Determining the Genetic Distances Between Sub-Populations of Aneides aeneus in the Westvaco Wildlife and Ecosystem Research Forest.

Andrew Johnson and Charles Somerville, Department of Biological Sciences, Marshall University.

The green salamander, Aneides aeneus, is found throughout the Appalachian Mountains from southwestern Pennsylvania to northern Alabama. Recent studies indicate that the number of individuals is declining, and A. aeneus has been listed as a species of concern in West Virginia. Green salamanders are common in the Westvaco Wildlife and Ecosystem Research Forest (WWERF), however; the population of the WWERF is fragmented by both natural and man-made features into several sub-populations. The purpose of this research is to determine if the physical separation of the salamanders has led to reproductive isolation that may impact the survival of the species. Tail samples were collected from eighteen salamanders at six sites within the forest. We are currently optimizing the preparation of DNA template from tail samples. A region of the mitochondrial cytochrome b gene will be amplified using the primers GAA CTA ATG GCC CAC ACW WTA CGN AA (MVZ15) and AAA TAG GAA RTA TCA YTC TGG TTT RAT (MVZ16; Moritz et al., Syst. Biol. 41:273). The primers were designed to produce an amplicon of 804 base pairs, which corresponds to approximately two-thirds of the cytochrome b gene. Aliquots of a single amplification reaction will be sent to three different sequencing facilities to determine if reliable sequence data can be generated directly from amplicon templates. Poor or inconsistent sequence data will indicate that the amplicons must be cloned prior to sequencing. Sequences will be aligned using Clustal X (Thompson et al., Nuc. Acids Res. 22: 4673), and phylogenetic distances and tree topologies will be determined using programs of the PHYLIP software package (Felsenstein, Cladistics 5:164).