Germination failure is not a critical stage of reproductive isolation between three congeneric orchid species.

PREMISE OF THE STUDY: In food-deceptive orchid species, postmating reproductive barriers (fruit set and embryo mortality) have been shown to be more important for reproductive isolation than premating barriers (pollinator isolation). However, currently there is very little knowledge about whether germination failure acts as a reproductive barrier in hybridizing orchid species. METHODS: In this study, we investigated germination and protocorm development of pure and hybrid seeds of three species of the orchid genus Dactylorhiza. To test the hypothesis that germination failure contributed to total reproductive isolation, reproductive barriers based on germination were combined with already available data on early acting barriers (fruit set and embryo mortality) to calculate the relative and absolute contributions of these barriers to reproductive isolation. KEY RESULTS: Protocorms were formed in all crosses, indicating that both hybrid and pure seeds were able to germinate and grow into protocorms. Also, the number of protocorms per seed packet was not significantly different between hybrid and pure seeds. High fruit set, high seed viability, and substantial seed germination resulted in very low reproductive isolation (average RI = 0.05). In two of six interspecific crosses, hybrids performed even better than the intraspecific crosses. CONCLUSIONS: Very weak postmating reproductive barriers were observed between our study species and may explain the frequent occurrence of first-generation hybrids in mixed Dactylorhiza populations. Germination failure, which is regarded as one of the most important bottlenecks in the orchid life cycle, was not important for reproductive isolation.

Variation in mycorrhizal associations with tulasnelloid fungi among populations of five Dactylorhiza species.

BACKGROUND: Orchid 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 FINDINGS: We 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/SIGNIFICANCE: We 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.

Reproductive isolation and hybridization in sympatric populations of three Dactylorhiza species (Orchidaceae) with different ploidy levels.

BACKGROUND AND AIMS: The potential for gene exchange between species with different ploidy levels has long been recognized, but only a few studies have tested this hypothesis in situ and most of them focused on not more than two co-occurring species. In this study, we examined hybridization patterns in two sites containing three species of the genus Dactylorhiza (diploid D. incarnata and D. fuchsii and their allotetraploid derivative D. praetermissa). METHODS: To compare the strength of reproductive barriers between diploid species, and between diploid and tetraploid species, crossing experiments were combined with morphometric and molecular analyses using amplified fragment length polymorphism markers, whereas flow cytometric analyses were used to verify the hybrid origin of putative hybrids. KEY RESULTS: In both sites, extensive hybridization was observed, indicating that gene flow between species is possible within the investigated populations. Bayesian assignment analyses indicated that the majority of hybrids were F(1) hybrids, but in some cases triple hybrids (hybrids with three species as parents) were observed, suggesting secondary gene flow. Crossing experiments showed that only crosses between pure species yielded a high percentage of viable seeds. When hybrids were involved as either pollen-receptor or pollen-donor, almost no viable seeds were formed, indicating strong post-zygotic reproductive isolation and high sterility. CONCLUSIONS: 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.

Patterns of hybridization between diploid and derived allotetraploid species of Dactylorhiza (Orchidaceae) co-occurring in Belgium.

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 Φ(ST) 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.