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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, 163.5–157.3 Ma) 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 66 mya. [ABSTRACT FROM AUTHOR]

The map turtles (genus Graptemys ) comprise a morphologically diverse clade that forms a major component of the southeastern US hotspot of chelonian diversity. Map turtles have experienced both recent and rapid diversification resulting in long-standing uncertainty regarding species boundaries and phylogenetic relationships within the genus as well as timing of their divergence. We present a phylogeny for the group that includes geographically representative sampling for all described species and subspecies. We make use of an empirical prior on rates of molecular evolution to estimate divergence times with a molecular clock under a coalescent framework. Together, the phylogeny and divergence time estimates suggest that diversification has been both more recent and more rapid than has so far been suspected. We provide a well-supported evolutionary framework for Graptemys that is necessary for understanding map turtle diversity, biogeography, and for conservation of this threatened clade of turtles. [ABSTRACT FROM AUTHOR]

Abstract: Turtles are one of Earth’s most instantly recognizable life forms, distinguished for over 200 million years in the fossil record. Even so, key nodes in the phylogeny of turtles remain uncertain. To address this issue, we sequenced >90% of the nuclear recombination activase gene 1 (RAG-1) for 24 species representing all modern turtle families. RAG-1 exhibited negligible saturation and base composition bias, and extensive base composition homogeneity. Most of the relationships suggested by prior phylogenetic analyses were also supported by RAG-1 and, for at least two critical nodes, with a much higher level of support. RAG-1 also indicates that the enigmatic Platysternidae and Chelydridae, often considered sister taxa based on morphological evidence, are not closely related, although their precise phylogenetic placement in the turtle tree is still unresolved. Although RAG-1 is phylogenetically informative, our research revealed fundamental conflicts among analytical methods for estimating phylogenetic hypotheses. Maximum parsimony analyses of RAG-1 alone and in combination with two mitochondrial genes suggest the earliest phylogenetic splits separating into three basal branches, the pig-nosed turtles (Carettochelyidae), the softshell turtles (Trionychidae), and a clade comprising all remaining extant turtles. Maximum likelihood and Bayesian analyses group Carettochelyidae and Trionychidae (=Trionychoidae) in their more traditional location as the sister taxon to all other hidden-necked turtles, collectively forming the Cryptodira. Our research highlights the utility of molecular data in identifying issues of character homology in morphological datasets, while shedding valuable light on the biodiversity of a globally imperiled taxon. [Copyright &y& Elsevier]

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 44 Ma, and the crown age of both contained subfamilies at roughly 30 Ma. One deirochelyine clade, which includes the genera Graptemys , Malaclemys , Pseudemys , and Trachemys and contains 11% of all turtle species, dates to 21 Ma 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. [ABSTRACT FROM AUTHOR]

Highlights: [•] We generate phylogenies from three mitochondrial genes and 10 nuclear loci for all species of Pseudemys. [•] No species of Pseudemys were recovered as monophyletic. [•] The phylogeny for Pseudemys depends upon the individual selected for analysis. [•] Population genetic analysis including all Pseudemys taxa identified three major lineages. [•] There are currently seven to nine recognized species of Pseudemys, but our analyses indicate that the genus may be oversplit. [ABSTRACT FROM AUTHOR]

Abstract: Past confusion about leopard frog (genus Rana) species composition in the Tri-State area of the US that includes New York (NY), New Jersey (NJ), and Connecticut (CT) has hindered conservation and management efforts, especially where populations are declining or imperiled. We use nuclear and mitochondrial genetic data to clarify the identification and distribution of leopard frog species in this region. We focus on four problematic frog populations of uncertain species affiliation in northern NJ, southeastern mainland NY, and Staten Island to test the following hypotheses: (1) they are conspecific with Rana sphenocephala or R. pipiens, (2) they are hybrids between R. sphenocephala and R. pipiens, or (3) they represent one or more previously undescribed cryptic taxa. Bayesian phylogenetic and cluster analyses revealed that the four unknown populations collectively form a novel genetic lineage, which represents a previously undescribed cryptic leopard frog species, Rana sp. nov. Statistical support for R. sp. nov. was strong in both the Bayesian (pp=1.0) and maximum-likelihood (bootstrap=99) phylogenetic analyses as well as the Structure cluster analyses. While our data support recognition of R. sp. nov. as a novel species, we recommend further study including fine-scaled sampling and ecological, behavioral, call, and morphological analyses before it is formally described. [Copyright &y& Elsevier]

Abstract: Advances in molecular biology have expanded our understanding of patterns of evolution and our ability to infer phylogenetic relationships. Despite many applications of molecular methods in attempts at resolving the evolutionary relationships among the major clades of turtles, some nodes in the tree have proved to be extremely problematic and have remained unresolved. In this study, we use 14 nuclear loci to provide an in depth look at several of these troublesome nodes and infer the systematic relationships among 11 of the 14 turtle families. We find strong support for two of the most problematic nodes in the deep phylogeny of turtles that have traditionally defied resolution. In particular, we recover strong support for a sister relationship between the Emydidae and the monotypic bigheaded-turtle, Platysternon megacephalum. We also find strong support for a clade consisting of sea turtles, mud and musk turtles, and snapping turtles. Within this clade, snapping turtles (Chelydridae) and mud/musk turtles (Kinosternidae) are sister taxa, again with strong support. Our results emphasize the utility of multi-locus datasets in phylogenetic analyses of difficult problems. [Copyright &y& Elsevier]

Abstract: We present a strategy for phylogenetic marker development in non-model systems. Rather than using the traditional approach of comparing distantly related taxa to develop conserved primers for unknown species, we explore an alternative strategy that builds primers directly from a single, relatively well characterized species and applies those primers to increasingly distantly related taxa. We develop and test our protocol with turtles. Using a single BAC end-sequence library consisting of 3461 sequences totaling 2.43 million base pairs of data, we outline a procedure to flag repeat elements, followed by a BLAST approach to categorize sequences into high, low, and no similarity compartments compared to GenBank sequences. We developed and tested a panel of 96 primer pairs with a set of turtle tissues that forms a series of increasingly distantly related taxa with respect to the BAC reference species. Finally, we sequenced 11 of these newly discovered markers across a diverse set of 18 turtle species that spans the 210 million years of chelonian crown-group history and that includes representatives of most of the major clades of extant turtles. Our results indicate that large numbers of new, phylogenetically informative markers can be developed quickly and inexpensively from a single BAC, EST, or similar genomic resource, and that those markers provide reliable phylogenetic information across both shallow and deep levels of phylogenetic history. Our results also highlight the importance of screening for and managing repetitive elements found in randomly sequenced DNA fragments. We presume that our strategy should work well across any similarly divergent clade, suggesting that many-marker datasets can be developed quickly and efficiently for phylogenetic analysis. [Copyright &y& Elsevier]

Introns have gained considerable popularity as markers for molecular phylogenetics. However, no primers exist for a nuclear intron that amplifies across all turtles. Available data from morphology and mitochondrial DNA have not unambiguously resolved relationships within the superfamily Trionychoidea and the family Chelidae, which together form a large portion of extant turtle diversity. We tested the phylogenetic utility of a novel intron from the RNA fingerprint protein 35 (R35) as applied to these two areas of turtle systematics. We found the intron to be a single-copy locus that provides excellent resolving power for lineages among turtles, though problems with alignment made it impossible to infer deeper amniote relationships. Maximum parsimony and maximum likelihood both demonstrated the polyphyly of Trionychoidea and the reciprocal monophyly of Australian/New Guinea and South American chelid turtles. This is the first study to resolve such relationships with strong statistical support, and we suggest that R35 holds great promise for resolving additional persistent problems in the phylogeny of living turtles. [Copyright &y& Elsevier]