Characterization of sympatric Platanthera bifolia and Platanthera chlorantha (Orchidaceae) populations with intermediate plants

Study species and sampling sites

Platanthera bifolia and P. chlorantha are two terrestrial orchids with a wide Eurasian distribution. The flowering period in Central Europe of both species occurs between May and July, partly overlapping in areas of sympatry (Delforge, 2006). The inflorescences of Platanthera display 10–25 white, hermaphroditic flowers, which open sequentially acropetally and possess a long, slender nectariferous spur as a backward extension of the lip. The length of the spur varies geographically in North-western Europe, and it is also positively correlated to the proboscis length of local pollinators (Darwin, 1862; Nilsson, 1985; Maad & Nilsson, 2004; Boberg & Ågren, 2009; Boberg et al., 2014). Also, the distance between the viscidia is an adaptation to various organs on the pollinator’s head (Nilsson, 1983).

Although the two species are very close genetically (Bateman, James & Rudall, 2012) and show the same diploid chromosome number (2n = 42) (Nilsson, 1983), a few floral traits, or combination thereof, allow separation of individuals into discrete groups matching the two species (Darwin, 1862; Nilsson, 1978; Nilsson, 1983; Nilsson, 1985). P. bifolia presents a small column with a narrow connective and anther pockets set almost parallel to each other, with a distance between the viscidia ranging between 0.2 and 1.1 mm, while the pollinium has a very short caudicle (0.2–0.5 mm); these characteristics imply that pollinaria will be attached to the pollinator’s proboscis (Figs. 1A and 1B). The species is predominantly pollinated by hawkmoths (Sphingidae) (Nilsson, 1983).

By contrast, the column of P. chlorantha is wide, with a broad connective and the anther pockets set strongly divergent at the base. The pollinium has a relatively long caudicle (1.2–2.2 mm) and the distance between the viscidia is between 2.3 and 4.9 mm (Figs. 1E and 1F). This is considered to be an adaptation for attachment to the eyes of pollinators, which are mostly noctuids (Noctuidae) (Nilsson, 1983). In the intermediate plants the distance between the viscidia is, on average, larger than in P. bifolia and smaller than in P. chlorantha (1.3–2.3 mm), while the caudicle length lies between 0.6 and 1.2 mm (Figs. 1C and 1D). We based a priori identification of the individuals on these values intervals for the two specified characters (caudicle length and distance between the viscidia).

This intermediate form of the gynostemium may induce an inadequate attachment of pollinaria to the hairy labial palps of the moths (Nilsson, 1978). Therefore, the pollen of putative hybrids will often be lost because it will not reach the stigmas of other Platanthera individuals. As a result, crossing between hybrid derivatives seems poorly effective (Nilsson, 1983). This process should contribute to pre-pollination isolation and help to maintain the genetic integrity of each species (Nilsson, 1983; Van der Cingel, 1995; Waser, 2001; Cozzolino, D’Emerico & Widmer, 2004; Scopece et al., 2007).

We investigated sympatric populations with P. bifolia, P. chlorantha and intermediate morphotypes in two sites in Southern Belgium. As shown in Table 1, the two populations were sampled in the Calestienne region, one on a calcareous grassland (Tienne de Botton) and the other on a light birch–ash wood (Bois Niau). In addition, two allopatric populations were sampled: in the Famenne region (Navaugle) for P. bifolia, in a semi-wet meadow on acidic soil, and in the Ardenne region (Transinne) in a semi-wet neutral meadow for P. chlorantha. In the sympatric sites, plants were classified based on the values firstly suggested by Nilsson (1983), which will be used as the starting point for the investigations to be conducted. In each site, plants showing good flowering conditions (i.e., fully flowering, with fresh flowers) were sampled randomly for the investigation. Selected individuals sampled for the morphological measurements were also subjected to genetic and chemical analyses (Table 1). Permissions to operate in the field in Nature Reserves were obtained from Walloon authorities in charge of Nature Protection (Département Nature et Forêts, Département de l’Étude du Milieu Naturel et Agricole). Photographic material of the studied populations was deposited in the herbarium of the Belgian National Botanic Garden (BR).

Floral morphology and reproductive success: statistical analyses

In order to characterize the floral morphology of the different populations, four floral traits were measured: spur length (mm), caudicle length (mm), distance between the viscidia (mm) and labellum length (mm) (Nilsson, 1983; Nilsson, 1985; Claessens & Kleynen, 2006).

To test the null hypothesis of no morphological differences among taxa, we first conducted a non-parametric Multiple Response Permutation Procedure (MRPP) using VEGAN package version 2.0–5, with the average Bray–Curtis distances among samples weighted to group size and 999 random permutations (Mielke & Berry, 2001; McCune & Grace, 2002). Then, an analysis of similarities (ANOSIM) was performed using the average Bray–Curtis distances among samples and 1,000 permutations with the VEGAN package (version 2.0–5; Oksanen et al., 2012) in R (R Core Team, 2015) as an alternative way to test statistically whether or not there is a significant difference in morphological traits.

Furthermore, we carried out a multiple comparison with the Kruskal–Wallis test to evaluate the degree of association between samples and the Dunn’s test (Dunn-Sidak-procedure) to determine which of the sample pairs are significantly different for each morphological trait.

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

Manual crosses and pre and post-pollination isolation index

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

To prevent the potential negative effects of over-pollination on fruit set and seed viability, a maximum of three flowers per individual were hand-pollinated. This experiment is based on Xu et al. (2011). To prevent insect visits after experimental crossings, each inflorescence was covered with a pollination bag (to prevent pollination by insects) before and after the cross-pollination. Fruit initiation and development were monitored until fruits were mature (about one month after pollination). All crossed capsules were collected from the two investigated sympatric species and stored in silica gel. In addition, eight allopatric and 12 sympatric individuals of P. bifolia and intermediate morphotypes (Table 1) were also covered with a pollination bag before anthesis to determine the degree of autonomous self-pollination.

Seeds produced by interspecific (hand pollinations), intraspecific crosses and also in the autonomous self-pollination treatment were harvested and brought to the laboratory. Seeds were observed under a microscope (100× magnification) to distinguish seeds containing one large viable embryo from non-viable seeds (i.e., small or aborted embryos or no embryo). Samples of 300 seeds per fruit were scored in order to estimate the percentage of viable seeds for each fruit (Xu et al., 2011). The significance of different seed viability among interspecies and intraspecies crosses was assessed using Student’s t-test, after normality testing of data distribution by the Shapiro test (Royston, 1982).

We also examined and quantified the effect of post-pollination barriers using indices of reproductive isolation (RI) (Kay, 2006). Based on the methods proposed in Scopece et al. (2007) and Marques et al. (2014), we estimated two measures of post-pollination reproductive isolation. We firstly estimated the post-pollination pre-zygotic isolation index as the proportion of fruits formed after interspecific crosses in relation to the proportion of fruits formed after intraspecific crosses: ${\displaystyle \text{RI post}-{\text{pollination}}_{\mathrm{pre}-\text{zygotic}}=1-\frac{\text{average fruit set after interspecific crosses}}{\text{average fruit set after intraspecific crosses}}.}$ Then, we calculated post-zygotic isolation index as the percentage of viable seeds from interspecific crosses in relation to the proportion of viable seeds obtained from intraspecific crosses, describing the embryo mortality: ${\displaystyle \text{RI post}-{\text{pollination}}_{\text{post}-\text{zygotic}}=1-\frac{\text{viable seeds formed after interspecific crosses}}{\text{viable seeds formed after intraspecific crosses}}.}$ In addition, since flowering time is known to contribute to the maintenance of phenotypic polymorphism, we estimated the strength of RI value, which corresponds to flowering phenology. The overall flowering period was recorded for both Platanthera species only at Botton site. Plants were checked every three days during one flowering season (2015). For the investigation of flowering phenology we examined: the beginning of blooming (first flower opened), the end of the flowering period (when the last flower opened). The RI phenology index was calculated as: RI_phenology = 1 − (overlapping flowering period between species (number of days)/flowering period (number of days)) (Ma et al., 2016).

DNA extraction and AFLP analysis

In each population, a leaf fragment of ca. 2 cm² was collected for 10–20 plants of each of the taxa (see Table 1), and the plant tissue was desiccated using silica gel in individually sealed plastic bags. Genomic DNA was extracted using a slight modification of the CTAB protocol of Doyle & Doyle (1987). Plant leaf material was macerated in 900 µL of standard CTAB buffer, incubated at 60 °C for 30 min, extracted twice with chloroform-isoamyl alcohol, precipitated with isopropanol and washed with 70% ethanol. Precipitated DNA was then resuspended in 30 µL of distilled water. We obtained AFLP fragments using the methods of Vos et al. (1995), with modifications as reported in Moccia, Widmer & Cozzolino (2007) using fluorescent dye-labeled primers. Approximately 250 ng of genomic DNA was digested with EcoRI and MseI restriction endonucleases, and then ligated with the appropriate adaptors. A pre-selective amplification of restriction fragments was conducted using a tem of 1 µL of restriction-ligation product and with EcoRIA + MseIA or MseIC as primers. After a preliminary screening for the variability and reproducibility, five selective combinations were chosen for this study: EcoRIA–MseICGG, EcoRIA–MseIACT, EcoRIA–MseICCAA, EcoRIA–MseICGTA, EcoRIA–MseIACTG.

The selective amplifications were conducted with 1 µL of a 1:10 dilution of pre-amplification product.

Separation and detection took place on a 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). GeneScan-500 LIZ (Applied Biosystems) was used as IS (internal standard). The electrophoregram generated by the sequencer was analysed using the GeneMapper version 3.7 software package (2004; Applied Biosystems). Clear and unambiguous peaks, between 50 and 500 bp, were considered as AFLP markers and scored as present or absent in order to generate a binary data matrix. DNA of both allopatric species was amplified and run in duplicate to validate repeatability. The AFLP analysis was performed considering two data sets: the first, contained the Botton plants group + allopatric (plate-Bt), and the second contained the Bois Niau plants group + allopatric (plate-BN). These two data sets were run and scored independently.

We calculated FST values to estimate the population differentiation using the software AFLP-SURV v. 1.0 (Vekemans, 2002). Genetic structure was explored using Principal Coordinates Analysis (PCoA) in GENALEX (Peakall & Smouse, 2006). We performed a Bayesian clustering analysis that allows to estimate the number of genetic clusters (i.e., populations), allele frequencies within clusters, and the genetic composition of individuals, by assigning the latter to different groups in which deviations from Hardy–Weinberg equilibrium and linkage equilibrium are minimized (Jacquemyn et al., 2012a). Data were analysed in STRUCTURE v. 2.3.1 (Pritchard, Stephens & Donnelly, 2000; Falush, Stephens & Pritchard, 2003) assuming an admixture model and correlated allele frequencies with 50,000 burn-in steps and 100,000 MCMC (Markov chain Monte Carlo) steps and K = 1–10, with ten independent runs per K. The goal was to estimate the K value that best fitted to our data.

The K value was assessed from the likelihood distribution (STRUCTURE output), which is the number of genetic clusters present in the data. K value fitting best with our data was selected using the ΔK statistic (Evanno, Regnaut & Goudet, 2005) produced by STRUCTURE HARVESTER (http://taylor0.biology.ucla.edu/struct_harvest/).

Finally, we used DISTRUCT (Rosenberg, 2004) to graphically display the output obtained with STRUCTURE.

NEWHYBRIDS (Anderson, 2008) was also performed to investigate the genetic profiles of the sympatric zone. We implemented a model that assumed two pure parental species and hybrids. This model assigns posterior probabilities for each individual to belong to one of the possibile six genotypic frequency classes: pure parental species, F1, F2, backcross to each parental species. A burn-in of 100,000 steps followed by run lengths of 1,000,000 steps was used (Jacquemyn et al., 2012a).

Moreover, the Hybrid index was estimated based in order to assess genome-wide admixture (Buerkle, 2005). This method calculated hybrid index (HI) based on a maximum likelihood and ranges between zero and one, corresponding to pure individuals of reference and alternative species, respectively. In our analyses, plants with a HI ranging between 0 and 0.2 were assigned to P. bifolia, whereas individuals with HI between 0.8 and 1 were assigned to P. chlorantha. We used AFLP data obtained from the allopatric P. bifolia and P. chlorantha individuals as parental data, while those obtained from the sympatric area were entered as putatively admixed individuals. This analysis was performed following the same parametric procedure proposed by Jacquemyn et al. (2012a). The plot was produced with the mk.image function in INTROGRESS. The hybrid index was estimated to assess genome-wide admixture using the est.h function ((Jacquemyn et al., 2012a) incorporated in the R program INTROGRESS (Gompert & Buerkle, 2010). Finally, we correlated the molecular hybrid index with morphological index obtained with the discriminant function (described previously) using Spearman’s rho method for non-normally distributed data (Jacquemyn et al., 2012a).

Volatile collection and analyses of floral scents

In the sympatric zone in Botton, we sampled the volatile compounds emitted by flowers (the entire inflorescence) (Table 1). Floral scents emitted by the sympatric Platanthera species and the intermediate morphotypes were sampled for chemical analyses in the same phenological flower state, using a dynamic headspace adsorption technique during peak flowering time (June–July) and between 21:00 and 01:00 h local time, thereby matching the peak feeding times of most nocturnal moths (Nilsson, 1978). The same individuals were used to sample plant material for genetic analyses. The intact inflorescences were carefully enclosed in modified polyacetate bags (Pingvin frying bags, Art.nr 352: Kalle Nalo GmbH, Wiesbaden, Germany). The air, together with volatiles, was drawn through the bag by a battery-operated membrane pump, with a flow of 100 ml/min, into Teflon-PTFE cartridges containing 85 mg of the adsorbent Tenax-GR, mesh 60/80 (Andersson et al., 2002) for 60 min. Trapped scent compounds were eluted with 100 µL of cyclohexan and all samples were stored at −20 °C. Then, extracts were analysed by Gas Chromatography/Mass Spectrometry (GC-MS) on a Finnigan Trace Ultra GC coupled to a Finnigan POLARIS Q ion trap mass and equipped with a Restek RXI-5 MS column (30 m length × 0.25 mm diameter × 0.25 µm film thickness).

Aliquots of 1 µL of the extracts were injected in splitless mode first at 35 °C (4 min, followed by a programmed increase of oven temperature to 200 °C at a rate of 5 °C/min⁻¹) then at 200 °C for 1 min with an oven temperature to 270 °C at a rate of 10 °C/min. Helium was used as carrier. The proportional abundance of floral scent compounds (relative amounts with respect to aggregate peak areas, excluding contaminants) was calculated on the absolute amounts of compounds. Component peaks in the GC-MS chromatograms were quantified by integration of selected ion currents relative to one internal standard (IS) (2-phenylethanol, C₈H₁₀O). The Xcalibur™ Software was used and 2 µL of the internal standard was added for quantification of five samples of each group randomly chosen. Components were identified by their mass spectral patterns and chromatographic retention data (retention time and relative retention time). Furthermore, components were identified by comparing recorded mass spectra with the NIST08 and Wiley275 spectral databases with a probability of match >90%.

Morphology and fruit set

A preliminary MRPP analysis indicated that the floral morphology was significantly different between allopatric and sympatric groups in Botton (A = 0.605, δ_obs = 0.025 δ_exp = 0.065 P < 0.001) and Bois Niau (A = 0.626, δ_obs = 0.026δ_exp = 0.069 P < 0.001). The ANOSIM analysis was in agreement with the results of the MRPP, since it also showed a significant difference between those groups (Botton: R = 0.762, P < 0.001; Bois Niau R = 0.779, P < 0.001). The results of the multiple comparisons with the Kruskal–Wallis and the Dunn’s test displayed which trait differed between groups (reported as letters in Fig. 2; Table 2).

Intermediate morphotypes in both sites did not show any significant differences compared to sympatric P. bifolia, although values of morphological traits were slightly higher. Contrarily, comparing to allopatric P. bifolia, intermediate morphotypes showed significant differences for all morphological traits. On the other hand, intermediate morphotypes in Botton displayed significantly smaller values than those of sympatric P. chlorantha, with the exception of spur length; intermediate morphotypes in Bois Niau had significant differences only for viscidia distance and caudicle length.

Nonetheless, comparing intermediate morphotypes of both sites to allopatric P. chlorantha, all traits were significantly different, with the exception of labellum length for Bois Niau plants. Multiple comparisons (Kruskal Wallis and Dunn’s test) did not show significant differences between intermediate morphotypes and sympatric P. bifolia for viscidia distance and caudicle length. The differences in these floral traits may be hidden by the extreme morphological values displayed by allopatric populations. We observed that caudicle length and viscidia distance in allopatric P. bifolia ranged from 0.1 to 0.3 mm and 0.1 to 0.58 mm, respectively, whereas the sympatric P. bifolia form had caudicle lengths varying between 0.3 and 0.9 mm and viscidia distances varying between 0.6 and 1.75 mm (results obtained considered both sites). It thus appears that those morphological characters are significantly different between allopatric and sympatric populations: these correspond to the two ecotypes as mentioned above (introduction).

The discriminant analysis using allopatric populations produced the function D = 0.680 (caudicle length), +0.689 (viscidia distance), (eigenvalue = 57.119; χ² = 150.112 canonical correlation = 0.991; P < 0.001). Based on these functions, P. bifolia individuals received negative scores (−7.36) and P. chlorantha positive scores (7.36).

Principal components analysis of flower traits explained 89% of the variance along its two main axes between allopatric and Botton populations; the first principal component accounted for 63.31% of the species variation and had high positive loadings with viscidia distance, caudicle length and labellum length. The length of the spur was positively correlated with the second axis and the variation was 26%. The proportion of the explained variance between allopatric and Bois Niau populations was 92% (Fig. 3) and, the first principal component accounted for 62.97% of the variation and showed high positive loadings with viscidia distance, caudicle length and labellum length, whereas spur length accounted for 29% (Fig. 3) on the second axes.

Fruit set differed significantly between sympatric species and intermediate morphotypes in both sites (Botton: χ² = 12.34 P < 0.001; Bois Niau: χ² = 9.07P < 0.05). Moreover, we observed a fruit-set advantage of P. chlorantha compared to P. bifolia in Botton (Dunn’s test: P < 0.01) (Fig. 4A) and Bois Niau (Dunn’s test: P < 0.05) (Fig. 4B), whereas a significant difference between P. bifolia and intermediate morphotypes was observed only in Bois Niau (Dunn’s test: P < 0.05) (Fig. 4B).

Manual crosses and pre and post-zygotic isolation index

The proportion of viable seeds obtained from interspecific crosses was not different between P. bifolia and P. chlorantha and between intraspecific crosses (allopatric populations). The average percentage of viable seeds was: 40% ± 33.8% SD for P. bifolia and 52.67 ± 37.38% SD for P. chlorantha. For intraspecific crosses we obtained for P. bifolia a mean of: 41.93% ± 25.68% SD, and for P. chlorantha: 38.07% ± 30.15% SD. The proportion of viable seeds was not significantly different between interspecific crosses (P = 0.56) and intraspecific crosses (P = 0.32). For bi-directional crosses the post-pollination pre-zygotic isolation indices were negative (P. bifolia: −0.43; P. chlorantha: −0.14), which indicated that interspecies crosses performed better than intraspecies ones. Similarly, the post-pollination post-zygotic isolation indices were also weak, 0.22 for P. bifolia/P. chlorantha, and −0.17 for P. chlorantha/P. bifolia.

Self-pollination tests revealed a very low level of autonomous self-pollination in allopatric and sympatric populations analysed, since we observed just one flower forming a fruit on a sympatric individual of P. bifolia (out of 12 plants), but without viable seeds.

The overall period of flowering between P. bifolia and P. chlorantha largely coincided. For 21 days P. bifolia and P. chlorantha were flowering at the same time. More precisely, P. bifolia flowered from 4th of June to 29th of June, while the flowering period of P. chlorantha lasted from 22th of May to 24th of June. Therefore, P. bifolia flowered for 26 days and 34 for P. chlorantha. Therefore, RI_phenology = 1 − (21∕26) = 0.19 for P. bifolia, and RI_phenology = 1 − (21∕34) = 0.38 for P. chlorantha.

Genetic diversity and differentiation

For the genetic analyses we excluded samples with a high number of missing values. Therefore, these analyses were carried out only on 28 allopatric individuals, 46 sympatric individuals in plate-Bt and 33 allopatric individuals, 43 sympatric individuals in plate-Bn. A total of 87 (plate-Bt) and 100 (plate-BN) polymorphic bands were detected in this study. The mean percentage of polymorphic loci was 79% for Botton and 76% for Bois Niau. We identified six and eight species-specific polymorphic bands in plate-Bt and plate-BN respectively; intermediate morphotypes showed the same polymorphism of P. bifolia for these species-specific sites (Table 3).

Pairwise F_ST values were high between the intermediate morphotypes and P. chlorantha populations (F_ST = 0.14 plate-Bt and F_ST = 0.16 plate-Bn), but considerably lower between the intermediate population and P. bifolia (F_ST = 0.01 plate-Bt and F_ST = 0.02 plate-Bn).

In the principal coordinates analysis (PCoA) the first two axes explained 54% of variance for Botton and 57% of variance for Bois Niau population (Fig. 5). The PCoA clearly separated P. bifolia from P. chlorantha along the first axis, although there is a very slight overlap in the case of Botton plants. Moreover, PCoA plots for both plates identified two groups: (1) the intermediate morphotypes with allopatric and sympatric P. bifolia, and (2) the sympatric and allopatric P. chlorantha (Fig. 5).

The results obtained from the Bayesian admixture analyses with STRUCTURE (Fig. 6) showed that the likelihood (LnP(D)) increased greatly at K = 2 which, together with the fact that ΔK reached its maximum at K = 2, suggests the existence of only two genetic clusters for both plates (Fig. 6). Population clustering showed a consistent pattern indicating two independent genetic clusters: P. bifolia and the intermediate morphotypes formed a single cluster separated from P. chlorantha (Fig. 6).

NEWHYBRIDS yielded similar results by assigning the intermediate morphotypes to the group of P. bifolia and revealing a low proportion of admixture genome in sympatric populations. Considering a threshold q-value of 0.9, we observed that 83% and 93% of individuals sampled in Botton and Bois Niau respectively were unequivocally assigned to P. bifolia and P. chlorantha and only four plants in Botton and one plants in Bois Niau that were identified as intermediate morphotype, had an admixed gene pool (Fig. 7).

In Bois Niau sympatric population all plants identified morphologically as P. bifolia and intermediate morphotypes had an hybrid index ranging between 0 and 0.2 supporting the previous results, whereas in Botton population most of them had an hybrid index ranging between 0 and 0.2 and only four individuals showed an hybrid index ranging between 0.3 and 0.4. Individuals firstly morphologically identified as P. chlorantha showed a hybrid index ranging between 0.4 and 0.7 in both sympatric populations.

Finally, molecular and morphological hybrid indices were significantly (P < 0.001) correlated in both sympatric sites (Fig. 8).

Floral scents

Based on the group of compounds that dominated a scent profile, the bouquets emitted by the inflorescences of P. bifolia, P. chlorantha and the intermediate morphotypes included a total of ten volatile compounds: two benzenoids and eight monoterpenoids (Table 4).

The following classes could be distinguished: lilac aldehydes, alcohol compounds, geraniolic compounds, and benzenoid compounds. Compared with P. bifolia, the scent patterns within P. chlorantha populations were less variable. Nevertheless, individuals of the two species showed a divergent chemical pattern. Specifically, the mean of relative percentage of lilac aldehyde in P. chlorantha was higher compared with the sympatric P. bifolia (Table 4), in contrast with the results of Tollsten & Bergström (1993) where P. chlorantha contained a higher percentage of lilac aldehyde. Furthermore, among the ten volatile compounds identified, the relative amount of three compounds (ocimene, 1,2-hexanediol-2-benzoate and santolina triene) was emitted in high percentage only by P. bifolia and the intermediate morphotypes compared with the P. chlorantha population. Also other compounds were dominant, such as benzenoids in P. bifolia compared with P. chlorantha (Table 4), which was in accordance with the results of Tollsten & Bergström (1993). By contrast, the floral compound 3,7-Dimethyl-1,3,6-octatriene is present in a high percentage in P.bifolia compared to the intermediate morphotypes and P. chlorantha species. This compound was observed to be pheromone involved in social regulation in a honeybee colony (Maisonnasse et al., 2010) but not directly involved in attraction of nocturnal moths (http://www.pherobase.com/database/compound/compounds-detail-cis-beta-ocimene.php).

MRPP analysis indicates that floral scents were significantly differentiated among groups in floral scent composition (A = 0.551, δobs = 0.262, δexp = 0.583, P < 0.001). The ANOSIM analysis was in accordance with the result of MRPP analysis, which showed a significant difference between Platanthera groups (R = 0.748, P < 0.001). Moreover, Tukey’s Honest Significant Differences and post-hoc test revealed that there was a significant difference in the variance dispersion of floral scents between P. bifolia and P. chlorantha (P = 0.002) and between P. chlorantha and intermediate morphotypes (P = 0.011).

The analysis of overall floral odour similarity with HCAST produced a dendrogram which shows that investigated sympatric populations are resolved into two clusters that were supported statistically (AU values > 80%) (Fig. 9). The first cluster contained the sympatric P. chlorantha and the second two subclusters contained intermediate and P. bifolia individuals sympatric with them. The results of this analysis show a significant similarity of chemical patterns in floral scent composition with P. bifolia of all intermediate morphotypes sampled (Fig. 9).