Toads of the genus Bufo synthesize and accumulate bufadienolides (BDs) in their parotoid glands. BDs are cardiotonic steroids that play an important role in defense against the toads’ predators. Three bufonid taxa occur in mainland Japan, Bufo japonicus formosus, B. j. japonicus, and B. torrenticola. The chemical structures of BDs isolated from B. j. formosus were studied several decades ago, but there is no further information on the toxic components of Japanese toads and their metabolism. In this study, we analyzed BDs of toads from throughout Japan and compared the BD profiles by liquid chromatography/mass spectrometry (LC/MS) and hierarchical cluster analysis (HCA). We observed BDs in three taxa of Japanese toads, and identified five of the most common BDs by nuclear magnetic resonance (NMR) analyses. Of the five BDs, only bufalin was detected in all individuals. HCA of individual BD profiles divided the three taxa into five primary clusters and several subclusters. This result indicates that BD profiles differ both among and within the taxa. The clustering pattern of BDs is generally concordant with a phylogenetic tree reconstructed from the mitochondrial cytochrome b gene of Japanese toads. Our results suggest that the BDs of Japanese toads have diversified not in response to specific selective pressures, but simply due to population structuring over evolutionary time.

蟾蜍属的蟾蜍在其腮腺中合成并积累了丁二烯内酯（BDs）。BD是强心类固醇，在防御蟾蜍的天敌中起着重要作用。三bufonid类群发生在日本本土，蟾蜍刺参福尔摩，B.学家 japonicus和B. torrenticola。从B. j。中分离出来的BDs的化学结构。mos几十年前进行了研究，但没有关于日本蟾蜍的毒性成分及其代谢的进一步信息。在这项研究中，我们分析了来自日本各地的蟾蜍的BD，并通过液相色谱/质谱（LC / MS）和层次聚类分析（HCA）比较了BD的分布。我们在日本蟾蜍的三个类群中观察了BD，并通过核磁共振（NMR）分析确定了五个最常见的BD。在五个BD中，在所有个体中仅检测到bufalin。单个BD配置文件的HCA将三个分类群分为五个主要簇和几个子簇。该结果表明，BD类在类群之间和之内都不同。BD的聚类模式通常与从线粒体细胞色素b重建的系统发育树一致日本蟾蜍的基因。我们的研究结果表明，日本蟾蜍的BD并非针对特定的选择压力而多样化，而仅仅是由于进化时期的种群结构。