Phylogeny and classification of the East Asian Amitostigma alliance (Orchidaceae: Orchideae) based on six DNA markers

Ying Tang, Tomohisa Yukawa, Richard M Bateman, Hong Jiang, Hua Peng

Tribe Orchideae dominates the orchid flora of the temperate Northern Hemisphere but its representatives in East Asia had been subject to less intensive phylogenetic study than those in Eurasia and North America. Although this situation was improved recently by the molecular phylogenetic study of Jin et al., comparatively few species were analyzed from the species-rich and taxonomically controversial East Asian Amitostigma alliance. Here, we present a framework nrITS tree of 235 accessions of Orchideae plus an in-depth analysis of 110 representative accessions, encompassing most widely recognized species within the alliance, to elucidate their relationships. We used parsimony, likelihood and Bayesian approaches to generate trees from data for two nuclear (rITS, low-copy Xdh) and four chloroplast (matK, psbA-trnH, trnL-F, trnS-trnG) markers. Nuclear and plastid data were analyzed separately due to a few hard incongruences that most likely reflect chloroplast capture. Our results suggest key phylogenetic placements for Sirindhornia and Brachycorythis, and confirm previous assertions that the Amitostigma alliance is monophyletic and sister to the Eurasian plus European clades of subtribe Orchidinae. Seven robust clades are evident within the alliance, but none corresponds precisely with any of the traditional genera; the smaller and more morphologically distinct genera Tsaiorchis, Hemipilia, Neottianthe and Hemipiliopsis are monophyletic but each is nested within a polyphyletic plexus of species attributed to either Ponerorchis or the most plesiomorphic genus, Amitostigma. Two early-divergent clades that escaped analysis by Jin et al. undermine their attempt to circumscribe an expanded monophyletic genus Ponerorchis. We provide a new framework on the complex phylogenetic relationships between Amitostigma and other genera traditionally included in its alliance; based on which, we combine the entire Amitostigma alliance into a morphologically and molecularly circumscribed Amitostigma sensu latissimo that also contains seven molecularly circumscribed sections. Our molecular trees imply unusually high levels of morphological homoplasy, but these will need to be quantified via a future group-wide review of the alliance based on living plants if morphology is to be fully integrated into our classification.