Characterization of sympatric Platanthera bifolia and Platanthera chlorantha

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.

128 attached to the pollinator's proboscis ( Figure 1A and 1B). The species is predominantly pollinated by 129 hawkmoths (Sphingidae) (Nilsson 1983). By contrast, the column of P. chlorantha is wide, with a broad 130 connective and the anther pockets set strongly divergent at the base. The pollinium has a relatively long 131 caudicle (1.2-2.2 mm) and the distance between the viscidia is between 2.3 and 4.9 mm ( Figure 1E and 132 1F).

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This is considered to be an adaptation for attachment to the eyes of pollinators, which are mostly 134 noctuids (Noctuidae) (Nilsson 1983). In the intermediate plants the distance between the viscidia is, on 135 average, larger than in P. bifolia and smaller than in P. chlorantha (1.3-2.3 mm) ( Figure 1C and 1D).

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This intermediate form of the gynostemium may induce an inadequate attachment of pollinaria to 137 the hairy labial palps of the moths (Nilsson 1978). Therefore, the pollen of putative hybrids will often be 138 lost because it will not reach the stigmas of other Platanthera individuals. As a result, crossing between 139 hybrid derivatives seems poorly effective (Nilsson 1983). This process should contribute to pre-140 pollination isolation and help to maintain the genetic integrity of each species (Nilsson 1983

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We investigated sympatric populations with P. bifolia, P. chlorantha and intermediate 143 morphotypes in two sites in Southern Belgium. As shown in Table 1, the two populations were sampled 144 in the Calestienne region, one on a calcareous grassland (Tienne de Botton) and the other on a light birch-145 ash wood (Bois Niau). In addition, two allopatric populations were sampled: in the Famenne region 146 (Navaugle) for P. bifolia, in a semi-wet meadow on acidic soil, and in the Ardenne region (Transinne) in 147 a semi-wet neutral meadow for P. chlorantha. In the sympatric sites, plants were classified based on the 148 values firstly suggested by Nilsson (1983), which will be used as the starting point for the investigations 149 to be conducted. In each site, plants showing good flowering conditions (i.e., fully flowering, with fresh 150 flowers) were sampled randomly for the investigation. Selected individuals sampled for the 151 morphological measurements were also subjected to genetic and chemical analyses (Table 1) In order to characterize the floral morphology of the different populations, four floral traits were 157 measured: spur length (mm), caudicle length (mm), distance between the viscidia (mm) and labellum 158 length (mm) (Nilsson 1983;1985;Claessens & Kleynen 2006).

159
To test the null hypothesis of no morphological differences among taxa, we first conducted a non-

169
In addition, we performed a canonical discriminant analysis using the morphological data. We 170 applied a stepwise method with an F value of 3.84 to enter a variable, and F value of 2.71 to remove it 171 (Moccia et al. 2007;Jacquemyn et al. 2012a). The discriminant function was derived using trait 172 measurements from the two allopatric Platanthera populations. Then, we used the function to estimate the 173 average floral morphology of each plant present in the sympatric zone (Moccia et al. 2007) that was used 174 as morphological index. This analysis was conducted using the SPSS 21.0 statistical package (SPSS Inc., 175 Chicago, IL). We also performed a multivariate analysis (PCA) on correlation matrix, using the function 176 prcomp, to summarize the information of morphological data. In addition, to compare fruit set (number of 177 fruits/number of flowers) between Platanthera groups in both sympatric sites a multiple comparison with 178 the Kruskal-Wallis test coupled with Dunn's test (Dunn-Sidak-procedure) was performed. These 179 statistical analyses were performed in the software environment R version 3.2.1 (R Core Team 2015).

182
To determine the level of compatibility between species, experimental crosses were carried out in 183 the sympatric area of Botton. Fresh flowers with intact pollinaria were randomly selected. Interspecific 184 hand-pollinations were performed by removing pollinaria through touching the viscidia with a plastic 185 toothpick and placing them on the stigmas of plants of the other species. Crossing combinations were 186 performed bi-directionally (P. bifolia/P. chlorantha and P. chlorantha/P. bifolia) with each plant 187 providing and receiving pollen, and included control-treatments (Table 1).

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To prevent the potential negative effects of over-pollination on fruit set and seed viability, a 189 maximum of three flowers per individual were hand-pollinated. This experiment is based on Xu et al. 190 (2011). To prevent insect visits after experimental crossings, each inflorescence was covered with a 191 pollination bag (to prevent pollination by insects) before and after the cross-pollination. Fruit initiation

221 DNA extraction and AFLP analysis
In each population, a leaf fragment of ca. 2 cm 2 was collected for 10-20 plants of each of the taxa 223 (see Table 1), and the plant tissue was desiccated using silica gel in individually sealed plastic bags.
224 Genomic DNA was extracted using a slight modification of the CTAB protocol of Doyle & Doyle (1987

266
Moreover, the Hybrid index was estimated based in order to assess genome-wide admixture 267 (Buerkle 2005). This method calculated hybrid index (HI) based on a maximum likelihood and ranges 268 between zero and one, corresponding to pure individuals of reference and alternative species, respectively. 269 In our analyses, plants with a HI ranging between 0 and 0.2 were assigned to P. bifolia, whereas 270 individuals with HI between 0.8 and 1 were assigned to P. chlorantha. We used AFLP data obtained from   The proportion of viable seeds obtained from interspecific crosses was not different between P.
353 For intraspecific crosses we obtained for P. bifolia a mean of: 41.93% ± 25.68% SD, and for P.

385
The results obtained from the Bayesian admixture analyses with STRUCTURE ( Figure 6) showed 386 that the likelihood (LnP(D)) increased greatly at K = 2 which, together with the fact that ΔK reached its 387 maximum at K = 2, suggests the existence of only two genetic clusters for both plates (Figure 6).

509
Given all these considerations, we may speculate that the low rate of introgressed genotypes 510 found in sympatry could be due to a combination of several pre-pollination isolation barriers, and their 511 potentially complex interactions. In Orchidaceae, pre-pollination mechanisms seem generally to be 512 particularly good at achieving isolation in sympatry despite often sharing pollinators (Dressler 1968; 513 Cozzolino (Table 4 -except for 3,7-523 dimethyl-1, 3,6-octatriene which is in common with P. chlorantha). These findings of were broadly in 524 agreement with previous studies on floral scent composition of Platanthera (Nilsson 1983;1985).
525 Monoterpenes were, indeed, the most abundant compounds in the floral bouquets of all studied 526 populations for P. chlorantha individuals; the floral scent was essentially composed of lilac aldehydes 527 and alcohols. In contrast, a mixture of monoterpenes and aromatic esters was observed in P. bifolia.
528 Nilsson (1983) suggested that the presence of lilac compounds in P. chlorantha could be an adaptation to 529 noctuid moths, and aromatic esters in P. bifolia to sphingid moth pollination.

556
Moreover, we may also speculate that among P. bifolia plants, the individuals tending towards P.
557 chlorantha's phenotype may be positively selected in order to attract and exploit the pollinators of this 558 species. The analysis of female success, indeed, showed a significantly higher fruit set in P. chlorantha 559 compared to P. bifolia in both sympatric sites (Figure 4).

560
These results may also suggest a slightly reproductive advantage of intermediate forms compared 561 to P. bifolia group ( Figure 4B). Indeed, since reproductive success depends on the interaction between 562 pollinators and column length, a better fit between them, may influence fruit rate through better pollinaria 563 removal and deposition, and can shape the evolution of interspecific floral variation. Particularly, 564 considering that pollinators penetrate the spur via its entrance, the distance separating the viscidia will be 565 a crucial trait that dictate which potential pollinator will be the most efficient in transferring pollen among 566 flowers, by influencing strongly the reproductive success. Moreover, in a previous study conducted by 567 Hapeman & Inoue (1997), the column morphology of Platanthera is considered evolutionary labile and 568 easily shifted when subjected to pollinator-mediated selection.

589
In this study, we investigated two sympatric contact zones between two closely related 590 Platanthera species and we observed that individuals with intermediate morphology were genetically 591 belonging to P. bifolia group. However, the assignment of these individuals as P. bifolia species has been 592 more reliable only by providing a much more detailed picture of the genetic structure of a sympatric zone 593 through the use of genome wide analysis.

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Overall, we found a low rate of hybridization/introgression, together with an apparent lack of 595 strong post-pollination isolation mechanisms, which allow us to speculate that it could be due to a 596 combination of pre-pollination isolation barriers. Thus, it would be interesting to explore if variation in 597 gynostemium morphology among species is the simple result of plasticity or may reflect adaptive 598 evolution in response to pollinator-mediated selection.     Figure 1 Pictures showing flowers of the plants investigated (all pictures D. Tyteca, except Figure   1F: F. Esposito).