First DNA barcode reference library for the identification of South American freshwater fish from the lower Paraná River

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.Para citar este articulo: Díaz J, Villanova GV, Brancolini F, del Pazo F, Posner VM, Grimberg A, et al. (2016) First DNA Barcode Reference Library for the Identification of South American Freshwater Fish from the Lower Paraná River. PLoS ONE 11(7): e0157419. doi:10.1371/journal.pone.0157419Fil: Díaz, Juan. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Villanova, Gabriela Vanina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Biología; Argentina.Fil: Villanova, Gabriela Vanina. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática (LMBA - UNR - Ministerio de Ciencia, Tecnología e Innovación productiva de Santa Fe); Argentina.Fil: Brancolini, Florencia. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología “Dr. Raúl A. Ringuelet” (ILPLA - CONICET); Argentina.Fil: Del Pazo, Felipe. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Biología; Argentina.Fil: Posner, Victoria María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Biología; Argentina.Fil: Grimberg, Alexis. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática (LMBA - UNR - Ministerio de Ciencia, Tecnología e Innovación productiva de Santa Fe); Argentina.Fil: Arranz, Silvia Eda. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Arranz, Silvia Eda. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Biología; Argentina.Fil: Arranz, Silvia Eda. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática (LMBA - UNR - Ministerio de Ciencia, Tecnología e Innovación productiva de Santa Fe); Argentina

Minimum and maximum genetic distances of Genus with more than one species.

<p>Minimum and maximum genetic distances of Genus with more than one species.</p

The K2P/ML tree of 308 COI sequences for 79 morphologically identified freshwater fish species from Lower Paraná River in Argentina.

<p>NJ tree was divided into two parts from top to bottom in order from left to right. Bootstrap values >90 for 1000 replicates are shown at each branch. The number of specimens analyzed for each species is shown between brackets. Solid triangles represent clusters of multiple specimens, with height proportional to specimen number and the horizontal width proportional to the genetic variation within each cluster. In gray are shown branches of species with high intraspecific genetic divergence and species with overlapping clusters. Columns next to the tree represent presence of recognition for each clustering method while boxes highlights the differences among methods. Specimens of <i>Brycon orbignyanus</i> with high genetic divergence that were re-classified as <i>B</i>. <i>cf</i>. <i>hilarii</i> are highlighted with "*".</p

Box plots of K2P distances at different taxonomic levels.

<p>(A) within-species variation; (A’) within-species variation excluding the four species with high genetic divergence (<i>H</i>. <i>malabaricus</i>, <i>B</i>. <i>orbignyanus</i>, <i>P</i>. <i>motoro</i> and <i>Megalonema argentinum</i>); (B) variation at genus level; (B’) variation at genus level excluding genus with low genetic divergence (<i>Odontesthes</i>), (C) variation at Family level. The box comprise 25–75th percentiles of the data set. Whiskers show the lowest and highest values. Points represent outliers. Grey bar indicates ‘barcoding gap’ between intra and interspecific distances.</p

K2P- NJ trees of species with high conspecific genetic divergence.

<p>On the left, K2P- NJ trees calculated with specimens of this project. On the right, K2P-NJ trees calculated with all the South American published sequences of each genus. Specimens of this project are shown in bold and clusters indicated with A and B. BIN numbers are shown in brackets. Pictures represent morphology of typical specimens for each branch. Specimen number on collapsed branches is shown in brackets. Bootstrap values >50 for 1000 replicates are shown at each branch. Specimens of <i>Brycon orbignyanus</i> with high genetic divergence that were re-classified as <i>B</i>. <i>cf</i>. <i>hilarii</i> are highlighted with "*". LPR: Lower Paraná River, UPR: Upper Paraná River, AMAZ: Amazonas River, SR: Salado River and SFR: Sao Francisco River.</p

Scatterplot showing the overlap of the max intraspecific distances vs. the interspecific (Nearest Neighbour) distances.

<p>Specimens of <i>Brycon orbignyanus</i> with high genetic divergence that were re-classified as <i>B</i>. <i>cf</i>. <i>hilarii</i> are highlighted with "*".</p

3D histogram showing the number of Families, Genus and species by Order.

<p>3D histogram showing the number of Families, Genus and species by Order.</p

Sample sites map.

<p>Map of the Lower Parana River Basin showing the 50 sampling points at sites 1 and 2.</p

List of species with “true” discordance BINs found by the ‘BIN Discordance Report’ sequence analysis tool.

<p>Only BINs with species level conflicts are shown.</p