Rhodeus ocellatus kurumeus is a small cyprinid fish endemic to the Japanese archipelago. This fish frequently hybridizes with an exotic subspecies Rhodeus ocellatus ocellatus, and non-introgressed populations of R. o. kurumeus have greatly decreased. Previous studies based on mitochondrial and nuclear DNA identified introgressed populations and inferred phylogenetic relationships, but these approaches may lead to underestimates of introgression or ambiguous results owing to the small number of genetic markers used. The present study applied double digest restriction site-associated DNA (ddRAD) sequencing to assignment tests and phylogenetic inferences. Data matrices can vary in size and content depending on the strategies used to process ddRAD sequences; therefore, we prepared 25 data matrices with various processing strategies. The assignment tests based on the 25 data matrices resulted in similar assignment patterns and provided evidence for introgression in some populations that had been identified as non-introgressed populations in previous studies. The maximum-likelihood (ML) phylogenetic trees differed in robustness and topology among the 25 matrices; interestingly, ML trees with low statistical support reflected geographical distributions of fish better than ML trees with high statistical support. These results indicate that ddRAD sequencing can detect introgression with greater sensitivity than conventional DNA markers, that ddRAD sequencing is useful for phylogenetic inference among closely related populations within a subspecies, and that statistically robust ML trees do not necessarily reflect the true phylogeny. Application of ddRAD data to conservation genetics and evolutionary history of this subspecies are also discussed.

Rhodeus ocellatus kurumeus是日本群岛特有的小型塞浦路斯鱼类。该鱼经常与外来亚种Rhodeus ocellatus ocellatus和非渗入R. o。种群杂交。久留米大大减少了。先前基于线粒体和核DNA的研究确定了渗入群体并推断了系统发生关系，但是由于使用的遗传标记物数量少，这些方法可能导致渗入低估或结果含糊。本研究将双酶切限制性酶切位点相关的DNA（ddRAD）测序应用于分配测试和系统发育推论。数据矩阵的大小和内容可能会有所不同，具体取决于用于处理ddRAD序列的策略。因此，我们准备了25种具有不同处理策略的数据矩阵。基于25个数据矩阵的分配测试得出相似的分配模式，并为在先前研究中已确定为非渗入群体的某些群体提供了渗入证据。在25个矩阵中，最大似然（ML）系统发育树的健壮性和拓扑结构有所不同。有趣的是，统计支持率低的ML树比统计支持率高的ML树更好地反映了鱼类的地理分布。这些结果表明，ddRAD测序比常规DNA标记具有更高的敏感性，ddRAD测序可用于亚种内密切相关种群之间的系统发育推断，统计上可靠的ML树不一定反映真实的系统发育。还讨论了ddRAD数据在该亚种的保护遗传学和进化史中的应用。具有低统计支持的ML树比具有高统计支持的ML树更好地反映了鱼的地理分布。这些结果表明，ddRAD测序比常规DNA标记具有更高的敏感性，ddRAD测序可用于亚种内密切相关种群之间的系统发育推断，统计上可靠的ML树不一定反映真实的系统发育。还讨论了ddRAD数据在该亚种的保护遗传学和进化史中的应用。具有低统计支持的ML树比具有高统计支持的ML树更好地反映了鱼的地理分布。这些结果表明，ddRAD测序比常规DNA标记具有更高的敏感性，ddRAD测序可用于亚种内密切相关种群之间的系统发育推断，统计上可靠的ML树不一定反映真实的系统发育。还讨论了ddRAD数据在该亚种的保护遗传学和进化史中的应用。ddRAD测序可用于在亚种内密切相关的种群之间进行系统发育推断，并且统计上可靠的ML树不一定反映真实的系统发育。还讨论了ddRAD数据在该亚种的保护遗传学和进化史中的应用。ddRAD测序可用于在亚种内密切相关的种群之间进行系统发育推断，并且统计上可靠的ML树不一定反映真实的系统发育。还讨论了ddRAD数据在该亚种的保护遗传学和进化史中的应用。