Historical Genetic Analysis Of Horned Lark (Eremophila Alpestris) Specimens From The Prairie Pothole Region

The Horned Lark (Eremphila alpestris) is a generalist bird species with a global distribution. A previous phylogeographic study of this species from western North America has been completed and identified three distinct mitochondrial DNA clades. This study aims to genetically characterize Horned Larks from the North Dakota region using the mtDNA NADH dehydrogenase subunit 2 (ND2) gene. Horned Larks were sampled from museum collections, with ages ranging from 13 - 120 years old. 5mm x 1mm feather cuttings were removed using a non-invasive feather sampling method from each specimen for genetic analysis.This sampling method was verified on 15 total museum specimens of Horned Larks, Red-winged Blackbirds (Agelaius phoeniceus) and Franklin's Gulls (Leucophaeus pipixcan) from the University of North Dakota Vertebrate Museum Collection. Sampling methodology was investigated by using three mechanisms of DNA isolation: EDTA with proteinase K followed by column-based purification, chelating resin with dithiothreitol, and a Direct PCR method without DNA purification. Three fragments of mitochondrial DNA loci were amplified including control region, cytochrome c oxidase I and NADH dehydrogenase subunit 2. Correct sequencing results indicated that this method is viable for amplification of museum DNA, while at the same time limiting physical damage to the museum specimen. Arsenic found in low concentrations from arsenic test strips, was not a factor seriously hindering the success of PCR.Phylogenetic reconstruction of historic Horned Lark genetic information using Bayesian Inference and Maximum Likelihood algorithms revealed the existence of a Nearctic South clade, and reclassifies the previously established Pacific Northwest clade as a broader Nearctic North clade. The use of sequences with uneven preservation from museum specimens had no effect on overall tree topology. Horned Larks from the North Dakota region population had higher nucleotide diversity than other populations, and were all part of the Nearctic Northern clade. Five new haplotypes and three new SNPs were identified from museum specimens in this study.