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      DNA Barcode Identification of Freshwater Snails in the Family Bithyniidae from Thailand

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          Abstract

          Freshwater snails in the family Bithyniidae are the first intermediate host for Southeast Asian liver fluke ( Opisthorchis viverrini), the causative agent of opisthorchiasis. Unfortunately, the subtle morphological characters that differentiate species in this group are not easily discerned by non-specialists. This is a serious matter because the identification of bithyniid species is a fundamental prerequisite for better understanding of the epidemiology of this disease. Because DNA barcoding, the analysis of sequence diversity in the 5’ region of the mitochondrial COI gene, has shown strong performance in other taxonomic groups, we decided to test its capacity to resolve 10 species/ subspecies of bithyniids from Thailand. Our analysis of 217 specimens indicated that COI sequences delivered species-level identification for 9 of 10 currently recognized species. The mean intraspecific divergence of COI was 2.3% (range 0-9.2 %), whereas sequence divergences between congeneric species averaged 8.7% (range 0-22.2 %). Although our results indicate that DNA barcoding can differentiate species of these medically-important snails, we also detected evidence for the presence of one overlooked species and one possible case of synonymy.

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          DNA barcoding and taxonomy in Diptera: a tale of high intraspecific variability and low identification success.

          DNA barcoding and DNA taxonomy have recently been proposed as solutions to the crisis of taxonomy and received significant attention from scientific journals, grant agencies, natural history museums, and mainstream media. Here, we test two key claims of molecular taxonomy using 1333 mitochondrial COI sequences for 449 species of Diptera. We investigate whether sequences can be used for species identification ("DNA barcoding") and find a relatively low success rate (< 70%) based on tree-based and newly proposed species identification criteria. Misidentifications are due to wide overlap between intra- and interspecific genetic variability, which causes 6.5% of all query sequences to have allospecific or a mixture of allo- and conspecific (3.6%) best-matching barcodes. Even when two COI sequences are identical, there is a 6% chance that they belong to different species. We also find that 21% of all species lack unique barcodes when consensus sequences of all conspecific sequences are used. Lastly, we test whether DNA sequences yield an unambiguous species-level taxonomy when sequence profiles are assembled based on pairwise distance thresholds. We find many sequence triplets for which two of the three pairwise distances remain below the threshold, whereas the third exceeds it; i.e., it is impossible to consistently delimit species based on pairwise distances. Furthermore, for species profiles based on a 3% threshold, only 47% of all profiles are consistent with currently accepted species limits, 20% contain more than one species, and 33% only some sequences from one species; i.e., adopting such a DNA taxonomy would require the redescription of a large proportion of the known species, thus worsening the taxonomic impediment. We conclude with an outlook on the prospects of obtaining complete barcode databases and the future use of DNA sequences in a modern integrative taxonomy.
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            Extreme diversity of tropical parasitoid wasps exposed by iterative integration of natural history, DNA barcoding, morphology, and collections.

            We DNA barcoded 2,597 parasitoid wasps belonging to 6 microgastrine braconid genera reared from parapatric tropical dry forest, cloud forest, and rain forest in Area de Conservación Guanacaste (ACG) in northwestern Costa Rica and combined these data with records of caterpillar hosts and morphological analyses. We asked whether barcoding and morphology discover the same provisional species and whether the biological entities revealed by our analysis are congruent with wasp host specificity. Morphological analysis revealed 171 provisional species, but barcoding exposed an additional 142 provisional species; 95% of the total is likely to be undescribed. These 313 provisional species are extraordinarily host specific; more than 90% attack only 1 or 2 species of caterpillars out of more than 3,500 species sampled. The most extreme case of overlooked diversity is the morphospecies Apanteles leucostigmus. This minute black wasp with a distinctive white wing stigma was thought to parasitize 32 species of ACG hesperiid caterpillars, but barcoding revealed 36 provisional species, each attacking one or a very few closely related species of caterpillars. When host records and/or within-ACG distributions suggested that DNA barcoding had missed a species-pair, or when provisional species were separated only by slight differences in their barcodes, we examined nuclear sequences to test hypotheses of presumptive species boundaries and to further probe host specificity. Our iterative process of combining morphological analysis, ecology, and DNA barcoding and reiteratively using specimens maintained in permanent collections has resulted in a much more fine-scaled understanding of parasitoid diversity and host specificity than any one of these elements could have produced on its own.
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              Extraction of high molecular weight DNA from molluscs.

              (1993)
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                4 November 2013
                : 8
                : 11
                : e79144
                Affiliations
                [1 ]Food-Borne Parasite Research Group, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
                [2 ]Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
                [3 ]Center of Excellence for Research in Biomedical Sciences, and Thailand Center of Excellence on Drug Discovery and Development, Thammasat University, Klongluang, Pathumthani, Thailand
                [4 ]Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
                Australian Museum, Australia
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JK ST WR TB VV JR. Performed the experiments: JK. Analyzed the data: JK SP JR TB PP. Contributed reagents/materials/analysis tools: JK SP PP PDNH. Wrote the manuscript: JK ST VV PP PDNH. Collected specimens: SP. Commentation: WR TB.

                Article
                PONE-D-12-38385
                10.1371/journal.pone.0079144
                3817070
                9d11b7a4-f47b-4307-854f-c2ed24e074e5
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 3 December 2012
                : 19 September 2013
                Funding
                The study was supported by the Higher Education Research Promotion and National Research University Project (NRU) of Thailand, and the Office of the Higher Education Commission, Ministry of Education of Thailand and through the Health Cluster (SHeP-GMS), Khon Kaen University, Thailand. Jutharat Kulsantiwong thanks the Office of the Higher Education Commission for supporting her PhD program (CHE) in the Department of Parasitology, Faculty of Medicine, Khon Kaen University, Thammasat University, and UdonThani Rajabhat University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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