Genetic diversity and phylogeny of E. chinense based on COI
The partial fragment of COI, 595bp in length, was sequenced from 103 E. chinense individuals collected from the eight sites in South Korea (Table 1). These sequences were aligned together with 37 COI haplotype sequences retrieved from the NCBI GenBank. Hence, 140 COI haplotype sequences of E. chinense were analyzed, representing 18 collection sites at the nine populations in South Korea and Japan (Table 1). Based on the alignment set (no indels) of these 140 COI sequences (Data S1), we obtained a total of 58 COI haplotypes, of which 43 were singleton, appeared in only a single site (Table 2). The novel 58 COI haplotype sequences obtained were registered under the GenBank accession nos. MW265437−MW265477. According to the sequence alignment of the 58 COI haplotypes (Fig. S2; Data S2), there were 71 polymorphic sites and 31 parsimoniously informative sites (Fig. 1c), among which four adenine/guanine hotspots at 207, 282, 354, and 420 were ascertained to articulately divide the haplotypes of E. chinense into four meaningful phylogenetic groups: (a) A(207)-A(282)-A(354)-A(420), (b) A-A-G-A, (c) G-A-G-A, and (d) G-G-G-G.
Based on the COI haplotype sequence alignment (Fig. S2; Data S2), we reconstructed a ML tree using Ellobium aurisjudae as an outgroup. In the resultant tree topology (Fig. S3), it was confirmed that E. chinense appeared as a monophyletic group, but no distinction between the haplotypes from each geographical population was observed. To define detailed relationships among the COI haplotypes, the outgroup was removed and then an unrooted ML tree (Fig. 1d) was reconstructed. The resultant tree showed two distinctive phylogenetic groups, namely A-A-A-A and the other groups (including at least one G or more in the four positions), regardless of collection localities. The A-A-A-A group included 35 of the 58 COI haplotypes. The others could be divided into the A-A-G-A group (N=12: ECH11, 12, 15, 16, 18, 23, 27, 28, 32, 33, 36, and 49), the G-A-G-A group (N=1: ECH35), and the G-G-G-G group (N=8: ECH01, 07, 19, 29, 41, 45, 48, and 54).
As shown in Table S2 and Fig. S3, ECH01 was a dominant member of the G-G-G-G group with the most individuals (27), which appeared across all the South Korean populations examined here. As shown in Fig. 1d, the A-A-A-A group is likely to be an ancestral type because it was most frequently found in the other species within Ellobiidae (data not shown) and its haplotype diversity was the highest among the four genetic groups. Given that the G-G-G-G group exhibited much lower haplotype diversity than the A-A-A-A group, and was not observed in any other ellobiid species (data not shown), it is reasonable to suggest that the G-G-G-G group is a derived rather than an ancestral type. Thus, as shown in Fig. 1d, it is conceivable that unidirectional and stepwise A→G transition events from A-A-A-A to G-G-G-G may have been occurred in E. chinense. Within the A-A-A-A and A-A-G-A groups, parsimoniously informative A→G transition event was found at the sites 120 (ECH12, 15, 16, 18, 23, 38, 40, 44, and 49) and 183 (ECH3, 9, 10, 20, 43, 50, and 55), with a few exceptional cases, for example, G→A at the sites 216 (ECH12, 15, 16, 18, and 23) and 372 (ECH12, 15, and 23), and 429 (ECH37, 38, 46, and 47; ECH19 found in the G-G-G-G group).
As indicated in Table 2, the nucleotide diversity (π) is relatively low among the nine populations of E. chinense, ranging from 0.00749 (population BG) to 0.01042 (SC) with an average of 0.00865, whereas the haplotype diversity was very high across these populations, ranging from 0.924 (YG) to 1.000 (SC and JB) with an average of 0.939). All values of Tajima's D and Fu's FS were congruently negative, with averages of -1.87100 (P < 0.05) and -2.75886 (not statistically significant), respectively, indicating that the signature of demographic expansions recently occurred in E. chinense.
Population genetic structureof E. chinense based on COI
As shown in Table S3, the pairwise FST values between nine E. chinense populations were highly low and statistically non-significant, ranging from -0.21177 (JB and GY) to 0.09091 (JB and JK), indicating the lack of genetic structure. Analysis of molecular variance (AMOVA) for the nine populations of E. chinense in South Korea and Japan (Table S4) revealed that 101.05% of the variance was allocated into the level of individuals within each population, while differentiation between populations (-1.05%) did not appear to contribute to the overall variance.
Upon the results of TCS network analysis, all the COI haplotypes were connected by forming a single, large network, conforming that no distinct genetic structure existed among populations (Fig. S4a). The G-G-G-G group, with its representative member ECH01, was located rather at a distal position on the TCS network, which implies that these haplotypes may have recently emerged during the expansion of northwestern Pacific E. chinense populations. The phylogenetic network analysis (Fig. S4b) also indicated that a distinct genetic structure representing the geographical distributions of E. chinense did not exist. Indeed, all haplotypes were grouped into a single broad cluster with overlaps between populations, which is in agreement with the results of the TCS network analysis (Fig. S4a). In addition, the mismatch distribution analysis showed a clearly unimodal curve (Fig. S4c), evidently implying the metapopulation of E. chinense that may have experienced a recent demographic expansion12,13 or through a range expansion with high levels of migration between neighboring demes14,15.
The PCoA shown in Fig. 2a provided additional evidence that, regardless of the geographical distribution of Northwestern Pacific E. chinense, there was consistent support for the existence of four distinct phylogenetic groups, i.e., A-A-A-A, A-A-G-A, G-A-G-A, and G-G-G-G (as also shown in Fig. 1). In this analysis, the plausible ancestral A-A-A-A group (with the highest degree of haplotype diversity) was placed mainly around the bottom of the second quadrant, whereas the A-A-G-A group was in the left of the fourth quadrant, the G-A-G-A group (ECH35 only) in the right middle of the fourth quadrant, and the G-G-G-G group (represented by ECH01) was in the first quadrant. The three variation types within the A-A-A-A group, namely A-A-A-G, A-G-A-A, and A-G-A-G, appeared in the ML tree (Fig. 1d; Fig. S3) and were spatially separated from the remaining orthodox members of A-A-A-A, which were located along the Y axis between the first and second quadrants. Based on the results of PCoA (Fig. 2a) and the ML tree (Fig. 1a), we confidently suggest that the four phylogenetic groups of E. chinense arose as a result of serial, stepwise A→G transition events in the following order: A-A-A-A (ancestral) → A-A-Ğ-A → Ğ-A-Ğ-A → G-Ğ-G-Ğ (derived). The other local A→G transition events occurred within the A-A-A-A group (A-A-A-Ğ, A-Ğ-A-A, and A-Ğ-A-Ğ) (Fig. 1d; Fig. S3); these could be considered the fifth spatially separated phylogenetic group of E. chinense, based only on PCoA (Fig. 2a), being located between the phylogenetic groups G-G-G-G and A-A-A-A but apart from A-A-A-A.
Covariance of four COI hotspots linked to the conformation of four G-quadruplexes
Similar sequence interval lengths existed between the four adenine/guanine hotspots of COI characterized by unidirectional A→G transitional changes: G-(75 bp)-G-(72 bp)-G-(66 bp)-G. Additionally, the covariance analysis indicated that there was a high degree of covariance among these (see the four thick and bold linkage lines in Fig. 2b): out of 58 haplotypes, 44 were covariate between 207 and 282 sites, 44 between 207 and 420 sites, 41 between 282 and 420 sites, 37 between 207 and 354 sites, 32 between 282 and 354 sites, and 32 between 282 and 420 sites (Data S3 in detail). Given the similar intervals and covariance characteristics, we used QGRS-Conserve to search for sequence motifs that could possibly indicate the formation of G-quadruplex structures in relation to the four hotspots on the CDS of the COI barcoding region. Our results revealed possible sequences associated with the conformation of four different G-quadruplexes represented by each of the four hotspots (Fig. 2c). The first hotspot located at site 207 may be involved in forming a G-quadruplex structure such as G2-N6-G2-N15-G2-N11-ĞA; the second hotspot at site 282 may be involved in forming G2-N2-G2-N5-G2-N5-ĞT or G2-N2-G2-N4-Ğ2-N1-GA; the third hotspot at site 354 may be involved in forming G2-N1-G2-N4-G2-N18-CĞ; and the fourth hotspot, found at site 420, may be involved in the formation of TĞ-N8-G2-N10-G2-N21-CG.
G-quadruplex structures generally have 1-7 nucleotides between the four guanine-tracks each consisting of two or three guanines, e.g., G2-3-N1-7-G2-3-N1-7-G2-3-N1-7- G2-3; two (for G2) or three (for G3) G-quadruplexes can form a stacked structure. Considering the typical features of G-quadruplex structures, each of the putative motifs searched for G-quadruplex structure formation is likely to exhibit two G-quadruplexes stacked together (Figs 2c and d). However, the number of nucleotides between the G-tracks is much larger than usual for some sites, and some G tracks consist of only one G (not two or three). Thus, the G-quadruplex structures formed from the putative motifs are expected to be relatively weak. Assuming that all four putative motifs found in our analyses are capable of forming G-quadruplex structures, the four consecutive G-quadruplexes with intervals of 57/60, 39/41, and 65 bp in order might play a role in reducing or inhibiting transcription or translation of COI expression, i.e., they may be an exemplar of downregulation of gene expression; the ribosome stalling process (Fig. 2d).
Genetic diversity of E. chinense inferred from microsatellite markers
The 10 polymorphic microsatellite loci were amplified from 54 individuals collected from the four natural populations (BG, YG, HK, and HS) of E. chinense in South Korea (Table 1), and the PCR products were then sequenced and analyzed to elucidate the level of genetic diversity and the pattern of genetic structure of E. chinense. Fig. 3a shows the microsatellite diversity indices related to genetic diversity for the four E. chinense populations. The number of observed alleles per locus (NA) varied greatly among loci, from 14 (ECHm41) to 29 (ECHm36). The number of effective alleles per locus (NE) ranged from 5.377 (ECHm24) to 11.858 (ECHm40), with the mean of 9.495. Overall, the effective number of alleles (NE = 9.495) was smaller than the mean number of alleles (NA = 13.4). The expected mean heterozygosity (HE) per locus ranged from 0.793 (ECHm24) to 0.913 (ECHm40), while the average value was 0.883. The observed heterozygosity per locus (HO) was from 0.533 (ECHm24) to 0.969 (ECHm40), with a mean value of 0.806. Thus, the expected heterozygosity (HE = 0.883) was larger than the observed heterozygosity (HO = 0.806). The average of the genetic differentiation coefficient FST was estimated relatively low to be 0.038; it ranged from 0.020 (ECHm40) to 0.051 (ECHm27). Table S5 provides more detail on the genetic diversity estimates within each of the four populations (BG, YG, HK, and HS) of E. chinense.
Genetic structure of E. chinense based on microsatellite markers
According to Table S6, the estimated FST values, showing the degree of genetic differentiation among pairs of populations, were statistically significant (P < 0.05) only between the populations BG and HS (FST = 0.120) and HK and HS (FST = 0.0184). In contrast, pairwise RST was not significant among populations. Generally, pairwise genetic differentiation among populations was low, indicating that the E. chinense populations were not genetically differentiated from each other.
The distributions of molecular genetic variation among and within populations, as estimated by AMOVA, are shown in Table S7; only 0.85% of the total molecular variation was attributed to interpopulation differentiation, whereas 86.40% of the variation was within populations. This supports the conclusion that significant genetic differences did not exist among the four examined E. chinense populations.
Based on microsatellite markers, the 54 E. chinense individuals were further examined to determine population stratification. The optimal number of clusters, K, according to the method of Evanno et al. (2005)16, was 2 (Fig. 3b). However, with this method, it is only possible to infer with confidence about clusters ≥2. Indeed, the LnP (D) plot shows a strong drop off in model fit only after K=2 (data not shown); thus, the ancestry proportions were plotted for each individual given two clusters. All individuals were equally admixed (Fig. 3b) across the range, supporting a single genetic cluster. IBD analysis indicated that the pairwise observed genetic differences were not correlated with the pairwise geographical distances among the four examined E. chinense populations (Fig. 3c). Consistently, the PCoA results (Fig. 3d) showed that a distinct genetic structure did not exist and that all populations were clustered into a single genetic group. Taken together, the Bayesian clustering approach, IBD analysis, and PCoA provided consistent results that indicated a lack of spatial genetic clustering among the four E. chinense populations from South Korea and Japan. These results are coincident with the metapopulation of E. chinense through recent expansion, as inferred from COI haplotypes (Figs 1 and 2).