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.
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