Designing primers for DNA barcoding is a significant challenge for the rich Neotropical fish fauna, which is comprised of ∼6000 species. Previously, researchers required multiple pairs of PCR primers or primer cocktails to obtain standard COI (i.e., mitochondrial cytochrome c oxidase subunit I) barcode sequences from assemblages of freshwater fish in this region. To simplify DNA barcoding and metabarcoding studies of Neotropical freshwater fish, we present a new pair of COI primers, which have yielded high quality barcodes across six teleost orders—Characiformes, Cichliformes, Cyprinodontiformes, Gymnotiformes, Siluriformes, and Synbranchiformes—native to South America. Following previous fish barcoding studies, we also tailed our primers with M13 forward and reverse primers to facilitate the DNA sequencing process. Although this practice generates primer dimers, we obtained complete and high quality COI barcode sequences for all samples. We discuss the problem of primer dimers and suggest strategies for neutralizing their influence on data quality.

The diversity and geographic distribution of Phalloceros Eigenmann, 1907 in the Ilha Grande Bay Hydrographic Region, in southeastern Brazil, is investigated. Examination of 81 samples revealed the presence of 5 species in the region: Phalloceros anisophallos Lucinda, 2008, P. aspilos Lucinda, 2008, P. enneaktinos Lucinda, 2008, P. harpagos Lucinda, 2008, and P. tupinamba Lucinda, 2008. Phalloceros harpagos and P. tupinamba are recorded for the first time in the area. The geographic ranges of P. anisophallos and P. enneaktinos are expanded. The 5 species belong to 3 distinct morphologies of the female urogenital papilla. Species from these distinct morphological lineages may occur sympatrically, but species within the same lineage are always allopatric. A key for the identification of these species is provided.

A new species of Rineloricaria is described from the Iguaçu, Magé, Saracuruna, Guapiaçu, Macacu, Macaé, and Paraíba do Sul River basins in southeastern Brazil based on morphological and molecular data. The new species is diagnosed among congeners by the combination of the following characters: absence of plates in most of the ventral surface of pectoral girdle; snout tip with elliptical naked area reaching posteriorly the anteriormost pore of the infraorbital ramus of sensory canal; dorsal-fin spinelet present; supraoccipital and predorsal plates with smooth keels; five series of lateral plates below the dorsal fin; median and mid-ventral series of plates with well-developed keels. The new species is distinguished from congeners that inhabit the rivers draining the Serra do Mar in Rio de Janeiro state by the partial plate coverage of the ventral surface of the pectoral girdle and the abdomen (vs. pectoral girdle and abdomen completely covered by plates in Rineloricaria nigricauda, R. steindachneri, and R. zawadzkii). The new species shares the absence of plates on the abdominal surface with other species from the Paraná, Uruguay, and other Brazilian coastal river basins.