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

Chu-Chih Chen,Teng-Lang Yu,Li-Hsuen Chen,Yi-Wen Tong,Kao-Jean Huang,Ching-Feng Weng,Kou-Wei Li,Pei-Yuan Sheu

doi:10.1186/1810-522x-52-48

Abstract

Abstract 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 Bam HI and Tsp RI. Following Bam HI 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 Bam HI, while it was cut by Tsp RI 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 Bam HI and cut by Tsp RI) is most likely B. gargarizans, a second one (western group 2, uncut by both Bam HI and Tsp RI) is B. bankorensis, and a third one (eastern clade, cut by Bam HI but not cut by Tsp RI) could be a new subspecies. All three phylogroups were found in some areas, suggesting that they are sympatric, not allopatric.

Highlights

Bufo bankorensis is an endemic species in Taiwan, and its populations are geographically and reproductively isolated
As is known from the literature, Bufo bufo is in Europe, Bufo gargarizans is in mainland Asia, Bufo japonicus is restricted to Japan, and Bufo miyakonis is found in Miyako Island, Japan (Igawa et al 2006)
B. bankorensis is placed in the B. gargarizans species complex, but no morphological distinction exists (Inger 1972; Matsui 1984; Liu et al 2000; Fu et al 2005)

Summary

Introduction

Bufo bankorensis is an endemic species in Taiwan, and its populations are geographically and reproductively isolated. In the Bufonidae, only two species (Bufo bankorensis and Bufo melanostictus) are found in Taiwan (Li et al 2006). Based on reproductive isolation mechanisms elucidated by crossing experiments, toads from Japan, China, and Taiwan are classified as the subspecies group Bufo gargarizans japonicus (Kawamura et al 1980, 1982). B. bankorensis was reclassified as a distinct endemic species in Taiwan similar to allopatric populations of Bufo andrewsi (Matsui 1986). B. bankorensis is one of three clades of B. japonicus (the other two are Bufo japonicus miyakonis in Miyako Island, Japan and the eastern and western groups of the Japanese Bufo japonicus japonicus subspecies group, and B. gargarizans in China) (Igawa et al 2006). The Bufo taxa of Taiwan, B. bankorensis and B. gargarizans, remain unclear

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