Supplementary Material for: Evolution of Sex Chromosome Heteromorphy in Geographic Populations of the Japanese Tago’s Brown Frog Complex

The sex chromosomes of most anuran amphibians are characterized by homomorphy in both sexes, and evolution to heteromorphy rarely occurs at the species or geographic population level. Here, we report sex chromosome heteromorphy in geographic populations of the Japanese Tago’s brown frog complex (2<i>n</i> = 26), comprising <i>Rana sakuraii</i> and <i>R. tagoi</i>. The sex chromosomes of <i>R. sakuraii</i> from the populations in western Japan were homomorphic in both sexes, whereas chromosome 7 from the populations in eastern Japan were heteromorphic in males. Chromosome 7 of <i>R. tagoi</i>, which is distributed close to <i>R. sakuraii</i> in eastern Japan, was highly similar in morphology to the Y chromosome of <i>R. sakuraii</i>. Based on this and on mitochondrial gene sequence analysis, we hypothesize that in the <i>R. sakuraii</i> populations from eastern Japan the XY heteromorphic sex chromosome system was established by the introduction of chromosome 7 from <i>R. tagoi</i> via interspecies hybridization. In contrast, chromosome 13 of <i>R. tagoi</i> from the 2 large islands in western Japan, Shikoku and Kyushu, showed a heteromorphic pattern of constitutive heterochromatin distribution in males, while this pattern was homomorphic in females. Our study reveals that sex chromosome heteromorphy evolved independently at the geographic lineage level in this species complex.