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Following sequencing, SNP discovery and genotyping were performed using a network based analysis pipeline, UNEAK (Universal Network Enabling Analysis Kit), a protocol developed for species without a reference genome (Lu et al. The UNEAK pipeline is designed to overcome complexities that impede SNP discovery, such as repeats, paralogs, and sequencing errors.

Briefly, sequence reads were trimmed to 64 bp, and then all identical reads were pooled to form distinct tags.

Samples were then pooled, cleaned, and amplified with PCR to increase fragment concentration.

Samples were then run on a partial lane (48 of 96 samples) of a 100 bp single-end Illumina Hiseq 2000.

Therefore, the current taxonomy used to guide management decisions may misrepresent the true evolutionary history for the species.

We sequenced 86 individuals for ND2 and genotyped 48 individuals for genome-wide SNPs to identify distinct lineages within Bell’s Vireo.

Species or subspecies delimited solely on the basis of non-genetic data can mislead conservation efforts by misrepresenting the underlying intraspecific variation and evolutionary history (Zink ).

These clades diverged from each other approximately 1.11–2.04 mya.The branch connecting the eastern and western clades (broken branch) represents 27 mutational steps.Circle size is proportional to the number of individuals sharing that haplotype with the largest western haplotype representing 21 individuals and the largest eastern haplotype representing 9 individuals. Total genomic DNA was extracted with Qiagen DNeasy extraction kits following the manufacturer’s protocol.Our genetic evidence, together with information from morphology and behavior, suggests that the Bell’s Vireo complex involves two species, each containing two separate subspecies. An inhabitant of some of North America’s most threatened habitat types, including riparian and grassland-shrub habitats, Bell’s Vireo has declined at a rate of approximately 2.7 % per year since the mid-1960s (Sauer et al. Habitat modification associated with agriculture, grazing, urbanization, flood management and wildfire has destroyed, degraded and fragmented vireo breeding habitat, while brood parasitism by the Brown-headed Cowbird ( is state threatened in Minnesota. S., Bell’s Vireo is identified as a high priority conservation target by the Audubon Society’s “Red Watchlist” of species of highest national concern, and by Partners in Flight U. as a “Bird of Conservation Concern” in 14 Bird Conservation Regions.This new information has implications for the federal, state and other listing status of Bell’s Vireo throughout its range.) is a species of conservation concern throughout its U. Thus, a variety of federal and state laws, as well as non-governmental programs, guide the conservation of Bell’s Vireo throughout its range.), no previous genetic studies have focused on the Bell’s Vireo, despite its conservation significance.

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