Environmental DNA characterization of amphibian communities in the Brazilian Atlantic forest: Potential application for conservation of a rich and threatened fauna
Introduction
The Brazilian Atlantic forest is home to a high diversity of amphibians, harboring over 500 species (approximately 7% of all known species) of which 88% are endemic to this biome (Haddad et al., 2013). Distributed along the coast of eastern Brazil, the Atlantic forest covers mountainous terrains with a large number of high-gradient streams (Morellato and Haddad, 2000). These freshwater environments harbor a high diversity of amphibians, including habitat specialists with most or all life cycle stages depending on lotic waters (Haddad et al., 2013). The Atlantic forest is also one of the most threatened tropical ecosystems in the world (Myers et al., 2000) with only 16% of its original vegetation cover remaining (Ribeiro et al., 2009), posing a high risk to habitat specialists endemic to this biome. Several other factors, such as invasive species, and emergent infectious diseases (Both et al., 2011, Carvalho et al., 2017, Eterovick et al., 2005) also potentially contribute to observed amphibian declines in Atlantic forest. Not surprisingly, approximately 88% of the threatened anuran species in Brazil are from the Atlantic forest (ICMBio, 2016a) and most of the documented frog declines in Brazil include species closely associated with streams (Eterovick et al., 2005). Furthermore, over one fifth of Atlantic forest amphibians that are stream specialists lack baseline data and are listed as Data-Deficient according to the International Union for Conservation of Nature (IUCN, 2015, ICMBio, Instituto de Conservação Chico Mendes da Biodiversidade, 2016b). Thus, it is critical to describe the occurrence and distribution of stream frog species in the Atlantic forest and any method that makes this task easier and more precise will be of crucial importance for amphibian conservation.
Field techniques traditionally used to assess amphibian occurrence require a large investment in fieldwork and come with their own sources of error (Heyer et al., 1993). For instance, frog detectability through acoustic survey are highly variable among species, some species call only a few days per year making their detection less likely, and all types of traps used to sample amphibians (e.g., pitfall, funnel, pipe) provide biased results (Dodd, 2010, Petitot et al., 2014). Recently, the analysis of environmental DNA (eDNA) has emerged as a promising alternative approach for detecting aquatic species. The term eDNA refers to DNA shed by an organism in the environment, such as water, soil, or even air (Taberlet et al., 2012a), which can be then sampled for sequencing and species identification (Bohmann et al., 2014). The use of eDNA has recently gained widespread attention because it allows researchers to detect species even at low abundances (Dejean et al., 2012) and during short term field samplings (Lopes et al., 2017), making eDNA ideal for applications in conservation biology.
The number of ecological studies applying eDNA analysis to monitor species is increasing, yet few studies have sampled eDNA from aquatic environments to perform broad community surveys (but see Shaw et al., 2016, Valentini et al., 2016). Most eDNA efforts have focused on detection of a particular species using species-specific molecular markers (Thomsen and Willerslev, 2015). These efforts, while valuable, do not assess species diversity. If the goal is to survey the biodiversity of particular taxa and assess community composition, one can use DNA-based identification of a group of species at a locality with universal primers, an approach known as “DNA metabarcoding” (Taberlet et al., 2012b).
Here we report the results of a short-term eDNA metabarcoding study applied to a system of Atlantic forest streams where amphibian community composition was well known through a long-term traditional survey. By comparing our eDNA results with our traditional survey, we assess the utility of the eDNA metabarcoding for the characterization of tropical frog communities from a megadiverse region. If one or a few eDNA samples can accurately describe amphibian community composition in tropical streams, instead of long-term, time and labor-expensive traditional surveys, this method has high potential for use in conservation studies of Atlantic forest frogs.
Section snippets
Study site
Our study site is located within the Parque Estadual da Serra do Mar, Núcleo Picinguaba, municipality of Ubatuba, São Paulo, southeastern Brazil (see Fig. 1 in Lopes et al., 2017). The climate is tropical (Köppen, 1948) with mean annual air temperature of 26.7 °C, high and constant air humidity (monthly means 85–90%), and an annual average rainfall of 2650 mm (CIIAGRO, 2016). We collected data from four freshwater mountain streams in separate drainages. During water sampling for eDNA analyses,
Results
During five years of traditional surveys at the four focal streams we found a total of 19 amphibian species representing 11 families and 17 genera (Table 1). Ten of the 19 species found have at least one life stage occurring adjacent to or within streams (HI > 0; hereafter stream species; Table 1). The remaining nine species have no association with streams (HI = 0; hereafter non-stream species; Table 1). Among stream species, the riparian habitat index ranged from 1 to 6 (Table 1).
We detected
Discussion
Our study showed that eDNA metabarcoding is a reliable method for detecting a large proportion of the amphibian communities of stream frogs in the Atlantic forest, but it is not perfect. Remarkably, with a single four-day sampling we detected DNA of all stream species found across all four streams during our five-year traditional survey, with the exception of a single uncommon species (Proceratophrys belzebul). However, at a finer within-stream scale, a single eDNA sample failed to detect
Conclusions
Freshwater ecosystems are essential for a large percentage of the world's amphibian species (e.g., Stuart et al., 2008). Despite their value, riparian habitats are being severely disturbed and declines in freshwater biodiversity are far greater than in terrestrial ecosystems (Dudgeon et al., 2006). The conservation management required is limited by the paucity of rigorous species occurrence data or the difficulty in surveying species in nature. In light of the added sensitivity of
Acknowledgements
This work was supported by São Paulo Research Foundation (FAPESP) grants #2006/58011-4, #2010/50146-3, and #2013/50741-7, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grant #401729/2013-3. We thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and FAPESP (grants #2014/06795-8 and #2016/14054-3 to T.S.L. and C.M.L., respectively) for scholarships provided during this project. C.M.L., K.R.Z., M.M. and C.F.B.H. thank CNPq for the research fellowships. The
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