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The pantropical orchid genus Polystachya comprises approximately 230 species and shows variation in ploidy. For ongoing phylogenetic investigations in this genus, a knowledge of ploidy is necessary. We also looked for a correlation between genome size and guard cell length, so that we could estimate ploidy from preserved material and plants containing high levels of mucilage that are difficult to analyse by flow cytometry (FC). For estimates of ploidy, we measured genome size using FC. For investigations of guard cell lengths and densities, tangential cuts and nail varnish impressions were made. Diploid, tetraploid and hexaploid individuals with genome sizes ranging from 0.58 to 1.80 pg (1C-values) were found. Guard cell length ranged from 15 to 39 µm. The minimum guard cell length was greater in polyploids, but diploid species had a large range of guard cell lengths that encompassed the size range found in polyploids. Although previous studies have found that guard cell length can be used to estimate ploidy in cases in which the genome size range is large, this is not applicable to genera with a small genome size, such as Polystachya. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 223–233.

Here evidence for reticulation in the pantropical orchid genus Polystachya is presented, using gene trees from five nuclear and plastid DNA data sets, first among only diploid samples (homoploid hybridization) and then with the inclusion of cloned tetraploid sequences (allopolyploids). Two groups of tetraploids are compared with respect to their origins and phylogenetic relationships.Sequences from plastid regions, three low-copy nuclear genes and ITS nuclear ribosomal DNA were analysed for 56 diploid and 17 tetraploid accessions using maximum parsimony and Bayesian inference. Reticulation was inferred from incongruence between gene trees using supernetwork and consensus network analyses and from cloning and sequencing duplicated loci in tetraploids.Diploid trees from individual loci showed considerable incongruity but little reticulation signal when support from more than one gene tree was required to infer reticulation. This was coupled with generally low support in the individual gene trees. Sequencing the duplicated gene copies in tetraploids showed clearer evidence of hybrid evolution, including multiple origins of one group of tetraploids included in the study.A combination of cloning duplicate gene copies in allotetraploids and consensus network comparison of gene trees allowed a phylogenetic framework for reticulation in Polystachya to be built. There was little evidence for homoploid hybridization, but our knowledge of the origins and relationships of three groups of allotetraploids are greatly improved by this study. One group showed evidence of multiple long-distance dispersals to achieve a pantropical distribution; another showed no evidence of multiple origins or long-distance dispersal but had greater morphological variation, consistent with hybridization between more distantly related parents.

The pantropical orchid genus Polystachya is the subject of ongoing taxonomic work. We inferred phylogenetic relation¬ships in the genus using 5.3 kb of plastid DNA, for 83 out of ca. 240 species and 12 out of 15 sections, as well as five outgroup species. We also collected ploidy data using chromosome counts and genome size estimates. Bayesian and parsimony trees were congruent with each other and well resolved. Polystachya appears monophyletic based on current sampling, provided that the name P. neobenthamia is used instead of Neobenthamia gracilis for that species. The current sectional classification does not define monophyletic groups, but the present study can be used as the basis for a future sectional classification. Areas postulated as Pleistocene refugia for wet tropical forests in Africa also form centres of diversity for the genus. Biogeographical analyses using DIVA and Lagrange show an early radiation in eastern Africa, followed by separate radiations in eastern and western Africa. Subsequent dispersal from western to eastern Africa has occurred at a much higher rate than from east to west. Dispersal to the Neotropics occurred more than once, and one lineage has spread recently and rapidly throughout the tropics. Polyploidy has occurred several times during the diversification of the genus, most notably in association with the recent pantropical dispersal.