DISEASES OF AQUATIC ORGANISMS Dis Aquat Org Vol. 124: 109–116, 2017 https://doi.org/10.3354/dao03114 Published April 20 INTRODUCTION Amphibian fungal pathogens of the genus Batra- chochytrium are linked to global population declines in anurans and salamanders through the infectious disease chytridiomycosis (Berger et al. 1998, Lips et al. 2008, Martel et al. 2013). The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) has been acknowledged as one of the most destructive patho- gens to wildlife (Fisher & Garner 2007, Skerratt et al. 2007), causing population declines in a large fraction of infected species. Bd is a host-generalist pathogen among amphi bians (Valencia-Aguilar et al. 2015), infecting anurans and salamanders from tropical and temperate regions (Lips et al. 2006, 2008, Vreden- burg et al. 2010, Cheng et al. 2011). Reports of Bd infection in caecilians (Gymnophiona), however, are rare in the literature. To our knowledge, only 4 © Inter-Research 2017 · www.int-res.com*Corresponding author: lambertini.carol@gmail.com Spatial distribution of Batrachochytrium dendrobatidis in South American caecilians Carolina Lambertini1,*, C. Guilherme Becker2, Cecilia Bardier1,3, Domingos da Silva Leite4, Luís Felipe Toledo1 1Laboratório de História Natural de Anfíbios Brasileiros (LaHNAB), Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo 13083-862, Brazil 2Departamento de Zoologia, Universidade Estadual Paulista, Rio Claro, São Paulo 13506-900, Brazil 3Laboratorio de Sistemática e Historia Natural de Vertebrados, Instituto de Ecología y Ciencias ambientales, Universidad de la República, Montevideo 11400, Uruguay 4Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo 13083-862, Brazil ABSTRACT: The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) is linked to population declines in anurans and salamanders globally. To date, however, few studies have attempted to screen Bd in live caecilians; Bd-positive caecilians have only been reported in Africa and French Guiana. Here, we performed a retrospective survey of museum preserved specimens to (1) describe spatial patterns of Bd infection in Gymnophiona across South America and (2) test whether areas of low climatic suitability for Bd in anurans predict Bd spatial epidemiology in caecilians. We used quantitative PCR to detect Bd in preserved caecilians collected over a 109 yr period, and performed autologistic regressions to test the effect of bioclimatic metrics of tempera- ture and precipitation, vegetation density, and elevation on the likelihood of Bd occurrence. We detected an overall Bd prevalence of 12.4%, with positive samples spanning the Uruguayan savanna, Brazil’s Atlantic Forest, and the Amazon basin. Our autologistic models detected a strong effect of macroclimate, a weaker effect of vegetation density, and no effect of elevation on the likelihood of Bd occurrence. Although most of our Bd-positive records overlapped with reported areas of high climatic suitability for the fungus in the Neotropics, many of our new Bd-positive samples extend far into areas of poor suitability for Bd in anurans. Our results highlight an impor- tant gap in the study of amphibian chytridiomycosis: the potential negative impact of Bd on Neotropical caecilians and the hypothetical role of caecilians as Bd reservoirs. KEY WORDS: Chytrid infection dynamics · Gymnophiona · Life history · Environmental variables · Museum specimens Resale or republication not permitted without written consent of the publisher Dis Aquat Org 124: 109–116, 2017 reports of Bd from wild-caught caecilians are avail- able to date (Doherty-Bone et al. 2013, Gower et al. 2013, Hydeman et al. 2013, Rendle et al. 2015). The first study screening for Bd in caecilians re - ported 53 Bd-positive caecilians out of 85 tested individuals from Cameroon (Doherty-Bone et al. 2013). In the same year, Gower et al. (2013) reported the first case of lethal chytridiomycosis in caecilians from Cameroon and Tanzania. They reported that wild-caught specimens of Geotrypetes seraphini that tested positive for Bd died in captivity with signs of chytridiomycosis. Bd infections were also confirmed in an endemic caecilian (Schistome- topum thomense) from an island of the archipelago of São Tomé and Príncipe (Hydeman et al. 2013). The only record of Bd infecting caecilians outside Africa was recently published for a wild-caught specimen from French Guiana (Rendle et al. 2015). Despite the observed high Bd prevalence in African caecilians, sampling efforts to detect Bd in Gymnophiona have been made in other regions but without any Bd-positive samples from specimens collected in the wild in peninsular Malaysia (n = 2; Savage et al. 2011), Colombia (n = 1; Vásquez- Ochoa et al. 2012), West Africa (n = 6; Penner et al. 2013), and the Seychelles archipelago (n = 78; Labisko et al. 2015). The small sample sizes of these published reports reflect the difficulty and/or lack of effort in working with this generally inconspicuous taxon (e.g. Gower & Wilkinson 2005); thus, it cannot be ruled out that Gymnophiona are infected with Bd as extensively as anurans are. Furthermore, con- spicuous die-offs due to chytridiomycosis, such as those observed in anurans, are relatively unlikely to be observed in caecilians (Gower et al. 2013) due to their fossorial or fully aquatic life styles (Wells 2007, Vitt & Caldwell 2014). Caecilians are broadly distri - buted in the tropics (Taylor 1968, Frost 2016), often co-occurring with anuran populations heavily im - pac ted by chytridiomycosis (Seimon et al. 2007, Gower et al. 2012, Bataille et al. 2013, James et al. 2015) and inhabiting microhabitats within Bd’s opti- mal growth conditions of temperature and humidity (Piotrowski et al. 2004). Determining whether cae- cilians are af fected by chytridiomycosis as much as anurans and salamanders are, and whether caecil- ians are serving as pathogen reservoirs, is relevant for amphibian conservation (Gower & Wilkinson 2005). Several environmental factors influence Bd infec- tion in anurans. Infection prevalence and zoospore loads are often positively correlated with elevation (Brem & Lips 2008, Gründler et al. 2012, Catenazzi et al. 2013), vegetation density (Puschendorf et al. 2009, Becker & Zamudio 2011, Becker et al. 2016), and preci pitation (Becker & Zamudio 2011), and neg- atively correlated with temperature (Becker & Zamu- dio 2011, Ruggeri et al. 2015, Becker et al. 2016). Because most caecilians are fossorial, they are likely exposed to lower microclimatic fluctuations dictated by land cover, insolation, and humidity. Therefore, microclimatic optima/averages for caecilians might fall within the optimal growth conditions of Bd, allowing Bd to persist in areas where it would other- wise not endure year-round. Here, we performed a retrospective survey of mu - seum preserved specimens to (1) describe spatial patterns of Bd infection in Gymnophiona in South America and (2) test whether areas of low climatic suitability for Bd in anurans predict spatial epidemio - logy of Bd in caecilians. We used quantitative PCR to detect Bd in preserved caecilians collected over a 109 yr period, and performed autologistic regressions to test the effect of bioclimatic metrics of temperature and precipitation, vegetation density, and elevation on the likelihood of Bd occurrence. Our results pro- vide novel information on Bd spatial epidemiology and suggest that caecilians could be potentially threatened or serve as Bd reservoirs in regions where anurans are not infected during most of the year. MATERIALS AND METHODS Species sampling We sampled 193 museum-preserved specimens of Gymnophiona: 160 from Brazil (Caeciliidae, Sipho no - pidae, and Typhlonectidae) and 33 from Uruguay (Typhlonectidae; Table 1). We screened specimens from 3 out of 4 South American caecilian families housed at the following herpetological collections: Museu Paraense Emílio Goeldi (MPEG), Museu de Zoologia prof. Adão José Cardoso, Universidade Estadual de Campinas (ZUEC), Museu Nacional, Universidade Federal do Rio de Janeiro (MNRJ), Coleção de Anfíbios Célio F. B. Haddad, Universi- dade Estadual Paulista (CFBH), and Colección de Vertebrados de la Universidad de La Republica, Mon- tevideo (ZVCB) (see Table S1 in the Supplement at www. int-res. com/ articles/ suppl/ d124p109 _ supp. pdf). For standardization purposes, we did not in clude in our analyses published Bd data from wild-caught caecilians from French Guiana, Guyana, and Colom- bia (Vásquez-Ochoa et al. 2012, Gower et al. 2013, Rendle et al. 2015). We gathered GPS coordinates in 110 http://www.int-res.com/articles/suppl/d124p109_supp.pdf Lambertini et al.: B. dendrobatidis in South American caecilians decimal degrees for each sampled specimen based on museum data. We used the geographic centroid of municipalities as an approximation when precise geographic coordinates were not available. We did not consider records of Bd from captive and live spec- imens for methodological consistency (e.g. Raphael & Pramuk 2007, Churgin et al. 2013). Bd detection Retrospective sampling of museum specimens has been widely used to determine historical dynamics of Bd across space and time (Weldon et al. 2004, Ouellet et al. 2005, Soto-Azat et al. 2010, Cheng et al. 2011, Rodriguez et al. 2014, Courtois et al. 2015, Talley et al. 2015, Becker et al. 2016). We swabbed individual specimens on the head, anal disc, and dorsal and ventral surfaces with a single swab per individual, following Rendle et al. (2015), and stored each sam- ple in a 1.5 ml dry sterile tube. We extracted DNA from each swab using 50 µl PrepMan ULTRA® and proceeded with molecular detection with the TaqMan® qPCR assay (Life Techno - logies), using strain CLFT 023 as a quantita- tive standard for the reactions diluted from 103 to 10−1 zoospore genomic equivalents (GE; Boyle et al. 2004, Lambertini et al. 2013). We considered Bd-positive samples with GE ≥ 1 (Kriger et al. 2007). Statistical analyses We described patterns of Bd infection in Caecilians from Brazil and Uru guay (propor- tion of Bd-infected individuals ± binomial 95% CI). We classified species based on their predominant life history (aquatic or fossor- ial) and reported the proportion (± binomial 95% CI) of infected individuals for each life- history category and ecoregion. We also de - scribed spatio temporal patterns of Bd infec- tions from 1905 to 2014; 28 specimens lacking in formation for their collection year were ex - cluded from these calculations. Furthermore, we conducted multi- model inference using autologistic re gressions to test for the effect of bioclimatic variables, vegetation density, and elevation on the likelihood of Bd infection while ac counting for the effects of spatial autocorrelation (Rangel et al. 2010). For each sampling loc - ation, we extracted 19 bioclimatic variables of tem- perature and precipitation averaged over a pe riod of 50 yr (Hijmans et al. 2005), vegetation density (FAO 2010), and elevation, using Arc Map v.10.1 (ESRI). We used a model averaging procedure, in - clu ding Bd as the res ponse variable (presence vs. ab sence) and the aforementioned environmental factors as explanatory variables. Our model averag- ing ranked all possible models based on Akaike’s information criterion (AIC) and averaged beta coef- ficients of variables present in 90% of models within ΔAIC < 2. We reported the strength and the direc- tion that each environmental variable influenced Bd. We used SAM v4.0 to perform spatial statistics (Rangel et al. 2010). RESULTS Our qPCR reactions detected Bd in 24 out of 193 screened specimens (12.4%, 95% CI = 0.08−0.17; Fig. 1). Infected individuals belonged to the families 111 Family Habit Habitat Proportion infected Caeciliidae Caecilia gracilis Fossorial Rainforest 0/1 Siphonopidae Luetkenotyphlus brasiliensis Fossorial Rainforest 3/9 Siphonops annulatus Fossorial Rainforest 1/12 Siphonops cf. annulatus Fossorial Rainforest 1/1 Siphonops paulensis Fossorial Rainforest 2/8 Siphonops cf. paulensis Fossorial Rainforest, 0/15 Grassland Siphonops sp. (aff. paulensis) Fossorial Grassland 0/2 Siphonops hardyi Fossorial Rainforest 0/2 Siphonops sp. Fossorial Rainforest, 1/7 Grassland Typhlonectidae Atretochoana eiselti Aquatic Rainforest 0/4 Chthonerpeton braestrupi Aquatic Rainforest 0/6 Chthonerpeton indistinctum Aquatic Grassland 4/33 Chthonerpeton indistinctum Aquatic Rainforest 0/1 Chthonerpeton noctinetes Aquatic Rainforest 0/8 Chthonerpeton sp. Aquatic Rainforest 0/1 Chthonerpeton tremembe Aquatic Grassland 0/4 Chthonerpeton viviparum Aquatic Rainforest 0/8 Potamotyphlus kaupii Aquatic Rainforest 9/39 Typhlonectes compressicauda Aquatic Rainforest 3/32 Total 24/193 Table 1. Families, species, environment, and country where individual cae cilians were captured, and proportion of animals infected by Batra - cho chytrium dendrobatidis (as the number of positives/total number screened). All species were collected in Brazil, apart from C. indistinctum (collected in Uruguay) Dis Aquat Org 124: 109–116, 2017 Siphonopidae (n = 8 individuals) and Typhlonecti- dae (n =16) distributed across the Uruguayan sa - vanna (proportion of infected individuals = 12.1%, 95% CI = 0.03−0.28, n = 33 total individuals sam- pled), the Amazon Basin (16%, 95% CI = 0.08−0.26, n = 75), and Brazil’s Atlantic Forest (13.5%, 95% CI = 0.06 −0.24, n = 59). We did not detect Bd in individuals of Caeciliidae or in samples from the Brazilian Cerrado, Caatinga, and Pantanal, but our sampling sizes in these ecoregions were small (n = 2, 22, and 2, respectively). We detected a proportion of infected individuals of 11.7% in aquatic species (95% CI = 0.06−0.18, n = 136) and 14% in terres- trial (typically fossorial) species (95% CI = 0.06− 0.25, n = 57). Although our sampling spanned 109 yr, most of our Bd-positive specimens (n = 16) were collected after 1994. Only 5 samples before this period tested posi- tive for Bd, and were collected from the wild between 1965 and 1994, and 3 positive samples had no collect- ing date. However, 21 samples known to have been collected prior to 1971 were screened, and they all tested negative for Bd. Our spatial regression models indicated a signifi- cant effect of macroclimate on the likelihood of Bd occurrence (Table 2). Our autologistic model averag- ing showed a negative effect of maximum tempera- ture of the warmest month and precipitation in the wettest quarter, and a positive effect of annual pre- cipitation on Bd infection likelihood; the full set of significant variables is provided in Table 2. Vegeta- tion density had a weak negative effect on Bd occur- rence, and elevation was not a significant variable in our models (Table 2, Table S2). DISCUSSION Seasonal variations in temperature and precipita- tion strongly mediate Bd infections by changing optimal physiological conditions of hosts and patho- gen (Piotrowski et al. 2004, Becker & Zamudio 2011, Ruggeri et al. 2015). These environmental con- straints are revealed in several environmental niche models, in dicating that much of South America is unsuitable for Bd during at least part of the year 112 Fig. 1. Batrachochytrium dendroba- tidis sampling in museum- preserved caecilians from Brazil and Uru guay (positive samples: red circles; nega- tive samples: white circles), and wild-caught caecilians (positive sam- ple: red cross; negative samples: white crosses; Vásquez- Ochoa et al. 2012, Gower et al. 2013, Rendle et al. 2015, present study). Siphonops paulensis is a fossorial terrestrial species found in eastern Brazil, and Chthonerpeton indistinctum is an aquatic species that is the only cae- cilian species found in Uru guay. Photo graphs: Daniel Loebmann. Service layer source: US National Park Service Lambertini et al.: B. dendrobatidis in South American caecilians (Rödder et al. 2009, Liu et al. 2012, James et al. 2015, Becker et al. 2016). Although most of the Bd- positive records overlapped with reported areas of high climatic suitability for the fungus in the Neo - tropics (Rödder et al. 2009, Liu et al. 2012, James et al. 2015, Becker et al. 2016), several Bd-positive records extend far into areas of poor suitability for Bd in anurans (e.g. central Amazon: see Becker et al. 2016). Our data points to widespread Bd infec- tions in Neotropical caecilians, and that this taxon may serve as an environmental reservoir, perhaps because hosts are able to avoid harsh seasonal ex - tremes where Bd would otherwise not persist year- round. These results, combined with a recent report of lethal chytridiomycosis in wild-caught caecilians (Gower et al. 2013), indicate that Gymno phiona are potentially experiencing silent population declines in the wild due to Bd. Spatial regressions are also consistent with the ob - served associations between macroclimate and Bd infection in anuran species (Becker & Zamudio 2011, James et al. 2015, Becker et al. 2016). Specifically, we detected a positive effect of precipitation and a neg- ative effect of temperature variables on the likeli- hood of Bd infection in caecilian hosts. Vegetation density, which is often positively associated with Bd infection in anurans (Raffel et al. 2010, Becker & Zamudio 2011, Becker et al. 2012), showed a weak negative effect on Bd in caecilians. This finding might be due to the high degree of fossoriality of terrestrial caecilian species, which spares them from the direct or indirect effects of habitat quality, with downstream shifts in both macro- and micro- climates. Elevation, which is often positively associ- ated with Bd infection in anurans (Walker et al. 2010, Piovia-Scott et al. 2011, Gründler et al. 2012) due to optimal growth conditions in highlands (Piotrowski et al. 2004), showed no effect on Bd in Caecilians. Although our sampling spanned 1000 m in elevation, most of our samples were collected at lower altitudes. This uneven sampling across the elevation gradient may have thus impacted our ability to detect a signif- icant effect of elevation in our analyses. Although large-scale climate may play a role in Bd epizoo - tiology of caecilians, these results indicate that in - fection dynamics in caecilians and anurans might be different. Although our results suggest that caecilians could act as pathogen reservoirs in environments or peri- ods of harsh microclimatic conditions for Bd, limited natural history information is available for most cae- cilian species (Gower & Wilkinson 2005, Vitt & Caldwell 2014). Basic information on foraging behavior, population densities, and breeding habits that would be key to quantifying transmission dy - namics between Gymnophiona and Anura are typi- cally lacking. It is known that 5 out of 10 families of caecilians are found in South America (Wilkinson et al. 2011, Frost 2016), and that these 5 families range from being obligate fossorial to completely aquatic (Haddad et al. 2013, Vitt & Caldwell 2014). We pre- dict that fossorial and fully aquatic species are less likely to be exposed to environmental and climatic fluctuations than terrestrial anurans because they spend longer periods of time underground or under- water. Future studies of caecilian foraging behavior and habitat use may help test the link between habitat use and temperature variability. Lower tem- perature extremes and variability are linked to higher Bd growth and persistence in amphibian hosts both in the wild and in the laboratory (Pounds et al. 2006, Raffel et al. 2013, 2015). Because Bd is a waterborne fungus (Longcore et al. 1999, Kilpatrick et al. 2010), we also expect fully aquatic caecilians to be exposed to the pathogen not only during their early life stages. Therefore, fossorial and aquatic life styles observed in caecilians are life history traits that likely make an efficient host reservoir, espe- 113 Variable Rank Beta 95% importance coefficient CI Bio 5 43 −0.038 0.002 Bio 12 32 0.003 0.001 Bio 16 22 −0.009 0.004 Bio 3 12 0.076 0.032 Bio 13 11 0.039 0.011 Bio 17 10 0.002 0.008 Bio 15 9 −0.029 0.006 Bio 14 9 −0.027 0.023 Bio 18 7 0.003 0.001 Bio 19 7 0.002 0.001 Vegetation density 6 −0.011 0.003 Bio 10 4 −0.034 0.003 Bio 9 3 −0.011 0.006 Table 2. Model averaging results ranking significant envi- ronmental variables explaining Batrachochytrium dendro- batidis occurrence in South American caecilians. Rank im- portance corresponds to the number of models in which each variable was present (also see Table S2 in the Supple- ment). CI: confidence interval. Bio 3: isothermality, Bio 5: tem- perature of warmest month, Bio 9: mean temperature of dri- est quarter, Bio 10: mean temperature of warmest quarter, Bio 12: annual precipitation, Bio 13: precipitation of wettest month, Bio 14: precipitation pf driest month, Bio 15: precipi- tation seasonality, Bio 16: precipitation of wettest quarter, Bio 17: precipitation of driest quarter, Bio 18: precipitation of warmest quarter, Bio 19: precipitation of coldest quarter Dis Aquat Org 124: 109–116, 2017 cially in areas where Bd does not persist in anuran hosts year-round. In areas of low predicted suitability for Bd such as the Amazon basin (Ron 2005, James et al. 2015, Becker et al. 2016), we detected an infection preva- lence of 16%, which is surprisingly high compared to the observed ~3% in museum-preserved anurans in this region (Becker et al. 2016). In contrast, the pro- portion of infected caecilians in the Atlantic Forest was slightly lower than what has been observed for preserved anurans (~23%) in this ecoregion (Rodri - guez et al. 2014). Nonetheless, limited sample sizes for caecilians prior to the 1970s precludes us from making any concrete spatio-temporal comparison between Bd in caecilians and in anurans from both the Atlantic Forest (Rodriguez et al. 2014) and the Amazon basin (Becker et al. 2016). Although most aquatic caecilians included in this study were sam- pled from the Amazon basin and most terrestrial cae- cilians from the Atlantic Forest, we did not detect a significant effect of host life style (aquatic vs. terres- trial) on the likelihood of Bd infection. These results further indicate that Bd infection dynamics in Gymnophiona might experience a lower pressure from macroclimate than in Anura. Our results highlight an important gap in the study of amphibian chytridiomycosis: the possible impact of Bd on Neotropical caecilians and the hypothetical role of caecilians as Bd reservoirs. To date, infor - mation on the susceptibility of caecilian hosts to Bd infection is still lacking. Therefore, Bd genotypes detected in caecilians may present different adapta- tions to host histophysiology or microclimates, which provides a key opportunity to isolate and genotype new Bd isolates from live caecilians and test the viru- lence of these new isolates in anurans. Because Bd has a disproportionately higher impact in tropical amphibians, a better understanding of Bd infection dynamics in Gymnophiona may increase our knowl- edge about chytridiomycosis and ad vance our con- servation efforts in the wild. Acknowledgements. We thank Adriano O. Maciel, Alexan- dre F. R. Missassi, Manoela Woitovicz Cardoso, Nadya C. Pupin, and Tamilie Carvalho for help with swabbing and providing museum specimens. Ana L. C. Prudente (MPEG), Célio F. B. Haddad (CFBH), José P. Pombal Jr. (MNRJ), and Raúl Maneyro (ZVCB) allowed access to museum speci- mens. We thank David J. Gower and 2 anonymous review- ers for constructive feedback on our manuscript. This work was funded by Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Council of Technological and Scientific Development (CNPq 405285/ 2013-2; 312895/2014-3) and the São Paulo Research Foun- dation (FAPESP 2014/23388-7). LITERATURE CITED Bataille A, Fong JJ, Cha M, Wogan GO and others (2013) Genetic evidence for a high diversity and wide distribu- tion of endemic strains of the pathogenic chytrid fungus Batrachochytrium dendrobatidis in wild Asian amphi - bians. Mol Ecol 22: 4196−4209 Becker CG, Zamudio KR (2011) Tropical amphibian popula- tions experience higher disease risk in natural habitats. 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