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Previous phylogenetic analyses of the grass-specialist leafhopper tribe Chiasmini have resolved relationships among genera but have included few representatives of individual genera. Here the phylogeny of 20 Chinese species belonging to 8 chiasmine genera was investigated by combining DNA sequence data from two mitochondrial genes (COI, 16S) and two nuclear genes (H3, 28S). In both maximum likelihood (ML) and Bayesian inference (BI) analyses, relationships among genera were largely consistent with prior analyses, with most members of the tribe placed into two sister clades: (Exitianus + Nephotettix) and the remaining five sampled genera. To examine morphology-based species definitions in the taxonomically difficult genus Exitianus Ball, 1929, one mitochondrial gene (COI) and one nuclear gene (ITS2) were used to infer the phylogenetic relationships and status of two common and widespread species and compare the performance of different molecular species-delimitation methods. These analyses divide the included populations into two well-supported clades corresponding to current morphological species concepts but some inconsistencies occurred under the jMOTU, ABGD and bPTP methods depending on the which gene and analytical parameter values were selected. Considering the variable results yielded by methods employing single loci, the BPP method, which combines data from multiple loci, may be more reliable in Exitianus.
Two new species, Hyphessobrycon frickei Guimarães, Brito, Bragança, Katz & Ottoni sp. nov. and H. geryi Guimarães, Brito, Bragança, Katz & Ottoni sp. nov., are herein described, based on seven different and independent species delimitation methods, and on molecular and morphological characters, making the hypothesis of these new species supported from an integrative taxonomy perspective. They belong to the “Rosy tetra” clade, which is mainly characterized by the presence of a dark brown or black blotch on the dorsal fin and the absence of a midlateral stripe on the body. These two new species are distinguished from the other members of this clade mainly by the arrangement, shape and color pattern of humeral and dorsal-fin spots, as well as by other characters related to scale counts and body pigmentation. The placement of the new species within the “Rosy tetra” clade was based on the combination of morphological character states mentioned above and corroborated by a molecular phylogenetic analysis using the mitochondrial gene cytochrome oxidase subunit 1. In addition, a new clade (here termed Hyphessobrycon copelandi clade) within the “Rosy tetra” clade is proposed based on molecular data, comprising H. copelandi, H. frickei sp. nov., H. geryi sp. nov. and a still undescribed species. Our results corroborate the occurrence of hidden species within the “Rosy tetra” clade, as suggested by previous studies.