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      Assembling a DNA barcode reference library for the spiders (Arachnida: Araneae) of Pakistan

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          Abstract

          Morphological study of 1,795 spiders from sites across Pakistan placed these specimens in 27 families and 202 putative species. COI sequences >400 bp recovered from 1,782 specimens were analyzed using neighbor-joining trees, Bayesian inference, barcode gap, and Barcode Index Numbers (BINs). Specimens of 109 morphological species were assigned to 123 BINs with ten species showing BIN splits, while 93 interim species included representatives of 98 BINs. Maximum conspecific divergences ranged from 0–5.3% while congeneric distances varied from 2.8–23.2%. Excepting one species pair ( Oxyopes azhariOxyopes oryzae), the maximum intraspecific distance was always less than the nearest-neighbor (NN) distance. Intraspecific divergence values were not significantly correlated with geographic distance. Most (75%) BINs detected in this study were new to science, while those shared with other nations mainly derived from India. The discovery of many new, potentially endemic species and the low level of BIN overlap with other nations highlight the importance of constructing regional DNA barcode reference libraries.

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            Comprehensive DNA barcode coverage of North American birds

            DNA barcoding seeks to assemble a standardized reference library for DNA-based identification of eukaryotic species. The utility and limitations of this approach need to be tested on well-characterized taxonomic assemblages. Here we provide a comprehensive DNA barcode analysis for North American birds including 643 species representing 93% of the breeding and pelagic avifauna of the USA and Canada. Most (94%) species possess distinct barcode clusters, with average neighbour-joining bootstrap support of 98%. In the remaining 6%, barcode clusters correspond to small sets of closely related species, most of which hybridize regularly. Fifteen (2%) currently recognized species are comprised of two distinct barcode clusters, many of which may represent cryptic species. Intraspecific variation is weakly related to census population size and species age. This study confirms that DNA barcoding can be effectively applied across the geographical and taxonomic expanse of North American birds. The consistent finding of constrained intraspecific mitochondrial variation in this large assemblage of species supports the emerging view that selective sweeps limit mitochondrial diversity.
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              Counting animal species with DNA barcodes: Canadian insects

              Recent estimates suggest that the global insect fauna includes fewer than six million species, but this projection is very uncertain because taxonomic work has been limited on some highly diverse groups. Validation of current estimates minimally requires the investigation of all lineages that are diverse enough to have a substantial impact on the final species count. This study represents a first step in this direction; it employs DNA barcoding to evaluate patterns of species richness in 27 orders of Canadian insects. The analysis of over one million specimens revealed species counts congruent with earlier results for most orders. However, Diptera and Hymenoptera were unexpectedly diverse, representing two-thirds of the 46 937 barcode index numbers (=species) detected. Correspondence checks between known species and barcoded taxa showed that sampling was incomplete, a result confirmed by extrapolations from the barcode results which suggest the occurrence of at least 94 000 species of insects in Canada, a near doubling from the prior estimate of 54 000 species. One dipteran family, the Cecidomyiidae, was extraordinarily diverse with an estimated 16 000 species, a 10-fold increase from its predicted diversity. If Canada possesses about 1% of the global fauna, as it does for known taxa, the results of this study suggest the presence of 10 million insect species with about 1.8 million of these taxa in the Cecidomyiidae. If so, the global species count for this fly family may exceed the combined total for all 142 beetle families. If extended to more geographical regions and to all hyperdiverse groups, DNA barcoding can rapidly resolve the current uncertainty surrounding a species count for the animal kingdom. A newly detailed understanding of species diversity may illuminate processes important in speciation, as suggested by the discovery that the most diverse insect lineages in Canada employ an unusual mode of reproduction, haplodiploidy. This article is part of the themed issue ‘From DNA barcodes to biomes’.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SoftwareRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draft
                Role: Data curationRole: InvestigationRole: MethodologyRole: Resources
                Role: InvestigationRole: MethodologyRole: Resources
                Role: Investigation
                Role: Methodology
                Role: Investigation
                Role: MethodologyRole: Resources
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                22 May 2019
                2019
                : 14
                : 5
                : e0217086
                Affiliations
                [1 ] Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada
                [2 ] Department of Zoology, GC University, Lahore, Pakistan
                [3 ] Department of Biotechnology, University of Sargodha, Sargodha, Pakistan
                [4 ] Department of Zoology, University of Sargodha, Sargodha, Pakistan
                [5 ] Directorate of Entomology, Ayub Agricultural Research Institute, Faisalabad, Pakistan
                [6 ] Department of Zoology, University of the Punjab, Lahore, Pakistan
                [7 ] National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan
                Chang Gung University, TAIWAN
                Author notes

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

                Author information
                http://orcid.org/0000-0003-1351-4126
                Article
                PONE-D-19-01391
                10.1371/journal.pone.0217086
                6530854
                31116764
                5ae711ec-1b3d-4948-a831-469c76229030
                © 2019 Ashfaq et al

                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
                : 15 January 2019
                : 4 May 2019
                Page count
                Figures: 7, Tables: 2, Pages: 21
                Funding
                This study was enabled by grant 106106-001 “Engaging Developing Nations in iBOL” from the International Development Research Centre in Canada and by grant HEC No. 20-1403/R& D/09 “Sequencing DNA Barcodes of Economically Important Insect Species from Pakistan” from the Higher Education Commission of Pakistan awarded to MA. Sequence analysis was made possible by a grant from the Government of Canada through Genome Canada and Ontario Genomics in support of the International Barcode of Life (iBOL) project awarded to PDNH. This is a contribution to the Food From Thought project supported by the Canada First Research Excellence Fund awarded to PDNH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Arthropoda
                Arachnida
                Spiders
                People and Places
                Geographical Locations
                Asia
                Pakistan
                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
                Research and analysis methods
                Database and informatics methods
                Bioinformatics
                Sequence analysis
                DNA sequence analysis
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Processes
                Speciation
                Cryptic Speciation
                Biology and life sciences
                Genetics
                DNA
                DNA libraries
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA libraries
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Systematics
                Phylogenetics
                Phylogenetic Analysis
                Biology and Life Sciences
                Taxonomy
                Evolutionary Systematics
                Phylogenetics
                Phylogenetic Analysis
                Computer and Information Sciences
                Data Management
                Taxonomy
                Evolutionary Systematics
                Phylogenetics
                Phylogenetic Analysis
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Custom metadata
                Collection data, a photograph, a taxonomic assignment, and DNA barcode (COI-5p) sequence for each specimen are available in the public dataset, "DS-MASPD DNA barcoding spiders of Pakistan" on the Barcode of Life Data System (BOLD) ( www.boldsystems.org). ( dx.doi.org/10.5883/DS-MASPD).

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