Date of Award
Master of Science (MS)
Thomas P. Husband
The New England cottontail (Sylvilagus transitionalis), New England’s endemic cottontail, has been experiencing dramatic population declines and is estimated to exist in only 14% of its historical range. The New England cottontail is currently a candidate for endangered species listing under the Endangered Species Act of 1973. New England cottontails may be distinguished accurately from eastern cottontails (S. floridanus) with the use of non-invasive genetic techniques (e.g., fecal sample collection) that allow sampling of large geographical areas with minimal cost. A restriction enzyme technique has been published based on NlaIII (New England Biolabs Inc, Massachusetts) cut sites within the control region of the mitochondrial genome (mtDNA); this technique assumes that variation among and between species does not interrupt cut patterns and relies on qualitative identification (visual interpretation of gel bands) with no positive control that true mtDNA has been amplified. Phylogenetic analyses of the New England cottontails, eastern cottontails and snowshoe hares (Lepus americanus) in northeastern states may indicate the reason for New England cottontail decline. Because eastern cottontails were stocked from several locations they may have increased genetic variability, especially when compared to New England cottontails, which could be an indication of hybrid vigor. Furthermore, phylogeographic patterns may help infer introduction and spread patterns of eastern cottontails. I sequenced 1,773 fecal and tissue samples from CT, MA, NH, NY, and RI. I identified 12 New England cottontail, 101 eastern cottontail, and eight snowshoe hare haplotypes. Eastern cottontails exhibited a larger number of haplotypes compared to New England cottontails; however, there was no geographic pattern to haplotype occurrences. I analyzed all haplotypes using the restriction enzyme technique to test digestion site reliability and found 19 instances of conflicting cut sites between haplotypes and previously published cut sites. I also found two haplotypes that appeared to be non-mitochondrial in origin and can be preferentially amplified in some samples when the mammalian reverse primer used in the restriction enzyme method was used for amplification. To provide an unambiguous and reliable identification method I created a “barcode” for the mitochondrial control region of these three species and developed a rabbit specific reverse primer. I found 13 diagnostic characters for New England cottontail, 18 diagnostic characters for eastern cottontail, and 36 diagnostic characters for snowshoe hare. I propose that the use of the barcode and the rabbit specific reverse primer described here provides a reliable and inexpensive method for species identification. Furthermore, sequencing Polymerase Chain Reaction (PCR) product provides an opportunity to detect nonmitochondrial sequences and provides information for further analyses such as phylogeographic studies.
Sullivan, Mary E., "Comparative Phylogeography and a Mitochondrial DNA Barcode for Identifying Three Sympatric Lagomorphs in the Northeastern United States" (2013). Open Access Master's Theses. Paper 65.