The rbcL gene region of each specimen was amplified via a two-step PCR protocol with a primary amplification with tailed primers (rbcLaf + adaptor, rbcLr506 + adaptor) followed by a second round of amplification to anneal NexteraXT indices. Extracts were quantified and shipped to the School of Environmental and Forest Sciences Genetics Lab at the University of Washington for PCR amplification and NexteraXT library preparation for sequencing. Geological Survey Alaska Science Center, employing a CTAB-PVP protocol modified from Stewart and Via as reported by Muñiz-Salazar et al. Plant specimens were obtained from herbarium specimens collected from the various arctic or alpine sites across mainland Alaska (Additional file 1). We then sequenced the rbcL gene of the plant chloroplast genome, which is one of the most commonly used barcoding regions for plants. To improve capabilities for diet analysis of Dall’s sheep and other arctic herbivores, we used a python script to identify gaps in archived nucleotide sequence data for species known to comprise the diet of Dall’s Sheep, then obtained specimens of 16 species of arctic/alpine vascular plants for which sequence information was missing or underrepresented in publicly archived databases. Lack of sequence data for some arctic/alpine plants known to be grazed upon by Dall’s sheep currently limits the development and application of metabarcoding for alpine herbivore diet studies. However, metabarcoding has not yet been used to assess the diet of Dall’s sheep. Next generation sequencing of DNA from fecal samples has been successfully used to characterize diets of a variety of species, including ungulates. DNA barcoding includes a reference database of potential diet components, providing the capability to identify diet items to a desirable taxonomic resolution, ensuring that all components will be detected and assigned. Specifically, DNA metabarcoding uses universal primers for multispecies identification to mass-amplify DNA barcodes using PCR that are then read using next generation sequencing and assigned to the appropriate taxon. DNA based tools can infer diet composition with higher resolution and reduces cost, time, and effort compared to observational, morphological, and microhistological methods. The level of taxonomic resolution of items consumed in a diet study greatly affects ecological analysis. However, the diet of Dall’s sheep remains relatively poorly characterized and represents a gap in understanding how climate change is affecting plant-animal interactions in alpine ecosystems. Dall’s sheep have a generalist plant diet they were observed eating 110 different plant species in the Yukon Territory, Canada through traditional observational methods. Climate change may be altering alpine plant communities and contributing to these declines. ![]() Dall’s sheep ( Ovis dalli dalli) are endemic to alpine ecosystems of northwestern North America, and their populations have been declining in recent decades.
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