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The closely related species of the genus Ficus with continuous variation have been confusing taxonomists, co-evolutionary researchers, and other related scientists. The boundary between species in the complex of F. auriculata, F. oligodon, F. hainanensis, F. beipeiensis, and F. variegata is still unclear. To clarify this problem, the nuclear loci ITS and G3pdh; chloroplast loci trnH-psbA, trnL-trnF, trnS-trnG, and psbK-psbI; and 15 pairs of SSR markers were used to reconstruct the phylogenetic relationship and clarify the species boundaries. The results of the present study indicated that F. variegata should be an independent species in Ficus sect. Sycomorus subsect. Neomorphe, which together with F. auriculata, F. oligodon, F. hainanensis, and F. beipeiensis compose a monophyletic group. The last four species of this complex are with small genetic distances, shared haplotypes, and overlapped geographic distribution, and should be treated as a single species.

Environmental conditions in coastal salt marsh habitats have led to the development of specialist genetic adaptations. We evaluated six DNA barcode loci of the 53 species of Poaceae and 15 species of Chenopodiaceae from China's coastal salt marsh area and inland area. Our results indicate that the optimum DNA barcode was ITS for coastal salt-tolerant Poaceae and matK for the Chenopodiaceae. Sampling strategies for ten common species of Poaceae and Chenopodiaceae were analyzed according to optimum barcode. We found that by increasing the number of samples collected from the coastal salt marsh area on the basis of inland samples, the number of haplotypes of Arundinella hirta, Digitaria ciliaris, Eleusine indica, Imperata cylindrica, Setaria viridis, and Chenopodium glaucum increased, with a principal coordinate plot clearly showing increased distribution points. The results of a Mann-Whitney test showed that for Digitaria ciliaris, Eleusine indica, Imperata cylindrica, and Setaria viridis, the distribution of intraspecific genetic distances was significantly different when samples from the coastal salt marsh area were included (P < 0.01). These results suggest that increasing the sample size in specialist habitats can improve measurements of intraspecific genetic diversity, and will have a positive effect on the application of the DNA barcodes in widely distributed species. The results of random sampling showed that when sample size reached 11 for Chloris virgata, Chenopodium glaucum, and Dysphania ambrosioides, 13 for Setaria viridis, and 15 for Eleusine indica, Imperata cylindrica and Chenopodium album, average intraspecific distance tended to reach stability. These results indicate that the sample size for DNA barcode of globally distributed species should be increased to 11–15.