Alba Navarro Lozano Ecology and Biogeography of Batrachochytrium dendrobatidis in the Brazilian Atlantic Forest São José do Rio Preto 2018 Campus de São José do Rio Preto Alba Navarro Lozano Ecology and Biogeography of Batrachochytrium dendrobatidis in the Brazilian Atlantic Forest Tese apresentada como parte dos requisitos para obtenção do título de Doutor em Biologia Animal, junto ao Programa de Pós-Graduação em Biologia Animal, Área de Concentração – Ecologia e Comportamento, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquite Filho”, Campus São José do Rio Preto. Financiadora: Bolsista AUIP-PAEDEX- UNESP. FAPESP 2014/23677-9. Orientador: Prof. Dr. Ricardo Jannini Sawaya Co-orientador: Prof. Dr. Eduardo Alves de Almeida. São José do Rio Preto 2018 Navarro Lozano, Alba. Ecology and Biogeography of Batrachochytrium dendrobatidis in the Brazilian Atlantic Forest / Alba Navarro Lozano. -- São José do Rio Preto, 2018 90 f. : il., tabs. Orientador: Ricardo Jannini Sawaya Coorientador: Eduardo Alves de Almeida Tese (doutorado) – Universidade Estadual Paulista (Unesp), Instituto de Biociências, Letras e Ciências Exatas, São José do Rio Preto 1. Ecologia animal. 2. Anfíbios. 3. Girino. 4. Fungos. 5. Conservação biológica. I. Título. CDU – 597.6 Ficha catalográfica elaborada pela Biblioteca do IBILCE UNESP – Câmpus de São José do Rio Preto Alba Navarro Lozano Ecology and Biogeography of Batrachochytrium dendrobatidis in the Brazilian Atlantic Forest Tese apresentada como parte dos requisitos para obtenção do título de Doutor em Biologia Animal, junto ao Programa de Pós-Graduação em Biologia Animal, Área de Concentração – Ecologia e Comportamento, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquite Filho”, Campus São José do Rio Preto. Comissão Examinadora Prof. Dr. Ricardo Jannini Sawaya UFABC – São Bernardo do Campo, Brasil Orientador Prof. Dr. Fernando Rodrigues da Silva UFSCAR – Sorocaba, Brasil Prof. Dr. Leandro Reverberi Tambosi UFABC – São Bernando do Campo, Brasil Prof. Dr. Carlos Arturo Navas USP – São Paulo, Brasil Prof. Dr. Marcio Roberto Costa Martins USP – São Paulo, Brasil São José do Rio Preto 27 de Março de 2018 i A mis padres Eleuterio y Francisca por enseñarme a mirar el mundo. A mi hermano David por hacérmelo más bonito. ii “It doesn’t matter whether you’re the lion or a gazelle, when the sun comes up, you’d better be running”. Cristopher McDougall iii AGRADECIMIENTOS A lo largo de estos años son muchas las personas e instituciones que han participado en este trabajo, y es a ellas a quienes quiero expresar mi gratitud en este apartado por el apoyo y la confianza que me han ofrecido. Minhas primeiras linhas, sim ou sim, são dedicadas aos dois eixos principais deste trabalho: Ricardo J. Sawaya, gracias por sua orientação desde o início, pelo apoio incondicional e a confiança que sempre mostrou ter em mim. Gracias por ter respondido ao e-mail de uma ‘gringa’ como eu, e por ter-me dado a oportunidade de trabalhar ao seu lado. Denise C. Rossa-Feres, gracias por me receber com os braços abertos desde o primeiro minuto em que cheguei em Rio Preto, por o tempo que você me dedicou para me ensinar sobre aquelas maravilhosas criaturas que são os girinos; e por me mostrar como enfrentar os obstáculos desde a melhor perspectiva possível. Denise e Ricardo, MUCHAS GRACIAS por me ajudar a crescer, não só como bióloga, também como pessoa, gracias pelo carinho e amizade que vocês sempre me mostrarem. Vocês são claros exemplos do que eu quero alcançar na minha vida. Si hay algo claro, es que este trabajo no podría haberlo realizado sin la ayuda de mi familia. Quiero agradecer a mi madre, Francisca Lozano Ferris, y a mi padre, Eleuterio Navarro Moya, por haberme dado la oportunidad de formarme como bióloga. Gracias por vuestro esfuerzo y constancia desde mis inicios. A mi hermano, David Navarro Lozano, por la comprensión, el cariño y la música. A mi compañero de viaje, David Sánchez Domene, me gustaría agradecerle el apoyo, la confianza, el amor, el cariño y la enorme paciencia que me ha mostrado desde que empecé este trabajo. Gracias por la ayuda en campo, en laboratorio, en la vida... Pero sobre todo, mil gracias por comprarme casquinhas e açaís en los últimos meses hiperestresantes de redacción. También quiero agradecer: A la Asociación Universitaria Iberoamericana de Posgrado (AUIP) por la beca de doctorado que me fue otorgada. Ao projeto temático: “Novas abordagens de ecología e conservação: diversidade filogenética e funcional de anfíbios e serpentes da Mata Atlântica brasileira” (proceso FAPESP 2014/23677- 9), sob a coordenação de Ricardo J. Sawaya, pelo apoio financeiro para as viagens de campo, e estágio no exterior do Brasil. A la Fundación BBVA, por el apoyo financiero ofrecido para la realización de análisis moleculares, siempre iv bajo coordinación de Jaime Bosch. Ao profesor Eduardo A. Almeida e o laboratório de Biomarcadores de Contaminação Aquática pelo apoio financeiro para a compra de produtos de laboratório. Ao laboratorio de Ecologia Teorica de Ibilce (UNESP) e sua coordinadora, Denise C. Rossa-Feres, por proporcionar parte do material usado nas coletas, assim como oferecer o espaço fisico onde a ciência e a amizade estavam sempre de mãos dadas. Ao ICMBio/SISBIO e o COTEC/IF por disponibilizar as autorizações para coletar no Núcleo Curucutu. Aos profissionais da Secção de Pós-Graduação de Ibilce pela presteza e atenção com que sempre fui tratada. À todos os membros (alunos e professores) do Programa de Pós-Graduação de Biologia Animal (IBILCE), pela convivência e amizade durante estes quatro anos. Especialmente as professoras Lilian Casatti e Eliane G. Freitas, pela paciência, respeito e carinho que sempre me ofereceram, e por me permitir viver esta experiência da melhor maneira possível. A Jaime Bosch, pues sus artículos fueron los que despertaron mi interés por la quitridiomicosis cuando tan sólo era una estudiante de segundo año de carrera. Gracias por la enorme ayuda ofrecida durante este trabajo, por haberme abierto las puertas de tu laboratorio y por soportar mis lloriqueos tontos hasta en días festivos. Tu trabajo y saber estar en todo momento son, y serán, fuente de inspiración para mí. A Camino Monsalve por el tiempo que dedicó a mi trabajo y el cariño que me ofreció desde el minuto uno; y por la inflá a bombones que nos pegamos. La Caja Roja y Melendi me persiguen… À Maria Stela M. Castilho-Noll e Angélica M. Otero pela ajuda material e metodológica oferecida durante as coletas de campo e na identificação do zooplâncton. À Josiani Pereira e Pedro Lucas pelo apoio nos testes de laboratorio e boas conversas. To Matthew Fisher and Pria Ghosh for their good advice and suggestions. À Carlos E. Sousa, Daniel G. Chagas, David Sánchez, Fabiane Annibale e Leo R. Malagoli por querer perder-se comigo durante varias semanas no mato buscando girinos. Especialmente a Leo R. Malagoli por ter me mostrado seu canto favorito no bioma mais lindo do mundo. À Carlos Navas e Ananda Brito pela ajuda com os primeiros testes de laboratório. v Aos colegas-amigos do meu laboratorio (porque o sinto como meu): Carlos E. de Sousa, Fabiane Annibale, Mainara Jordani, Katiuce Picheli, Rodolfo Pelinson, Fernanda Simioni, Lilian Sayuri e Cristiano Ferreira, gracias pela oportunidade de convivência e aprendizagem. À Fabi, Katiuce e Mainara, gracias uma e mil vezes pela força, incentivo e AMIZADE. A meus amigos Leandro R. Tambosi e Mariana Vidal pela ajuda incansável que vocês nos deram em todos nossos passos (antes e depois de chegar ao Brasil), e pelo enorme carinho que sempre nos mostrarem. A mi capu-amigo, Daniel Coáguila, por su inmensa paciencia durante mis primeros meses de aprendizaje con SDMs. Gracias amigo por aguantar a esta española mal hablada. Ao gestor do Núcleo Curucutu, Marcelo Gonçalves, assim como a todos os trabalhadores do Núcleo, por facilitar a minha estadia na unidade para a realização deste trabalho. À Wesley Pereira por guiar-nos através da floresta. Aos índios Guarani da aldeia Rio Branco por permitirem o acesso ás suas terras e guiar-nos através da floresta. À Eduardo A. Almeida e sua mulher Beatriz por nos ajudar e facilitar a nossa permanência no Brasil. Aos colegas que conheci durante todos estes anos no Laboratório de Ecologia e Evolução da UNIFESP, Thiago A. Oliveira, Leo R. Malagoli, Thiago A. Pires, José Thales Portillo, Maria Carolina R. Manzano, Marcela B. Godinho e Daniel G. Chagas, pelas boas discussões e reuniões que desfrutei com vocês. Aos professores de Português Língua Estrangeira. À minhas colegas de zumba por terem tornado as semanas tão divertidas. A todos los alumnos extranjeros de IBILCE por enriquecer mi estancia en Brasil, y por montar los mejores ‘churrascos’. Especialmente, a mis amigos y hermanos, Mariana Molina y Arturo Solís, por darme los mejores años de mi estancia en Brasil. E finalmente, a minha segunda casa… Brasil. Infinitamente grata. vi INDEX Resumo ....................................................................................................... 1 Abstract ...................................................................................................... 3 Introduction ............................................................................................... 4 References ............................................................................................... 11 Chapter I Batrachochytrium dendrobatidis infection of frogs is driven by different biotic and abiotic factors in ponds and streams of the megadiverse Atlantic Forest of Brazil ............................................................................. 14 Abstract .................................................................................................... 16 Resumen .................................................................................................. 17 Introduction .............................................................................................. 18 Materials and methods Study site and sampling .......................................................................... 19 Prevalence and intensity of Bd................................................................. 20 Data collection ....................................................................................... 21 Data analysis ......................................................................................... 22 Results ..................................................................................................... 23 Discussion ................................................................................................ 25 Acknowledgments .................................................................................... 28 Literature cited ......................................................................................... 29 Supplementary material ........................................................................... 31 Extra supplementary material................................................................... 36 Chapter II Global warming could decrease potential geographic distributon of amphibian-killing fungus in Braziian Atlantic Forest .............................. 38 Abstract .................................................................................................... 39 Introduction .............................................................................................. 41 vii Methods Data collection ....................................................................................... 42 Species distribution modelling ................................................................. 43 Results ..................................................................................................... 46 Discussion ................................................................................................ 50 Acknowledgments .................................................................................... 51 References ............................................................................................... 53 Supporting Information ............................................................................. 56 Chapter III Are oral deformities in tadpoles accurate indicators of anuran chytridiomycosis? ...................................................................................... 61 Abstract .................................................................................................... 62 Introduction .............................................................................................. 64 Materials and methods Ethics Statement .................................................................................... 65 Study animals ........................................................................................ 66 Identification of oral deformities ............................................................... 66 Batrachochytrium dendrobatidis detection................................................. 68 Statistical analyses ................................................................................. 68 Results ..................................................................................................... 69 Discussion ................................................................................................ 73 Acknowledgments .................................................................................... 75 References ............................................................................................... 76 Chapter IV Batrachochytrium dendrobatidis in Brazil: www.quitribrasil.com ......... 78 Webpage impact ...................................................................................... 82 Acknowledgments .................................................................................... 87 References ............................................................................................... 87 Conclusions ............................................................................................ 89 http://www.quitribrasil.com/ A b s t r a c t | 1 RESUMO O fungo quitridio Batrachochytrium dendrobatidis (Bd) é considerado uma das principais causas do declínio global dos anfíbios. Bd já foi encontrado em quase 700 espécies de anfíbios de todo o mundo. Embora muitos estudos tenham sido publicados desde a sua descrição em 1999, ainda há muito a ser conhecido, particularmente em relação à sua ecologia. Nossos objetivos neste estudo foram: (i) analisar os fatores bióticos e abióticos que regulam a prevalência e a intensidade de infecção de Bd em girinos que habitam poças e riachos no Núcleo Curucutu do Parque Estadual da Serra do Mar, sudeste da Mata Atlântica brasileira; (ii) compreender qual será o efeito das mudanças climáticas na distribuição potencial de Bd na Mata Atlântica brasileira; (iii) avaliar o uso de deformidades orais em girinos como indicadores confiáveis para a determinação de infecção por Bd; e (iv) criar uma ferramenta de divulgação para promover a transferência de conhecimento sobre Bd entre a universidade e a sociedade brasileira. Detectamos que a prevalência e a intensidade de Bd não diferiu entre poças e riachos, mas são regulados por diferentes fatores ambientais. Em riachos, a velocidade da água e a profundidade explicaram a variabilidade na prevalência de Bd, mas a intensidade permaneceu inexplicada. Por outro lado, a densidade do zooplâncton foi o fator chave para explicar a prevalência e a intensidade em poças. Estimamos que, hoje, cerca do 60% da área da Mata Atlântica brasileira apresenta condições climáticas adequadas para a ocorrência de Bd, porém diferentes cenários de aquecimento global para o ano de 2070 diminuiriam essas áreas entre um 27.5 e 42.6%. Também demonstramos que as deformidades orais em girinos não são um indicador confiável para o diagnóstico da quitridiomicose em girinos de anfíbios anuros, e que não devem ser utilizadas como ferramenta de diagnóstico isoladamente. Desenvolvimos uma página de internet (www.quitribrasil.com) para a divulgação de todos os casos de Bd relatados no Brasil. As informações geradas por estes trabalhos podem melhorar a compreensão da ecologia e distribuição de Batrachochytrium dendrobatidis em um dos biomas mais ricos e com as maiores taxas de endemismo de anfíbios no mundo. A b s t r a c t | 2 Palavras-chave: Batrachochytrium dendrobatidis, Amphibia, Girino, Ecologia, Conservação, Distribuição geográfica, Mudança climática, Mata Atlântica, Morfologia A b s t r a c t | 3 ABSTRACT The chytrid fungus Batrachochytrium dendrobatidis (Bd) is considered one of the main cause of global amphibian declines. Bd has been reported in nearly 700 species of amphibian worldwide. Although many studies have been published since its description in 1999, there is still much to be known, particularly regarding to its ecology. In this study, we aim to: i) analize biotic and abiotic factors that regulate prevalence and intensity of Bd infection in tadpoles inhabiting lentic and lotic habitats, in Nucleo Curucutu of the Parque Estadual da Serra do Mar, southeastern Brazilian Atlantic Forest; ii) understand the effect of climate change in potential future distribution of Bd in the Brazilian Atlantic Forest; iii) evaluate the use of oral deformities in tadpoles as reliable proxies for the determination of infection by Bd; and iv) create an outreach tool to communicate the knowledge of Bd from university to Brazilian society. We detected that the prevalence and intensity of Bd did not differ between ponds and streams but were driven by different environmental factors in the two habitats. In streams, water velocity and depth explained variability in Bd prevalence, but Bd intensities remained unexplained. On the other hand, zooplankton density was the key factor in explaining Bd prevalence and intensity in ponds. We estimated that currently, about 60% of the Atlantic Forest area shows climatic suitability for Bd occurrence; but in global warming scenarios for 2070 those suitable areas will decrease by between 27.5 and 42.6%. We also showed that oral deformities in tadpoles are not a trustworthy indicator for chytridiomycosis diagnosis in tadpoles of anuran amphibians, and should not be used in isolation as a diagnosis tool. We developed and launched one internet webpage (www.quitribrasil.com) to communicate all Bd cases reported in Brazil. Information generated by our study could improve the knowledge of ecology and distribution of Batrachochytrium dendrobatidis in one of the richest biomes with greatest values of endemism of anuran amphibians in the world. Keywords: Batrachochytrium dendrobatidis, Amphibia, Tadpole, Ecology, Conservation, Geograohic distribution, Climatic change, Atlantic Forest, Morphology http://www.quitribrasil.com/ I n t r o d u c t i o n | 4 INTRODUCTION Global loss of biodiversity is one of the most serious problem of our time (Baillie et al., 2010). Data from the International Union for Conservation of Nature (IUCN) warns that approximately 41% of the world's amphibians species are endangered, making them the most vulnerable group among vertebrates (IUCN, 2016). Whereas it is true that amphibian populations present natural abundance fluctuations (Pechmann et al., 1991; Alford & Richards, 1999), population declines occurred in the last decades have not being balanced with equivalent increases (Houlahan et al., 2000). Different studies have shown that amphibian population declines are the outcome of complex interactions of factors such as habitat loss and land-use changes (Bradford et al., 1993; Stuart et al., 2004; Cushmann, 2006), exotic species introduction (Fisher & Shaffer, 1996; Bradford 1989; Kiesecker et al., 2001), environmental contamination (Harte & Hoffman, 1989; Rohr et al., 2004; Blaustein et al., 2003), UV-radiation increase (Blaustein et al 1994, 1997; Anzalone et al 1998; Lizana & Pedraza 1998), climatic changes (Heyer et al., 1988; Laurance et al., 1996; Pounds et al, 1999, 2006; Pounds, 2001), and the emergence infectious diseases (Berger et al., 1998, 1999; Lips, 1999, 1998). From the end of 1970’s and during the 1990’s, dramatic amphibian death events occurred in Central America and Australia (Laurence et al., 1996; Campbell et al., 1999; Pounds et al., 1997; Crump et al., 1992). What researchers studying these cases found most striking was that both events occurred in well conserved pristine forest areas. They suggested that climate change was responsible for them (Pounds et al., 1999). However, at the end of I n t r o d u c t i o n | 5 the 1990’s it was detected a link between mass mortality events in Central America and Australia: the presence of fungal sporangium in upper layers of amphibians skin, a, infection called chytridiomycosis (Berger et al., 1998). Shortly after this discovery, Longcore et al. (1999) described Batrachochytrium dendrobatidis (Bd), a chytrid fungus responsible for those death events. Since Bd was first described, several dramatic declines of amphibian populations worldwide have been explained by Bd infections (e.g. Berger et al, 1998, 1999; Lips, 1999, 1998; Bosch et al., 2001; Ouellet et al., 2005; Weldon et al., 2004; Garner et al., 2005; Lips et al., 2006). Nowadays is known that Bd is spread worldwide, already found in 56 countries and affecting nearly 700 species of all three amphibian orders (http://www.bd-maps.net). Who is Batrachochytrium dendrobatidis? And, how does it affect amphibians? Bd is a non-hyphal zoosporic chytrid aquatic fungus belonging to the class Chytridiomycetes and order Rhizophydiales (Longcore et al., 1999; Letcher et al., 2006). Although a saprobic phase has been suggested for Bd, to BOX 1 In 2013, Martel et al. discovered a second species of chytrid fungus, Batrachochytrium salamandrivorans (Bsal), which was causing lethal infections and population declines in the European Salamandra salamandra. Shortly after, the same research group pointed Oriental Asia as the origin of Bsal, since unlike with Bsal in Europe, the presence of the fungus do not seem to be affecting Asian urodels (Martel et al., 2014). To date, Bsal only occurs in urodeles (newts and salamanders) and not in anurans (frogs and toads) or caecilians. I n t r o d u c t i o n | 6 date only two life cycle phases has been described, a free-swimming zoosporic phase that allows Bd to meet a host, and a sporangial phase that results from the encystment of the zoospore into keratinizing tissue of a host, or those tissues fated to keratinize, where matures and produce new zoospores (Longcore et al., 1999; Fellers et al., 2001; Berger et al., 2005). Bd infection is restricted to the oral region in tadpoles, the only keratinized part of this life cycle phase (Marantelli et al., 2004; Berger, 1998, 1999; Fellers et al., 2001). In adults, Bd colonize keratinized cells of the stratum corneum and granulosum of the epidermis, with higher concentrations in ventral and pelvic regions, hind limbs and feet (Longcore et al., 1999; Pessier et al., 1999). Superficial pathologies associated with the infection vary from swollen and dekeratinization of the oral disk (Berger et al., 1999; Fellers et al., 2001; Navarro-Lozano et al., 2018), to hyperkeratosis, hyperplasia, ulceration, depigmentation, slower rehydration and sloughing of metamorph skin (Berger et al., 1998; Voyles et al., 2009; Carver et al., 2010), as well as behavioral abnormalities such as inappetance, lethargy and loss of reflex and escape response in adults (Berger et al., 1998, 1999; Nichols et al., 2001; Pessier et al., 1999, Voyles et al., 2009). In has also been described pathophysiologies caused by the impaired performance of the amphibians skin such as significant reductions in osmolarity and concentrations of Na+, K+ and Cl− in plasma, which may result in death by cardiac arrest (Voyles et al., 2009; Marcum et al., 2010). In addition, it has been demonstrated that Bd produces mycotoxins that block the amphibian immune response, inhibiting lymphocyte proliferation and causing cell apoptosis (Fites et al., 2013). I n t r o d u c t i o n | 7 Not all species from affected regions have the same likelyhood to be infected (Blaustein et al., 2005; Searle et al., 2011; Lips 2006, Gahl 2012). Some species of salamanders show low susceptibility to Bd infection which has been linked to the presence of bacterial strains on their skin (Harris et al. 2006). Indeed, the bacteria Janthinobacterium lividum, found in the skin of some salamander species, produces lethal concentrations of antifungal metabolites for Bd (Brucker et al., 2008). Functional traits can also determine the likelyhood of species to be infected. Species inhabiting permanent waterbodies seem to be more infected by Bd than those inhabiting temporal waterbodies (Kriger & Hero, 2007). In turn, among species that share the same habitat, those occuping warmer and dryer microhabitats are also less likely to be infected (Rowley & Alford, 2013). BOX 2 Despite Bd came into the spotlight for its detrimental effect on amphibians, this fungus has been found in other animals that share amphibian habitats as: • Lizards: Anolis humilis and Anolis lionotus (Kilburn et al., 2011). • Snakes: Pliocercus euryzonus, Imantodes cenchoa and Nothopsis rugosus (Kilburn et al., 2011). • Birds: Branta canadensis and Anser domesticus (Garmyn et al., 2012); Anas falvirostris, Anas georgica, Fulica ardesiaca, Fulica gigantean, Lophonetta specularioides, Plegadis ridgwayi, Rollandia Rolland and Spatula puna (Burrowes & De la Riva, 2017a). • Arthropods: Procambarus spp. and Orconectes virilis (McMahon et al. 2013). • Fishes: Danio rerio (Liew et al., 2017). I n t r o d u c t i o n | 8 Origin of Batrachochytrium dendrobatidis Since chytridiomycosis was first reported, there has been a great debate to explain the origin of this disease. And although it has taken almost two decades of studies, a research with more than 200 Bd isolates, collected from various regions of the planet, has managed to point to East Asia as the geographical origin of Bd (Dr. Jaime Bosch's personal communication). This recent research by Dr. Jaime Bosch and other 52 authors, which will be published in Nature this year, highlights the presence of four divergent main lineages of Bd: Bd-Asia1, Bd-Asia2/Brazil, Bd-Cape and Bd-GPL (Global Panzootic Lineage). It is Bd-GPL who stands out over the rest of lineages for its hypervirulence and widely distribution, being associated with species extinction and mass mortality events of amphibians occurred in Central America, Australia and Spain (Farrer et al., 2011; Schloegel et al., 2012). From this research it is also concluded that Bd-GPL has a recent origin, and that its global expansion took place until the first half of the 20th century, coinciding in time with the commercialization of amphibians for scientific and feed purposes (Dr. Jaime Bosch's personal communication). I n t r o d u c t i o n | 9 Eighteen years after the discovery of Bd, many studies have been published. However, there is still much to be known, particularly regarding to its ecology. This doctoral dissertation presents a study of the distribution and ecology of Bd in the Brazilian Atlantic forest, which stands out as an ideal place for the study of Bd due to its importance as a global hotspot (Myers, 2000; Jenkins et al., 2015) and to the absence of harmful pathologies, despite of the large number of amphibian species Bd-infected. The dissertation includes four chapters: (1) Batrachochytrium dendrobatidis infection of frogs is driven by different biotic and abiotic factors in ponds and streams of the megadiverse Atlantic Forest of Brazil, in which biotic and abiotic environmental factors that influence the dynamic of Bd infection (prevalence and intensity) in a pristine area of the Brazilian Atlantic Areas are studied; (2) Global warming could decrease potential geographic distributon of amphibian-killing fungus in Braziian Atlantic Forest, in which we have analyzed the effect of climate change over Bd distribution in the Brazilian BOX 3 Until today, the international trade of amphibian species seems to be the main source of dispersion of the fungus around the world (Laurance et al., 1996; Fisher & Garner, 2007; Schloegel et al. 2012). Why? For example, Xenopus laevis and Lithobates catesbeiana, commonly commercialized for consumption, as pets or for laboratory use, can accommodate high loads of Bd without succumbing to the disease (Mazzoni et al., 2003; Weldon et al., 2004; Daszak et al., 2004). The lack of signs of disease and the ineffectiveness of transcontinental biosecurity have turned these species into perfect vectors for the global dissemination of Bd. Specimens of L. catesbeiana infected by the fungus been reported in markets in Asia, South and North America, and Europe (Garner et al., 2006; Bai et al., 2010). I n t r o d u c t i o n | 10 Atlantic Forest; (3) Are oral deformities in tadpoles accurate indicators of anuran chytridiomycosis?, in which the relationship between oral deformities and Bd infection in tadpoles of different species and is potential use as a proxy for the determination of Bd infection is studied; and (4) Batrachochytrium dendrobatidis in Brazil: www.quitribrasil.com, in which we present the webpage www.quitribrasil.com, where information of all Bd cases reported in Brazil is provided. Finally, the conclusions section highlights the main findings that our studies have revealed. http://www.quitribrasil.com/ I n t r o d u c t i o n | 11 REFERENCES Alford RA, Richards SJ. 1999. Global amphibian declines: a problem in applied ecology. Annual Review of Ecology and Systematics 30: 133-165. Anzalone CR, Kats LB, Gordon MS. 1998. Effects of solar UV-B radiation on embryonic development in Hyla cadaverina, Hyla regilla, and Taricha torosa. Conservation Biology. 12: 646-653. Bai C, Garner TWJ, Li Y. 2010. First evidence of Batrachochytrium dendrobatidis in China: discovery of chytridiomycosis in introduced American bullfrogs and native amphibians in the Yunnan Province, China. EcoHealth. 7:127-134. Baillie JEM, Griffiths J, Turvey ST, Loh J, Collen B. 2010. Evolution Lost. Status and trends of the world’s vertebrates. Zoological Society of London, United Kingdom. Bataille A, Fong JJ, Cha M, Wogan GOU, Baek HJ, Lee H, et al. 2013. Genetic evidence for a high diversity and wide distribution of endemic strains of the pathogenic chytrid fungus Batrachochytrium dendrobatidis in wild Asian amphibians. Mol. Ecol. 22:4196–4209. Berger L, Speare R, Daszak P et al. 1998. Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proceedings of the National Academy of Sciences, USA, 95, 9031–9036. Berger L, Speare R, Hyatt A. 1999. Chytrid fungi and amphibian declines: Overview, implications and future directions. En: Campbell A (ed). Declines and disappearances of Australian frogs. Canberra, Australia: Biodiversity Group Environment Australia. Berger, L.; Marantelli, G.; Skerrat, L.F.; Speare, R. 2005. Virulence of the amphibian chytrid fungus Batrachochytrium dendrobatidis varies with the strain. Diseases of Aquatic Organisms, v. 68, n. 1, p.47-50. Blaustein AR, Hoffman PD, Hokit DG, Kiesecker JM, Walls SC, Hays JB. 1994. UV repair and resistance to solar UV-B in amphibian eggs: A link to population declines? Proceedings of the National Academy of Sciences USA. 91: 1791-1795. Blaustein AR, Kiesecker JM, Chivers DP, Anthony RG. 1997. Ambient UV-B radiation causes deformities in amphibian embryos. Proc Natl Acad Sci USA. 94:13735-13737. Blaustein AR, Romansic JM, Kiesecker JM, Hatch AC. 2003. Ultraviolet radiation, toxic chemicals and amphibian population declines. Diversity and Distribution 9: 123-140. Blaustein AR, Romansic JM, Scheessele EA, Han BA, Pessier AP, Longcore JE. 2005. Interespecific variation in susceptibility of frog tadpoles to the pathogenic fungus Batrachochytrium dendrobatidis. Conservation Biology. 1460-1468. DOI: 10.1111/j.1523-1739.2005.00195.x Bosch J, Martinez-Solano I, Garcia-Paris M. 2001. Evidence of a chytrid fungus infection involved in the decline of the common midwife toad (Alytes obstetricans) in protected areas of central Spain. Biological Conservation. 97: 331-7. Bosch J, Carrascal LM, Durán L, Walker S, Fisher MC. 2007. Climate change and outbreaks of amphibian chytridiomycosis in a montane area of Central Spain; is there a link? Proceedings of Royal Society B. 274: 253-260. Bradford D. 1989. Allotopic distribution of native frogs and introduced fishes in high Sierra Nevada lakes of California: implication of the negative effect of fish introductions. Copeia. 1989: 775-778. Bradford DF, Tabatabai F, GraberDM. 1993. Isolation of remaining populations of the native frog, Rana muscosa, by introduced fishes in Sequoia and Kings Canyon National Parks, California. Conservation Biology. 7: 882-888. Brucker RM, Harris RN, Schwantes, Gallaher TN, Flaherty DC, Lam BA, Minbiole KPC. 2008. Amphibian chemical defense: antigunal metabolites of the microsymbiont Janthinobacterium lividum on the salamander Plethodon cinereus. J Chem Ecol. 341:1422-1429. Burrowes PA, De la Riva I. 2017a. Detection of the amphibiand chytrid fungus Batrachochytrium dendrobatidis in museum specimens of Andean aquatic birds: implications for pathogen dispersal. Journal of Wildlife Diseases. doi: 10.7589/2016-04-074 Burrowes PA, De la Riva I. 2017b. Unraveling the historical prevalence of the invasive chytrid fungus in the Bolivian Andes: Implications in recent amphibian declines. Biological Invasions, doi: 10.1007/s10530- 017-1390-8. Campbell A (ed). 1999. Declines and disappearances of Australian frogs. Canberra, Australia: Biodiversity Group Environment Australia. Published by Environment Australia. Carver S, Bell BD, Waldman B. 2010. Does chytridiomycosis disrupt amphibian skin function? Copeia, 3:487-495. Coutinho SDA, Burke JC, de Paula CD, Rodrigues MT, Catão-Dias JL. 2015. The use of singleplex and nested PCR to detect Batrachochytrium dendrobatidis in free- living frogs. Brazilian Journal of Microbiology, 46;2: 551-555. Crump ML, Hensley F, Clark K. 1992. Apparent declines of the Golden toad: underground or extinct? Copeia. 413-420. Cushmann SA. 2006. Effects of habitat loss and fragmentation on amphibians: a review and prospectus. Biological Conservation 128: 231-240. Daszak P, Berger L, Cunningham AA, Hyatt AD, Green DE, Speare R. 1999. Emerging infectious disease and amphibian population declines. Emerging Infectious Diseases. 5: 735-748. Daszak P, Cunningham AA, Hyatt AD. 2003. Infectious disease and amphibian population declines. Diversiy and distributions. 9: 141-150. Daszak P, Strieby A, Cunningham AA, Longcore JE, Brown CC, Porter D. 2004. Experimental evidence that the bullfrog (Rana catesbeiana) is a potential carrier of chytridiomycosis, an emerging fungal disease of amphibians. Herpetological Journal. 14: 201-207. de Paula CD. Patologia comprada de infecções selecionadas de anfíbios anuros de vida livre do bioma da Mata Atlântica: estudo prospectivo. Sc. Thesis, USP. 2011. I n t r o d u c t i o n | 12 Farrer RA, Weinert LA, Bielby J et al. 2011. Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage. Proceedings of the National Academy of Sciences, USA, 108, 18732–18736. Fellers GM, Green DE, Longcore JE. 2001. Oral chytridiomycosis in the mountain yellow-legged frog (Rana muscosa). Copeia. 2001: 945-953. Fisher MC, Garner TWJ. 2007. The relationship between the emergence of Batrachochytrium dendrobatidis, the international trade in amphibians and introduced amphibian species. Fungal Biology Reviews. 21:2-9. Fisher RN, Shaffer HB. 1996. The decline of amphibians in California's Great Central Valley. Conservation Biology. 10:1387-1397. Fites JS, Ramsey JP, Holden WM, Collier SP, Sutherland DM, Reinert LK, et al. 2013. The invasive chytrid fungus of amphibians paralyzes lymphocyte responses. Science. 342:366-369. Gahl MK, Longcore JE, Houlahan JE. 2012. Varying responses of northeastern North American amphibians to the chytrid pathogen Batrachochytrium dendrobatidis. Conserv. Biol. 26:135–141. Garmyn A, Van Rooij P, Pasmans F, Hellebuyck T, Van Den Broeck W, et al. 2012. Waterfowl: potential environmental reservoirs of the chytrid fungus Batrachochytrium dendrobatidis. PLoS ONE 7(4): e35038. doi:10.1371/journal.pone.0035038 Garner TWJ, Walker S, Bosch J, Hyatt A, Cunningham AA, Fisher MC. 2005. Chytrid fungus in Europe. Emerging Infectious Diseases. 11(10): 1639-1641. Garner TWJ, Perkins MW, Govindarajulu P et al. 2006. The emerging amphibian pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana. Biology Letters. 2:455–459. Goka K, Yokoyama J, Une Y, Kuroki T, Suzuki K, Nakahara M, Kobayashi A, et al. 2009. Amphibian chytridiomycosis in Japan: distribution, haplotypes and possible route of entry into Japan. Molecular Ecology, 18:4757-4774. Harris RN, James TY, Lauer A, Simon MA, Patel A. 2006. Amphibian pathogen Batrachochytrium dendrobatidis is inhibited by the cutaneous bacteria of amphibian species. EcoHelth. 3:53-56. Harte J, Hoffman E. 1989. Possible effects of acidic deposition on a Rocky Mountain population of the tiger salamander Ambystoma tigrinum. Conservation Biology. 3:149-158 Heyer RW, Rand SA, Cruz CAG, Peixoto OL. 1988. Decimations, extinctions, and colonizations of frog populations in southeast Brazil and their evolutionary implications. Biotropica. 20:230–235. Houlahan JE, Findlay CS, Schmidt BR, Meyer AH, Kuzmin SL. 2000. Quantitative evidence for global amphibian population declines. Nature. 404:752-755. IUCN – International Union for Conservation of Nature, Red List of Species. Link: http://www.iucnredlist.org/initiatives/amphibians/analysi s James TY, Toledo LF, Röoder D, Leite DSL, et al. 2015. Disentangling host, pathogen, and environmental determinants of a recently emerged wildlife disease: lessons from the first 15 years of amphibian chytridiomycosis research. Ecology and Evolution. doi: 10.1002/ece3.1672 Jenkins CN, Alves MAS, Uezu A, Vale MM. 2015. Patterns of Vertebrate Diversity and Protection in Brazil. PLoS ONE 10(12): e0145064. doi:10.1371/ journal.pone.0145064 Kielgast J, Rödder D, Veith M, Lötters S. 2009. Widespread occurrence of the amphibian chytrid fungus in Kenya. Animal Conservation. 13(1): 36-43. Kiesecker JM, Blaustein AR, Miller C. 2001. Potencial mechanisms underlying the displacement of native red-legged frogs by introduced Bullfrogs. Ecology. 82:1964-1970. Kilburn VL, Ibáñez R, Green DM. 2011. Reptiles as potential vectors and hosts of the amphibian pathogen Batrachochytrium dendrobatidis in Panama. 97: 127- 134. Kolby JE, Smith KM, Berger L, Karesh WB, Preston A, et al. 2014. First Evidence of Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) and Ranavirus in Hong Kong Amphibian Trade. PLoS ONE 9(3): e90750. doi:10.1371/journal.pone.0090750 Kriger KM, Hero JM. 2007. The chytrid fungus Batrachochytrium dendrobatidis is non-randomly distributed across amphibian breeding habitats. Diversity and Distribution. 13:781-788. Laurence WF. 1996. Catastrophic declines of Australian rainforest frogs: Is unusual weather responsible? Biological Conservation. 77: 203-212. Letcher PM, Powell MJ, Churchull PF, Chambers JG. 2006. Ultrastructural and molecular phylogenetic delineation of a new order, the Rhizophydiales (Chytridiomycota). Mycological Research. 110:898- 915. Liew N, Mazon MJM, Wierzbicki CJ, Hollinshead M, Dillon MJ, Thomton CR, et al. 2017. Chytrid fungus infection in zebrafish demonstrates that the pathogen can parasitize non-amphibian vertebrate hosts. Nature. DOI: 10.1038/ncomms15048 Lips KR. 1998. Decline of a tropical montane amphibian fauna. Conservation Biology. 12:106–117. Lips KR. 1999. Mass mortality and population declines of anurans at upland site in western Panama. Conservation Biology. 13:117-125. Lips K, Brem F, Brenes R, Reeve JD, Alford R, Voyles J, Carey C, Livo L, Pessier A, Collins JP. 2006. Emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community. Proceeding of the National Academy of Sciences of the United States of America. 103: 3165-3170. Lizana M, Pedraza EM. 1998. The effects of UV-B radiation on toad mortality in mountainous areas of central Spain. Conservation Biology. 12: 703-707. Longcore JC, Pessier AP, Nichols DK. 1999. Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia. 91: 219- 227. Marantelli G, Berger L, Speare R, Keegan L. 2004. Distribution of the amphibian chytrid Batrachochytrium dendrobatidis and keratin during tadpole development. Pacific Conservation Biology. 10:173-179. Marcum RD, St-Hilaire S, Murphy PJ, Rodnick KJ. 2010. Effects of Batrachochytrium dendrobatidis infection on ion concentrations in the boreal toad Anaxyrus (Bufo) boreas boreas. Dis Aquat Org. 91:17-21. Martel A, Spitzen-van der Sluijs A, Blooi M, Bert W, Ducatelle R, Fisher MC, et al. 2013. Batrachochytrium http://www.iucnredlist.org/initiatives/amphibians/analysis http://www.iucnredlist.org/initiatives/amphibians/analysis I n t r o d u c t i o n | 13 salamandrivorans sp. nov. causes lethal chytridiomycosis in amphibians. Proceedings of the National Academy of Sciences of the United States of America 110, 15325e15329. Martel A, Blooi M, Adriaensen C, Van Rooij P, Beukema W, et al. 2014. Recent introduction of chytrid fungus endangers Western Palearctic salamanders. Science 346:630-631. Mazzoni R, Cunningham AA, Daszak P, Apolo A, Perdomo E, Speranza G. 2003. Emerging pathogen of wild amphibians in frogs (Rana catesbeiana) farmed for international trade. Emerging Infectious Diseases. 9(8):995-998. McMahon TA, Brannelly LA, Chatfield MWH, Johnson PTJ, Joseph MB, McKenzie VJ, et al. 2013. Chytrid fungus Batrachochytrium dendrobatidis has nonamphibian hosts and releases chemicals that cause pathology in the absence of infection. PNAS. 110(1): 210-215. Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J. 2000. Biodiversity hotspots for conservation priorities. Nature, 403, 853–858. Navarro-Lozano A, Sánchez-Domene D, Rossa-Feres D, Bosch J, Sawaya RJ. 2018. Are oral deformities in tadpoles accurate indicators of anuran chytridiomycosis? PLoS ONE 13(1):e0190955. https://doi.org/10.1371/journal.pone.0190955 Nichols DK, Lamirande EW, Pessier AP, Longcore JE. 2001. Experimental transmission of cutaneous chytridiomycosis in dendrobatid frogs. Journal of Wildlife Diseases. 37(1): 1-11. Ouellet M, Mikaelian I, Pauli B, Rodrigue J, Green DM. 2005. Historical evidence of widespread chytrid infection in North America amphibian populations. Conservation Biology 19: 1431-1440. Pechmann JHK, Scott DE, Semlitsch RD, Caldwell JP, Vitt LJ, Gibbons W. 1991. Declining amphibian populations: the problem of separating human impacts from natural fluctuations. Science 253: 892-895. Pessier AP, Nichols DK, Longcore JE, Fuller MS. 1999. Cutaneous chytridiomycosis in poison dart frogs (Dendrobates spp.) and White's tree frogs (Litoria caerulea). Journal of Veterinary Diagnostic Investigation. 11: 194-199. Piotrowski JS, Annis SL, Longcore JE. 2004. Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians. Mycologia. 96(1): 9-15. Pounds JA, Fogden MPL, Savage JM, Gorman GC. 1997. Tests of nulls models for amphibian declines on a tropical mountain. Conservation Biology. 11(6):1307- 1322. Pounds JA, Fogden MPL, Campbell JH. 1999. Biological response to climate change on a tropical mountain. Nature. 398: 611-615. Pounds JA. 2001. Climate and amphibian declines. Nature.410: 639-640. Pounds JA, Bustamante MR, Coloma LA et al. 2006. Widespread amphibian extinctions from epidemic disease driven by global warming. Nature, 439, 161– 167. Rachowicz LJ, Hero J-M, Alford RA et al. 2005. The novel and endemic pathogen hypotheses: competing explanations for the origin of emerging infectious diseases of wildlife. Conservation Biology, 19, 1441– 1448. Rodriguez D, Becker CG, Pupin NC, Haddad CFB, Zamudio KR. 2014. Long-term endemism of two highly divergent lineages of the amphibian-killing fungus in the Atlantic Forest of Brazil. Molecular Ecology, 23: 774-787. Rohr JR, Elskus AA, Shepherd BS, Crowley PH, McCarthy TM, Niedzwiecki JH, Sager T, Sih A, Palmer BD. 2004. Multiple stressors and salamanders: effects of an herbicide, food limitation, and hydroperiod. Ecological Applications 14: 1028-1040. Rosenblum EB, James TY, Zamudio KR, Poorten TJ, Ilut D, et al. 2013. Complex history of the amphibian-killing chytrid fungus revealed with genome resequencing data. Proceedings of the National Academy of Sciences, USA, 10, 9385–9390. Rowley JJL, Alford RA. 2013. Hot bodies protect amphibians against chytrid infection in nature. Scientific reports. 3:1515. DOI: 10.1038/srep01515 Schloegel LM, Picco AM, Kilpatrick AM et al. 2009. Magnitude of the US trade in amphibians and presence of Batrachochytrium dendrobatidis and ranavirus infection in imported North American bullfrogs (Rana catesbeiana). Biological Conservation, 142, 1420–1426. Schloegel LM, Toledo LF, Longcore JE et al. 2012. Novel, panzootic and hybrid genotypes of amphibian chytridiomycosis associated with the bullfrog trade. Molecular Ecology, 21, 5162–5177. Searle CL, Fervasi SS, Hua J, Hammond JI, Relyea RA, Olson DH, Blaustein AR. 2011. Differential host susceptibility to Batrachochytrium dendrobatidis, an emerging amphibian pathogen. Conservation Biology. 25(5):965-974. Solís R, Lobos G, Walker SF, Fisher M, Bosch J. 2010. Presence of Batrachochytrium dendrobatidis in feral populations of Xenopus laevis in Chile. Biol Invasions. 12:1641-1646. Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, Waller RW. 2004. Status and trends of amphibian declines and extinctions worldwide. Science 306: 1783-1786. Talley BL, Muletz CR, Vredenburg VT, Fleischer RC, Lips KR. 2015. A century of Batrachochytrium dendrobatidis in Illinois amphibians. Biological Conservation. 182: 254-261. Tarrant J, Cilliers D, du Preez LH, Weldon C. Partial assessment of amphibian chytrid fungus (Batrachochytrium dendrobatidis) in South Africa confirms endemic and widespread infection. PLoS One. 2013; 8: e69591. Voyles J, Young S, Berger L, Campbell C, Voyles WF, Dinudom A, et al. 2009. Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian declines. Science. 326:582-585. Voyles J, Johnson LR, Rohr J, Kelly R, Barron C, Miller D, et al. 2017. Diversity in growth patterns among strains of the lethal fungal pathogen Batrachochytrium dendrobatidis across extended thermal optima. Oecologia. DOI 10.1007/s00442-017-3866-8 Vredenburg VT, Felt SA, Morgan EC et al. 2013. Prevalence of Batrachochytrium dendrobatidis in Xenopus collected in Africa (1871–2000) and in California (2001–2010). PLoS ONE, 8, e63791 Weldon C, du Preez LH, Hyatt AD, Muller R, Spears R. 2004. Origin of the amphibian chytrid fungus. Emerging Infectious Diseases, 10, 2100–2105 C h a p t e r I | 14 Chapter I Batrachochytrium dendrobatidis infection of frogs is driven by different biotic and abiotic factors in ponds and streams of the megadiverse Atlantic Forest of Brazil Chapter submitted to Journal of Herpetology on 07/12/2017 This chapter has been formatted following the journal authors’ guide C h a p t e r I | 15 Batrachochytrium dendrobatidis infection of frogs is driven by different biotic and abiotic factors in ponds and streams of the megadiverse Atlantic Forest of Brazil Alba Navarro-Lozano1,6, David Sánchez-Domene2, Jaime Bosch3, Denise C. Rossa-Feres1, Leo R. Malagoli4, Ricardo J. Sawaya5. 1Departamento de Zoologia e Botânica. Universidade Estadual Paulista, São José do Rio Preto, São Paulo, Brazil. 2Instituto de Pesquisa em Bioenergia, Universidade Estadual Paulista, Rio Claro, São Paulo, Brazil. 3Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain 4Departamento de Zoologia e Centro de Aquicultura. Universidade Estadual Paulista, Rio Claro, São Paulo, Brazil. 5Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, São Bernardo do Campo, São Paulo, Brazil. 6Corresponding author. E-mail: alba.navarro.lozano@gmail.com LRH: A. Navarro-Lozano et al. RRH: Bd infection ecological driving factors C h a p t e r I | 16 Abstract.—We assessed how abiotic and biotic factors regulate the prevalence and intensity of Batrachochytrium dendrobatidis (Bd) infection in tadpoles inhabiting ponds and streams in the Atlantic Forest of southeastern Brazil. The prevalence and intensity of Bd did not differ between ponds and streams, but were driven by different environmental factors in the two habitats. In streams, water velocity and depth explained variability in Bd prevalence, but Bd intensities remained unexplained. On the other hand, zooplankton density was the key factor in explaining Bd prevalence and intensity in ponds. Our study contributes to the ecological knowledge of Bd in tropical forests, which will be useful to researchers and wildlife managers in future scientific studies and conservation actions for amphibians. Key-words: Amphibians; Atlantic Forest; Chytrid fungus; Tadpoles; Zooplankton. C h a p t e r I | 17 Resumen.— Se evaluó cómo los factores abióticos y bióticos regulan la prevalencia e intensidad de la infección de Batrachochytrium dendrobatidis (Bd) en renacuajos que habitan charcas y arroyos en la Selva Atlántica del sureste de Brasil. Aunque la prevalencia e intensidad de Bd no difirió entre charcas y arroyos, se observó que estas estaban impulsadas por diferentes factores ambientales en los dos hábitats. En arroyos, la velocidad y la profundidad del agua explicaron la variabilidad en la prevalencia de Bd, sin embargo no fue posible explicar la variabilidad en la intensidad. Por otra parte, la densidad del zooplancton fue el factor clave para explicar la prevalencia e intensidad de Bd en estanques. Con este estudio contribuimos al entendimiento de la ecología de Bd en las selvas tropicales, lo cual será útil para investigadores y gestores en el desarrollo de futuros estudios y planes de conservación de anfibios. Palabras clave: Anfibios; Hongo quitridio; Renacuajos; Selva Atlántica; Zooplancton. C h a p t e r I | 18 INTRODUCTION Parasite virulence, host susceptibility and environmental conditions are all regulatory factors of infection dynamics (Begon et al., 2007). Batrachochytrium dendrobatidis (Bd) is an aquatic parasitic fungus that infects amphibians worldwide and can lead to chytridiomycosis. Both hypo- and hypervirulent lineages of the fungus have been recorded, with the latter associated with the onset of epizootics in America, Australia and Europe (Farrer et al., 2011). However, the presence of this hypervirulent lineage is not always linked to amphibian population declines, with anuran species behavior and life history seemingly involved in regulating infection dynamics (Bielby et al., 2008; Rowley et al., 2013; Blooi et al., 2017). Environmental characteristics, such as temperature, precipitation or moisture, are important regulators of amphibian-Bd interactions (Piotrowski et al., 2004; Fisher et al., 2009; Rohr et al., 2010). Temperature has been identified as one of the most important factors regulating Bd biology, being directly related to seasonality and the distribution of infections in various regions (Daszak et al., 2003; Pounds et al., 2006; Bosch et al., 2007; Kielgast et al., 2009). For instance, increases in seasonal temperature have been associated with decreased prevalence and intensity of Bd infections in amphibian populations (Berger et al., 2004; Phillott et al., 2013). In addition to abiotic factors, biotic interactions seem to regulate Bd infection. Low susceptibility to Bd infection of some species of amphibians has been linked to the presence of bacterial symbionts on their skin (Harris et al., 2006; Brucker et al., 2008). Zooplankton has also been shown to play a regulatory role in Bd infection. C h a p t e r I | 19 Laboratory and mesocosm experiments have demonstrated that zooplankton regulates Bd prevalence by consuming its free-swimming infectious zoospores, thus drastically decreasing tadpole infection prevalence (Hamilton et al., 2012; Searle et al., 2013; Schmeller et al., 2014). We aimed to identify how abiotic and biotic factors regulate the prevalence and intensity of Bd infection in streams and ponds in one of the most preserved and megadiverse regions of the Atlantic Forest in southeastern Brazil. We attempted to answer three main questions: (1) do abiotic characteristics of the environment influence the prevalence and intensity of Bd infection? (2) do communities of zooplankton regulate the prevalence and intensity of Bd infection? and (3) do these regulatory factors act differently in ponds versus streams? MATERIAL AND METHODS Study Site and Sampling.—We sampled 19 permanent water bodies (11 streams and 8 ponds; see figures 1 to 6 in Extra Supplementary Material) in Núcleo Curucutu, a poorly studied area of old growth forest with patches of grassland in Parque Estadual da Serra do Mar in the Atlantic Forest of southeastern Brazil (Garcia and Pirani, 2005). This area serves as a refuge for one of the most biodiverse amphibian faunas of any single locality in the Atlantic Forest, with 66 species from 12 families having already been recorded (Malagoli, 2013). Since Bd prevalence and intensity has been shown to be greater at higher elevations (Gründler et al., 2012), the selected water bodies were at higher elevations (750 to 880 m) of Núcleo Curucutu. C h a p t e r I | 20 Tadpoles between Gosner stages 25 and 40 were sampled in February of 2016 by dip netting. We selected to use individuals within this range of stages because their oral discs are normally completely keratinized and Bd infection occurs only in keratinized tissues (Berger et al., 1998; McDiarmid and Altig, 1999; Berger et al., 1999; Nichols et al., 2001; Marantelli et al., 2004). Once sampled, individuals were euthanized by immersion in lidocaine solution and preserved in 90% alcohol solution for subsequent analyses. Species identification was made by direct comparison of external morphology and tooth row formulae with tadpole specimens deposited in the Amphibia - Tadpoles collection of the Department of Zoology and Botany of UNESP – São José do Rio Preto (DZSJRP-Amphibia-Tadpoles), and the use of dichotomous keys (Heyer et al., 1990; Frost, 2004). The tadpoles collected belonged to six species: Crossodactylus caramaschii (Hylodidae); Physalaemus cuvieri (Leptodactylidae); and Aplastodiscus aff albosignatus, Boana albopunctata (=Hypsiboas albopunctatus), B. faber (=H. faber), and Scinax hayii (Hylidae) (Table S1). Prevalence and Intensity of Bd.—Oral discs of tadpoles were excised (Hyatt et al., 2007) and analyzed individually using real-time PCR following Boyle et al. (2004). DNA extraction was made using PrepMan Ultra (Applied Bioysistems) and amplified using a CFX96TM Real-Time PCR Detection System (Bio-Rad) with a Bd-specific Taqman Assay (Boyle et al., 2004). Each 96-well assay plate included a negative control and four different standards containing DNA from 100, 10, 1 and 0.1 Bd genome equivalents. Samples, negative control and standards were run in duplicate. When both duplicate analyses C h a p t e r I | 21 revealed Bd zoospore genome equivalents >0.1 and amplification curves demonstrated the typical sigmoidal shape, Bd was considered present. When only one of the replicates met these criteria we re-ran the other replicate, and if it still did not comply we considered the sample negative for Bd. Samples that showed signs of inhibition (nonsigmoidal amplification) were further diluted to 1:100 and re-analyzed. The prevalence of Bd (hereafter prevalence) in each water body was determined by dividing the number of infected individuals by the total number of individuals analyzed, which was expressed as a percentage. Numbers of zoospore genomic equivalents recovered from the oral discs are reported as infection intensity. Infection intensity in different water bodies was estimated as mean zoospore load of infected tadpoles in each water body (hereafter intensity), excluding non-infected tadpoles. Data Collection.—The abiotic variables measured in streams were: (1) velocity (average of three measures of water velocity; cm/seg), (2) depth (maximum depth; cm), (3) width (maximum width; m), and (4) vegetation cover (percentage canopy cover measured at five points – two points in each stream margin and one in its center – with a concave densiometer and expressed as the average of the measures). Abiotic variables were measured along 15-m river sections. The abiotic variables measured for ponds were: (1) depth (maximum depth; cm), (2) area (m2), and (3) vegetation cover (both canopy cover and vegetation covering the surface of the water). In order to standardize measurements all abiotic variables were measured by the same person. Zooplankton samples were collected in each water body by filtering (mesh size 45 µm) 20 liters of water to estimate population density. The C h a p t e r I | 22 samples were fixed in 4% formaldehyde and the density of zooplankton (sum of cladocerans, rotifers and copepods; ind/m3) was determined at Laboratório de Ecologia de Zooplâncton da UNESP (São José do Rio Preto, São Paulo, Brazil). We used the sum of densities of these three zooplankton groups, and not the composition of the zooplankton community, because low Bd prevalence has been linked with total zooplankton density (Hite et al., 2016). Data Analysis.—Differences in the prevalence and intensity of Bd infection between ponds and streams were assessed with one-way analysis of variance (ANOVA). Multiple linear regression analyses were used to evaluate the influence of abiotic and biotic parameters on the prevalence and intensity of Bd infection in both kinds of habitats. Prior to regression analyses, multicollinearity among independent variables was assessed using the Variance Inflation Factor (VIF) of the usdm package (Naimi, 2017) of R software (R Core Team, 2016), with a VIF > 3 indicating multicollinearity (Zuur et al., 2010). In addition, prevalence values were logit transformed (Warton and Hui, 2011) in the faraway package (Faraway, 2016) and the explanatory variables were standardized using the “decostand” function included in the vegan package (Oksanen et al., 2017). VIF analysis led to the exclusion of “maximum width” from the set of explanatory variables for streams, and “vegetation cover” from the set of explanatory variables for ponds. Thus, the global model for ponds comprised depth, area, and zooplankton; and the global model for streams comprised velocity, depth, vegetation cover, and zooplankton. For each response variable (prevalence and intensity), we generated all possible models, including a null model (without a predictor variable and considering only the C h a p t e r I | 23 intercept), global models, models with a single variable, and complex models including two or three variables. Sixteen models were thus generated for streams and eight for ponds. In order to better understand which variables best explained both prevalence and intensity, Akaike’s Information Criterion corrected for small samples sizes (AICc) was used to rank all models, with only those with AICc of less than 2.0 being considered for interpretation (Burnham and Anderson, 2002). The likelihood of the models was also assessed using Akaike weights, which quantify the probability that a given model is the best among those selected (Burnham and Anderson, 2002). Normality and homoscedasticity of residuals were visually confirmed to meet the assumptions for parametric statistical testing. Analyses were performed in R-3.3.1 (R-Core Team, 2016). RESULTS Infected tadpoles were found in thirteen of the nineteen water bodies studied (eight streams and five ponds). Prevalence varied from 0% to 67% in ponds and from 0% to 84% in streams; however, one-way ANOVA revealed no significant differences between habits (mean ± standard deviation; 35.00 ± 26.90 in ponds and 48.64 ± 24.22 in streams; F1,11 = 0.89, p = 0.36) (Fig. 1). The intensity of infection also did not different between ponds and streams (127.87 ± 113.12 in streams and 151.86 ± 91.36 in ponds; F1,11 = 0.16, p = 0.69) (Fig. 1, Table S2). C h a p t e r I | 24 Figure 1. (A) Variation in the prevalence (F1,11 = 0.89, p = 0.36) and (B) intensity (F1,11 = 0.16, p = 0.69), of Batrachochytrium dendrobatidis infection in lentic and lotic habitats of Núcleo Curucutu in the Atlantic Forest of southeastern Brazil. Boxes correspond to the 25th to 75th percentiles; bold lines within boxes indicate medians while whiskers indicate minimum and maximum values; empty dots correspond to outliers; black dots are average values. Among the models fitted to assess the influence of abiotic and biotic factors on prevalence of Bd infection (see supplementary Table S3), only one (zooplankton) was considered for ponds, and two (Velocity + Depth and Velocity) for streams (Table 1). Zooplankton density showed a negative relationship with Bd prevalence in ponds, and explained 85% of the variation in prevalence (Table 1). In turn, both velocity and depth explained 91% of the variation in Bd prevalence in streams (velocity in isolation explained up to 86%), with velocity showing a negative relationship and depth a positive relationship (Table 1). Regarding intensity, only the zooplankton model was considered for ponds, while none of the models were better than the null model for streams (Table 1 and Table S4). Again, zooplankton was highlighted as a regulatory factor for Bd infection in ponds, explaining 50% of the variation in intensity of infection. B A C h a p t e r I | 25 Table 1. Models considered in examining the relationships between abiotic and biotic factors of ponds and streams, and the prevalence and intensity of Batrachochytrium dendrobatidis infection in Núcleo Curucutu in the Atlantic Forest of southeastern Brazil. Models ΔAICc df wAICc R2 p β0 β1 β2 STREAMS Prevalence Velocity + Depth 0 4 0.506 0.91 <0.001 -2.56 -4.71 1.17 Velocity 0.5 3 0.391 0.86 <0.001 -2.56 -4.08 - NULL 19.4 2 <0.001 - - - - - Intensity NULL 0 2 0.532 - - - - - PONDS Prevalence Zooplankton 0 3 0.954 0.85 <0.001 -4.05 -4.16 - NULL 11.1 2 0.004 - - - - - Intensity Zooplankton 0 3 0.589 0.50 0.03 94.9 -79.03 - NULL 1.2 2 0.329 - - - - - DISCUSSION Despite numerous studies reporting that amphibians inhabiting tropical streams are more severely impacted by Bd (Laurence, 1996; Berger et al., 1998, 1999; Lips, 1999; Kriger and Hero, 2007; Blooi et al., 2017), neither prevalence nor intensity of Bd infection differed between streams and ponds in C h a p t e r I | 26 the present study. This discrepancy could be due to the fact that, for the most part, these other studies were performed on adult individuals, while our study focused on tadpoles, and it is well known that Bd affects the different life phases of amphibians differentially (Berger et al., 2016). Therefore, studies of the adult amphibian community at Núcleo Curucutu are needed to confirm whether they have the same prevalence and intensity of Bd infection in streams and ponds, or whether they follow the pattern observed in adults studied at other locations. The present study found that in streams, Bd prevalence was negatively related to water velocity. Piotrowski et al. (2004) reported that Bd zoospores loose their capacity for motility after 24 hours, and during that period the zoospores reached distances of less than 2 cm. Therefore, water velocity could be acting as a barrier to dispersion, and thus preventing encystment of zoospores in the oral discs of tadpoles. This possibility was also suggested by Medina et al. (2015), who pointed out that increased water flow could reduce the density of infectious particles, reducing the likelihood of successful Bd transmission. Since no data related to Bd zoospore resistance to water currents could be found, we encourage future research to study zoospores performance in order to better understanding their behavior in the field. Although the fastest streams exhibited a negative effect on the prevalence of Bd, those with greater depths seemed to favor the prevalence of Bd. The explanation for this may be that the deepest zones of streams possess lower temperatures and lower water velocity (Tarbuck and Lutgens, 2005), which could propitiate the presence of microhabitats that serve as refugia from the current for both Bd zoospores and tadpoles, and thus promote host-parasite interactions and increase Bd prevalence among tadpoles. C h a p t e r I | 27 On the other hand, when zooplankton density was higher, the prevalence of Bd decreased. Indeed, the three ponds with the highest zooplankton density did not possess tadpoles infected with Bd. Furthermore, zooplankton density also explained a significant amount of the variation in infection intensity, with lower infection intensities occurring in tadpoles inhabiting ponds with higher densities of zooplankton. A similar trend was found in lentic habitats of Spain (high mountain ponds) and Honduras (bromeliads) (Schmeller et al., 2014; Hite et al., 2016; Blooi et al., 2017). These studies, and ours, demonstrate that biotic factors may be important regulators of Bd in the wild. Furthermore, they support the results of laboratory and mesocosm experiments that showed zooplankton to be predators of Bd zoospores [e.g. Hamilton et al., 2012; Searle et al., 2013; Schmeller et al., 2014). None of the models assessed met our selection criteria for the analysis of the intensity of streams. We consider there to be three non-mutually exclusive explanatory hypotheses. First, we may have failed to select key explanatory variables to study. Second, infection intensity could be species dependent (host susceptibility), which, if true, would mean that environmental factors are acting as filters for the presence of Bd in habitats, but once an individual becomes infected, the susceptibility of the particular species would become an additional key factor for the intensity of infection. However, our ANOVA analyses that assessed differences in Bd intensity among the studied species do not support this proposal (see supplemental Table S5). Third, the intensity of infection is dependent on the particular Bd isolate present in each stream. Indeed, a high level of phenotypic diversity among isolates in proximate streams was documented in Serra do Mar (Lambertini et al., 2016). Thus, the confirmation of C h a p t e r I | 28 the existence of different isolates in our study area is the only way to test this hypothesis. We conclude that, in a megadiverse region of the Brazilian Atlantic Forest, Bd infection is driven by biotic and abiotic factors, but in different ways in ponds and streams. We also identify zooplankton as an important factor in the ecology of Bd in lentic habitats, and so we encourage researchers to investigate in-depth which zooplankton species are potentially responsible for this Bd regulation. This study contributes to understanding the ecology of Bd in tropical forests, which has great significance for amphibian conservation actions. Acknowledgments.—We thank Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) and ComissãoTécnico-Científica do Instituto Florestal (COTEC; a committee of Instituto Florestal, the state research agency responsible for the reserve) for the collection permits that they provided us (#47148-2 and SMA #260108-001.809/2015, respectively). We thank Asociación Universitaria Iberoamericana de Postgrado (AUIP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for doctoral fellowships. We also thank Fundação de Amparo à Pesquisa do Estado de São Paulo (#2010/52321-7; #2014/23677-9) and Fundación BBVA for finnacial support. We are in debt to F.S. Annibale, C.E. Sousa and D. Garcia for help with fieldwork; A. Otero for her help with zooplankton sampling; M.S.M. Castilho-Noll for help with zooplankton identification; P. Ghosh for help in designing the sampling protocol; and C. Monsalve for help with qPCR analyses. C h a p t e r I | 29 LITERATURE CITED Begon, M., C. R. Townsend, and J. L. Harper. 2007. Ecologia. De Indivíduos a Ecossistemas. 4th ed. Artmed Editora. Berger, L., R. Speare, P. Daszak, D. E. Green, A. A. Cunningham, C. L. Goggin, R. Slocombe, M. A. Ragan, A. D. Hyatt, K. R. McDonald, H. B. Hines, K. R. Lips, G. Marantelli, and H. Parkes. 1998. Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proceedings of National Academy of Sciences USA 95:9031–9036 Berger, L., R. Speare, and A. Hyatt. 1999. Chytrid fungi and amphibian declines: Overview, implications and future directions. Pp. 23-33 in A. Campbell (Ed.), Declines and disappearances of Australian frogs. Canberra, Australia Berger, L., R. Speare, H. B. Hines, G. Marantelli, A. D. Hyatt, K. R. McDonald, L. F. Skerratt, V. Olsen, J. M. Clarke, G. Gillespie, M. Mahony, N. Sheppard, C. Williams, and M. J. Tyler. 2004. Effect of season and temperature on mortality in amphibians due to chytridiomycosis. Australian Veterinary Journal 82:434-439. Berger, L., A. A. Roberts, J. Voyles, J. E. Longcore, K. A. Murray, and L. F. Skerratt. 2016. History and recent progress on chytridiomycosis in amphibians. Fungal Ecology 19:89-99. Bielby, J., N. Cooper, A. A. Cunningham, T. W. J. Garner, and A. Purvis. 2008. Predicting susceptibility to future declines in the word’s frogs. Conservation Letters 1:82-90. Blooi, M., A. E. Laking, A. Martel, F. Haesebrouck, M. Jocque, T. Brown, S. Green, M. Vences, M. C. Bletz, and F. Pasmans. 2017. Host niche may determine disease-driven extinction risk. PLoS ONE 12:e0181051. https://doi.org/10.1371/journal.pone.0181051 Bosch, J., L. M. Carrascal, L. Durán, S. Walker, and M. C. Fisher. 2007. Climate change and outbreaks of amphibian chytridiomycosis in a montane area of Central Spain; is there a link? Proceedings of Royal Society B 274:253-260. Boyle, D. G., D. B. Boyle, V. Olsen, J. A. T. Morgan, and A. D. Hyatt. 2004. Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Diseases of Aquatic Organisms 60:141-148. Brucker, R. M., R. N. Harris, C. R. Schwantes, T. N. Gallaher, D. C. Flaherty, B. A. Lam, K. P. C. Minbiole. 2008. Amphibian chemical defense: antigunal metabolites of the microsymbiont Janthinobacterium lividum on the salamander Plethodoncinereus. Journal of Chemical Ecology 341:1422-1429. Burnham, K. P., and D. R. Anderson. 2002. Model selection and multimodel inference: a practical information-theoretic approach. New York, USA. Daszak, P., A. A. Cunningham, and A. D. Hyatt. 2003. Infectious disease and amphibian population declines. Diversity and Distribution 9:141-150. Faraway, J. 2016. Package “faraway”. Functions and datasets for books by Julian Faraway. Available at: https://CRAN.R-project.org/package=faraway Farrer, R.A., L. A. Weinert, J. Bielby, T. W. J. Garner, F. Balloux, F. Clare, J. Bosch, A. A. Cunningham, C. Weldon, L. H. Preez, L. Anderson, S. L. K. Pond, R. Shahar-Golan, D. A. Henk, and M.C. Fisher. 2011. Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage. Proceedings of National Academy of Sciences USA 108:18732– 18736. Fisher, M.C., T. W. J. Garner, and S. F. Walker. 2009. Global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time and host. Annual Review of Microbiology 63:291-310. Frost, D. R. 2004. Amphibian species of the world: an online reference. Available at http://research.amnh.org/herpetology/amphibia/index. html. Accessed on 24 July 2017. Garcia, R. J. F., and J. R. Pirani. 2005. Análise sobre a interferência antrópica na origem dos campos do Núcleo Curucutu, Parque Estadual da Serra do Mar, São Paulo. Paisagem Ambiente: ensaios 20:131-151. Gründler, M. C., L. F. Toledo, G. Parra-Olea, C. F. B. Haddad, L. O M. Giasson, R. J. Sawaya, C. P. A. Prado, O. G. S. Araujo, F. J. Zara, F. C. Centeno, and K. R. Zamudio. 2012. Interaction between breeding habitat and elevation affects prevalence but not infection intensity of Batrachochytrium dendrobatidis in Brazilian anuran assemblages. Diseases of Aquatic Organisms 97:173-184. Hamilton, P. T., J. M. L. Richardson, and B. R. Anholt. 2012. Daphnia in tadpole mesocosms: trophic links and interactions with Batrachochytrium dendrobatidis. Freshwater Biology 57: 676-683. Harris, R. N., T. Y. James, A. Lauer, M. A. Simon, and A. Patel. 2006. Amphibian pathogen Batrachochytrium dendrobatidis is inhibited by the cutaneous bacteria of amphibian species. EcoHealth 3:53-56. Heyer, W.R., A. S. Rand, C. A. G. da Cruz, O. L. Peixoto, and C. E. Nelson. 1990. Frogs of Boracéia. São Paulo, Brazil. Hite, J. L., J. Bosch, S. Fernández-Beaskoetxea, D. Medina, and S. R. Hall. 2016. Joint effects of habitat, zooplankton, host stage structure and diversity on amphibian chytrid. Proceedings of Royal Society B 283:20160832. http://dx.doi.org/10.1098/rspb.2016.0832 Hyatt, A. D., D. G. Boyle, V. Olsen, D. B. Boyle, L. Berger, D. Obendorf, A. Dalton, K. Kriger, M. Hero, H. Hines, R. Phillott, R. Campbell, G. Marantelli, F. Gleason, and A. Colling. 2007. Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 73:175-192. Kielgast, J., D. Rödder, M. Veith, and S. Lötters. 2009. Widespread occurrence of the amphibian chytrid fungus in Kenya. Animal Conservation. 13:36-43. Kriger, K. M., and J. M. Hero. 2007. The chytrid fungus Batrachochytrium dendrobatidisis non-randomly distributed across amphibian breeding habitats. Diversity and Distribution 13:781-788. Lambertini, C., C. G. Becker, T. S. Jenkinson, D. Rodriguez, D. S. Leite, T. Y. James, K. R. Zamudio, and L. F. Toledo. 2016. Local phenotypic variation in https://doi.org/10.1371/journal.pone.0181051 https://cran.r-project.org/package=faraway http://research.amnh.org/herpetology/amphibia/index.html http://research.amnh.org/herpetology/amphibia/index.html C h a p t e r I | 30 amphibian-killing fungus predicts infection dynamics. Fungal Ecology 20:15-21. Laurence, W.F. 1996. Catastrophic declines of Australian rainforest frogs: Is unusual weather responsible? 1996. Biological Conservation 77:203-212. Lips, K. R. 1999. Mass mortality and population declines of anuran at an upland site in western Panama. Conservation Biology 13:117-125. Malagoli, L.R. 2013. Diversidade e distribuição dos anfíbios anuros do Núcleo Curucutu, Parque Estadual da Serra do Mar, SP. M. Sc. Thesis, Universidade Estadual Paulista, Brazil. Marantelli, G., L. Berger, R. Speare, and L. Keegan. 2004. Distribution of the amphibian chytrid Batrachochytrium dendrobatidis and keratin during tadpole development. Pacific Conservation Biology 10:173-79. McDiarmid, R. W., and R. Altig. 1999. Tadpoles: the biology of anuran larvae. Chicago, USA. Medina, D., T. W. J. Garner, L. M. Carrascal, and J. Bosch. 2015. Delayed metamorphosis of amphibian larvae facilitates Batrachochytrium dendrobatidis transmission and persistence. Diseases of Aquatic Organisms 117: 85-92. Naimi, B. 2017. Package “usdm”. Uncertainty Analysis for Species Distribution Models. Available at https://CRAN.R-project.org/package=usdm Nichols, D. K., E. W. Lamirande, A. P. Pessier, and J. E. Longcore. Experimental transmission of cutaneous chytridiomycosis in dendrobatid frogs. Journal of Wildlife Diseases. 37:1-11. Oksanen, J., F. G. Blanchet, M. Friendly, R. Kindt, P. Legendre, D. McGlinn, P. R. Minchin, R. B. O’Hara, G. L. Simpson, P. Solymos, M. H. H. Stevens, E. Szoecs, and H. Wagner. 2017. Package “vegan”. Community ecology package. Available at https://CRAN.R-project.org/package=vegan Phillott, A. D., L. F. Grogan, S. D. Cashins, K. R. McDonald, L. Berger, and L. F. Skerratt. 2013. Chytridiomycosis and seasonal mortality of tropical stream-associated frogs 15 years after introduction of Batrachochytrium dendrobatidis. Conservation Biology 0:1-11. Piotrowski, J. S., S. L. Annis, and J. E. Longcore. 2004. Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians. Mycologia 96:9-15. Pounds, J. A., M. R. Bustamante, L. A. Coloma, J. A. Consuegra, M. P. L. Fogden, P. N. Foster, E. La Marca, K. L. Masters, A. Merino-Viteri, R. Puschendorf, S. R. Ton, G. A. Sánchez-Azofeifa, C. J. Still, and B. E. Young. 2006. Widespread amphibian extinctions from epidemic disease driven by global warming. Nature 439:161–167. R Core Team. 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing. Available from: https://www.R- project.org/. Rohr, J. R., and T. R. Raffel. 2010. Linking global climate and temperature variability to widespread amphibian declines putatively caused by disease. Proceedings of National Academy of Sciences USA 107:8269- 8274. Rowley, J. J. L., and R. A. Alford. 2013. Hot bodies protect amphibians against chytrid infection in nature. Nature 3:1515. doi:10.1038/srep01515 Schmeller, D. S., M. Blooi, A. Martel, T. W. J. Garner, M. C. Fisher, F. Azemar, F. C. Clare, C. Leclerc, L. Jäger, M. Guevara-Nieto, A. Loyau, and F. Pasmans. 2014. Microscopic aquatic predators strongly affect infection dynamics of a globally emerged pathogen. Current Biology 24:176-180. Searle, C. L., J. R. Mendelson, L. E. Green, and M. A. Duffy. 2013. Daphnia predation on the amphibians chytrid fungus and its impacts on disease risk in tadpoles. Ecology and Evolution 3:4129-4138. doi:10.1002/ece3.777 Tarbuck, E. J., and F. K. Lutgens. 2005. Ciencias de la Tierra. Una introducción a la geología física. Madrid, Spain. Warton, D.I., and F. K. C. Hui. 2011. The arcsine is asinine: the analysis of proportions in ecology. Ecology 92:3-10. Zuur, A. F., E. N. Ieno, and C. S. Elphick. 2010. A protocol for data exploration to avoid common statistical problems. Methods in Ecology and Evolution 1:3-14. https://cran.r-project.org/package=usdm https://cran.r-project.org/package=vegan C h a p t e r I | 31 SUPPLEMENTARY MATERIAL TABLE S1. Species sampled in Núcleo Curucutu. N = number of individuals; ST = streams; and PO = ponds. Habitat N Family Hylodidae Crossodactylus caramaschii ST 34 Family Leptodactylidae Physalaemus cuvierii PO 20 Family Hylidae Aplastodiscus aff. albosignatus ST/PO 143/54 Boana albopunctata PO 63 Boana faber PO 33 Scinax hayii PO 32 C h a p t e r I | 32 TABLE S2. Sample of tadpoles used to determine the prevalence and intensity of Batrachochytrium dendrobatidis infection at the study sites. N = number of tadpoles analyzed; Ni = number of tadpoles infected; Pv = prevalence per site; Int = intensity (average zoospores load per site). N Ni Pv (%) Int Pond_1 29 0 0.0 0 Pond_2 33 1 3.0 59.9 Pond_3 18 0 0.0 0 Pond_4 39 12 30.8 82.3 Pond_5 5 0 0.0 0 Pond_6 12 2 16.7 255.4 Pond_7 45 25 57.8 118.8 Pond_8 21 14 66.7 242.9 Stream_1 4 0 0.0 0 Stream_2 35 20 60.0 160.2 Stream_3 38 31 84.2 83.3 Stream_4 20 12 60.0 61.3 Stream_5 36 23 66.7 74 Stream_6 17 9 52.9 79 Stream_7 10 1 20.0 145.3 Stream_8 6 1 16.7 387.5 Stream_9 7 2 28.6 32.4 Stream_10 1 0 0.0 0 Stream_11 3 0 0.0 0 C h a p t e r I | 33 TABLE S3. Models fitted to examine relationships among biotic and abiotic factors and the prevalence of Batrachochytrium dendrobatidis. ΔAICc = difference in corrected Akaike’s information criteria; df = degrees of freedom; wAICc = weights of corrected Akaike’s information criteria. Models ΔAICc df wAICc STREAMS Velocity + Depth 0 4 0.5062 Velocity 0.5 3 0.3910 Velocity + Zooplankton 5.1 4 0.0392 Velocity + Canopy Cover 5.4 4 0.0345 Velocity + Zooplankton + Depth 7.1 5 0.0142 Velocity + Depth + Canopy Cover 7.2 5 0.0135 Velocity + Zooplankton + Canopy Cover 12 5 0.0012 Canopy Cover + Velocity + Depth + Zooplankton 18 6 <0.001 NULL 19.4 2 <0.001 Canopy Cover 21.5 3 <0.001 Depth 22.2 3 <0.001 Zooplankton 23.2 3 <0.001 Canopy Cover + Depth 25.6 4 <0.001 Zooplankton + Canopy Cover 26.7 4 <0.001 Zooplankton + Depth 27.5 4 <0.001 Canopy Cover + Zooplankton + Depth 32.9 5 <0.001 PONDS Zooplankton 0 3 0.9544 Zooplankton + Depth 6.9 4 0.0308 Zooplankton + Area 9 4 0.0104 NULL 11.1 2 0.0038 Depth 15.8 3 <0.001 Area 16.6 3 <0.001 Area + Depth 25 4 <0.001 Area + Depth + Zooplankton 25.4 5 <0.001 C h a p t e r I | 34 TABLE S4. Model sets fitted to examine the relationship between biotic and abiotic factors and the intensity of Batrachochytrium dendrobatidis infections. ΔAICc = difference in corrected Akaike’s information criteria; df = degrees of freedom; wAICc = weights of corrected Akaike’s information criteria. Models ΔAICc df wAICc STREAMS NULL 0 2 0.532 Velocity 2.8 3 0.128 Cover 3.4 3 0.096 Zooplankton 3.5 3 0.093 Depth 3.9 3 0.077 Cover + Velocity 6.6 4 0.019 Depth + Velocity 7 4 0.015 Zooplankton + Velocity 7.8 4 0.010 Cover + Zooplankton 8.1 4 0.009 Zooplankton + Depth 8.5 4 0.007 Cover + Depth 8.6 4 0.007 Cover + Depth + Velocity 12 5 0.001 Cover + Zooplankton + Velocity 13.6 5 <0.001 Zooplankton + Depth + Velocity 13.8 5 <0.001 Cover + Zooplankton + Depth 15.2 5 <0.001 Cover + Depth + Velocity + Zooplankton 22.1 6 <0.001 PONDS ΔAICc df wAICc Zooplankton 0 3 0.589 NULL 1.2 2 0.329 Area 6.1 3 0.027 Zooplankton + Area 6.2 4 0.026 Depth 6.6 3 0.022 Zooplankton + Depth 9.3 4 0.005 Area + Depth 15.4 4 <0.001 Zooplankton + Depth + Area 24.8 5 <0.001 C h a p t e r I | 35 TABLE S5. Intensity of Batrachochytrium dendrobatidis infection per species in streams and ponds (mean of zoospores and standard deviation), and the results of analyses of variance among species sharing each habitat. Aplastodiscus aff. albosignatus Boana albopunctata Boana faber Scinax hayii Crossodactylus caramaschii Physalaemus cuvieri ANOVA p-value F Ponds 118.8 ± 88.8 76.5 ± 51.3 94.4 ± 44.9 244.5 ± 256.4 - 0 0.26 1.37 Streams 94 ± 88.1 - - - 161.1 ± 163.1 - 0.48 0.49 C h a p t e r I | 36 EXTRA SUPPLEMENTARY MATERIAL Figures 1-3. Examples of lentic localities sampled in Núcleo Curucutu, Parque Estadual da Serra do Mar, São Paulo state, southeastern Brazil. C h a p t e r I | 37 Figures 4-6. Examples of lotic localities sampled in Núcleo Curucutu, Parque Estadual da Serra do Mar, São Paulo state, southeastern Brazil. C h a p t e r I I | 38 Chapter II Global warming could decrease potential geographic distribution of amphibian-killing fungus in Brazilian Atlantic Forest This chapter will be submitted to Diversity and Distributions, and will be exposed in the IBS Climate Change Biogeography Meeting (Évora – Portugal, March 2018) This chapter has been formatted following the journal authors’ guide. C h a p t e r I I | 39 Global warming could decrease potential geographic distribution of amphibian-killing fungus in Brazilian Atlantic Forest Alba Navarro-Lozano1*, David Sánchez-Domene2, Jaime Bosch3, Daniel N. Coáguila4, Denise C. Rossa-Feres1, Eduardo A. Almeida5, Ricardo J. Sawaya6 1Departamento de Zoologia e Botânica. Universidade Estadual Paulista, São José do Rio Preto, São Paulo, Brazil. 2Instituto de Pesquisa em Bioenergia, Universidade Estadual Paulista, Rio Claro, São Paulo, Brazil. 3Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain. 4Instituto de Ensino Superior de Rio Verde, Goias, Brazil. 5Department of Natural Sciences, Regional University of Blumenau, Blumenau, Santa Catarina, Brazil. 6Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, São Bernardo do Campo, São Paulo, Brazil. *Correspondence: Alba Navarro-Lozano, Dpto Zoologia e Botânica, Universidade Estadual Paulista, São José do Rio Preto, São Paulo, Brazil. E-mail: alba.navarro.lozano@gmail.com ABSTRACT To better understand the possible effects of climate change in the potential geographic distribution in the Brazilian Atlantic Forest of Batrachochytrium dendrobatidis (Bd), we constructed species distribution models for Bd for the present and future climatic scenarios. For present time, 58.8% of the Brazilian Atlantic Forest present suitable areas for Bd. Future scenarios for year 2070 forecast a reduction of these areas by between 27.5% and 42.6%. Future projections also indicate that Bd suitable areas will remain mainly in highlands of the southern half of the biome, tending to disappear from lowlands and low latitudes. In the light of these results, we recommend monitoring amphibian populations inhabiting areas that could correspond as refuges for Bd in the near future, which would enable prompt responses from possible outbreak of the disease in those areas. C h a p t e r I I | 40 Key words Batrachochytrium dendrobatidis, Brazilian Atlantic Forest, Conservation, Climate Change, Global Warming, Species Distribution Model C h a p t e r I I | 41 INTRODUCTION The aquatic chytrid fungus Batrachochytrium dendrobatidis (Bd), described at the end of the last century (Berger et al., 1998; Longcore et al., 1999) has been identified as responsible for the decline and extinction of amphibian populations in different parts of the world (Stuart et al., 2004; Skerratt et al., 2007; Olson et al., 2013). Chytridiomycosis, name given to the infectious disease caused by this fungus, is considered the biggest disease-driven vertebrate biodiversity loss ever recorded (Berger et al, 1998, 1999; Lips, 1998, 1999; Carnaval et al., 2005; Skerratt et al., 2007; Bosch et al., 2001; Bai et al., 2010; Kielgast et al., 2009; http://www.bd-maps.net). Since the disease was highlighted as the main cause of global amphibian declines, Bd has become one of the best studied wildlife pathogens by the scientific community and has also meant an increase in the role given to infectious diseases in species conservation plans (Berger et al., 2016). The increment in virulence of a local Bd isolates (Fisher et al., 2009, Phillips et al., 2013); the arrival of a novel Bd isolate from Asia, probably belonging to Bd-GPL (Global Panzootic Lineage), the most virulent Bd linage that has been associated with amphibian population declines worldwide (Farrer et al., 2011); and, collateral effects of climate change (Pounds et al., 1999, 2006; Bosch et al., 2007; Rohr et al., 2010), are the hypotheses that the scientific community proposed to explain these declines. Recents studies have pointed that the arrival of the Bd-GPL lineage, possibly by the trade of species, could be the begginig of the important population declines that occurred in Central America, Australia and Spain (Dr. Jaime Bosch personal communication, unpublished data), and that climate change could be responsible of increased http://www.bd-maps.net/ C h a p t e r I I | 42 chytridiomycosis epizootics in regions where the fungus is already present (Raffel et al., 2013, 2015). Therefore, understanding the effects of climatic change on Bd potential geographic distribution is essential for prediction of future chytridiomycosis epizootics. The use of Species Distribution Models (SDMs; see Franklin, 2010; Guisan et al, 2017) to predict potential geographic distribution of Bd has been applied in different regions affected by chytridiomycosis (e.g. Ron et al., 2005; Rödder et al., 2009; Liu et al., 2012; James et al., 2015). However, the knowledge about future potential distributions associated to climate change scenarios are still poorly explored. The chytridiomycosis is affecting more severely pristine areas characterized by short-range temperatures, abundant rainfalls and high amphibian richness (Olson et al., 2013). Although the Brazilian Atlantic Forest (BAF) does not escape from Bd (Navarro-Lozano, 2016), amphibian population declines linked to chytridiomycosis have not been demonstrated for this bioma. Therefore, to better understand the potential implications of climate change over Bd presence in BAF, we build species distribution models to analyze current and future potential distribution of chytrid fungus in this high diverse area. METHODS Data collection Our data correspond to positive cases of Bd-infection combining both published scientific reports (198 localities; see Navarro-Lozano, 2016) and our own field samples (five localities). Our field study were conducted in Nucleo Curucutu (NC), Parque Estadual da Serra do Mar (São Paulo state, southeastern Brazil). This old growth area shows low human activity and is one C h a p t e r I I | 43 of the less studied regions in São Paulo state Atlantic Forest (Garcia & Pirani, 2005). Twenty-one adults were captured in nine different locations of NC during February 2016, and swabbed gently with sterile cotton swabs following Hyatt et al. (2007) procedure, and released back into their place of capture. Swabs preserved in alcohol 90% were analyzed in duplicate to detect presence of Bd following the methodology described by Boyle et al. (2004). We considered Bd presence when both analysis performed to each swab detected Bd, zoospore genome equivalents were >0.1, and the amplification curves have sigmoidal shape. The analyses determined Bd presence in six adults from five different locations, all of them with no apparent signs of chytridiomycosis (see Appendix S1 in Supporting Information). Species Distribution Modeling Our predictor variables correspond to four bioclimatic variables of present time with a spatial resolution of 2.5 min, downloaded from WorldClim databased (v1.4; Hijmans et al., 2005). This set of predictor variables, Mean diurnal range (Bio2; Mean of monthly (maximum temperature - minimum temperature)), Maximum temperature of warmest month (Bio5), Annual Precipitation (Bio 12), and Precipitation of warmest quarter (Bio18) were selected based on their relevance to Bd biology in both, field (Kriger & Hero, 2007; Kriger et al., 2007; Kriger, 2009) and laboratory (Piotrowski et al., 2004; Woodhams et al., 2008) studies. For future projections, we inlcuded the same set of predictor variables for the year 2070 by four different climatological research centers: The Beijing Climate Center Climate System Model (BCC-CSM1-1), The Hadley Centre Global Environment Model (HadGEM2-ES), The Institute Pierre Simon Laplace C h a p t e r I I | 44 (IPSL-CM5A), and the Community Climate System Model (CCSM4). All future climatic models were obtained from WorldClim database (v1.4; Hijmans et al., 2005). Each of the four Atmosphere-Ocean General Circulation Model (AOGCM) of future climatic models was composed by four different scenarios of Representative Concentration Pathways (RCPs) 2.6, 4.5, 6.0 and 8.5 (see Wayne, 2013 and Vuuren et al., 2011 for details). To create the potential current distribution model of Bd in BAF, we implemented a multi-model ensemble strategy, including four different algorithms, Maxent, BRT, GAM and GLM, in the R package “sdm” (Naimi & Araújo, 2016). In turn, future potential projections were made by a consensus of the four different AOGCMs for each RCPs. Previously to the modeling, we assessed spatial autocorrelation of the data by a Mantel test in package ecospat (Broennimann et al. 2016) in R (R Core Team, 2016). Distances equal or greater than to 55 km among localities did not present spatial autocorrelation; therefore to avoid spurious results, occurrence data was rarefied in spThin (Aiello-Lammens et al., 2014), considering only localities separated by at least 55 km. Then, from the 203 original records, a set of 60 localities remained for modeling procedures. Models were calibrated a random 75% of the final data set (training sample) and evaluated by the remaining 25% (test data). Performance of models were evaluated by the area under the Receiver Operating Characteristic curve (AUC), and the true skill statistic (TSS). AUC is a common method for evaluating the accuracy (ability of the model to classify correctly a specie as present or absent) of classification models (Fielding & Bell, 1997), and varies between 0 and 1, with 1 indicating a perfect prediction (model discriminates C h a p t e r I I | 45 perfectly between presence and absence records) and values below 0.5 indicating random prediction (Hanley & McNeil, 1982). TSS is defined as sensitivity (correctly classified presences) + specificity (correctly classified absences) -1 (Allouche et al., 2006). TSS varies between -1 and +1, where -1 indicates a performance worse than those randomly expected and +1 indicates a perfect fit (Allouche et al. 2006). Models with AUC values over 0.80 and TSS values over 0.50 are considered high-accurate and with strong predictive power (Araujo et al., 2005; Allouche et al., 2006), only the algorithms with equal or higher of these values were included in the consensus model. The background of the geographic studied area impacts on the appropriateness and accuracy of the model prediction when extrapolating the species distribution in space (VanDerWal et al., 2009; Elith et al., 2011). In order to check the effect of spatial extent in our models, four different geographic backgrounds where tested: BAF and BAF plus buffer areas of 100, 200 and 300 km (see Appendix S2). In turn, since there is no consensus in a number of pseudo-absences to be used in SDMs of presence-ony datasets, and given that the relationship between the amount of occurrence data and the absence data influence the accuracy of the model (McPherson et al., 2004; Barbet-Massin et al., 2012), we assessed four different amounts of pseudo- absences for each of the study backgrounds: same pseudo-absences than occurrences and five, ten and one hundred times more pseudo-absences than occurrences. Preliminary analyses made us to choose the universe BAF plus buffer area of 300 Km with five times more pseudo-absences than occurrences (see results of this preliminary analysis in Appendix S3). C h a p t e r I I | 46 All projections of potential Bd presence were reclassified in five classes for easier interpretation using ArcGis® 10.0 (ArcMapTM) ESRI. The first of this five classes represents Bd absence in the maps. The Bd positive locality with the lower occurrence probability value was used as turning limit for Bd presence, that is, all areas with lower values were assumed as free of Bd. The remaining four classes represented different probability ranges of Bd occurrence (see Fig 1). RESULTS The Bd positive locality with lower value of suitability in models for present time had a value of 0.124, and was used as turning limit between presence and absence for current and future maps (first class in the maps of the Fig 1), with which were calculated the potential occurrence areas for Bd. Present time potential occurrence area for Bd in BAF varied from 617,787 Km2 (GLM) to 717,579 Km2 (GAM) (see details in Table 1). All SDMs algorithms presented high-accuracy and strong predictive power (AUC ≥ 0.84, TSS ≥ 0.6). Therefore, all of them were included in the consensus current potential distribution model which estimated an area of 665,769.2 km2 of potential occurrence for Bd (58.8% of the BAF territory). The BAF areas with higher probabilities of Bd occurrence are largely concentrated in its southern half, although there is a smalle area with moderate probabil