Cytonuclear Discordance and Historical Demography

13 Prior studies of mitochondrial genomic variation reveal that the Japanese brown frog Rana 14 tagoi comprises a complex of cryptic species lineages, and that R. sakuraii arose from within 15 this complex. Neither species forms a monophyletic group on the mitochondrial haplotype tree, 16 precluding a simple explanation for the evolutionary origins of R. sakuraii. We present a more 17 complete sampling of mitochondrial haplotypic variation (from the ND1 and 16S genes) plus 18 DNA sequence variation for five nuclear loci (from the genes encoding NCX1, NFIA, POMC, 19 SLC8A3, and TYR) to resolve the evolutionary histories of these species. We test hypotheses of 20 population assignment (STRUCTURE) and isolation-with-migration (IM) using the more 21 slowly evolving nuclear markers. These demographic analyses of nuclear genetic variation 22 confirm species-level distinctness and integrity of R. sakuraii despite its apparent polyphyly on 23 the mitochondrial haplotype tree. Divergence-time estimates from both the mitochondrial 24 haplotypes and nuclear genomic markers suggest that R. sakuraii originated approximately one 25 million years ago, and that incomplete sorting of mitochondrial haplotype lineages best explains 26