Sample collection of Liolophura japonica and the genetic diversity of COI barcoding region
To examine genetic lineage divergence within L. japonica on the northwestern Pacific coast, we newly collected a total of 342 L. japonica samples from 12 sampling localities in the intertidal coasts of the Korean Peninsula and Japanese Archipelago (Fig. 1; Table S1). 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 based on COI sequences obtained from 342 individuals of L. japonica were detected via the present study (Table S2). In addition to this, we extracted 31 COI haplotypes based on COI sequences from 127 individuals of L. japonica (also known as Acanthopleura japonica) previously reported in the NCBI GenBank database, consisting of two Japanese and 29 Chinese COI haplotypes. Finally, we gathered 106 COI haplotypes from 469 L. japonica individuals collected in 15 localities of South Korea, Japan, and southern China (Tables S1, S2). The average haplotype (h) and nucleotide diversities (π) were 0.808 and 0.04936, respectively; the highest haplotype diversity was observed in Tsushima (TS; h = 0.963), and the highest nucleotide diversity was found in Wando (WD; π = 0.04581), located in the South Sea of the Korean Peninsula. As shown in Table S3, the population distribution pattern of COI haplotypes revealed that all collection sites had site-specific haplotype(s) except for Busan (BS), Wando (WD), Sinan (SA), and Jeju Island (JJ). The most abundant haplotype was A1, which was found in 170 (39.4%) out of the COI sequences obtained from 469 L. japonica individuals.
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).
Phylogenetic and population genetic analyses based on COI
We constructed a nucleotide sequence alignment set with 106 COI haplotypes of L. japonica (Data S1), and identified 95 polymorphic sites (15.0%, Table S4) and 68 parsimoniously informative sites (10.7%). To elucidate phylogenetic relationships among the populations of L. japonica, we performed molecular phylogenetic analyses, including maximum likelihood (ML), Bayesian inference (BI), and neighbor-joining (NJ) analyses, based on these 106 COI haplotypes with the outgroup Acanthopleura spinosa (Fig. 2a, Figs. S1, S2). The resultant phylogenetic trees clearly revealed the existence of three distinct genetic lineages within the monophyletic group of L. japonica (100 BP in ML, 1.00 BPP in BI, and 100 BP in NJ): Lineage N (91 BP, 1.00 BPP, and 100 BP), Lineage S1 (79 BP, 0.82 BPP, and 98 BP), and Lineage S2 (98 BP, 1.00 BPP, and 100 BP). Among these three genetic lineages, Lineages S1 and S2 were grouped with high node confidence values (94 BP, 1.00 BPP, and 95 BP). We additionally conducted a phylogenetic network analysis using a neighbor net algorithm without an outgroup (Fig. 2b), which confirmed that these sequences were distinctly divided into three genetic lineages, in agreement with the topology of the rooted phylogenetic trees (Fig. 2a, Fig. S1).
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.
Consistently, the TCS network analysis (Fig. 2c) and principal coordinate analysis (PCoA) (Fig. 2d) showed the existence of three distinguished genetic groups among L. japonica, in accordance with the phylogenetic analyses (Fig. 2a − b). The TCS network (Fig. 2c) revealed that Lineages S1 and S2 were separated by 18 mutation steps, which is far shorter than the distance between Lineages N and S1 (37 mutation steps) or between Lineages N and S2 (60 mutation steps), indicating that Lineages S1 and S2 have a close affinity and had only recently diverged from each other. The overwhelming dominancy of the A1 haplotype implies a recent and rapid population expansion of Lineage N. In addition to A1, it was found that haplotypes B2 for Lineage S1 and C21 for Lineage S2 were dominant. In the PCoA plot (Fig. 2d), the three genetic groups of L. japonica were also observed, as in the phylogenetic (Fig. 2a,b) and TCS network (Fig. 2c) analyses. Lineage N was distantly located from Lineages S1 and S2, while Lineages S1 and S2 were spatially much closer.
Sample collection of L. japonica and the genetic diversity of 16S rRNA
The 342 individuals of L. japonica from 12 localities in the intertidal coasts on the Korean Peninsula and Japanese Archipelago (Fig. 1) were subjected to PCR amplification of a partial region of 16S rRNA (506 bp) (Tables S5, S6). Of these, only 299 samples were successfully amplified and sequenced. Based on 299 individual 16S rRNA sequences, a total of 23 16S rRNA haplotypes of L. japonica were detected (Tables S5, S6). Combined with 11 haplotypes extracted from 16S rRNA sequences of 125 L. japonica individuals known previously in southern China, we totaled 34 16S rRNA haplotypes from 425 L. japonica individuals in 13 collection localities. The average haplotype (h) and nucleotide (π) diversities were 0.702 and 0.02093, respectively; the highest haplotype diversity was found in Geojedo (GJ; h = 0.833), and the highest nucleotide diversity in Wando (WD; π = 0.02244), located in the South Sea of the Korean Peninsula. Overall, the average haplotype and nucleotide diversities of 16S rRNA were lower than those of COI (Table S1). As shown in Table S7, the population distribution pattern of 16S rRNA haplotypes revealed that most of the collection sites had site-specific haplotype(s), except for BS, GJ, WD, SA, JJ, and TT. The most abundant haplotype was RA1, which was found in 186 (48.1%) out of the 16S rRNA sequences obtained from 425 L. japonica individuals.
Phylogenetic and population genetic analyses based on 16S rRNA
We constructed a nucleotide sequence alignment set with 34 16S rRNA haplotypes of L. japonica (Data S2), and identified 35 polymorphic sites (6.9%; Table S8) and 24 parsimoniously informative sites (4.7%). Phylogenetic analyses, including ML, BI, and NJ analyses, were conducted with the outgroup Acanthopleura echinata (Table S6). The resultant phylogenetic trees (Fig. S2) and unrooted phylogenetic network (Fig. 3a) consistently supported the three distinct genetic lineages of L. japonica, with the phylogenetic relationship between Lineages S1 and S2 being much closer than those inferred from the results of COI (Fig. 2a,b; Fig. S1). The TCS network (Fig. 3b) revealed that Lineages S1 and S2 were closely connected with only with 4–5 mutation steps between them, while Lineages N and S1 or Lineages N and S2 were remotely distanced by 18 mutation steps. Also, the overwhelming dominance of the RA1 haplotype implied a recent and rapid population expansion of Lineage N. In addition to RA1, haplotypes RB1 for Lineage S1 and RC1 and RC2 for Lineage S2 were dominant (Fig. 3b; Table S7). Consistent with this, in the PCoA plot (Fig. 3c), the three genetic groups of L. japonica were spatially separated. Lineage N was distantly located apart from Lineages S1 and S2, while Lineages S1 and S2 were spatially much closer.
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.
Examination of species discrimination of L. japonica based on COI and 16S rRNA
Using the Automatic Barcode Gap Discovery (ABGD), we performed distribution of pairwise genetic divergences, ranked pairwise difference, and automatic partition analyses based on COI and 16S rRNA of L. japonica, respectively (Fig. 4a–c), which confirmed that there were distinct barcoding gaps between intraspecific and interspecific variations, strongly supporting the possibility of species discrimination of L. japonica. the COI-based analysis yielded two different barcoding gaps, while the 16S rRNA-based analysis revealed only a single barcoding gap (Fig. 4a–c). The results of automatic partition at each value of the prior intraspecific divergence (P) divided L. japonica into three groups by COI and two groups by 16S rRNA, respectively (Fig. 4a–c). We also implemented two DNA taxonomy approaches to evaluate the possibility of species discrimination based on COI: the general mixed Yule coalescent (GMYC) approach (Fig. S3) and a Bayesian implementation of a Poisson Tree Processes model (bPTP) (Fig. S4). The results consistently and robustly supported the possibility that L. japonica can be divided into three different species, as shown in the results of ABDG (Fig. 4a–c).
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.
The molecular variance analyses using analysis of molecular variance (AMOVA), based on COI and 16S rRNA, were conducted to evaluate the degree of genetic differentiation among Lineages N, S1, and S2 (Tables S9, S10). According to the results, supposing that there are three genetic lineages (N, S1, and S2) or two genetic lineages (N and S1/S2), almost all variation in both cases is attributed to variation among groups (= among lineages), whereas variations within populations (within lineages) exhibit negative values in common. We confirmed that there was a high degree of genetic differentiation among Lineages N, S1, and S2, which supports the results of the COI barcoding gap analysis shown in Fig. 4a–c, although this was not statistically significant (P > 0.05; Table S9). When we assumed only two genetic groups, Lineages N and S1/S2, the genetic differentiation between the two groups was statistically significant (P < 0.001) in COI but not in 16S rRNA (P > 0.05) (Tables S9 and S10). The discrepancy between the number of barcoding gaps inferred from COI and 16S rRNA may have been affected by different gene evolutionary rates of the molecular markers11; nucleotide substitution rate of 16S rRNA is known to be generally slower than that of COI (which is especially fast in the third codon positions: 105 out of 127 polymorphic sites). When an ML tree was constructed based on 22 polymorphic sites, which are found only in the first and second codon positions of COI that are much more conserved than the third codon position, the three genetic lineages were retained in the resultant tree (Fig. S5), but Lineage S2 was nested within Lineage S1, as in the trees inferred from 16S rRNA (Fig. S2). Reflecting the powerful resolution of the COI barcoding marker well known from animals12 and the high degree of variation among the three genetic lineages (Fig. 4, Figs. S3, S4), we suggested that L. japonica could be categorized into three different species: L. koreana, Yeo and Hwang, sp. nov. for Lineage N, L. japonica for Lineage S1, and L. sinensis Choi, Park, and Hwang, sp. nov. for Lineage S2. To examine whether it is reasonable to give these a species-level taxonomic status, as shown in Fig. 4d, we reconstructed a COI-based NJ tree with one congeneric species L. tenuispinosa13, which was originally described as a subspecies-level taxon of L. japonica14,15 and was then revised as an independent species closely related to L. japonica by Saito & Yoshioka16 in 1993. The resultant tree (Fig. S6 and Data S3) showed that L. tenuispinosa forms a sister group with L. japonica (Lineage S1). This likely indicates that L. koreana and L. sinensis have taxonomic status as independent species.
Morphological comparison and geographical distribution of the three Liolophura species
We compared morphological characteristics among Liolophura koreana, sp. nov. (Lineage N), L. japonica (Lineage S1), and L. sinensis, sp. nov. (Lineage S2). Their morphological appearances are shown in Fig. 5a–c, which indicated that black spots on the tegmentum (Fig. 5d–e) and shapes of spicules on the perinotum (Figs. 5f–k, 6e–f) represent key morphological characteristics to distinguish them from each other. Although black dots in pleural areas, which are between the middle and lateral areas of the tegmentum on valves II–VII (or VIII), are commonly shared in all three lineages (Fig. 5a–c), other black spots on the valves exhibit a high degree of variation in morphology (Fig. 5a–c, Fig. S7). Herein, we described a new species of genus Liolophura, that is, L. koreana Yeo and Hwang from South Korea and Japan, with detailed descriptions of morphological characteristics observed by light microscopy (M205, Leica Camera AG, Germany) and FE-SEM (SU8220, Hitachi, Japan). In addition, we suggested the divergence of a new species, L. sinensis Choi, Park, and Hwang from southern China, with simple remarks based on distinct genetic difference (mainly COI barcoding gaps), with possible unique morphological characteristics as follows.
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).
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).
Liolophura koreana Yeo and Hwang, sp. nov. (Figs. 5, 6; Figs. S7, S8)
(urn:lsid:zoobank.org:act:4418355E-F55C-44FA-B4CE-585589FDCD23).
Type specimens examined
[Holotype] SOUTH KOREA: 1 specimen, Jeju-do, Seogwipo-si, Seongsan-eup, Seopjikoji, 3.XI.2020, UW Hwang, B Park & CR Shin (LEGOM040501); [Paratypes] SOUTH KOREA: 1 specimen, Gyeongsangbuk-do, Pohang-si, Guryongpo-eup, Janggil-ri, 27.VII.2008, UW Hwang (LEGOM040502); 3 specimens, Gyeongsangbuk-do, Ulleung-gun, Seo-myeon, Namyang-ri, Ulleungdo Island, Namtong tunnel, 12.VI.2007, UW Hwang (LEGOM040503–0505); 1 specimen, Gyeongsangbuk-do, Ulleung-gun, Namyang-ri, Ulleungdo Island, Namyang tunnel, 5.X.2007, UW Hwang (LEGOM040506); 2 specimens, Gyeongsangnam-do, Geoje-si, Nambu-myeon, Dapo-ri, 28.IV.2009, MY Yeo (LEGOM040507,0508); 4 specimens, same data as the holotype (LEGOM040509–0512); 2 specimens, Jeollanam-do, Yeosu-si, Hwajeong-myeon, Sado-ri, Sado Island, 8.IV.2008, MY Yeo (LEGOM040513,0514); 4 specimens, same data as the holotype (LEGOM040515–0518); JAPAN: 6 specimens, Tottori Prefecture, Hakuto, 24.V.2009, UW Hwang (LEGOM040519–0524); 1 specimen, Tottori Prefecture, Iwato, 25.V.2009, UW Hwang (LEGOM040525).
Description
Body small-sized and broad oval- to oval-shaped (Fig. 5a; Fig. S7); length 3.9 (1.9–12.3) mm and width 2.4 (1.2–7.1) mm. Tegmentum entirely brown (dark brown or black entirely, or each valve with black line anteriorly or white line laterally), with black dots on the pleural areas of valves II–VII (or VIII) (Fig. 5a,d, Fig. S7); articulamentum entirely black (dark brown); whitish and blackish spicules on the perinotum scattered irregularly, sometimes forming a band besides each valve (Fig. 5a, Fig. S7). Surface of the tegmentum in middle and lateral areas as in Fig. 6a,b and Fig. S8; posterior margin of the head valve nearly straight; dorsal shape of intermediate valves round-backed and side slopes slightly convex; the posterior valve margin with a distinct central apex, its shape subtriangular to triangular (rounded or linear), particularly valve II, mainly with a strong projection (Fig. 6a). Perinotum covered with large, solid, slightly curved, and obtusely pointed spicules (rarely with smooth and radial ribbed spicules apically), its density relatively lower than that of L. japonica (Figs. 5f,h,i, 6e).
Distribution
South Korea, Japan; avobe 33°24′ N (Seogwipo, JJ) in South Korea and TT and MY in Japan (Fig. 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).
Habitat
This new species appears to be attached to rocks in coastal areas with strong waves, or a calm inner shore in the northwestern Pacific Ocean (Fig. S9).
Etymology
The species is named per the locality of the new species.
Remarks
We found that Liolophura koreana, sp. nov. has no black spots on lateral areas of the tegmentum (Fig. 5d), and large, slightly curved, and obtusely pointed spicules on the perinotum compared to those of L. japonica (Figs. 5f,h,i, 6e). On the other hand, L. japonica from southern South Korea and southern Japan has two black spots on the lateral areas of valves II–VII (or VIII) (Fig. 5e), and small, almost straight, and cylindrical spicules compared to those of L. koreana (Figs. 5g,j,k, 6f).
As shown in Fig. 7, L. koreana (Lineage N) was observed in all the South Korean and Japanese populations examined here, except for the EH population in Japan (refer to Tables S1, S2), which were found from JJ at 33°24′ N to MY at 38°32′ N. On the other hand, L. japonica (Lineage S1) was found from the southern coastal areas of South Korea and Japan, which were found only between JJ at 33°24′ N and TT at 35°53′ N. Interestingly, we found only L. koreana north from the latitude of 35°10′ N (BS) such as UL/DD (37°24′ N) and PH (36°02′ N) in South Korea. In Japan, there was found only L. koreana at MY (38°32′ N) too, but it remains to be explored to clarify its distribution range in Japan with much more sample collections covering northern Japanese coastal areas through further study. It was also confirmed that L. koreana and L. japonica show a sympatric distribution pattern between JJ at 33°24′ N and TT at 35°53′ N on the southern coastal area of South Korea and Japan.
Liolophura sinensis Choi, Park, and Hwang, sp. nov. (Fig. 5c).
(urn:lsid:zoobank.org:act:72DF7E75-1853-4F23-AC12-3AB8CD054187).
Type specimens examined
[Holotype] CHINA: 1 specimen, Zhejiang Province, Dongtou Island, 27°49′57.44″ N, 121°10′19.13″ E, 2017; [Paratypes] CHINA: 1 specimen, Beiji Island, 27°37′08.82″ N, 121°11′47.82″ E, 2017; 1 specimen, Beilongshan Island, 27°40′08.56″ N, 121°58′51.56″ E, 2017; 1 specimen, Chaishi Island, 27°25′40.36″ N, 121°04′54.45″ E, 2017; 1 specimen, Daleishan Island, 27°29′39.48″ N, 121°05′24.50″ E, 2017; 1 specimen, Daqu Island, 27°47′29.92″ N, 121°05′23.97″ E, 2017; 1 specimen, Dazhushi Island, 27°49′12.87″ N, 121°12′48.74″ E, 2017; 1 specimen, Dongce Island, 27°45′32.04″ N, 121°09′01.38″ E, 2017; 1 specimen, Dongxingzai Island, 27°02′40.36″ N, 121°02′47.98″ E, 2017; 1 specimen, Houjishan Island, 27°28′26.96″ N, 121°07′40.71″ E, 2017; 1 specimen, Luxi Island, 27°59′33.43″ N, 121°12′50.70″ E, 2017; 1 specimen, Nanji Island, 27°27′30.57″ N, 121°03′06.28″ E, 2017; 1 specimen, Nanpanshan Island, 28°00′15.29″ N, 121°15′33.62″ E, 2017.
Distribution
Southern China; Zhejiang Province around ca. 27°02′–28°00′ N (Fig. 7).
Habitat
This new species appears to be attached to rocks in coastal areas of the northwestern Pacific Ocean.
Etymology
The species is named after its locality.
Remarks
L. sinensis, sp. nov. was examined and established mainly by molecular data such as the COI barcoding gap (Fig. 4a–c) presented in this study and photos provided from Prof. Yong-Pu Zhang (Wenzhou University, Zhejiang Province, China) without any direct real sample observation. The morphology of this new species is very similar to that of a previously known species, L. japonica. L. sinensis from southern China has black spots on the lateral areas of valves II–VII similar to L. japonica, but black bowtie-shaped spots anterior to valves II–VII (Fig. 5c) are a unique characteristic for L. sinensis. L. sinensis (Lineage S2) was found in Zhejiang Province (ZJ) in southern China, around ca. 27°02′–28°00′ N (Fig. 7; Tables S1, S5).
Demographic history and divergence time estimation analyses
Mismatch distribution analyses (MDA) based on COI were performed for L. koreana, L. japonica, and L. sinensis, respectively. The MDA results (Fig. 8a) showed a unimodal curve for each of the three lineages. In addition, when neutrality tests were performed with COI and 16S rRNA (Table S11), all three showed statistically significant negative values in both Tajima’s D and Fu’s Fs, except for the Tajima’s D values in COI (L. sinensis) and 16S rRNA (L. japonensis and L. sinensis), implying that these had experienced population expansions. Bayesian skyline plot (BSP) analyses with COI (Fig. 8b) were performed to examine the fluctuation patterns in effective population sizes for L. koreana, L. japonica, and L. sinensis, respectively. The effective population sizes of L. japonica and L. sinensis had gradually grown between ca. 100 Ka and ca. 50 Ka, while those of L. japonica had grown between ca. 80–50 Ka, L. sinensis had grown between 100–60 Ka, and that of L. koreana had begun to rapidly expand ca. 85 Ka and ceased ca. 75 Ka. This indicated that population expansion had occurred more dramatic in L. koreana than in L. japonica and L. sinensis, following the last interglacial age, called the Eemian (129–116 Ka). As shown in Fig. 8c and Fig. S10, according to the molecular clock analysis by the BEAST program, it was estimated that L. japonica and L. koreana shared their most recent common ancestor about 3.37 Ma, around the mid-Pliocene warm period (3.30–3.00 Ma), before the extensive glaciation in the late Pliocene (ca. 3.00 mya). L. japonica and L. sinensis likely diverged around 1.84 Ma, around the beginning stage of the Early Pleistocene Transition (EPT; 1.85–1.66 mya). The augmentation of haplotype diversity in L. japonica, L. sinensis, and L. koreana might have intensified in the interglacial stages during the late-middle (0.35–0.126 Ma) and late Pleistocene (0.126–0.012 Ma), before the last glacial maximum (LGM: 0.026–0.019 Ma).
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).
Source: Ecology - nature.com
