1. Figure 1. Map depicting the supposed evolutionary history of terrestrial leeches in North America. Shaded area represents the Appalachian Range. (●) Haemopis ottorum, ( ) Haemopis terrestris, ( ) Haemopis septagon (Wirchansky and Shain, 2010). There are geographic barriers, such as water bodies and mountain ranges, that keep organisms from either side of the barrier from exchanging genetic information. In allopatric speciation, the geographic isolation causes the separated groups to become too genetically distinct to be considered members of the same species. While gene flow between certain species is restricted, the geographic barriers might not have such a large effect on other species, allowing them to be able to cross the barriers and mate with different populations. The Appalachian Mountains can be considered a geographic dispersal barrier for many organisms, resulting in high levels of biodiversity and endemic species. Morphological features alone are often not sufficient in the distinction of different species in this region and genetic analyses are used to recognize obscure lineages. This was observed in a study comparing different populations of Brownback Salamanders in the southern Appalachian region (Timpe et al. 2009). The series of alternating ridgelines and valleys of the Appalachian Mountain Range creates numerous barriers that many organisms are unable to cross. Helobdella stagnalis is an aquatic leech that lives in lakes, ponds, and rivers in parts of North America and Europe. This leech species demonstrates a passive dispersal mechanism in which they latch onto rocks, fish, plants, humans, birds, and other substrates (biotic and abiotic), which can then be used to transport the leech to new habitats. By using passive dispersal to move from one habitat to another instead of active dispersal, in which an organism uses its own energy to physically move itself to a new habitat, Helobdella stagnalis is able to cross many geographic barriers and exchange genetic information with species on the other side. (Wirchansky and Shain, 2010) With the Appalachian Mountains acting as a geographical barrier to East-West dispersal, Haemopis terrestris had to actively disperse south along the mountain range and circle around the southern end, where it eventually speciated to form Haemopis septagon (Figure 1). The lineage continued northward, with Haemopis ottorum representing the leading edge of the northern expansion. This pattern suggests an active dispersal mechanism since passive dispersal should have permitted colonization of all three Haemopis species on either side of the Appalachian Range. The Effects of the Appalachian Mountain Range on Gene Flow for the Aquatic Leech Helobdella stagnalis Katherine Willever Department of Biological Sciences, York College of Pennsylvania (Timpe et al. 2009) Due to similar body type and overlapping habitats, Eurycea aquatica (Brownback Salamander) was considered a local ecomorph of E. cirrigera, but genetic analyses recognized E. aquatica as a distinct species. Lineages of E. aquatica from three geographically different locations showed low genetic divergence and diversity, suggesting recent regional spread or high levels of gene flow among populations. (Sipe and Browne, 2004) Due to the mountainous nature of the Appalachian Mountain Range, there should be an increasing number of potential barriers to dispersal as geographic distance increases. Because dispersal barriers cause a reduction in the free movement of individuals, there should be a marked reduction in gene flow between separated populations of species that are unable to overcome the barriers. Populations that have the greatest geographic distance between them tend to exhibit the greatest genetic dissimilarity. The Appalachian Mountain Range does not act as a barrier to gene flow for Helobdella stagnalis because this species has a passive dispersal mechanism. DNA is able to be exchanged from either side of the mountains by the use of other organisms, such as plants, birds, fish, and humans, and other substrates. Because of this enabled exchange, collected samples on either side are not substantially different from each other (Figure 2). Observe if the Appalachian Mountain Range acts as a barrier to gene flow for Helobdella stagnalis by analyzing DNA sequences of collected samples from various locations on either side of the mountain range. Elayna, D. 2008. Helobdella stagnalis. Virtual Zoo. Retrieved 4 March 2012 from. Siddall, M.E. and Borda, E. 2003. Phylogeny and revision of the leech genus Helobdella (Glossiphoniidae) based on mitochondrial gene sequences and morphological data and a special consideration of the triserialis complex. Zoologica Scripta 32: 23-33. Sipe, T.W. and Browne, R.A. 2004. Phylogeography of Masked (Sorex cinereus) and Smoky shrews (Sorex fumeus) in the Southern Appalachians. Journal of Mammalogy 85(5):875-885. Timpe, E.K., Graham, S.P., and Bonett, R.M. 2009. Phylogeography of the Brownback Salamander reveals patterns of local endemism in Southern Appalachian springs. Molecular Phylogenetics and Evolution 52:368-376. Wirchansky, B.A. and Shain, D.H. 2010. A new species of Haemopis (Annelida: Hirudinea): Evolution of North American terrestrial leeches. Molecular Phylogenetics and Evolution 54: 226-234. Collect samples of H. stagnalis from either side of and within the Appalachian Mountain Range Run PCR amplifications and gel electrophoresis to amplify nuclear 18S and 28S rDNA and mitochondrial 16S rRNA, cytochrome c oxidase subunit 1 (COI), and NADH dehydrogenase subunit 1 (ND-I) fragments Extract tissue from caudal sucker and solubilize with Proteinase K enzyme Sequence DNA Use PAUP computer program to perform maximum parsimony and maximum likelihood analyses Use MrBayes v.3.1 computer program to perform Bayesian Inference Choose the best rooted phylogenetic tree based upon results of sequence analysis and interpret results Haementeria gracilis H.stagnalis West sample 4 H.stagnalis East sample 17 H.stagnalis West sample 1 H.stagnalis West sample 8 H.stagnalis East sample 9 H.stagnalis East sample 14 H.stagnalis West sample 5 H.stagnalis West sample 7 H.stagnalis East sample 11 H.stagnalis East sample 16 H.stagnalis West sample 3 H.stagnalis East sample 10 H.stagnalis East sample 13 H.stagnalis East sample 15 H.stagnalis West sample 2 H.stagnalis East sample 12 H.stagnalis West sample 6 Figure 2. Phylogenetic tree showing expected results from 17 H. stagnalis samples collected from either side of Appalachian Mountain Range. Bayesian posterior probability indicated above internode branch, and bootstrap values indicated below branch. 1.00 100 Image of the key identification characteristic for H. stagnalis, the scute, located on somite VIII (Siddall and Borda, 2003). Helobdella stagnalis (Elayna 2008) Some examples of substrates H. stagnalis can use to disperse into new habitats Acknowledgments I would like to thank Dr. Kleiner and Dr. Hagerty for their continuous support and help in the development of my research project. Introduction Review of Literature Review of Literature (continued)Objective Research Design Expected Results Literature Cited Perform bootstrap analysis on phylogenetic trees Record morphological features of collected specimens