Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo, Kyoto, Kyoto, 606-8502, Japan.
Department of Bioscience and Biotechnology, Kyoto University of Advanced Science, 1-1 Nanjo Otani, Sogabe, Kameoka, Kyoto, 621-8555, Japan.
J Chem Ecol. 2020 Oct;46(10):997-1009. doi: 10.1007/s10886-020-01217-y. Epub 2020 Sep 30.
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.
蟾蜍属的蟾蜍在其耳后腺中合成和积累蟾蜍毒素（BDs）。BDs 是强心甾类化合物，在防御蟾蜍的捕食者方面发挥着重要作用。在日本大陆有三种蟾蜍，即日本蟾蜍 Formosus、日本蟾蜍 japonicus 和 Torrenticola。几十年前就研究了从 B. j. formosus 中分离出的 BDs 的化学结构，但关于日本蟾蜍的有毒成分及其代谢物尚无进一步的信息。在这项研究中，我们分析了日本各地蟾蜍的 BDs，并通过液相色谱/质谱（LC/MS）和层次聚类分析（HCA）比较了 BDs 图谱。我们观察到了三种日本蟾蜍的 BDs，并通过核磁共振（NMR）分析鉴定了五种最常见的 BDs。在这五种 BDs 中，只有 bufalin 存在于所有个体中。个体 BDs 图谱的 HCA 将这三个分类群分为五个主要聚类和几个子聚类。这一结果表明，BDs 图谱不仅在分类群之间存在差异，而且在分类群内部也存在差异。BDs 的聚类模式与从日本蟾蜍的线粒体细胞色素 b 基因重建的系统发育树基本一致。我们的结果表明，日本蟾蜍的 BDs 多样化不是由于特定的选择压力，而是由于进化过程中的种群结构造成的。
