Plant materials and sequencing
A total of 631 A. corniculatum individuals from 18 sites in the Indo-West Pacific region were collected (Figure 1a, Table 1). The sampled individuals were at least 5 m apart, and one leaf was collected from each individual. DNA of each individual was extracted using the cetyltrimethylammonium bromide (CTAB) method (Doyle & Doyle, 1987).
Referencing the transcriptome of an individual from Hainan, we newly designed 93 pairs of primers anchored at the nuclear genome of A. corniculatum , with each pair of primers anchoring at exons but spanning at least one intron. Six genes (A189, A245, A383, A414, A440, and C058) were amplified in 10 to 37 individuals from the 18 populations (423 in total) (Figure 1, Table 1). The amplicons were sequenced using the Sanger method on an ABI 3730 platform at Huada Genomic Institute (BGI) (Shenzhen). The PCR procedure for amplification was as follows: 4 min at 94°C; 30 cycles of 15 s at 94°C, 15 s of annealing at 53°C, and extension at 72°C for 2 min; and a final 10 min extension at 72°C. The reactions were held at 10 ℃ before the PCR products were subjected to electrophoresis on 1% agarose gels.
To corroborate the pattern uncovered by the six Sanger-sequenced genes, we sequenced all 93 genes against 11 populations (491 individuals in total) on an Illumina GA platform at Huada Genomic Institute (BGI) (Shenzhen). For each population, we pooled equal amounts of DNA from all the individuals of the population. For each of the 93 genes, we amplified the pooled DNA using the same procedure described above, and the purified PCR products of all 93 nuclear loci from one population were further pooled in equal quantities to reach a total of 10 μg for sequencing.