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Evaluation of insertion/deletion (InDel) regions by comparative chloroplast DNA (cpDNA) analysis of two endemic moth orchids, Phalaenopsis aphrodite and P. equestris, identified 15 highly variable cpDNA regions that could be served as useful markers for interspecific differentiation of moth orchids. In this study, we evaluated 31 cpDNA regions carrying simple sequence repeats (SSRs) or InDels from two moth orchids to reveal intraspecific markers for distinguishing the cultivars of P. equestris. In total, 18 of the cpDNA markers were variable among cultivars of P. equestris; 9 cpDNA markers were highly variable, with polymorphic information content ≥5.5. The petA-psbJ, psbB-psbT and rps16-trnQ markers showed the best discriminatory power; 11 P. equestris cultivars could be separated into at least five groups. From the sequences for six selected cpDNA regions, the rps16-trnQ and psbB-psbT intergenic spacers were the best DNA barcodes; 11 P. equestris cultivars could be separated into six distinct groups. Phylogenetic analysis revealed that P. equestris cultivars have evolved into two major lineages, and the species in the Stauroglottis and Amboinenses sections are in a non-monophyletic relationship. We have revealed a set of cpDNA markers that could be used for intraspecific identification and phylogenetic study of P. equestris cultivars.

Comparative analysis of chloroplast DNA (cpDNA) between two endemic moth orchids in Taiwan, Phalaenopsis aphrodite subsp. formosana and P. equestris, previously revealed many evolutionary hotspots. In this study, we explored 27 pre-screened cpDNA regions to evaluate molecular markers for distinguishing Phalaenopsis species. Fifteen cpDNA markers were highly variable among moth orchids, with polymorphic information contents ≥8.0. The rps16-trnQ marker showed the best discrimatory power which 15 endemic moth orchids could be successfully separated into 13 groups. From the sequences of the seven selected cpDNA regions, the rps16-trnQ intergenic spacer was the best DNA barcode which 19 endemic moth orchids could be completely distinguished. The petN-psbM intergenic spacer could be better used as a DNA barcode to differentiate P. aphrodite subsp. formosana and P. amabilis. Additionally, the inheritance mode of cpDNA in moth orchids was maternally predominated. Phylogenetic analysis revealed non-monophyletic relationships below the Phalaenopsis genus level. In summary, we have revealed a set of cpDNA markers that could be used for identification and phylogenetic study of Phalaenopsis orchids.