The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity assessment in the region. Here, we developed a mitochondrial 12S ribosomal DNA reference database for 67 fish species, representing 54 genera, 25 families, and six major Neotropical orders. We aimed to develop mini-barcode markers (i.e. amplicons with less than 200 bp) suitable for DNA metabarcoding by evaluating the taxonomic resolution of full-length and mini-barcodes and to determine a threshold value for fish species delimitation using 12S. Evaluation of the target amplicons demonstrated that both full-length library (565 bp) and mini-barcodes (193 bp) contain enough taxonomic resolution to differentiate all 67 fish species. For species delimitation, interspecific genetic distance threshold values of 0.4% and 0.55% were defined using full-length and mini-barcodes, respectively. A custom reference database and specific mini-barcode markers are important assets for ecoregion scale DNA based biodiversity assessments (such as environmental DNA) that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.

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Hypomasticus copelandii is a Neotropical freshwater fish widely distributed across coastal drainages of southeastern Brazil, a highly impacted region of South America. The interspecific phylogenetic relationships within the genus and the taxonomic status of the species remain uncertain. Using two mitochondrial and one nuclear locus, we performed a phylogenetic, species delimitation, and time-calibrated analyses to test the hypothesis that H. copelandii is a species complex currently delimited by different Atlantic coastal systems. Results indicate that H. copelandii presents two well-delimited genetic lineages: one in the northern drainages of the Jucuruçu, Mucuri and Doce rivers, and the other in the southern region represented by the Paraíba do Sul River Basin. The time-calibrated phylogeny indicated a split between the two genetic lineages at around 2.8 million years ago (Ma), which might be related to headwater capture events during the Plio-Pleistocene. The discovery of a distinct genetic lineage for H. copelandii suggests distinct management plans for the northern and southern drainages. Such hidden diversity within the H. copelandii provides useful information for taxonomy and conservation across a severely impacted region of Brazil.