Historical Distribution of Batrachochytrium Dendrobatidis in Brazil”

UNIVERSIDADE ESTADUAL DE CAMPINAS

Instituto de Biologia

Tamilie Carvalho

“Distribuição histórica de Batrachochytrium dendrobatidis no Brasil”

“Historical distribution of Batrachochytrium dendrobatidis in Brazil”

Campinas 2016 Tamilie Carvalho

“Distribuição histórica de Batrachochytrium dendrobatidis no Brasil”

“Historical distribution of Batrachochytrium dendrobatidis in Brazil”

Dissertação apresentada ao Instituto de Biologia da Universidade Esta- dual de Campinas como parte dos requisitos exigidos para a obtenção do título de Mestra em Ecologia

Dissertation presented to the Instituto de Biologia of the Univer- sity of Campinas in partial fulfill- ment of the requirements for the Master degree in Ecology

Orientador: PROF. DR. LUÍS FELIPE DE TOLEDO RAMOS PEREIRA

ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELA ALUNA TAMILIE CARVALHO E ORIENTADA PELO PROF. DR. LUÍS FELIPE DE TOLEDO RAMOS PEREIRA.

Campinas 2016 Agência(s) de fomento e nº(s) de processo(s): CAPES; FAPESP, 2014/23388-7

Ficha catalográfica Universidade Estadual de Campinas Biblioteca do Instituto de Biologia Mara Janaina de Oliveira - CRB 8/6972

Carvalho, Tamilie, 1989- C253d CarDistribuição histórica de Batrachochytrium dendrobatidis no Brasil / Tamilie Carvalho. – Campinas, SP : [s.n.], 2016.

CarOrientador: Luís Felipe de Toledo Ramos Pereira. CarDissertação (mestrado) – Universidade Estadual de Campinas, Instituto de Biologia.

Car1. Batrachochytrium dendrobatidis. 2. Declínio de anfíbios. 3. Epidemiologia. 4. Relação hospedeiro-parasito. I. Toledo, Luís Felipe,1979-. II. Universidade Estadual de Campinas. Instituto de Biologia. III. Título.

Informações para Biblioteca Digital

Título em outro idioma: Historical distribution of Batrachochytrium dendrobatidis in Brazil Palavras-chave em inglês: Batrachochytrium dendrobatidis Amphibian declines Epidemiology Host-parasite relationships Área de concentração: Ecologia Titulação: Mestra em Ecologia Banca examinadora: Luís Felipe de Toledo Ramos Pereira [Orientador] Fernando Rodrigues da Silva Delio Pontes Baeta da Costa Data de defesa: 18-07-2016 Programa de Pós-Graduação: Ecologia

Campinas, 18 de Julho de 2016 Comissão Examinadora

Prof. Dr. Luís Felipe de Toledo Ramos Pereira

Prof. Dr. Fernando Rodrigues da Silva

Prof. Dr. Delio Pontes Baeta da Costa

Profa. Dra. Luciana Bolsoni Lourenço

Prof. Dr. André Victor Lucci Freitas

Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se en- contra no processo de vida acadêmica do aluno.

Este trabalho é dedicado a minha família, pelo incentivo, apoio, ensinamentos e amor a mim dedicados.

Agradecimentos

Ao meu orientador Prof. Dr. Luís Felipe Toledo, pelo qual tenho grande admira-

ção como pessoa e como profissional, por aceitar me orientar, por toda a ajuda, conversas e amizade, sem o qual este trabalho não aconteceria.

Ao C. Guilherme Becker, pela imensa ajuda neste trabalho, atenção e ensinamentos.

A toda família do LaHNAB, fonoteca e Unicamp, com um agradecimento especial aos meus queridos Carolina Lambertini, Carlos Henrique LN Almeida, Luiz Fer- nando M de Lima, Joice Ruggeri Gomes, Camila Z Torres, Anat Belasen, Pedro Peres,

Pedro Joaquim Bergamo, Verônica Bernardino, Meghi Nogueira de Souza, Leandro

Tacioli e Javier Ibarra Isassi, por todas as conversas, companheirismo e carinho, que me ajudam a seguir em frente.

Às minhas grandes amigas Lígia Dominiquini, Lívia Tibério, Fernanda Oliveira de Souza, Flávia Gimenez de Fávari, Nayara Vanti e ao Thiago de Oliveira Pires. Não conseguiria expressar todos os motivos de agradecimentos, gratidão por existirem na minha vida, vocês são uma das melhores partes de mim.

A Natalia Bergamo de Sá, uma grande companheira que em anos nunca mediu esforços em me ajudar, dedicando seu carinho, apoio e atenção a mim e a minha família.

Espero poder retribuir ao menos um pouco o bem que você nos faz.

A minha família, Silvana C Carvalho Rossi, Sandro Rossi, Aparecida LR Carva- lho, Thiago D Carvalho, Agnaldo J Carvalho e Manoel R Carvalho, por me darem a vida, amor, carinho, compreensão e educação. Vocês são meu norte, minha base e meu orgulho. Amores de sempre e para sempre.

Ao Programa de Pós Graduação em Ecologia da Unicamp.

Ao Fundo de Apoio ao Ensino, à Pesquisa e Extensão e a Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior – PROEX, pelo financiamento deste trabalho através da concessão de bolsas de estudos.

A Deus.

Gratidão

Resumo

Os anfíbios são o grupo de vertebrados mais ameaçados em todo o mundo. As principais ameaças ao grupo são a perda de habitat e a quitridiomicose, uma doença infecciosa emergente causada pelo fungo Batrachochytrium dendrobatidis (Bd). O fungo se distribui por todos os continentes do mundo levando populações de anfíbios ao declínio ou extinção, e no Brasil sua distribuição é pouco compreendida. Muitos declí- nios foram registrados no sudeste do Brasil, principalmente nas décadas de 1970 e 1980, e as causas para esses declínios permanecem pouco esclarecidas. Em nosso trabalho analisamos aparatos bucais de mais de 30 mil girinos de todo o Brasil preservados em museus. Com isso, fomos capazes de identificar a distribuição do Bd em todo o territó- rio nacional e correlacionar positivamente o aumento da prevalência da quitridiomicose com os declínios registrados na Mata Atlântica.

Palavras Chave: Batrachochytrium dendrobatidis; declínios de anfíbios; epidemiologia espacial; dinâmica patógeno-hospedeiro

Abstract

Amphibians are the most threatened group of vertebrates in the world. The main threats to the group are habitat loss and chytridiomycosis, an emerging infectious disease caused by the fungus Batrachochytrium (Bd). The fungus is spread over all conti- nents of the world leading amphibian populations to decline or extinction, and in Brazil its distribution is poorly understood. Many declines were recorded in southeastern Bra- zil, mainly in the 70’s and 80’s, and the causes for these declines remain unclear. In our work we analyze buccal apparatus of more than 30 thousand tadpoles from all over Bra- zil preserved in museums. Thus, we were able to identify the distribution of Bd throughout the country and positively correlate the increased prevalence of chytrid with the declines recorded in the Atlantic Forest.

Keywords: Batrachochytrium dendrobatidis; amphibian declines; spatial epidemiology; host-pathogen dynamics

Sumário

Introdução Geral ...... 11

Capítulo I: Historical Amphibian Declines in Brazil Linked to Chytridiomycosis .. 14

Introduction ...... 16

Methods ...... 18

Results ...... 22

Discussion ...... 23

Considerações Finais ...... 48

Referências ...... 49

Introdução Geral

As causas do declínio ou extinção de diversas espécies animais ainda permanecem desconhecidas (Stuart et al. 2004). Isto é particularmente importante no caso dos anfíbios, que é o grupo de vertebrados mais ameaçado (Stuart et al. 2004; Hoffmann et al. 2010; Monastersky 2014), sendo atualmente pelo menos 228 espécies desaparecidas

(Moore & Lewis 2012), e há previsão de que mais 7% dos anfíbios sejam extintos no próximo século (Alroy 2015). Sem dúvidas, a principal causa dos declínios dos anfíbios são as alterações da paisagem (correspondendo a cerca de 42%), mas 48% dos declínios ocorreram por causas enigmáticas (Stuart et al. 2004). Assim, diversas hipóteses foram levantadas sobre as causas dos declínios e extinções dos anfíbios no mundo, como mu- danças no uso do solo, mudanças climáticas, contaminantes ambientais, introdução de espécies invasoras, uso comercial de animais e doenças infecciosas emergentes (EDIs)

(Young et al. 2001; Collins et al. 2003; Young et al. 2005; Becker et al. 2007; Collins et al. 2010; Alroy 2015), podendo essas agirem sozinhas ou em sinergia (Hayes et al.

2010; Hof et al. 2011; Altizer et al. 2013). Entender as causas é fundamental pra cons- trução de planos de ação para conservação (Silvano & Segalla 2005; Verdade et al.

2012), já que os anfíbios são apontados como bioindicadores de qualidade ambiental, apresentam fundamental papel em redes tróficas, controle de pragas e são fonte de fár- macos (Oza 1990; Camargo 2005, Pukala et al. 2006; Raghavendra et al. 2008; Toledo

2009).

Se no mundo a conservação dos anfíbios é necessária, no Brasil, a situação é a- inda mais alarmante, por este ser o país detentor da maior riqueza de anfíbios do mundo

(Segalla et al. 2014), correspondendo à cerca de 14% da riqueza global (Frost 2016).

Dentre os biomas brasileiros, a Mata Atlântica se destaca por ser um dos hotspots de biodiversidade do mundo (Myers 2003), em especial de anfíbios (Haddad et al. 2013), 12 contendo 92% das espécies nacionais ameaçadas (DOU 2014) e quase todos os registros de declínios populacionais ou extinções do país (Heyer et al. 1988; Weygoldt 1989;

Eterovick et al. 2005). Tais declínios no sudeste do Brasil foram atribuídas a razões climáticas, como a poluição e severas geadas (Heyer et al. 1988), fogo e perda de habitat (Papp & Papp 2000; Heyer et al. 1988), ou mesmo EIDs foram apontados como uma causa potencial no Espírito Santo (Weygoldt 1989). Além de concentrados na Mata A- tlântica, grande parte dos declínios relatados no Brasil se enquadra temporalmente dis- tribuídos entre 1978 e 1988 e até o momento as causas destes declínios não foram reve- ladas com clareza.

Dentre as causas enigmáticas (Stuart 2004), a quitridiomicose tem sido revelada como a principal agente (Alroy 2015). Por exemplo, o quitrídio (= Bd) causou declínio ou extinção de diversas espécies em Serra Nevada, Califórnia (Vredenburg et al. 2010), no leste da Austrália (Berger 1998), na América Central (Cheng et al. 2011; Lips et al.

2006, 2008), e nos Andes da América do Sul (Catenazzi et al. 2011). Entretanto, a pre- sença do Bd não necessariamente implica em declínio de anfíbios (Toledo et al. 2006;

Rodriguez et al. 2014). Para relacionar o declínio dos anfíbios com o Bd, são necessá- rios estudos de monitoramento (Lips et al. 2008; Martel et al. 2014) ou estudos históri- cos com dados de coleções científicas (e.g., Rodriguez et al. 2014; Talley et al. 2015;

Becker et al. 2016). Os dois trabalhos históricos desenvolvidos no Brasil mostram que o fungo pode ser encontrado em espécimes muito antigos, desde 1894 na Mata Atlântica

(Rodriguez et al. 2014) e 1935 na Amazônia (Becker et al. 2016). Um dos trabalhos tentou correlacionar a presença do Bd com declínios de anfíbios na porção sul da Mata

Atlântica (Rodriguez et al. 2014). Neste, utilizaram ca. 2.800 amostras de adultos, mas não encontrou correlação entre o fungo (sua presença e intensidade de infecção) e o declínio dos anfíbios. Desta forma, pretendemos testar a mesma hipótese (de que o fun- 13 go estaria ligado aos declínios de anfíbios no Brasil) utilizando um banco de dados mui- to maior, mais de 30 mil amostras de todo o Brasil, sendo ca. de 15 mil na Mata Atlânti- ca, e exclusivamente de girinos. Com estes dados fomos capazes de correlacionar a in- fecção com os declínios no Brasil, tanto espacialmente quanto temporalmente.

Historical Amphibian Declines in Brazil

Linked to Chytridiomycosis

Tamilie Carvalho1, C. Guilherme Becker2, Luís Felipe Toledo1,3

1 Laboratório de História Natural de Anfíbios Brasileiros (LaHNAB), Departamento de

Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas,

São Paulo, 13083-862, Brazil

2 Departamento de Zoologia, Universidade Estadual Paulista, Rio Claro, São Paulo,

13506-900, Brazil

3 Author for correspondence: Luís Felipe Toledo: [email protected]

Abstract

The enigmatic amphibian declines and extinctions in Brazil are a matter of continued debate. Most declines occurred in pristine environments and disproportionately affected stream-breeding species, which led to the hypothesis that Brazil experienced one of the first outbreaks of chytridiomycosis in recent history. Here, we quantified depigmentation in tadpole mouthparts as a proxy for chytridiomycosis and tested for an association between disease and the observed enigmatic declines after the late 1970s. We examined over 30,000 tadpole-preserved specimens collected across six Brazilian biomes between

1930 and 2015 and used spatial statistics to detect clusters of disease. Both niche model- ing and spatial autoregressions underscored that to both chytridiomycosis and tadpole mouthpart depigmentations are linked to the same macroenvironmental variables. We detected a significant increase in the proportion of diseased tadpoles during the onset of the reported amphibian declines. Furthermore, we revealed a significant spatiotemporal aggregation of diseased tadpoles in the Atlantic Forest coinciding with 21 out of 25 sites of declines. The long-term presence of Bd in Brazil without conspicuous amphibian die- offs, combined with a spatiotemporal increase in prevalence in areas of declines, highlight that host-pathogen dynamics in the Atlantic Forest might fundamentally differ from those observed in Central America and Australia.

Keywords: Batrachochytrium dendrobatidis; spatial epidemiology; host-pathogen dynamics; disease distribution; spatiotemporal analysis.

Introduction

For over thirty years herpetologists attempted to understand the enigmatic amphibian declines and extinctions in seemingly pristine forests of Brazil. Dozens of amphibian species experienced drastic reductions in population sizes, with a number of species going locally extinct in the Atlantic Forest during the early 1980s (Heyer et al. 1989,

Weygoldt 1989, Eterovick et al. 2005). Amphibians declined in dozens of sites, but the extinctions reported in the protected areas of Boraceia, Serra dos Orgãos, Itatiaia, and

Santa Teresa are among the most widely known in the scientific community. Specifical- ly, population declines were reported for over 13 species at Estação Biológica de

Boraceia, state of Sao Paulo, after 1979 (Heyer et al. 1989). Declines were also observed during the same period of time at Parque Nacional da Serra dos Orgãos and

Parque Nacional do Itatiaia (Brazil’s first national park), both in the state of Rio de

Janeiro (Heyer et al. 1988; Weygoldt 1989; Guix et al. 1998). Peter Weygoldt not only reported amphibian declines at Reserva Ecológica Santa Lúcia, state of Espírito Santo, after 1981 (Weygoldt 1989), but also speculated about a potential disease-causing agent.

What these declines have in common is that they all occurred in pristine environments, within the same timeframe (between 1978 and 1988), and disproportionately affecting species with aquatic larvae.

Chytridiomycosis, a disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has been linked to declines of hundreds of amphibian species in pristine tropical forests (IUCN et al. 2016). Dozens of amphibians from the genus

Atelopus declined or disappeared along streams after an epidemic of chytridiomycosis swept through Central America and the tropical Andes (Lips et al. 2008). Similarly, several frogs with aquatic larval development went extinct in the wild in eastern Aus- tralia, with die-offs also attributed to the emergence of Bd in natural environments (Ber- 17 ger 1998; Hero & Gillespie 2003; Schloegel et al. 2006). The similarities among the enigmatic amphibian declines observed in Brazil more than three decades ago and the more recent declines in other tropical regions led to the hypothesis that Brazil experienced one of the first outbreaks of chytridiomycosis in recent history.

Retrospective surveys of museum preserved specimens have been widely used to describe historical Bd dynamics in several regions (Weldon et al. 2004, Ouellet et al.

2005, Soto-Azat et al. 2010, Cheng et al. 2011, Vredenburg et al. 2013, Rodriguez et al.

2014, Courtois et al. 2015, Talley et al. 2015, Becker et al. 2016). Rodriguez et al.

(2014) recently reported the long-term presence of Bd in museum preserved specimens collected across the Atlantic Forest. In that study, Bd prevalence of adult frogs remained consistently low during the last 100 years, with no signs of increased prevalence during the years of reported declines. Becker et al. (2016) also failed to detect an increase in Bd prevalence in adult frogs from both the Amazon and the Brazilian Cerrado after the late

1970s. Therefore, none of these studies corroborated the hypothesis of Bd as one of the causal agents of local amphibian declines and extinctions in Brazil.

Thus far, retrospective studies focused on adult frogs, despite the fact that tadpoles are the life stage most exposed to waterborne Bd (Longcore 1999). Bd attacks keratinized tissue, which in tadpoles is concentrated in the mouthparts (Berger et al.

1998). Bd infection in tadpoles consequently causes depigmentation in both jaw sheath and tooth rows (Lips 1999; Fellers et al. 2001; Rachowicz & Vredenburg 2004; Knapp

& Morgan 2006; Garner et al. 2009; Vieira et al. 2013). Although depigmentation may also result from exposure to environmental contaminants (Boyle et al. 2004) or to very low temperatures (Rachowicz 2002), the depigmentation pattern due to Bd infection is unequivocal; Bd causes patchy depigmentation with complete loss of keratin in local- ized areas compared to fully keratinized surrounding areas (Rachowicz & Vredenburg 18

2004). Recent studies found an overwhelming proportion of tadpoles with highly depigmented mouthparts attributed to Bd; infection prevalence was estimated at 95% in

Atlantic Forest torrent frog Hylodes cf. ornatus and in American bullfrogs Lithobates catesbeianus (Vieira et al. 2013), 100% in Rana muscosa (Knapp & Morgan 2006), and

96% in several amphibian species from Australia (Obendorf & Dalton 2006). In addi- tion, more than 100 Bd strains were isolated from tadpoles with depigmented mouthparts across Brazil’s Atlantic Forest (Jenkinson et al. 2016). Therefore, patterns of mouthpart depigmentation can be effectively used as a proxy for Bd infections in Brazil.

Here, we quantified depigmentation in tadpole mouthparts to test for a potential association between disease and the historical declines in Brazil. We examined over

30,000 museum specimens collected in Brazil between 1930 and 2015 and quantified the proportion of affected tadpoles across space and time. We used a combination of

SATSCAN spatiotemporal cluster analysis and autologistic regressions to both detect spatiotemporal aggregations of disease and test the impact of macro-environmental variables on diseased tadpoles. Our results shed new light on the potential role of chytridiomycosis as the leading cause of amphibian declines observed in Brazil after the late 1970s.

Methods

Sampling

We analyzed 32,545 tadpoles from 5,597 lots collected between the years of 1930 and

2015. The number of specimens contained in each collection lot ranged from 1 to 60.

We obtained data from 13 families across 923 localities spanning the six Brazilian biomes. The collections and museums from which samples were collected included: Co- leção de Anuros Célio F. B. Haddad, Universidade Estadual Paulista, Rio Claro (CF- 19

BH); Coleção Científica de Anfíbios do Departamento de Zoologia e Botânica, Univer- sidade Estadual Paulista, São José do Rio Preto (DZSJRP); Coleção de Anfíbios e Rép- teis, Instituto Nacional de Pesquisa da Amazônia, Manaus (INPA); Museu Nacional,

Rio de Janeiro (MNRJ); Museu Paraense Emílio Goeldi, Belém (MPEG); Museu de

Zoologia, Universidade de São Paulo, São Paulo (MZUSP); Museu de Ciência e Tecno- logia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre (MCP);

Coleção Zoológica de Vertebrados, Universidade Federal de Mato Grosso, Cuiabá

(CZV-UFMT); Coleção Científica do Laboratório de Herpetologia, Universidade Fede- ral de Santa Maria, Santa Maria (ZUFSM), and Museu de Zoologia “Prof. Adão José

Cardoso”, Universidade Estadual de Campinas, Campinas (ZUEC). We obtained precise geographic coordinates of collection locality for the majority of analyzed specimens. For those specimens without precise locality information, we extracted the municipality’s geographic centroid using Geonames (http://www.geonames.org/).

Disease assessment

We categorized tadpole mouthparts as normal or abnormal. We performed visual inspections on the buccal apparatus of specimens using a stereoscopic microscope

(Lambertini et al. 2013). All visualizations were performed by the same person (T.

Carvalho) for standardization purposes. We considered specimens normal if they exhibited fully pigmented tooth rows and jaw sheaths. We considered specimens to be abnormal if they exhibited patterns of full or partial depigmentation of the buccal apparatus compatible with depigmentation due to Bd infection (Rachowicz & Vredenburg

2004; Obendorf & Dalton 2006; Vieira et al. 2013; Valencia-Aguilar et al. 2016). In case of uncertainties during visual inspection of tadpole mouthpart, we performed histo- logical sections of the buccal apparatus as described by Lambertini et al. (2013). We did 20 not include specimens that exhibited jaw sheaths or tooth rows with slight depigmentation or with tooth rows that were easily detached after gentle manipulation. We did not use qPCR detection methods due to low detectability of Bd in formalin preserved specimens such as tadpoles (Adams et al. 2015); adult frogs are usually preserved in EtOH.

We included in the analyses specimens at Gosner stages ranging from 25 to 40 (Gosner

1960).

Proportion of tadpoles with depigmented mouthparts

Brazil harbors six biomes: Amazon, Cerrado, Pantanal, Caatinga, Atlantic Forest, and

Pampas (IBGE 2015). Sample size in each biome ranged from 178 to 19,174 specimens

(mean = 5,425.16 ± 7,044.2 sd). We calculated the proportion of tadpoles with depigmented mouthparts for each biome (± 95% binomial confidence intervals).

The majority of the sampled tadpoles were collected in the Atlantic Forest, thus we performed a finer-scale analysis for this biome. We calculated the proportion of tadpoles with depigmented mouthparts (± 95% of binomial confidence intervals) for five of the 12 Atlantic Forest ecoregions according to Olson et al. (2001) (i.e., Bahia Interior

Forests, Araucaria Moist Forests, Serra do Mar Coastal Forests, Alto Paraná Atlantic

Forests, and Bahia Coastal Forests). We excluded Pernambuco Coastal Forests (n = 7 tadpoles) and Pernambuco Interior Forests (n = 5 tadpoles) due to reduced sample sizes.

Southern Atlantic Mangroves and Atlantic Coast Restingas were grouped to their re- spective adjacent ecoregion, as they have a relative small area (10,025 and 7,850 km2, respectively). We did not obtained records from Northeastern Brazil Restingas and

Caatinga Enclaves Moist Forests. Atlantic Dry Forests was grouped to the Cerrado biome, thus, not considered for this analysis. The sample size in each Atlantic Forest 21 ecoregion ranged from 358 to 7,348 tadpoles, totalizing 15,981 specimens (mean =

3.200 ± 2.800 sd).

Differences in the total number of specimens for the Atlantic forest when com- paring biomes (19,174) and ecoregions (15,981) reflects different biome limits in these two approaches.

Environmental Analyses

We used autologistic regression models (Rangel et al. 2010) to test for the effects of the following environmental variables on tadpole mouthpart depigmentation: human footprint, vegetation density, precipitation, temperature, topographic complexity, and elevation. We extracted data on 11 temperature variables (bio1-bio11) and 8 precipitation variables (bio12-bio19) from worldclim/bioclim (Hijmans et al. 2005). These metrics were calculated based on a dense network of climatic stations throughout the world (i.e., precipitation data from 47,554 locations and temperature data from 24,542 locations).

We also extracted data on human footprint (Sanderson et al. 2002), topographic complexity (Jarvis et al. 2009), elevation, and vegetation density (USGS & FAO 2000) for each sampling location using Arc Map v. 10.1 (Esri 2012). All rasters were generated at a scale of 1 km. We used principal component analysis (PCA) to consolidate climatic variables owing to their high cross-correlation, and used the scores of the first principal component axes (PC) depicting temperature and precipitation as explanatory variables in downstream model selection. We classified models according to the Akaike Infor- mation Criterion (AIC) and reported the most parsimonious model. We conducted model selection analyses both for our country-wide data and for the subset of samples within the Atlantic Forest. To account for potential misidentification issues during mouthpart inspections, we also conducted independent analyses where we randomly selected 10 % 22 of tadpoles with depigmented mouthparts and treated them as normal tadpoles. The influence of taxonomy was not considered, as depigmentation proportion increased with the number individuals in each sampled family (linear regression statistics F(11,12) =

79.00, r2 = 0.88, P < 0.001; see also Valencia-Aguilar et al. 2015, Figure S1 and Table

S1).

Spatiotemporal scan analysis

We performed a spatiotemporal analysis to detect clusters of diseased tadpoles across the Atlantic Forest using SATSCAN v. 9.1.1 (Kulldorff 2009). We preformed space- time scan analyses by applying Kulldorff’s clustering algorithm (Kulldorff 1997) under a Bernoulli probability model. For the space-time analysis, we set the maximum temporal cluster size at 50 % of the population and time aggregation at 21 years (see Becker et al. 2016). This method places a variable circular window on each collection locality, and then the algorithm evaluates positive and negative counts in a space-time cluster.

The clusters are compared to the entire landscape using the maximum-likelihood ratio statistic to infer statistical significance for the most likely clusters.

We categorized time into four intervals (1931-1952, 1953-1973, 1974-1994, and

1995-2015) and calculated the proportion of tadpoles with depigmented mouthparts in our entire database.

Results

We found that Brazil’s Atlantic Forest had the highest proportion of tadpoles with depigmented mouthparts when compared to Cerrado, Pampas, Caatinga, Amazon and

2 Pantanal (χ = 2469.65; n = 32545; P < 0.0001; Figure 1). Within Atlantic Forest ecoregions, Bahia Interior Forest and Araucaria Moist Forest showed the highest pro- 23

2 portion of affected tadpoles (χ = 247.549; n = 15710; P < 0.0001; Figure 2). We also detected a significant increase in the proportion of diseased tadpoles after the mid-1970s

2 (χ = 816.482; n = 15048; P < 0.0001; Figure 3A). This temporal aggregation included a large cluster of tadpoles with altered mouthparts in the southeastern Atlantic Forest between the years of 1974 and 2015 and four additional recent and small clusters in southern Brazil (Figure 4; Table 1).

Our autologistic regression that best explained the variation in tadpole mouthpart depigmentation included the following explanatory variables: vegetation, topographic complexity, annual precipitation, and human footprint, all positively associated, and annual temperature, negatively associated with mouthpart depigmentation (Table 2). For the Atlantic Forest, the best model included vegetation density, elevation and human footprint (P < 0.001; Table 3), all of which were positively associated with mouthpart depigmentation. Our results remained unaltered after including a margin of error of 10% for inaccurately detecting depigmentation when analyzing data from both the Atlantic

Forest (Tables S2) and the entire country (Table S3).

Discussion

Our research is the first to provide strong evidence that chytridiomycosis caused the historical amphibian declines observed in Brazil. The significant increase in the proportion of tadpoles with depigmented mouthparts after the mid-1970s,combined with the large spatial aggregation of affected tadpoles coinciding with areas of historical declines, support the hypothesis that Bd played an important role in amphibian declines across the Atlantic Forest. The most significant spatiotemporal cluster of affected tadpoles included 21 out of the 25 areas with reported amphibian declines after the late

1970s (Figure 4; Table S4). We did not detect any spatial aggregation of tadpoles with 24 depigmented mouthparts in areas with no reports of amphibian declines, such as northeastern Brazil (which includes the two Bahia ecoregions). The additional four narrow clusters detected in the southern range of the Atlantic Forest coincided with areas of extensive bullfrog farming (Both et al. 2011). This observed spatiotemporal overlap provides additional evidence that invasive bullfrog populations could maintain and/or spread Bd in southern Brazil as suggested in previous studies (Schloegel et al. 2009;

Rödder et al. 2013; Jenkinson et al. 2016). Our data point to strong spatiotemporal association between the historical amphibian declines in Brazil and the 19-fold increase in tadpole mouthpart depigmentation, likely caused by Bd.

The strength of our results is supported by a combination of environmental niche models and spatial regressions. We detected higher proportions of tadpoles with depigmented mouthparts in areas where previous environmental niche models estimated high likelihood of Bd persistence, such as the Atlantic Forest (Ron 2005; Rödder et al.

2009; Hof et al. 2011; Liu et al. 2013; James et al. 2015; Becker et al. 2016). Further- more, our interpretation is also strongly supported by our spatial regression models.

Specifically, we found mouthpart depigmentation to be positively associated with elevation, vegetation density, rainfall, human footprint, and negatively associated with temperature across Brazil. These results are in agreement with previous studies that found comparable effects of the same macro-environmental variables and Bd (Liu et al. 2013;

Becker & Zamudio 2011, Becker et al. 2016). Our results provide further evidence that

Bd is the cause of mouthpart depigmentation, which is spatiotemporally linked to the historical amphibians population declines in Brazil.

There are at least three hypotheses to explain the significant increase in tadpole mouthpart depigmentation after late 1970s, including: i) the arrival of a novel strain of the Bd-GPL clade (the most virulent clade associated with declines on different conti- 25 nents: Berger et al. 1998; Lips et al. 2008; Vredenburg et al. 2010; James et al. 2015); ii) a sudden increase in virulence of a local strain, through genetic muta- tion/recombination (Fisher et al. 2009; Phillips & Puschendorf 2013; Rosenblum et al.

2013) or phenotypic adaptations (Lambertini et al. 2016); and iii) a synergetic effect of multiple impacts (Hayes et al. 2010; Hof et al. 2011; Altizer et al. 2013), such as climate change, invasive species, pollution, which contribute to an increase in disease in- cidence following the long-term presence of mainly enzootic Bd. These three hypotheses are not mutually exclusive; further studies are needed to investigate mechanisms responsible for the observed shift in disease dynamics. However, we believe that the arrival of a novel strain of the Bd-GPL clade or an increase in virulence of a local strain unlikely caused the observed historical declines, because Bd usually takes years to spread through relatively short distances (Lips et al. 2008). It is possible that environmental change, coupled with a long-term presence of Bd, is responsible for the amphibian declines observed in the Atlantic Forest (see Pounds 2006).

Contrary to our findings, previous work on the historical distribution of Bd in

Brazil failed to correlate amphibian declines with the presence of the pathogen (Rodri- guez et al. 2014; Becker et al. 2016). Our study differs from previous research in that we focused on tadpoles while other historical studies focused on post-metamorphic amphibians (Rodriguez et al. 2014; Talley et al. 2015; Becker et al. 2016). All anuran life stages can be infected with Bd, and although tadpoles rarely die (Rachowicz &

Vredenburg 2004; Garner et al. 2009) recently metamorphosed froglets are the most affected life stage (Garner et al. 2009). Thus, many tadpoles may not survive up to the adult stage, and therefore, studies focused solely on adult frogs may miss important host-pathogen dynamics and may not detect the disease-causing agent. Bd infection in tadpoles could lead to decreases in population fitness, as infected tadpoles forage less, 26 grow less, and have reduced size after metamorphosis (Garner et al. 2009; Venesky et al. 2010; DeMarchi et al. 2015). Decrease in tadpole fitness may thus cause downstream effects on population persistence, leading to slow pace (and silent) population declines.

We conducted a comprehensive analysis of Brazilian amphibians and detected mouthpart depigmentation in tadpoles in 5 out of 6 biomes, covering almost the entire geographic extent of the country. Furthermore, we showed that disease is not uniformly distributed across biomes or within the same biome. Our results coincided in large part with our predictions and previous models of environmental suitability for Bd (Becker et al. 2016; Jenkinson et al. 2016). However, we found unexpectedly high proportion of diseased tadpoles in Bahia Interior Forests, a region with low risk of chytridiomycosis according to niche models (Liu et al. 2013; Becker et al. 2016). This result might be explained by sampling bias, with more tadpoles collected in areas of high elevation and optimal climatic conditions for Bd, such as the national parks of Serra do Caparaó, state of Espírito Santo, and Serra do Cipó, state of Minas Gerais.

Our study presents novel historical information about the global emergence of

Bd. Our data provide overwhelming evidence for one of the earliest reports of amphibian declines linked to chytridiomycosis. Recent studies focused on Bd diversification point to Brazil as a potential point of origin (Rosenblum et al. 2013; James et al. 2015).

Therefore, further studies focused on Bd strain diversification and spatial turnover in areas of observed extinctions such as Boraceia, Serra dos Orgãos, and Santa Teresa

(Table S4) could help elucidate the origins of Bd virulent strains and understand the roots of the Bd pandemic. Our data highlight that Bd infection in tadpoles may indeed cause population declines, even without the conspicuous die-offs that are often observed in adult amphibians in areas of epidemic chytridiomycosis such as Central America, the 27 tropical Andes, and eastern Australia (Berger 1998; Lips et al. 2008). Understanding historical outbreaks of chytridiomycosis may guide efforts to prevent future die-offs and extinctions.

Acknowledgements

Célio F. B. Haddad, Denise de Cerqueira Rossa-Feres, Richard Vogt, José Pombal Jr.,

Ana Prudente, Taran Grant, Gláucia Maria Funk Pontes, Felipe Franco Curcio, and

Sonia Cechin allowed access to museum specimens. Leandro Tacioli and David Rodri- guez helped with data handling. Carlos H. L. Nunes-de-Almeida with figure editing.

Carolina Lambertini with museum sampling. Sergio Potsch de Carvalho e Silva, Itamar

Alves Martins, and João Luiz R. Gasparini provided data on declining populations from the states of Rio de Janeiro, São Paulo and Espírito Santo, respectively. Fernanda O. de

Souza and Anat M. Belasen reviewed earlier versions of the manuscript. Our study was funded by grants and fellowships from Fundo de Apoio ao Ensino, à Pesquisa e

Extensão (FAEPEX: #1105/13 to L.F.T), São Paulo Research Foundation (FAPESP

#2014/23388-7 to L.F.T), Coordination for the Improvement of Higher Education Per- sonnel (PROEX-CAPES to T.C.), and National Council for Scientific and Technologi- cal Development (CNPq #302589/2013-9; #405285/2013-2; #312895/2014-3 to L.F.T and C.G.B).

Table 1. Spatiotemporal clusters of diseased tadpoles obtained with 21-year temporal

aggregations. Abbreviations as follows: temporal aggregation (AG), cluster radius in

km (Radius), number of locations (NL), log-likelihood ratio (LLR), observed number of

positive samples (O), expected positive cases (E) and relative risk (RR).

Cluster AG Latitude Longitude Radius Period NL LLR P value O E O/E RR

1 21 23.895 S 46.425 W 368.69 1974-2015 6207 336.5 < 0.001 1630 1028.09 1.59 2.52

2 21 29.242 S 50.449 W - 1974-2015 772 167.9 < 0.001 338 127.87 2.64 2.88

3 21 27.243 S 53.953 W - 1995-2015 30 29.3 < 0.001 24 4.97 4.83 4.86

4 21 26.564 S 52.556 W 33.44 1995-2015 13 23.4 < 0.001 13 2.15 6.04 6.06

5 21 27.737 S 51.442 W 44.63 1995-2015 14 13.0 < 0.001 11 2.32 4.74 4.76

Table 2. Best model found for proportion of tadpoles with depigmented mouthparts in

Brazil. Variables with significant effect are highlighted in bold.

Variable Coefficient Std Error Std Error T P Value

Constant -2.02 0 0.334 -6.048 <0.001 yW 4.853 2.912 0.205 23.717 <0.001

Vegetation Density 0.003 0.361 0.001 2.461 0.014

Complexity Topographic <.001 0.381 <.001 3.763 <0.001

Annual Precipitation <.001 0.202 <.001 1.544 0.123

Annual Temperature -0.009 -0.983 0.001 -6.795 <0.001

Human Footprint 0.012 0.721 0.002 4.844 <0.001

* yW stands for spatial autocovariate term; Whole-model test: χ2: 1189.85; n: 5597; P <.0001

Table 3. Best model found for proportion of tadpoles with depigmented mouthparts in

the Atlantic Forest. Variables with significant effect are highlighted in bold.

Variable Coefficient Std Coeff. Std Error T P Value

Constant -3.108 0 0.207 -14.983 <0.001

yW 4.238 2.337 0.244 17.377 <0.001

Vegetation Density 0.003 0.266 0.002 1.796 0.072

Elevation <.001 0.49 <.001 3.617 <0.001

Footprint 0.006 0.313 0.003 2.065 0.039

* yW stands for spatial autocovariate term; Whole-model test: χ2: 494.164; n: 2802; P: < .0001

Figures

Figure 1. Geographical distribution of specimens with depigmented mouthparts (orange dots) and specimens with pigmented mouthparts (black dots) collected between 1930 and 2015 (A). Map of Brazil colored according to the proportion of affected tadpoles in each biome, indicating higher proportion with darker colors (B). Depigmentation prevalence in each Brazilian biome with binomial confidence interval of 95 % (C).

Figure 2. Map of Atlantic Forest colored according to the proportion of affected tadpoles in each ecoregion; higher disease shown in darker shades (A). Depigmentation prevalence in each ecoregion of the Atlantic Forest with binomial confidence interval of

95 % (B).

Figure 3. Recorded population declines (grey bars) and proportion of diseased tadpoles

(solid orange line) in each time interval (matching the SATSCAN intervals) with binomial confidence interval of 95 % (A). Maps with temporal divisions of Atlantic Forest, presenting the geographical distribution of specimens with depigmented mouthparts

(orange dots) and healthy specimens (black dots), collected between 1931 and 2015 (B).

Figure 4. Significant clusters found in the Atlantic Forest and sites of declines of amphibians. Decline sites are: Morretes, Antonina, and São João da Graciosa (1); Botucatu

(2); Queimada Grande Island (3); Santos, Campo Grande da Serra, Paranapiacaba,

Mongaguá and old road between São Paulo and Santos (4); Boracéia (5); São Sebastião

(6); Campos do Jordão (7); Itatiaia, São José do Barreiro, Brejo da Lapa, and Itamonte

(8); Itaguaí and Engenheiro Paulo de Frontin (9); Teresópolis, Serra dos Orgãos, and

Rio de Janeiro (10); Santa Teresa (11); Serra do Cipó (12); Lauro Müller (13); and 32

Cambará do Sul (14). We did not plot two sites in the map: municipality of Pacoti, state of Ceará, as it is outside of the Atlantic Forest, and municipality of Linhares, state of

Espírito Santo as the cause of decline was known to be fire (i.e., not related to our study).

Figure 1

Figure 2

Figure 3

Figure 4

Table S1. Museum specimens grouped by taxonomic family, sample size, number of Bd+, and prevalence with binomial confidence interval of 95 % for each group.

Family Bd+ / Sample size Prevalence / CI 95 % Alsodidae 0 / 120 0.000 (0.000 – 0.030) Bufonidae 8 / 877 0.009 (0.004 – 0.018) Centrolenidae 0 / 179 0.000 (0.000 – 0.020) Ceratophryidae 0 / 10 0.000 (0.000 – 0.308) Cycloramphidae 1 / 317 0.003 (0.000 – 0.017) Dendrobatidae 2 / 445 0.004 (0.001 – 0.016) Hemiphractidae 4 / 52 0.076 (0.021 – 0.185) Hylidae 2563 / 17387 0.147 (0.142 – 0.152) Hylodidae 643 / 2023 0.318 (0.289 – 0.339) Leptodactylidae 46 / 5896 0.008 (0.006 – 0.010) Odontophrynidae 36 / 1045 0.034 (0.024 – 0.047) Phyllomedusidae 27 / 928 0.029 (0.019 – 0.042) Ranidae 11 / 201 0.055 (0.028 – 0.096) 38

Table S2. Best models found for Bd occurrence in Brazil.

Data source Models AIC Delta Without error Vegetation, Complexity Topographic, Annual Precipitation, Annual Temperature, Footprint 3466.395 0.0000 Vegetation, Complexity Topographic, Annual Temperature, Elevation, Footprint 3466.396 0.0007 Vegetation, Complexity Topographic, Annual Precipitation, Annual Temperature, Elevation, Footprint 3466.61 0.2150 Vegetation, Complexity Topographic, Annual Temperature, Footprint 3466.77 0.3746 Error 10% Vegetation, Complexity Topographic, Annual Precipitation, Annual Temperature, Footprint 3440.616 0.0000 Vegetation, Complexity Topographic, Annual Temperature, Footprint 3441.191 0.5746 Vegetation, Complexity Topographic, Annual Precipitation, Annual Temperature, Elevation, Footprint 3441.398 0.7825 Vegetation, Complexity Topographic, Annual Temperature, Elevation, Footprint 3441.433 0.8168

Table S3. Best models found for Bd occurrence in the Atlantic Forest.

Data source Models AIC Delta Without error Vegetation, Elevation, Footprint 2434.2299 0.0000 Vegetation, Annual Precipitation, Elevation, Footprint 2434.6140 0.3841 Annual Precipitation, Elevation, Footprint 2435.0951 0.8652 Elevation 2435.3077 1.0778 Elevation, Footprint 2435.4654 1.2355 Annual Precipitation, Elevation 2436.1528 1.9229 Vegetation, Complexity Topographic, Elevation, Footprint 2436.2262 1.9963 Vegetation, Annual Temperature, Elevation, Footprint 2436.2286 1.9987 Error 10% Vegetation, Elevation, Footprint 2399.678 0.0000 Vegetation, Annual Precipitation, Elevation, Footprint 2400.089 0.4104 Annual Precipitation, Elevation, Footprint 2400.866 1.1879 Elevation 2401.042 1.3643 Elevation, Footprint 2401.232 1.5543 Vegetation, Annual Temperature, Elevation, Footprint 2401.659 1.9804 Vegetation, Complexity Topographic, Elevation, Footprint 2401.678 1.9995

Table S4. Species, site (number in parenthesis stands for the number of the site in figure 4), probable period of population decline or extinction (some data refers to the last specimen collected and deposited in museums; though we did not have access to all collected specimens), suggested causes of threat, and references.

Family / Species Site, State Date Decline / Ex- Suggested Cause Reference tinction Aromobatidae Allobates capixaba Santa Teresa, ES (11) 1988 Decline Pollution, climatic changes Weygoldt 1989 and epidemic diseases Allobates olfersioides Engenheiro Paulo de Frontin, 1987 Extinct Unknown Izecksohn & Carvalho e RJ (9) Silva 2001; DOU 2014; data based on the museum record MNRJ 40580 Bufonidae Melanophryniscus moreirae Itatiaia, RJ (8) 1975-1994 Decline Ultraviolet radiation and Guix et al. 1998 infectious diseases Brachycephalidae Ischnocnema oea Santa Teresa, ES (11) 1942-1984 Decline Unknown JL Gasparini, person. comm.; Heyer 1984 Ischnocnema paranaensis Antonina, PR (1) 1988-1999 Extinct Unknown Eterovick et al. 2005; Bornschein et al. 2015 Ischnocnema parva Boraceia, SP (5) 1979-1983 Decline Pollution and heavy frost Heyer et al. 1988 Ischnocnema sp. (aff. guentheri) Boraceia, SP (5) 1979-1983 Decline Pollution and heavy frost Heyer et al. 1988 Centrolenidae Vitreorana eurygnatha Santa Teresa, ES (11) 1981-1987 Extinct Pollution, climatic changes Weygoldt 1989 and epidemic diseases Vitreorana eurygnatha Boraceia, SP (5) 1979-1982 Decline Pollution and heavy frost Heyer et al. 1988 41

Vitreorana uranoscopa Rio de Janeiro, RJ (10) 1951 Decline Unknown Izecksohn & Carvalho e Silva 2001; between 1951 (MNRJ) and 2011 (MNRJ) no individuals have been collected. Craugastoridae Holoaden bradei Itamonte, MG (8) 1978 Extinct Habitat degradation and Stuart et al. 2008; DOU heavy frost 2014; data based on the museum record ZUEC 8138-45 Cycloramphidae Cycloramphus boraceiensis Boraceia, SP (5) 1979-1982 Extinct Pollution and heavy frost Heyer et al. 1988 Cycloramphus duseni Morretes, PR (1) 1982 Extinct Pollution and heavy frost Eterovick et al. 2005 Cycloramphus eleutherodactylus Rio de Janeiro, RJ (10) 1949 Extinct Unknown Izecksohn & Carvalho e Silva 2001; Last specimens collected in 1949: MNRJ 60503 Cycloramphus fuliginosus Santa Teresa, ES (11) 1981-1987 Extinct Pollution, climatic changes Weygoldt 1989 and epidemic diseases Cycloramphus fuliginosus Rio de Janeiro, RJ (10) 1985 Extinct Unknown Izecksohn & Carvalho e Silva 2001; Last specimens collected in 1985: MNRJ 76112-13 Cycloramphus granulosus Itatiaia, RJ (8) 1979-1982 Extinct Pollution and heavy frost Heyer et al. 1988 Cycloramphus mirandaribeiroi São João da Graciosa, PR (1) 1978-1986 Extinct Unknown Eterovick et al. 2005 Cycloramphus ohausi Serra dos Órgãos, RJ (10) 1942 Extinct Unknown DOU 2014; Last specimens 42

collected in 1942: MNRJ 78796-97, MNRJ 78800-01 Cycloramphus semipalmatus Boraceia, SP (5) 1979-1982 Extinct Pollution and heavy frost Heyer et al. 1988 Cycloramphus stejnegeri Serra dos Órgãos, RJ (10) 1979 Extinct Unknown Garcia et al. 2010; DOU 2014; Last specimens collected in 1979: MNRJ 57052 Cycloramphus valae Lauro Müller, SC (13) 1982 Extinct Unknown Heyer 1983 Cycloramphus valae Cambará do Sul, RS (14) 1976 Extinct Unknown Garcia & Vinciprova 1998 Thoropa taophora Boraceia, SP (5) 1979-1982 Extinct Pollution and heavy frost Heyer et al. 1988 Thoropa petropolitana Serra dos Órgãos, RJ (10) 1982 Extinct Pollution and heavy frost Heyer et al. 1988; DOU 2014; MNRJ 61403-4 Thoropa lutzii Serra dos Órgãos, RJ (10) 1951 Extinct Unknown Izecksohn & Carvalho e Silva 2001; MNRJ 23484-5 Dendrobatidae Ameerega flavopicta Serra do Cipó, MG (12) 1974-1996 Decline Fire, human settlement and Eterovick et al. 2005 habitat fragmentation Eleutherodactylidae Adelophryne baturitensis* Pacoti, CE 1993-1994 Extinct Unknown Eterovick et al. 2005 Hemiphractidae Fritziana ohausi Boraceia, SP (5) 1979-1982 Decline/ Ex- Lack of breeding site, due to Heyer et al. 1988 tinct Bamboo reproductive cycle Hylidae Aplastodiscus flumineus Serra dos Órgãos, RJ (10) 2003 Decline Unknown SP Carvalho e Silva, persn. comm.; UNIRIO 1589; 1736 43

Aplastodiscus musicus Serra dos Órgãos, RJ (10) 1986 Extinct Unknown SP Carvalho e Silva, persn. comm. Bokermannohyla circumdata Rio de Janeiro, RJ (10) n/a Decline Unknown Izecksohn & Carvalho e Silva 2001 Bokermannohyla clepsydra São José do Barreiro, SP (8) 1968 Extinct Unknown Garcia et al. 2010; DOU 2014; Last specimens collected in: 1968: ZUEC 15937. Bokermannohyla izecksohni Botucatu, SP (2) 1993 Extinct Habitat destruction, pollu- Machado et al. 2005; Last tion specimens collected in 1993: MNRJ 61401, MNRJ 79116 Bokermannohyla langei Morretes, PR (1) 1946-1986 Extinct Unknown Eterovick et al. 2005 Dendropsophus ruschii Santa Teresa, ES (11) 1982-1987 Decline Pollution, climatic changes Weygoldt 1989 and epidemic diseases Hypsiboas cymbalum Campo Grande da Serra, SP 1963*** Extinct Habitat loss and Stuart et al. 2008; Garcia et (4) chytridiomycosis al. 2010; DOU 2014 Hypsiboas prasinus Boraceia, SP (5) 1965 Extinct Competition with H. Heyer et al. 1988 albopunctatus Ololygon cf. perpusillus Boraceia, SP (5) 1979-1982 Decline Pollution and heavy frost Heyer et al. 1988 Ololygon peixotoi Queimada Grande Island, SP 2007*** Decline/ Ex- Unknown DOU 2014 (3) tinct Ololygon heyeri Santa Teresa, ES (11) 1982-1987 Decline Pollution, climatic changes Weygoldt 1989 and epidemic diseases

Phyllodytes luteolus** Linhares, ES 1993-1995 Extinct Fire Eterovick (1999); Papp & Papp (2000) 44

Xenohyla truncata Itaguaí, RJ (9) 1967 Decline Unknown Stuart et al. 2008; DOU 2014; Last specimens collected in 1967: MNRJ 74019-20 Hylodidae Crossodactylus dispar Boraceia, SP (5) 1979-1982 Extinct Pollution and heavy frost Heyer et al. 1988; Last specimens collected in 1977: USNM 318225, 318227, 318230 Crossodactylus cf. gaudichaudii Boraceia, SP (5) 1979-1994 Extinct Progressive aridity of the Heyer et al. (1988), climate Bertoluci & Heyer (1995) Crossodactylus cf. gaudichaudii Santa Teresa, ES (11) 1982-1988 Extinct Pollution, climatic changes Weygoldt 1989 and epidemic diseases Crossodactylus timbuhy Santa Teresa, ES (11) 1982-1988 Decline Pollution, climatic changes Weygoldt 1989 and epidemic diseases Crossodactylus trachystomus Serra do Cipó, MG (12) 1974-1996 Decline Habitat reduction and frag- Eterovick et al. 2005 mentation Crossodactylus werneri Itatiaia, RJ (8) 1978 Extinct Unknown Pimenta et al. 2014 Hylodes asper Boraceia, SP (5) 1979-1982 Extinct Pollution and heavy frost Heyer et al. 1988 Hylodes babax Santa Teresa, ES (11) 1982-1987 Decline Pollution, climatic changes Weygoldt 1989 and epidemic diseases Hylodes babax Santa Teresa, ES (11) 1988 Extinct Pollution, climatic changes Weygoldt 1989 and epidemic diseases Hylodes glaber Itatiaia, RJ (8) 1979-1984 Extinct Pollution and heavy frost Heyer et al. 1988 Hylodes lateristrigatus Santa Teresa, ES (11) 1982-1988 Extinct Pollution, climatic changes Weygoldt 1989 and epidemic diseases 45

Hylodes mertensi Old road between São Paulo 1956 Extinct Unknown Bokermann 1956 and Santos, SP (4) Hylodes phyllodes Boraceia, SP (5) 1979-1982 Decline Pollution and heavy frost Heyer et al. 1988 Megaelosia bocainensis São José do Barreiro, SP (8) 1968 *** Extinct Unknown Giaretta et al. 1993; Holotype: MNRJ 15900. Leptodactylidae Adenomera marmorata Boraceia, SP (5) 1979-1983 Decline Pollution and heavy frost Heyer et al. 1988 Paratelmatobius lutzii Itatiaia, RJ (8) 1978 Extinct Unknown Pombal & Haddad 1999; DOU 2014 Paratelmatobius gaigeae Paranapiacaba, SP (4) / 1931 Extinct Habitat degradation Pombal & Haddad 1999; Boraceia, SP (5) Zaher et al. 2005 Paratelmatobius mantiqueira Campos do Jordão, SP (7) 1953 Extinct Unknown Pombal & Haddad 1999; Vrcibradic et al. 2010 Physalaemus jordanensis Campos do Jordão, SP (7) 2007 Extinct Unknown IA Martins, persn. comm.

Odontophrynidae Proceratophrys moratoi Botucatu, SP (2) n/a Extinct Habitat destruction, pollu- Machado et al. 2005 tion Phyllomedusidae Phasmahyla guttata Teresópolis, RJ (10) 1979-1984 Decline Pollution and heavy frost Heyer et al. 1988 Phasmahyla guttata Rio de Janeiro, RJ (10) 1969-2010 Decline Unknown Izecksohn & Carvalho e Silva 2001; Between 1969 (MNRJ 49235-36) and 2010 (MNRJ 78409-10) specimens were collected only in 1985 (MNRJ 81341-42) 46

Phasmahyla exilis Santa Teresa, ES (11) 1982-1987 Decline Pollution, climatic changes Weygoldt 1989 and epidemic diseases Phasmahyla exilis Santa Teresa, ES (11) 1988 Extinct Pollution, climatic changes Weygoldt 1989 and epidemic diseases Phrynomedusa appendiculata Paranapiacaba, SP (4) 1966 Extinct Unknown Garcia el al. 2004 Phrynomedusa bokermanni Mongaguá, SP (4) 1978*** Extinct Unknown Present study Phrynomedusa fimbriata Paranapiacaba, SP (4) 1923*** Extinct Unknown Machado et al. 2005; Stuart et al. 2008; Garcia et al. 2010; DOU 2014 Phrynomedusa marginata Santa Teresa, ES (11) 1974*** Extinct Unknown Present study Phrynomedusa vanzolinii Boraceia, SP (5) 1929*** Extinct Unknown Cruz 1991; Garcia et al. 2010; DOU 2014 Phrynomedusa vanzolinii Serra dos Órgãos, RJ (10) 1929*** Extinct Unknown Cruz 1991; Garcia et al. 2010; DOU 2014

Both species indicated with asterisks were not mapped, either because it does not occurs in the Atlantic Forest “*” or the causes of decline are

well known and do not relate to Bd “**”. UNIRIO: Universidade Federal do Rio de Janeiro; USNM: National Museum of Natural History; SP:

State of São Paulo, RJ: State of Rio de Janeiro, MG: State of Minas Gerais, ES: State of Espírito Santo, PR: State of Paraná, SC: State of Santa

Catarina, RS: State of Rio Grande do Sul. n/a = not available. Three asterisks “***” indicates the year of description, and that no other individual

was found after that. 47

Figure S1. Correlation between the total number of depigmented specimens and number of specimens analyzed per family. The families are presented by the abbreviations:

Als (Alsodidae), Buf (Bufonidae), Cen (Centrolenidae), Cer (Ceratophryidae), Cyc

(Cycloramphidae), Den (Dendrobatidae), Hem (Hemiphractidae), Hyl (Hylidae), Hlo

(Hylodidae), Lep (Leptodactylidae), Odo (Odontophrynidae), Phy (Phyllomedusidae), and Ran (Ranidae).

Considerações Finais

Em nosso trabalho elucidamos a distribuição e prevalência da quitridiomicose em todos os biomas do Brasil, e em todas as ecorregiões da Mata Atlântica, demons- trando que a doença não esta uniformemente distribuída entre os biomas e dentro de um mesmo bioma. Nossa analise espaço-temporal apontou agregados de quitridiomicose e um aumento de prevalência que coincidem com pontos históricos de declínios ou extin-

ções populacionais de anfíbios. Ainda, nosso trabalho destaca a importância do estudo e monitoramento dos girinos em populações de anfíbios.

Os resultados desse estudo tem relevância tanto para a comunidade científica quanto para os tomadores de decisão acerca da conservação da biodiversidade, particularmente por apontar a quitridiomicose como principal causa dos declínios ou extinções de populações de anfíbios ocorridos no sudeste do Brasil, os quais tinham suas causas apontadas como enigmáticas. Ainda, nos apresentamos uma nova informação histórica sobre a quitridiomicose (1930-2015), a qual complementa pesquisas sobre a origem do

Bd no mundo.

A partir de nossas contribuições com este trabalho, surgem perguntas que podem ser utilizadas como base de futuras pesquisas, tais como:

 Quais os fatores responsáveis pelo aumento da prevalência de Bd nos lo-

cais de declínio?

 O fungo realmente está ausente no Pantanal?

 Por ter reportado a maior prevalência de Bd, a ecorregião Bahia Interior

Forest merece um maior esforço amostral e de monitoramento. Será que

já ocorreram declínios nesta ecorregião?

 Declínios por Bd deixa algum sinal que podemos detectar ao nível de

comunidade? 49

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