Mycorrhizal associations and reproductive isolation in three closely related Orchis species

doi:10.1093/aob/mcq248

Ann Bot. 2011 March; 107(3): 347–356.

Published online 2010 December 24. doi: 10.1093/aob/mcq248

PMCID: PMC3043927

Mycorrhizal associations and reproductive isolation in three closely related Orchis species

Hans Jacquemyn,¹^* Rein Brys,² Bruno P. A. Cammue,³ Olivier Honnay,¹ and Bart Lievens^4,⁵

¹Laboratory of Plant Ecology, Department of Biology, KULeuven, 3001 Leuven, Belgium

²Research Institute for Nature and Forest, 1070 Brussels, Belgium

³Centre of Microbial and Plant Genetics (CMPG), Department of Microbial and Molecular Systems, KULeuven, 3001 Leuven, Belgium

⁴Scientia Terrae Research Institute, 2860 Sint-Katelijne-Waver, Belgium

⁵Laboratory for Process Microbial Ecology and Bioinspirational Management, Lessius University College, Campus De Nayer, Consortium for Industrial Microbiology and Biotechnology (CIMB), Department of Microbial and Molecular Systems, KULeuven Association, 2860 Sint-Katelijne-Waver, Belgium

^*For correspondence. E-mail hans.jacquemyn/at/bio.kuleuven.be

Received July 8, 2010; Revised September 30, 2010; Accepted November 15, 2010.

This article has been cited by other articles in PMC.

Abstract

Background and Aims

The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid–mycorrhizal associations may be involved in acting as a post-mating barrier.

Methods

We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology.

Results

Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents.

Conclusions

Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.

Keywords: DNA array, gene flow, hybrid zones, mycorrhizal associations, reproductive barriers, seed germination

INTRODUCTION

The maintenance of species integrity depends largely on the strength of reproductive barriers (Coyne and Orr, 2004). These barriers act sequentially and determine whether gene flow may occur between species (pre-mating barriers) and whether viable progeny may be produced (post-mating barriers) (Mayr, 1942; Ramsey et al., 2003). The relative contribution of pre- and post-mating barriers in maintaining species integrity, however, remains largely unknown (Coyne and Orr, 1998; Schemske, 2000) and has been shown to vary greatly among taxa, depending on the characteristics of the reproductive biology of the species involved (Rieseberg et al., 2006).

In orchids of the genera Anacamptis, Neotinea and Orchis, most of which are food-deceptive species, high levels of hybridization have been documented among species and genera (reviewed in Cozzolino and Widmer, 2005; Kretzschmar et al., 2007). Although frequent occurrence of hybridization suggests that pre- and/or post-mating barriers should be weak, experimental hand-pollinations have shown that post-mating barriers (fruit set, seed viability and hybrid sterility) can be strong, particularly in species that are genetically distant (Scopece et al., 2007, 2008). Investigation of pollinator assemblages, on the other hand, revealed that most orchid species of these genera are pollinated by generalist pollinators and that they show considerable overlap in their pollinator assemblages (Cozzolino et al., 2005), resulting in weak pre-mating barriers (Scopece et al., 2007). Although chromosomal differences have been suggested to play an important role in species isolation (e.g. Moccia et al., 2007), it has been suggested that strong post-mating pre-zygotic isolation may also have evolved in these species (Cozzolino and Scopece, 2008).

Because mycorrhizal fungi are needed for seed germination and subsequent seedling establishment in orchids (Rasmussen, 1995; Smith and Read, 2008), it can be hypothesized that orchid–mycorrhizal associations may be involved in acting as a post-mating barrier (Scopece et al., 2008). At present, however, little is known about the genetic basis of orchid–mycorrhizal associations and the mechanisms of cellular signalling among orchid seedlings and their fungal partners (Rasmussen and Rasmussen, 2009), and it remains unclear whether the specificity of orchid–mycorrhizal interactions can prevent hybrid seeds from successful seed germination or seedling establishment. There are only a few studies to date that have investigated how mycorrhizal associations in hybrid seeds compare with those of pure plants. Investigation of mycorrhizal associations in hybridizing species of the genus Caladenia showed that fungi from one or both parents can stimulate germination of hybrid seeds, and that hybrids associate with fungi that are genetically different from those associating with the parents (Hollick et al., 2005). Schatz et al. (2010) showed that adult individuals of O. anthropophora, O. simia and their hybrid (O. bergonii) associate with taxonomically similar symbionts, but that fungi associating with the hybrids show less divergent internal transcribed spacer (ITS) sequences than those associating with the parents. However, because the diversity and identity of mycorrhizal fungi associating with orchids can be strongly affected by the life stage of an individual (Bidartondo and Read, 2008), ideally mycorrhizal associations should be investigated at both the seed germination and adult stages in order to be able to obtain more detailed insights into the role of mycorrhizal associations in affecting germination and establishment of hybrid plants.

The strength of reproductive barriers and mycorrhizal associations was studied in three species of the genus Orchis: O. anthropophora, O. militaris and O. purpurea. Hybridization between all three species has been described previously, but the prevalence of hybridization differs between species (Kretzschmar et al., 2007). Hybridization between O. purpurea and O. militaris occurs frequently and can give rise to hybrid swarms, whereas hybrids between O. anthropophora and the other two species are rare (Kretzschmar et al., 2007). More specifically, we aimed at (a) investigating the strength of reproductive barriers up to the seed germination stage; (b) determining mycorrhizal associations in adult individuals of the three species; and (c) comparing mycorrhizal associations of adult individuals with those observed in protocorms originating from both pure and hybrid crosses.

MATERIALS AND METHODS

Study species

Three Orchis species that are known to hybridize in nature were the subject of this study: O. anthropophora, O. purpurea and O. militaris. Despite pronounced morphological differences (see below), recent phylogenetic analyses (Bateman et al., 2003) have placed O. anthropophora, O. purpurea and O. militaris in a clade representing the genus Orchis. Previously O. anthropophora was assigned by some authors to the genus Aceras, of which it was the sole member (Kretzschmar et al., 2007). These three species have slightly different habitat preferences and also differ in their distribution area. Whereas O. anthropophora and O. purpurea clearly have an Atlantic–Mediterranean distribution, O. militaris shows a more continental distribution, spreading as far east as Mongolia and Siberia (Kretzschmar et al., 2007). Orchis anthropophora and O. purpurea also favour warmer conditions for growth and survival than O. militaris, which prefers damp to wet or seasonally wet grasslands (Kretzschmar et al., 2007). Despite these different preferences, several sites are known where the three species can be found together.

All three species flower during about the same period, from the beginning of May to the end of May–beginning of June. Floral morphology differs substantially between the three species (Fig. 1). Whereas O. militaris and O. purpurea have a short spur, there is no spur in O. anthropophora. The length, width and colour of the sepals and petals also differ greatly between the three species (Fig. 1). Whereas the lip and side lobes in O. anthropophora are dirty yellow, orange or brown, those of O. purpurea and O. militaris vary from white through pale pink to light purple (O. purpurea) and from whitish to intense pink (O. militaris) (Fig. 1). Flowers of O. militaris and O. purpurea are nectarless, whereas in O. anthropophora nectar is secreted from two small glands at the basis of the labellum (van der Cingel, 1995). All three species are allogamous and depend on pollinators to guarantee successful pollination. Little, however, is known about the pollinators of the species (van der Cingel, 1995). Orchis anthropophora has been reported to be pollinated by two species of sawflies, Tenthredopsis sp. and Arge thoracia (both Hymenoptera; Tenthredinidae), and by two beetle species, Cantharis rustica (Coleoptera: Cantharidae) and Cidnopus pilosus (Coleoptera: Elateridae), by Schatz (2006). In western France (Brittany), the beetle Isomira murina (Coleoptera: Elateridae) has been reported as the sole pollinator of O. anthropophora. Few data are available on the pollination biology of O. militaris and O. purpurea (van der Cingel, 1995), but our own observations and those reported in Vöth (1987) and Farrell (1985) indicate that both species are regularly visited by generalist pollinators, including bumble-bees, bees and butterflies, which may serve as occasional pollinators for these species. In particular, members of Andrenidae and Halictidae were frequently observed pollinating flowers of O. militaris (Vöth, 1987).

Fig. 1.

Flower morphology of the three investigated Orchis species. (A) Side view of the helmet, (B) front view of the lip and (C) the pollinium of O. anthropophora, O. purpurea and O. militaris.

Experimental crosses and seed viability

Experimental crosses were conducted to assess the magnitude of post-mating barriers between the different study species. To exclude the possibility that pollinations were performed on hybrid plants, all experimental crosses were performed on plants from pure populations. All sites were located in the eastern part of Belgium and consisted of calcareous grasslands, which were not more than 20 km apart. The O. anthropophora population was located in a species-rich calcareous grassland on a steep slope (inclination >25°) and a very shallow soil. Plants of O. militaris were selected in the largest population known in the area, where several thousands of plants co-occur with other orchid species. Finally, O. purpurea was studied in a calcareous grassland immediately bordering a beech forest.

For each species, 30 plants were randomly selected within the population and covered with nylon bags prior to pollination. Three treatments (ten plants per treatment) were applied: pollination with pollen of one of the two other species and pollination with pollinia from the same species. From each plant ten flowers were pollinated, resulting in a total of 900 experimental pollinations (3 species × 3 treatments × 10 plants × 10 flowers). Pollinia for crosses were collected with a toothpick from up to ten donor plants selected at random within each population. Subsequently, the collected pollinia were placed on the stigmatic surface of receptor plants after they were first emasculated. All pollinations were conducted at peak flowering (mid May 2008). After pollination, plants were bagged again to avoid supplemental pollination by insects.

After fruit maturation, the number of fruits that had successfully developed from flowers was counted for each plant. All fruits were harvested and brought to the laboratory for further analysis. For each fruit, the proportion of viable seeds was determined. To distinguish viable from non-viable seeds, a batch of approx. 200 seeds was stained with tetrazolium using the modified staining technique of Van Waes and Debergh (1984). The remaining seeds were used for seed germination experiments. After staining, seeds were mounted on a glass, and digital photographs (Leica) were taken. Finally, the proportion of viable seeds was determined on the screen, by counting the number of viable (with a coloured embryo) and non-viable (lacking a viable embryo) seeds.

Seed germination

Seed germination experiments were conducted at a site where all three species occurred together. At this site, hybridization between O. militaris and O. purpurea could be observed, but not between O. anthropophora and the other two species (H. Jacquemyn, unpubl. res.). For each experimental cross, seeds that were not used for viability analysis were mixed to generate a random sample of seeds within each cross. These seed mixtures were brought to a site where all three species co-occurred. Seeds were buried in the ground using the modified seed package method of Rasmussen and Whigham (1993), in which the orchid seeds are subjected to the physical and chemical conditions of the soil, and contact with small soil organisms (micro-organisms) is possible, but the seeds are protected from larger soil inhabitants such as millipedes and earthworms (Whigham et al., 2006). In August 2008, approx. 250 seeds were placed within a square of 53 µm mesh phytoplankton netting, enclosed within a Polaroid slide mount. Seed packages were buried along four transects, that were 7·5 m apart. Each transect consisted of ten sample points that were 2 m apart. At each point, nine seed packets (one for each cross) were placed vertically in the ground, leading to a total of 360 seed packages that were left in the ground for about 2 years. In April 2010, seed packages were retrieved, gently washed and maintained moist in paper towels for 1 d until examination. Packages were first rinsed with tap water and then opened with a mini-knife under a dissecting microscope connected to a digital camera. As orchid seed germination stages can be variable (Ramsay et al., 1986), germination was considered when a seed had developed into the protocorm stage, clear signs of mycorrhiza formation were present and the leaf primordia had developed (stage 3 sensu Ramsay et al., 1986). Photographs were taken for each package and the percentage of seed germination was determined by counting the number of seeds that had successfully developed into protocorms and the total number of seeds that had been put in the package. Where seed germination had taken place, protocorms were further assessed for identification of mycorrhizal fungi. To compare mycorrhizal associations of protocorms with those of adult plants, for each species roots were also collected from five adult plants.

Assessment of mycorrhizal fungi

At the site where the seed germination experiment was conducted, mycorrhizal associations were determined for both adult individuals of all three Orchis species and protocorms obtained from the seed packages. For the adults, five individuals were randomly selected for each species and small parts of the roots were cut for mycorrhizal analysis. Roots were surface sterilized (30 s submergence in 1 % sodium hypochlorite, followed by three 30 s rinses in sterile distilled water) and microscopically checked for mycorrhizal colonization. Subsequently, DNA was extracted from 0·5 g of mycorrhizally colonized root pieces per plant using the UltraClean Plant DNA Isolation Kit as described by the manufacturer (Mo Bio Laboratories Inc., Solana Beach, CA, USA). For the protocorms, all protocorms were gently removed from the seed package using sterile forceps, rinsed in sterile distilled water and individually subjected to DNA extraction using the phenol–chloroform extraction method described by Lievens et al. (2003). DNA yield and purity were measured spectrophotometrically. In order to circumvent potential PCR inhibition, extracts were diluted ten times prior to PCR amplification.

The mycorrhizal fungi colonizing the roots or protocorms were assessed using a basidiomycete-centred DNA array enabling the simultaneous detection and identification of 11 operational taxonomic units (OTUs) with ≥97 % ITS sequence similarity (Table 1; Jacquemyn et al., 2010; Lievens et al., 2010). These OTUs, representing different members of Tulasnellaceae (eight OTUs), Thelephoraceae (one OTU), Cortinariaceae (one OTU) and Ceratobasidiaceae (one OTU), had previously been found to associate with Orchis species, including O. anthropophora, O. militaris and O. purpurea. The phylogenetic relationships between these OTUs were presented previously (Jacquemyn et al., 2010; Lievens et al., 2010). Additional sequencing did not yield any new fungal OTUs, suggesting that the previously found mycorrhizal OTUs were representative for the site. DNA arrays were produced according to the DNA array template used by Jacquemyn et al. (2010). DNA array production, PCR amplification and labelling, hybridization, post-hybridization processing and signal detection were performed as described previously (Jacquemyn et al., 2010). However, whereas PCR amplification using samples from adult individuals was performed using the basidiomycete-specific primer pair ITS1-OF and ITS4-OF (Taylor and McCormick, 2008), an additional pair of primers [ITS1 (White et al., 1990) and PH-R1 (Lievens et al., 2010)] was added to the reaction mixture for the protocorm samples to increase the sensitivity of the assay (semi-nested PCR). All DNA samples (1 µl) were amplified according to the same thermal cycling profile: initial denaturation at 94 °C for 2 min, followed by 35 cycles of 45 s at 94 °C, 45 s at 58 °C and 45 s at 72 °C, with a final elongation step at 72 °C for 10 min. Hybridizations were performed twice, but in all cases yielded similar results, demonstrating the robustness of the technique.

Table 1.

Targets of the DNA array used in this study to assess mycorrhizal associations

Data analysis

Determining the extent of post-zygotic reproductive barriers

The effects of pollination treatments on fruit set, seed viability and germination percentages were tested using one-factor fixed effects model analysis of variance (ANOVA). To test whether asymmetries in reproductive barriers occurred between the different species pair combinations, for each pair differences in fruit set, seed viability and germination percentages were tested using t-tests. All data were arcsin transformed prior to analyses.

Components of reproductive isolation (RI) due to sequential post-zygotic barriers were determined using methods outlined in Ramsey et al. (2003). We first computed for each component RI_x as 1 minus the ratio of the fitness of F₁ hybrids to the fitness of the parents. This measure of hybrid fitness varies between 0 and 1, except for comparisons in which the hybrids are fitter than the respective parents (Ramsey et al., 2003). Because crosses were performed reciprocally, for each species combination, two measures for each component of RI were obtained. Finally, for each cross, total post-mating RI was calculated as (Ramsey et al., 2003):

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(1)

where

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(2)

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(3)

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(4)

The subscripts in eqns (1)–(4) refer to RI due to fruit set (1), seed viability (2) and seed germination (3), respectively. Finally, the relative contribution of each of these barriers was calculated as RC_n = AC_n/RI_tot.

Comparison of mycorrhizal associations

After identification of the fungal OTUs associating with adults and protocorms, we first investigated whether the number of fungal OTUs identified differed between adults and protocorms. To do this, the total and average number of fungal OTUs detected in protocorms originating from the different crosses and in adult individuals of each species was first determined. One-factor fixed effects model ANOVA was used to compare fungal OTU richness between adults and protocorms. The same analysis was used to investigate whether fungal richness, i.e. the number of OTUs detected simultaneously in a single adult or protocorm, differed between protocorms obtained from different crosses. Finally, a χ²-test was used to see whether the fungal community differed between protocorms originating from different crosses.

RESULTS

Reproductive barriers

In general, interspecific pollinations generated high levels of fruit set (>80 %; Table 2), indicating that pre-zygotic post-mating isolation was weak. Only in interspecific crosses between O. purpurea^(♀) and O. anthropophora^(♂) was fruit set significantly reduced (<20 %) (Table 2). In contrast, the proportion of viable seeds varied significantly between crosses (F_8,71 = 10·78, P < 0·001), varying between 79·44 % in pure O. anthropophora crosses and 27·47 % in crosses in which O. purpurea acted as receptor plant and O. anthropophora as donor plant (Table 2). Results were also highly asymmetric (Table 2). Seed viability of crosses involving O. militaris as receptor plant was invariably high, whereas in crosses with O. purpurea acting as receptor plant seed viability was always low (<40 %).

Table 2.

Fruit set, seed viability and germination rate of nine different experimental crosses between O. anthropophora (A), O. militaris (M) and O. purpurea (P): the first letter denotes the receptor species, whereas the second letter denotes the donor species (more ...)

The proportion of seed packages in which at least one protocorm was observed varied between 0·39 and 0·96 (Table 2). Seed germination percentages within seed packages (i.e. the number of protocorms relative to the total number of seeds) varied between 0·4 and 5·0 %, corresponding to an average of one and 12 protocorms per paramount slide. Germination rates were particularly low in crosses in which O. anthropophora acted as receptor plant. Even in combination with O. anthropophora as a donor plant seed, germination was not high (germination proportion 0·64) compared with other crosses between two plants from the same species. In contrast, crosses between O. purpurea^(♀) and O. anthropophora^(♂) resulted in the highest germination rates (0·95; Table 2), indicating that low percentages of viable seeds do not necessarily translate into low germination rates. Again, there were strong asymmetries in germination proportions and percentages between crosses.

Components of RI due to fruit set, seed viability and seed germination varied between 0·01 [O. militaris^(♀) × O. purpurea^(♂)] and 0·81 [O. purpurea^(♀) × O. anthropophora^(♂)], between 0·01 [O. militaris^(♀) × O. purpurea^(♂)] and 0·47 [O. purpurea^(♀) × O. anthropophora^(♂)] and between 0·01 [O. purpurea^(♀) × O. anthropophora^(♂)] and 0·41 [O. anthropophora^(♀) × O. militaris^(♂)], respectively. Total post-mating RI calculated using eqns (1)–(4) varied between 0·18 [O. militaris^(♀)× O. purpurea^(♂)] and 0·90 [O. purpurea^(♀) × O. anthropophora^(♂)], indicating that in none of the species crosses was the investigated species completely reproductively isolated from another species due to the investigated post-mating reproductive barriers.

Mycorrhizal associations

Roots of adult individuals of the pure Orchis species were heavily colonized by mycorrhizal fungi. Out of 11 fungal OTUs identified previously (Jacquemyn et al., 2010; Lievens et al., 2010), nine were observed in the investigated adult plants (Fig. 2). Eight were related to Tullasnellaceae, and one to Ceratobasidiaceae (Table 1). The total number of OTUs observed in the five investigated individuals of O. anthropophora, O. militaris and O. purpurea was seven, seven and four, respectively (Fig. 2). On average, adult plants associated with 2·6 (±1·34), 2·6 (±0·89) and 2·2 (±0·84) fungal OTUs simultaneously in O. anthropophora, O. militaris and O. pupurea, respectively.

Fig. 2.

Mycorrhizal associations identified in (A) adults and (B) protocorms obtained from different crosses between O. anthropophora (A), O. militaris (M) and O. purpurea (P). In the experimental crosses, the first letter denotes the receptor species and the (more ...)

All seed packages were colonized by fungal hyphae, and rhizomorphs and protocorms retrieved were heavily colonized by mycorrhizal fungi (Fig. 3). In total, genomic DNA was isolated from 40 protocorms, distributed across all but one cross. Similar to the adult individuals, the vast majority of fungal OTUs detected in protocorms were related to Tullasnellaceae, although OTU 11, which represents Ceratobasidiaceae, was found in >50 % of the crosses (Fig. 2). Both the total and average numbers of fungi observed in protocorms were in general a subset of those observed in adult plants. The total number of fungal OTUs observed in protocorms originating from the different crosses varied between one and four [average: 2·4 (± 0·92)] (Fig. 2). For crosses between O. anthropophora^(♀) and O. purpurea^(♂), no hybridization signals were obtained, not even when using semi-nested PCR amplification. This could be due to the fact that the amount of isolated fungal DNA was below the detection limit of the assay. Another explanation would be that mycorrhizal fungi other than those that could be detected by the DNA array (Table 1) were present. Nevertheless, analysis of a DNA sample from the nylon mesh in which the seeds were packed resulted in positive signals (data not shown), favouring the first explanation.

Fig. 3.

Protocorms retrieved in seed packages originating from crosses between (A) O. militaris^(♀) and O. purpurea^(♂), (B) O. purpurea^(♀) and O. anthropophora^(♂), and (C) O. anthropophora^(♀) and O. militaris^(♂) (more ...)

The different crosses showed significantly different association patterns (χ² = 99·77, d.f. = 42, P < 0·001). Interspecific crosses involving O. militaris and O. anthropophora as acceptor plant always associated with OTU 6, whereas interspecific crosses with O. purpurea as acceptor plant lacked OTU 6. The most prominent OTUs that were observed in protocorms of crosses involving O. militaris^(♀) were OTU 6 and OTU 11, which were also the dominant OTUs in adult plants on the same study site. OTU 7, which was frequently observed in O. anthropophora adults, was also present in crosses in which O. anthropophora acted as receptor plant. OTU 4, which is a regular associate in adult plants of O. purpurea, was also frequently observed in crosses with O. purpurea^(♀), except in pure crosses. In contrast, protocorms of pure O. purpurea crosses associated with OTU 6 and OTU 7, which were lacking in adult individuals (Fig. 2).

DISCUSSION

Reproductive barriers

The study of reproductive barriers (fruit set, seed viability and seed germination) in three related species of Orchis confirmed two important features of reproductive isolation in Mediterranean deceptive orchids that were previously highlighted by Scopece et al. (2007): (1) components of reproductive isolation are asymmetric and (2) reproductive isolation increases with increasing genetic distance between species. Incorporation of seed germination did not alter these general conclusions. However, in contrast to results reported by Scopece et al. (2007) and despite the fact that seed germination was incorporated in our analysis, in none of the crosses was total post-mating reproductive isolation found (minimum RI, 0·18; maximum RI, 0·90). These results indicate that the three species are not completely reproductively isolated from each other, confirming reported evidence (Kretzschmar et al., 2007) of hybridization between all three species in natural populations (see further).

Our results showed that reproductive isolation was asymmetric, and this adds to a growing body of evidence that this is the case in many plant and animal species (Tiffin et al., 2004). Although it remains unclear what may have caused these asymmetries, Scopece et al. (2007) suggested differences in gynostemium length and differential fruit abortion to be important factors. Results from our crossing studies showed that post-mating barriers were mainly due to post-zygotic factors (i.e. seed viability) and less to pre-zygotic factors (fruit set), suggesting that differential fruit abortion is unlikely to explain the observed asymmetries. More probably, substantial differences in the size of pollinia between species (Fig. 1) have contributed to asymmetric isolation patterns. However, it should be stressed that experimental pollinations were performed in pure populations, which may have affected the strength of pre-zygotic post-mating barriers. Because this barrier has been shown to evolve independently of genetic divergence (a proxy of divergence time), it is possible that allopatric populations did not experience the same selective pressures that might have been present in sympatric populations.

Although the association between reproductive isolation and genetic distance tends to vary among species groups (Moyle et al., 2004), a significant positive relationship has recently been reported for a large guild of Mediterranean food-deceptive orchid species (Scopece et al., 2007). Because O. purpurea and O. militaris are regarded as sister species (Bateman et al., 2003), weak reproductive isolation was expected between these two species. Moreover, hybrid swarms are often observed at sites where both species co-occur (Kretzschmar et al., 2007), indicating that hybrids are fertile and that reproductive isolation should be weak. On the other hand, O. anthropophora is genetically more distant from the other two species. In the cladogram of Bateman et al. (2003), it is the first-branching species

Mycorrhizal associations

Whereas the importance of pre- and post-mating barriers has been extensively studied in orchids, particularly in Mediterranean species (Scopece et al., 2007), the role of mycorrhizal associations in contributing to post-mating barriers has not yet been studied in detail, despite their importance in affecting germination and establishment in orchid species (Bidartondo and Read, 2008; Smith and Read, 2008). It can be hypothesized that high specificity and divergent association patterns between species could lead to an effective barrier to hybridization due to incompatibilities between orchid and mycorrhizal fungi. If, on the other hand, orchid species share most of their mycorrhizal fungi, no such incompatibilities are to be expected, and post-mating barriers at the seed germination stage should be weak, implying that mycorrhizal associations only play a minor role in affecting hybridization between species.

Hollick et al. (2005) found that species of the Australian genus Caladenia showed high specificity towards their mycorrhizal partners, as in vitro seed germination trials showed that parental species used different fungi from each other. Hybrids, on the other hand, shared the fungi of one parental species. Recently, Schatz et al. (2010) also documented no detectable differences in mycorrhizal associations between hybrid plants and pure plants of O. simia and O. anthropophora, but no data on germination were reported. Here, we have shown that adult plants of the three species associated with several different fungal OTUs at the same time. However, the relative frequency of the different OTUs differed significantly between species. Consistent with our previous analyses (Jacquemyn et al., 2010; Lievens et al., 2010), O. militaris predominantly associated with OTU 6 and 11, O. anthropophora with OTU 7 and 10, and O. purpurea with OTU 4 and 5.

Protocorms, on the other hand, associated with significantly fewer fungal OTUs, supporting previous results of Bidartondo and Read (2008) that mycorrhizal specificity is higher in the protocorm stage than in the adult stage, in which individual plants associate with a much wider range of mycorrhizal fungi. Our results further corroborate earlier findings of Hollick et al. (2005) that hybrid seeds tend to share fungi of one parental species, most often the receptor plant. For example, all protocorms originating from crosses with O. militaris as receptor plant associated with the most frequently observed fungi in adult specimens of O. militaris. Similarly, the most frequent mycorrhizal fungi observed colonizing protocorms originating from crosses in which O. anthropophora acted as receptor plant were similar to those observed in adult plants. Because our germination experiments were conducted in situ, the results might to some extent have been affected by the spatial distribution of mycorrhizal fungi across the landscape (Jacquemyn et al., 2007). To present a more coherent picture of mycorrhizal specificity, in situ experiments should ideally be complemented with in vitro germination experiments (Swarts et al., 2010). However, despite numerous trials, culturing isolated Tulasnella fungi has proved unsuccessful.

The extent of hybridization between O. anthropophora, O. militaris and O. purpurea in natural populations

Hybridization between O. militaris and O. purpurea has been frequently reported, and in sites where both species occur together hybrid swarms are often observed (Kretzschmar et al., 2007). In contrast, hybrids between O. anthropophora and the other two species appear to be rare, but, in contrast to what is indicated by previous results (Scopece et al., 2007), are most probably the result of pre- rather than post-mating isolation. Our results showed that none of the post-mating barriers up to the seed germination stage led to total reproductive isolation of one of the species, and in all cases in which O. anthropophora acted as receptor plant relatively low levels of post-mating reproductive isolation were observed.

Orchis anthropophora is primarily pollinated by Cidnopus beetles (Schatz, 2006), whereas O. militaris and probably also O. purpurea are predominantly pollinated by short-tongued bees (Vöth 1987). Moreover, the flowers of O. anthropophora differ substantially from those of O. purpurea and O. militaris in colour, shape (Fig. 1) and, probably, scent profile (Schatz et al., 2010). Thus it is not unlikely that differences in pollinator assemblages may have contributed to rarity of hybrids between O. anthropophora on the one hand and O. militaris or O. purpurea on the other hand. To investigate the role of pre-mating barriers on total reproductive isolation, we therefore calculated total reproductive isolation resulting from both pre- and post-mating barriers using similar methods outlined in Scopece et al. (2007) (see Appendix for a list of pollinators). From the results it becomes clear that pre-mating barriers indeed appear to be the most important factor restricting hybridization between O. anthropophora on the one hand and O. militaris or O. purpurea on the other (Fig. 4).

Fig. 4.

Total reproductive isolation incorporating both pre- and post-mating barriers up to seed germination and relative contributions of reproductive barriers of reciprocal crosses between O. anthropophora, O. militaris and O. purpurea. In all crosses, the (more ...)

CONCLUSIONS

Although our sampling size was still rather small, our results indicate that seedlings originating from crosses between different species showed significantly different mycorrhizal associations. However, because mycorrhizal fungi detected in seedlings were a subset of those observed in adult plants, they probably contribute little to reproductive isolation. On the other hand, because seed germination in orchids is likely to be sensitive to the contingencies of spatial distribution of suitable mycorrhizal fungi (Diez, 2007; Jacquemyn et al., 2007), seed germination of hybrid seeds and thus hybridization rates in natural populations are most probably also indirectly affected by mycorrhizal fungi. Future research should therefore incorporate the spatial extent of mycorrhizal availability in natural populations to grasp fully the role of mycorrhizal fungi in affecting hybridization in orchid species.

ACKNOWLEDGEMENTS

This work was funded by the Flemish Fund for Scientific Research (grant: G.0592·08). H.J. acknowledges funding from the European Research Council (ERC starting grant 260601 – MYCASOR). Two anonymous reviewers provided very useful comments on a previous draft of this manuscript.

APPENDIX

List of different insects observed visiting flowers of O. purpuea, O. anthropophora and O. militaris.

Orchid species Orchis purpurea	Insect species Epicometis hirta	Insect family Coleoptera	Source http://perso.normandnet.fr/sfonormand/pollinisateurs.htm
	Bee	Long-tongued bees	Lang (2004)
	Odynerus parietus	Long-tongued bees	Lang (2004)
	Pieris brassicae	Lepidoptera	H. Jacquemyn (pers. obs.)
	Pieris napi	Lepidoptera	H. Jacquemyn (pers. obs.)
	Bombus sp.	Long-tongued bees	H. Jacquemyn (pers. obs.)
	Flies	Diptera	H. Jacquemyn (pers. obs.)
	Andrena sp.	Short-tongued bees	H. Jacquemyn (pers. obs.)
	Ants	Other Hymenoptera	H. Jacquemyn (pers. obs.)
Orchis anthropophora	Cidnopus minutus	Coleoptera	Van der Cingel (1995)
	Tenthredopsis sp.	Other Hymenoptera	Schatz (2006)
	Arge sp.	Other Hymenoptera	Schatz (2006)
	Cidnopus sp.	Coleoptera	Van der Cingel (1995)
	Cantharis sp.	Coleoptera	Schatz (2006)
	Cidnopus sp.	Coleoptera	Schatz (2006)
	Hoverflies	Diptera	Lang (2004)
Orchis militaris	Leucozona lucorum	Diptera	Farrell (1985)
	Melanostoma mellinum	Diptera	Farrell (1985)
	Onesia sp.	Diptera	Farrell (1985)
	Opomyza germinationis	Diptera	Farrell (1985)
	Platycheirus scutatus	Diptera	Farrell (1985)
	Rhingia campestris	Diptera	Farrell (1985)
	Scatophagia stercoraria	Diptera	Farrell (1985)
	Thricops semicinereus	Diptera	Farrell (1985)
	Bombus lapidarius	Long-tongued bees	Farrell (1985)
	Bombus lucorum	Long-tongued bees	Farrell (1985)
	Myrmica ruginodis	Other Hymenoptera	Farrell (1985)
	Pieris brassicae	Lepidoptera	Farrell (1985)
	Pieris napi	Lepidoptera	Farrell (1985)
	Andrena curvungulata	Short-tongued bees	Farrell (1985)

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