Assessing the links among environmental contaminants, endocrinology, and parasites to understand amphibian declines in montane regions of Costa Rica.

Amphibians inhabiting montane riparian zones in the Neotropics are particularly vulnerable to decline, but the reasons are poorly understood. Because environmental contaminants, endocrine disruption, and pathogens often figure prominently in amphibian declines it is imperative that we understand how these factors are potentially interrelated to affect montane populations. One possibility is that increased precipitation associated with global warming promotes the deposition of contaminants in montane regions. Increased exposure to contaminants, in turn, potentially elicits chronic elevations in circulating stress hormones that could contribute to montane population declines by compromising resistance to pathogens and/or production of sex steroids regulating reproduction. Here, we test this hypothesis by examining contaminant levels, stress and sex steroid levels, and nematode abundances in male drab treefrogs, Smilisca sordida, from lowland and montane populations in Costa Rica. We found no evidence that montane populations were more likely to possess contaminants (i.e., organochlorine, organophosphate and carbamate pesticides or benzidine and chlorophenoxy herbicides) than lowland populations. We also found no evidence of elevational differences in circulating levels of the stress hormone corticosterone, estradiol or progesterone. However, montane populations possessed lower androgen levels, hosted more nematode species, and had higher nematode abundances than lowland populations. Although these results suggested that nematodes contributed to lower androgens in montane populations, we were unable to detect a significant inverse relationship between nematode abundance and androgen level. Our results suggest that montane populations of this species are not at greater risk of exposure to contaminants or chronic stress, but implicate nematodes and compromised sex steroid levels as potential threats to montane populations.

Species tree estimation of North American chorus frogs (Hylidae: Pseudacris) with parallel tagged amplicon sequencing.

The field of phylogenetics is changing rapidly with the application of high-throughput sequencing to non-model organisms. Cost-effective use of this technology for phylogenetic studies, which often include a relatively small portion of the genome but several taxa, requires strategies for genome partitioning and sequencing multiple individuals in parallel. In this study we estimated a multilocus phylogeny for the North American chorus frog genus Pseudacris using anonymous nuclear loci that were recently developed using a reduced representation library approach. We sequenced 27 nuclear loci and three mitochondrial loci for 44 individuals on 1/3 of an Illumina MiSeq run, obtaining 96.5% of the targeted amplicons at less than 20% of the cost of traditional Sanger sequencing. We found heterogeneity among gene trees, although four major clades (Trilling Frog, Fat Frog, crucifer, and West Coast) were consistently supported, and we resolved the relationships among these clades for the first time with strong support. We also found discordance between the mitochondrial and nuclear datasets that we attribute to mitochondrial introgression and a possible selective sweep. Bayesian concordance analysis in BUCKy and species tree analysis in (*)BEAST produced largely similar topologies, although we identify taxa that require additional investigation in order to clarify taxonomic and geographic range boundaries. Overall, we demonstrate the utility of a reduced representation library approach for marker development and parallel tagged sequencing on an Illumina MiSeq for phylogenetic studies of non-model organisms.