) (13‒121) [79] [6] [25] [48] - [11] [86] Call duration (s) 3.2 ± 1.2 5.5 ± 0.5 4.3 ± 0.7 6.4 ± 2.5 - 4.1 ± 1.0 4.9 ± 1.9 (1.9‒6.3) (4.7‒6.0) (3.5‒6.2) (4.8‒11.6) (2.0‒13.0) (2.2‒5.7) (1.4‒10.1) [79] [6] [25] [6] - [11] [52] Calls/min 17.5 ± 10.4 ------(6.4‒41.2) ------Intercall interval (s) 14.6 ±5.8 ------(8.3‒25.8) ------Note duration (ms) 18.2 ± 5.0 - 28.0 ± 4.0 12.0 ± 2.6 - 22.0 ± 7.0 14.0 ± 5.0 (11.0‒27.0) - (3.0‒40.0) (6.0‒20.0) (40.0‒50.0) (9.0‒61.0) (1.0‒33.0) [2478] [1074] - [1601] [284] - [289] Internote interval (ms) 157.0 ± 43.8 - 40.0 ± 3.0 123.0 ± 13.4 - 100.0 ± 28.0 92.0 ± 21.0 (128.0‒278.0) - (29.0‒65.0) [1576] (90.0‒184.0) (40.0‒50.0) (17.0‒249.0) [279] (56.0‒265.0) [1042] - [284] [2392] Notes/s 6.3 ± 1.2 ------(3.2‒7.5) ------Minimum frequency (kHz) 1.7 ± 0.2 ~1.6 - - - 1.5 ± 0.3 - (1.2‒2.3) - (0.7‒1.2) - (2.0‒5.5) (1.0‒2.0) - [915] - [25] - - [11] - Dominant frequency (kHz) 3.3 ± 0.2 ~5.0 4.9 ± 0.6 3.4 ± 2.8 - 3.3 ± 0.6 3.8 ± 0.5 (3.0‒3.6) - (3.4‒5.4) (2.3‒4.0) - (2.0‒4.2) (1.8‒4.8) [79] - [25] [284] - [11] [1535] Call Rise time (%) 70.3 ± 20.0 ------(20.1‒94.4) ------
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Table 2. Morphometric traits (in mm) for adult specimens of Crossodactylus werneri from new localities in the state São Paulo and topotypes (Parque Nacional do Itatiaia). Values are expressed as Mean ± SD (range).
C. werneri (Serra das Cabras) C. werneri (Mococa) C. werneri (Valinhos) C. werneri (topotypes)
Males (N = 13) Females (N =10) Males (N = 2) Females (N = 6) Females (N = 4) Males (N = 6) Females (N = 1) 22.1 ± 1.2 23.1 ± 1.7 20.3 ± 0.3 22.1 ± 1.7 23.4 ± 3.2 21.1 ± 1.1 SVL 24.0 (20.9‒25.4) (20.4‒25.3) (20.0‒20.5) (19.6‒23.8) (19.2‒26.7) (19.9‒22.7) 7.7 ± 0.6 7.7 ± 0.5 7.2 ± 0.2 7.6 ± 0.4 7.5 ± 0.6 7.4 ± 0.5 HL 8.9 (6.9‒8.7) (6.7‒8.4) (7.1‒7.4) (7.1‒8.3) (6.7‒8.1) (6.8‒8.2) 7.8 ± 0.3 7.9 ± 0.4 6.8 ± 0.1 7.2 ± 0.9 7.6 ± 0.5 7.1 ± 0.6 HW 9.2 (7.3‒8.3) (7.2‒8.7) (6.7‒7.0) (5.6‒8.5) (6.7‒7.9) (6.5‒7.9) 10.7 ± 0.6 11.2 ± 0.6 10.1 ± 0.2 10.4 ± 0.5 11.0 ± 1.3 10.1 ± 0.5 TBL 11.5 (9.6‒11.6) (10.4‒12.6) (9.9‒10.2) (9.5‒11.0) (9.7‒12.7) (9.5‒10.8) 10.1 ± 0.7 10.3 ± 0.6 9.2 ± 0.2 8.8 ± 0.3 9.8 ± 1.1 8.8 ± 0.3 THL 9.5 (8.5‒11.7) (9.3‒11.4) (9.1‒9.4) (8.4‒9.4) (8.6‒11.1) (8.4‒9.1) 16.6 ± 0.9 18.1 ± 1.0 15.8 ± 0.6 16.6 ± 0.8 16.9 ± 2.1 16.3 ± 0.9 FL 18.6 (15.4‒18.7) (16.8‒19.3) (15.4‒16.3) (15.8‒17.9) (14.2‒18.9) (15.0‒16.6) 1.5 ± 0.1 1.7 ± 0.3 1.5 ± 0.1 1.7 ± 0.1 1.6 ± 0.2 1.4 ± 0.1 TD 1.4 (1.2‒2.1) (1.2‒2.3) (1.5‒1.6) (1.5‒1.8) (1.4‒1.9) (1.4‒1.5) 2.5 ± 0.1 2.6 ± 0.1 2.2 ± 0.1 2.6 ± 0.2 2.6 ± 0.1 2.3 ± 0.1 ED 2.5 (2.2‒2.8) (2.3‒2.9) (2.2‒2.3) (2.4‒2.9) (2.5‒2.9) (2.2‒2.5) 1.5 ± 0.1 1.5 ± 0.1 1.8 ± 0.1 1.7 ± 0.2 1.7 ± 0.1 1.5 ± 0.1 END 1.8 (1.3‒1.8) (1.3‒1.9) (1.7‒1.9) (1.4‒1.8) (1.7‒1.9) (1.4‒1.8) 0.7 ± 0.1 0.7 ± 0.1 0.8 ± 0.2 0.9 ± 0.3 0.9 ± 0.1 0.9 ± 0.1 NSD 0.9 (0.5‒1.0) (0.6‒0.9) (0.7‒1.0) (0.7‒1.2) (0.7‒1.1) (0.7‒1.0) 2.5 ± 0.1 2.5 ± 0.2 2.4 ± 0.2 2.3 ± 0.1 2.4 ± 0.2 2.5 ± 0.1 IND 2.9 (2.3‒2.8) (2.1‒2.9) (2.3‒2.6) (2.1‒2.4) (2.0‒2.6) (2.4‒2.6) 1.9 ± 0.1 2.1 ± 0.1 1.9 ± 0.1 2.0 ± 0.1 2.2 ± 0.3 2.1 ± 0.1 IOD 2.5 (1.8‒2.3) (1.9‒2.3) (1.8‒2.0) (1.9‒2.2) (1.8‒2.5) (2.0‒2.2)
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Table 3. Morphometric traits (mm) for tadpoles of Crossodactylus werneri (stages 31 and 34) from Serra das Cabras (Campinas, São Paulo, Brazil).
Measurements Stage 31 (N = 16) Stage 34 (N = 2) TL 30.5 ± 2.4 (27.4‒33.2) 36.0 ± 1.4 (35.0‒37.9) BL 11.6 ± 1.0 (10.6‒13.1) 14.0 ± 0.0 (14.0‒14.1) BW 7.8 ± 0.5 (7.1‒8.6) 10.2 ± 0.1 (10.1‒10.3) BW 7.1 ± 0.5 (6.5‒7.9) 9.0 ± 0.1 (8.9‒9.1) TL 20.3 ± 2.3 (17.5‒22.7) 24.8 ± 0.2(24.7‒25.0) TH 6.9 ± 0.4 (6.1‒7.4) 8.1 ± 0.2 (7.9‒8.3) DFH 2.5 ± 0.2 (2.2‒2.9) 3.1 ± 0.1 (3.1‒3.2) VFH 1.3 ± 0.9 (1.5‒2.1) 2.5 ± 0.1 (2.3‒2.4) IND 2.2 ± 0.1 (2.1‒2.6) 2.5 ± 0.1 (2.5‒2.6) IOD 3.2 ± 0.1 (3.1‒3.5) 3.6 ± 0.1 (3.5‒3.7) ED 1.2 ± 0.1 (1.1‒1.4) 1.6 ±0.1 (1.6‒1.7) ND 0.3 ± 0.1 (0.1‒0.4) 0.4 ± 0.1 (0.4‒0.5) END 1.7 ± 0.3 (1.4‒2.5) 1.8 ± 0.1 (1.8‒1.9) NSD 1.3 ± 0.1 (1.3‒1.5) 1.7 ± 0.1 (1.7‒1.8) ESD 3.0± 0.4 (2.5‒3.6) 3.0 ± 0.1 (2.9‒3.1) SSD 6.4 ± 0.3 (6.1‒6.9) 6.8 ± 0.1 (6.7‒6.9) ODW 2.1 ± 0.1 (1.9‒2.4) 2.3 ± 0.1 (2.3‒2.4)
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Table 4. Uncorrected molecular distances (p-distance) for 16S rRNA gene among Crossodactylus werneri, C. aeneus, C. caramaschii, and C. schmidti.
C. werneri C. werneri C. werneri C. aeneus C. schmidti C. caramaschii C. caramaschii (KU21590 (KU21590 (KU21590 (KM390791 (AY843579 (KJ961569) (AY263235) 0) 1) 2) ) )
C. werneri (KU215900)
C. werneri (KU215901) 0.00%
C. werneri (KU215902) 0.00% 0.00%
C. aeneus (KM390791) 8.19% 8.19% 8.19%
C. caramaschii (KJ961569) 4.57% 4.57% 4.57% 7.43%
C. caramaschii (AY263235) 4.19% 4.19% 4.19% 7.81% 2.67%
C. schmidti (AY843579) 4.95% 4.95% 4.95% 6.67% 5.33% 5.33%
C. schmidti (HQ290948) 4.95% 4.95% 4.95% 6.67% 5.33% 5.33% 0.00%
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4.1.9.2 Figures
Figure 1. (A) From top to bottom: oscillogram of a 29-note advertisement call of Crossodactylus werneri preceded by five isolated notes (second note identified by a red arrow); spectrogram of three median notes and respective oscillogram. Sound file: Crossod_werneriSousasSP1aAAGm671. (B) Spectrogram and respective oscillogram of the territorial call of Crossodactylus werneri. Sound file: Crossod_werneriSousasSP1eAAGm671. Further information on these recordings are provided in Appendix III
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Figure 2. (A) Adult male specimens of Crossodactylus werneri in life from Serra das Cabras, Campinas, São Paulo: above—AAG-UFU 0981, SVL 21.7 mm; below—AAG-UFU 0982, SVL 21.6 mm. (B) Adult males of Crossodactylus werneri in dorsal view. Left—Serra das Cabras, Campinas, state of São Paulo (AAG-UFU 1880; SVL 23.0 mm); right—Parque Nacional do Itatiaia, Itatiaia, state of Rio de Janeiro (topotype ZUEC-AMP 7981; SVL 22.1 mm). (C) Adult males of Crossodactylus werneri from Serra das Cabras, Campinas, São Paulo, depicting variation in size and in the degree of reticulation on belly (right—AAG-UFU 1875, SVL 21.57 mm; left—AAG-UFU 1878, SVL 25.05 mm).
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Figure 3. Density plot of the first discriminant axis (DAPC) on morphological traits adult males of Crossodactylus werneri. Note the low discrimination among topotypes (blue) and specimens from Serra das Cabras (red). Six principal component axes were retained explained 95% total variance.
.
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Figure 4. The tadpole (stage 31) of Crossodactylus werneri. From top to bottom: lateral view; dorsal and ventral views of body (on left); on right: oral disc. Specimen from Serra das Cabras, Campinas, state of São Paulo (lot AAG-UFU 5236).
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Figure 5. Geographic distribution of Crossodactylus werneri in southeastern Brazil. Red circle: type locality (Itatiaia, between the limits of the Brazilian states of Rio de Janeiro, São Paulo, and Minas Gerais); white circles: localities reported by Pimenta et al. (2014); white triangles: new records (present study) in Mococa (above) and Campinas/Valinhos (below).
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4.2 Manuscrito II (Formatado nos moldes do periódico Zootaxa). A Morphological, Acoustic and Genetic Evaluation of the Specific Identity of a
Crossodactylus Duméril & Bibron, 1841 (Anura: Hylodidae) Population from the
Mantiqueira Range
Abstract Crossodactylus Duméril & Bibron currently comprises 14 species typically associated with
Atlantic Forest and Campos Rupestres rivulets. All the Crossodactylus species have been diagnosed based mostly on adult morphology, and an integrative approach, including different lines of evidences has not yet been made for comparison purpose. Herein, we evaluate the issue of the identity of a Crossodactylus population from Atibaia, until now attributed to C. caramaschii, integrating morphological, acoustic and genetic evidences. Analyzes on morphological and acoustic evidences suggest a strong discrimination between Atibaia specimens and C. caramaschii, from Apiai. Insights under genetic divergence in mithocondrial 16S are inconclusive. However, Atibaia specimens appear to be co-specific to
C. caramaschii from Itanhaem (Sao Paulo state), which seems to compose a new clade close- related to C. caramaschii.
Keywords: Advertisement call, Amphibia, Atlantic Forest, spinethumb frogs, torrent frogs.
4.2.2 Introduction Crossodactylus Duméril & Bibron currently comprises 14 species distributed from
Northeastern to Southern Brazil, and Southern Paraguay to Northern Argentina (Caldart et al.
2013; Frost 2017), typically associated with Atlantic Forest and Campos Rupestres (montane rocky field) rivulets (Nascimento et al. 2005; Giaretta & Facure 2008).
Despite some degree of morphological variability and consequent taxonomic
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difficulties (Heyer et al. 1990; Izecksohn & Carvalho-e-Silva 2001; Haddad et al. 2003;
Ribeiro et al. 2005; Pimenta et al. 2008, 2014), all the Crossodactylus species have been diagnosed based mostly on adult morphology (e.g. Caramaschi & Sazima 1985; Nascimento et al. 2005; Pimenta et al. 2014, 2015). Data on acoustics are known for only seven species
(Weygoldt & Carvalho-e-Silva 1992; Bastos & Pombal 1995; Nascimento et al. 2005;
Pimenta et al. 2008, 2015; Caldart et al. 2011; Vidigal et al. 2017, in press), and genetic differentiation are yet poorly explored. So, an integrative approach, including different lines of evidences has not yet been made for comparison purpose within Crossodactylus.
Based on adult morphology, Pimenta et al. (2014) reviewed the taxonomy of some problematic or misidentified Crossodactylus populations distributed in Southeast and South
Brazil recognizing, for example, three different species under the name Crossodactylus dispar
Lutz, 1925. In addition, they attributed some of all analyzed populations to C. caramaschii, one of them cited by Giaretta et al. (1999) as unidentified, in Atibaia (Sao Paulo state) assuming a range extension of about 280 Km to northeast from its type locality and the presence of this species in the Mantiqueira Range.
To better evaluate the issue of the identity the Crossodactylus from Atibaia, we carried out fieldworks around the type locality of C. caramaschii and Atibaia to collect specimens’ tissues and calls. We also examined specimens in zoological collections and data from literature, to complement conclusions regarding the Atibaia population.
4.2.3 Materials and Methods Specimens and calls were collected in Parque Florestal do Itapetinga (23º15'S;
46º45'W, 1250 m asl), municipality of Atibaia, São Paulo state. We statistically compared morphological and acoustic datasets from these specimens with C. caramaschii type series and newly gathered specimens from around type locality (Apiaí, Sao Paulo state). Besides, we have considered other specimens housed in collections as well as data from literature in order
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to make comparisons (external morphology) among other species of Crossodactylus (e.g.
Bastos & Pombal 1995; Pimenta et al. 2014, 2015). Analyzed specimens (Appendix I) are housed in the Collection of Amphibians of the Museu de Biodiversidade do Cerrado at the
Universidade Federal de Uberlândia (AAG-UFU), Uberlândia, Minas Gerais state, Brazil; at
Museu de Zoologia da Universidade Estadual de Campinas (ZUEC-AMP), Campinas, São
Paulo state, Brazil; at Collection Célio Fernando Baptista Haddad (CFBH) at the
Universidade Estadual Paulista, Rio Claro, São Paulo state, Brazil; at Museu de Zoologia da
Universidade de Sao Paulo (MZUSP), Sao Paulo, Sao Paulo state, Brazil and at Museu
Nacional do Rio de Janeiro (MNRJ), Rio de Janeiro, Rio de Janeiro state.
Twelve morphometric traits were measured on adult males, following Pimenta et al.
(2014): snout-vent length (SVL), head length (HL), head width (HW), tibia length (TBL), thigh length (THL), foot length (FL), tympanum diameter (TD), eye diameter (ED), eye- nostril distance (END), nostril-snout distance (NSD), internarial distance (IND), interorbital distance (IOD). These morphometric measurements were taken using a Mitutoyo Absolute digital caliper CD-6‖ CSX, except for tympanum diameter, eye diameter, eye-nostril distance, nostril-snout distance, internarial distance and interorbital distance which were measured under a stereomicroscope coupled to an ocular micrometer. Vocal sac characterization follows
Elias-Costa et al. (2017).
Advertisement calls were recorded with Sennheiser K6/ME67 directional microphones coupled to Marantz PMD 671 digital recorders set at a sampling rate of 48.0 kHz and a resolution of 16 bits. We applied a high band pass filter (cutoff frequency = 150 Hz) to recordings with background noise levels. Acoustic variables were analyzed with Raven Pro
1.4 software, 64-bit version (Bioacoustics Research Program 2012) with the following settings: window type = Hann, window size = 256 samples, 3dB filter bandwidth = 270 Hz, overlap = 85%, hop size = 0.79 ms, DFT size = 1024 samples, grid spacing = 46.9 Hz. Sound
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figures were generated with Seewave, version 1.6 (Sueur et al. 2008) in R (v.2.13.0), with
FFT size of 256 and 85% overlap. Acoustic terminology follows the ―call-centered approach‖
(Köhler et al. 2017) and variables are explained in Vidigal et al. (2017, in press).
Morphometric and acoustic datasets were tested separately for inter-population variability, considering both the Discriminant Analysis of Principal Components [DAPC
(Jombart et al. 2010); package adegenet 1.4-2 (Jombart 2008)] and the non-parametric
Random Forests model [(Breiman 2001); package randomForest 4.6-10 (Liaw & Wiener
2002)]; both performed in R (R Development Core Team 2015). To those features indicated as important by the discriminant methods, significance of the differences was evaluated according to the ―wilcox_test‖ function (Exact Wilcoxon Mann Whitney Rank Sum Test) of the package coin 1.1-2 (Hothorn et al. 2008) in R.
The genomic DNA was isolated from muscle preserved in ethanol 100%. Tissue samples were digested with proteinase K, following a modified salt-extraction protocol
(Aljanabi & Martinez 1997). Molecular variation was assessed for 16S rRNA mitochondrial gene by PCR amplification using the primers 16Sar-L and 16Sbr-H (Palumbi et al. 1991) according to Goebel et al. (1999). The PCR products were purified using Qiagen PCR
Purification Kit (Qiagen) and were sequenced by Macrogen Inc. (Seoul, South Korea).
Sequences obtained were subjected to comparisons in GenBank 16S rRNA sequences available for Crossodactylus species (C. caramaschii Bastos & Pombal, 1995 – KJ961569; C. schmidti Gallardo, 1961 – HQ290948; C. werneri Pimenta et al. 2014 – KU215900-02).
Molecular distances between Crossodactylus species were computed as uncorrected p- distances (Yang 2006) using Mega 6 software (Tamura et al. 2013). Sequences obtained in this study were deposited in GenBank under the accession numbers MG019408−11 (Atibaia specimens., from Parque Florestal do Itapetinga), and MG019412 (Crossodactylus caramaschii, from Apiaí).
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4.2.4 Results and Discussion
Atibaia species
Crossodactylus sp. (Giaretta et al. 1999)
Crossodactylus caramaschii (―part‖ Pimenta et al. 2014)
Main reference specimen — AAG-UFU 5200, adult male (Figure 1), collected in
Parque Florestal do Itapetinga (23º15’S; 46º45’W, 1250 m asl), municipality of Atibaia, Sao
Paulo state, in October 2015, by A. A. Giaretta.
Other reference specimens — Nine adult males, all collected at the type locality:
AAG-UFU 3914 collected in September 2014; AAG-UFU 523739 and AAG-UFU 524042 collected in February 2016; all by A. A. Giaretta.
Diagnosis — (1) body slender; (2) head nearly as long as wide; (3) snout nearly pentagon-shaped in dorsal view, protruding in lateral view; (4) canthus rostralis poorly marked; (5) tympanum distinct; (6) vocal sac subgular, bilobate on external view; (7) thumb spines small or absent; (8) developed fringes on toes and tarsus, reduced in females; (9) finger tips dilated; (10) toe tips dilated and truncate; (11) postrictal tubercle in small granules; (12) discrete glandular crest on anterior surface of the arm absent; (13) dorsal skin posteriorly granular; (14) dorsolateral glandular ridges well marked; (15) white or cream stripe from the snout to the shoulder, with brown blots near to oral gap; (16) presence of white blot on inguinal region; (17) belly reticulated.
Description of the reference specimen — Body slender. Head nearly as long as
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wide; nostrils prominent, closer to the tip of snout than to the eye. Snout nearly pentagon?- shaped in dorsal view, protruding in lateral view. Canthus rostralis poorly defined; loreal region slightly concave. Tympanum distinct, with a well-marked supratympanic fold, extending from the posterior corner of the eye to the end of tympanum (Figure 2). Vocal sac bilobate, subgular. Upper lip bordered by many small white spines. No vomerine teeth.
Tongue large, ovoid, covering almost the whole mouth floor, not attached behind. Slender arms; upper arms same thickness the forearms. Finger tips dilated; finger lengths II~IV In preservative (70% ETOH), main reference specimen presents light brown 66 tympanum, stripe from the snout to the shoulder and inguinal blot cream. Arms and legs with darker transversal bars. Ventral surface, throat, chest and belly with gray reticulate on a white background. Measurements of the reference specimen: SVL = 19.9; HL = 7.3; HW = 6.7; TBL = 11.1; THL = 9.9; FL = 16.4; TD = 1.6; ED = 2.7; END = 1.6; NSD = 1.0; IND = 2.6; IOD = 2.6. Variation: Three or four spines on thumb, not related to sex. Lateral stripe can be a white blot on inguinal region or extends to middle portion of the body. Vertebral glandular ridge may be continuous or interrupted. Upper lip spines absent in some specimens. Postrictal tubercle anastomosed in some specimens by the preservative action. Comparisons with Crossodactylus caramaschii Bastos & Pombal, 1995 and other species Morphometric comparisons (Table 1; Figures 1−5) — (N = 12 males from Atibaia, and N = 16 from Apiai and type series). Most important variables allowing differentiation between C. caramaschii and the Atibaia specimens were snout vent length, foot length, head width and eye-nostril distance (Figure 2). Both, the RandomForests model and DAPC (Figure 3) discriminated both populations. Most remarkable differences in morphology between C. caramaschii and the Atibaia specimens were observed in shape of toe tips (truncate in C. caramaschii; dilated and truncate in Atibaia the specimens), shape of inner metatarsal tubercle (more elongated in C. caramaschii than Atibaia specimens), development of subarticular tubercles on toes (more developed in Atibaia specimens than in C. caramaschii), lateral view of snout (more protrunding in C. caramaschii than Atibaia specimens), canthus rostralis (sharp in C. caramaschii and poorly marked in Atibaia specimens), lateral stripe (extensive in C. caramaschii and a little white blot on inguinal region in Atibaia specimens) and forearms 67 (thicker than arms in C. caramaschii, and in the same thickness in Atibaia specimens.). Besides, C. caramaschii presents an ornamentation in dorsal view of snout, which is absent in Atibaia specimens. Comparative morphology on head and appendixes are presented in Figures 4 and 5, respectively. All specimens from Apiai showed the diagnostic features of C. caramaschii, except for one feature: in original description of C. caramaschii (Bastos & Pombal 1995), one of the diagnostic parameters is short snout, however all specimens collected by us have protrunding snout in lateral view, as well as noted by Pimenta et al. (2014). This feature was even seen in the type series. Such confusion on morphology statements might be due to different terminologies that have been used in original description and in recent studies (see Pimenta et al. 2014). Besides, another feature seems controversial in literature: Pimenta et al. (2014) attributes immaculate belly to C. caramaschii, perhaps due to preservative state of the specimens examined by them. All specimens from Apiai (freshly preserved) present brown reticulations on belly, which is in accordance with original description. In a general comparison to other Crossodactylus species (character states in parentheses), Atibaia specimens differs from C. boulengeri, C. caramaschii, C. cyclospinus, C. franciscanus, C. grandis, C. timbuhy, C. trachystomus and C. werneri due to its head nearly as long as wide (longer than wide; wider in C. grandis). Snout nearly pentagon shaped in dorsal view distinguishes from C. aeneus, C. dispar, C. franciscanus, C. gaudichaudii, C. grandis, C. trachystomus and C. werneri (rounded; variable in C. aeneus and C. gaudichadii); and in lateral view is protruding, which is different in C. dispar, C. franciscanus, and C. grandis (rounded). Sharp canthus rostralis distinguishes from C. dispar, C. grandis and C. werneri (rounded). Tympanum is distinct in all species except for C. grandis (poorly defined). Postrictal tubercle in small granules separates Atibaia specimens from C. boulengeri, C. franciscanus, C. timbuhy, C. trachystomus and C. werneri (continuous postrictal tubercle). 68 Fingers tips are dilated in Atibaia specimens, which differs from C. dispar, C. franciscanus and C. grandis (undilated); and thumb spines are small or even absent (developed or strongly developed in C. boulengeri, C. dispar, C. franciscanus, C. grandis and C. werneri; or variable in C. trachystomus). Atibaia specimens present a glandular crest on anterior surface of its arms differing in C. aeneus, C. caramaschii, C. cyclospinus, C. gaudichaudii and C. schmidti (absent). Males present developed fringes on tarsus and toes. In females, such trait is reduced and in some other species, as in C. dispar and C. grandis, fringes are not extensively developed (moderate). Dilated and truncate toe tips is distinguished in C. aeneus, C. caramaschii, C. dantei, C. dispar, C. franciscanus, C. gaudichaudii, C. grandis, C. lutzorum and C. schmidti (dilated and rounded in C. aeneus, C. dantei, C. gaudichaudii and C. lutzorum; undilated and rounded in C. dispar, C. grandis and C. schmidti; undilated and truncate in C. caramaschii and C. franciscanus). Presence of dorsolateral glandular ridges separates Atibaia specimens from C. dantei and C. lutzorum (absent). All species present glandular ridges in posterior dorsal skin being more evident in C. timbuhy. A white blot on inguinal region distinguishes Atibaia specimens from C. boulengeri, C. caramaschii, C. dantei, C. dispar, C. franciscanus, C. grandis, C. lutzorum, C. timbuhy and C. werneri (white lateral stripe well marked in C. franciscanus; absent in C. dantei, C. dispar, C. grandis, C. lutzorum and C. timbuhy; and variable in C. boulengeri, C. caramaschii and C. werneri). Occurrence of a white or cream stripe from the snout to the shoulder with brown spots on upper surface of the mouth differs in C. boulengeri, C. caramaschii, C. dispar, C. franciscanus, C. grandis, C. timbuhy, C. trachystomus and C. werneri (white stripe without brown spots in C. caramaschii, C. cyclospinus, C. schmidti, C. trachystomus; poorly marked in C. boulengeri, C. dispar, C. franciscanus, C. grandis, C. timbuhy; and variable in C. werneri). Reticulated belly distinguishes from C. dispar, C. franciscanus, and C. grandis 69 (immaculate). Advertisement calls and acoustic comparisons (Table 2; Figures 6−9) — Acoustic data for Atibaia specimens, C. caramaschii and other comparative Crossodactylus species are in Table 2. Analyzed sound files in Appendix II. Atibaia specimens (N = 9 males recorded; 29 calls, 2280 notes analyzed): Advertisement call resembles trills. In a call-centered approach, advertisement call consisted of a train of about 80 pulsatile notes, with around three dense harmonics (Figure 6). Amplitude ascends until the middle of the call, when it stabilizes. Call duration lasted around 8s, emitted at the highest rate of two calls per minute and 12 notes per second. Note duration lasted around 30 ms; note interval around 50 ms. Call dominant frequency was about 5800 Hz, fundamental frequency around 1600 Hz. Peak time around 70% of call duration. Crossodactylus caramaschii (N = 9 males recorded; 55 calls, 2697 notes analyzed): Advertisement call resembles trills, consisted of about 50 pulsatile notes, presenting dense harmonics (Figure 7). Amplitude increases over the call reaching its maximum on last third portion, however keeping irregular. Call duration lasted around 6s, emitted at the rate around of three calls per minute and eight notes per second. Note duration around of 40 ms, and about 80 ms of silence period intercalating them. Dominant frequency was about 4700 Hz, fundamental frequency around 2400 Hz. Peak time around 80% of each call. Most important advertisement calls’ variables distinguishing the Atibaia specimens from C. caramaschii were notes per call, dominant frequency and fundamental frequency (Figure 8). Both RandonForests model and DAPC resulted in a total discrimination between these two populations (Figure 9). In a general comparison with the others described calls, advertisement call of Atibaia specimens is promptly distinguished from the calls of its congeners by having more notes per 70 call, longer call duration and higher peak frequency. Similarly to C. caramaschii, it has long note duration. Such values appear to be slightly higher than those of C. cyclospinus, C. schmidti, C. trachystomus and C. werneri. As described by Pimenta et al. (2008) and Pimenta et al. (2015) for C. trachystomus and C. franciscanus Pimenta, Caramaschi & Cruz, the advertisement call of Atibaia specimens gradually increases in amplitude, reaching maximum amplitude about midway during the call duration (Figure 6). Detailed comparisons with the call of C. timbuhy (Pimenta et al. 2015; C. cf. dispar in Weygoldt 1986) were not possible due to its poor characterization. As well as other described advertisement calls to congeners and other hylodids, Atibaia specimens presents tonal structures on its notes (well-marked harmonic components). Such feature has been related to environments with stream noise, where high-frequency signals could be a solution to minimize masking interference of the low-frequency noisy. Genetic divergence analyses — (Table 3) Sequences of the 16S rRNA mitochondrial gene of Atibaia specimens from Parque Florestal do Itapetinga had some intra-group variation (0.03%). Interspecific comparisons resulted in 2.6−2.9%, 1.8−2.4%, 3.8−4.9%, and 2.8−5.6% of divergence from C. caramaschii (Apiaí – MG019412), C. caramaschii (Itanhaém – KJ961569), C. schmidti (Misiones, Argentina – HQ290948), and C. werneri (Serra das Cabras, Campinas – KU215900-02) respectively. Conclusions Morphological and acoustic evidences showed a strong discrimination between Atibaia specimens and C. caramaschii. Presently these two populations are not connected by suitable habitats, and probably they do not represent interbreeding lineages. Despite the lack of major qualitative differences 71 in their advertisement calls (which must be due to closely evolutionary history) and the lack of information on motivation, quantitative variables used on differentiation, such as dominant frequency and notes per call, are considered very static and important on mating recognition. Insights under genetic divergence in mithocondrial 16S are inconclusive. However, Atibaia specimens appear to be co-specific to C. aff. caramaschii from many localities in São Paulo state), which seems to compose a new clade close-related to C. caramaschii (topotypes) (Montesinos, R.; pers. communication, based on molecular evidence). 4.2.5 Acknowledgements Financial support by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). A research grant to AAG by CNPq. Fellowships by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; IV). Collection permit was granted by Instituto Chico Mendes (ICMBio/SISBIO 02015.008064/02–51). 72 4.2.6 References Aljanabi, S. M. & Martinez, I. (1997) Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Research, 25, 4692–4693. http://dx.doi.org/10.1093/nar/25.22.4692 Bastos, R.P. & Pombal, J.P. 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Figure 2. Most important morphometric traits in discrimination between Atibaia specimens and C. caramaschii. *(p<0.05) Figure 3. Scatterplot of the first discriminant axis (DAPC) (above) and proximityplot (RandomForests model) on morphological traits of adult males of Atibaia specimens and C. caramaschii, showing a high discrimination. Eight principal component (95% variance) axes were in DAPC. Figure 4. Dorsal and lateral views of head of Crossodactylus caramaschii from Apiaí (AAG- UFU 5218) (A and B), São Paulo state, and Atibaia specimen (main reference specimen: AAG-UFU 5200) (C and D). Scale bar = 5 mm. Figure 5. Ventral view of the foot and dorsal and ventral views of the hand of Crossodactylus caramaschii from Apiaí (AAG-UFU 5218) (A, B and C), and Atibaia specimen (main reference specimen AAG-UFU 5200) (D, E and F). Scale bar = 5 mm. Figure 6. Top: Oscillogram of a 108-notes advertisement call of Atibaia specimen Spectrogram (middle) and corresponding oscillogram (bottom) of five median notes from the advertisement call. Sound file: Crossod_AtibaiaSP2cAAGb.wav. See appendix II for further information. Figure 7. Top: Oscillogram of a 53-notes advertisement call of C. caramaschii from Apiai, São Paulo state. Spectrogram (middle) and corresponding oscillogram (bottom) of five median notes from the advertisement call. Sound file: Crossod_caramaschiiApiaiSP1cAAGm671.wav. See appendix II for further information. Figure 8. Most important acoustical traits in discrimination between Atibaia specimens and C. caramaschii. *(p<0.05) Figure 9. Scatterplot of the first discriminant axis (DAPC) (above) and proximityplot (RandomForests model) (below) on acoustical traits of adult males of Atibaia specimens and C. caramaschii, showing total discrimination. Six principal component axes were used in DAPC. 78 4.2.8 Appendices Appendix I. Examined adult specimens of Crossodactylus. Crossodactylus aeneus: RIO DE JANEIRO: Guapimirim: ZUEC-AMP 20974, 20459; Duque de Caxias: ZUEC-AMP 17578–88. Crossodactylus boulengeri: SÃO PAULO: Capão Bonito: CFBH 35180; Paranapiacaba: MZUSP 111047, 111049, 111052–53. Crossodactylus caramaschii: SÃO PAULO: Iporanga: ZUEC-AMP 8255–58 (paratypes). Crossodactylus cyclospinus: MINAS GERAIS: Cristália: MNRJ 40220. Crossodactylus dantei: ALAGOAS: Murici: MNRJ 39443, 39445. Crossodactylus dispar: RIO DE JANEIRO: Campo de Fruticultura da Bocaina: MZUSP 109674, 109679–80, 109682, 109684–85, 109701, 109691, 109699. Crossodactylus franciscanus: MINAS GERAIS: Parque Nacional da Serra da Canastra: ZUEC-AMP 4343; São Roque de Minas (Serra da Canastra): ZUEC-AMP 8382. Crossodactylus gaudichaudii: RIO DE JANEIRO: Rio de Janeiro: ZUEC-AMP 13552–54, ZUEC-AMP 17569–77. Crossodactylus grandis: RIO DE JANEIRO: Lagoa Azul (Parque Nacional do Itatiaia): ZUEC-AMP 10; SÃO PAULO: Campos do Jordão: ZUEC-AMP 991 Crossodactylus lutzorum: BAHIA: Valença: MNRJ 4761–62. Crossodactylus schmidti: ARGENTINA: GUARANY: San Vicente: CFBH 9495–98 Crossodactylus trachystomus: MINAS GERAIS: Santana do Riacho: ZUEC-AMP 2287; Jaboticatubas: ZUEC-AMP 2531–32, ZUEC-AMP 2540–41. Crossodactylus timbuhy: ESPIRITO SANTO: Santa Teresa: MZUSP 23821, 23823, 69113, 69115, 69122–23, 69125, 69130–31. Crossodactylus werneri: BRAZIL: SÃO PAULO: Campinas: AAG-UFU 0848, AAG- 79 UFU 0981–0982, AAG-UFU 1875–1885, AAG-UFU 3912–3913, AAG-UFU 4612, ZUEC- AMP 3188, ZUEC-AMP 8922–8925; Mococa: ZUEC-AMP 8187, ZUEC-AMP 8189, ZUEC- AMP 8191–8195, ZUEC-AMP 8198; Valinhos: ZUEC-AMP 10867-69, ZUEC-AMP 10875; RIO DE JANEIRO: Itatiaia, Parque Nacional do Itatiaia (topotypes): ZUEC-AMP 7981– 7982; Itatiaia (near Parque Nacional do Itatiaia, BR 354, km 9): ZUEC-AMP 8310–8313, ZUEC-AMP 10126–10127, CFBH 9411–16. 80 Appendix II. Analyzed sound files for specimens from Parque Florestal do Itapetinga, municipality of Atibaia, and Crossodactylus caramaschii, from Apiai, both in Sao Paulo state, Brazil. For each recorded male, the date of recording, water and air temperatures, and voucher males (AAG- UFU accession numbers) are provided. Sound files: Crossod_AtibaiaSP1AAGm671 [one recording for male 1]; Crossod_AtibaiaSP2AAGm671 (one recording for male 2); Crossod_AtibaiaSP3AAGm671 (one recording for male 3); Crossod_AtibaiaSP4a-cAAGm671 [three recordings (―a-c‖) for male 4]; Crossod_AtibaiaSP5AAGm671 [one recording for male 5]; Crossod_AtibaiaSP6a-cAAGm671 [three recordings (―a-c‖) for male 6]; Crossod_AtibaiaSP7a-dAAGm671 [four recordings (―a-d‖) for male 7]; Crossod_AtibaiaSP8a-bAAGm671 [two recordings (―a-b‖) for male 8]; Crossod_AtibaiaSP9AAGm671 [one recording for male 9]; Crossod_caramaschiiApiaiSP1a-cAAGm671 [three recordings (―a-c‖) for male 1]; Crossod_caramaschiiApiaiSP2a-bAAGm671 [two recordings (―a-b‖) for male 2]; Crossod_caramaschiiApiaiSP3a-bAAGm671 [two recordings (―a-b‖) for male 3]; Crossod_caramaschiiApiaiSP4a-bAAGm671 [two recordings (―a-b‖) for male 4]; Crossod_caramaschiiApiaiSP5AAGm671 [one recording for male 5]; Crossod_caramaschiiApiaiSP6a-bAAGm671 [two recordings (―a-b‖) for male 6]; Crossod_caramaschiiApiaiSP7a- cAAGm671 [three recordings for male 7]; Crossod_caramaschiiApiaiSP8a-bAAGm671 [two recordings (―a-b‖) for male 8]; Crossod_caramaschiiApiaiSP9i-jIVm671 [three recordings (―i-l‖) for male 9]. Date Voucher male Recorded male Water (°C) Air (°C) Atibaia specimens. 81 29/Jan/05 - Male 1 (one recording) 18 19 30/Jan/05 - Male 2 (one recording) 17 18 11/Sep/14 - Male 3 (one recording) 18 23 11/Sep/14 - Male 4 (three recordings) 18 23 12/Sep/14 AAG-UFU 3914 Male 5 (one recording) 18 23 10/Oct/15 AAG-UFU 5200 Male 6 (three recordings) 18 22 31/Oct/15 - Male 7 (four recordings) 18 22 02/Feb/16 AAG-UFU 5245 Male 8 (two recordings) 20 22 02/Feb/16 - Male 9 (one recording) 20 22 Crossodactylus caramaschii 02/Dec/15 AAG-UFU 5211 Male 1 (three recordings) 19 21 02/Dec/15 Male 2 (two recordings) 19 21 02/Dec/15 AAG-UFU 5212 Male 3 (two recordings) 19 21 02/Dec/15 Male 4 (two recordings) 19 21 02/Dec/15 Male 5 (one recording) 19 21 02/Dec/15 Male 6 (two recordings) 19 21 03/Dec/15 Male 7 (three recordings) 19 22 03/Dec/15 Male 8 (two recordings) 19 22 03/Dec/15 Male 9 (three recordings) 18 20 82 4.2.9 Tables Table 1. Morphometric traits (in mm) for adult specimens from Parque Florestal do Itapetinga, municipality of Atibaia, São Paulo state; Crossodactylus caramaschii from Apiai, Sao Paulo state, and type series (only specimens deposited on ZUEC collection). Values are expressed as Mean ± SD (range). Atibaia specimens. (Parque Florestal do Crossodactylus caramaschii Crossodactylus caramaschii (type series – Eldorado, Itapetinga) (Apiaí) Iporanga and Pariquera-Açu) Males (N = 12) Females (N =3) Males (N = 8) Males (N = 8) 21.7 ± 1.7 22.2 ± 0.2 23.6 ± 0.7 24.1 ± 0.6 SVL (19.9‒23.6) (21.9‒22.5) (22.2‒24.8) (23.2‒25.1) 7.6 ± 0.4 7.3 ± 1.0 7.9 ± 0.3 8.2 ± 0.4 HL (7.1‒8.7) (6.1‒8.4) (7.3‒8.5) (7.2‒8.5) 7.1 ± 0.3 7.1 ± 0.2 7.6 ± 0.1 7.7 ± 0.2 HW (6.6‒7.6) (7.0‒7.4) (7.5‒8.0) (7.5‒8.1) 11.3 ± 0.3 11.7 ± 0.4 12.7 ± 0.4 12.5 ± 0.6 TBL (10.8‒11.8) (11.2‒12.3) (12.1‒13.3) (11.9‒13.4) 10.2 ± 0.5 10.5 ± 0.5 11.5 ± 0.6 10.9 ± 0.6 THL (9.4‒11.4) (10.1‒11.1) (10.7‒12.4) (10.1‒12.2) 16.3 ± 0.6 15.7 ± 1.3 18.7 ± 0.6 18.7 ± 0.4 FL (15.1‒17.2) (14.2‒17.4) (17.8‒20.0) (18.3‒19.3) 1.6 ± 0.2 1.5 ± 0.2 1.8 ± 0.1 1.8 ± 0.2 TD (1.3‒1.9) (1.3‒1.7) (1.6‒1.9) (1.4‒2.0) 2.6 ± 0.1 2.3 ± 0.4 2.8 ± 0.1 3.0 ± 0.2 ED (2.4‒3.1) (1.8‒2.6) (2.7‒3.0) (2.6‒3.3) 1.6 ± 0.1 1.6 ± 0.1 1.7 ± 0.2 1.9 ± 0.1 END (1.5‒1.7) (1.5‒1.7) (1.5‒1.9) (1.7‒2.1) 0.8 ± 0.1 0.9 ± 0.1 0.9 ± 0.2 1.2 ± 0.3 NSD (0.7‒1.1) (0.8‒1.1) (0.6‒1.3) (0.6‒1.5) 2.7 ± 0.1 2.63 ± 0.04 2.8 ± 0.2 2.1 ± 3.2 IND (2.5‒3.1) (2.6‒2.7) (2.4‒3.1) (2.5‒3.1) 83 2.6 ± 0.3 2.4 ± 0.1 2.7 ± 0.2 2.6 ± 0.4 IOD (2.3‒3.5) (2.3‒2.6) (2.5‒2.9) (1.9‒3.1) 84 Table 2. Acoustic traits for the advertisement call of Atibaia specimens, Crossodactylus caramaschii (data from Apiaí, São Paulo state, and original description) and those of the other six congeners with acoustic data available. Data provided as mean ± SD (range) (sample size for the variable). - = unavailable data. Brazilian state abbreviations: SP (São Paulo) Atibaia specimens C. caramaschii C. caramaschii C. werneri C. cyclospinus C. franciscanus C. gaudichaudii C. schmidti C. trachystomus N = 9 males Apiaí, SP Bastos & Pombal Vidigal et al. Nascimento et al. Pimenta et al. (2015) Weygoldt & Caldart et al. Pimenta et al. Call traits N = 9 males (1995) (submitted) (2005) Carvalho-e-Silva (2011) (2008) N = 10 males (1992) Notes/call 80.7± 19.1 48.1± 19.1 56.8± 5.4 19.9 ± 4.3 63.3 ± 11.9 48.0 ± 18.2 - 35.0 ± 6.64 58.8 ± 26.2 (53‒156) (37‒70) (49‒69) (12‒25) (35‒98) (36‒84) (25‒130) (13‒45) (13‒121) (29) (55) (6) (79) (25) (48) - (11) (86) Call duration (s) 7.7 ± 1.9 5.7 ± 0.4 5.5 ± 0.5 3.2 ± 1.2 4.3 ± 0.7 6.4 ± 2.5 - 4.1 ± 1.0 4.9 ± 1.9 (5.4‒11.7) (4.7‒6.5) (4.7‒6.0) (1.9‒6.3) (3.5‒6.2) (4.8‒11.6) (2.0‒13.0) (2.2‒5.7) (1.4‒10.1) (29) (55) (6) (79) (25) (6) - (11) (52) Calls/min 1.5 ± 0.8 3.2 ± 0.5 - 17.5 ± 10.4 - - - - - (0.7‒3.2) (2.0‒4.2) - (6.4‒41.2) - - - - - Intercall interval (s) 77.7 ±35.8 33.8 ±11.4 - 14.6 ±5.8 - - - - - (23.6‒136.7) (12.9‒56.0) - (8.3‒25.8) - - - - - Note duration (ms) 39.2 ± 5.4 41.5 ± 5.0 - 18.2 ± 5.0 28.0 ± 4.0 12.0 ± 2.6 - 22.0 ± 7.0 14.0 ± 5.0 (33.1‒49.7) (30.8‒47.1) - (11.0‒27.0) (3.0‒40.0) (1601) (6.0‒20.0) (40.0‒50.0) (9.0‒61.0) (1.0‒33.0) (2478) (2280) (2697) - (1074) (284) - (289) Internote interval 56.0 ± 9.4 76.3 ± 7.8 - 157.0 ± 43.8 40.0 ± 3.0 123.0 ± 13.4 - 100.0 ± 28.0 92.0 ± 21.0 (ms) (47.7‒76.9) (61.6‒86.5) - (128.0‒278.0) (29.0‒65.0) (1576) (90.0‒184.0) (284) (40.0‒50.0) (17.0‒249.0) (56.0‒265.0) (2250) (2638) - (1042) (279) (2392) Notes/s 10.5 ± 1.3 8.3 ± 0.6 - 6.3 ± 1.2 - - - - - (8.1‒12.1) (7.3‒9.2) - (3.2‒7.5) - - - - - Fundamental 1.7 ± 0.4 2.4 ± 0.2 ~1.6 1.7 ± 0.2 - - - 1.5 ± 0.3 - frequency (kHz) (1.2‒2.5) (2.2‒2.8) - (1.2‒2.3) (0.7‒1.2) - (2.0‒5.5) (1.0‒2.0) - (2280) (2697) - (915) (25) - - (11) - Dominant frequency 5.7 ± 0.2 4.7 ± 0.2 ~5.0 3.3 ± 0.2 4.9 ± 0.6 3.4 ± 2.8 - 3.3 ± 0.6 3.8 ± 0.5 (kHz) (5.3‒6.2) (4.4‒5.0) - (3.0‒3.6) (3.4‒5.4) (2.3‒4.0) - (2.0‒4.2) (1.8‒4.8) (29) (55) - (79) (25) (284) - (11) (1535) Peak time (%) 75.7 ± 11.2 84.3 ± 11.8 - 70.3 ± 20.0 - - - - - (51.1‒90.1) (61.7‒97.3) - (20.1‒94.4) - - - - - 85 Table 4. Uncorrected molecular distances (p-distance) for 16S rRNA gene among Crossodactylus werneri, C. caramaschii, and C. schmidti, and Atibaia specimens. Atibaia Atibaia Atibaia Atibaia C. C. caramaschii C. schmidti C. werneri C. werneri C. werneri specimen specimen specimen specimen caramaschii (MG019412) (HQ290948) (KU215900) (KU215901) (KU215902) (MG019408) (MG019409) (MG019410) (MG019411) (KJ961569) Atibaia specimen (MG019408) Atibaia specimen (MG019409) 0.03% Atibaia specimen (MG019410) 0.00% 0.03% Atibaia specimen (MG019411) 0.00% 0.03% 0.00% C. caramaschii (MG019412) 2.60% 2.90% 2.60% 2.60% C. caramaschii (KJ961569) 1.80% 2.10% 1.80% 1.80% 2.40% C. schmidti (HQ290948) 4.50% 4.90% 4.50% 4.50% 4.70% 3.80% C. werneri (KU215900) 5.20% 5.60% 5.20% 5.20% 6.00% 6.00% 6.70% C. werneri (KU215901) 2.80% 3.10% 2.80% 2.80% 3.30% 3.30% 4.00% 0.00% C. werneri (KU215902) 4.50% 4.80% 4.50% 4.50% 4.70% 4.70% 5.40% 0.00% 0.00% 86 4.2.9.1 Figures Figure 1. Main reference specimen: adult male in life from Atibaia (AAG-UFU 5200; SVL 19.97 mm). Photo: Ariovaldo A. Giaretta. 87 Figura 2. Most important morphometric traits in discrimination between Atibaia specimens and C. caramaschii. *(p<0.05) 88 Figura 3. Scatterplot of the first discriminant axis (DAPC) (above) and proximityplot (RandomForests model) on morphological traits of adult males of Atibaia specimens and C. caramaschii, showing the high discrimination. Eight principal component (95% variance) axes were in DAPC. 89 Figure 4. Dorsal and lateral views of Crossodactylus caramaschii from Apiaí (AAG-UFU 5218) (A and B), São Paulo state, and Atibaia specimen. (main reference specimen: AAG-UFU 5200) (C and D). Scale bar = 5 mm. Photo: Yuji Kodato. 90 Figure 5. Ventral view of the foot and dorsal and ventral views of the hand of Crossodactylus caramaschii from Apiaí (AAG-UFU 5218) (A, B and C), and Atibaia specimen. (main reference specimen AAG-UFU 5200) (D, E and F). Scale bar = 5 mm. Photo: Yuji Kodato. 91 Figure 6. Top: Oscillogram of a 108-notes advertisement call of Atibaia specimen. Spectrogram (middle) and corresponding oscillogram (bottom) of five median notes from the advertisement call. Sound file: Crossod_AtibaiaSP2cAAGb.wav. See appendix II for further information. 92 Figure 7. Top: Oscillogram of a 53-notes advertisement call of C. caramaschii from Apiai, São Paulo state. Spectrogram (middle) and corresponding oscillogram (bottom) of five median notes from the advertisement call. Sound file: Crossod_caramaschiiApiaiSP1cAAGm671.wav. See appendix II for further information. 93 Figure 8. Most important acoustical traits in discrimination between Atibaia specimens and C. caramaschii. *(p<0.05) 94 Figure 9. Scatterplot of the first discriminant axis (DAPC) (above) and proximityplot (RandomForests model) (below) on acoustical traits of adult males of Atibaia specimens. and C. caramaschii, showing total discrimination. Six principal component axes were used in DAPC, 95% of explained variance retained. 95 5 CONCLUSÕES GERAIS Nossos resultados corroboram a relevância do uso integrado de diferentes bases de dados para a catalogação da biodiversidade. Conseguimos com este tipo de abordagem, contribuir especificamente com o conhecimento da anurofauna brasileira, obtendo os seguintes resultados específicos: 1. Confirmação da identidade da primeira população (Crossodactylus werneri, na Serra das Cabras, município de Campinas, estado de São Paulo), com base em evidências de morfologia externa e morfometria, além de contribuir para o conhecimento do repertório acústico, larva, história natural e genética para esta população; 2. Sugerimos uma nova espécie para o gênero, de ocorrência na Serra da Mantiqueira, de perto relacionada à C. caramaschii, com base em discriminação de dados morfométricos e acústicos, além da diagnose da morfologia externa. 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