Evol Ecol DOI 10.1007/s10682-010-9356-7

ORIGINAL PAPER

Absence of pollinator-mediated premating barriers in mixed-ploidy populations of Gymnadenia conopsea s.l. (Orchidaceae)

Jana Jersa´kova´ • Sı´lvia Castro • Nicole Sonk • Kathrin Milchreit • Iva Scho¨delbauerova´ • Till Tolasch • Stefan Do¨tterl

Received: 28 August 2009 / Accepted: 11 January 2010 Ó Springer Science+Business Media B.V. 2010

Abstract Polyploidy has played a key role in plant evolution and diversification. Despite this, the processes governing reproductive isolation among cytotypes growing in mixed- ploidy populations are still largely unknown. Theoretically, coexistence of diploid and polyploid individuals in sympatric populations is unlikely unless cytotypes are prezygoti- cally isolated through assortative pollination. Here, we investigated the pre-mating barriers involved in the maintenance of three co-occurring cytotypes from the genus Gymnadenia (Orchidaceae): tetraploid and octoploid G. conopsea and tetraploid G. densiflora.We assessed differences in flowering phenology, floral morphology, and visual and olfactory cues, which could lead to assortative mating. Gas chromatography coupled with

Electronic supplementary material The online version of this article (doi:10.1007/s10682-010-9356-7) contains supplementary material, which is available to authorized users.

J. Jersa´kova´ (&) I. Scho¨delbauerova´ Department of Theoretical Ecology, Institute of System Biology and Ecology AS CR, Na Sa´dka´ch 7, 37005 Cˇ eske´ Budeˇjovice, Czech Republic e-mail: [email protected]

J. Jersa´kova´ I. Scho¨delbauerova´ Faculty of Science, University of South Bohemia, Branisˇovska´ 31, 37005 Cˇ eske´ Budeˇjovice, Czech Republic

S. Castro Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, PO Box 3046, 3001401 Coimbra, Portugal

S. Castro Institute of Botany, Academy of Sciences, Za´mek 1, 25243 Pru˚honice, Czech Republic

N. Sonk K. Milchreit S. Do¨tterl Department of Plant Systematics, University of Bayreuth, Universita¨tsstrasse 30, 95447 Bayreuth, Germany

T. Tolasch Institut fu¨r Zoologie, Fg. Tiero¨kologie, Universita¨t Hohenheim, Garbenstraße 30, Stuttgart 70593, Germany 123 Evol Ecol electroantennographic detection was used to identify scent compounds with physiological activity in the two main pollinators, Deilephila porcellus and Autographa gamma. The importance of olfactory cues was also assessed in the field by analysing the moths’ responses to the olfactory display of the plants, and by following the pollinator’s behaviour on artificial arrays. Our complex approach demonstrated that the coexistence of Gymnadenia cytotypes in mixed-ploidy populations was only partly explained by differences in floral phenology, as cytotypes with overlapping flowering (i.e., octoploid G. conopsea and tetraploid G. densiflora) might freely exchange pollen due to only 1 mm differences in spur lengths and the lack of assortative behaviour of pollinators. While floral colour among the cytotypes was similar, floral scent differed significantly. Though both pollinator species seemed to phys- iologically detect these differences, and the floral scent alone was sufficient to attract them, pollinators did not use this cue to discriminate the cytotypes in the field. The absence of pre- mating barriers among cytotypes, except partial temporal segregation, suggests the existence of other mechanisms involved in the cytotypes’ coexistence. The genetic differences in ITS sequences among cytotypes were used to discuss the cytotype’s origin.

Keywords Cytotypes Floral volatiles Fragrant orchid Gas chromatography– electroantennographic detection Matting barriers Polyploidy

Introduction

Unlike other speciation processes, polyploidization is often assumed to confer immediate reproductive isolation and it is widely recognized as a major mode of sympatric speciation in flowering plants (Coyne and Orr 2004; Soltis et al. 2009). In the early stages of establishment, a neopolyploid suffers from its minority in a population (Levin 1975; Felber 1991) until the evolution of breeding barriers increases the probability of its successful mating (Rieseberg and Willis 2007). The reproductive barriers involved in the coexistence of closely related cytotypes include microhabitat differentiation resulting in spatial separation at small scale (Felber-Girard et al. 1996), differences in floral morphology resulting in species-specific placement of pollen on the pollinator’s body (Grant 1994), flowering time divergence leading to non-overlapping phenologies (Petit et al. 1997; Nuismer and Cunningham 2005), or dif- ferent pollinator behaviours leading to pollinator-mediated isolation (Husband and Schemske 2000; Thompson and Merg 2008). Such pre-mating barriers may be especially advantageous in reducing the reproductive cost associated with pollen wasted to stigmas of incompatible cytotypes (Harder and Wilson 1998), stigma clogging with incompatible pollen (Lloyd and Webb 1986), and subsequent ovule discounting (Barrett et al. 1996). Although pre-zygotic isolation via assortative mating has been documented as a major breeding barrier in closely related plant taxa (Jones 1978; Johnston 1991; Grant 1992), its role in polyploid systems is largely unexplored (but see Husband and Sabara 2004; Kennedy et al. 2006). For assortative mating to develop, the divergent cytotypes need to adapt to different pollinator taxa or need to diverge sufficiently for individual pollinators to use their variable trait(s) as a basis for constancy or labile preference, thereby improving pollen targeting (Jones 2001). Floral preference occurs when a pollinator visits a specific cytotype regardless of its proportion in the environment, while floral constancy results from a learned tendency of an individual pollinator to visit the same cytotype consecutively on a short term basis, skipping the alternative cytotypes present in the population (Waser 1986; Dafni et al. 2005). Assortative mating via non-random foraging strategies is only possible when cytotypes differ in floral traits that can be recognized and used by pollinators. Features such as floral 123 Evol Ecol rewards, floral display size or floral scent composition have already been observed to be involved in non-random mating in several plant taxa (e.g., Ellis and Johnson 1999; Riffell et al. 2008). Nevertheless, in polyploid complexes the information is rather scarce. The available studies revealed different foraging patterns for pollinators in mixed-ploidy popu- lations (Husband and Schemske 2000; Kennedy et al 2006) and a significant role of floral morphology in pollinator preferences (Cook and Soltis 1999; Segraves and Thompson 1999). To date, however, little is known about the importance of other floral traits that may even evolve primarily to improve pollen targeting rather than to increase pollinator visitation rates. In this study we used a complex of Gymnadenia conopsea s.l. orchids as a model to investigate pre-mating barriers involved in the maintenance of polyploids. This complex is a good model to study this issue as it comprises different cytotypes (namely, tetraploids and octoploids) growing intermingled in mixed-ploidy populations (apparently without spatial segregation, Suda et al. pers. comm.), exhibiting high variation in floral phenology, morphology and scent composition, and pollinated by a large spectrum of diurnal and nocturnal moths (Vo¨th 2000; Marhold et al. 2005). Specifically, we asked the following questions: (1) Are there differences in floral phenology leading to temporal segregation of the cytotypes? (2) Are there morphological, visual and/or olfactory differences among G. conopsea s.l. cytotypes? (3) Do the pollinators respond to the differences in adver- tisement among the cytotypes? And (4) do the differences in floral traits correspond to genetic differentiation of the cytotypes? To address these questions we evaluated pheno- logical, morphological, visual and olfactory features of the flowers from each cytotype through morphometric, optical and scent analyses. Scent compounds were identified and their physiological activity in pollinators assessed through electroantennographic analyses. Natural behaviour of the pollinators to the olfactory display and to artificial arrays com- posed of different cytotypes was assessed in the field. Finally, we used DNA markers to explore the genetic relationship among the cytotypes.

Materials and methods

Study system

Gymnadenia conopsea (L.) R.Br. s.l. is a perennial orchid widely distributed through Europe and Asia (Hulte´n and Fries 1986). The plants grow in open meadows from lowland up to subalpine levels. The most recent classification recognizes two taxa within the complex: G. conopsea (L.) R.Br. s.s., comprising both tetraploid (4xGc) and octoploid individuals (8xGc), and G. densiflora A. Dietr. comprising only tetraploid plants (4xGd). In Central Europe the three cytotypes occur in pure and mixed populations growing intermingled in variable proportions (Marhold et al. 2005; Suda et al. pers. comm.). The complex is highly variable with regards to morphology (Dworschak 2001, Sup- plement 1), flower colour, scent production and flowering phenology (Marhold et al. 2005). The available information in the literature indicates that 4xGcflowers earlier in the spring (May–June), while 4xGdand 8xGcflower later (July–August). The scent of the flowers perceived by humans has also been used as a discriminatory character, with 4xGcand 8xGcproducing a slightly unpleasant vanilla-like scent, and 4xGdpresenting a pleasant spicy to carnation-like odour (Marhold et al. 2005). The flowers are arranged in a fairly dense spike with 10–80 flowers varying from pale pink to violet. The flowers are self-compatible, yet spontaneous autogamy or apomixis is absent, and significant inbreeding depression was detected in artificially selfed seeds 123 Evol Ecol

(Jersa´kova´, unpublished data). The abundant nectar and strong scent produced by the inflorescences attract a diverse range of diurnal and nocturnal moths (Vo¨th 2000). In the study populations the most frequent pollinators were the nocturnal hawkmoth Deilephila porcellus L. and the noctuid moth Autographa gamma L. (Supplement 1). Floral nectar is abundant (standing crop ranges from 0 up to 4.5 ll) accumulating in a long slender spur with no major differences in sugar (sucrose) concentration of nectar among the cytotypes (mean ± SE: 4xGc= 27.3% ± 0.9, n = 60; 8xGc= 25.8% ± 0.9, n = 51; 4xGd= 27.8% ± 1.1, n = 38; one-way ANOVA of log-transformed data F2,146 = 1.05, P = 0.35). Despite the differences in scent and phenology, recognition of the cytotypes in the field is rather difficult due to large morphological variability. Fortunately, flow cytometry turned out to be a useful tool for cytotype identification producing species-specific histograms (Dolezˇel et al. 2007).

Study sites

We focused on two mixed-ploidy and three pure populations of G. conopsea s.l. occurring in the White Carpathians situated in the southeast of the Czech Republic from seasons 2006 until 2008 (for details see Table 1). Scent and genetic samples were collected in all populations, and plant phenology, floral traits and pollinator behaviour were studied in the mixed-ploidy populations.

Flowering phenology

To follow the flowering time in detail, we marked 201 and 100 inflorescences in bud in NR Zahrady pod Ha´jem and NR Jazevcˇ´ı, respectively. The number of open flowers per inflorescence was censused once a week from 11 June till 12 July in 2006. Each plant was assigned to a cytotype by flow cytometric analysis. To compare the differences in the flowering curves among the cytotypes, we fitted the number of open flowers of individual plants with a Gaussian curve to assess the flowering peak. The differences in flowering peak were tested by one-way ANOVA, followed by a Tukey test (all statistical analyses were performed with STATISTICA unless otherwise stated). In addition, we

Table 1 Description of study populations of Gymnadenia conopsea s.l. cytotypes Site description, village Coordinates Cytotype composition

4xGc 8xGc 4xGd

Nature reserve Zahrady pod Ha´jem (Velka´ nad 48°53021.4800N 32% (14, 2) 42% (10, 1) 26% (9, 1) Velicˇkou) 17°31053.8700E Nature reserve Jazevcˇ´ı (Javornı´k) 48°52027.8300N 58% (9, 1) 42% (9, 1) 17°34014.3600E Nature reserve Javorˇina (Stra´nı´)48°51013.8000N 100% (3, 1) 17°40005.1300E Nature reserve Kamenny´ vrch (Kurdeˇjov) 48°57049.7800N 100% (7, 1) 16°45021.700E Nature monument Hrncˇa´rky (Stra´nı´)48°54030.7400N 100% (3, 2) 17°40033.9200E Populations are characterized by cytotype composition (given in percentages). The numbers in the brackets denote the number of scent and genetic samples analyzed, respectively. 4xGc– tetraploid G. conopsea, 8xGc– octoploid G. conopsea,4xGd– tetraploid G. densiflora 123 Evol Ecol calculated an index of overlap by first expressing the number of open flowers per cytotype at each census as a percentage of the total number of open flowers observed for that cytotype across all census periods, and then for each census date taking the value for the cytotype that had the lower percentage of open flowers and summing these values across all census dates. This index represents the proportion of the flowering curves that overlap and will equal 0 when flowering is completely asynchronous, or 1 when there is complete overlap (modified from Husband and Schemske 2000).

Floral morphology

Due to the importance of spur length in plant evolution and pollen transfer efficiency (Johnson and Steiner 1997; Young 2008), we measured and compared spur length among cytotypes in the two mixed-ploidy populations. Spur length was considered to be the distance between the stigma and the tip of the spur. Differences in spur length among three cytotypes were assessed by one-way ANOVA, followed by a Tukey test. The interactions between factors (cytotype and population) were evaluated using a two-way ANOVA, excluding 4xGdas it grows only in one of the studied populations.

Floral spectral reflectance

To obtain an accurate estimate of similarity in flower colour among cytotypes, spectral reflectance over the UV–visible range (300–700 nm) was measured for 12 flowers of each cytotype using an Ocean Optics S2000 spectrometer and an Ocean Optics DT-mini light source (200–1100 nm; Dunedin, Florida, USA). Readings were taken through a fiber-optic reflection probe (UV/VIS 400 lm) held at 45° and about 5 mm from the surface of the labellum.

Floral scent collection

The selection of plants for floral scent analyses was based on previous cytometrical screening, plant morphology and flowering phenology. The floral scent was sampled through headspace sorption for 2–3 h between 20:00 and 23:00 h (GMT?1), corresponding with the maximum release of floral scent and peak in pollinator activity. The number of sampled plants per cytotype and population is presented in Table 1. Inflorescences were inserted into PET cooking bags (Toppits, Germany), and a glass cartridge filled with 30 mg of Porapak Q (Alltech Associates, USA) was connected to the bag. Air passed through the filter at a rate of approximately 200 ml min-1 using a PAS-500 personal air sampler (Spectrex Corp., USA). Before use, the Porapak was cleaned with 1 ml of acetone (Merck, Suprasolv). During cytotype sampling, ambient air was also collected to control for background contamination. After odor collection, adsorbed volatiles were eluted with 100 ll of acetone and stored at -80°C until analysis. The extracts were used for two purposes: (1) for gas chromatography coupled to mass spectrometry (GC-MS; all samples), and (2) for gas chromatography coupled to electroantennographic detection (GC-EAD; nine samples).

GC-MS analyses

Samples were analyzed on a Saturn 2000 mass spectrometer (MS) coupled to a Saturn 3800 gas chromatograph (GC) using a 1079 injector (all Varian, USA), fitted with the ChromatoProbe kit (Do¨tterl et al. 2005b). One microlitre of each sample was filled in a 123 Evol Ecol quartz vial, which was placed in the injector port by means of the ChromatoProbe, and analysed on a ZB 5 column (Phenomenex) with the same GC and MS settings as described by Do¨tterl et al. (2005b). Separation of vicinal dimethyl thioderivatives of extract compounds, produced for the determination of double bond positions, was carried out on a 30 m9 0.25 mm i.d., 0.25 lm film thickness HP5-MS fused silica capillary column (Agilent Technologies, Santa Clara, CA, USA), starting at 60°C for 3 min, then programmed to 300°C at a rate of 3°C min-1. The final temperature was held for 70 min. Reaction of double bonds was determined using dimethyl disulfide (DMDS) (Buser et al. 1983). Analysis of the data was performed using the Saturn Software package 5.2.1. To identify the floral scent compounds of the GC-MS spectra, the databases NIST 02 and MassFinder 3 were used, and identifications were confirmed by comparison of Kovats retention indices with published data (Adams 2007). Identification of some compounds was also verified by comparison of mass spectra and retention times with those of authentic standards. Scent samples were used to determine the compounds emitted, and the contribution of the single compounds to the total scent (percentage amount). Scent quantity (total amount of scent emitted) was not determined. To test for differences in scent among cytotypes and populations (some of them were pure and contained only one cytotype, while others were mixed, see Table 1) we calculated a Bray-Curtis similarity matrix based on the percentage amount of compounds in the samples, and used this (semi)quantitative matrix to run a PERMANOVA analysis (Anderson et al. 2008) with the fixed factors cytotype and population in a crossed design. PERMANOVA is a technique for testing the simultaneous response of one or more variables to one or more factors in an ANOVA experimental design on the basis of a (dis)similarity matrix, using permutation methods. A SIMPER analysis was used to determine the compounds being most typical (high percentage amount and presence) for individual cytotypes and being responsible for dif- ferences among them. This test was run in a one-way design as we found no population effect on scent of the different cytotypes (see results). All analyses were performed in Primer version 6.1.11 (Clarke and Gorley 2006).

Electrophysiological analyses

To determine which floral scent compounds can be detected by the antennae of the most abundant visitors of G. conopsea s.l., we performed GC-EAD with Autographa gamma and Deilephila porcellus. We tested 3 scent samples of each cytotype totaling 25 runs (9 runs with 4xGcand 4xGdeach; 7 runs with 8xGc) on the antennae of 11 A. gamma and 11 D. porcellus individuals (one antenna per individual). In three of the D. porcellus indi- viduals both antennae were used for measurements. Insects were trapped at NR Zahrady pod Ha´jem (Table 1) and on a clover meadow near village Dobrsˇ´ın in South Bohemia (49°150N, 13°330E), and sent by overnight express to Bayreuth, where measurements were conducted. The analyses (1 ll of an acetone-floral scent sample was injected in splitless mode) were performed with the same GC-EAD system and same settings as described by Do¨tterl et al. (2005a). The system consisted of a gas chromatograph (Vega 6000 Series 2, Carlo Erba, Rodano, Italy) equipped with a flame ionization detector (FID), and an EAD setup (heated transfer line, 2-channel USB acquisition controller) provided by Syntech (Hil- versum, Netherlands). For the EAD, both sides of an excised antenna were plugged into 123 Evol Ecol glass micropipette electrodes filled with ringer solution (8.0 g l-1 NaCl, 0.4 g l-1 KCl, -1 4gl CaCl2) and connected to silver wires. To identify the structure of the compounds eliciting signals in the insect antennae, the chromatograms obtained from the FID of the GC-EAD system were compared with the results obtained by GC-MS.

Pollinator behaviour

To evaluate the occurrence of floral preference and constancy between Gymnadenia cytotypes we assessed the pollinator’s behaviour in artificial arrays constructed within the mixed-ploidy population of NR Zahrady pod Ha´jem. The artificial arrays were composed of a total of 12 inflorescences from the 8xGcand 4xGdcytotypes that overlapped in flowering phenology. The inflorescences were cut in the evening, placed in test tubes with water and fixed in the soil. Each inflorescence was used over two nights to minimize the number of cut plants. The comparison of scent samples collected from freshly cut, 1-day old and non-cut inflorescences revealed no significant changes in the scent composition, except for the amount of scent, which was lower in 1-day old inflorescences (data not shown). The inflorescences of each cytotype were displayed alternately in two rows and separated from direct neighbors by 30 cm. Two to three arrays were displayed from 20:30 until 22:30 h (GMT?1), during the main foraging activity of the pollinators. The obser- vations were performed during 8 days in 2008, summing a total of 16 h of net observation. During the observation periods, insects interacting efficiently with the inflorescences of the array and their movements were recorded with a digital voice-recorder. Indices of floral preference and constancy for a specific Gymnadenia cytotype were calculated for each pollinator that efficiently visited more than four inflorescences during a bout. Floral preference was calculated as the proportion of visits to 4xGd. Values of 0.5 indicate no preference by a pollinator, while values of 0 or 1 reveal a preference for a specific cytotype (namely for 8xGcor 4xGd, respectively). Floral constancy was calcu- lated as a ratio between the number of conspecific movements and the total number of movements during a bout. Values of 0 indicate alternating foraging, 0.5 represents random foraging and 1 represents constant foraging. Significant deviations of a pollinator’s behaviour from 0.5 were tested by using a One Sample Wilcoxon Sign Rank Test. The differences in the foraging behaviour between hawkmoths and noctuids were assessed using the non-parametric Mann–Whitney U-test. To determine whether pollinators respond to floral scent in the field we performed an experiment in which we removed the visual cues of a Gymnadenia cytotype, but allowed fragrance to still be perceived. We constructed an array composed of 6 inflorescences covered in black cloth. Half of inflorescences contained a flowering spike of 8xGc, the second half served as an empty control. The inflorescences were displayed for one night only in an alternated manner as in the previous experiment. We observed pollinator behaviour over 3 days in 2008. As pollinators rarely approached more than one inflores- cence within an array, we used a Chi-square test to compare the observed and expected frequencies of initial choice of inflorescence.

Genetic analyses

One to two individuals of each cytotype per study site were randomly chosen for sequence analyses using nuclear and chloroplast DNA markers (Table 1, Supplement 2). Total DNA was extracted from leaf material stored in silica gel using a DNA isolation kit (Invisorb 123 Evol Ecol

Spin Plant Mini Kit, INVITEK, Germany). PCR was performed using TC-XP Bioerg and Biometra T3000 cyclers (Spectrex Corp., USA) in a total reaction volume of 25 ll, con- taining 1 ll template DNA, 19 Plain PP Master Mix (Top Bio) and 0.3 lmol/l of each primer. ITS sequences were amplified using combinations of nrDNA primers ITS1P ? ITS4 (Selosse et al. 2002), and cpDNA primers ndhJ (Shaw et al. 2007) with TabE (Taberlet et al. 1991), psbA ? trnH (Aldrich et al. 1988) and sak23F ? sak24R (Watts et al. 2008). The primer ITS1P was used instead of the standard universal ITS1, as Gymnadenia leaves are often contaminated by fungal DNA (Kuba´tova´, pers. comm.). The PCR cycling conditions of individual primer sets followed those of the above-mentioned studies exactly. The quality of the PCR products was checked on 1.5% agarose gels before purification with ExoSAP-IT (USB Corporation) according to the manufacturer’s instructions. The sequencing reactions were performed at Macrogen Inc. (Seoul, South Korea) using a Big DyeTM terminator cycle sequencing kit on an ABI3730XL sequencer (Applied Biosystems, USA). Sequences were edited and assembled using ChromasPro, version 1.41. The boundaries of the ITS1 and ITS2 regions were determined from orchid sequences published in Carlsward et al. (2006).

Results

Differences in floral traits among cytotypes

The study of floral phenology revealed significant differences in flowering peaks among Gymnadenia cytotypes (one-way ANOVA: F2,295 = 728.7, P = 0.0001): the 4xGc flowered significantly sooner than both 8xGcand 4xGd(Fig. 1). At NR Jazevcˇ´ı, the flowering curves of 4xGcand 8xGcoverlapped by 12%. At NR Zahrady pod Ha´jem, the

1.0 4x Gc (Z) 8x Gc (Z) 4x Gd (Z) 4x Gc (J) 0.8 8x Gc (J)

0.6

0.4 Proportion of open flowers 0.2

0.0 10.6. 18.6. 26.6. 4.7. 12.7. Date

Fig. 1 Flowering phenology of Gymnadenia cytotypes (4xGc– tetraploid G. conopsea,8xGc– octoploid G. conopsea and 4xGd– tetraploid G. densiflora) in two mixed-ploidy populations (Z – NR Zahrady pod Ha´jem, J – NR Jazevcˇ´ı) recorded as the proportion of open flowers from 11 June until 12 July 2006 123 Evol Ecol

flowering curves of 4xGcoverlapped by 19% with 8xGcand by 6% with 4x Gd, while the curves of the two cytotypes flowering later (8xGcand 4xGd) overlapped by 77%. In addition, the flow cytometric screening revealed the occurrence of three and one hybrid plants (hexaploids) at sites NR Zahrady pod Ha´jem and NR Jazevcˇ´ı, respectively. The flowering time of hybrids overlapped by 90% with that of 8xGc. All cytotypes significantly differed from each other in floral spur length (mean ± SE: 4xGc= 15.5 ± 0.15 mm, n = 116; 8xGc= 17.5 ± 0.12 mm, n = 211; 4xGd= 16.5 ± 0.19 mm, n = 80; one-way ANOVA: F2,404 = 55.8, P = 0.0001), and the dif- ference between 8xGcand 4xGcwas significant for both populations studied (two-way ANOVA, interaction cytotype 9 population: F1,323 = 0.30, P = 0.58). Regarding floral colour, the flowers of the cytotypes presented similar spectral reflec- tance (95% confidence intervals of the mean curves substantially overlapped), with only slight differences in the overall brightness (Fig. 2). In total, 112 compounds were found in the 64 scent samples analyzed, and 76 of them could be identified (Supplement 3). The samples collected from 49 Gc contained on average 44, from 89 Gc 49, and from 49 Gd 36 compounds. The dominant compound classes were benzenoids (20 compounds), fatty acid derivatives (48), and phenylpropa- noids (20), and an additional few monoterpenoids, sesquiterpenoids, and nitrogen-bearing compounds were found. The most commonly occurring compounds were the phenyl- propanoids elemicin, (E)-isoelemicin (found in all samples), (E)-methylisoeugenol (63 samples), and eugenol (61 samples). The most abundant compounds (percentage amount) over all the samples were eugenol (median 26%), indole (9%), elemicin (7%), and methyleugenol (6%). On a qualitative level, major differences were found between G. conopsea and G. densiflora. Both cytotypes of G. conopsea emitted several fatty acid derivatives, which were completely absent in G. densiflora (Supplement 3), and on the other hand G. densiflora emitted some benzenoids and phenyl propanoids, which were not found in G. conopsea. Qualitative differences between 4xGcand 8xGcwere comparatively small, and these cytotypes emitted nearly the same spectrum of compounds. On a semiquantitative level, significant differences were found in the relative amount (percentage) of compounds emitted by the different cytotypes (Fig. 3; PERMANOVA: Pseudo-F = 16.72, P \ 0.001), with no significant population effect (PERMANOVA: Pseudo-F = 1.39, P = 0.13). Further, there was no significant interaction between the factors cytotype and population (PERMANOVA: Pseudo-F = 1.34, P = 0.23), indicating

100 4x Gc 8x Gc 4x Gd 80

60

40

20

0

Spectral reflectance (%) 300400 500 600 300 400 500 600300 400 500 600 700 Wavelength (nm)

Fig. 2 The spectral reflectance of flowers of three Gymnadenia cytotypes: (4xGc– tetraploid G. conopsea, 8xGc– octoploid G. conopsea and 4xGd– tetraploid G. densiflora). Grey lines depict individual flowers; black line indicates a mean 123 Evol Ecol

Cytotypes 4x Gc mixed 1 4x Gc pure 8x Gc mixed 3 8x Gc pure 4x Gd mixed 4x Gd pure

1: Benzyl benzoate 2: Eugenol 2 3: Elemicin 4: Indole 4

Fig. 3 Nonmetric multidimensional scaling of the floral scent profile of scent samples collected from Gymnadenia plants of the three different cytotypes (4xGc– tetraploid G. conopsea,8xGc– octoploid G. conopsea and 4xGd– tetraploid G. densiflora)) growing in mixed and pure populations based on Bray-Curtis similarities, which were built on the basis of the percentage amount of the compounds in the single samples. Samples placed close to each other in two-dimensional odor space were similarly scented, samples placed far away had quite different scent patterns. Compounds most responsible for the ordination of samples along the axes are also presented that the significant cytotype effect occurred in all populations regardless their cytotype composition. A post-hoc test revealed that all three cytotypes differed from each other (P \ 0.001). However, differences were bigger between G. densiflora and G. conopsea than between 4xGcand 8xGc(Fig. 3). A SIMPER analysis and a comparison of the relative amounts of compounds revealed the compounds being characteristic for a specific cytotype and also the compounds being responsible for the differences among the cytotypes. The most characteristic compound in 4xGcwas indole. It occurred in all 26 samples, and was most abundant (median 19%). Two further compounds contributed with a median of more than 5% to the total scent of this species, i.e., (Z)-7-dodecenyl acetate, and dodecyl acetate, and these compounds were also frequently found (25 and 26 samples, respectively). Altogether, these three com- pounds explained more than half of the similarity found within this cytotype. In 8xGc, eugenol was the most typical compound. It occurred in all 26 samples, and contributed in the median 36% to the total scent. Elemicin reached in the median 13% (26 samples), methyleugenol and indole 8% each (25 and 26 samples, respectively), and (Z)-7- dodecenyl acetate 5% (26 samples). Together, these five compounds explained more than 80% of the similarity in this cytotype. Eugenol was also the most characteristic compound in 4xGd(41%, all 12 samples), where also benzyl benzoate (26%, 12 samples), benzyl acetate (8%, 12 samples), and methyleugenol (6%, 11 samples) were found in relative amounts of more than 5%. These four compounds explained almost 90% of the similarity among the samples of this cytotype. Therefore, compounds being most responsible for the differences among the cytotypes were, according to SIMPER: eugenol, which dominated the samples of 8xGcand 4xGd, but not the samples of 4xGc; indole, which dominated the samples of 4xGc, and which was found in lower amounts in 8xGc and only in trace amounts in very few 4xGd samples; benzyl benzoate, which was found in high amounts in 4xGdand absent in the

123 Evol Ecol other cytotypes; and elemicine, which was found in high amounts in 8xGc, and in lower amounts in 4xGcand 4xGd. The antennae of A. gamma and D. porcellus responded to 24 compounds emitted by the different cytotypes, among them several benzenoids, fatty acid derivatives, and phenyl- propanoids. Both moth species responded to a similar compound spectrum, and responses were elicited to the most abundant Gymnadenia compounds (e.g., eugenol, indole), but also to minor compounds. Antennae responded to a similar compound spectrum when testing them on 4xGcand 8xGc, but there were clear differences between samples of G. conopsea and G. densiflora (Table 2; Fig. 4).

Pollinator behaviour

Mean values for the index of floral preference were 0.51 and 0.50 for hawkmoths and noctuids, respectively, and were not significantly different from 0.5 (Table 3). This revealed that both groups of pollinators had no preference for a specific cytotype when foraging in the experimental arrays of 89 Gc and 49 Gd (Table 3). However, the indices of floral constancy were significantly different between pollinator groups (M–W U test: Z = 2.51, df = 25, P = 0.011), indicating differences in pollinator’s behavior. Hawk- moths had a mean index of floral constancy significantly lower than 0.5, corresponding to an alternating behaviour when foraging in the experimental array (Table 3); while noctuids showed a more diverse behaviour with a mean index of floral constancy not significantly different from 0.5, corresponding to a random behaviour when foraging in the experi- mental array (Table 3). It is also interesting to note that hawkmoths visited a larger amount of inflorescence during a bout (on average 10 plants in the array; n = 16) than noctuids (mostly 3 plants; n = 59). The field experiment with bagged inflorescences confirmed that floral scent alone is sufficient to attract pollinators in the absence of visual cues. During their initial choice, 2 hawkmoths and 15 noctuids approached the cloth-bagged natural flowers, only 1 noctuid approached an empty control (Chi-square = 12.5, df = 1, P = 0.0004).

Genetic differentiation

Sequencing analysis of 3 non-coding cpDNA regions revealed no interspecific variability among the Gymnadenia cytotypes, while the sequences of ITS 1 and ITS2 regions sepa- rated 4xGdfrom both 4xGcand 8xGc, through 12 variable nucleotide sites (Table 4). The ITS sequences of both G. conopsea cytotypes were identical.

Discussion

Contact zones of different cytotypes of a species or a species complex provide natural laboratories for studying evolutionary transitions (Lexer and van Loo 2006). Recently, with the development of flow cytometry as a tool for assessing DNA content and ploidy in plants, large-scale assessment of cytotype distribution patterns has became possible (Kron et al. 2007) revealing surprisingly widespread co-occurrence of several cytotypes (Keeler 2004; Halverson et al. 2008; Stahlberg 2009) despite theoretical assumptions that without reproductive isolation, local cytotype co-occurrence is unstable and frequency-dependent selection will exclude the cytotype in minority (Levin 1975; Felber 1991). 123 Evol Ecol

Table 2 Floral scent compounds of three Gymnadenia cytotypes (tetraploid and octoploid G. conopsea, and tetraploid G. densiflora) eliciting antennal responses in the noctuid Autographa gamma and the hawkmoth Deilephila porcellus Compounds ID 4x G. conopsea 8x G. conopsea 4x G. densiflora

A. gamma D. porcellus A. gamma D. porcellus A. gamma D. porcellus

Benzenoids Benzyl alcohol 99 Benzyl acetate 9 Vanillin 9 Benzyl benzoate 9 99 Fatty acid derivates (Z)-5-Decenyl acetate 4 99 99 Decyl acetate 9 Dodecanal 9 (Z)-5-Dodecenyl acetate 6 99 9 (Z)-7-Dodecenyl acetate 7 99 99 9a 9a Dodecyl acetate 8 99 99 (Z)-7-Tetradecenyl 10 99 acetate Tetradecyl acetate 9 Phenylpropanoids (E)-Cinnamic alcohol 99 Eugenol 3 99 99 99 Methyleugenol 9999 (E)-Cinnamyl acetate 99 (E)-Isoeugenol 99 99 (E)-Methylisoeugenol 99 9 Elemicin 5 99 99 9 N-bearing compounds 2-Aminobenzaldehyde 1 99 99 Indole 2 99 99 Terpenoids m/z: 91,79,43,77,67,105 9 Unknown compounds m/z: 69,110,82,53,52,39 9 m/z: 43,69,41,39,67,61 99 No. of compounds 13 11 15 12 7 8

The ID numbers correspond to the responses shown in Fig. 4 a Antennae were more sensitive than the flame ionization detector and mass spectrometer as no clear peak in 4xGd samples corresponding to that antennal response was found. As the time of antennal response coincided with antennal responses to (Z)-7-dodecenyl acetate (in 4xGcand 8xGcsamples), which was sensitively detected by the moths, we assume that this compound also occurred in 4xGdbut in amounts too low for detection by GC-FID and GC-MS

The occurrence of early and late flowering variants of Gymnadenia conopsea s.l. dif- fering in morphologic and scent characteristics has been reported repeatedly for many areas of its distribution (Heusser 1938; Gustafsson and Lo¨nn 2003;Vo¨th and Sontag 2006). 123 Evol Ecol

Fig. 4 Examples of antennal responses of Autographa gamma moths to an 8xGcand a 4xGdscent sample, respectively. The numbers correspond to the ID numbers (compounds) presented in Table 2. Many compounds occurred only in trace amounts in the samples, and the small peaks of these compounds are not visible

Table 3 Indices of floral preference and floral constancy of hawkmoths and noctuids (main pollinator groups of Gymnadenia conopsea s.l. at the study sites) facing artificial arrays composed of inflorescences of octoploid G. conopsea and tetraploid G. densiflora Index Pollinators N Median ± SE Range Difference from 0.5

Floral preference Hawkmoths 11 0.51 ± 0.02 0.33–0.66 Z = 0.98, P = 0.32 Noctuids 14 0.50 ± 0.06 0.00–0.66 Z = 0.81, P = 0.41 Floral constancy Hawkmoths 11 0.14 ± 0.02 0.06–0.28 Z = 2.93, P = 0.003 Noctuids 14 0.46 ± 0.09 0.00–1.00 Z = 0.35, P = 0.72

Floral preference: 0.5 – no preference by a pollinator, 0 – preference for 8x G. conopsea, 1 – preference for 4x G. densiflora. Floral constancy: 0 – alternating foraging, 0.5 – random foraging, 1 – constant foraging

Table 4 Positions and base substitutions in ITS 1 and 2 in the studied Gymnadenia cytotypes: tetraploid and octoploid G. conopsea, and tetraploid G. densiflora Position and base ITS 1 (239 bp) ITS 2 (239 bp) Genbank substitution accession 64 108 178 187 202 54 75 77 140 180 186 193

4x G. conopsea TAAAGCCGATACFJ751757 8x G. conopsea TAAAGCCGATACFJ751758 4x G. densiflora CG G C C TTTT A T – FJ751759

Recently, Marhold et al. (2005) suggested that the variability in Gymnadenia complex might be underlined by differences in the ploidy level. Indeed, mixed-ploidy populations of G. conopsea s.l. are quite common in Central Europe, with populations frequently har- bouring two or more cytotypes growing without any spatial arrangement (Suda et al. pers. comm.) and thus representing an excellent model system to study the mechanisms involved in the maintenance of several cytotypes in mixed-ploidy populations. Specifically, this

123 Evol Ecol study was designed to investigate the role of pre-mating reproductive isolation regulating the coexistence of tetraploid and octoploid individuals of G. conopsea s.l. Our results showed that pre-mating barriers are insufficient to prevent gene flow between co-flowering 8xGcand the 4xGd, while flowering phenology largely restricts gene flow between the 4xGcand the remaining cytotypes. Though the co-flowering cytotypes differ significantly in scent composition and pollinators are most likely able to detect these differences, both hawkmoths and noctuids showed no preference or constancy to a particular cytotype in the field. The absence of pre-mating barriers among cytotypes, except partial temporal seg- regation, suggests the existence of other mechanisms involved in the cytotypes’ coexis- tence. The significance of each studied pre-mating barrier is discussed below.

Phenological segregation

Polyploidization in plants frequently results in a shift in the timing of flowering (Tothill and Hacker 1976; Petit et al. 1997; Husband and Schemske 2000). Within the study populations, the early flowering 4xGcmarkedly differed phenologically from the other cytotypes (6–19% overlap only), presumably resulting in a strong decrease in inter-cyto- type pollen exchange. On the contrary, both late flowering 8xGcand 4xGdsubstantially overlapped in their phenologies (77% overlap), showing an opportunity for hybridization. Such phenological separation may arise due to genetic differences among cytotypes gen- erated by chromosome doubling (Lewis and Suda 1976) or by an occupation of slightly different microenvironments within the population which might affect timing and growth rates (Lumaret et al. 1987). Furthermore, selection can operate on flowering time variation and thus increase divergence (van Dijk and Bijlsma 1994; Nuismer and Cunningham 2005). Although possible, microhabitat effects are unlikely to produce the observed dif- ferences among Gymnadenia cytotypes, as seen in the studied mixed-ploidy populations in which the cytotypes grow intermingled (frequently less then 1 m apart), lacking obvious differences in microhabitat requirements. Regarding the genetic bases of the differences, it has been suggested that higher DNA content and thus cell size, resulting, for example, from chromosome doubling, may be associated with slower growth and later flowering (Stebbins 1950). In Gymnadenia, following this theory and assuming the autopolyploid origin of 8xGcfrom the 4xGc(see the results on scent composition and genetics), it is possible to consider that the oldest taxa 4xGdand 4xGcmay have evolved flowering displacement as a result of selective pressure to avoid hybridization, while a most recent event of poly- ploidization produced the later flowering taxon 8xGcdue to genetic shifts caused by the polyploidization process. These ideas are, however, hypothesized and further studies are being undertaken to address the origin and evolutionary relationship among Gymnadenia cytotypes.

Mechanical segregation

Differences in floral morphology have been shown to result in species-specific placement of pollen on a pollinator’s body (reviewed in Grant 1994), leading to mechanical isolation, and thus minimizing undesirable inter-specific pollen flow. In orchids adapted to Lepi- dopteran pollination, the column structure ensures that pollinia are placed either on the proboscis or the eyes of the pollinator (Nilsson 1983). The nectar spurs act as pollinator filters as only those pollinators possessing tongues long enough to reach the base of the spur can access the nectar reward. 123 Evol Ecol

In G. conopsea s.l., the column morphology is unlikely to influence pollen flow among cytotypes, because the cytotypes present similar column structures and pollinia are always placed on the pollinator’s tongue. The measurements of spur length revealed significant differences among the cytotypes which could promote their reproductive isolation. As we did not track pollen flow directly by labelling orchid pollinia by histochemical or fluo- rescent dyes, nor we used a phenotypic selection approach, we cannot fully evaluate if 1 mm difference in spur length is large enough to promote assortative mating. Our survey of published studies on both evolutionary and ecological consequences of variability in spur length within and between species shows that (1) changes in spur length play an important role in plant diversification, (2) these changes have considerable consequences for pollen tranfer, and (3) species/ecotypes used in those studies differed by 5 up to 40 mm in spur length (e.g., Nilsson 1983; Nilsson 1988; Anderson and Johnson 2009; Anderson et al. 2009). As we failed to find a study showing assortative mating in species with less than 5 mm difference in spur length, we believe that the 1 mm difference in spur length among Gymnadenia cytotypes is too small to promote assortative pollen flow within mixed-ploidy populations, yet this issue needs further research.

Assortative pollinator behaviour

Animal pollinated plants advertise themselves by various visual, olfactory and tactile stimuli presented simultaneously to the pollinators. The conspicuousness of the stimulus will depend on signals such as flower size, colour and shape and strength of volatile emissions, as well as on the ability of pollinators to perceive these signals. Petal colour is an important visual cue, and shifts in floral colour have been shown to promote speciation through reduced gene flow between colour morphs in close association with congruent changes in pollinator identity (e.g., Bradshaw and Schemske 2003). Gymnadenia conopsea s.l. is pollinated by a wide assembly of noctuids and hawkmoths (Vo¨th 2000), but in our study populations Deilephila porcellus and Autographa gamma were the main floral visitors. Recent colour vision tests with nocturnal hawkmoth species showed that they could discriminate floral colours at starlight intensities when humans and honeybees are colourblind (Kelber et al. 2003). Facing these observations, colour vision in crepuscular and nocturnal species may be more important than previously considered (Raguso and Willis 2005). Spectral reflectance curves of G. conopsea s.l. flowers showed high variability within cytotypes and also a wide overlap among them, with major dif- ferences being mainly in corolla brightness and not in colour hue. Thus, despite the fact that hawkmoths may use these differences (Kelber et al. 2003), the overlap in the spectral reflectance curves of Gymnadenia cytotypes is presumably too large for enabling its dif- ferentiation by moths (Kelber pers. comm.), and this floral trait may be excluded as a cue involved in cytotype recognition by pollinators. Even more important than the visual floral display may be the olfactory cues, i.e., floral scent, for attraction of crepuscular and nocturnal pollinators (Brantjes 1978; but see Raguso and Willis 2005), and indeed pollinators have been attracted in the present study to Gymnadenia inflorescences (tested for 8xGc) even when excluding the visual cue and offering only the olfactory display. The plants of all three cytotypes analysed emitted a complex mixture of compounds, most of which are well known in floral scents (Knudsen et al. 2006), and several of which were repeatedly found in plant species pollinated by nocturnal moths (Dobson 2006). Some of these compounds were also found by Kaiser (1993) and Huber et al. (2005) in the scent of G. conopsea. Though both studies did not discriminate between G. conopsea subsp. conopsea and G. densiflora, their compound 123 Evol Ecol spectra are more similar to the spectrum we found in 4xGdthan to those in 4xGcand 8xGc. Several of the compounds found in these two studies, such as benzyl benzoate, benzyl butyrate, cinnamyl aldehyde and alcohol isomers, were only detected in 4xGd, but not in the other two cytotypes. Our finding of several fatty acid derivatives in both 4xGc and 8xGcis curious, such as (Z)-7-dodecenyl acetate and (Z)-9-tetradecenyl acetate, which were not reported as floral scent before (e.g., Knudsen et al. 2006), but are well known female moth sex pheromones occurring in species of different Lepidoptera families, especially in noctuids (El-Sayed 2008). (Z)-7-dodecenyl acetate, the most abundant fatty acid derivative in both cytotypes is the most important male attractant in the female sex pheromone of Autographa gamma (Dunkelblum and Gothilf 1983; Mazor and Dunkelblum 1992), one of the main pollinators in the present study. (Z)-9-tetradecenyl acetate, how- ever, is known to have strong negative effects on attraction of A. gamma males when offering it together with (Z)-7-dodecenyl acetate (Mazor and Dunkelblum 1992). It remains to be studied whether these unusual floral scent compounds play a role in the attraction of moths, and whether 4xGcand 8xGcattracts male moths by means of sexual drive. As we wanted to know, whether moths could detect the differences in scent among the cytotypes, which is an important prerequisite for when scent acts as a pre-mating barrier, we tested the scent of each cytotype on the antennae of the most important moth pollin- ators. Pollinators typically do not respond to all compounds emitted by plants, and such measurements are needed to determine compounds from the complex scent blend for which pollinators have olfactory receptors (Schiestl and Marion-Poll 2001). Antennae of both moth species responded to several compounds emitted from the different Gymnadenia cytotypes, many of which (e.g., indole, eugenol, benzyl benzoate) are already known to be detected by nocturnal moths (e.g., Raguso et al. 1996; Raguso and Light 1998; Huber et al. 2005;Do¨tterl et al. 2009). A few compounds, however, (e.g., elemicin) are described here for the first time to be physiologically active in moths. All these active compounds are potential mediators in the interaction of Gymnadenia with their most important pollinators, and a few of these compounds (e.g., benzyl acetate, benzyl benzoate) are already known to be not only physiologically but also behaviourally active (Meagher 2002; Plepys et al. 2002). For most of the physiologically active compounds, however, among them the abundant eugenol and indole, it is unclear whether they elicit behavioural responses in moths. Interestingly, moths responded in the electroantennographic measurements to all of the compounds that are characteristic for the three different cytotypes, and that are responsible for the main differences (qualitatively and/or semi quantitatively) in scent among the cytotypes. Therefore, it seems that the moths have the olfactory capabilities to differentiate the cytotypes based on their scent, and especially between co-flowering 8xGcand 4xGd, which show considerable differences in their scent bouquet (Fig. 3). However, our observations of pollinator behaviour on artificial arrays composed of the two co-flowering Gymnadenia cytotypes showed that there was neither preference nor constancy for a cytotype, indicating that pollinators did not use scent for cytotype discrimination in the field. Regardless of its potential importance, studies up to date exploring pollinator behaviour in mixed-ploidy populations and their role in mediating assortative mating are scarce. The little evidence that exists indicates that pollinators with a high degree of flower constancy can facilitate polyploid establishment. Different foraging patterns have been already observed in sympatric diploid-tetraploid populations of Chamerion angustifolium (Husband and Schemske 2000; Kennedy et al 2006), Heuchera grossulariifolia (Segraves and Thompson 1999; Thompson and Merg 2008) and Tragopogon mirus (Cook and Soltis 123 Evol Ecol

1999). Although there were no differences in overall pollinator assemblage, each species contributed differently to the reproductive fitness of the cytotypes. Pollinators tended to visit a disproportionately higher number of flowers/inflorescences from a specific cytotype, leading to assortative mating in varying degrees. On the contrary, in our study, both hawkmoths and noctuids showed no assortative visitation behaviour, preventing a contri- bution to pre-zygotic reproductive isolation.

Consequences of the lack of pre-mating barriers

Reproductive isolation is a necessary condition for mixed-ploidy populations to be maintained in equilibrium and this can be achieved by various pre- and/or post-zygotic mating barriers (e.g., Levin 1975). Pre-zygotic breeding barriers prevent individuals from wasting their gametes in the formation of unviable seeds or unfit hybrid individuals, bearing the potential to undergo natural selection and reinforcing any pre-existing post- zygotic mating barriers (Dobzhansky 1940; Howard 1993). In our study we investigated various pre-zygotic barriers, except spatial separation of cytotypes or the cryptic repro- ductive barriers acting after pollination but before fertilization as a result of pollen com- petition and interactions between male gametophytes and female reproductive tissues (gametic isolation, Howard et al. 1998). Furthermore, this mechanism of sexual isolation may be asymmetrical, such that the gametic interactions depend on the direction of mating between the parental taxa (Rieseberg et al. 1995; Arnold et al. 1996). For example, Husband et al. (2002) investigated the role of gametic barriers operating between diploid and tetraploid Chamerion angustifolium and showed an enhancement in siring success of the tetraploids as a result of pollen competition. Our preliminary data on pollen tube growth in experimental crosses with Gymnadenia cytotypes, together with the general occurrence of strong post-zygotic barriers reducing hybrid fitness and preventing gene introgression in terrestrial orchids (Moccia et al. 2007) suggest that gametic isolation is unlikely to play an important role in pre-zygotic isolation in G. conopsea s.l. The effect of spatial segregation on cytotypes’ coexistence is currently being studied by Suda et al. (pers. comm.), whose preliminary results indicate no spatial arrangement of the cytotypes within mixed-ploidy populations, thus downplaying its role as a pre-zygotic barrier for pollen exchange among Gymnadenia cytotypes. In mixed-ploidy G. conopsea s.l. populations, the lack of pre-mating barriers will lead to high rates of inter-cytotype pollen flow and this can generate different scenarios. If post- zygotic mating barriers exist, cytotypes will experience high levels of pollen and ovule wastage, while further mating barriers limit the production of unfit hybrids. This will select against hybrids and enable cytotype co-existence through the evolution of prezygotic isolation. If no post-mating barriers exist, a high proportion of hexaploid hybrids and, to less extent, tetraploid hybrids will also form. Again, newly formed polyploid establishment will occur only if a diverse array of ecological features and breeding barriers increases their probability of successful mating, such as the capability of self-fertilisation (Vamosi et al. 2007). Our cytometrical screening of 301 plants in two mixed-ploidy populations of G. conopsea s.l. revealed only 4 hybrids indicating the existence of post-mating barriers. Studies are now being developed to assess the levels of pollen flow among the cytotypes and the existence of post-zygotic mating barriers acting in these populations.

Acknowledgments We thank I. Jongepierova´ for locating plant populations, F. Schiestl for helpful comments, A. Kelber for advises on moth vision and S.-L. Steenhuisen for English corrections. We also thank two anonymous reviewers for their helpful suggestions improving the manuscript. The work was

123 Evol Ecol

financially supported by the GA ASCR No. KJB600870601 to J.J., MSM 6007665801 to the Faculty of Science of University of South Bohemia and the Portuguese Foundation for Science and Technology (SFRH/BPD/41200/2007) to S.C.

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