Early stages of speciation with gene flow in the Amazilia Hummingbird ( Amazilis amazilia ) subspecies complex of Western South America.

Disentangling the factors underlying the diversification of geographically variable species with a wide geographical range is essential to understanding the initial stages and drivers of the speciation process. The Amazilia Hummingbird, Amazilis amazilia , is found along the Pacific coast from northern Ecuador down to the Nazca Valley of Peru, and is currently classified as six phenotypically differentiated subspecies. We aimed to resolve the evolutionary relationships of the six subspecies, to assess the geographical pattern and extent of evolutionary divergence, and to test for introgression using both a mtDNA marker and a genome-by-sequencing dataset from 86 individuals from across the species range. The consensus phylogenetic tree separated the six subspecies into three distinct clades, corresponding with the Ecuador lowlands ( A .  amazilia dumerilii ), the Ecuador highlands ( A .  amazilia alticola and A .  amazilia azuay ), and the Peruvian coast ( A .  amazilia leucophoea , A .  amazilia amazilia , and A .  amazilia caeruleigularis ). However, an unresolved mtDNA network suggests that the diversification of the subspecies was recent and rapid. We found evidence of gene flow among the subspecies A .  amazilia dumerilii , A .  amazilia alticola , and A .  amazilia leucophoea , with strong genetic isolation of the subspecies A .  amazilia azuay in the isolated Yunguilla Valley of Ecuador. Finally, environmental data from each subspecies' capture locations were concordant with the three distinct clades. Overall, our results suggest that both expansions into new habitats and geographic isolation shaped the present-day phylogeny and range of the A .  amazilia subspecies, and that A .  amazilia azuay may be genetically divergent enough to be considered a separate species.