The genus Prochilodus comprises 13 species distributed throughout South American rivers. Among these, P. magdalenae (Colombia) and P. reticulatus (Colombia and Venezuela) have been historically identified relying on geographical distribution, range, and modal values for certain meristic characteristics. Nevertheless, morphological similarities have made species differentiation a challenging task. Species differentiation at the molecular level has also proven challenging, due to an apparent lack of phylogenetic differentiation between these two species, across a set of mitochondrial and nuclear marker genes, suggesting the possibility of a single P. magdalenae-reticulatus species with allopatric differentiation. The limited availability of complete mitogenome sequences for these species in public databases further limits their phylogenetic characterization. This study aimed to resolve the taxonomic ambiguity between Prochilodus magdalenae and Prochilodus reticulatus through mitogenome analysis and phylogenetic characterization. In a prior effort, we assembled and annotated a P. magdalenae mitogenome (Pmag_1). Here a second P. magdalenae mitogenome was assembled from ONT sequences (Pmag_2). We present the first complete mitochondrial genome sequence of P. reticulatus, along with a comprehensive phylogenetic analysis that resolves the apparent taxonomic ambiguity between these two species. The mitochondrial genomes of three P. reticulatus individuals (Pret_1, Pret_2, and Pret_3) collected at the Catatumbo municipality in Zulia, Venezuela were assembled from Illumina sequences. Mitogenomes were annotated (Fig.1) and phylogenetic analyses were performed for the complete mitochondrial sequences and partial mitochondrial gene sequences (Cox1 -COI, Cytb, ATPase8, tRNA-Pro, and 16S rRNA9).
P. magdalenae and P. reticulatus complete mitochondrial genome sequences separate forming distinct well-supported clades (Fig. 2). We found significant differences in partial Cox1, Cytb, ATPase8, tRNA-Pro, and 16S rRNA9 sequences between P. reticulatus and P. magdalenae. Interestingly, the few Cox1 and Cytb sequences available in GenBank for P. reticulatus share complete identity with P. magdalenae, while ATPase8 and tRNA-Pro sequences are identical to our P. reticulatus sequences. Our results prove these species can be readily identified using complete mitogenomes or partial mitochondrial gene sequences. We propose that some of the mitochondrial gene sequences currently labeled as P. reticulatus in the databases correspond to P. magdalenae. This study is part of the program formulated by ISAGEN S.A., within the PMA, to protect fish and fishing resources in the Sogamoso River.