Morphological characters, sampling and DNA extraction
A. m. marina is widely distributed from eastern Africa, through the Middle East, South Asia, Southeast Asia and north to South China. It is also found in western Australia. A. m. eucalyptifolia is mainly distributed in northern Australia and extends to southern Philippines, western Indonesia and the Southwestern Pacific islands. There is a significant range overlap of the two groups in western Australia. The third putative subspecies A. m. australasica is restricted to south-eastern Australia and northern New Zealand (Figure 1). A. m. australasica can be morphologically distinguished from the other two groups by its fully pubescent calyx lobes and bracts (Duke, 2006). These structures are more glabrous in the other groups. The bark of A. m. australasica is grey fissured, with short longitudinal fissures or reticulate lines, while barks of the other two subspecies are smooth green or chalky white with flaky patches. A. m. eucalyptifolia is mainly distinguished by its lanceolate leaves (as opposed to ovate to elliptic), as well as the style in open flowers which are positioned level with upper edges of anthers (instead of the lower edges of anthers) (Duke, 2006). A. m. marina may also be distinguished by its larger flowers and thicker leaves. However, these distinctions in morphological characters may be inconclusive where two putative subspecies coexist (Duke, 2006).
We sampled 16 populations, 577 individuals (16 to 40 individuals per population) from East Africa, South China, Southeast Asia, Australia to New Zealand, covering A. marina ’s range (Table 1, Figure 1). Leaves of each individual were dried, labeled, and stored for DNA extraction. DNA content of each extraction was measured by NanoDrop 2000. We pooled equal amounts of DNA of individuals from the same population to make one DNA mixture. In total, 16 DNA mixtures were used in the following experiments.