SummaryBetween 1959 and 1988, three populations of purple-flowered terrestrial orchids attributable to Dactylorhiza subgenus Dactylorhiza were discovered in Canada. The populations at Timmins, Ontario, and St John's, Newfoundland were strongly marked on both flowers and leaves, in contrast with the anthocyanin-deficient population at Tilt Cove, Newfoundland. All three populations have since experienced a wide range of taxonomic assignments; debates are also ongoing regarding their origin and most appropriate conservation status. Here, we address these questions by combining detailed in situ morphometric analyses based on 52 characters with allozyme profiles and data from nrITS, 15 plastid microsatellites and seven nuclear microsatellites. The allozyme data alone are sufficient to both confirm allopolyploidy and categorically refute past assignments of these populations to D. incarnata, D. maculata, D. fuchsii, D. majalis or D. purpurella. Several morphometric characters, nuclear microsatellites and nrITS all reliably distinguish each of the three study populations, whereas the two sampled subpopulations from St John's proved near-identical morphologically. In contrast, morphological variation within each of the three populations is strikingly low, particularly in characters other than those influenced by plant vigour. Similarly, compared with 14 European populations, the three Canadian populations proved genetically impoverished (two were near-invariant) and likely experienced recent, extreme genetic bottlenecks during establishment. The three populations differ substantially, both morphologically and molecularly, therefore probably representing independent immigration events. Although clearly attributable to D. praetermissa, all three populations deviate significantly in morphology and DNA data from comparable populations sampled across Europe, preventing identification of their precise geographic origins. Any attempt to determine their mode or origin — through natural long-distance transport, or accidental or deliberate introduction by humans – is challenged to explain why three lineages of a single European Marsh-orchid species, each in different ways atypical of that species, arrived independently in North America whereas no other European dactylorchid species has become established there.

Pollinators represent one of the main agents of selection on floral traits. Here, we estimated phenotypic selection on floral morphology and phenology in a sympatric population of two orchid species, Platanthera bifolia and P. chlorantha, including enigmatic individuals with intermediate column morphology (as reflected by the distance between viscidia and caudicle length, two traits involved in assortative mating and reproductive isolation among Platanthera species), but genetically indistinguishable from P. bifolia. Our aim was to clarify whether the occurrence of intermediate phenotypes could be explained by the presence of selective pressures exerted by pollinators. Simple linear and quadratic regressions together with univariate and multivariate analyses were used to evaluate the strength of directional, disruptive and stabilizing selection. We found that selection on phenotypic traits varied between groups and sex functions. Contrary to our hypothesis, selection on the viscidia distance and caudicle length appeared to be consistent in the two P. bifolia groups. Interestingly, the viscidia distance was under significant stabilizing selection through female reproductive success in intermediate individuals. Based on these results, we conclude that, despite a significant selective pressure on some phenotypic traits, the presence of individuals with intermediate phenotype is not due to selection. Stabilizing selection on distance between viscidia in intermediate individuals may suggest that assortative mating play a role in the maintenance of this phenotypic polymorphism.

Plant density varies naturally, from isolated plants to clumped individuals, and this can influence pollinator foraging behaviour and plant reproductive success. In addition, the effect of conspecific density on reproduction may depend on the pollination system, and deceptive species differ from rewarding ones in this regard, a high density being often associated with low fruit set in deceptive plants. In our study, we aimed to determine how local conspecific density and floral display size (i.e. number of flowers per plant) affect fruit set in a deceptive orchid ( Orchis militaris ) through changes in pollinator visitation. We measured fruit set in a natural population and recorded pollinator abundance and foraging behaviour within plots of different O. militaris densities. Detailed data were recorded for the most abundant potential pollinators of O. militaris , i.e. solitary bees. Floral display size was negatively correlated to fruit set in medium‐density plots, but uncorrelated in low‐ and high‐density plots. Plot density had no effect on solitary bee abundance and visitation, which may be due to low pollinator abundance within the study site. The proportion of visited flowers per inflorescence was negatively influenced by floral display size, which is in line with previous studies. In addition, solitary bees spent decreasing time in successive flowers within an inflorescence, and the time spent per flower was negatively affected by ambient temperature. Our results suggest that pollinator behaviour during visitation is poorly linked to pollen deposition and reproductive success in O. militaris .

Platanthera bifolia and P. chlorantha are terrestrial and rewarding orchids with a wide Eurasian distribution. Although genetically closely related, they exhibit significant morphological, phenological and ecological differences that maintain reproductive isolation between the species. However, where both species co-occur, individuals with intermediate phenotypic traits, often considered as hybrids, are frequently observed. Here, we combined neutral genetic markers (AFLPs), morphometrics and floral scent analysis (GC-MS) to investigate two mixed Platanthera populations where morphologically intermediate plants were found. Self-pollination experiments revealed a low level of autogamy and artificial crossings combined with assessments of fruit set and seed viability, showed compatibility between the two species. The results of the genetic analyses showed that morphologically intermediate plants had similar genetic patterns as the P. bifolia group. These results are corroborated also by floral scent analyses, which confirmed a strong similarity in floral scent composition between intermediate morphotypes and P. bifolia. Therefore, this study provided a much more detailed picture of the genetic structure of a sympatric zone between two closely allied species and supports the hypothesis that intermediate morphotypes in sympatry could reflect an adaptive evolution in response to local pollinator-mediated selection.

Background and aims - About one third of orchid species do not produce any floral reward but have developed various strategies to attract pollinators. The most common system is food deception, which takes place through two types of attraction mechanisms: Batesian floral mimicry and generalised food deception. In rewardless species, fruit set is generally low and various factors, such as floral display size or density, are known to positively or negatively influence fruit set. This study aimed to clarify the deceptive pollination system in Orchis militaris and to investigate the effect of individual, population and environmental factors on fruit set in this orchid species. Methods - The effects of floral display size, con-and heterospecific density and rewarding species diversity were studied both at the scale of the individual plant (fourteen sites) and of the population (22 sites). Generalised linear (mixed) models and multivariate analysis were performed to examine which factors affect fruit set. Key Results - At the individual scale, the probability of setting fruit was influenced by several factors and their interaction, including floral display size, conspecific density, and heterospecific total density and richness. Fruit set was higher in medium-height vegetation than in short vegetation. Globularia bisnagarica was the only species whose density was significantly (and negatively) correlated with fruit set. At the population scale, none of the studied factors was significantly correlated to fruit set. Conclusions - In Orchis militaris, generalised food deception is the most likely strategy of pollinator attraction. This orchid species may benefit from the proximity of rewarding plants through the magnet-species effect, depending on the size of its floral display. The effect of con-and heterospecific density and rewarding species richness seem scale-dependent.

Zoophilous flowering plants communicate with pollinators to ensure pollen transfer. Pin-pointing which species are effective pollinators is not only essential to better understand plant-pollinator networks, but equally so to better understand the potential of hybridization in plant systems, such as in orchids. As a case study, we studied two sympatric populations of the congeneric orchids Platanthera bifolia and P. chlorantha in order to assess their nocturnal pollinators by checking which moth species carried pollinaria, and of which orchid species. Moths carrying Platanthera pollinaria were photographed and identified. The carried pollinaria were identified and counted, and their attachment position on the moth’s head was scored. Based on these observations we show that three species of noctuid moths visited the Platanthera inflorescences. Although Noctua pronuba visited P. chlorantha, only Cucullia umbratica and Autographa gamma turned out to be potential pollinators for both orchid species. As such, we here demonstrate that the latter two noctuids have high potential to facilitate hybridization among these two orchid species, especially so in sympatric populations.

As part of a research project on the food deception strategy in Orchis militaris (L.), the objective of this study was to identify insect visitors and potential pollinators of this orchid species in Belgium. In 2013, insects were collected over 2 d per site in five localities distributed in Southern Belgium (Wallonia). A total of 104 insects belonging to 49 species were caught. Dipterans were the most abundant visitors (50% of total specimens), followed by Hymenopterans (32%). Rhingia campestris Meigen, Bombylius venosus Mikan, Apis mellifera (L.), and Bombus lapidarius (L.) were the most abundant species. Only five specimens bore one to more than 10 pollinia: four honeybees (A. mellifera) and one bumblebee worker (B. lapidarius). These two species should be considered as potential pollinators in the study area, but probably not confirmed ones.

While it is generally acknowledged that orchid species rely on mycorrhizal fungi for completion of their life cycle, little is yet known about how mycorrhizal fungal diversity and community composition vary within and between closely related orchid taxa. In this study, we used 454 amplicon pyrosequencing to investigate variation in mycorrhizal communities between pure (allopatric) and mixed (sympatric) populations of two closely related Platanthera species (Platanthera bifolia and P. chlorantha) and putative hybrids. Consistent with previous research, the two species primarily associated primarily with members of the Ceratobasidiaceae and, to a lesser extent, with members of the Sebacinales and Tulasnellaceae. In addition, a large number of ectomycorrhizal fungi belonging to various families were observed. Although a considerable number of mycorrhizal fungi were common to both species, the fungal communities were significantly different between the two species. Individuals with intermediate morphology showed communities similar to P. bifolia, confirming previous results based on the genetic architecture and fragrance composition that putative hybrids essentially belonged to one of the parental species (P. bifolia). Differences in mycorrhizal communities between species were smaller in mixed populations than between pure populations, suggesting that variation in mycorrhizal communities was largely controlled by local environmental conditions. The small differences in mycorrhizal communities in mixed populations suggests that mycorrhizal fungi are most likely not directly involved in maintaining species boundaries between the two Platanthera species. However, seed germination experiments are needed to unambiguously assess the contribution of mycorrhizal divergence to reproductive isolation.

Orchid species are critically dependent on mycorrhizal fungi for completion of their life cycle, particularly during the early stages of their development when nutritional resources are scarce. As such, orchid mycorrhizal fungi play an important role in the population dynamics, abundance, and spatial distribution of orchid species. However, less is known about the ecology and distribution of orchid mycorrhizal fungi. In this study, we used 454 amplicon pyrosequencing to investigate ecological and geographic variation in mycorrhizal associations in fourteen species of the orchid genus Dactylorhiza. More specifically, we tested the hypothesis that variation in orchid mycorrhizal communities resulted primarily from differences in habitat conditions where the species were growing. The results showed that all investigated Dactylorhiza species associated with a large number of fungal OTUs, the majority belonging to the Tulasnellaceae, Ceratobasidiaceae and Sebacinales. Mycorrhizal specificity was low, but significant variation in mycorrhizal community composition was observed between species inhabiting different ecological habitats. Although several fungi had a broad geographic distribution, Species Indicator Analysis revealed some fungi that were characteristic for specific habitats. Overall, these results indicate that orchid mycorrhizal fungi may have a broad geographic distribution, but that their occurrence is bounded by specific habitat conditions.

Plusieurs arguments montrent que les sous-genres Orchis et Masculae sont nettement separes. Dans cet article, tout en discutant notamment les arguments recemment proposes par Bateman (2012), nous passons d’abord en revue des etudes recentes dont les resultats vont dans ce sens. Ceux-ci incluent l’isolation reproductive, telle qu’exprimee par des barrieres prezygotiques et postzygotiques, la phylogenie moleculaire, la micromorphologie des graines, ainsi que l’inexistence d’hybrides produits naturellement ou artificiellement entre les deux sous-genres. L’article presente egalement les resultats de recherches complementaires sur la morphometrie. Des analyses statistiques multivariees montrent qu’il existe davantage de similarite entre le sous-genre Masculae et le genre Anacamptis (ce dernier represente par le groupe d’A. morio) qu’entre chacun d’eux et le sous-genre Orchis. Pour completer la discussion, nous comparons la paire de (sous-) genres Orchis / Androrchis a deux autres paires analogues, Anacamptis / Serapias et Dactylorhiza / Gymnadenia, sur les plans de la compatibilite genetique, la biologie de la pollinisation, l’ecologie et la phenologie, et concluons que pour des raisons de simple coherence, Orchis et Androrchis meritent d’etre vus comme genres distincts, comme l’ont deja fait plusieurs chercheurs.

Floral nectar of animal-pollinated plants is commonly infested with microorganisms, yet little is known about the microorganisms inhabiting the floral nectar of orchids. In this study, we investigated microbial communities occurring in the floral nectar of seven Epipactis (Orchidaceae) species. Culturable bacteria and yeasts were isolated and identified by partially sequencing the small subunit (SSU) ribosomal RNA (rRNA) gene and the D1/D2 domains of the large subunit (LSU) rRNA gene, respectively. Using three different culture media, we found that bacteria were common inhabitants of the floral nectar of Epipactis. The most widely distributed bacterial operational taxonomic units (OTUs) in nectar of Epipactis were representatives of the family of Enterobacteriaceae, with an unspecified Enterobacteriaceae bacterium as the most common. In contrast to previous studies investigating microbial communities in floral nectar, very few yeast species (mainly of the genus Cryptococcus) were observed, and most of them occurred in very low densities. Total OTU richness (i.e., the number of bacterial and yeast OTUs per orchid species) varied between 4 and 20. Cluster analysis revealed that microbial communities of allogamous species differed from those of autogamous and facultatively autogamous species. This study extends previous efforts to identify microbial communities in floral nectar and indicates that the floral nectar of the orchids investigated mainly contained bacterial communities with moderate phylogenetic diversity.

Populations of two critical Ophrys taxa from the O. fusca and O. omegaifera groups were studied and examined using multivariate morphometric analyses. They are described as new species. Ophrys pintoi looks like a miniature O. fusca and grows, sometimes in extensive populations, in central Portugal. Ophrys lenae is probably a paleohybrid originating from O. fusca and O. dyris; it grows in a restricted area in central Portugal

Circumscription of Orchis (Orchidaceae, Orchidinae) has undergone many changes in the last few decades. However, delimitation of the genus is still subject to debate. In its present state, Orchis s.str. appears to be composed of two well-differentiated, monophyletic groups of species, now treated as subgenera (Orchis s.s. and Masculae). Here we present detailed morphological measurements of floral and vegetative traits collected for a large number of species of the two subgenera and the related genus Anacamptis to provide further insights in the taxonomy of this genus. Using multivariate analyses, we show that three groups of species are clearly separated morphologically and that this separation is more pronounced between the two subgenera than between Masculae and Anacamptis. This morphological variation is also supported by significant differences in seed micromorphology of the two subgenera. Review of the literature further provided significant differences in pollinator assemblages between the two subgenera, suggesting substantial pre-zygotic reproductive isolation, and a significant difference in diversity of fungal associates with species of the two subgenera, possibly contributing to post-zygotic reproductive isolation. The fact that no viable hybrid between the two subgenera has ever been observed under artificial or natural conditions is in line with these findings. Based on these findings, an appropriate taxonomic rank for the two groups is discussed.

The specificity of orchids for their fungi can vary substantially, from highly specialist interactions to more generalist interactions, but little is known about the evolutionary history of the mycorrhizal specificity of orchids.Here, we used a network analysis approach to investigate orchid mycorrhizal associations in 16 species of the genus Orchis sampled across 11 different regions in Europe. We first examined in detail the structure of the network of associations and then tested for a phylogenetic signal in mycorrhizal specificity and identified the fungi with which the orchids associated.We found 20 different fungal lineages that associated with species of the genus Orchis, most of them being related to members of the Tulasnellaceae (84.33% of all identified associations) and a smaller proportion being related to members of the Ceratobasidiaceae (9.97%). Species associations formed a nested network that is built on asymmetric links among species. Evolution of mycorrhizal specificity in Orchis closely resembles a Brownian motion process, and the interaction between Orchis and Tulasnellaceae fungi is significantly influenced by the phylogenetic relationships between the Orchis species.Our results provide evidence of the presence of phylogenetic conservatism in mycorrhizal specificity in orchids and demonstrate that evolutionary processes may be an important factor in generating patterns of mycorrhizal associations.

Conclusion du long travail d’inventaire des stations d’orchidees de France, cet ouvrage fondamental de 400 pages, au format A4, presente, a travers 159 monographies superbement illustrees de 380 photographies et 154 cartes, un etat des lieux de la repartition de ces plantes fascinantes. Cet Atlas est une Publication scientifique du Museum national d’Histoire naturelle (MNHN), en coedition avec l’editeur Biotope. Quatorze auteurs et de nombreux photographes ont mene a bien cette entreprise ; Francois Dusak et Daniel Prat, de la SFO, etant les coordinateurs de cette equipe. Commande par le Ministere charge de l’environnement, l’inventaire, fruit du travail de plus de 3 000 observateurs ou organismes (dont les Conservatoires botaniques nationaux), regroupe, sur deux decennies, plus de 420 000 donnees transmises par la SFO au MNHN (Service du patrimoine naturel) en 2008. Les parties introductives de l’ouvrage detaillent la genese de la cartographie, les implications de cet Atlas sur la gestion des populations d’orchidees, les differentes zones biogeographiques deduites de leur repartition et des donnees climatiques, et font le point sur les risques auxquels elles sont exposees. Pour chaque taxon, sont indiques en plus d’une carte detaillee, la repartition globale, l’ecologie et le classement liste rouge nationale selon les criteres de l’Union Internationale pour la Conservation de la Nature (UICN). L’Atlas procure des informations de reference pour definir les priorites de protection et les actions a mener en faveur des especes classees en danger (EN) ou vulnerables (VU) telles que le Sabot de Venus plante spectaculaire, le Liparis de Loesel espece fragile des milieux humides, et l’Ophrys de l’Aveyron qui occupe une aire tres restreinte. Ce dernier est une des rares especes de la flore francaise proposee pour inscription sur la liste rouge mondiale de l’UICN. Dans la lignee des ouvrages naturalistes edites recemment par Biotope et le MNHN, cet Atlas offre un outil important pour l’evaluation de la biodiversite francaise. Il permet de mieux apprehender les difficultes de la conservation de nos orchidees et de leurs habitats et de comprendre les menaces qui pesent sur elles ; de ce fait il est, pour les nombreux passionnes, un complement indispensable des livres existants axes sur l’identification. Enfin, la repartition des orchidees, servira tous ceux qui interviennent dans la gestion du territoire !