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Jodrell Laboratory, Royal Botanic Gardens Kew, Richmond, Surrey, TW9 3AB, UK School of Plant Sciences, University of Reading, Whiteknights, Reading, RG6 6AS, UK John Innes Centre, Norwich Research Park, Colney Lane, Norwich, NR4 7RH, UK Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK Radagatan 8, SE 531-52, Lidkoping, Sweden Department of Botany, University of Debrecen, Egyetem ter 1, Debrecen, H-4010, Hungary

Labellum micromorphology was imaged via scanning electron and light microscopy in 32 microspecies and one artificial hybrid of the European terrestrial orchid genus Ophrys, together representing all ten macrospecies circumscribed in the genus via molecular phylogenetics. Imaging of homologous regions of the adaxial surface, paying particular attention to the diagnostic feature of the comparatively reflective speculum, revealed the presence of between three and seven epidermal cell types on each labellum, the less complex labella being plesiomorphic. Epidermal protuberances range from short, domed papillae to long, twisted unicellular filaments. Multiple origins are inferred for pale labellar margins, large yellow appendices (both putative osmophores exuding pseudopheromones), broad flat labella and long lateral horns. Homoplasy in the speculum is manifested in unusually complex or simple outlines and the presence or absence of a pale margin or iridescence. The reflectivity of the speculum is caused by a combination of chemical and physical colour, whereas iridescence can be caused only by physical properties. The specula of most microspecies studied bear striated trichomes, albeit maturing comparatively late in ontogeny and being sufficiently narrow to allow light to reach the flat polygonal trichome bases. Reflectivity appears to be negatively correlated with the convexity and degree of cuticular corrugation shown by these epidermal cells. Two clades (the Speculum + Tenthredinifera + Bombyliflora group and Bertolonii subgroup of the Sphegodes group) have lost specular trichomes and include the most iridescent species; their flat, polygonal, nonstriated cells resemble those observed on the paired pseudoeyes that bracket the stigmas of all Ophrys except the Fusca group. The smooth thick-layered cuticle and dense layers of organelles and starch bodies revealed by preliminary transmission electron microscopy study provide alternative candidates for the primary reflective surface of the speculum; in contrast, the trichomes and conical cells that dominate Ophrys labella, and occur on the specula of all but the most reflective species, absorb and/or diffuse light. Multiple MYB family genes are hypothesized to control epidermal micromorphology. The relative contributions of olfactory, visual and tactile cues to the sophisticated pseudocopulatory pollination mechanism that characterizes Ophrys remain unclear, but the degree of reproductive isolation achieved, and thus the speciation rate, have certainly been greatly exaggerated by most observers. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162, 504–540.

Floral nectar spurs are widely considered to influence pollinator behaviour in orchids. Spurs of 21 orchid species selected from within four molecularly circumscribed clades of subtribe Orchidinae (based on Platanthera s.l., Gymnadenia-Dactylorhiza s.l., Anacamptis s.l., Orchis s.s.) were examined under light and scanning electron microscopes in order to estimate correlations between nectar production (categorized as absent, trace, reservoir), interior epidermal papillae (categorized as absent, short, medium, long) and epidermal cell striations (categorized as apparently absent, weak, moderate, strong). Closely related congeneric species scored similarly, but more divergent species showed less evidence of phylogenetic constraints. Nectar secretion was negatively correlated with striations and positively correlated with papillae, which were especially frequent and large in species producing substantial reservoirs of nectar. We speculate that the primary function of the papillae is conserving energy through nectar resorption and explain the presence of large papillae in a minority of deceit-pollinated species by arguing that the papillae improve pollination because they are a tactile expectation of pollinating insects. In contrast, the prominence of striations may be a 'spandrel', simply reflecting the thickness of the overlying cuticle. Developmentally, the spur is an invagination of the labellum; it is primarily vascularized by a single 'U'-shaped primary strand, with smaller strands present in some species. Several suggestions are made for developing further, more targeted research programmes. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 369-387 ADDITIONAL KEYWORDS: deceit - epidermal striations - nectar secretion - papillae - pollination - resorption - reward.

We report the first confirmed occurrence in Britain of Orchis × angusticruris Franch. ex Rouy, a hybrid between two closely related orchid species of anthropomorphic Orchis (O. purpurea Huds. × O. simia Lam.) that hybridize frequently in Continental Europe. Seven individual hybrids, most likely F1 plants representing a single interspecific pollination event, first flowered with both parents in May 2006 at a nature reserve in the Chiltern Hills near Goring, Oxfordshire. Univariate and multivariate morphometric analyses (43 characters plus 12 indices), internal transcribed spacer sequencing, plastid microsatellites and amplified fragment length polymorphism (AFLP) analyses together readily separate the parents and confirm that O. purpurea was the ovule parent and O. simia the pollen parent, presumably reflecting the greater frequency and/or later flowering period of the latter at the site. This study reinforces a more general observation that, in most orchids, the ovule parent contributes substantially more to the hybrid phenotype than does the pollen parent, perhaps reflecting cytoplasmic inheritance. In contrast, the hybrids are placed closer to O. simia than to O. purpurea in the AFLP tree. Apparently recent arrivals, the few O. purpurea plants at Goring contrast genetically with the two other small populations of this species known in the Chilterns, but rather are consistent with relatively uncommon Continental populations. This suggests that the plants may have been deliberately introduced at Goring by man, although transport from the Continent in high-level air currents cannot be ruled out. The Goring population of O. simia is likely to have become genetically impoverished through (1) preferential removal of many relatively fit plants to herbaria in the 19th century and/or (2) a catastrophic population crash in the first half of the 20th century. However, both our re-examination of herbarium specimens and our population genetic data indicate past hybridization among anthropomorphic Orchis species occurring naturally in the Chilterns. Thus, we tentatively recommend retention of the hybrid plants at Goring, despite their likely anthropogenic origin from Continental material and the partial viability of their pollen and seeds, which offers opportunities for future introgression. Although the Goring hybrids broadly resemble morphologically O. militaris, another anthropomorphic Orchis still found at two Chiltern localities, sufficient morphological and molecular differences were observed to strongly refute our initial hypothesis that O. militaris could have originated through hybridization between ancestors that resembled O. purpurea and O. simia. The comparatively complex genetic properties evident in both O. simia and O. purpurea merit further study. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157, 687–711.

Thoroughly sampled molecular phylogenies of the dominantly European orchid subtribe Orchidinae were used to identify a pair and a triplet of recently diverged species in which: (1) divergence involved substantial changes in floral morphology, particularly in the labellar lobes and spur; and (2) the polarity of those changes could be inferred phylogenetically. Floral ontogeny in the selected species was documented in detail through macromorphological, light microscopic, and scanning electron microscopic study of a wide range of ontogenetic stages. All study species showed differentiation of perianth segments earlier than the gynostemium. Unsurprisingly, component parts of the basic floral organs (gymnostemial auricles and rostellum, labellar lateral lobes, and spur) were initiated relatively late, the spur and ovary continuing to expand beyond anthesis. The predominant evolutionary pattern identified in the two case studies was paedomorphosis via progenesis (earlier offset of growth); this credibly explained the reduction in spur size and lateral lobing of the labellum in Gymnadenia odoratissima and, especially, G. austriaca relative to G. conopsea. Loss of resupination in G. austriaca was best viewed as the deletion of a formerly terminal ontogenetic stage. Radical reduction of the spur of Dactylorhiza viridis relative to D. fuchsii was also attributed to progenesis, although the long, narrow outline and relatively short central lobe of its labellum were attributed to increased growth of the lateral lobes (i.e. hypermorphosis resulting in peramorphosis). Microscopic study of epidermal cell types on the labellum and spur suggested a degree of decoupling of micromorphological from macromorphological transitions, although both were subject to heterochronic shifts. Each of the two case studies was consistent with, but not proof of, saltational macroevolution operating via functional changes in one or more key developmental genes. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157, 429–454.

Internal transcribed spacer (ITS nuclear rDNA) data have been obtained from 190 terrestrial orchid species, encompassing all genera and the great majority of the widely recognized species of Orchidinae, a heterogeneous selection of species of Habenariinae, and single species of Satyriinae and Disinae (the latter serving as outgroup). The resulting parsimony-based phylogeny reveals 12 well-resolved clades within the Orchidinae, based on Anacamptis s.l., Serapias, Ophrys, Steveniella–Himantoglossum s.l. (including ‘Comperia’ and ‘Barlia’, most species being 2n = 36), Neotinea s.l., Traunsteinera–Chamorchis, Orchis s.s., Pseudorchis–Amerorchis–Galearis–Neolindleya–Platanthera s.l. (most 2n = 42), Dactylorhiza s.l., Gymnadenia s.l. (most 2n = 40, 80), Ponerorchis s.l.–Hemipilia s.l.–Amitostigma–Neottianthe, and Brachycorythis (most 2n = 42). Relationships are less clearly resolved among these 12 clades, as are those within Habenariinae; the subtribe appears either weakly supported as monophyletic or as paraphyletic under maximum parsimony, and the species-rich genus Habenaria is clearly highly polyphyletic. The triphyly of Orchis as previously delimited is confirmed, and the improved sampling allows further generic transfers to Anacamptis s.l. and Neotinea s.l. In addition, justifications are given for: (1) establishing Steveniella as the basally divergent member of an appreciably expanded Himantoglossum that incorporates the former genera ‘Barlia’ and ‘Comperia’, (2) reuniting ‘Piperia’ with a broadly defined Platanthera as section Piperia, necessitating ten new combinations, (3) broadening Ponerorchis to include Chusua, and Hemipilia to include single ‘orphan’ species of Ponerorchis and Habenaria, and (4) recognizing ‘Gymnadenia’camtschatica as the monotypic Neolindleya camtschatica within the Pseudorchis∼Platanthera clade. Few further generic transfers are likely in Orchidinae s.s., but they are anticipated among habenariid genera, on acquisition of additional morphological and molecular evidence; one probable outcome is expansion of Herminium. Species-level relationships are also satisfactorily resolved within most of the major clades of Orchidinae, with the notable exceptions of Serapias, the derived sections of Ophrys, Himantoglossum s.s., some sections within Dactylorhiza, the former genus ‘Nigritella’ (now tentatively placed within Gymnadenia s.l.), Hemipilia s.l., and possibly Ponerorchis s.s. Relationships among the 12 major clades broadly accord with bona fide records of intergeneric hybridization. Current evidence supports the recently recognized 2n = 36 clade; it also indicates a 2n = 40 clade that is further diagnosed by digitate root-tubers, and is derived relative to the recently recognized clade of exclusively Asian genera (Ponerorchis s.l.–Hemipilia s.l.–Amitostigma–Neottianthe). This in turn appears derived relative to the Afro-Asiatic Brachycorythis group; together, these two clades identify the plesiomorphic chromosome number as 2n = 42. If the African genus Stenogolottis is correctly placed as basally divergent within a monophyletic Habenariinae, the tribe Orchideae and subtribes Orchidinae and Habenariinae could all have originated in Africa, though in contrast the Asiatic focus of the basally divergent members of most major clades of Orchidinae suggests an Asiatic radiation of the subtribe. Morphological characters informally ‘mapped’ across the molecular phylogeny and showing appreciable levels of homoplasy include floral and vegetative pigmentation, flower shape, leaf posture, gynostemium features, and various pollinator attractants. Qualitative comparison of, and reciprocal illumination between, degrees of sequence and morphological divergence suggests a nested set of radiations of progressively decreasing phenotypic magnitude. Brief scenarios, both adaptive and non-adaptive, are outlined for specific evolutionary transitions. Recommendations are made for further species sampling, concentrating on Asian Orchidinae (together with the Afro-Asiatic Brachycorythis group) and both Asian and Southern Hemisphere Habenariinae, and adding plastid sequence data. Taxonomic changes listed are: Anacamptis robusta (T.Stephenson) R.M.Bateman, comb. nov., A. fragrans (Pollini) R.M.Bateman, comb. nov., A. picta (Loiseleur) R.M.Bateman, comb. nov., Neotinea commutata (Todari) R.M.Bateman, comb. nov., N. conica (Willdenow) R.M.Bateman, comb. nov., Platanthera elegans Lindley ssp. maritima (Rydberg) R.M.Bateman, comb. nov., P. elegans Lindley ssp. decurtata (R.Morgan & Glicenstein) R.M.Bateman, comb. nov., P. elongata (Rydberg) R.M.Bateman, comb. nov., P. michaelii (Greene) R.M.Bateman, comb. nov., P. leptopetala (Rydberg) R.M.Bateman, comb. nov., P. transversa (Suksdorf) R.M.Bateman, comb. nov., P. cooperi (S.Watson) R.M.Bateman, comb. nov., P. colemanii (R.Morgan & Glicenstein) R.M.Bateman, comb. nov., P. candida (R.Morgan & Ackerman) R.M.Bateman, comb. nov. and P. yadonii (R.Morgan & Ackerman) R.M.Bateman, comb. nov. © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40.

BATEMAN, R. M. & FARRINGTON, O. S., 1989.Morphometric comparison of populations of Orchis sitnia Lam. (Orchidaceae) from Oxfordshire and Kent. Univariate and multivariate morphometric analyses of Orchis simia Lam. from its two remaining naturally-founded populations in Britain (Goring, Oxfordshire and Faversham, Kent) reveal only minor morphological differences between the populations and suprisingly low levels of intra-population variation. This probably reflects recent founding from Continental seed at Faversham and penecontemporaneous, human-induced, catastrophic contraction of the gene pool at Goring. The apparently restricted gene pools of these populations may adversely effect their long-term survival.