Authors: Ainsley E Parkins, Sylvia Lee, Elizabeth J Heath, Thomas Petitjean, Kyle Cink, Kyle Kittelberger, Cagan Sekercioglou, Rodolfo S Probst
Mentors: Rodolfo S Probst
Insitution: University of Utah
Biodiversity loss poses a significant threat, requiring accurate species assessment and conservation strategies. Non-invasive and cost-effective techniques should be prioritized, especially when dealing with threatened vertebrate fauna. By focusing on cryptic Eastern European species within the genus Emberiza (Passeriformes: Emberizidae), we designed an affordable molecular pipeline (including DNA primers and bioinformatics) integrating DNA barcoding with Nanopore sequencing to address species boundaries and provide conservation information for avifauna diversity. We conducted non-invasive DNA extractions from individual feathers of 192 specimens collected in the Aras River Riparian Corridor (ARRC), an important ecological hotspot in Turkey. By targeting the cytochrome oxidase subunit I (CO1) region, our goal was to differentiate Emberiza taxa and identify potential cryptic lineages while using cutting-edge next-generation sequencing (NGS) of multiplexed specimens with portable Oxford Nanopore Technologies. Our focal study examined boundaries in Emberiza subspecies (E. shoeniclus shoeniclus and E. shoeniclus caspia) with subtle morphological differences, utilizing DNA barcoding and Nanopore sequencing. We obtained 182 Emberiza DNA barcodes. Clustering analysis recovered a paraphyletic assemblage (n=32) and a clade, the latter recovering majority of the barcodes (n=150), separated by at least 8% genetic difference. In both cases, we observed a mixture of barcodes from the nominal subspecies and Caspian subspecies, suggesting that cryptic morphological characteristics are not reflected in genetic separation. Our results also suggest putative cryptic Emberiza species, highlighting the importance of the ARRC for conserving avian diversity. We demonstrate that our pipeline, integrating real-time Nanopore sequencing with non-invasive DNA barcoding, can be used for conservation initiatives (particularly aiding cryptic species identification). This study expands our knowledge of avian diversity and highlights the potential of NGS DNA barcodes as a powerful tool for aiding conservation efforts amidst biodiversity loss.