Liolophura species discrimination with geographical distribution patterns and their divergence and expansion history on the northwestern Pacific coast

Abstract

The chiton Liolophura japonica (Lischke 1873) is distributed in intertidal areas of the northwestern Pacific. Using COI and 16S rRNA, we found three genetic lineages, suggesting separation into three different species. Population genetic analyses, the two distinct COI barcoding gaps albeit one barcoding gap in the 16S rRNA, and phylogenetic relationships with a congeneric species supported this finding. We described L. koreana, sp. nov. over ca. 33°24' N (JJ), and L. sinensis, sp. nov. around ca. 27°02'-28°00' N (ZJ). We confirmed that these can be morphologically distinguished by lateral and dorsal black spots on the tegmentum and the shape of spicules on the perinotum. We also discuss species divergence during the Plio-Pleistocene, demographic expansions following the last interglacial age in the Pleistocene, and augmentation of COI haplotype diversity during the Pleistocene. Our study sheds light on the potential for COI in examining marine invertebrate species discrimination and distribution in the northwestern Pacific.

Figure 1

A map showing sampling localities and photos of a habitat landscape and wild samples of Liolophura japonica inhabiting coastal areas of the Korean Peninsula (N = 249), the Japanese Archipelago (N = 57), and southern China (N = 125) in the northwestern Pacific Ocean. (a) A map showing twelve direct sampling localities for L. japonica in coastal areas of the northwestern Pacific Ocean. The sampling localities of one southern Chinese (ZJ) and two Japanese (EH and MY) previously catalogued haplotype sequencing studies retrieved from NCBI are also depicted. Table S1 and S2 contain more accurate information on the populations and individuals. The basic map is from a free map providing site (https://d-maps.com), which is modified with Adobe Illustrator v.25.2. (https://www.adobe.com). (b) Photos of a habitat landscape and wild samples of L. japonica, taken from Seogwipo-si, Jeju Island, South Korea, photographed by Mi Young Yeo, Bia Park, and Cho Rong Shin. The photos were edited using Adobe Photoshop v.22.2 (https://www.adobe.com).

Figure 2

Phylogenetic, TCS network, and PCoA analyses based on 106 COI haplotypes from 469 individuals of Liolophura japonica inhabiting coastal areas of the northwestern Pacific Ocean, suggesting the existence of the three different genetic lineages: Lineage N, Lineage S1, and Lineage S2. (a) Maximum likelihood tree showing the three different genetic lineages for L. japonica: Lineage N members are most likely from the populations inhabiting a wide range of South Korea and Japan, Lineage S1 members from the populations inhabiting southern coastal areas of South Korea and Japan only, and Lineage S2 members from the southern Chinese population. As shown in Fig. S1, Acanthopleura spinosa was used as an outgroup. Numbers on branches indicate node confidence values: BP in ML, BPP in BI, and BP in NJ in order. (b) A phylogenetic network reconstructed using the neighbor net algorithm without an outgroup, showing three different genetic lineages for L. japonica inhabiting the northwestern Pacific coast: Lineages N, S1, and S2. The COI sequence alignment set used is shown in Data S1. Detailed information of the 106 COI haplotypes used in this phylogenetic analysis is summarized in Table S1 and S2. (c) An unrooted TCS network showing three distinct genetic clusters, corresponding to Lineages N, S1, and S2. Three different genetic groups correspond to the three genetic lineages shown in the phylogenetic tree (a), respectively. The haplotype frequency is displayed by the circle size. (d) A two-dimensional PCoA plot showing the three distinct genetic groups corresponding to Lineages N, S1, and S2 shown in the phylogenetic tree (a). The score on the first two axes (Axis 1 = 79.05% and Axis 2 = 15.32%) from the matrix of genetic distances estimated with the 106 COI haplotypes are indicated.

Figure 3

The results of phylogenetic and population genetic analyses based on 34 16S rRNA haplotypes from 425 individuals of Liolophura japonica inhabiting coastal areas of the northwestern Pacific Ocean. (a) Phylogenetic network reconstructed using the neighbor net algorithm, showing three different genetic lineages for L. japonica: Lineage N, Lineage S1, and Lineage S2. The 16S rRNA sequence alignment set used is shown in Data S2. Detailed information of 34 16S rRNA haplotypes used in these analyses is summarized in Table S5 and S6. (b) An unrooted TCS network. There are distinctly observed three different genetic groups, corresponding to the three genetic lineages shown in the phylogenetic network (a). The haplotype frequency is displayed by the circle size. (c) A two-dimensional PCoA plot showing the three distinct genetic groups, corresponding to Lineage N, Lineage S1, and Lineage S2. The score on the first two axes (Axis 1 = 87.77% and Axis 2 = 4.4%) from the matrix of genetic distances estimated with the 34 16S rRNA haplotypes are indicated.

Figure 4

Distribution of pairwise genetic divergences, ranked pairwise difference, and automatic partition based on COI and 16S rRNA haplotypes of Liolophura japonica and a COI-based NJ tree showing the phylogenetic relationship with a congeneric species L. tenuispinosa. (a) Distribution patterns of pairwise genetic divergences observed in COI and 16S rRNA for L. japonica. The horizontal axis represents intervals of pairwise Kimura-2-parameter (K2P) genetic distance in percentage, and the vertical axis represents the number of individuals associated with each distance interval. (b) The results of ranked pairwise differences based on COI and 16S rRNA, ranked by ordered value, which is similar to the distribution of pairwise genetic divergence in (a). The horizontal axis indicates a ranked ordered value based on K2P genetic distance, and the vertical axis represents the K2P genetic distance in percentage. (c) The results of automatic partition analyses based on COI and 16S rRNA. The horizontal axis represents the prior maximum intraspecific divergence (P), and the vertical axis represents the number of groups inside the partitions (primary and recursive). (d) A COI-based NJ tree with L. tenuispinosa. Refer to Fig. S3 and Data S3.

Figure 5

Morphological comparison of Liolophura koreana, sp. nov., L. japonica, and L. sinensis, sp. nov. (a–c) Photos of dorsal views of the individuals belonging to L. koreana (Lineage N), L. japonica (Lineage S1), and L. sinensis (Lineage S2) in order. (d,e) Morphological comparison of pleural and lateral black spots on valves III and IV of the tegmentum of L. koreana (d; holotype) and L. japonica (e). (f,g) Morphological comparison of spicules on the perinotum of L. koreana (f; holotype) and L. japonica (g). (h–k) Morphological comparison of the spicule of L. koreana (h,i; paratype) and L. japonica (j,k) in lateral and dorsal views. The scale bar marks 2.0 mm (d,e), 1.0 mm (f,g), and 0.5 mm (h–k). The photos were edited using Adobe Photoshop v.22.2 (https://www.adobe.com).

Figure 6

Microstructural comparison of Liolophura koreana, sp. nov. and L. japonica using field emission scanning electron microscopy (FE-SEM). (a,b) Middle and lateral areas on the tegmentum of the holotype of L. koreana. (c,d) Middle and lateral areas on the tegmentum of L. japonica. Arrows indicate that morphological difference of the posterior valve margin of the valve II between two species. The scale bar marks 1.0 mm. (e,f) The occurrence frequency, and shape and structure differences of the spicules on the perinotum between the holotype of L. koreana (e) and L. japonica (f). The scale bar marks 1.0 mm and 0.2 mm, respectively. The photos were edited using Adobe Photoshop v.22.2 (https://www.adobe.com).

Figure 7

Geographical distribution of Liolophura koreana, sp. nov., L. japonica, and L. sinensis, sp. nov. inhabiting coastal areas of the northwestern Pacific Ocean. A COI-based map showing geographical distribution of L. koreana, L. japonica, and L. sinensis on the northwestern Pacific coast. L. koreana are found in a wide range of South Korea and Japan above ca. 33°24′ N (JJ), L. japonica in mainly southern coastal areas of South Korea and Japan below ca. 35°53′ N (TT), and L. sinensis in ZJ of southern China around ca. 27°02′ N–28°00′ N. The sympatric distribution of L. koreana and L. japonica is shown between 33°24′ and 35°53′ N. Table S1–S3 contain the full names of localities and detailed haplotype information. The question mark indicates that collection of Liolophura samples from such coastal areas in Japan is required to clarify distribution patterns of L. koreana and L. japonica in the East Sea (= Sea of Japan). The basic map was obtained from a free map-providing site (https://d-maps.com), which was modified using Adobe Illustrator v.25.2. (https://www.adobe.com).

Figure 8

The results of mismatch distribution analyses (MDA), Bayesian skyline plots (BSPs), and molecular clock analysis performed with COI haplotypes for Liolophura koreana, sp. nov., L. japonica, and L. sinensis, sp. nov. (a) MDA plots resulting in a unimodal curve for L. koreana, L. japonica, and L. sinensis. Dotted lines indicate the observed distribution of mismatches, and solid lines represent the expected distribution under a demographic expansion model. (b) BSP results showing the demographic history of population expansions of L. koreana, L. japonica, and L. sinensis. The graph in gray depicts sea level changes during the last 330 Ka. (c) Time-calibrated Bayesian tree reconstructed using BEAST with the inference of ancestral areas under the Bayesian binary MCMC (BBM) model implemented in RASP ver 3.2. Ancestral areas were hypothesized based on the distribution range of the fossil records of Mopalia and the contemporary distribution of L. koreana, L. japonica, and L. sinensis. LGM indicates the last glacial maximum (0.026–0.019 Ma; blue vertical bar) and three interglacial periods are indicated by light green boxes during the late-middle and late Pleistocene. The pictures were edited using Adobe Illustrator v.25.2. (https://www.adobe.com).