Reproductive Isolating Mechanisms in the Bangladesh Coastal Bullfrog Hoplobatrachus litoralis and Its Congeneric Species Revealed by Crossing Experiments and Examination on Spermatogenesis of the Hybrids

To elucidate reproductive isolating mechanisms in the Bangladesh coastal bullfrog Hoplobatrachus litoralis and its congeneric species, we performed crossing experiments using three species： H. litoralis, H. tigerinus and H. rugulosus. In addition, we conducted histological observations on spermatogenesis of the hybrids. The reciprocal hybrids between H. litoralis and H. tigerinus developed normally with somewhat lower viability at the metamorphosis stage compared with the controls. Most of the metamorphosed frogs became mature. On the other hand, almost all hybrids between female H. rugulosus and male H. litoralis or H. tigerinus died of underdevelopment at the tadpole stage, and only a few hybrids metamorphosed normally and survived to maturity. The inner structures of the testes of the control H. litoralis and H. tigerinus were completely normal, with seminiferous tubules filled with compact bundles of normal spermatozoa. Those of the reciprocal hybrids between H. litoralis and H. tigerinus were almost normal or slightly abnormal, with seminiferous tubules that contained pycnotic nuclei in addition to normal bundles of normal spermatozoa, which demonstrates slight abnormalities in spermatogenesis. In contrast, the hybrids between female H. rugulosus and male H. litoralis or H. tigerinus had no bundles of spermatozoa nor spermatids in the seminiferous tubules, which indicates entirely abnormal spermatogenesis. Meiotic chromosome figures in the reciprocal hybrids between H. litoralis and H. tigerinus showed slight abnormalities, with the occurrence of univalents and increase of rod-shaped bivalents. These results indicate that H. litoralis and H. tigerinus are not isolated from each other by hybrid inviability nor by hybrid sterility, although the hybrids showed somewhat abnormal spermatogenesis in hybrids and that H. rugulosus is isolated from both H. litoralis and H. tigerinus by incomplete hybrid inviability and complete hybrid sterility.