Phylogenomic Reconstruction of the Neotropical Poison Frogs (Dendrobatidae) and Their Conservation

The evolutionary history of the Dendrobatidae, the charismatic Neotropical poison frog family, remains in flux, even after a half-century of intensive research. Understanding the evolutionary relationships between dendrobatid genera and the larger-order groups within Dendrobatidae is critical for making accurate assessments of all aspects of their biology and evolution. In this study, we provide the first phylogenomic reconstruction of Dendrobatidae with genome-wide nuclear markers known as ultraconserved elements. We performed sequence capture on 61 samples representing 33 species across 13 of the 16 dendrobatid genera, aiming for a broadly representative taxon sample. We compare topologies generated using maximum likelihood and coalescent methods and estimate divergence times using Bayesian methods. We find most of our dendrobatid tree to be consistent with previously published results based on mitochondrial and low-count nuclear data, with notable exceptions regarding the placement of Hyloxalinae and certain genera within Dendrobatinae. We also characterize how the evolutionary history and geographic distributions of the 285 poison frog species impact their conservation status. We hope that our phylogeny will serve as a backbone for future evolutionary studies and that our characterizations of conservation status inform conservation practices while highlighting taxa in need of further study

Phylogenetic relationships and systematics of the Amazonian poison frog genus Ameerega using ultraconserved genomic elements

The Amazonian poison frog genus Ameerega is one of the largest yet most understudied of the brightly colored genera in the anuran family Dendrobatidae, with 30 described species ranging throughout tropical South America. Phylogenetic analyses of Ameerega are highly discordant, lacking consistency due to variation in data types and methods, and often with limited coverage of species diversity in the genus. Here, we present a comprehensive phylogenomic reconstruction of Ameerega, utilizing state-of-the-art sequence capture techniques and phylogenetic methods. We sequenced thousands of ultraconserved elements from over 100 tissue samples, representing almost every described Ameerega species, as well as undescribed cryptic diversity. We generated topologies using maximum likelihood and coalescent methods and compared the use of maximum likelihood and Bayesian methods for estimating divergence times. Our phylogenetic inference diverged strongly from those of previous studies, and we recommend steps to bring Ameerega taxonomy in line with the new phylogeny. We place several species in a phylogeny for the first time, as well as provide evidence for six potential candidate species. We estimate that Ameerega experienced a rapid radiation approximately 7–11 million years ago and that the ancestor of all Ameerega was likely an aposematic, montane species. This study underscores the utility of phylogenomic data in improving our understanding of the phylogeny of understudied clades and making novel inferences about their evolution

Sequence Capture and Phylogenetic Utility of Genomic Ultraconserved Elements Obtained from Pinned Insect Specimens.

Obtaining sequence data from historical museum specimens has been a growing research interest, invigorated by next-generation sequencing methods that allow inputs of highly degraded DNA. We applied a target enrichment and next-generation sequencing protocol to generate ultraconserved elements (UCEs) from 51 large carpenter bee specimens (genus Xylocopa), representing 25 species with specimen ages ranging from 2-121 years. We measured the correlation between specimen age and DNA yield (pre- and post-library preparation DNA concentration) and several UCE sequence capture statistics (raw read count, UCE reads on target, UCE mean contig length and UCE locus count) with linear regression models. We performed piecewise regression to test for specific breakpoints in the relationship of specimen age and DNA yield and sequence capture variables. Additionally, we compared UCE data from newer and older specimens of the same species and reconstructed their phylogeny in order to confirm the validity of our data. We recovered 6-972 UCE loci from samples with pre-library DNA concentrations ranging from 0.06-9.8 ng/μL. All investigated DNA yield and sequence capture variables were significantly but only moderately negatively correlated with specimen age. Specimens of age 20 years or less had significantly higher pre- and post-library concentrations, UCE contig lengths, and locus counts compared to specimens older than 20 years. We found breakpoints in our data indicating a decrease of the initial detrimental effect of specimen age on pre- and post-library DNA concentration and UCE contig length starting around 21-39 years after preservation. Our phylogenetic results confirmed the integrity of our data, giving preliminary insights into relationships within Xylocopa. We consider the effect of additional factors not measured in this study on our age-related sequence capture results, such as DNA fragmentation and preservation method, and discuss the promise of the UCE approach for large-scale projects in insect phylogenomics using museum specimens

Phylogenomic Reconstruction of the Neotropical Poison Frogs (Dendrobatidae) and Their Conservation

The evolutionary history of the Dendrobatidae, the charismatic Neotropical poison frog family, remains in flux, even after a half-century of intensive research. Understanding the evolutionary relationships between dendrobatid genera and the larger-order groups within Dendrobatidae is critical for making accurate assessments of all aspects of their biology and evolution. In this study, we provide the first phylogenomic reconstruction of Dendrobatidae with genome-wide nuclear markers known as ultraconserved elements. We performed sequence capture on 61 samples representing 33 species across 13 of the 16 dendrobatid genera, aiming for a broadly representative taxon sample. We compare topologies generated using maximum likelihood and coalescent methods and estimate divergence times using Bayesian methods. We find most of our dendrobatid tree to be consistent with previously published results based on mitochondrial and low-count nuclear data, with notable exceptions regarding the placement of Hyloxalinae and certain genera within Dendrobatinae. We also characterize how the evolutionary history and geographic distributions of the 285 poison frog species impact their conservation status. We hope that our phylogeny will serve as a backbone for future evolutionary studies and that our characterizations of conservation status inform conservation practices while highlighting taxa in need of further study

Comparison of means by age group.

<p>Table comparing group means for specimens aged under 20 years and over 20 years for pre-PCR library concentration (ng/μL), post-PCR library concentration (ng/μL), UCE mean contig length (bp) and UCE locus count (n). Significance was tested with a Welsh two sample t-test. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161531#pone.0161531.g003" target="_blank">Fig 3</a>.</p

Correlation between specimen age, library preparation and sequence capture statistics.

<p><b>(A)</b> pre-library DNA concentration (ng/μL) with specimen age (years); (<b>B)</b> post-library DNA concentration (ng/μL) with specimen age (years); (<b>C)</b> UCE contig length (bp) with specimen age (years); (<b>D)</b> raw read count (million bp) with specimen age (years); (<b>E)</b> specimen age (years) with UCE locus count (n loci); (<b>F)</b> raw read count (million bp) with UCE locus count (n loci); (<b>G)</b> pre-library DNA concentration (ng/μL) with UCE locus count (n loci); (<b>H)</b> post-library DNA concentration (ng/μL) with UCE locus count (n loci). Correlations were tested with the Pearson’s correlation coefficient; black lines represent linear regressions; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161531#pone.0161531.t002" target="_blank">Table 2</a> for specification of results. Blue lines in panels A, B and C represent piecewise regression lines, and red line with diamond represents the estimated breakpoint with 95% confidence interval around it (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161531#pone.0161531.t003" target="_blank">Table 3</a>).</p

Boxplots comparing means of several library preparation and UCE capture statistics by age group.

<p>We compared means of two age groups, above 20 years (N = 41) and below 20 years (N = 10), with a Welsh two sample t-test for (<b>A)</b> pre-library DNA concentration (ng/μL), (<b>B)</b> post-library DNA concentration (ng/μL), (<b>C)</b> UCE contig length (bp), and (<b>D</b>) UCE locus count (n loci). ** = p<0.001, * = p<0.05; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161531#pone.0161531.t003" target="_blank">Table 3</a> for full results of t-tests.</p

Correlation between selected UCE capture statistics.

<p>Correlation between specimen age, DNA concentration (pre and post library preparation), raw sequence read counts, reads on UCE targets, UCE mean contig length, UCE locus count, as well body size (LMT and HW). Calculated as Pearson's product-moment correlation. ** = p <0.001, * p <0.05. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161531#pone.0161531.g002" target="_blank">Fig 2</a>.</p

Histograms for specimen age and UCE locus count for the 51 taxa included in the study.

<p><b>(A)</b> Specimen age distribution among sampled specimens; <b>(B)</b> Distribution of UCE loci capture.</p

Phylogeny of <i>Xylocopa</i> study specimen based on the 50% complete data set.

<p>Maximum likelihood best tree including 49 <i>Xylocopa</i> specimens, based on 774 UCE loci and 258,013 bp (50% matrix). Values from bootstrap analysis are mapped onto the tree. Only bootstrap values > 50 are shown. Taxon labels in red font indicate taxa with a different position in the analysis of the 70% complete data set (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161531#pone.0161531.g004" target="_blank">Fig 4</a>). Scale bars represent nucleotide substitutions per base pair; tree is unrooted and displayed using midpoint rooting.</p