Two frog species or one? A multi-marker approach to assessing the distinctiveness of genetic lineages in the Northern Leopard Frog, Rana pipiens

A genetic boundary at the Mississippi River, USA, has been suggested for the Northern Leopard Frog, Rana pipiens, which was recently proposed for listing as federally threatened in the western USA. This suggestion was made on the basis of limited geographic sampling of a mitochondrial gene. However, mitochondrial DNA represents a very small part of the genome and is not necessarily indicative of patterns in nuclear DNA. We tested the hypothesis that eastern and western populations are separated by a distinct genetic boundary by sequencing mitochondrial DNA more extensively across the range, including focused sampling in the zone of hypothetical introgression, and by analyzing four nuclear sequences and seven microsatellite loci. We confirmed previous results that eastern and western populations have unique mitochondrial sequences that are deeply divergent (3.8 %) and which overlap only in a narrow region around the Mississippi River. Nuclear sequences also show divergent eastern and western lineages in some cases but with a broader zone of geographic overlap. Microsatellite data correspond closely to mitochondrial data, differing between east and west and changing abruptly near the Mississippi River. These data collectively demonstrate that eastern and western clades of this species introgress considerably in some markers but are distinct and defined by clear and narrow boundaries in others. We demonstrate that the Mississippi River forms an important, albeit somewhat permeable, boundary between genetic lineages in this species. This genetic boundary coincides with previously described discontinuities in morphological features.