DNA barcoding cannot discriminate between Sardinella tawilis and S. hualiensis (Clupeiformes: Clupeidae)

Sardinella tawilis, the only known freshwater sardinella in the world, is endemic to Taal Lake, Philippines. Previous studies found the Taiwan sardinella, S. hualiensis, to be morphologically very similar to S. tawilis and identified it as the marine sister species of S. tawilis. In this study, DNA barcoding using the mitochondrial cytochrome c oxidase I (COI) gene was carried out to analyze species demarcation in the Sardinella genus, focusing primarily on the relationship between S. tawilis and S. hualiensis. The neighbour-joining (NJ) tree that was constructed using Kimura 2-parameter (K2P) model showed a single clade for the two species with 100% bootstrap support. K2P interspecific genetic divergence ranged from 0% to 0.522%, which is clearly below the suggested 3–3.5% cutoff for species discrimination. Recombination activating gene 1 (RAG1), mitochondrial control region (CR), cytochrome b, 16S rRNA, and S7 markers were used to further validate the results. Sardinella tawilis and S. hualiensis clustered together with a bootstrap support of 99–100% in each of the NJ trees. Low interspecific genetic distances between S. tawilis and S. hualiensis for all the markers except CR could be attributed to incipient allopatric speciation

DNA analyses of large pangolin scale seizures: Species identification validation and case studies

Pangolins are the mosttrafficked mammal in theworld, and all eightspecies are listed under CITESAppendix I.DNAbased wildlife forensic techniques are recognized as an important component of investigating a pangolin seizure. In particular, determining the species of pangolin in a seizure will 1) confirm the presence of pangolin to establish the legality of any trade, and 2) ensure appropriate laws are applied to theirfullest extentin a prosecution. Furthermore, valuable intelligence data, such as determining the geographic provenance of samples, can be produced through analysis of pangolin seizures. Despite the immense scale of the pangolin trade, standardized wildlife forensic techniquesfortesting pangolin seizures are in theirinfancy. To addressthis, here, we present a standardized genetic marker suitable for species identification of all eight pangolin species, and outline practical strategies for sampling large-volume pangolin scale seizures. We assessed the repeatability, reproducibility, robustness, sensitivity and phylogenetic resolution of this species identification test. Critically, the assay was tested in four wildlife forensic laboratories involved in testing pangolins. Additionally, we demonstrated the test’s utility to conduct geographic provenance analysis of Phataginus tricuspis samples. We analysed five large-volume pangolin scale seizures in Malaysia, which elucidated key targetspecies, poaching hotspots, and trafficking routes. Phataginustricuspis wasthe most commonly identified species(88.8%)from the seizure samples, and 84.3% of these P. tricuspisindividuals were likely sourced from western central Africa. We expect the im

Conservation Genetics of the Philippine Tarsier: Cryptic Genetic Variation Restructures Conservation Priorities for an Island Archipelago Primate

Acknowledgments We thank the Philippine Department of Environment and Natural Resources for facilitating research, sample collection, and export permits (in particular T. M. Lim, C. Custodio, J. deLeon, and A. Tagtag) necessary for this and related research. Sampling protocols were approved by the University of Kansas Animal Care and Use Committee (IACUC 158-01 to RMB) and protocols to capture, sedate, and harvest ear biopsies from wild tarsiers were approved by the Dartmouth Animal Care and Use Committee (IACUC 10-11-12 and 11-06-06AT to NJD). Thanks are due to J. Quilang for assistance with data and comments on the manuscript. We thank N. Antoque, J. Cantil, and V. Yngente for assistance in the field and anonymous reviewers for comments on previous versions of the manuscript.Author Contributions Conceived and designed the experiments: RMB JAW CDS JAE MS MLD ACD. Performed the experiments: RMB MRMD LVD INA JAE NJD PSO AL MLD ACD CDS. Analyzed the data: KVO AJB CDS. Contributed reagents/materials/analysis tools: RMB KVO AJB CDS JAE LVD GLM. Wrote the paper: RMB JAW CDS JAE MS GLM. Obtained permission and executed field surveys: RMB MRMD LVD MS INA JAE NJD PSO GLM AL MLD ACD CDS.Establishment of conservation priorities for primates is a particular concern in the island archipelagos of Southeast Asia, where rates of habitat destruction are among the highest in the world. Conservation programs require knowledge of taxonomic diversity to ensure success. The Philippine tarsier is a flagship species that promotes environmental awareness and a thriving ecotourism economy in the Philippines. However, assessment of its conservation status has been impeded by taxonomic uncertainty, a paucity of field studies, and a lack of vouchered specimens and genetic samples available for study in biodiversity repositories. Consequently, conservation priorities are unclear. In this study we use mitochondrial and nuclear DNA to empirically infer geographic partitioning of genetic variation and to identify evolutionarily distinct lineages for conservation action. The distribution of Philippine tarsier genetic diversity is neither congruent with expectations based on biogeographical patterns documented in other Philippine vertebrates, nor does it agree with the most recent Philippine tarsier taxonomic arrangement. We identify three principal evolutionary lineages that do not correspond to the currently recognized subspecies, highlight the discovery of a novel cryptic and range-restricted subcenter of genetic variation in an unanticipated part of the archipelago, and identify additional geographically structured genetic variation that should be the focus of future studies and conservation action. Conservation of this flagship species necessitates establishment of protected areas and targeted conservation programs within the range of each genetically distinct variant of the Philippine tarsier.Support for fieldwork was provided by the University of Kansas Biodiversity Institute and Department of Ecology and Evolutionary Biology (to RMB, CDS, and JAE), the National Geographic Society (NGS 8446-08 to RMB, JAW, MS and INA), funds from the Primate Action Fund (to MS), Ewha Womans University (Ewha Global Top5 Grant 2013 to MS), the David and Lucile Packard Foundation (Fellowship in Science and Engineering no. 2007-31754, to NJD), and U.S. National Science Foundation (DEB 0743491 to RMB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Yeshttp://www.plosone.org/static/editorial#pee

Data from: Conservation genetics of the Philippine tarsier: cryptic genetic variation restructures conservation priorities for an island archipelago primate

Establishment of conservation priorities for primates is a particular concern in the island archipelagos of Southeast Asia, where rates of habitat destruction are among the highest in the world. Conservation programs require knowledge of taxonomic diversity to ensure success. The Philippine tarsier is a flagship species that promotes environmental awareness and a thriving ecotourism economy in the Philippines. However, assessment of its conservation status has been impeded by taxonomic uncertainty, a paucity of field studies, and a lack of vouchered specimens and genetic samples available for study in biodiversity repositories. Consequently, conservation priorities are unclear. In this study we use mitochondrial and nuclear DNA to empirically infer geographic partitioning of genetic variation and to identify evolutionarily distinct lineages for conservation action. The distribution of Philippine tarsier genetic diversity is neither congruent with expectations based on biogeographical patterns documented in other Philippine vertebrates, nor does it agree with the most recent Philippine tarsier taxonomic arrangement. We identify three principal evolutionary lineages that do not correspond to the currently recognized subspecies, highlight the discovery of a novel cryptic and range-restricted subcenter of genetic variation in an unanticipated part of the archipelago, and identify additional geographically structured genetic variation that should be the focus of future studies and conservation action. Conservation of this flagship species necessitates establishment of protected areas and targeted conservation programs within the range of each genetically distinct variant of the Philippine tarsier

Tarsier_Convert_input

Tarsier microsatelite

Uncorrected mitochondrial sequence divergence (%) among Philippine tarsier (<i>Tarsius syrichta</i>) evolutionary lineages shown below diagonal.

<p>Percentages on the diagonal represent intraspecific (or within clade) genetic diversity. Mean inferred migration rates (inferred in BAYESASS) between regionally partitioned diversity shown above diagonal. Migration rates above the 0.05 threshold bolded for emphasis.</p

DISTRUCT visualization of STRUCTURE analyses (A) assigning individuals to major population groupings (genetically distinct evolutionary lineages) for Philippine tarsier demes (<i>K</i> = 2 and 3 populations).

<p>Mindanao faunal region (B; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104340#pone-0104340-g001" target="_blank">Fig. 1</a>, inset) with sampling (17 sites, 66 individuals) labeled with letters corresponding to full localities listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104340#pone.0104340.s002" target="_blank">Appendix S2</a>, protected areas shaded red. SplitsTree gene network (C; numbers at internodes = ML bootstrap replicates), and results of GMYC analyses (red asterisks denote lineages delineated by the Yule-coalescent), with numbers at tips corresponding to individual samples in Structure plots (A) and cluster shading corresponding to islands on map (B).</p

Models of evolution selected by AIC and applied for partitioned, phylogeographic analyses¹.

1<p>The model GTR + Γ was used for partitioned RAxMLHPC analyses.</p

Microsatellite variation in 66 individuals of <i>Tarsius syrichta</i> from 17 localities (Fig. 2).

<p>Abbreviations include: <i>N</i>, number of samples; <i>H</i>_O, observed heterozygosity; <i>H</i>_E, expected heterozygosity; <i>L</i>_P, number of polymorphic loci; <i>F</i>_IS inbreeding coefficient.</p

Phylogeographic relationships of <i>Tarsius syrichta</i> (see Appendix S1 for taxonomic summary) estimated from a combined, partitioned, RAxML ML analysis of mitochondrial (12S, CytB, ND2) gene fragments.

<p>Black circles at nodes correspond to ML bootstraps ≥70% and Bayesian PP ≥95%.</p