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Hybridization experiments to elucidate reproductive isolating mechanisms

(a) Reproductive isolating mechanisms among species and populations
Hybridization experiments on various species and local populations collected from Palearctic and Nearctic regions were performed in order to confirm the existence of reproductive isolating mechanisms among these species and populations, based on the definition of the biological species concept. Most Bufo species were found to be reproductively isolated from one another by gametic isolation, hybrid inviability at the embryonic or tadpole stage, and male hybrid sterility. However, several lineages classified as separate species were found not to be reproductively isolated from one another, and in fact seemed to belong to a single species (Fig.9).

(b) Incipient speciation in Rana japonica
The eastern and western groups of Rana japonica were reproductively isolated from each other by incomplete male hybrid sterility due to abnormal spermatogenesis (Fig.10, Fig.11). The differentiation between the eastern and western groups of Rana japonica was considered to be an incipient speciation of this species (Fig.12).

(c) Sex-linked genes and linkage maps
While a good number of linkage groups have been conserved in amphibians, there does not seem to be a common ancestral or conserved sex-linkage group (Fig. 13). Comparisons of amphibian linkage maps with those of fishes and mammals reveal several synthetic associations that apparently have been conserved over a very long period of vertebrate divergence.

(d) Color mutants and their loci in Rana japonica
Three color mutant strains were established in the laboratory, and the mode of inheritance and the linkage relationships of their loci were analyzed (Fig. 14). The black-eyed mutant locus (Blk) was found to link with the AAT-1 locus.

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