Among-group genetic divergence
We obtained 76 to 87 kb of DNA sequence covering 88 to 94 genes from each population (Table 1). By mapping short reads to reference sequences, we identified 74 to 1657 segregating sites (Table 1). We calculated among-population pairwise DXY values to assess genetic divergence and used the resulting distance matrix to construct a neighbor-joining tree (Supplementary Figure 1). TheDXY matrix showed clear divergence between the three varietal groups previously designated as subspecies (Figure 2b), the maBB is an outlier discussed below. The largestDXY values were observed between the A. m. australasica populations and the other two subspecies, ranging from 7.7 to 9.9/kb (Supplementary Table 4). Relatively lower divergence was observed between A. m. eucalyptifolia and A. m.marina populations, with DXY values between 6.5 and 7.4/kb. By pooling populations within putative subspecies, we estimated the DXY to be 8.2/kb between A. m. eucalyptifolia and A. m. australasica , 6.7/kb between A. m. marina and A. m. eucalyptifolia and 9.1/kb between A. m. marina and A. m. australasica .
Genetic divergences were generally lower among populations than among subspecies. The two A. m. australasica populations diverged little from each other (DXY =2.2/kb), while theA. m. eucalyptifolia pair diverged more but still less than differentiation among subspecies (DXY = 5.48/kb). Within A. m. marina , we see two major groups, one containing maMC, maLS, and maPN (west of the Malay Peninsula), the other maTN, maBK, maSS, maSY, maWC, maSB, maCB, and maBL (east of the Malay Peninsula, Supplementary Figure 2). DXY per kb ranges from 1.27 to 3.75 within the first and from 0.94 to 4.69 within the second group. Between the two groups, DXYranges from 4.32 to 5.69, still lower than between subspecies. The maBB population is an outlier and has diverged far from other A. m. marina populations (DXY = 7.76-8.43/kb), to a level comparable with among subspecies differentiation.
Genetic divergences can also be observed by comparing the polymorphism frequencies within populations. By plotting principal components of the allele frequency matrix, we again see clear differences among the three subspecies. The two A. m. eucalyptifolia populations also show obviously different polymorphism frequency patterns (Figure 2a).DXY quantifies the absolute divergence between groups, while FST reflects how much genetic divergence is fixed in each subspecies (Cruickshank & Hahn, 2014). The 120 values of pairwise FST calculated for the 16 populations are generally high, with the average value of 0.61 (first and third quartiles are 0.50 and 0.76 respectively).FST values among five A. m. marinapopulations from the South China Sea, i.e. maTN, maBK, maSS, maSY and maWC cluster at the bottom of the distribution. If we exclude the ten values or pairs of these five populations, the average of remaining 110 pairs is elevated to 0.66 (first and third quartiles 0.54 and 0.77). Notably, the FST values among populations fromA. m eucalyptifolia and A. m. australasica are 0.19 and 0.42 respectively. The prevalence of such highFST value indicates that genetic polymorphisms are fixed to a large extent between subspecies as well as between some populations within A. m. marina .
Both the nucleotide diversity (π) and Watterson’s estimator of nucleotide polymorphism (θ) identified different levels of within-population genetic variation. The two A. m.eucalyptifolia populations have the highest genetic diversity, with average θ (across segments) = 2.82 and 3.94/kb and average π = 3.41 and 4.06/kb (Figure 3a). In contrast, A. m. marina populations are low in genetic diversity, with average θ ranging from 0.21 to 0.91/kb and average π ranging from 0.15 to 1.39/kb (Table1, Figure 3a). The two A. m. australasica populations have very different levels of genetic diversity (Table1, Figure 3a). The very low level of diversity in the auAK population is likely due to its marginal location. Marginal populations of A. m. marina such as maWC and maSY are also very monomorphic. The auBS population of A. m. australasicahas an intermediate level of genetic diversity, higher than A. m. marina but lower than A. m. eucalyptifolia .