Tiny animals in various metazoan phyla inhabit the interstices between sand and/or gravel grains, and adaptive traits in their body plan, such as simplification and size reduction, have attracted research attention. Several possible explanations of how such animals colonized interstitial habitats have been proposed, but their adaptation to this environment has generally been regarded as irreversible. However, the actual evolutionary transitions are not well understood in almost all taxa. In the present study, we show reversible evolutionary shifts from interstitial to epibenthic habitats in the lineage of the polyclad flatworm genus Boninia. In addition, we establish two new species of this genus found from different microhabitats on a single beach in Okinawa Island, Japan: (i) the interstitial species Boninia uru sp. nov. from gravelly sediments and (ii) the epibenthic species Boninia yambarensis sp. nov. from rock undersurfaces. Our observations suggest that rigid microhabitat segregation exists between these two species. Molecular phylogenetic analyses based on the partial 18S and 28S rDNA sequences of the new Boninia species and four other congeners, for which molecular sequences were available in public databases [Boninia antillara (epibenthic), Boninia divae (epibenthic), Boninia neotethydis (interstitial), and an unidentified Boninia sp. (habitat indeterminate)], revealed that the two interstitial species (B. neotethydis and B. uru sp. nov.) were not monophyletic among the three epibenthic species. According to ancestral state reconstruction analysis, the last common ancestor of the analyzed Boninia species inhabited interstitial realms, and a shift to the epibenthic environment occurred at least once. Such an "interstitial to noninterstitial" evolutionary route seems to be rare among Animalia; to date, it has been reported only in acochlidian slugs in the clade Hedylopsacea. Our phylogenetic tree also showed that the sympatric B. uru sp. nov. and B. yambarensis sp. nov. were not in a sister relationship, indicating that they colonized the same beach independently rather than descended in situ from a common ancestor that migrated and settled at the beach.
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