Among-subspecies genetic divergence
We obtained 76 to 87 kb of DNA sequence covering 88 to 94 genes (Table
1). By mapping short reads to reference sequences, we identified 74 to
1657 segregating sites within each population (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. The DXY matrix shows clear
divergence between the three subspecies, with the BB population the sole
exception (Figure 2b). The largest DXY values
were observed between the australasica populations and the other
two subspecies, ranging from 7.7 to 9.9/kb (Table S6). Lower divergence
was observed between eucalyptifolia and marinapopulations, with DXY values between 6.5 and
7.4/kb. By pooling populations within each subspecies, we estimated theDXY to be 8.2/kb between eucalyptifoliaand australasica , 6.7/kb between marina andeucalyptifolia and 9.1/kb between marina andaustralasica .
Genetic divergence was generally lower among populations than among
subspecies (Fig. 2d). The two australasica populations diverged
little from each other (DXY =2.2/kb). The pair ofeucalyptifolia populations diverged more but still less than
among subspecies (DXY = 5.48/kb). Withinmarina , we see two major geographical groups: one containing MC,
LS, and PN (west of the Malay Peninsula) and the other TN, BK, SS, SY,
WC, SB, CB, and BL (east of the Malay Peninsula, Figure S1).DXY per kb ranges from 1.27 to 3.75 within the
first and from 0.94 to 4.69 within the second geographical group.
Between the two geographical groups, DXY ranges
from 4.32 to 5.69, still lower than between subspecies. The BB
population is an outlier and has diverged far from other marinapopulations (DXY = 7.76-8.43/kb), to a level
among subspecies. The AMOVA indicates 65.1% of genetic divergences
(DXY ) is accounted by subspecies division. In
contrast, 50.8% of the DXY variance is accounted
by geographical division.
DXY provides a measurement of how far the
populations diverged from each other. We also measured the extent of
divergence by comparing the allele frequencies of polymorphisms within
populations (Cruickshank & Hahn, 2014). Plotting principal components
of the allele frequency matrix, populations of each subspecies generally
cluster together but diverged from other subspecies at PC2, except that
the DW population (eucalyptifolia ) is close to marinapopulations and the BB population (marina ) is again different
from all the other marina populations (Figure 2c). In PC1, only
population DW diverges largely from all other populations. In addition,
the CA population (eucalyptifolia ) diverges from other
populations largely in PC3 and PC4 (Figure S2).
The FST statistic quantifies these genetic
differences. The 120 values of pairwise FSTestimates 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). Populations from the South China Sea, i.e. TN, BK, SS,
SY, and WC (Figure S1), have relatively low pairwise differentiation.FST between the two populations on the west coast
of Malay Peninsula (LS and TN) are also low (Figure S1).
The Mantel tests show a significant relationship (P value
<0.01) between genetic differentiation and geographic
distance. This is regardless of whether the geographic distance was
estimated using the spherical or coastline method (Figure S3, see
Methods for details). All four tests have P values less than 0.01and
survive a multiple-test correction. This correlation indicates that
geographical distance contributes, at least partly, to the high level of
genetic differentiation among A. marina populations. However, the
two geographical groups around the Malay Peninsula show genetic
differentiation greater than what we would expect from the distance
separating them, indicating that other factors are also important
(Figure S3).
The BARRIER analysis reveals that major barriers (with
>80% bootstrap support) roughly lie along the Sunda shelf
and between Australasia and Southeast Asia. Minor barriers are also
identified between Africa and Southeast Asia, as well as between Western
Australia and Northern Australia. The major barrier in the historic
Sunda Land corresponds to the obvious deviation ofFST values from the expectation based on distance
alone (Figure S3 & S4).
Isolation among subspecies indicated by high divergence and inferred
barriers may influence genetic diversity within populations. Both the
nucleotide diversity (π) and Watterson’s estimator of nucleotide
polymorphism (θ) show different levels of within-population genetic
variation. The two eucalyptifolia populations have the highest
genetic diversity, on average θ (across segments) = 2.82 and 3.94/kb and
π = 3.41 and 4.06/kb (Figure 3). In contrast, marina populations
are low in genetic diversity, with average θ ranging from 0.21 to
0.91/kb and π from 0.15 to 1.39/kb (Table1, Figure 3). The BS population
(australasica ) has intermediate diversity, while the AK
population (australasica ) is unusually monomorphic (Table1,
Figure 3). The very low diversity of the AK population is likely due to
its marginal location, similar to WC and SY.