Phylogeographic Analyses of Obligate and Facultative Cave Crayfish Species on the Cumberland Plateau of the Southern Appalachians

PHYLOGEOGRAPHIC ANALYSES OF OBLIGATE AND FACULTATIVE CAVE CRAYFISH SPECIES ON THE CUMBERLAND PLATEAU OF THE SOUTHERN APPALACHIANS by Jennifer E. Buhay Department of Integrative Biology Doctor of Philosophy Cave systems and their unique biota are widely viewed as highly endangered, yet very little is known about basic life history, ecology, distributions, habitat requirements, and evolutionary relationships of subterranean species. The crux of the problem in cave studies is the assumption that traditionally defined morpho-species represent distinct evolutionary lineages. Convergence is exhibited in the morphologies of many animal groups, vertebrate and invertebrate, which leads to confusion in diagnosing species’ boundaries, geographic distributions, gene flow routes, and imperilment. This dissertation research includes phylogeographic analyses of freshwater cave-dwelling crayfishes in the Southern Appalachians, a global hotspot of subterranean biodiversity. By examining population structure in light of habitat, geology, geography, and hydrology, we can better provide conservation direction for these groundwater species. Chapter one introduces a method, Nested Clade Phylogeographic Analysis (NCPA), used to investigate hypotheses about historical and current population structures within species. Using a statistically-testable framework, NCPA can elucidate historical speciation patterns and current routes of gene flow using genetic sequence data of thoroughly-sampled species. Using diverse examples, the chapter details the methodology of building haplotype networks, performing the geographic analyses, inferring past and contemporary evolutionary patterns and processes, and delineating species’ boundaries. Chapter two examines two competing hypotheses regarding conservation status of cave-dwelling species using a wide-ranging group of obligate subterranean crayfish species on the Cumberland Plateau’s western escarpment. Using a population genetic approach, cave crayfish exhibited moderate to high levels of genetic diversity and attained large population sizes over their evolutionary histories. Phylogeographic analyses revealed that this crayfish assemblage originated along the northern end of the Cumberland Plateau and in leading-edge small steps, colonized southward and accumulated diversity along the way. Current species’ boundaries do not match traditional morpho-species designations and also do not match current hydrological units. Chapter three explores phylogeography and habitat differences within the facultative cave-dwelling crayfish species Cambarus tenebrosus. This freshwater species is unique in that it inhabits surface and subsurface karst environments, has an unusually large distribution, and exhibits troglomorphism with reduced eyes and elongated limbs. Using sequence data from over 100 sampled localities, mostly along the Cumberland Plateau, C. tenebrosus appears to have inhabited surface and subsurface biomes throughout its evolutionary history. Additionally, this species shows extremely high levels of genetic diversity and NCA revealed significant phylogeographic structure within the species, but there was no significant relationship between habitat and genetic structure. Chapter four examines the obligate cave crayfish assemblage, genus Cambarus, subgenus Aviticambarus, which ranges across the southernmost area of the Southern Appalachians, which is known to contain the highest species diversity of obligate terrestrial animals in the United States. The Aviticambarus assemblage is only currently known from 58 caves in Alabama and Tennessee, and with samples from half of the known sites, this study uncovered additional lineages previously obscured by convergent morphology. These species show low levels of genetic diversity and populations that do not appear to be expanding. Species’ boundaries are supported by geologic and phylogeographic information, but not current drainage basin boundaries.