BackgroundOrchid species rely on mycorrhizal symbioses with fungi to complete their life cycle. Although there is mounting evidence that orchids can associate with several fungi from different clades or families, less is known about the actual geographic distribution of these fungi and how they are distributed across different orchid species within a genus.Methodology/Principal FindingsWe investigated among-population variation in mycorrhizal associations in five species of the genus Dactylorhiza (D. fuchsii, D. incarnata, D. maculata, D. majalis and D. praetermissa) using culture-independent detection and identification techniques enabling simultaneous detection of multiple fungi in a single individual. Mycorrhizal specificity, determined as the number of fungal operational taxonomic units (OTUs), and phylogenetic diversity of fungi were compared between species, whereas discriminant analysis was used to compare mycorrhizal spectra across populations and species. Based on a 95% cut-off value in internal transcribed spacer (ITS) sequence similarity, a total of ten OTUs was identified belonging to three different clades within the Tulasnellaceae. Most OTUs were found in two or more Dactylorhiza species, and some of them were common and widespread, occurring in more than 50% of all sampled populations. Each orchid species associated with at least five different OTUs, whereas most individuals also associated with two or more fungal OTUs at the same time. Phylogenetic diversity, corrected for species richness, was not significantly different between species, confirming the generality of the observed orchid mycorrhizal associations.Conclusions/SignificanceWe found that the investigated species of the genus Dactylorhiza associated with a wide range of fungal OTUs from the Tulasnellaceae, some of which were widespread and common. These findings challenge the idea that orchid rarity is related to mycorrhizal specificity and fungal distribution.

Strong post-mating reproductive barriers prevent local breakdown of species boundaries in Dactylorhiza despite frequent hybridization between parental species. However, the presence of triple hybrids indicates that in some cases hybridization may extend the F(1) generation.

Colonization is crucial to habitat restoration projects that rely on the spontaneous regeneration of the original vegetation. However, as a previously declining plant species spreads again, the likelihood of founder effects increases through recurrent population founding and associated serial bottlenecks. We related Amplified Fragment Length Polymorphism markers genetic variation and fitness to colonization history for all extant populations of the outcrossing terrestrial orchid Dactylorhiza incarnata in an isolated coastal dune complex. Around 1970, D. incarnata suffered a severe bottleneck yet ultimately persisted and gradually spread throughout the spatially segregated dune slacks, aided by the restoration of an open vegetation. Genetic assignment demonstrated dispersal to vacant sites from few nearby extant populations and very limited inflow from outside the spatially isolated reserve. Results further indicated that recurrent founding from few local sources resulted in the loss of genetic diversity and promoted genetic divergence (F(ST) = 0.35) among populations, but did not influence population fitness. The few source populations initially available and the limited inflow of genes from outside the study reserve, as a consequence of habitat degradation and spatial isolation, may have magnified the genetic effects of recurrent population founding.

• Premise of the study: Although the potential for gene flow between species with large differences in chromosome numbers has long been recognized, only few studies have thoroughly investigated in situ hybridization across taxa with different ploidy levels. We combined morphological, cytological, and genetic marker data with pollination experiments to investigate the degree, direction, and spatial pattern of hybridization between the diploid Dactylorhiza incarnata and its tetraploid derivative, D. praetermissa. • Methods: To identify hybrids, 169 individuals were genotyped using AFLPs and morphologically characterized. Individuals were clustered on the basis of their AFLP profile using the program Structure. To reduce the dimensionality of the plant‐trait matrix, PCA was applied. The origin of suspected hybrid individuals was verified using flow cytometry. An AMOVA and spatial autocorrelation analysis were used to indirectly infer the extent of gene flow. • Key results: Only five individuals were regarded as putative hybrids on the basis of the AFLP data; all had been assigned to the D. praetermissa morphotype. Only one had deviating DNA content and was presumably a triploid. High ΦST values between different subpopulations and significant spatial genetic structure were observed, suggesting localized gene flow. • Conclusions: Using combined data to study hybridization between D. incarnata and D. praetermissa, very few unequivocal hybrids were observed. We propose several non‐mutually exclusive explanations. Localized pollen flow, in combination with different microhabitat preferences, is probably one of the reasons for the low frequency of hybrids. Also, the triploid first‐generation hybrids may experience difficulties in successful establishment, as a result of genic incompatibilities.

Background and aims -Understanding how plant traits mediate the relationship between plant population size, fitness and genetic diversity is fundamental to conservation. Many orchid species are threatened, possibly due to their susceptibility to the consequences of habitat fragmentation and the associated decreasing population size. One third of all orchids do not produce nectar to entice pollinators, and this is believed to promote gene flow and outcrossing. Therefore, we hypothesised that deceptive orchids are less susceptible to reductions in population size than rewarding species. Methods -We reviewed the available literature and used traditional and phylogenetically independent meta-analyses to explore relationships between population size, fitness (fruit set) and genetic diversity. Key results -Results showed that orchids in general are highly susceptible to the consequences of small population size. Fruit set significantly decreased in small populations of rewarding, but not in deceptive species. Deceptive orchids were, however, as likely to lose genetic diversity in small populations, even though significantly less genetic differentiation (Fst) was present between populations of deceptive orchids. Finally, we found that homozygosity did not relate with population size, which may indicate a selection process against less fit homozygotes in small orchid populations. Conclusions -We conclude that reductions in population size most likely resulting from habitat loss and fragmentation represent major threats to the population viability of orchids. Fitness of rewarding orchids declines through ecological mechanisms, whereas deceptive orchids may be more susceptible to genetic loss as they are more susceptible to decreased gene flow.