Lack of Genetic Variation in Cytochrome bin a Population of Smooth Softshell Turtles

An important issue in intraspecific molecular phylogenetic studies concerns distribution of genetic variation within and among populations and, hence, within-population sample sizes used in analyses. To address this sampling issue, we sequenced a 795 base pair (bp) segment of the mitochondrial cytochrome b gene from 19 unrelated individuals from a Louisiana population of the smooth softshell turtle (Apalone mutica LeSueur). We found a complete lack of within-population variation in this large segment of mtDNA. This result supports the use of minimal within-population sample sizes in intraspecific molecular phylogenetic studies of Apalone using cytochrome b

Genomic Data Reveal Conserved Female Heterogamety in Giant Salamanders with Gigantic Nuclear Genomes

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Systems of genetic sex determination and the homology of sex chromosomes in different taxa vary greatly across vertebrates. Much progress remains to be made in understanding systems of genetic sex determination in non-model organisms, especially those with homomorphic sex chromosomes and/or large genomes. We used reduced representation genome sequencing to investigate genetic sex determination systems in the salamander family Cryptobranchidae (genera Cryptobranchus and Andrias), which typifies both of these inherent difficulties. We tested hypotheses of male- or female-heterogamety by sequencing hundreds of thousands of anonymous genomic regions in a panel of known-sex cryptobranchids and characterized patterns of presence/absence, inferred zygosity, and depth of coverage to identify sex-linked regions of these 56 gigabase genomes. Our results strongly support the hypothesis that all cryptobranchid species possess homologous systems of female heterogamety, despite maintenance of homomorphic sex chromosomes over nearly 60 million years. Additionally, we report a robust, non-invasive genetic assay for sex diagnosis in Cryptobranchus and Andrias which may have great utility for conservation efforts with these endangered salamanders. Co-amplification of these W-linked markers in both cryptobranchid genera provides evidence for long-term sex chromosome stasis in one of the most divergent salamander lineages. These findings inform hypotheses about the ancestral mode of sex determination in salamanders, but suggest that comparative data from other salamander families are needed. Our results further demonstrate that massive genomes are not necessarily a barrier to effective genome-wide sequencing and that the resulting data can be highly informative about sex determination systems in taxa with homomorphic sex chromosomes.Cryptobranchid Interest GroupFresno Chaffee ZooSaint Louis Zoo WildCare InstituteUniversity of Kentucky G.F. Ribble FundNational Science Foundation (DEB-0949532 to D.W.W. and DGE-3048109801 to P.M.H.

A support vector machine based test for incongruence between sets of trees in tree space

BACKGROUND: The increased use of multi-locus data sets for phylogenetic reconstruction has increased the need to determine whether a set of gene trees significantly deviate from the phylogenetic patterns of other genes. Such unusual gene trees may have been influenced by other evolutionary processes such as selection, gene duplication, or horizontal gene transfer. RESULTS: Motivated by this problem we propose a nonparametric goodness-of-fit test for two empirical distributions of gene trees, and we developed the software GeneOut to estimate a p-value for the test. Our approach maps trees into a multi-dimensional vector space and then applies support vector machines (SVMs) to measure the separation between two sets of pre-defined trees. We use a permutation test to assess the significance of the SVM separation. To demonstrate the performance of GeneOut, we applied it to the comparison of gene trees simulated within different species trees across a range of species tree depths. Applied directly to sets of simulated gene trees with large sample sizes, GeneOut was able to detect very small differences between two set of gene trees generated under different species trees. Our statistical test can also include tree reconstruction into its test framework through a variety of phylogenetic optimality criteria. When applied to DNA sequence data simulated from different sets of gene trees, results in the form of receiver operating characteristic (ROC) curves indicated that GeneOut performed well in the detection of differences between sets of trees with different distributions in a multi-dimensional space. Furthermore, it controlled false positive and false negative rates very well, indicating a high degree of accuracy. CONCLUSIONS: The non-parametric nature of our statistical test provides fast and efficient analyses, and makes it an applicable test for any scenario where evolutionary or other factors can lead to trees with different multi-dimensional distributions. The software GeneOut is freely available under the GNU public license

Species delimitation in lemurs: multiple genetic loci reveal low levels of species diversity in the genus Cheirogaleus

<p>Abstract</p> <p>Background</p> <p>Species are viewed as the fundamental unit in most subdisciplines of biology. To conservationists this unit represents the currency for global biodiversity assessments. Even though Madagascar belongs to one of the top eight biodiversity hotspots of the world, the taxonomy of its charismatic lemuriform primates is not stable. Within the last 25 years, the number of described lemur species has more than doubled, with many newly described species identified among the nocturnal and small-bodied cheirogaleids. Here, we characterize the diversity of the dwarf lemurs (genus <it>Cheirogaleus</it>) and assess the status of the seven described species, based on phylogenetic and population genetic analysis of mtDNA (<it>cytb </it>+ <it>cox2</it>) and three nuclear markers (<it>adora3</it>, <it>fiba </it>and <it>vWF</it>).</p> <p>Results</p> <p>This study identified three distinct evolutionary lineages within the genus <it>Cheirogaleus</it>. Population genetic cluster analyses revealed a further layer of population divergence with six distinct genotypic clusters.</p> <p>Conclusion</p> <p>Based on the general metapopulation lineage concept and multiple concordant data sets, we identify three exclusive groups of dwarf lemur populations that correspond to three of the seven named species: <it>C. major</it>, <it>C. medius </it>and <it>C. crossleyi</it>. These three species were found to be genealogically exclusive in both mtDNA and nDNA loci and are morphologically distinguishable. The molecular and morphometric data indicate that <it>C. adipicaudatus </it>and <it>C. ravus </it>are synonymous with <it>C. medius </it>and <it>C. major</it>, respectively. <it>Cheirogaleus sibreei </it>falls into the <it>C. medius </it>mtDNA clade, but in morphological analyses the membership is not clearly resolved. We do not have sufficient data to assess the status of <it>C. minusculus</it>. Although additional patterns of population differentiation are evident, there are no clear subdivisions that would warrant additional specific status. We propose that ecological and more geographic data should be collected to confirm these results.</p

Concatenation and Concordance in the Reconstruction of Mouse Lemur Phylogeny: An Empirical Demonstration of the Effect of Allele Sampling in Phylogenetics

The systematics and speciation literature is rich with discussion relating to the potential for gene tree/species tree discordance. Numerous mechanisms have been proposed to generate discordance, including differential selection, longbranch attraction, gene duplication, genetic introgression, and/or incomplete lineage sorting. For speciose clades in which divergence has occurred recently and rapidly, recovering the true species tree can be particularly problematic due to incomplete lineage sorting. Unfortunately, the availability of multilocus or “phylogenomic” data sets does not simply solve the problem, particularly when the data are analyzed with standard concatenation techniques. In our study, we conduct a phylogenetic study for a nearly complete species sample of the dwarf and mouse lemur clade, Cheirogaleidae. Mouse lemurs (genus, Microcebus) have been intensively studied over the past decade for reasons relating to their high level of cryptic species diversity, and although there has been emerging consensus regarding the evolutionary diversity contained within the genus, there is no agreement as to the inter-specific relationships within the group. We attempt to resolve cheirogaleid phylogeny, focusing especially on the mouse lemurs, by employing a large multilocus data set. We compare the results of Bayesian concordance methods with those of standard gene concatenation, finding that though concatenation yields the strongest results as measured by statistical support, these results are found to be highly misleading. By employing an approach where individual alleles are treated as operational taxonomic units, we show that phylogenetic results are substantially influenced by the selection of alleles in the concatenation process. Includes supplementary materials

Sal-Site: Integrating new and existing ambystomatid salamander research and informational resources

Salamanders of the genus Ambystoma are a unique model organism system because they enable natural history and biomedical research in the laboratory or field. We developed Sal-Site to integrate new and existing ambystomatid salamander research resources in support of this model system. Sal-Site hosts six important resources: 1) Salamander Genome Project: an information-based web-site describing progress in genome resource development, 2) Ambystoma EST Database: a database of manually edited and analyzed contigs assembled from ESTs that were collected from A. tigrinum tigrinum and A. mexicanum, 3) Ambystoma Gene Collection: a database containing full-length protein-coding sequences, 4) Ambystoma Map and Marker Collection: an image and database resource that shows the location of mapped markers on linkage groups, provides information about markers, and provides integrating links to Ambystoma EST Database and Ambystoma Gene Collection databases, 5) Ambystoma Genetic Stock Center: a website and collection of databases that describe an NSF funded salamander rearing facility that generates and distributes biological materials to researchers and educators throughout the world, and 6) Ambystoma Research Coordination Network: a web-site detailing current research projects and activities involving an international group of researchers. Sal-Site is accessible at

From Biomedicine to Natural History Research: EST Resources for Ambystomatid Aalamanders

BACKGROUND: Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum) and Eastern tiger salamander (A. tigrinum tigrinum), species with deep and diverse research histories. RESULTS: Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human - Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species. CONCLUSIONS: Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research

Amplicon sequencing of 42 nuclear loci supports directional gene flow between South Pacific populations of a hydrothermal vent limpet

In the past few decades, population genetics and phylogeographic studies have improved our knowledge of connectivity and population demography in marine environments. Studies of deep‐sea hydrothermal vent populations have identified barriers to gene flow, hybrid zones, and demographic events, such as historical population expansions and contractions. These deep‐sea studies, however, used few loci, which limit the amount of information they provided for coalescent analysis and thus our ability to confidently test complex population dynamics scenarios. In this study, we investigated population structure, demographic history, and gene flow directionality among four Western Pacific hydrothermal vent populations of the vent limpet Lepetodrilus aff. schrolli. These vent sites are located in the Manus and Lau back‐arc basins, currently of great interest for deep‐sea mineral extraction. A total of 42 loci were sequenced from each individual using high‐throughput amplicon sequencing. Amplicon sequences were analyzed using both genetic variant clustering methods and evolutionary coalescent approaches. Like most previously investigated vent species in the South Pacific, L. aff. schrolli showed no genetic structure within basins but significant differentiation between basins. We inferred significant directional gene flow from Manus Basin to Lau Basin, with low to no gene flow in the opposite direction. This study is one of the very few marine population studies using >10 loci for coalescent analysis and serves as a guide for future marine population studies

Phylogenomics Reveals Ancient Gene Tree Discordance in the Amphibian Tree of Life

Molecular phylogenies have yielded strong support for many parts of the amphibian Tree of Life, but poor support for the resolution of deeper nodes, including relationships among families and orders. To clarify these relationships, we provide a phylogenomic perspective on amphibian relationships by developing a taxon-specific Anchored Hybrid Enrichment protocol targeting hundreds of conserved exons which are effective across the class. After obtaining data from 220 loci for 286 species (representing 94% of the families and 44% of the genera), we estimate a phylogeny for extant amphibians and identify gene tree–species tree conflict across the deepest branches of the amphibian phylogeny. We perform locus-by-locus genealogical interrogation of alternative topological hypotheses for amphibian monophyly, focusing on interordinal relationships. We find that phylogenetic signal deep in the amphibian phylogeny varies greatly across loci in a manner that is consistent with incomplete lineage sorting in the ancestral lineage of extant amphibians. Our results overwhelmingly support amphibian monophyly and a sister relationship between frogs and salamanders, consistent with the Batrachia hypothesis. Species tree analyses converge on a small set of topological hypotheses for the relationships among extant amphibian families. These results clarify several contentious portions of the amphibian Tree of Life, which in conjunction with a set of vetted fossil calibrations, support a surprisingly younger timescale for crown and ordinal amphibian diversification than previously reported. More broadly, our study provides insight into the sources, magnitudes, and heterogeneity of support across loci in phylogenomic data sets

Digits Lost or Gained? Evidence for Pedal Evolution in the Dwarf Salamander Complex (Eurycea, Plethodontidae)

Change in digit number, particularly digit loss, has occurred repeatedly over the evolutionary history of tetrapods. Although digit loss has been documented among distantly related species of salamanders, it is relatively uncommon in this amphibian order. For example, reduction from five to four toes appears to have evolved just three times in the morphologically and ecologically diverse family Plethodontidae. Here we report a molecular phylogenetic analysis for one of these four-toed lineages – the Eurycea quadridigitata complex (dwarf salamanders) – emphasizing relationships to other species in the genus. A multilocus phylogeny reveals that dwarf salamanders are paraphyletic with respect to a complex of five-toed, paedomorphic Eurycea from the Edwards Plateau in Texas. We use this phylogeny to examine evolution of digit number within the dwarf−Edwards Plateau clade, testing contrasting hypotheses of digit loss (parallelism among dwarf salamanders) versus digit gain (re-evolution in the Edwards Plateau complex). Bayes factors analysis provides statistical support for a five-toed common ancestor at the dwarf-Edwards node, favoring, slightly, the parallelism hypothesis for digit loss. More importantly, our phylogenetic results pinpoint a rare event in the pedal evolution of plethodontid salamanders