Overcoming challenges to morphological and molecular identification of Empidonax flycatchers: a case study with a Dusky Flycatcher

Flycatchers in the genus Empidonax are among the most difficult avian taxonomic groups to identify to species. Observers often rely on calls or songs in the field or detailed morphometrics in the hand to identify species. In January and February 2013, we twice captured an Empidonax flycatcher at the Virginia Zoo in Norfolk, Virginia. After being unable to identify the flycatcher to species level using morphometrics and photographs, we extracted DNA from two tail feathers collected during the second encounter to identify the individual genetically. Comparison of cytochrome c oxidase I (COI) with reference sequences in the Barcode of Life Database (BOLD) suggested that the specimen had a >99.8% probability of placement as a Dusky Flycatcher ( Empidonax oberholseri ). Additional comparisons of NADH dehydrogenase subunit 2 (ND2) to reference sequences in GenBank, however, suggested that the specimen was a Pine Flycatcher ( Empidonax affinis ), a species not represented in BOLD and confined geographically to a small area in Mexico and Guatemala. After analyzing both COI and ND2 from additional vouchered specimens, the bird caught in Virginia was determined to be a Dusky Flycatcher. We also suspect that some of the sequences in GenBank might derive from incorrectly identified specimens or otherwise could represent overlooked pseudogenes. Because the putative identification, based on GenBank sequences, would have represented the first record of Pine Flycatcher from the United States, our results reinforce the need for carefully vetted and taxonomically comprehensive molecular databases to allow definitive conclusions about sample identity. Further molecular phylogeographic review of this genus is warranted to resolve haplotype ambiguities.