Genetic structure of Bufo bankorensis distinguished by amplified restriction fragment length polymorphism of cytochrome b

Background: Bufo bankorensis is an endemic species in Taiwan, and its populations are geographically and reproductively isolated. However, the distinction of Taiwanese B. bankorensis as a separate species from the Chinese Bufo gargarizans remains in dispute. Results: A primer set was designed to explore the mitochondrial (mt) DNA cytochrome (Cyt) b sequence (700 bp) of B. bankorensis in 148 individuals collected from 12 locations in Taiwan. After a polymerase chain reaction and sequencing, we found that the nucleotide sequence of Cyt b contained two restricted enzyme sites of BamHI and TspRI. Following BamHI enzyme digestion, samples of B. bankorensis were divided into two clades: western (which were undigested) and eastern (which were digested) clades. Additionally, Cyt b of the western clade of B. bankorensis was not cut by BamHI, while it was cut by TspRI into two sublineages. The result infers that at least two broadly divergent phylogroups of B. bankorensis exist in Taiwan and are not morphologically distinguishable. Based on the divergent sequence of Cyt b and cutting restriction enzymes, these populations were classified into three distinct phylogroups. Conclusion: Genetically, one (western group 1, uncut by BamHI and cut by TspRI) is most likely B. gargarizans, a second one (western group 2, uncut by both BamHI and TspRI) is B. bankorensis, and a third one (eastern clade, cut by BamHI but not cut by TspRI) could be a new subspecies. All three phylogroups were found in some areas, suggesting that they are sympatric, not allopatric.