More than 85 species of cave-obligate (troglobiotic) millipede have been described from North America. Understanding the patterns and processes that determine their distribution in this region is an area of recent research. Here, we present the first molecular phylogeographic study of troglobiotic millipedes. Millipedes of the genus Tetracion Hoffman, 1956 (Callipodida: Abacionidae) inhabit caves on the Cumberland Plateau in Tennessee and Alabama, a global hotspot for cave biodiversity. Three species have been described: T. jonesi Hoffman, 1956, T. antraeum Hoffman, 1956, and T. tennesseensis Causey, 1959. To examine genetic divergence within and between species of Tetracion we sequenced part of the mitochondrial cytochrome oxidase 1 gene from 53 individuals from eleven caves across the range of T. tennesseensis and in the northern part of the range of T. jonesi. We found: (1) little variation within species (six haplotypes in T. tennesseensis and four haplotypes in T. jonesi, with a maximum of 1.4% intraspecific divergence between haplotypes), (2) that gene flow between caves is limited (7 of 10 haplotypes were restricted to a single cave, and FST > 0.80 and P < 0.05 for fifteen of eighteen comparisons between caves), and (3) significant genetic divergence between species (8.8% between T. tennesseensis and T. jonesi). Our results are consistent with previous morphology-based species definitions showing T. tennesseensis and T. jonesi belonging to distinct taxa. Our research contributes to the growing body of phylogeographic information about cave species on the Cumberland Plateau, and provides a point of comparison for future studies of troglobionts and millipedes.
