Please refer to Choi et al. (2021): "Choi, E.H., Yeo, M.Y., Kim, G. et al. Liolophura species discrimination with geographical distribution patterns and their divergence and expansion history on the northwestern Pacific coast. Sci Rep 11, 17602 (2021).

The chiton Liolophura japonica (Lischke, 1873) is widely distributed in intertidal coastal areas of the northwestern Pacific. Here we show species discrimination of L. japonica into two species and one subspecies based on COI and 16S rRNA; L. koreana, sp. nov. was mainly distributed at ca. 33°24'–38°32' N, L. japonica at ca. 33°24'–35°53' N, and L. j. sinensis, ssp. nov. at ca. 27°02'–28°00' N. These species were morphologically distinguishable by black spots on the tegmentum and the shape of spicules on the perinotum. In addition, we have discussed their molecular divergence times (3.37 mya for L. koreana and L. japonica, around the mid Pliocene warm period; 1.84 mya for L. japonica and L. j. sinensis), demographic expansion events following the last interglacial age, called the Eemian (129–116 kya), and augmentation of COI haplotype diversity during the late-middle to late Pleistocene. Their latitudinal geographical distribution gradients may be helpful for monitoring the migration of marine 38 invertebrates north, fostered by global warming in the northwestern Pacific.

To examine population genetic structure and the possibility of species discrimination of L. japonica on the northwestern Pacific coast, we collected a total of 304 L. japonica samples from nine sampling localities in the intertidal coasts of the Korean Peninsula and Japanese Archipelago). From the collected L. japonica samples, we amplified the COI barcoding region using PCR, and then sequenced the 635-bp PCR products. As a result, a total of 75 COI haplotypes of L. japonica were detected through the present study. To create a COI haplotype dataset including previously reported data, we retrieved 31 COI haplotypes of L. japonica (also known as Acanthopleura japonica) from the NCBI GenBank database, consisting of two Japanese and 29 Chinese COI haplotypes. Finally, we gathered 106 COI haplotypes from 431 L. japonica individuals in twelve localities of South Korea, Japan, and southern China.

In the case of 16S rRNA genes, the 304 samples of L. japonica from nine localities in the intertidal coasts on the Korean Peninsula and Japanese Archipelago were subjected to PCR amplification of a partial region of 16S rRNA (506 bp). Of these, only 261 samples were successfully amplified and sequenced. As a result, a total of 23 16S rRNA haplotypes of L. japonica were detected. Combined with 11 previously known southern Chinese haplotypes of 16S rRNA from 126 individuals, we totaled 34 16S rRNA haplotypes from 387 L. japonica individuals in ten localities.

Supplementary Data S1: Nucleotide sequence alignment of 106 COI haplotypes of Liolophura japonica with an outgroup.

Supplementary Data S2: Nucleotide sequence alignment of 34 16S rRNA haplotypes of Liolophura japonica with an outgroup.

Supplementary Data S3: Nucleotide sequence alignment of 106 COI haplotypes of Liolophura japonica, Liolophura koreana sp. nov., and Liolophura sinensis sp. nov., one COI haplotype of Liolophura tenuispinosa, and 14 COI haplotypes of eight Acanthopluera congeneric species with an outgroup Tonicia forbesii.