Fabiane Santana Annibale Variação Geográfica no Canto de Anúncio de Dendropsophus nanus (Anura, Hylidae) São José do Rio Preto 2015 Fabiane Santana Annibale Variação Geográfica no Canto de Anúncio de Dendropsophus nanus (Anura, Hylidae) Dissertação apresentada como parte dos requisitos para obtenção do título de Mestre em Biologia Animal, junto ao Programa de Pós-Graduação em Biologia Animal, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de São José do Rio Preto. Orientador: Prof. Dr. Fernando Rodrigues da Silva São José do Rio Preto 2015 Fabiane Santana Annibale Variação Geográfica no Canto de Anúncio de Dendropsophus nanus (Anura, Hylidae) Dissertação apresentada como parte dos requisitos para obtenção do título de Mestre em Biologia Animal, junto ao Programa de Pós-Graduação em Biologia Animal, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de São José do Rio Preto. Comissão Examinadora Prof. Dr. Fernando Rodrigues da Silva UFSCar – Campus Sorocaba Orientador Prof. Dr. Rogério Pereira Bastos UFG – Campus II Prof. Dr. Fausto Nomura UFG – Campus II São José do Rio Preto 06 de fevereiro de 2015 Dedico este trabalho a meu primo Fábio, cuja brevidade da vida nunca superará a eternidade do sorriso. AGRADECIMENTOS Entrar no mundo da bioacústica e concluir esse trabalho foi um desafio muito interessante. A oportunidade de estudar a natureza e adentrar, ainda que pouco, no universo da física pelo estudo do som, foi pra mim uma experiência incrível, pois unifica dois mundos que cresceram juntos dentro da mesma casa. Agradeço minha família pelo apoio incondicional: meus pais que me ensinaram a respeitar e admirar a vida em todas as suas diferentes formas; meu irmão, que sempre me pede um exército de sapos para combater os insetos no calorão brasileiro; minha avó, que ainda tem certo receio da herpetofauna, mas apoia minhas decisões; e meu “primirmão” Mário, que se dispõe a me ouvir falar de sapos em longas e divertidas caminhadas. Às minhas familiares, também conhecidas como amigas: Alessandra, Thalita, Silmara, Jurema, Graziela e Meggie, que estiveram ao meu lado ainda que distantes fisicamente e sem as quais minha vida seria muito menos valiosa e divertida! Ao membro da família que eu conheci correndo (literalmente) quando estava começando a etapa do mestrado, Hederson. O meu querido genético que esteve comigo nessa jornada, me ouvindo (muito), incentivando e que saiu de seu habitat, que é o laboratório, para trabalhar comigo em vários momentos! À professora Denise, com quem simpatizei desde o primeiro e-mail trocado, que me aceitou em seu laboratório de braços abertos, que me cedeu material, indicou trabalhos, aconselhou e se preocupou quando parti pra campo, que dividiu conhecimento, risadas e conversas animadas. A generosidade e a alegria fazem com que a sua bagagem de conhecimento seja ainda mais respeitada e admirada! Ao Carlos, que é o técnico que resolve os nossos problemas com a maior facilidade, o companheiro de laboratório com quem aprendemos muito, e uma pessoa que transforma simples conversas em grandes momentos! Ir pra campo nunca foi tão incrível! Trabalhar na Coleção de adultos nunca foi tão engraçado! E andar de carrinho empurrado a mão... que aventura! À Verônica pela sua paciência, que eu considero um fenômeno, porque me aguentou em todo esse processo de aprendizado! E também porque me aguentou em campo! Na verdade, aguentou o Carlos e eu juntos em campo! É, a Verônica merece um prêmio! Aliás, em campo formamos um trio de coletas com eficiência, cumplicidade, responsabilidade e claro, muitas risadas! Obrigada por ter dividido comigo seu conhecimento, parado pra raciocinar comigo, me dado liberdade para expor as ideias, me guiado à distância e ainda descontrair quando era preciso! Que as conversas intermináveis continuem por tempos intermináveis! Aos colegas de laboratório: Paquito, MaRinara, Sayuri, Alba, David, Fernanda, Geise, Katiuce, Lara, Camila, Cássia, Estela, Curita e Thiago (Albine capivara), que me ajudaram indo pra campo ou indicando contatos para coletas, que leram meu trabalho para sugerir ideias, assistiram minhas apresentações, discutiram artigos e me deram força sempre. Juntos fizemos do nosso laboratório um ambiente acolhedor, divertido e um tanto quanto sonoro. Obrigada a cada um! Aos agregados do nosso lab, alguns que nem biólogos são, e que me ajudaram quando precisei de material de estudo, ou coletar ou ainda conversar sobre o trabalho: Fagner, Juliane, Cintya, Leonardo Gedraite, André Pansonato e o professor Florindo. Aos colegas de outras universidades, sem os quais não teria conseguido fazer o trabalho em localidades fora do estado de São Paulo e que me receberam com alegria, foram atenciosos e trabalharam comigo com grande força de vontade: Priscilla Gambale, Sabine Rocha, Fernando Martins, Claudia Ferreira e “Guile”. Às Babuinis, que me acolheram temporariamente por período indeterminado, que assistiram minha apresentação (em treinamento e valendooo), que me deram a maior força e muitas alegrias. A casa de você é certamente especial! E Daya, muito obrigada pela comida maravilhosa, pelos momentos de reflexão e... vídeo show! Aos meus dois orientadores, pois ambos me guiaram e me ensinaram muito sobre bioacústica e ecologia! Obrigada, Kit, por ter aceitado o desafio de orientar à distância e se dispor a conversar comigo pela internet por todo o tempo necessário. E obrigada Fernando, ou melhor, Nandão, por topar entrar no mundo da bioacústica e acreditar no meu trabalho! Às bancas de qualificação e defesa, pelas sugestões, ideias e correções. O trabalho ficou ainda melhor com a contribuição dos professores Itamar Martins, Lilian Casatti, Fausto Nomura e Rogério Bastos! Agradeço também aos donos das propriedades rurais onde realizei meu trabalho pela compreensão, por abrirem as portas de suas casas com curiosidade e confiança, e por ajudarem sempre que possível com informações e conhecimento. Esse trabalho é fruto não apenas do meu esforço individual, mas de um conjunto de pessoas que tive o privilégio de encontrar durante a vida! A todos: muito obrigada! “It’s not easy being GREEN.” Kermit the Frog RESUMO Anuros transmitem informações para coespecíficos principalmente através de sinais acústicos, cuja função principal é a reprodução. O sistema de comunicação pode apresentar variação geográfica devido à ação de diferentes forças evolutivas. O objetivo deste estudo foi testar as hipóteses de processos neutros, temperatura do ar e tamanho corpóreo para tentar entender quais variáveis influenciam o canto de anúncio de Dendropsophus nanus, uma espécie generalista com ampla distribuição na América do Sul. Foram amostradas nove populações em áreas de Cerrado no Brasil. O canto de anúncio da espécie é composto por dois tipos de notas: tipo A, relacionadas à competição entre machos e tipo B, que podem estar relacionadas à atração de fêmeas para reprodução assim, analisamos o efeito das variáveis sobre cada nota. Distância geográfica e temperatura do ar foram as principais variáveis que explicaram a variação em ambas as notas. Também observamos diferenças entre as populações de localidades leste e oeste. Como a temperatura está estruturada no espaço, a distância geográfica pode produzir diferença de temperatura ao longo de um gradiente longitudinal, refletindo a variação observada nos parâmetros entre populações. Tamanho corporal teve explicação apenas para as notas tipo B e também que indivíduos de populações que ocorrem em localidades mais quentes apresentaram menor tamanho corporal, assim como o oposto. Sugerimos que a variação nos parâmetros acústicos pode ser resultado indireto do efeito da temperatura sobre o tamanho do corpo. Finalmente, as populações de D. nanus amostradas neste estudo apresentam variação geográfica no canto de anúncio devido a processos neutros e ecológicos. Palavras-chave: ecologia de populações, bioacústica, variação intraespecífica, processos neutros, pressões seletivas, forças evolutivas, temperatura. ABSTRACT Anurans communicate information for conspecifics mainly through acoustic signals, whose principal function is reproduction. However, communication system may present geographic variation due to different evolutionary forces. In this study we aimed to test three non-exclusive hypotheses: random drift, environment temperature and body size hypothesis to understand which variables influenced variation in advertisement call of Dendropsophus nanus, a generalist species with large distribution in South America. We sampled nine populations in Cerrado domain in Brazil. The advertisement call of this species is composed by two types of notes: type A, related to male-male competition and type B that may attract females for reproduction so, we analyzed the effect of explanatory variables on each note. Geographical distance and temperature were the main variables explaining variation on both notes. Also we observed differences in the advertisement call among populations between western and eastern localities. As temperature is spatially structured, geographic distance may produce variation in temperature along the longitudinal gradient, reflecting the observed variation in bioacoustic parameters among populations. Body size had effect in variation of type B notes and also we observed that populations occurring in warmer localities presented smaller individuals such as the opposite. We suggest that variation in acoustic parameters may be an indirect result of temperature acting on body size. Therefore we found that the advertisement call of D. nanus present geographic variation due to neutral and ecological processes. Keywords: population ecology, bioacoustics, intraspecific variation, random drift, selective pressures, evolutionary forces, temperature. 11 SUMÁRIO Introduction....................................................................................................... 12 Methodology ..................................................................................................... 15 Sampling areas .......................................................................................... 15 Study species ............................................................................................. 15 Field work ................................................................................................... 16 Bioacoustic analysis ................................................................................... 17 Statistical analysis ...................................................................................... 17 Results ............................................................................................................. 18 Discussion ........................................................................................................ 19 Conclusion........................................................................................................ 21 Tables .............................................................................................................. 23 Figures ............................................................................................................. 26 Bibliography ...................................................................................................... 31 12 INTRODUCTION Populations of species with wide distributional range relative to its dispersal capacity are subjected to different evolutionary forces which can produce greater variation among than within populations (JANG et al., 2011; LAMPERT et al., 2005; WILKINS; SEDDON; SAFRAN, 2013). In animals, geographic variation in communication systems can be important for speciation through pre-mating, and hence pre-zygotic, isolation of populations (PROHL et al., 2006) since signals play an important role in mate choice and species recognition in a broad range of taxa (WILKINS; SEDDON; SAFRAN, 2013). Variation in intraspecific communication system is a good model for studying signal evolution because it is possible to quantify differences among individuals. Furthermore, this variation, even in short evolutionary timescales, may have impact on receiver response (CAMPBELL et al., 2010). Variation in acoustic signals may arise from the action of selective and/or random evolutionary forces (CAMPBELL et al., 2010; MCLEAN; STUART-FOX, 2014; WILKINS; SEDDON; SAFRAN, 2013) among which the principals: i) ecological selection – morphological traits (i.e. body mass) are predicted to covary with the degree of acoustic differentiation among individuals (an inverse relationship between body size and advertisement call frequency has been found in several frog species; HOSKIN; JAMES; GRIGG, 2009); ii) sexual selection – the acoustic divergence is due to female preferences (studies have showed that females prefer males with greater signal complexity; e.g. BOUL et al., 2007) and iii) random drift - divergence on acoustic traits increases linearly with geographic distance (i.e. females mate with males from their natural regions; KLYMUS; HUMFELD; GERHARDT, 2012). 13 Both selective and neutral forces are important when studying geographic variation in communication system: environment may influence acoustic signals directly (BLAIR, 1958; ZWEIFEL, 1959) or indirectly (OLALLA- TÁRRAGA; RODRÍGUEZ, 2007), while random drift may produce differences among populations despite natural selection. Among selective forces, temperature can be a fundamental factor influencing variation in anuran communication. As ectothermic animals, anurans obtain heat from environment and their physiological processes, including the production of vocal signal (NAVAS, 1996), are temperature-dependent. The parameters of a call (i.e. call rate, note duration and dominant frequency; BLAIR, 1958; ZWEIFEL, 1959) may change with temperature (NARINS; MEENDERINK, 2014) as trunk muscles contraction speed increases with temperature, leading to an increase in velocity of airflow through vocal cords (MCLISTER, 2001). Considering a long period of time, temperature also plays an important role on morphology of anurans, leading to differences in animals size among populations. Body size is important when studying vocal communication system because production of sound is determined by the shape and mass of the laryngeal apparatus: an increase in size of vocal cords, cartilage and resonating box in larger males may lead to calls with lower frequencies (see DUELLMAN; TRUEB, 1986; WELLS, 2007). On the other hand, random drift may also be important to explain intraspecific variation: when populations are isolated by geographic distance (or barriers), gene flow among them decreases, so more distant populations will accumulate more variation than closer populations (BERNAL; GUARNIZO; LÜDDECKE, 2005; HUTCHISON; TEMPLETON; URL, 1999; JANG et al., 2011; LIN et al., 2014). Therefore, geographic variation may be 14 better explained by the balance of neutral and selective forces (ENDLER, 1977; JANG et al., 2011). Anurans are able to communicate different information to other individuals, accounting for a repertoire of calls (TOLEDO et al., 2014). Nevertheless, the advertisement call is the most common vocalization and is related to male-male competition and to attract females for reproduction. This type of call is simple and highly stereotyped when compared to bird songs or mammals communication (CAMPBELL et al., 2010; GERHARDT, 1994) what makes it a good object to test the role of ecological selection and random drift in the processes of acoustic divergence. Also, it is recognized that advertisement call is influenced by ambient temperature (BLAIR, 1958; ZWEIFEL, 1959) and anuran body size (BEE; GERHARDT, 2001; COCROFT; RYAN, 1995; RYAN; PERRILL; WILCZYNSKI, 1991; SULLIVAN; MALMOS; GIVEN, 1996). In this study, we aimed to test three non-exclusive hypotheses to understand the variables influencing the variation in advertisement call of Dendropsophus nanus (Boulenger, 1889) from nine populations distributed in central and south areas in Brazil: i) random drift hypothesis – we predicted that advertisement call of closer populations would be more similar than more distant populations; ii) ambient temperature hypothesis - we predicted that advertisement call would be more similar among populations in localities with similar air temperature and more different among populations with more differences in air temperature and iii) body size hypothesis - we predicted that body size represents a potential driver of acoustic divergence with populations exhibiting a negative relationship between body size and the advertisement call. Thus, we expect that our results may contribute to comprehend the evolutionary 15 forces influencing intraspecific variation in communication system of an anuran species with wide geographical distribution. METHODOLOGY Sampling Areas The study was conducted in nine ponds located in four Brazilian states: São Paulo, Goiás, Mato Grosso do Sul and Mato Grosso (Fig. 1, Table 1). We selected ponds localized in open areas of farmlands, with vegetal formation of Cerrado and/or Mesophitic Semidecidual Forest (Atlantic Forest Domain), where climate type is similar, with two well-marked seasons: a hot and rainy summer, and a dry and cold winter [35]. Study species Dendropsophus nanus is an anuran species from Hylidae family. This species has a wide distribution along South America (FROST, 2014) being abundant in open areas of tropical forest formation, and can be found even in areas disturbed by human activities (LANGONE; BASSO, 1987; REICHLE et al., 2004). The species breeds in permanent or temporary ponds during the rainy season (MENIN, 2002; VASCONCELOS; ROSSA-FERES, 2005). Males aggregate and emit the advertisement call (Fig. 2) to conspecifics which was characterized by Martins & Jim (2003) as pulsed notes emitted in series and compound by two types of notes: the introductory notes (A type) and secondary notes (B type). Type A notes are longer in duration with lower repetition rates and higher pulse number. Males emit these notes most in the beginning of their vocalization activity or when they are distant from the aggregation. Also, this 16 type of note inform other males about the emissary position and competitive ability so they are able to establish territories avoiding fights (SOUSA, 2012). Type B notes are shorter in duration, have higher repetition rates and lower pulse number. These notes were hypothesized to be used by males to attract females for reproduction (I. Martins, pers. comm.). Field work Field work was carried out from November 2013 to March 2014 at night (around 8:00 p.m. to 12:00 a.m.), when individuals were active. We recorded calls of about 9 individuals in each pond (Ntotal = 82 males). Recordings started after establishment of the species aggregation in the pond: males started the calling activity emitting A notes at sunset, while choosing a calling site, and about 01h30min hour later, when males’ abundance and calling sites were practically established, they started emit mainly the advertisement call (F.S.A., pers. obs.). Recording of calls were obtained through standardized methodology: a semi-directional microphone (Seinnheiser ME66/K3U) was positioned 01m from the focal animal and 01m from the substrate (land/water), always using the same volume entry (27 dB) in a digital recorder (Ediroll R- 09HR). After each recording, the focal male was captured and kept in a plastic bag to avoid recapture. We considered the local population as the set of individuals breeding in a determined pond. Males were measured (snout-vent length, SVL) with a digital caliper. They were all released at the end of measurements. Abundance of D. nanus was estimated in the end of all procedures descripted, by active search on 17 perimeter of the water body, counting males in calling activity by hearing and non-calling males when sighted (HUNTER; KREBS, 1979). Bioacoustic analysis Recordings were sampled at a rate of 44.1 kHz and 24 bit resolution in mono pattern, saved in uncompressed wave files. Bioacoustic analyses were performed in Raven Software Pro 1.4 (CORNELL BIOACOUSTICS RESEARCH PROGRAM, 2008) with the following configuration: window Hann 1024 sampled size, DFT size 1024 samples; 3 dB filter, bandwidth 248 Hz; time grid overlap 50%. We analyzed 05 A notes and 05 B notes randomly per individual. Acoustic parameters were analyzed to each note (A and B) – fundamental frequency (FF), note duration (ND), note repetition rate (NRR) and sound pressure level (SPL): as SPL was not measured on field, we back- calculated SPL values by using RMS (root-mean-square) amplitude values, obtained through Raven software, by the equation y = 33.698e0.1129x (where x represents SPL values and y the RMS amplitude values). We used averaged values (Tables 2 and 3) of the call parameters within and between individuals to minimize intraspecific variation (BOSCH; DE LA RIVA, 2004). Statistical analysis In order to reduce data dimensionality and multicollinearity of acoustic parameters, we performed a principal component analysis (PCA) based on a correlation matrix of the data considering FF, SPL, ND and NRR separately for A and B notes. PCA axes were used as response variables in subsequent analyses. We performed a variation partitioning test to determine the relative 18 importance of geographic distance (distance-based Moran’s Eigenvector Maps - dbMEMs; (DRAY et al., 2012; LEGENDRE; LEGENDRE, 2012), body size (mean snout-vent length [SVL] of D. nanus in each pond) and ambient temperature (annual mean temperature of each locality – obtained from WorldClim database (HIJMANS et al., 2005)) on variation of acoustic parameters (BORCARD; LEGENDRE; DRAPEAU, 1992). Simple linear regression was used to verify the relationship among explanatory (geographic distance, SVL and ambient temperature) and response variables (first axes of PCA). Statistical analyses were performed in R software 3.1 (R DEVELOPMENT CORE TEAM, 2014). RESULTS We used the first axes of PCA in variation portioning test and linear regressions as they explained 50% and 55% of the variation in acoustic parameters for A and B notes respectively. The results of the variation partitioning test showed that the variance in acoustic parameters of A notes can be explained mostly by the effect of the temperature on this note (Fig. 3). For B notes, temperature was important to explain variation in acoustic parameters, however the combination of all factors (geographical distance, environmental temperature and SVL) explained a relatively large proportion of the variation in acoustic parameters on this type of note (Fig.3). For notes type A, we found a positive relationship only between acoustic parameters and ambient temperature (F1,7 = 23.88, p = 0.001, Fig. 4). On the other hand, for notes type B, acoustic parameters showed: i) a positive relationship with geographical distance (F1,7 = 19.61, p < 0.01, Fig. 5); ii) a 19 positive relationship with ambient temperature (F1,7 = 98.67, p < 0.001, Fig. 5); and iii) a negative relationship with SVL (F1,7 = 8.02, p = 0.02, Fig. 5). Furthermore it is possible to observe that differences in advertisement call are more evident when considering longitude than latitude directions: there are more differences between western (Mato Grosso and Mato Grosso do Sul) and eastern localities (São Paulo and Goiás) (Fig. 5). DISCUSSION Our results indicate that random drift, ambient temperature and body size hypotheses seem to be synergistic forces driving variation of acoustic parameters of the advertisement call in Dendropsophus nanus. Although, it is recognized that geographic variation of advertisement call can be influenced by both attraction of females and male-male interactions, the influence of notes related to mate choice seem to be more relevant promoting intraspecific variation in advertisement call of D. nanus. Similar results were found to other hylid frogs with advertisement calls composed by two types of notes (for instance, Dendropsophus ebraccatus and Dendropsophus microcephalus; SCHWARTZ, 1986; WELLS; SCHWARTZ, 1984). As proposed by Wright (1943), for some species, the increase in distance separating populations results in more interpopulation variation because they get separated by longer genetic and phenotypic distances. Differences among populations may arise due to neutral processes as shown for greenish warblers (IRWIN; THIMGAN; IRWIN, 2008), singing mice (CAMPBELL et al., 2010) and frogs (AMÉZQUITA et al., 2009; BERNAL; GUARNIZO; LÜDDECKE, 2005) and also may reflect historical processes which can decrease migration rates and 20 even lead to isolation of populations. Our results show that differences in advertisement call increase with geographical distance and are more evident when considering longitude than latitude directions. Furthermore, through variation partitioning, we observed that when geographical distance is considered with temperature, the percentage of explanation is higher. Because temperature distribution is spatially structured (i.e. sites near to each other show have more similar values of temperature than sites distant of each other) and the relationship between temperature and acoustic parameters showed a positive correlation, we argue that the variation in temperature with geographical distance, along a longitudinal gradient, produces variation in bioacoustic parameters among western and eastern populations. Besides geographic distance and ambient temperature, we found that body size also contributes to the variation of notes related to attraction of female in a negative correlation (Fig.5). It is recognized that temperature is related to physiology and body size of anurans (DUELLMAN; TRUEB, 1986; OLALLA- TÁRRAGA; RODRÍGUEZ, 2007; OLALLA-TÁRRAGA et al., 2009). According to the hypothesis of heat balance (OLALLA-TÁRRAGA; RODRÍGUEZ, 2007), populations in cold areas would present individuals with larger body sizes than warm areas. This pattern usually explains evolutionary responses to minimize heat loss in cold climates. In this study, we observed that populations occurring in warmer localities presented individuals with the smallest SVL measure while populations occurring in cold ones presented the highest SVL (Table 1). Thus, we suggest that variation in acoustic parameters may be an indirect result of temperature acting on body size. 21 Although, we have not tested sexual selection, our results indicate that variation in acoustic signals in D. nanus is a good model to test whether females prefer local male signals to foreign ones because we found more variation in type B notes, which probably are related to reproduction. Selective forces as sexual selection may act on individuals from a determined population leading to differences in vocal signals which are preferred by females so, signals must not only be optimized to transmit well in environment avoiding sound degradation (BONCORAGLIO; SAINO, 2007; ERDTMANN; LIMA, 2013; RYAN, 1990; WILCZYNSKI; RYAN, 1999) but also must contain information interesting to females from a specific population (PANHUIS et al., 2001; PROHL et al., 2006). For example, Boul et al. (2007) showed that female preferences for calls between neighboring populations have promoted divergence in male mating calls of Physalaemus petersi. CONCLUSION Our results showed that neutral and selective forces are non-exclusive to explain variation in advertisement call among populations of Dendropsophus nanus. This was expected since frog calls are subjected to strong constraining and/or diversifying pressures (AMÉZQUITA et al., 2011; HANSEN; ORZACK, 2005; WILCZYNSKI; RYAN, 1999). In this study we also observed that notes related to reproduction presented more variation in acoustic parameters, reflecting plasticity in this type of note. Plasticity in behavior is of a huge importance for animals to adapt to changes and to novel conditions (FISCHER et al., 2004; FORSMAN, 2014), playing an important role in species evolutionary processes. Therefore, studying geographic variation in 22 communication system of species with low dispersal ability, as anurans, and the evolutionary forces that may cause intraspecific variation contributes to understand the wide distribution of generalist species. 23 TABLES Table 1: Data of each locality studied: geographical coordinates (WGS84), annual mean temperature (AMT) and, mean and standard deviation of males’ body size (snout-vent length, SVL). State Locality Latitude Longitude AMT (°C) SVL (mm) São Paulo Macaubal -20.74 -49.93 22.53 20.64 ± 2.4 Nova Itapirema -21.07 -49.54 22.52 19.64 ± 1.0 Nova Granada -20.41 -49.27 23.39 20.31 ± 0.6 Goiás Bonfinópolis -16.57 -48.95 22 21.76 ± 1.3 Silvânia -16.67 -48.84 22.48 20.75 ± 0.7 Goiânia -16.52 -49.27 23.18 20.87 ± 0.9 Mato Grosso do Sul Piraputanga -20.46 -55.52 24.04 18.20 ± 0.9 Cachoeirão -20.43 -55.28 24.43 18.06 ± 0.6 Mato Grosso Cuiabá -15.61 -56.06 26.05 19.13 ± 1.1 24 Table 2: Mean and standard deviation of bioacoustic parameters of notes type A for each sampled population. State Locality FF (Hz) ND (s) SPL (dB) NRR (notes/s) São Paulo Macaubal 4386.61 ± 46.69 0.04 ± 0.004 88.03 ± 5.5 0.82 ± 0.3 Nova Itapirema 4212.09 ± 38.62 0.04 ± 0.006 77.78 ± 2.2 0.93 ± 0.8 Nova Granada 4382.43 ± 42.10 0.04 ± 0.005 78.90 ± 4.6 0.75 ± 0.3 Goiás Bonfinópolis 4250.69 ± 65.02 0.05 ± 0.004 65.16 ± 19.8 0.87 ± 0.2 Silvânia 4346.90 ± 41.47 0.03 ± 0.006 81.59 ± 3.0 0.79 ± 0.2 Goiânia 4310.07 ± 34.33 0.04 ± 0.004 81.98 ± 1.6 0.79 ± 0.4 Mato Grosso do Sul Piraputanga 4502.16 ± 35.75 0.04 ± 0.005 82.22 ± 2.3 1.18 ± 0.4 Cachoeirão 4671.84 ± 86.18 0.03 ± 0.005 79.62 ± 4.3 1.0 ± 0.4 Mato Grosso Cuiabá 4747.63 ± 65.16 0.03 ± 0.007 38.74 ± 3.2 0.68 ± 0.2 25 Table 3: Mean and standard deviation of bioacoustic parameters of notes type B for each sampled population. State Locality FF (Hz) ND (s) SPL (dB) NRR (notes/s) São Paulo Macaubal 4380.46 ± 24.23 0.02 ± 0.003 90.20 ± 5.6 5.46 ± 2.5 Nova Itapirema 4187.29 ± 65.95 0.02 ± 0.003 79.64 ± 2.4 4.66 ± 2.4 Nova Granada 4387.60 ± 44.88 0.02 ± 0.005 79.31 ± 5.0 6.29 ± 0.9 Goiás Bonfinópolis 4236.52 ± 73.89 0.03 ± 0.004 67.41 ± 20.1 4.19 ± 0.9 Silvânia 4327.69 ± 40.04 0.02 ± 0.003 83.19 ± 3.3 4.90 ± 0.6 Goiânia 4317.83 ± 35.42 0.02 ± 0.001 82.78 ± 1.9 5.94 ± 0.6 Mato Grosso do Sul Piraputanga 4501.29 ± 42.51 0.02 ± 0.002 84.85 ± 2.5 5.93 ± 2.9 Cachoeirão 4660.64 ± 70.67 0.02 ± 0.003 81.71 ± 4.8 5.64 ± 2.1 Mato Grosso Cuiabá 4747.63 ± 65.16 0.01 ± 0.002 41.42 ± 4.0 5.85 ± 3.2 26 FIGURES Figure 1. Distribution of nine populations of Dendropsophus nanus sampled in this study. 27 A B Figure 2. Waveform (A) and spectrogram (B) representing the two types of notes in the advertisement call of Dendropsophus nanus: A note in the left and B note in the right. Spectrogram parameters: Window type Hann, window size 1024 samples, 3 dB filter bandwidth - 248 Hz, DFT size 1024 samples. 28 Figure 3. Deviance partitioning analysis representing the deviance in acoustic parameters of notes type A and B of D. nanus. Light-dark gradient of the figure represents the low-high deviance explained by the predictors. 29 Figure 4. Relationship between acoustic parameters (first axis of the principal component analysis - PCA) of notes type A of the advertisement call of Dendropsophus nanus and: geographical distance (distance-based Moran’s Eigenvector Maps), environment temperature (annual mean temperature of each locality) and body size (mean of snout-vent length of all individuals recorded in each pond). Symbols: circles represent localities in São Paulo; triangles, Goiás; squares, Mato Grosso do Sul; and lozenge, Mato Grosso. Notes type A 30 Figure 5. Relationship between acoustic parameters (first axis of the principal component analysis - PCA) of notes type B of the advertisement call of Dendropsophus nanus and: geographical distance (distance-based Moran’s Eigenvector Maps), environment temperature (annual mean temperature of each locality) and body size (mean of snout-vent length of all individuals recorded in each pond). Symbols: circles represent localities in São Paulo; triangles, Goiás; squares, Mato Grosso do Sul; and lozenge, Mato Grosso. Notes type B 31 BIBLIOGRAFY AMÉZQUITA, A. et al. Calls, colours, shape, and genes: a multi-trait approach to the study of geographic variation in the Amazonian frog Allobates femoralis. Biological Journal of the Linnean Society, v. 98, n. 4, p. 826–838, 23 nov. 2009. AMÉZQUITA, A. et al. Acoustic interference and recognition space within a complex assemblage of dendrobatid frogs. PNAS, v. 108, n. 41, p. 17058– 17063, 2011. BEE, M. A.; GERHARDT, H. C. 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