Absence of Pollinator-Mediated Premating Barriers in Mixed-Ploidy Populations of Gymnadenia Conopsea Sl

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´ (&) Á I. Scho¨delbauerova´ Department of Theoretical Ecology, Institute of System Biology and Ecology AS CR, Na Sa´dkaćh 7, 37005 Cˇ eske´ Budeˇjovice, Czech Republic e-mail: [email protected] J. Jersa´kova´ Á 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 Á K. Milchreit Á 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 Á Floral volatiles Á Fragrant orchid Á Gas chromatography– electroantennographic detection Á Matting barriers Á 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 different 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 populations (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 (Hulteń 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

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