Annals of Botany 104: 1077–1084, 2009 doi:10.1093/aob/mcp208, available online at www.aob.oxfordjournals.org On the roles of colour and scent in a specialized floral mimicry system Nicolas J. Vereecken1,2,* and Florian P. Schiestl2 1Evolutionary Biology & Ecology, Free University of Brussels/Universite´ Libre de Bruxelles, Av. F.D. Roosevelt 50 CP 160/12, B-1050 Brussels, Belgium and 2Institute of Systematic Botany, University of Zu¨rich, Zollikerstrasse 107, CH-8008 Zu¨rich, Switzerland Received: 15 May 2009 Returned for revision: 29 June 2009 Accepted: 15 July 2009 Published electronically: 18 August 2009 † Background and Aims Sexually deceptive orchids achieve cross-pollination by mimicking the mating signals of female insects, generally hymenopterans. This pollination mechanism is often highly specific as it is based pri- marily on the mimicry of mating signals, especially the female sex pheromones of the targeted pollinator. Like many deceptive orchids, the Mediterranean species Ophrys arachnitiformis shows high levels of floral trait vari- ation, especially in the colour of the perianth, which is either green or white/pinkinsh within populations. The adaptive significance of perianth colour polymorphism and its influence on pollinator visitation rates in sexually deceptive orchids remain obscure. † Methods The relative importance of floral scent versus perianth colour in pollinator attraction in this orchid pol- linator mimicry system was evaluated by performing floral scent analyses by gas chromatography-mass spec- trometry (GC-MS) and behavioural bioassays with the pollinators under natural conditions were performed. † Key Results The relative and absolute amounts of behaviourally active compounds are identical in the two colour morphs of O. arachnitiformis. Neither presence/absence nor the colour of the perianth (green versus white) influence attractiveness of the flowers to Colletes cunicularius males, the main pollinator of O. arachnitiformis. † Conclusion Chemical signals alone can mediate the interactions in highly specialized mimicry systems. Floral colour polymorphism in O. arachnitiformis is not subjected to selection imposed by C. cunicularius males, and an interplay between different non-adaptive processes may be responsible for the maintenance of floral colour polymorphism both within and among populations. Key words: Colletes cunicularius, floral odour, floral colour polymorphism, mimicry, Ophrys arachnitiformis, pollination by sexual deception, pollinator attraction. INTRODUCTION than more common coexisting morphs, a process known as negative frequency-dependent selection (nFDS; Gigord et al., It is often assumed that many flowering plants owe much of 2001). This might ultimately promote the persistence of their floral polymorphism in colour, shape and odour to the phenotypic variance in floral traits involved in pollinator combined effects of attracting different pollinator species attraction (Smithson and Macnair, 1997; Ferdy et al., 1998; and to the spatio-temporal variation in the selection regimes Gigord et al., 2001). To date, very few studies have combined they are subjected to (Galen and Kevan, 1980; Pettersson, the quantitative measurement of different floral traits and 1991; Eckhart, 1992; Ollerton et al., 2006). If these determi- examined how variation in these traits may be linked to the nants of floral polymorphism hold for most cases where the preferences and learning of the pollinators. Among these pollinators obtain a reward from their pollination visits, the studies, high levels of floral odour variation have been found selection pressures at play may somewhat differ for plants in several deceptive orchids, leading to the suggestion that that benefit from pollinator services without offering a floral odour polymorphism was probably the result of the reward of any kind to their pollen vectors. Such cases of absence of selection rather than nFDS mediated by the pollina- ‘deceptive’ pollination have evolved multiple times within tors (Ackerman et al., 1997; Aragon and Ackerman, 2004; the angiosperms (Renner, 2006) and they are particularly Salzmann et al., 2007). Evolutionary forces other than nFDS well represented in the orchid family, where approximately may therefore be important in shaping the floral traits of poly- one-third of all 30 000 species known to science bear only morphic orchids. rewardless flowers (Dafni, 1984; Nilsson, 1992; Cozzolino Among deceptive orchids, those species that achieve cross- and Widmer, 2005; Schiestl, 2005; Tremblay et al., 2005; pollination by mimicking the mating signals of female insects Jersakova et al., 2006, 2009). Recent studies have reported stand out with their intimate relationships with only one or that pollinators visiting deceptive orchids are likely to leave few pollinator taxa (Kullenberg, 1961; Dafni, 1984; Nilsson, each inflorescence rapidly visited as no reward is available 1992). The specificity in this orchid–pollinator system is (Smithson and Macnair, 1996; Smithson and Gigord, 2003). based primarily on the mimicry of mating signals of female Hence, several authors have suggested that rare morphs insects, especially the sex pheromones, by the flower as pollina- within populations could experience higher visitation rates tor attractant (reviewed by Schiestl, 2005; Ayasse, 2006; Vereecken and Schiestl, 2008). Pollination in these orchids is * Corresponding author. E-mail [email protected] # The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] Downloaded from https://academic.oup.com/aob/article-abstract/104/6/1077/184726 by [email protected] user on 25 August 2018 1078 Vereecken & Schiestl — Floral colour and scent in mimicry F IG. 1. Perianth colour morphs of Ophrys arachnitiformis in the study area in southern France showing flowers with a green vs. a white perianth (photographs by N. J. Vereecken). effected as the male of the targeted insect species attempts copu- particularly suited for this study as a considerable body of lation with the female decoy of the flower, a phenomenon knowledge has accumulated over the past few years on its known as pseudocopulation (Kullenberg, 1961). Several sexu- floral scent and pollinator behaviour (Peakall and Schiestl, ally deceptive orchids show discrete intraspecific polymorph- 2004; Mant et al., 2005a; Vereecken et al., 2007; Vereecken ism in floral traits, yet contrary to their related food-deceptive and Schiestl, 2008). Furthermore, translocation experiments counterparts, a narrow range of insect taxa, sometimes of plants in pots and the monitoring of individual plants perhaps only a single species, ensures pollination (Schiestl, grown in different environments have shown that each colour 2005; Schiestl and Schlu¨ter, 2009; Vereecken, 2009). morph is stable, suggesting that the polymorphism observed Although a recent study on Ophrys heldreichii has reported on has a genetic basis. The frequency of each morph varies the synergistic effect of scent and floral perianth colour in pol- among populations, ranging from 100 % of green morphs in linator attraction (Spaethe et al., 2007), the extent to which these some sites of the Rhoˆne river basin to almost 100 % of results may be applicable to other species within the morpho- white morphs in some areas of south-east France and north- logically diverse genus Ophrys remains unknown. east Spain (N. J. Vereecken, pers. obs.). The present study investigated the adaptive significance of perianth colour polymorphism and its influence on pollinator visitation rates in the sexually deceptive orchid Ophrys arachniti- Sample collection formis, a taxon for which the perianth colour is either completely The floral odour of each colour morph [green (g) vs. white green or translucent (white; Fig. 1). A combination of chemical (w)] of O. arachnitiformis was sampled in two populations in analyses of floral odours by gas chromatography and mass spec- southern France [Montbazin: n(g) ¼ 10, n(w) ¼ 10; trometry (GC, GC-MS) along with behavioural bioassays per- Montferrier-sur-lez: n(g) ¼ 12, n(w) ¼ 13]. The two orchid formed in situ was employed in an experimental design to populations investigated for their floral odour were located dissect the floral phenotype and address two major questions. approx. 20 km apart. Epicuticular waxes of orchids were (1) Is floral colour polymorphism associated with differences in sampled by extracting fresh and unpollinated flower labella floral scent, especially in the patterns of behaviourallyactive com- in 200 mL n-hexane (HPLC grade) for 1 min. All extracts pounds? (2) To what extent are visual signals of the flowers, com- were then stored at –20 8C. pared with floral odour, involved in pollinator attraction? Chemical analyses MATERIALS AND METHODS All samples were analysed by GC on an Agilent 6890N chromatograph equipped with a HP-5 capillary column Study species (30 m  0.32 mm  0.25 mm). The injector temperature was The study species, Ophrys arachnitiformis Gren. & kept at 300 8C. Prior to GC analysis, 100 ng n-octadecane M.Philippe, grows in calcareous grasslands, garrigues and was added as internal standard to all samples. Aliquots of open woodlands. Its flowering period extends from the extracts (1 mL) were injected splitless at 50 8C (1 min), mid-February until early April and its geographical range followed by a programmed increase of oven temperature to extends along the Mediterranean coast from north-eastern