Molecular systematic analysis demonstrates that the threatened southern bell frog, Litoria raniformis (Anura: Pelodryadidae) of eastern Australia,comprises two sub-species

In south-eastern Australia, the pelodryadid Litoria aurea Group (sensu Tyler & Davies 1978)comprises three species: Litoria aurea (Lesson,1829), Litoria raniformis (Keferstein,1867), and Litoria castanea (Steindachner,1867). All three species have been subject to declines over recent decades andtaxonomic uncertainty persists among populations on the tablelands in New SouthWales. We address the systematics of the Group by analysing mitochondrial andnuclear DNA sequences to assess divergence in the Litoriaraniformis from across its current range in New South Wales (NSW),Victoria, South Australia (SA) and Tasmania. We also included samples of Litoria castanea from a recentlyrediscovered population in the southern tablelands of NSW. Our phylogenetic andpopulation genetic analyses show that Litoriaraniformis comprises northern and southern lineages, showing deepmitochondrial DNA sequence divergence (7% net average sequence divergence) andcan be diagnosed by fixed allelic differences at more than 4,000 SNP loci.Samples of the northern lineage were collected from the Murray-Darling Basinwhile those of the southern lineage were collected from south-eastern SouthAustralia, southern and south-eastern Victoria and Tasmania. Analysis of themorphology and bioacoustics did not unequivocally delineate the two lineages.The presence of a hybrid backcross individual in western Victoria at thenorthern margin of the southern lineage, leads us to assign sub-species statusto the two lineages, L. r.raniformis for the northern lineage and L.r. major for the southern lineage. Our data do not unequivocallyresolve the taxonomic status of L.castanea which will require molecular genetic analyses of museumvouchers from those parts of the range where L.castanea and L. raniformis areno longer extant. Our data also suggest that human mediated movement of frogsmay have occurred over the past 50 years. Our genotyping of vouchers collectedin the 1970s from the Mount Lofty Ranges in South Australia detectedmitochondrial haplotypes of both sub-species and SNP analysis showed that asingle Tasmanian specimen was a backcross with L.r. raniformis ancestry. Movement of L.r. raniformis into Tasmania and both sub-species into the Mount LoftyRanges are not likely due to passive movements of animals through agriculturalcommerce, but due to the attractiveness of the species as pets and subsequentescapes or releases, potentially of the larval life stage.