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      First DNA Barcode Reference Library for the Identification of South American Freshwater Fish from the Lower Paraná River

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          Abstract

          Valid fish species identification is essential for biodiversity conservation and fisheries management. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I for a valid identification of 79 freshwater fish species from the Lower Paraná River. Neighbour-joining analysis based on K2P genetic distances formed non-overlapping clusters for almost all species with a ≥99% bootstrap support each. Identification was successful for 97.8% of species as the minimum genetic distance to the nearest neighbour exceeded the maximum intraspecific distance in all these cases. A barcoding gap of 2.5% was apparent for the whole data set with the exception of four cases. Within-species distances ranged from 0.00% to 7.59%, while interspecific distances varied between 4.06% and 19.98%, without considering Odontesthes species with a minimum genetic distance of 0%. Sequence library validation was performed by applying BOLDs BIN analysis tool, Poisson Tree Processes model and Automatic Barcode Gap Discovery, along with a reliable taxonomic assignment by experts. Exhaustive revision of vouchers was performed when a conflicting assignment was detected after sequence analysis and BIN discordance evaluation. Thus, the sequence library presented here can be confidently used as a benchmark for identification of half of the fish species recorded for the Lower Paraná River.

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          Can DNA barcoding accurately discriminate megadiverse Neotropical freshwater fish fauna?

          Background The megadiverse Neotropical freshwater ichthyofauna is the richest in the world with approximately 6,000 recognized species. Interestingly, they are distributed among only 17 orders, and almost 80% of them belong to only three orders: Characiformes, Siluriformes and Perciformes. Moreover, evidence based on molecular data has shown that most of the diversification of the Neotropical ichthyofauna occurred recently. These characteristics make the taxonomy and identification of this fauna a great challenge, even when using molecular approaches. In this context, the present study aimed to test the effectiveness of the barcoding methodology (COI gene) to identify the mega diverse freshwater fish fauna from the Neotropical region. For this purpose, 254 species of fishes were analyzed from the Upper Parana River basin, an area representative of the larger Neotropical region. Results Of the 254 species analyzed, 252 were correctly identified by their barcode sequences (99.2%). The main K2P intra- and inter-specific genetic divergence values (0.3% and 6.8%, respectively) were relatively low compared with similar values reported in the literature, reflecting the higher number of closely related species belonging to a few higher taxa and their recent radiation. Moreover, for 84 pairs of species that showed low levels of genetic divergence ( 2%), pointing to at least 23 strong candidates for new species. Conclusions Our study is the first to examine a large number of freshwater fish species from the Neotropical area, including a large number of closely related species. The results confirmed the efficacy of the barcoding methodology to identify a recently radiated, megadiverse fauna, discriminating 99.2% of the analyzed species. The power of the barcode sequences to identify species, even with low interspecific divergence, gives us an idea of the distribution of inter-specific genetic divergence in these megadiverse fauna. The results also revealed hidden genetic divergences suggestive of reproductive isolation and putative cryptic speciation in some species (23 candidates for new species). Finally, our study constituted an important contribution to the international Barcoding of Life (iBOL.org) project, providing barcode sequences for use in identification of these species by experts and non-experts, and allowing them to be available for use in other applications.
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            Effects of phylogenetic reconstruction method on the robustness of species delimitation using single-locus data

            Coalescent-based species delimitation methods combine population genetic and phylogenetic theory to provide an objective means for delineating evolutionarily significant units of diversity. The generalised mixed Yule coalescent (GMYC) and the Poisson tree process (PTP) are methods that use ultrametric (GMYC or PTP) or non-ultrametric (PTP) gene trees as input, intended for use mostly with single-locus data such as DNA barcodes. Here, we assess how robust the GMYC and PTP are to different phylogenetic reconstruction and branch smoothing methods. We reconstruct over 400 ultrametric trees using up to 30 different combinations of phylogenetic and smoothing methods and perform over 2000 separate species delimitation analyses across 16 empirical data sets. We then assess how variable diversity estimates are, in terms of richness and identity, with respect to species delimitation, phylogenetic and smoothing methods. The PTP method generally generates diversity estimates that are more robust to different phylogenetic methods. The GMYC is more sensitive, but provides consistent estimates for BEAST trees. The lower consistency of GMYC estimates is likely a result of differences among gene trees introduced by the smoothing step. Unresolved nodes (real anomalies or methodological artefacts) affect both GMYC and PTP estimates, but have a greater effect on GMYC estimates. Branch smoothing is a difficult step and perhaps an underappreciated source of bias that may be widespread among studies of diversity and diversification. Nevertheless, careful choice of phylogenetic method does produce equivalent PTP and GMYC diversity estimates. We recommend simultaneous use of the PTP model with any model-based gene tree (e.g. RAxML) and GMYC approaches with BEAST trees for obtaining species hypotheses.
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              The complete sequence of the zebrafish (Danio rerio) mitochondrial genome and evolutionary patterns in vertebrate mitochondrial DNA.

              We describe the complete sequence of the 16,596-nucleotide mitochondrial genome of the zebrafish (Danio rerio); contained are 13 protein genes, 22 tRNAs, 2 rRNAs, and a noncoding control region. Codon usage in protein genes is generally biased toward the available tRNA species but also reflects strand-specific nucleotide frequencies. For 19 of the 20 amino acids, the most frequently used codon ends in either A or C, with A preferred over C for fourfold degenerate codons (the lone exception was AUG: methionine). We show that rates of sequence evolution vary nearly as much within vertebrate classes as between them, yet nucleotide and amino acid composition show directional evolutionary trends, including marked differences between mammals and all other taxa. Birds showed similar compositional characteristics to the other nonmammalian taxa, indicating that the evolutionary trend in mammals is not solely due to metabolic rate and thermoregulatory factors. Complete mitochondrial genomes provide a large character base for phylogenetic analysis and may provide for robust estimates of phylogeny. Phylogenetic analysis of zebrafish and 35 other taxa based on all protein-coding genes produced trees largely, but not completely, consistent with conventional views of vertebrate evolution. It appears that even with such a large number of nucleotide characters (11,592), limited taxon sampling can lead to problems associated with extensive evolution on long phyletic branches.
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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, CA USA )
                1932-6203
                21 July 2016
                2016
                : 11
                : 7
                : e0157419
                Affiliations
                [1 ]Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Rosario, Santa Fe, Argentina
                [2 ]Area Biología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina
                [3 ]Instituto de Limnología “Dr. Raúl A. Ringuelet” (ILPLA, CONICET-UNLP), La Plata, Buenos Aires, Argentina
                [4 ]Laboratorio de Biotecnología Acuática (FCByF-UNR/SeCTEI-Santa Fe), Acuario del río Paraná, Rosario, Santa Fe, Argentina
                University of Minnesota, UNITED STATES
                Author notes

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

                Conceived and designed the experiments: JD GVV FDP SEA. Performed the experiments: JD GVV VP AG. Analyzed the data: JD GVV VP FDP FB SAE. Contributed reagents/materials/analysis tools: SAE. Wrote the paper: JD GVV VP FDP SAE.

                Article
                PONE-D-16-04768
                10.1371/journal.pone.0157419
                4956254
                27442116
                4e88851b-2931-4431-810d-f6398e2193a1
                © 2016 Díaz 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
                : 2 February 2016
                : 31 May 2016
                Page count
                Figures: 6, Tables: 3, Pages: 20
                Funding
                Funded by: CONICET
                Award ID: Fondo iBOL
                Award Recipient :
                This work was supported by Fondo iBOL Argentina/CONICET (Argentina), project name: DNA barcoding of fishes from Lower Paraná River (2012-2014). GVV is a member of the carrier of scientific investigator of CONICET (Argentina). JD and FDP are Doctoral Fellows from CONICET (Argentina), http://www.conicet.gov.ar/. 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
                Animals
                Vertebrates
                Fishes
                Freshwater Fish
                Biology and Life Sciences
                Taxonomy
                Computer and Information Sciences
                Data Management
                Taxonomy
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                Sequence analysis
                DNA sequence analysis
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                Sequence analysis
                DNA sequence analysis
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Fishes
                Marine Fish
                Biology and Life Sciences
                Marine Biology
                Marine Fish
                Earth Sciences
                Marine and Aquatic Sciences
                Marine Biology
                Marine Fish
                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
                Biological Databases
                Sequence Databases
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Processes
                Speciation
                Cryptic Speciation
                Custom metadata
                Most relevant data are within the manuscript, and in its Supporting Information files. All DNA sequences are available from the GenBank database (accession numbers KU288760-KU289067). Specimen and collection data, specimen images, trace files, primer sequences, and GPS coordinates for all specimen collection localities are available for each record in the Public Data Portal in BOLD Systems ( www.boldsystems.org). Records can be accessed by BIN numbers or scientific names from the Public Data Portal ( www.boldsystems.org). Records from the “Fishes from the Lower Paraná River” project can be identified by the project code LAR.

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