Use of Genetic Markers to Verify the Distribution of Northern Leopard Frogs (Lithobates pipiens) and Southern Leopard Frogs (Lithobates sphenocephalus) in Kentucky Emily S. Gustin 1 and Stephen C. Richter Department of Biological Sciences, Eastern Kentucky University, Richmond, Kentucky 40475 ABSTRACT Understanding species distributions is important for determining whether conservation efforts are necessary. In central Kentucky, an area of potential range overlap occurs between northern leopard frogs (Lithobates pipiens), which is a species of special concern, and southern leopard frogs (L. sphenocephalus). Their distributions are not completely delineated because of their low abundance in central Kentucky and similarity in morphology. Thus, use of genetic markers is necessary for identification. We surveyed for new, and sampled known, populations with a focus on the area of overlap in central Kentucky. We sequenced a portion of the mitochondrial 16S rRNA gene for 55 individuals from 16 populations. Maximum parsimony analysis separated individuals into two completely unresolved lineages representing the two species. Species were distributed as predicted, and no areas of fine-scale overlap were detected, although we did find a population of southern leopard frogs 10 km north-northwest of a northern leopard frog population. It was difficult to find L. pipiens at historically recorded populations, but we documented them in two counties where identification was previously unknown because morphology was intermediate between the species. Although genetic data reliably distinguished the species, they were inconclusive about whether hybridization occurs; future work is necessary to address this question. KEY WORDS: Lithobates pipiens, Lithobates sphenocephalus, genetics, distribution, Kentucky INTRODUCTION Accurately determining a species distribu- tion and understanding the genetic relation- ships with surrounding heterospecifics can help clarify past evolutionary history, current status, and, if necessary, the need for future conservation efforts (Avise 2004). Several factors affect species distributions, including physical geographic barriers, environmental changes, interspecies competition, and hy- bridization (Bridle and Vines 2006). In Kentucky, two closely related frog species, northern leopard frogs (Lithobates pipiens) and southern leopard frogs (L. sphenocepha- lus), have distributions that are generally adjacent across their margins (Figure 1), but previous research has not determined distri- bution along these range margins in Kentucky, or whether these two species hybridize. The Lithobates pipiens species complex consists of more than 25 species of leopard frogs (Hillis 1988). Many species in this complex have overlapping distributions, sim- ilar morphology, and similar habitat and breeding requirements, which may lead to misidentification, inaccurate determination of species distributions, and possible hybridiza- tion of the species (Hillis et al. 1983; Hillis 1988). For example, L. pipiens and L. blairi (plains leopard frogs) hybridize in sympatric areas (Hillis 1988), and L. sphenocephalus and L. blairi are documented to hybridize in the laboratory (Parris 2000, 2001). However, no studies have examined whether L. pipiens and L. sphenocephalus hybridize. Lithobates pipiens is distributed across Canada and the northern United States and range as far south as northern Kentucky in the eastern portion of their distribution (Rora- baugh 2000). Lithobates sphenocephalus is found in the southern United States, ranging as far north as central Illinois and as far west as Texas (Butterfield et al. 2005). The ranges of L. pipiens and L. sphenocephalus typically do not overlap, but in Kentucky, the two distributions meet and potentially overlap along a narrow zone located primarily along the edge of the Outer Bluegrass Ecoregion (Woods et al. 2002). Prior to our study, Lithobates pipiens was documented in 23 counties in northern Kentucky (KDFWR 2005; Figure 1), whereas L. sphenocephalus had been found in 68 counties, primarily in 1 Corresponding author e-mail: gustin.emily@gmail.com J. Ky. Acad. Sci. 74(1–2):10–15. 2013. 10