DNA sequences from plastid rbcL and matK genes and the trnL‐F region, as well as the nuclear ribosomal ITS region, were used to evaluate monophyly and subtribal delimitation of Cranichideae and generic relationships in Spiranthinae. Cranichideae are moderately supported as monophyletic, with Chloraeinae and Pterostylis‐Megastylis indicated as their collective sisters. Within Cranichideae, Pachyplectroninae and Goodyerinae form a well‐supported monophyletic group sister to a “core spiranthid” clade that includes, according to their branching order, Galeottiellinae, Manniellinae, and a Prescottiinae‐Cranichidinae‐Spiranthinae subclade. Inclusion of Galeottiella in Spiranthinae, as in previous classifications, renders the latter paraphyletic to all other spiranthid subtribes. Cranichidinae and Spiranthinae (minus Galeottiella) are monophyletic and strongly supported, but Prescottiinae form a grade that includes a strongly supported prescottioid Andean clade and a weakly supported Prescottia‐Cranichidinae clade sister to Spiranthinae. Well‐supported major clades in Spiranthinae identified in this study do not correspond to previous alliances or the narrowly defined subtribes in which they have been divided recently. Morphological characters, especially those that have been used for taxonomic delimitation in Cranichideae, are discussed against the framework of the molecular trees, emphasizing putative synapomorphies and problems derived from lack of information or inadequate interpretation of the characters.

Phylogenetic relationships of subtribes Cranichidinae and Prescottiinae, two diverse groups of neotropical terrestrial orchids, are not satisfactorily understood. A previous molecular phylogenetic study supported monophyly for Cranichidinae, but Prescottiinae consisted of two clades not sister to one another. However, that analysis included only 11 species and eight genera of these subtribes. Here, plastid and nuclear DNA sequences are analysed for an enlarged sample of genera and species of Cranichidinae and Prescottiinae with the aim of clarifying their relationships, evaluating the phylogenetic position of the monospecific genera Exalaria, Ocampoa and Pseudocranichis and examining the value of various structural traits as taxonomic markers. Approx. 6000 bp of nucleotide sequences from nuclear ribosomal (ITS) and plastid DNA (rbcL, matK-trnK and trnL-trnF) were analysed with cladistic parsimony and Bayesian inference for 45 species/14 genera of Cranichidinae and Prescottiinae (plus suitable outgroups). The utility of flower orientation, thickenings of velamen cell walls, hamular viscidium and pseudolabellum to mark clades recovered by the molecular analysis was assessed by tracing these characters on the molecular trees. Spiranthinae, Cranichidinae, paraphyletic Prescottia (with Pseudocranichis embedded), and a group of mainly Andean ‘prescottioid’ genera (the ‘Stenoptera clade’) were strongly supported. Relationships among these clades were unresolved by parsimony but the Bayesian tree provided moderately strong support for the resolution (Spiranthinae–(Stenoptera clade-(Prescottia/Pseudocranichis–Cranichidinae))). Three of the four structural characters mark clades on the molecular trees, but the possession of a pseudolabellum is variable in the polyphyletic Ponthieva. No evidence was found for monophyly of Prescottiinae and the reinstatement of Cranichidinae s.l. (including the genera of ‘Prescottiinae’) is favoured. Cranichidinae s.l. are diagnosed by non-resupinate flowers. Lack of support from parsimony for relationships among the major clades of core spiranthids is suggestive of a rapid morphological radiation or a slow rate of molecular evolution.