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

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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.


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.


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.


There are many species of Anura in Taiwan. They are identified and classified into five families: the Bufonidae, Hylidae, Microhylidae, Ranidae, and Rhacophoridae. The Bufonidae is one of the most species-rich families of anurans with more than 550 species in approximately 50 recognized genera (Frost 2011). 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). Interestingly, in the Bufonidae, only two species (Bufo bankorensis and Bufo melanostictus) are found in Taiwan (Li et al. 2006). B. melanostictus is a common toad in Asia. B. bankorensis is widely distributed in the island of Taiwan at 0 ~ 3,000 m in elevation. According to the classification of previous studies (Kawamura et al. 1980 1982; Nishioka et al. 1990), two subspecies of B. gargarizans, i.e., B. gargarizans and B. bankorensis, are found in Taiwan. Typically, 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). The B. gargarizans complex is one of the most common and widely distributed amphibian groups in eastern Asia. 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 although similar to allopatric populations of Bufo andrewsi (Matsui 1986). Moreover, 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). Thus, the Bufo taxa of Taiwan, B. bankorensis and B. gargarizans, remain unclear.

Mitochondrial (mt)DNA can be a powerful molecular marker for reconstructing evolutionary lineages of animals (Avise 1994; Kocher and Stepien 1997; Zhao et al. 2011). Many recent phylogenetic studies also applied mtDNA markers to infer the histories of animals with respect to geography, geology, and paleoclimatology (Macey et al. 1998; Mulcahy and Mendelson 2000). Cytochrome (Cyt) b, a region of mtDNA, is used to determine phylogenetic relationships between organisms due to its sequence variability (Castresana 2001). In a phylogenetic study of B. bufo based on mtDNA (Cyt b, transfer (t)RNAs, 12S ribosomal (r)RNA, and 16S rRNA), gene sequences suggested that one group is B. bufo in Europe and the other is B. japonicus in the Far East. B. japonicus was later divided into four major clades corresponding to a group consisting of B. gargarizans in China, B. bankorensis in Taiwan, B. miyakonis in Miyako Island, and eastern and western groups of the Japanese B. j. japonicus subspecies group (Igawa et al. 2006). The taxonomic status of B. bankorensis has been widely debated, and various names, e.g., B. bufo, B. gargarizans, and Bufo vulgaris var. asiatica, have been either recognized as distinct species (Frost 1985; Matsui 1986; Zhao and Adler 1993) or synonymized with B. gargarizans which is widely distributed in China (Lue and Chen 1982). The taxonomic status and phylogenetic relationships among populations in eastern Asia are still unclear, and to understand the effects that past geological events had on the evolutionary history, further investigation is necessary (Fu et al. 2005). The debate as to whether the Taiwanese B. bankorensis is a species or subspecies, however, is still ongoing. This study was conducted to analyze the mtDNA Cyt b of B. bankorensis collected from various locations in Taiwan to verify the genetic structure and taxonomic status.


Animal collection

B. bankorensis and Buergeria robusta treatment and capture procedures were performed with permission from the Taroko National Park Administration (permit nos. 0990010921, 0990011963, 1000011365, and 1010011630), approved by the Committee for Animal Experimentation of National Dong Hwa University, and conformed to guidelines of the International Association for the Study of Pain. In total, 148 adult B. bankorensis were collected from 12 locations including Jhuzihu (n = 20), Chilan (n = 7), Guanwu (n = 8), Shueili (n = 6), Tungpu (n = 15), Tatachia (n = 7), Walami (n = 9), Motian (n = 21), Chukou (n = 8), Nanzihsien River (n = 3), Meishankou (n = 32), and Kenting (n = 12) (Table 1, Figure 1). In addition, Buergeria robusta and Rana swinhoana were collected from Shakadang Creek, a low-elevation area in Taroko National Park (Hualien, Taiwan). B. melanostictus was collected on our school campus in National Dong-Hwan University (Hualien, Taiwan). B. gargarizans was obtained from Shangai (China). Buergeria robusta and R. swinhoana were used as outgroups. In our previous study, we determined that the population of B. bankorensis could be divided into two major clades (western and eastern clades) following a phylogenetic analysis of haplotypes from a control region (D-loop) sequence (unpublished data).

Figure 1

Sampling locations of B. bankorensis in Taiwan.

Table 1 Summary of the sample size in each location

Preparation of genomic DNA by B. bankorensis mtDNA extraction

Animals were first placed on ice in a bucket causing them to pass out in accordance with the Animal Protection Law for animal welfare. Next, 20 ~ 30 mg of muscle tissue was excised from each animal. The QuickExtractTM DNA Extraction Solution (Epicentre, Madison, WI, USA) was used to extract the muscle tissue following the manufacturer's instructions. The muscle was cut into pieces in GT buffer, 20 μL of proteinase K was added, and then it heated to 60°C for 10 min. Next, 500 μL of GBT buffer was added to the vial and heated to 60°C for 10 min, followed by the addition of 500 μL of 100% absolute ethanol to precipitate the DNA. Finally, 750 μL of the mixture was loaded onto a GD column (Epicentre) for filtering, and the filtrate was centrifuged at 13,000 rpm for 1 min. The previous steps were repeated on the remaining mixture. Then, 400 μL of W1 buffer was added to the GD column, and the filtrate was centrifuged at 13,000 rpm for 30 s. Next, 600 μL of wash buffer (ethanol added) was added to the GD column, the filtrate was centrifuged at 13,000 rpm for 30 s again, and the mixture was subjected to further centrifugation at 13,000 rpm for 30 min. The filtrate of the GD column was put into a new 1.5-mL Eppendorf vial, and 100 μL of elution buffer or double-distilled (dd)H2O (previously preheated at 60°C) was added to dissolve the DNA. After standing for 5 min, the vial was centrifuged at 13,000 rpm for 30 s. The remaining solution contained the DNA. The DNA solution was stored at −20°C, or polymerase chain reaction (PCR) amplification was carried out immediately.

PCR amplification

In total, 25 μL in the PCR vial contained 1 μL of genomic DNA, 1 μL of 10 pmol primers (Table 2), 2.5 μL of 10× PCR buffer, 2 μL of dNTP, and 1 U Taq (R001A, Takara Bio, Otsu, Japan) with ddH2O added to reach 25 μL with adequate vortexing and centrifuging. The vial was heated to 94°C for 5 min, followed by 35 cycles of heat denaturation at 94°C for 30 s, primer annealing at 45°C for 30 s, and DNA extension at 72°C for 1 min in a PCR machine (ASTEC PC802, GMB, Banciao, New Taipei City, Taiwan), with a final amplification step at 72°C for 10 min. Moreover, 1.5% agarose gel electrophoresis was employed to analyze the products, which were visualized by SYBR Safe DNA gel stain (S33102, InvitrogenTM, Life Technologies, Taipei, Taiwan).

Table 2 Sequences of cytochrome (Cyt) b primers

Sequencing Cyt b

After gel electrophoresis, PCR products were extracted with a DNA gel extraction mini kit (Geneaid, Agoura Hills, CA, USA) and selected for DNA sequencing analysis (Genomics BioSci & Tech, Taipei, Taiwan). The isolated and sequenced nucleotide fragments (n = 148, 700 bp) of B. bankorensis were blasted by a Basic Local Alignment Search Tool (BLAST, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA) to confirm validation of the cloned sequences. Based on these sequences, restriction enzyme digestion of DNA (vers. 711, was used to find restriction sites such as Bam HI and Tsp RI.

BamHI and TspRI restriction sites confirmed with an agarose gel electrophoresis analysis

In total, 10 μL of enzymatic digestion reaction contained 4 μL of PCR products, 1 μL of 10× buffer E, l μL of 10× bovine serum albumin, and 1 μL of Bam HI (BioLab, Taigen, Taipei, Taiwan) or Tsp RI (BioLab), and ddH2O was added to reach 10 μL with vortexing. The digestion reaction was conducted at 37°C for 2 h (Bam HI) or 65°C for 4 h (Tsp RI). After digestion, 5 μL of products was electrophoresed on a 1.5% agarose gel for 30 min at 100 V, photographed, and archived.

Phylogenetic analysis

A haplotype genealogy was constructed using an unrooted neighbor-joining (NJ) algorithm and a bootstrap method with 1,000 replicates using the software MEGA vers. 4.0 (Biodesign Institute, Center for Evolutionary Functional Genomics, Tempe, AZ, USA). B. japonicus was used as an outgroup.


The Cyt b primer set was applied to run the PCR with DNA of B. bankorensis, B. melanostictus, B. gargarizans, Buergeria robusta, and R. swinhoana. Figure 2 illustrates alignments of nucleotide sequences of Cyt b of B. bankorensis, B. gargarizans, and Buergeria robusta. Only PCR products of B. bankorensis, B. gargarizans, and Buergeria robusta were amplified, and the size of the band was about 700 bp, but R. swinhoana was not amplified. Figure 3A presents the results of Cyt b PCR products. Based on the Cyt b sequence, the Bam HI restriction site was found at 410 ~ 420 of 700 bp (Figure 3B). After digestion with the Bam HI restriction enzyme, the mtDNA of B. bankorensis was divided into two types, and electrophoretic results are shown in Figure 3B. B. bankorensis populations were divided into two types based on whether the sequence was cut or not. B. bankorensis 1 (Bu1) and Bu3 were the eastern group, and Bu2 was the western group. The distinction between the eastern and western groups depended upon geography because samples were collected from both western to eastern parts of the island of Taiwan. The western group was not cut and had a size of about 700 bp, while the eastern group was digested into two bands of about 400 and 300 bp. In a comparison of Cyt b sequences among eastern and western groups of B. bankorensis with B. gargarizans, only the eastern group of B. bankorensis contained the Bam HI restriction site. Additionally, comparison of variations in Cyt b sequences revealed that 99% similarity was observed for western group 1 of B. bankorensis with B. gargarizans (Figure 4A), and 97% similarity was found for western group 2 of B. bankorensis with B. gargarizans (Figure 4B). The Tsp RI site of Cyt b among B. gargarizans and B. bankorensis is depicted in Figure 5A. B. gargarizans DNA was digested by the Tsp RI enzyme. Moreover, the western clade of B. bankorensis was further digested by Tsp RI and defined as western clade 1, while the other one (western clade 2) was not (Figure 5B). Therefore, the Cyt b sequence of B. bankorensis could be cut by Tsp RI which was 99% similar to B. gargarizans, whereas the uncut type was only 97% similar. Figure 6 illustrates phylogenetic relationships. Surprisingly, western group 1 differed from B. gargarizans and formed a cluster with B. gargarizans at 99% similarity. This result suggests that two B. bankorensis populations (western and eastern clades) exist in Taiwan. Moreover, the western clade had two phylogroups, one of which was possibly genetically related to B. gargarizans (western clade 1) but morphologically differed, and the other which was still named B. bankorensis (western clade 2). The eastern clade may be a new subspecies in the genetic structure.

Figure 2

Alignments of nucleotide sequence and the Bam HI site. Alignments of nucleotide sequence of Cyt b of B. bankorensis and B. gargarizans (A) and the Bam HI site (GGATCC) of the Cyt b sequence (B). The alignments were performed with DNA Star. B. bankorensis 1 and 2 are from the Walami area (labeled location number 7 in Table 1).

Figure 3

PCR products of various toads and frogs (A), and digestion of Bam HI (B). B. bankorensis (Bu), B. melanostictus (BM), B. gargarizans (BG), Buergeria robusta (B), and R. swinhoana (S). Buergeria robusta (B) and R. swinhoana (S) were used to test the specificity of the primer set. No specific bands were found for B. melanostictus or R. swinhoana, and no further sequencing was performed. Bu1 and Bu3 are the eastern clade. Bu2 is the western clade. Bu1, Bu2, Bu3, BM1, BM2, BM3, B1, B2, B3, S1, S2, S3, BG1, BG2, BG3, and BG4 represent different individual samples in each group.

Figure 4

Alignments of Cyt b sequences. Alignments of Cyt b sequences between western clade 1 of B. bankorensis and B. gargarizans (A), and western clade 2 of B. bankorensis and B. gargarizans (B). The alignment of nucleotide sequences was prepared using Mega 4 and NCBI blast.

Figure 5

The Tsp RI site and digestion of Tsp RI. (A) The Tsp RI site (NNCASTGNN in the green box; N = A, T, C, G; S = C, G) of cytochrome b in the western clade and B. gargarizans. (B) Digestion of Tsp RI in B. bankorensis (Bu) and B. gargarizans (BG). BG1, BG2, BG3, BG4, Bu7, Bu8, Bu9, Bu10, Bu11, and Bu12 represent different individual samples in each group. BG1, BG2, BG3, BG4 are from China. Bu7, Bu8, Bu9, Bu10, Bu11, and Bu12 are from the Jhuzihu area, Taiwan (labeled location number 1 in Table 1).

Figure 6

Phylogenetic tree. The neighbor-joining phylogenetic tree of cytochrome b of the eastern and western clades of B. bankorensis, B. gargarizans, and B. japonicus (sequence AB159256.1 from NCBI GenBank). The phylogenetic tree was analyzed by Mega 4. B. japonicus was used as an outgroup. B. gargarizans (BG1, BG2, BG3, and BG4) was from China. The eastern (Bu5 and Bu6) and western clades (Bu7, Bu8, Bu9, Bu10, Bu11, and Bu12) were from the Jhuzihu area, Taiwan (labeled location number 1 in Table 1).


After the Bam HI assay of the Cyt b sequence of B. bankorensis, the presence of two genotypes was observed: one was the Cyt b genotype of the western clade (which was not cut), and the other was the Cyt b genotype of the eastern clade (which was cut). This result was similar to that for the Japanese common toad B. japonicus which is classified into two subspecies, B. j. japonicus and Bufo japonicus formosus of western and eastern regions of Japan, respectively (Hase et al. 2012). Interestingly, the similarity of the Cyt b nucleotide sequence was 99% between the western clade 1 and B. gargarizans, and they were further digested by Tsp RI, whereas the other western clade 2 was 97% similar with B. gargarizans and was not cut by Tsp RI. Recent molecular phylogenetic studies (Liu et al. 2000; Fu et al. 2005) supported the two-species classification by synonymizing B. bankorensis with B. gargarizans. Our data (Figure 6) revealed that B. bankorensis was proposed to be B. japonicus which was divided into three major clades corresponding to a group consisting of Bufo japonicus gargarizans in China, Bufo japonicus bankorensis in Taiwan, and B. j. miyakonis in Miyako Island, Japan (Igawa et al. 2006); B. bankorensis was also postulated to be an endemic species in Taiwan (Fu et al. 2005). From ecological view, the range of certain species is associated with habitat conditions. In the current study, the size and age at metamorphosis of tadpoles vary among individuals from the same species in different habitats in a relatively small area, suggesting that distributions of different species depend upon adaptation to extreme conditions (Goldberg et al. 2012). Moreover, we found that bankorensis toads formed a complex from the restriction enzyme and phylogenetic relationship, but more detailed assessments including morphology and physiology need to be carried out in the future work to identify subspecies or new species existing.

A previous report found that B. japonicus in the Far East was divided into two groups, one of which became two subspecies: B. j. gargarizans in China and Taiwan and B. j. miyakonis in Miyako Island, Japan, while the other group became four subspecies on the eastern and western groups of Japanese B. j. japonicus, Hakodate, and Yaku Island, Japan (Nishioka et al. 1990). However, in past phylogenetic studies, sampling was limited to a small sample size (mostly one or two locations and did not include samples from eastern Taiwanese B. Bankorensis populations). Given that populations resembling B. gargarizans occur on an isolated island (Taiwan Island), there are two alternative hypotheses that might explain this pattern: (1) B. bankorensis populations are from a single lineage of B. gargarizans that subsequently dispersed to the island and became isolated, or (2) B. bankorensis independently evolved multiple times as B. gargarizans colonized the island. The present work proved the existence of two species (B. gargarizans and B. bankorensis) in Taiwan using a simple restriction enzyme (Tsp RI). From our data and according to previous reports, there are two subspecies, Bufo gargarizans gargarizans and Bufo gargarizans bankorensis, in Taiwan (Kawamura et al. 1980 1982; Nishioka et al. 1990). Furthermore, B. bankorensis was classified in the B. gargarizans species complex (Inger 1972; Matsui 1984;Liu et al. 2000; Fu et al. 2005), one of the most common and widely distributed amphibian groups in eastern Asia. The eastern and western groups of B. japonicus were divided into several subclades that tended to reflect the region-specific geographic distribution of all localities except B. j. japonicus from Hakodate, Japan (Igawa et al. 2006). The western clade of B. bankorensis can be distinguished by restriction fragment length polymorphism and the phylogenetic tree, but not by nucleotide homology. This viewpoint suggests that the tree represents homoplasy. This region-specific subclade of the toad, such as western groups 1 and 2, was also found in Taiwanese B. bankorensis. This finding is in agreement with a report by Hase et al. (2012) that an admixed population was observed in the urban Tokyo area that consisted of both native and non-native B. japonicus subspecies. Conversely, Bufo tibetanus is a morphologically identified species but not genetically diagnosable, which was inferred to be a junior synonym of B. gargarizans(Zhan and Fu 2011). Our results propose that two populations (western and eastern clades) of B. bankorensis appear in Taiwan and are classified into three distinct phylogroups: the first is genetically related to B. gargarizans, the second should be B. bankorensis, and the third may be considered a new subspecies. In the same area (Tungpu, Taiwan), we also identified two different clades: western clades 1 and 2. The molecular phylogenetic studies supported B. gargarizans and B. bankorensis being synonymized, and B. bankorensis only being a lineage of B. gargarizans (Liu et al. 2000 ; Fu et al. 2005). This reflects a previous study (Fu et al. 2005) showing that B. gargarizans and B. bankorensis may be subspecies if samples were collected at certain locations. The present study postulates that B. bankorensis (western clade 1) is only a lineage of B. gargarizans because they are only genetically identifiable in contrast to having different morphologies. Hence, B. bankorensis is one species and is restricted to Taiwan. Based on our data, all three distinct phylogroups were observed in the wild and were found in some locations, further suggesting that they are sympatric, not allopatric.


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. B. bankorensis is recognized as one species having different morphologies compared with B. gargarizans and is restricted to Taiwan. All three phylogroups were found in some areas, suggesting that they are sympatric, not allopatric.


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This study was supported by funding from Taroko and Yushan National Parks, Taiwan. We would like to thank Yushan National Park staff for their assistance and contributions to the laboratory work. Of course, this work also dedicates a memorial to the frontier heroes and animals during the development of the eastern-western region of Taiwan.

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Correspondence to Kao-Jean Huang or Ching-Feng Weng.

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The authors declare that they have no competing interests.

Authors’ contributions

C-CC, K-WL, P-YS, and Yi-WT carried out the molecular genetic studies and participated in the sequence alignment. T-LY and L-HC participated in the design of the study and performed the statistical analysis. K-JH and C-FW conceived the study and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.

Chu-Chih Chen, Kou-Wei Li, Kao-Jean Huang and Ching-Feng Weng contributed equally to this work.

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Chen, C., Li, K., Yu, T. et al. Genetic structure of Bufo bankorensis distinguished by amplified restriction fragment length polymorphism of cytochrome b . Zool. Stud. 52, 48 (2013) doi:10.1186/1810-522X-52-48

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  • Cytochrome b
  • Restriction enzyme
  • Sympatry
  • Bufo bankorensis
  • B. gargarizans