An empirical pipeline for choosing the optimal clustering threshold in RADseq studies.

Genomic data are increasingly used for high resolution population genetic studies including those at the forefront of biological conservation. A key methodological challenge is determining sequence similarity clustering thresholds for RADseq data when no reference genome is available. These thresholds define the maximum permitted divergence among allelic variants and the minimum divergence among putative paralogues and are central to downstream population genomic analyses. Here we develop a novel set of metrics to determine sequence similarity thresholds that maximize the correct separation of paralogous regions and minimize oversplitting naturally occurring allelic variation within loci. These metrics empirically identify the threshold value at which true alleles at opposite ends of several major axes of genetic variation begin to incorrectly separate into distinct clusters, allowing researchers to choose thresholds just below this value. We test our approach on a recently published data set for the protected foothill yellow-legged frog (Rana boylii). The metrics recover a consistent pattern of roughly 96% similarity as a threshold above which genetic divergence and data missingness become increasingly correlated. We provide scripts for assessing different clustering thresholds and discuss how this approach can be applied across a wide range of empirical data sets.

Genomic data recover previously undetectable fragmentation effects in an endangered amphibian.

A critical consideration when using molecular ecological methods to detect trends and parameterize models at very fine spatial and temporal scales has always been the technical limits of resolution. Key landscape features, including most anthropogenic modifications, can cause biologically important, but very recent changes in gene flow that require substantial statistical power to detect. The problem is one of temporal scale: Human change is rapid and recent, while genetic changes accumulate slowly. We generated SNPs from thousands of nuclear loci to characterize the population structure of New York-endangered eastern tiger salamanders (Ambystoma tigrinum) on Long Island and quantify the impacts of roads on population fragmentation. In stark contrast to a recent microsatellite study, we uncovered highly structured populations over an extremely small spatial scale (approximately 40 km2 ) in an increasingly human-modified landscape. Geographic distance and the presence of roads between ponds were both strong predictors of genetic divergence, suggesting that both natural and anthropogenic factors contribute to the observed patterns of genetic variation. All ponds supported small to modest effective breeding populations, and pond surface area showed a strong positive correlation with population size. None of these patterns emerged in an earlier study of the same system using microsatellite loci, and we determined that at least 300-400 SNPs were needed to recover the fine-scale population structure present in this system. Conservation assessments using earlier genetic techniques in other species may similarly lack the statistical power for small-scale inferences and benefit from reassessments using genomic tools.

Population genomic data reveal extreme geographic subdivision and novel conservation actions for the declining foothill yellow-legged frog.

Genomic data have the potential to inform high resolution landscape genetic and biological conservation studies that go far beyond recent mitochondrial and microsatellite analyses. We characterize the relationships of populations of the foothill yellow-legged frog, Rana boylii, a declining, "sentinel" species for stream ecosystems throughout its range in California and Oregon. We generated RADseq data and applied phylogenetic methods, hierarchical Bayesian clustering, PCA and population differentiation with admixture analyses to characterize spatial genetic structure across the species range. To facilitate direct comparison with previous analyses, we included many localities and individuals from our earlier work based on mitochondrial DNA. The results are striking, and emphasize the power of our landscape genomic approach. We recovered five extremely differentiated primary clades that indicate that R. boylii may be the most genetically differentiated anuran yet studied. Our results provide better resolution and more spatially consistent patterns than our earlier work, confirming the increased resolving power of genomic data compared to single-locus studies. Genomic structure is not equal across the species distribution. Approximately half the range of R. boylii consists of a single, relatively uniform population, while Sierra Nevada and coastal California clades are deeply, hierarchically substructured with biogeographic breaks observed in other codistributed taxa. Our results indicate that clades should serve as management units for R. boylii rather than previously suggested watershed boundaries, and that the near-extinct population from southwestern California is particularly diverged, exhibits the lowest genetic diversity, and is a critical conservation target for species recovery.

Phylogenomic analyses of 539 highly informative loci dates a fully resolved time tree for the major clades of living turtles (Testudines).

Accurate time-calibrated phylogenies are the centerpiece of many macroevolutionary studies, and the relationship between the size and scale of molecular data sets and the density and accuracy of fossil calibrations is a key element of time tree studies. Here, we develop a target capture array specifically for living turtles, compare its efficiency to an ultraconserved element (UCE) dataset, and present a time-calibrated molecular phylogeny based on 539 nuclear loci sequenced from 26 species representing the breadth of living turtle diversity plus outgroups. Our gene array, based on three fully sequenced turtle genomes, is 2.4 times more variable across turtles than a recently published UCE data set for an identical subset of 13 species, confirming that taxon-specific arrays return more informative data per sequencing effort than UCEs. We used our genomic data to estimate the ages of living turtle clades including a mid-late Triassic origin for crown turtles and a mid-Carboniferous split of turtles from their sister group, Archosauria. By specifically excluding several of the earliest potential crown turtle fossils and limiting the age of fossil calibration points to the unambiguous crown lineage Caribemys oxfordiensis from the Late Jurassic (Oxfordian, 163.5-157.3Ma) we corroborate a relatively ancient age for living turtles. We also provide novel age estimates for five of the ten testudine families containing more than a single species, as well as several intrafamilial clades. Most of the diversity of crown turtles appears to date to the Paleogene, well after the Cretaceous-Paleogene mass extinction 66mya.

Phylogeny and temporal diversification of the New World pond turtles (Emydidae).

We present a comprehensive multigene phylogeny and time tree for the turtle family Emydidae. Our phylogenetic analysis, based on 30 nuclear and four mitochondrial genes (23,330 total base pairs) sequenced for two individuals for each of the currently recognized species of the subfamily Emydinae and two species from each of the more species-rich Deirochelyinae genera, yielded a well-supported tree that provides an evolutionary framework for this well-studied clade and a basis for a stable taxonomy. We calibrated an emydid time tree using three well-vetted fossils, modeled uncertainty in fossil ages to reflect their accuracy in node dating, and extracted stem/crown ages of a number of key diversification events. We date the age of crown emydids at a relatively young 44Ma, and the crown age of both contained subfamilies at roughly 30Ma. One deirochelyine clade, which includes the genera Graptemys, Malaclemys, Pseudemys, and Trachemys and contains 11% of all turtle species, dates to 21Ma just prior to the mid-Miocene climatic optimum, suggesting a potential causal link between warm, moist conditions and rapid species accumulation of these highly aquatic turtles. Both nuclear DNA data alone and in combination with mitochondrial DNA support the monophyly of an inclusive genus Emys containing the old world species orbicularis and trinacris and the New World blandingii, marmorata and pallida. Given that all members of this group were originally aligned in the genus Emys and that the age of the clade is roughly equal to other emydine genera, we strongly support a classification that places these five species in a single genus rather than the alternative three-genus scheme (Emys (orbicularis, trinacris), Actinemys (marmorata, pallida), Emydoidea (blandingii)). The phylogeny and resulting time tree presented here provides a comprehensive foundation for future comparative analyses of the Emydidae that will shed light on the historical ecology and conservation prioritization of this diverse chelonian clade.

Exon capture optimization in amphibians with large genomes.

Gathering genomic-scale data efficiently is challenging for nonmodel species with large, complex genomes. Transcriptome sequencing is accessible for organisms with large genomes, and sequence capture probes can be designed from such mRNA sequences to enrich and sequence exonic regions. Maximizing enrichment efficiency is important to reduce sequencing costs, but relatively few data exist for exon capture experiments in nonmodel organisms with large genomes. Here, we conducted a replicated factorial experiment to explore the effects of several modifications to standard protocols that might increase sequence capture efficiency for amphibians and other taxa with large, complex genomes. Increasing the amounts of c0 t-1 repetitive sequence blocker and individual input DNA used in target enrichment reactions reduced the rates of PCR duplication. This reduction led to an increase in the percentage of unique reads mapping to target sequences, essentially doubling overall efficiency of the target capture from 10.4% to nearly 19.9% and rendering target capture experiments more efficient and affordable. Our results indicate that target capture protocols can be modified to efficiently screen vertebrates with large genomes, including amphibians.

Amphibian molecular ecology and how it has informed conservation.

Molecular ecology has become one of the key tools in the modern conservationist's kit. Here we review three areas where molecular ecology has been applied to amphibian conservation: genes on landscapes, within-population processes, and genes that matter. We summarize relevant analytical methods, recent important studies from the amphibian literature, and conservation implications for each section. Finally, we include five in-depth examples of how molecular ecology has been successfully applied to specific amphibian systems.

Conservation genetics and genomics of amphibians and reptiles.

Amphibians and reptiles as a group are often secretive, reach their greatest diversity often in remote tropical regions, and contain some of the most endangered groups of organisms on earth. Particularly in the past decade, genetics and genomics have been instrumental in the conservation biology of these cryptic vertebrates, enabling work ranging from the identification of populations subject to trade and exploitation, to the identification of cryptic lineages harboring critical genetic variation, to the analysis of genes controlling key life history traits. In this review, we highlight some of the most important ways that genetic analyses have brought new insights to the conservation of amphibians and reptiles. Although genomics has only recently emerged as part of this conservation tool kit, several large-scale data sources, including full genomes, expressed sequence tags, and transcriptomes, are providing new opportunities to identify key genes, quantify landscape effects, and manage captive breeding stocks of at-risk species.

Population structure and gene flow of the yellow anaconda (Eunectes notaeus) in northern Argentina.

Yellow anacondas (Eunectes notaeus) are large, semiaquatic boid snakes found in wetland systems in South America. These snakes are commercially harvested under a sustainable management plan in Argentina, so information regarding population structuring can be helpful for determination of management units. We evaluated genetic structure and migration using partial sequences from the mitochondrial control region and mitochondrial genes cyt-b and ND4 for 183 samples collected within northern Argentina. A group of landscape features and environmental variables including several treatments of temperature and precipitation were explored as potential drivers of observed genetic patterns. We found significant population structure between most putative population comparisons and bidirectional but asymmetric migration in several cases. The configuration of rivers and wetlands was found to be significantly associated with yellow anaconda population structure (IBD), and important for gene flow, although genetic distances were not significantly correlated with the environmental variables used here. More in-depth analyses of environmental data may be needed to fully understand the importance of environmental conditions on population structure and migration. These analyses indicate that our putative populations are demographically distinct and should be treated as such in Argentina's management plan for the harvesting of yellow anacondas.