The broad distribution of the Sceloporus magister species group (squamata: phrynosomatidae) throu... more The broad distribution of the Sceloporus magister species group (squamata: phrynosomatidae) throughout western North America provides an appropriate model for testing biogeographical hypotheses explaining the timing and origins of diversity across mainland deserts and the Baja California Peninsula. We inferred concordant phylogenetic trees describing the higher-level relationships within the magister group using 1.6 kb of mitochondrial DNA (mtDNA) and 1.7 kb of nuclear DNA data. These data provide strong support for the parallel divergence of lineages endemic to the Baja California Peninsula (S. zosteromus and the orcutti complex) in the form of two sequential divergence events at the base of the magister group phylogeny. A relaxed phylogenetic analysis of the mtDNA data using one fossil and one biogeographical constraint provides a chronology of these divergence events and evidence that further diversification within the Baja California clades occurred simultaneously, although patterns of geographical variation and speciation between clades differ. We resolved four major phylogeographical clades within S. magister that (i) provide a novel phylogenetic placement of the Chihuahuan Desert populations sister to the Mojave Desert; (ii) illustrate a mixed history for the Colorado Plateau that includes Mojave and Sonoran Desert components; and (iii) identify an area of overlap between the Mojave and Sonoran Desert clades near Yuma, Arizona. Estimates of bidirectional migration rates among populations of S. magister using four nuclear loci support strong asymmetries in gene flow among the major mtDNA clades. Based on the nonexclusivity of mtDNA haplotypes, nuclear gene flow among populations and wide zones of phenotypic intergradation, S. magister appears to represent a single geographically variable and widespread species.
Recent fieldwork in southern Tanintharyi revealed the presence of a small Green Crested Lizard in... more Recent fieldwork in southern Tanintharyi revealed the presence of a small Green Crested Lizard in the wet evergreen forest. We generated mtDNA sequence data (ND2) that demonstrates that this population's nearest relative is Bronchocela rayaensis Grismer et al., 2015 of Pulau Langkawi, northwestern Peninsular Malaysia and Phuket Island. Morphologically the Burmese Bronchocela shares many features with B. ray-aensis, which potentially would make this recently described Thai-Malay species a synonym of Bronchocela burmana Blanford, 1878; however, we interpret the genetic and morphological differences to reflect evolutionary divergence and recommend the recognition of both species.
Genomic samples of non-model organisms are becoming increasingly important in a broad range of st... more Genomic samples of non-model organisms are becoming increasingly important in a broad range of studies from developmental biology, biodiversity analyses, to conservation. Genomic sample definition, description, quality, voucher information and metadata all need to be digitized and disseminated across scientific communities. This information needs to be concise and consistent in today's ever-increasing bioinformatic era, for complementary data aggregators to easily map databases to one another. In order to facilitate exchange of information on genomic samples and their derived data, the Global Genome Biodiversity Network (GGBN) Data Standard is intended to provide a platform based on a documented agreement to promote the efficient sharing and usage of genomic sample material and associated specimen information in a consistent way. The new data standard presented here build upon existing standards commonly used within the community extending them with the capability to exchange data on tissue, environmental and DNA sample as well as sequences. The GGBN Data Standard will reveal and democratize the hidden contents of biodiversity biobanks, for the convenience of everyone in the wider biobanking community. Technical tools exist for data providers to easily map their databases to the standard.
The relationships between the number of trunk and caudal vertebrae, the number of ventral and sub... more The relationships between the number of trunk and caudal vertebrae, the number of ventral and subcaudal scales, and snout-vent length (SVL) and tail length were investigated in several species of Hypsiglena, including a few specimens from species contact zones. We confirm the presence of a 1:1 relationship between trunk vertebrae and ventral scales within Hypsiglena (99 specimens total; 50 males; 49 females). We also found a positive relationship between the number of ventral scales and SVL in adult specimens. We did not find a 1:1 relationship between caudal vertebrae and subcaudal scales, but there is a positive relationship between subcaudal scales and tail length. Because there is a positive relationship with the number of ventral scales and SVL, we infer-as general population trends-that snakes with more ventral scales have the capacity to grow to be larger than snakes with fewer ventral scales. Many other environmental factors influence body size, however, and this trend should be used only as a general comparison. Therefore, the number of ventral scales can be used as a proxy for general population trends in body size among species of Hypsiglena, as wide geographicranging species may be exposed to large-scale environmental gradients (e.g., lower temperatures at higher latitudes). Ventral and subcaudal scale counts may not be good diagnostic characters for assigning individual specimens to taxonomic groups or for diagnosing taxonomic groups, because species appear to show much overlapping variation. This variation may be good for examining different trends in body size between taxa across geographic landscapes.
Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebra... more Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.
Many authors have claimed that short branches in the Tree of Life will be very difficult to resol... more Many authors have claimed that short branches in the Tree of Life will be very difficult to resolve with strong support, even with the large multilocus data sets now made possible by genomic resources. Short branches may be especially problematic because the underlying gene trees are expected to have discordant phylogenetic histories when the time between branching events is very short. Although there are many examples of short branches that are difficult to resolve, surprisingly, no empirical studies have systematically examined the relationships between branch lengths, branch support, and congruence among genes. Here, we examine these fundamental relationships quantitatively using a data set of 20 nuclear loci for 50 species of snakes (representing most traditionally recognized families). A combined maximum likelihood analysis of the 20 loci gives strong support for 69% of the nodes, but many remain weakly supported, with bootstrap values for 20% ranging from 21% to 66%. For the combined-data tree, we find significant correlations between the length of a branch, levels of bootstrap support, and the proportion of genes that are congruent with that branch in the separate analyses of each gene. We also find that strongly supported conflicts between gene trees over the resolution of individual branches are common (roughly 35% of clades), especially for shorter branches. Overall, our results support the hypothesis that short branches may be very difficult to confidently resolve, even with large, multilocus data sets. Nevertheless, our study provides strong support for many clades, including several that were controversial or poorly resolved in previous studies of snake phylogeny.
Advances in biodiversity genomic sequencing will increasingly depend on the availability of DNA s... more Advances in biodiversity genomic sequencing will increasingly depend on the availability of DNA samples—and their quantifiable metadata—preserved in large institutional biorepositories that are discoverable to the scientific community. Improvements in sequencing technology constantly provide longer reads, such that longer fragment length, higher molecular weight, and overall " genome-quality " DNA (gDNA) will be desirable. Ideally, biorepositories should publish numerical scale measurements of DNA quality useful to the user community. However, the most widely used technique to evaluate DNA quality, the classic agarose gel, has yet to be quantified. Here we propose a simple and economical method using open source image analysis software to make gDNA gel images quantifiable, and propose percentage of gDNA " greater than X kb " as a standard of comparison, where X is a band from any widely used DNA ladder with desirably large band sizes. We employ two metadata standards (" DNA Threshold " and " Percent above Threshold ") introduced as part of the Global Genome Biodiversity Network (GGBN) Darwin Core extension. We illustrate the method using the traditionally used HindIII ladder and the 9,416 base-pair (bp) band as a standard. We also present data, for two taxa, a vertebrate (fish) and an invertebrate (crab), on how gDNA quality varies with seven tissue preservation methods, time since death, preservation method (i.e. buffers vs. cold temperatures), and storage temperature of various buffers over time. Our results suggest that putting tissue into a buffer prior to freezing may be better than directly into ultra-cold conditions.
Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increa... more Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phyloge-ny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies) and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological , and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia). These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses) without considering the possible impact of molecular data on their placement.
Aim In this study, we investigate phylogeographic structure in two different species groups of lo... more Aim In this study, we investigate phylogeographic structure in two different species groups of lowland toads. First, we further investigate strict parapatry of the Pliocene-vicariant Bufo valliceps/B. nebulifer species pair. Secondly, we test for similar phylogeographic structure in the distantly related toad B. marinus, a species we hypothesize will show a Pleistocene dispersal across the same area.
Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have becomeincreas... more Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have becomeincreasingly controversial. Squamates include >9000 species, making them the secondlargest group of terrestrial vertebrates. They are important medicinally and as modelsystems for ecological and evolutionary research. However, studies of squamate biologyare hindered by uncertainty over their relationships, and some consider squamate phylogenyunresolved, given recent conflicts between molecular and morphological results. To resolvethese conflicts, we expand existing morphological and molecular datasets forsquamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa,including a new set of 81 morphological characters and adding two genes from publishedstudies) and perform integrated analyses. Our results resolve higher-level relationships asindicated by molecular analyses, and reveal hidden morphological support for the molecularhypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades(Mosasauria and Polyglyphanodontia). These results further demonstrate the importance ofcombining fossil and molecular information, and the potential problems of estimating theplacement of fossil taxa from morphological data alone. Thus, our results caution againstestimating fossil relationships without considering relevant molecular data, and againstplacing fossils into molecular trees (e.g. for dating analyses) without considering the possibleimpact of molecular data on their placement.
Vertebrates are currently going extinct at an alarming rate, largely because of habitat loss, glo... more Vertebrates are currently going extinct at an alarming rate, largely because of habitat loss, global warming, infectious diseases, and human introductions. Island ecosystems are particularly vulnerable to invasive species and other ecological disturbances. Properly documenting historic and current species distributions is critical for quantifying extinction events. Museum specimens, field notes, and other archived materials from historical expeditions are essential for documenting recent changes in biodiversity. The Islas Revillagigedo are a remote group of four islands, 700-1100 km off the western coast of mainland México. The islands are home to many endemic plants and animals recognized at the specific-and subspecific-levels, several of which are currently threatened or have already gone extinct. Here, we recount the initial discovery of an endemic snake Hypsiglena ochrorhyncha unaocularus Tanner on Isla Clarió n, the later dismissal of its existence, its absence from decades of field surveys, our recent rediscovery, and recognition of it as a distinct species. We collected two novel complete mitochondrial (mt) DNA genomes and up to 2800 base-pairs of mtDNA from several other individuals, aligned these with previously published mt-genome data from samples throughout the range of Hypsiglena, and conducted phylogenetic analyses to infer the biogeographic origin and taxonomic status of this population. We found the Isla Clarió n population to be most closely related to populations in the Sonora-Sinaloa state border area of mainland México and Isla Santa Catalina, in the Gulf of California. Based on genetics, morphology, and geographic distributions, we also recognize these two other lineages as distinct species. Our study shows the importance of museum specimens, field notes, and careful surveys to accurately document biodiversity and brings these island endemics (Clarió n and Santa Catalina nightsnakes) and mainland population near the Sonora-Sinaloa state border to the attention of conservation biologists currently monitoring biodiversity in these fragile subtropical ecosystems.
Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebra... more Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.
The salamander family Hynobiidae contains over 50 species and has been the subject of a number of... more The salamander family Hynobiidae contains over 50 species and has been the subject of a number of molecular phylogenetic investigations aimed at reconstructing branches across the entire family. In general, studies using the greatest amount of sequence data have used reduced taxon sampling, while the study with the greatest taxon sampling has used a limited sequence data set. Here, we provide insights into the phylogenetic history of the Hynobiidae using both dense taxon sampling and a large mitochondrial DNA sequence data set. We report exclusive new mitochondrial DNA data of 2566 aligned bases (with 151 excluded sites, of included sites 1157 are variable with 957 parsimony informative). This is sampled from two genic regions encoding a 12S-16S region (the 3' end of 12S rRNA, tRNA VAl , and the 5' end of 16S rRNA), and a ND2-COI region (ND2, tRNA Trp , tRNA Ala , tRNA Asn , the origin for light strand replication-O L , tRNA Cys , tRNA Tyr , and the 5' end of COI). Analyses using parsimony, Bayesian, and maximum likelihood optimality criteria produce similar phylogenetic trees, with discordant branches generally receiving low levels of branch support. Monophyly of the Hynobiidae is strongly supported across all analyses, as is the sister relationship and deep divergence between the genus Onychodactylus with all remaining hynobiids. Within this latter grouping our phylogenetic results identify six clades that are relatively divergent from one another, but for which there is minimal support for their phylogenetic placement. This includes the genus Batrachuperus, the genus Hynobius, the genus Pachyhynobius, the genus Salamandrella, a clade containing the genera Ranodon and Paradactylodon, and a clade containing the genera Liua and Pseudohynobius. This latter clade receives low bootstrap support in the parsimony analysis, but is consistent across all three analytical methods. Our results also clarify a number of well-supported relationships within the larger Batrachuperus and Hynobius clades. While the relationships identified in this study do much to clarify the phylogenetic history of the Hynobiidae, the poor resolution among major hynobiid clades, and the contrast of mtDNA-derived relationships with recent phylogenetic results from a small number of nuclear genes, highlights the need for continued phylogenetic study with larger numbers of nuclear loci.
Recently, phylogenetics has expanded to routinely include estimation of clade ages in addition to... more Recently, phylogenetics has expanded to routinely include estimation of clade ages in addition to their relationships. Various dating methods have been used, but their relative performance remains understudied. Here, we generate and assemble an extensive phylogenomic data set for squamate reptiles (lizards and snakes) and evaluate two widely used dating methods, penalized likelihood in r8s (r8s-PL) and Bayesian estimation with uncorrelated relaxed rates among lineages (BEAST). We obtained sequence data from 25 nuclear loci ($500-1000 bp per gene; 19,020 bp total) for 64 squamate species and nine outgroup taxa, estimated the phylogeny, and estimated divergence dates using 14 fossil calibrations. We then evaluated how well each method approximated these dates using random subsets of the nuclear loci (2, 5, 10, 15, and 20; replicated 10 times each), and using $1 kb of the mitochondrial ND2 gene. We find that estimates from r8s-PL based on 2, 5, or 10 loci can differ considerably from those based on 25 loci (mean absolute value of differences between 2-locus and 25-locus estimates were 9.0 Myr). Estimates from BEAST are somewhat more consistent given limited sampling of loci (mean absolute value of differences between 2 and 25-locus estimates were 5.0 Myr). Most strikingly, age estimates using r8s-PL for ND2 were $68-82 Myr older (mean = 73.1) than those using 25 nuclear loci with r8s-PL. These results show that dates from r8s-PL with a limited number of loci (and especially mitochondrial data) can differ considerably from estimates derived from a large number of nuclear loci, whereas estimates from BEAST derived from fewer nuclear loci or mitochondrial data alone can be surprisingly similar to those from many nuclear loci. However, estimates from BEAST using relatively few loci and mitochondrial data could still show substantial deviations from the full data set (>50 Myr), suggesting the benefits of sampling many nuclear loci. Finally, we found that confidence intervals on ages from BEAST were not significantly different when sampling 2 vs. 25 loci, suggesting that adding loci decreased errors but did not increase confidence in those estimates.
We combine mitochondrial and nuclear DNA sequence data with non-molecular (morphological and natu... more We combine mitochondrial and nuclear DNA sequence data with non-molecular (morphological and natural history) data to conduct phylogenetic analyses and generate an evolutionary hypothesis for the relationships among nearly every species of Mesoamerican bufonid in the genus Incilius. We collected a total of 5,898 aligned base-pairs (bp) of sequence data from mitochondrial (mtDNA: 12S-16S, cyt b, ND2-CO1, including tRNAs TRP-TYR and the origin of light strand replication; total 4,317 bp) and nuclear (CXCR4 and RAG1; total 1,581 bp) loci from 52 individual toads representing 37 species. For the non-molecular data, we collected 44 characters from 29 species. We also include Crepidophryne, a genus that has not previously been included in molecular analyses. We present results of parsimony and Bayesian analyses for these data separately and combined. Relationships based on the non-molecular data were poorly supported and did not resolve a monophyletic Incilius (Rhinella marina was nested within). Our molecular data provide significant support to most of the relationships. Our combined analyses demonstrate that inclusion of a considerably smaller dataset (44 vs. 5,898 characters) of non-molecular characters can provide significant support where the molecular relationships were lacking support. Our combined results indicate that Crepidophryne is nested within Incilius; therefore, we place the former in the synonymy of the latter taxon. Our study provides the most comprehensive evolutionary framework for Mesoamerican bufonids (Incilius), which we use as a starting point to invoke discussion on the evolution of their unique natural history traits.
We investigated the evolutionary relationships among populations of two threatened Red Data Book ... more We investigated the evolutionary relationships among populations of two threatened Red Data Book fossorial skinks, Scelotes gronovii and Scelotes kasneri, along the Western Cape Coast of South Africa. The genus Scelotes shows considerable variation in limb and digit reduction. We sampled four localities purported to contain S. gronovii and seven of S. kasneri, encompassing all of each species' limited distribution. Each of these species lack forelimbs, and differ by the number of digits on the hind limbs, among other morphological characters; S. gronovii bears a single digit and S. kasneri bears two digits on the hind limbs. Sequence data obtained from three mtDNA (16S ribosomal RNA, cytochrome b, and nicotinamide adenine dinucleotide dehydrogenase 1 unit; 2035 bp ttl.) and two nuclear (dynein axonemal heavy chain 3 and the natural killer tumor recognition; 1848 bp ttl.) gene regions were used to reconstruct the evolutionary relationships among the two focal species and several other co-distributed species ( Scelotes bipes, Scelotes montispectus, and Scelotes sexlineatus). Phylogenetic results (Bayesian and parsimony) revealed that several populations previously considered S. kasneri actually belong to other species, and others are paraphyletic with respect to one another. Additionally, populations of S. gronovii were also found to be paraphyletic, with populations south of the Berg River supported as sister to S. bipes, and populations north of the Berg River sister the remaining sampled species. Our results require a redefinition of S. sexlineatus to encompass populations morphologically convergent with S. kasneri and restrict the ranges of the already threatened S. kasneri and S. gronovii even further. The paraphyly of S. gronovii and the placement of each clade as sister to clades of species bearing two digits on the hind limbs suggests that digit loss has occurred at least twice in this group.
In spite of Amazonia's large biodiversity, we still understand relatively little about how it evo... more In spite of Amazonia's large biodiversity, we still understand relatively little about how it evolved. Phylogeographic studies help us to understand the spatial and temporal context in the diversification of organisms, but only a few such studies have been done in Amazonia, where regional and local variation exists that may have been influenced by different events or similar events at different times. Here we test whether, and to what degree, the phylogeographic structure of faunal elements reflects a recently proposed geological scenario for the establishment of the lower Amazon-Tocantins basins in eastern Amazonia. We test an area cladogram that assumes that the Amazon River represents the first potential barrier ($3.6 million years ago [mya]), followed by an east-west separation south of the Amazon caused by the paleo-Tocantins River (,2.5 mya), and most recently by the shift of the lower Tocantins eastward to its present course (,6000-8000 yr ago). We examined mtDNA (,520 base-pairs [bp] of 16S, 815 bp of cytochrome b) of two widely distributed and distantly related lizard species, Gonatodes humeralis (Sphaerodactylidae) and Kentropyx calcarata (Teiidae). The phylogeographic structure of G. humeralis indicates that the species is characterized by deep splits between localities, preventing us from recovering well-supported relationships between populations. Kentropyx calcarata haplotype networks and our Bayesian mtDNA tree identify a number of clades that we compare through an AMOVA. The overall phylogeny of K. calcarata was partially congruent with the geological scenario, but some unforeseen relationships were also recovered. Our results reinforce the potential of phylogeographic studies to help understand the recent evolution of Amazonia, and at the same time point to the necessity of refined sampling and the use of multiple molecular markers in such studies.
The broad distribution of the Sceloporus magister species group (squamata: phrynosomatidae) throu... more The broad distribution of the Sceloporus magister species group (squamata: phrynosomatidae) throughout western North America provides an appropriate model for testing biogeographical hypotheses explaining the timing and origins of diversity across mainland deserts and the Baja California Peninsula. We inferred concordant phylogenetic trees describing the higher-level relationships within the magister group using 1.6 kb of mitochondrial DNA (mtDNA) and 1.7 kb of nuclear DNA data. These data provide strong support for the parallel divergence of lineages endemic to the Baja California Peninsula (S. zosteromus and the orcutti complex) in the form of two sequential divergence events at the base of the magister group phylogeny. A relaxed phylogenetic analysis of the mtDNA data using one fossil and one biogeographical constraint provides a chronology of these divergence events and evidence that further diversification within the Baja California clades occurred simultaneously, although patterns of geographical variation and speciation between clades differ. We resolved four major phylogeographical clades within S. magister that (i) provide a novel phylogenetic placement of the Chihuahuan Desert populations sister to the Mojave Desert; (ii) illustrate a mixed history for the Colorado Plateau that includes Mojave and Sonoran Desert components; and (iii) identify an area of overlap between the Mojave and Sonoran Desert clades near Yuma, Arizona. Estimates of bidirectional migration rates among populations of S. magister using four nuclear loci support strong asymmetries in gene flow among the major mtDNA clades. Based on the nonexclusivity of mtDNA haplotypes, nuclear gene flow among populations and wide zones of phenotypic intergradation, S. magister appears to represent a single geographically variable and widespread species.
Recent fieldwork in southern Tanintharyi revealed the presence of a small Green Crested Lizard in... more Recent fieldwork in southern Tanintharyi revealed the presence of a small Green Crested Lizard in the wet evergreen forest. We generated mtDNA sequence data (ND2) that demonstrates that this population's nearest relative is Bronchocela rayaensis Grismer et al., 2015 of Pulau Langkawi, northwestern Peninsular Malaysia and Phuket Island. Morphologically the Burmese Bronchocela shares many features with B. ray-aensis, which potentially would make this recently described Thai-Malay species a synonym of Bronchocela burmana Blanford, 1878; however, we interpret the genetic and morphological differences to reflect evolutionary divergence and recommend the recognition of both species.
Genomic samples of non-model organisms are becoming increasingly important in a broad range of st... more Genomic samples of non-model organisms are becoming increasingly important in a broad range of studies from developmental biology, biodiversity analyses, to conservation. Genomic sample definition, description, quality, voucher information and metadata all need to be digitized and disseminated across scientific communities. This information needs to be concise and consistent in today's ever-increasing bioinformatic era, for complementary data aggregators to easily map databases to one another. In order to facilitate exchange of information on genomic samples and their derived data, the Global Genome Biodiversity Network (GGBN) Data Standard is intended to provide a platform based on a documented agreement to promote the efficient sharing and usage of genomic sample material and associated specimen information in a consistent way. The new data standard presented here build upon existing standards commonly used within the community extending them with the capability to exchange data on tissue, environmental and DNA sample as well as sequences. The GGBN Data Standard will reveal and democratize the hidden contents of biodiversity biobanks, for the convenience of everyone in the wider biobanking community. Technical tools exist for data providers to easily map their databases to the standard.
The relationships between the number of trunk and caudal vertebrae, the number of ventral and sub... more The relationships between the number of trunk and caudal vertebrae, the number of ventral and subcaudal scales, and snout-vent length (SVL) and tail length were investigated in several species of Hypsiglena, including a few specimens from species contact zones. We confirm the presence of a 1:1 relationship between trunk vertebrae and ventral scales within Hypsiglena (99 specimens total; 50 males; 49 females). We also found a positive relationship between the number of ventral scales and SVL in adult specimens. We did not find a 1:1 relationship between caudal vertebrae and subcaudal scales, but there is a positive relationship between subcaudal scales and tail length. Because there is a positive relationship with the number of ventral scales and SVL, we infer-as general population trends-that snakes with more ventral scales have the capacity to grow to be larger than snakes with fewer ventral scales. Many other environmental factors influence body size, however, and this trend should be used only as a general comparison. Therefore, the number of ventral scales can be used as a proxy for general population trends in body size among species of Hypsiglena, as wide geographicranging species may be exposed to large-scale environmental gradients (e.g., lower temperatures at higher latitudes). Ventral and subcaudal scale counts may not be good diagnostic characters for assigning individual specimens to taxonomic groups or for diagnosing taxonomic groups, because species appear to show much overlapping variation. This variation may be good for examining different trends in body size between taxa across geographic landscapes.
Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebra... more Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.
Many authors have claimed that short branches in the Tree of Life will be very difficult to resol... more Many authors have claimed that short branches in the Tree of Life will be very difficult to resolve with strong support, even with the large multilocus data sets now made possible by genomic resources. Short branches may be especially problematic because the underlying gene trees are expected to have discordant phylogenetic histories when the time between branching events is very short. Although there are many examples of short branches that are difficult to resolve, surprisingly, no empirical studies have systematically examined the relationships between branch lengths, branch support, and congruence among genes. Here, we examine these fundamental relationships quantitatively using a data set of 20 nuclear loci for 50 species of snakes (representing most traditionally recognized families). A combined maximum likelihood analysis of the 20 loci gives strong support for 69% of the nodes, but many remain weakly supported, with bootstrap values for 20% ranging from 21% to 66%. For the combined-data tree, we find significant correlations between the length of a branch, levels of bootstrap support, and the proportion of genes that are congruent with that branch in the separate analyses of each gene. We also find that strongly supported conflicts between gene trees over the resolution of individual branches are common (roughly 35% of clades), especially for shorter branches. Overall, our results support the hypothesis that short branches may be very difficult to confidently resolve, even with large, multilocus data sets. Nevertheless, our study provides strong support for many clades, including several that were controversial or poorly resolved in previous studies of snake phylogeny.
Advances in biodiversity genomic sequencing will increasingly depend on the availability of DNA s... more Advances in biodiversity genomic sequencing will increasingly depend on the availability of DNA samples—and their quantifiable metadata—preserved in large institutional biorepositories that are discoverable to the scientific community. Improvements in sequencing technology constantly provide longer reads, such that longer fragment length, higher molecular weight, and overall " genome-quality " DNA (gDNA) will be desirable. Ideally, biorepositories should publish numerical scale measurements of DNA quality useful to the user community. However, the most widely used technique to evaluate DNA quality, the classic agarose gel, has yet to be quantified. Here we propose a simple and economical method using open source image analysis software to make gDNA gel images quantifiable, and propose percentage of gDNA " greater than X kb " as a standard of comparison, where X is a band from any widely used DNA ladder with desirably large band sizes. We employ two metadata standards (" DNA Threshold " and " Percent above Threshold ") introduced as part of the Global Genome Biodiversity Network (GGBN) Darwin Core extension. We illustrate the method using the traditionally used HindIII ladder and the 9,416 base-pair (bp) band as a standard. We also present data, for two taxa, a vertebrate (fish) and an invertebrate (crab), on how gDNA quality varies with seven tissue preservation methods, time since death, preservation method (i.e. buffers vs. cold temperatures), and storage temperature of various buffers over time. Our results suggest that putting tissue into a buffer prior to freezing may be better than directly into ultra-cold conditions.
Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increa... more Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phyloge-ny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies) and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological , and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia). These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses) without considering the possible impact of molecular data on their placement.
Aim In this study, we investigate phylogeographic structure in two different species groups of lo... more Aim In this study, we investigate phylogeographic structure in two different species groups of lowland toads. First, we further investigate strict parapatry of the Pliocene-vicariant Bufo valliceps/B. nebulifer species pair. Secondly, we test for similar phylogeographic structure in the distantly related toad B. marinus, a species we hypothesize will show a Pleistocene dispersal across the same area.
Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have becomeincreas... more Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have becomeincreasingly controversial. Squamates include >9000 species, making them the secondlargest group of terrestrial vertebrates. They are important medicinally and as modelsystems for ecological and evolutionary research. However, studies of squamate biologyare hindered by uncertainty over their relationships, and some consider squamate phylogenyunresolved, given recent conflicts between molecular and morphological results. To resolvethese conflicts, we expand existing morphological and molecular datasets forsquamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa,including a new set of 81 morphological characters and adding two genes from publishedstudies) and perform integrated analyses. Our results resolve higher-level relationships asindicated by molecular analyses, and reveal hidden morphological support for the molecularhypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades(Mosasauria and Polyglyphanodontia). These results further demonstrate the importance ofcombining fossil and molecular information, and the potential problems of estimating theplacement of fossil taxa from morphological data alone. Thus, our results caution againstestimating fossil relationships without considering relevant molecular data, and againstplacing fossils into molecular trees (e.g. for dating analyses) without considering the possibleimpact of molecular data on their placement.
Vertebrates are currently going extinct at an alarming rate, largely because of habitat loss, glo... more Vertebrates are currently going extinct at an alarming rate, largely because of habitat loss, global warming, infectious diseases, and human introductions. Island ecosystems are particularly vulnerable to invasive species and other ecological disturbances. Properly documenting historic and current species distributions is critical for quantifying extinction events. Museum specimens, field notes, and other archived materials from historical expeditions are essential for documenting recent changes in biodiversity. The Islas Revillagigedo are a remote group of four islands, 700-1100 km off the western coast of mainland México. The islands are home to many endemic plants and animals recognized at the specific-and subspecific-levels, several of which are currently threatened or have already gone extinct. Here, we recount the initial discovery of an endemic snake Hypsiglena ochrorhyncha unaocularus Tanner on Isla Clarió n, the later dismissal of its existence, its absence from decades of field surveys, our recent rediscovery, and recognition of it as a distinct species. We collected two novel complete mitochondrial (mt) DNA genomes and up to 2800 base-pairs of mtDNA from several other individuals, aligned these with previously published mt-genome data from samples throughout the range of Hypsiglena, and conducted phylogenetic analyses to infer the biogeographic origin and taxonomic status of this population. We found the Isla Clarió n population to be most closely related to populations in the Sonora-Sinaloa state border area of mainland México and Isla Santa Catalina, in the Gulf of California. Based on genetics, morphology, and geographic distributions, we also recognize these two other lineages as distinct species. Our study shows the importance of museum specimens, field notes, and careful surveys to accurately document biodiversity and brings these island endemics (Clarió n and Santa Catalina nightsnakes) and mainland population near the Sonora-Sinaloa state border to the attention of conservation biologists currently monitoring biodiversity in these fragile subtropical ecosystems.
Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebra... more Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.
The salamander family Hynobiidae contains over 50 species and has been the subject of a number of... more The salamander family Hynobiidae contains over 50 species and has been the subject of a number of molecular phylogenetic investigations aimed at reconstructing branches across the entire family. In general, studies using the greatest amount of sequence data have used reduced taxon sampling, while the study with the greatest taxon sampling has used a limited sequence data set. Here, we provide insights into the phylogenetic history of the Hynobiidae using both dense taxon sampling and a large mitochondrial DNA sequence data set. We report exclusive new mitochondrial DNA data of 2566 aligned bases (with 151 excluded sites, of included sites 1157 are variable with 957 parsimony informative). This is sampled from two genic regions encoding a 12S-16S region (the 3' end of 12S rRNA, tRNA VAl , and the 5' end of 16S rRNA), and a ND2-COI region (ND2, tRNA Trp , tRNA Ala , tRNA Asn , the origin for light strand replication-O L , tRNA Cys , tRNA Tyr , and the 5' end of COI). Analyses using parsimony, Bayesian, and maximum likelihood optimality criteria produce similar phylogenetic trees, with discordant branches generally receiving low levels of branch support. Monophyly of the Hynobiidae is strongly supported across all analyses, as is the sister relationship and deep divergence between the genus Onychodactylus with all remaining hynobiids. Within this latter grouping our phylogenetic results identify six clades that are relatively divergent from one another, but for which there is minimal support for their phylogenetic placement. This includes the genus Batrachuperus, the genus Hynobius, the genus Pachyhynobius, the genus Salamandrella, a clade containing the genera Ranodon and Paradactylodon, and a clade containing the genera Liua and Pseudohynobius. This latter clade receives low bootstrap support in the parsimony analysis, but is consistent across all three analytical methods. Our results also clarify a number of well-supported relationships within the larger Batrachuperus and Hynobius clades. While the relationships identified in this study do much to clarify the phylogenetic history of the Hynobiidae, the poor resolution among major hynobiid clades, and the contrast of mtDNA-derived relationships with recent phylogenetic results from a small number of nuclear genes, highlights the need for continued phylogenetic study with larger numbers of nuclear loci.
Recently, phylogenetics has expanded to routinely include estimation of clade ages in addition to... more Recently, phylogenetics has expanded to routinely include estimation of clade ages in addition to their relationships. Various dating methods have been used, but their relative performance remains understudied. Here, we generate and assemble an extensive phylogenomic data set for squamate reptiles (lizards and snakes) and evaluate two widely used dating methods, penalized likelihood in r8s (r8s-PL) and Bayesian estimation with uncorrelated relaxed rates among lineages (BEAST). We obtained sequence data from 25 nuclear loci ($500-1000 bp per gene; 19,020 bp total) for 64 squamate species and nine outgroup taxa, estimated the phylogeny, and estimated divergence dates using 14 fossil calibrations. We then evaluated how well each method approximated these dates using random subsets of the nuclear loci (2, 5, 10, 15, and 20; replicated 10 times each), and using $1 kb of the mitochondrial ND2 gene. We find that estimates from r8s-PL based on 2, 5, or 10 loci can differ considerably from those based on 25 loci (mean absolute value of differences between 2-locus and 25-locus estimates were 9.0 Myr). Estimates from BEAST are somewhat more consistent given limited sampling of loci (mean absolute value of differences between 2 and 25-locus estimates were 5.0 Myr). Most strikingly, age estimates using r8s-PL for ND2 were $68-82 Myr older (mean = 73.1) than those using 25 nuclear loci with r8s-PL. These results show that dates from r8s-PL with a limited number of loci (and especially mitochondrial data) can differ considerably from estimates derived from a large number of nuclear loci, whereas estimates from BEAST derived from fewer nuclear loci or mitochondrial data alone can be surprisingly similar to those from many nuclear loci. However, estimates from BEAST using relatively few loci and mitochondrial data could still show substantial deviations from the full data set (>50 Myr), suggesting the benefits of sampling many nuclear loci. Finally, we found that confidence intervals on ages from BEAST were not significantly different when sampling 2 vs. 25 loci, suggesting that adding loci decreased errors but did not increase confidence in those estimates.
We combine mitochondrial and nuclear DNA sequence data with non-molecular (morphological and natu... more We combine mitochondrial and nuclear DNA sequence data with non-molecular (morphological and natural history) data to conduct phylogenetic analyses and generate an evolutionary hypothesis for the relationships among nearly every species of Mesoamerican bufonid in the genus Incilius. We collected a total of 5,898 aligned base-pairs (bp) of sequence data from mitochondrial (mtDNA: 12S-16S, cyt b, ND2-CO1, including tRNAs TRP-TYR and the origin of light strand replication; total 4,317 bp) and nuclear (CXCR4 and RAG1; total 1,581 bp) loci from 52 individual toads representing 37 species. For the non-molecular data, we collected 44 characters from 29 species. We also include Crepidophryne, a genus that has not previously been included in molecular analyses. We present results of parsimony and Bayesian analyses for these data separately and combined. Relationships based on the non-molecular data were poorly supported and did not resolve a monophyletic Incilius (Rhinella marina was nested within). Our molecular data provide significant support to most of the relationships. Our combined analyses demonstrate that inclusion of a considerably smaller dataset (44 vs. 5,898 characters) of non-molecular characters can provide significant support where the molecular relationships were lacking support. Our combined results indicate that Crepidophryne is nested within Incilius; therefore, we place the former in the synonymy of the latter taxon. Our study provides the most comprehensive evolutionary framework for Mesoamerican bufonids (Incilius), which we use as a starting point to invoke discussion on the evolution of their unique natural history traits.
We investigated the evolutionary relationships among populations of two threatened Red Data Book ... more We investigated the evolutionary relationships among populations of two threatened Red Data Book fossorial skinks, Scelotes gronovii and Scelotes kasneri, along the Western Cape Coast of South Africa. The genus Scelotes shows considerable variation in limb and digit reduction. We sampled four localities purported to contain S. gronovii and seven of S. kasneri, encompassing all of each species' limited distribution. Each of these species lack forelimbs, and differ by the number of digits on the hind limbs, among other morphological characters; S. gronovii bears a single digit and S. kasneri bears two digits on the hind limbs. Sequence data obtained from three mtDNA (16S ribosomal RNA, cytochrome b, and nicotinamide adenine dinucleotide dehydrogenase 1 unit; 2035 bp ttl.) and two nuclear (dynein axonemal heavy chain 3 and the natural killer tumor recognition; 1848 bp ttl.) gene regions were used to reconstruct the evolutionary relationships among the two focal species and several other co-distributed species ( Scelotes bipes, Scelotes montispectus, and Scelotes sexlineatus). Phylogenetic results (Bayesian and parsimony) revealed that several populations previously considered S. kasneri actually belong to other species, and others are paraphyletic with respect to one another. Additionally, populations of S. gronovii were also found to be paraphyletic, with populations south of the Berg River supported as sister to S. bipes, and populations north of the Berg River sister the remaining sampled species. Our results require a redefinition of S. sexlineatus to encompass populations morphologically convergent with S. kasneri and restrict the ranges of the already threatened S. kasneri and S. gronovii even further. The paraphyly of S. gronovii and the placement of each clade as sister to clades of species bearing two digits on the hind limbs suggests that digit loss has occurred at least twice in this group.
In spite of Amazonia's large biodiversity, we still understand relatively little about how it evo... more In spite of Amazonia's large biodiversity, we still understand relatively little about how it evolved. Phylogeographic studies help us to understand the spatial and temporal context in the diversification of organisms, but only a few such studies have been done in Amazonia, where regional and local variation exists that may have been influenced by different events or similar events at different times. Here we test whether, and to what degree, the phylogeographic structure of faunal elements reflects a recently proposed geological scenario for the establishment of the lower Amazon-Tocantins basins in eastern Amazonia. We test an area cladogram that assumes that the Amazon River represents the first potential barrier ($3.6 million years ago [mya]), followed by an east-west separation south of the Amazon caused by the paleo-Tocantins River (,2.5 mya), and most recently by the shift of the lower Tocantins eastward to its present course (,6000-8000 yr ago). We examined mtDNA (,520 base-pairs [bp] of 16S, 815 bp of cytochrome b) of two widely distributed and distantly related lizard species, Gonatodes humeralis (Sphaerodactylidae) and Kentropyx calcarata (Teiidae). The phylogeographic structure of G. humeralis indicates that the species is characterized by deep splits between localities, preventing us from recovering well-supported relationships between populations. Kentropyx calcarata haplotype networks and our Bayesian mtDNA tree identify a number of clades that we compare through an AMOVA. The overall phylogeny of K. calcarata was partially congruent with the geological scenario, but some unforeseen relationships were also recovered. Our results reinforce the potential of phylogeographic studies to help understand the recent evolution of Amazonia, and at the same time point to the necessity of refined sampling and the use of multiple molecular markers in such studies.
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Papers by Dan Mulcahy