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.

Platanthera bifoliaandP. chloranthaare 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 mixedPlatantherapopulations 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 theP. bifoliagroup. These results are corroborated also by floral scent analyses, which confirmed a strong similarity in floral scent composition between intermediate morphotypes andP. 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.

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.