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      How many species and under what names? Using DNA barcoding and GenBank data for west Central African amphibian conservation

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          Abstract

          Development projects in west Central Africa are proceeding at an unprecedented rate, often with little concern for their effects on biodiversity. In an attempt to better understand potential impacts of a road development project on the anuran amphibian community, we conducted a biodiversity assessment employing multiple methodologies (visual encounter transects, auditory surveys, leaf litter plots and pitfall traps) to inventory species prior to construction of a new road within the buffer zone of Moukalaba-Doudou National Park, Gabon. Because of difficulties in morphological identification and taxonomic uncertainty of amphibian species observed in the area, we integrated a DNA barcoding analysis into the project to improve the overall quality and accuracy of the species inventory. Based on morphology alone, 48 species were recognized in the field and voucher specimens of each were collected. We used tissue samples from specimens collected at our field site, material available from amphibians collected in other parts of Gabon and the Republic of Congo to initiate a DNA barcode library for west Central African amphibians. We then compared our sequences with material in GenBank for the genera recorded at the study site to assist in identifications. The resulting COI and 16S barcode library allowed us to update the number of species documented at the study site to 28, thereby providing a more accurate assessment of diversity and distributions. We caution that because sequence data maintained in GenBank are often poorly curated by the original submitters and cannot be amended by third-parties, these data have limited utility for identification purposes. Nevertheless, the use of DNA barcoding is likely to benefit biodiversity inventories and long-term monitoring, particularly for taxa that can be difficult to identify based on morphology alone; likewise, inventory and monitoring programs can contribute invaluable data to the DNA barcode library and the taxonomy of complex groups. Our methods provide an example of how non-taxonomists and parataxonomists working in understudied parts of the world with limited geographic sampling and comparative morphological material can use DNA barcoding and publicly available sequence data (GenBank) to rapidly identify the number of species and assign tentative names to aid in urgent conservation management actions and contribute to taxonomic resolution.

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          Cryptic species as a window on diversity and conservation.

          The taxonomic challenge posed by cryptic species (two or more distinct species classified as a single species) has been recognized for nearly 300 years, but the advent of relatively inexpensive and rapid DNA sequencing has given biologists a new tool for detecting and differentiating morphologically similar species. Here, we synthesize the literature on cryptic and sibling species and discuss trends in their discovery. However, a lack of systematic studies leaves many questions open, such as whether cryptic species are more common in particular habitats, latitudes or taxonomic groups. The discovery of cryptic species is likely to be non-random with regard to taxon and biome and, hence, could have profound implications for evolutionary theory, biogeography and conservation planning.
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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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              Tropical forests were the primary sources of new agricultural land in the 1980s and 1990s.

              Global demand for agricultural products such as food, feed, and fuel is now a major driver of cropland and pasture expansion across much of the developing world. Whether these new agricultural lands replace forests, degraded forests, or grasslands greatly influences the environmental consequences of expansion. Although the general pattern is known, there still is no definitive quantification of these land-cover changes. Here we analyze the rich, pan-tropical database of classified Landsat scenes created by the Food and Agricultural Organization of the United Nations to examine pathways of agricultural expansion across the major tropical forest regions in the 1980s and 1990s and use this information to highlight the future land conversions that probably will be needed to meet mounting demand for agricultural products. Across the tropics, we find that between 1980 and 2000 more than 55% of new agricultural land came at the expense of intact forests, and another 28% came from disturbed forests. This study underscores the potential consequences of unabated agricultural expansion for forest conservation and carbon emissions.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: Project administrationRole: ResourcesRole: ValidationRole: Writing – review & editing
                Role: Formal analysisRole: MethodologyRole: ResourcesRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: ResourcesRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                13 November 2017
                2017
                : 12
                : 11
                : e0187283
                Affiliations
                [1 ] Center for Conservation and Sustainability, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC, United States of America
                [2 ] Global Genome Initiative, National Museum of Natural History, Smithsonian Institution, Washington, DC, United States of America
                [3 ] Department of Vertebrate Zoology, Division of Amphibians and Reptiles, National Museum of Natural History, Smithsonian Institution, Washington, DC, United States of America
                [4 ] Museum of Comparative Zoology, Harvard University, Cambridge, MA, United States of America
                [5 ] USGS, Patuxent Wildlife Research Center, National Museum of Natural History, Washington DC, United States of America
                Universitat Trier, GERMANY
                Author notes

                Competing Interests: While the field work presented in this paper was funded by Shell Gabon (listed in the financial disclosure section), representing private sector funds, the Smithsonian Institution maintains all intellectual property and rights in data, and we declare no competing interests exist.

                Author information
                http://orcid.org/0000-0002-2198-3515
                Article
                PONE-D-16-45461
                10.1371/journal.pone.0187283
                5683629
                29131846
                0f0a9d2a-2a2b-4cd7-b2ab-2afd46e2194a

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 15 November 2016
                : 6 September 2017
                Page count
                Figures: 5, Tables: 3, Pages: 38
                Funding
                Funded by: Shell Gabon
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100009198, Smithsonian Conservation Biology Institute;
                Award Recipient :
                Funded by: Smithsonian Institution's DNA Barcode Network
                Award Recipient :
                Shell Gabon funded the field work in the LMR study area. The Smithsonian Institution’s DNA Barcode Network provided funding for all laboratory work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Smithsonian Conservation Biology Institute and the National Museum of Natural History’s Laboratories of Analytical Biology provided in-kind support.
                Categories
                Research Article
                Biology and Life Sciences
                Taxonomy
                Computer and Information Sciences
                Data Management
                Taxonomy
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                DNA barcoding
                Research and analysis methods
                Molecular biology techniques
                DNA barcoding
                Biology and life sciences
                Evolutionary biology
                Evolutionary systematics
                Molecular systematics
                DNA barcoding
                Biology and life sciences
                Taxonomy
                Evolutionary systematics
                Molecular systematics
                DNA barcoding
                Computer and information sciences
                Data management
                Taxonomy
                Evolutionary systematics
                Molecular systematics
                DNA barcoding
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Amphibians
                Biology and Life Sciences
                Ecology
                Biodiversity
                Ecology and Environmental Sciences
                Ecology
                Biodiversity
                Biology and Life Sciences
                Ecology
                Ecological Metrics
                Species Diversity
                Ecology and Environmental Sciences
                Ecology
                Ecological Metrics
                Species Diversity
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Processes
                Speciation
                Species Delimitation
                Ecology and Environmental Sciences
                Conservation Science
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Databases
                Custom metadata
                16S and COI sequences have been deposited in GenBank under the accession numbers KY079387-KY080438. All COI and 16S records have also been deposited in BOLD (Process IDs: WAFRA001-14 to WAFRA542-14; West Central African amphibian barcode library for biodiversity).

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