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.

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 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. (Nilsson 1983;1985). These two closely related species exhibit not only 67 morphological differences, but also distinct ecological preferences (P. chlorantha favouring dry, 68 calcareous grasslands, while P. bifolia will be typically found in fresh to wet meadows on acidic soil). 80 Recently, a study on some Western-European Platanthera populations composed almost exclusively of 81 intermediate looking individuals, based on morphology and molecular markers, concluded that such 82 individuals were not hybrids, but constitute an independent lineage, distinct from both widespread species 83 (Durka et al. 2017). 84The level of geitonogamy was observed to be higher in P. bifolia than in P. chlorantha because 85 the latter has a pollinarium-bending mechanism that prevents deposition of the pollinia directly after 86 removal (Maad & Nilsson 2004;Maad & Reinhammar 2004). This process may also affect the probability 87 of hybrid formation (Ishizaki et al. 2013). An allopatric P. bifolia population with a high degree (i.e. 88 almost 60%) of self-pollination was found by Brzosko (2003), although self-pollination in Platanthera 89 species is considered generally rare (Nilsson 1983;Maad 2002). 90In the genus Platanthera, floral scent plays a crucial role in guiding pollinators to the flowers 91 (Nilsson 1983;1985;Tollsten & Bergström 1993). A strong fragrance is emitted after dusk, when 92 pollinators (nocturnal moths) are most active (Nilsson 1983;1985;Tollsten & Bergström 1993; Hapeman 93 & Inoue 1997;Plepys et al. 2002a;2002b). Floral fragrances of Platanthera have been classified into 94 linaloolic, lilac, geraniolic and benzenoic chemotypes depending on the main class of compounds present 95 in the blend (Tollsten & Bergström 1993;Plepys et al. 2002a;2002b (Plepys et al. 2002a;2002b). Furthermore, a change in floral scent composition has been suggested 98 by Nilsson (1983;1985) to prevent effective cross-pollination between both species (Nilsson 1978; 99 Tollsten & Bergström 1993), by acting as a reproductive barrier via ethological mechanisms. Tollsten & 100 Bergström (1993) discovered that important inter-individual and inter-population variation in floral scent 101 exists, and may act as an adapta...

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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 the same genetic patterns of morphologically intermediate individuals with 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.

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