The integration of genetic, morphological, behavioral, and ecological information in the analysis of species boundaries has increased, allowing integrative systematics that better reflect the evolutionary history of biological groups. In this context, the goal of this study was to recognize independent evolutionary lineages withinEuphonia affinisat the genetic, morphological, and ecological levels. Three subspecies have been described:E. affinis godmani, distributed in the Pacific slope from southern Sonora to Guerrero;E. affinis affinis, from Oaxaca, Chiapas and the Yucatan Peninsula to Costa Rica; andE. affinis olmecorumfrom Tamaulipas and San Luis Potosi east to northern Chiapas (not recognized by some authors). A multilocus analysis was performed using mitochondrial and nuclear genes. These analyses suggest two genetic lineages:E. godmaniandE. affinis, which diverged between 1.34 and 4.3 My, a period in which the ice ages and global cooling fragmented the tropical forests throughout the Neotropics. To analyze morphometric variations, six morphometric measurements were taken, and the Wilcoxon Test was applied to look for sexual dimorphism and differences between the lineages. Behavioral information was included, by performing vocalization analysis which showed significant differences in the temporal characteristics of calls. Finally, Ecological Niche Models were estimated with MaxEnt, and then compared using the method of Broennimann. These analyses showed that the lineage distributed in western Mexico (E. godmani) has a more restricted niche than the eastern lineage (E. affinis) and thus we rejected the hypotheses of niche equivalence and similarity. Based on the combined evidence from genetic, morphological, behavioral, and ecological data, it is concluded thatE. affinis(withE. olmecorumas its synonym) andE. godmanirepresent two independent evolutionary lineages.
