"Contribution to a reference library of DNA barcodes for Colombian freshwater fishes."

Refers to the COX1 sequences generated in this study of freshwater fish specimens catalogued in the Ichthyology Collection of the University of Antioquia, which have been reported in the Atrato, Cauca and Magdalena River Basins in Colombia.

Manuela Mejía Estrada (Project developer, Student), Iván D. Soto-Calderón (Project mentor), Luz Fernanda Jiménez Segura (Project coordinator).

Freshwater fish specimens were collected in the field using methods defined according to the habitat, including, but not limited to, drift nets, gill nets (1-12 cm, mesh size), hand nets, cast nets (0.5-3 cm mesh size), bottom trawls (0.5-3 cm, mesh size), seine nets, handline and electrofishing. They were also morphologically identified and DNA barcoded.

This project was funded by “Empresas Públicas de Medellin, EPM” through the agreement No. CT-2017-001714 with the University of Antioquia.

Atrato, Cauca and Magdalena Basins, Colombia.

The analysed material was collected in 138 different localities. Sampling was conducted between 2010 and 2020 on a wide range of habitats, using the different fishing arts mentioned before. Collected specimens were fixed and stored in alcohol and a portion of muscle or fin was stored in 96% ethanol for downstream molecular analysis. Morphological identification was performed, based on taxonomic keys and descriptions from literature (Suppl. material 1).

DNA was extracted from muscle and/or fin preserved in 96% alcohol using the QIAgen Dneasy Blood & Tissue kit ® (Hilden, Germany), following manufacturer’s protocol. A fragment of approximately 580 bp of the mitochondrial COX1 gene was amplified using the primers FishF1 (5´-TCAACCAACCACAAAGACATTGGCAC-3´) and FISHR1 (5´-TAGACTTCTGGGTGGCCAAAGAATCA-3´) (Ward et al. 2009). A 25 µl PCR cocktail included 2 µl of DNA, 2.5 µl of 10x Taq Buffer, 2.6 µl of 25 mM MgCl₂, 2.5 µl of 2 mM dNTPs, 1 µl of each primer 2 µM, 0.2 µl (1U) of Taq polymerase and 13.2 µl of water. The thermal profile of the PCR consisted of an initial denaturation step for 5' at 94°C, followed by 35 cycles of 1' at 94°C, 1' at 56°C and 1' at 72°C and then a final pass for 10' at 72°C. The amplification of the single fragment and within the expected size range was verified by 2% agarose electrophoresis. The products were then cleaned with Exonuclease I and Shrimp Alkaline Phosphatase (New England Biolabs, Ipswich, Massachusetts, USA) and sequenced by the standard Sanger method. The forward and reverse sequences were edited and assembled using Geneious Prime (2019) and inspected manually.

The sequences were translated into protein to verify the absence of stop codons and indel events that indicated errors in the sequence or the unintentional amplification of nuclear pseudogenes (numts). The sequences generated are available on the BOLD page (Ratnasingham and Hebert 2007) within the CIUA project.

Middle to lower portion of the Atrato, Cauca and Magdalena River Basins, continental Colombia (Fig. 1).

4.53888 and 8.89651 Latitude; -76.81916667 and -73.55097222 Longitude.

This dataset consists of data relating to 757 specimens of freshwater fishes occurring in Colombia; 673 specimens were identified to the species level and 84 to genus. Overall 96 species in 24 families are represented in the dataset (Suppl. material 2). The families Characidae, Astroblepidae and Loricariidae account for 71% of the total collected specimens. The family Sciaenidae is represented by one species with one sequence, whereas seven families (Apteronotidae, Aspredinidae, Callichthyidae, Cynolebiidae, Erythrinidae, Salmonidae (exotic) and Sciaenidae) are represented by a single species with more than one sequence (Suppl. material 3).