Detection of stream amphibians in Brazilian Atlantic Forest using environmental DNA

Abstract

Environmental DNA (eDNA) is the name assigned to fragments of nuclear or mitochondrial DNA present in environmental samples. Detection of macro-organisms DNA in water samples is a promising new method for documenting the presence of native and invasive species, at high or low-density, in freshwater environments. The advance of Next-Generation Sequencers (NGS) tools concomitantly with the development of DNA markers that can target degraded DNA opens up many possibilities for the applicability of eDNA information in ecology today, as for biodiversity monitoring, animal diet assessment, and conservations efforts. eDNA isolation from aquatic vertebrates had been recently proven as an effective method in wetland, ranging from ponds to lakes. However, the detectability of eDNA in fast-moving streams where DNA-material may travel rapidly away from their source has not been fully investigated. It also remains not well clarified whether eDNA concentrations reflect species abundance in natural freshwater systems. To test the efficacy of this method in detecting stream vertebrates, we will verify the presence of a short, species-specific mitochondrial DNA fragment (Cytochrome b, Cyt-b) for three stream amphibian species (Cycloramphus boraceiensis, Hylodes phyllodes e H. asper) in four rivers in Atlantic Rain Forest, Brazil. In the field, water samples will be collected in three sites in each river where the species had been documented. Data collection will take place in two seasons (July and August/2014; January and February/2015). For DNA amplification we will apply quantitative PCR, which provides additional specificity. Additionally, population density of the target species will be estimated by visual encounters. In follow-up analysis we will test for relatedness between eDNA measures and field-measured densities and how eDNA detection differ between seasons. The refinement of this technique is crucial for the relevance and reliability of eDNA detection across more environments and a wider range of taxonomic groups. (AU)