Arthropod-Plant Interactions DOI 10.1007/s11829-017-9512-x ORIGINAL PAPER Interpopulation variation in pollinators and floral scent of the lady’s-slipper orchid Cypripedium calceolus L. Herbert Braunschmid1 · Bernadette Mükisch1 · Thomas Rupp1 · Irmgard Schäffler1 · Pietro Zito2 · Daniele Birtele3 · Stefan Dötterl1 Received: 28 October 2016 / Accepted: 4 March 2017 © The Author(s) 2017. This article is an open access publication Abstract Floral scent is a key mediator in many plant– and octyl acetate, scents differed among populations. Simi- pollinator interactions. It is known to vary not only among larly, there were strong differences in flower visitor spec- plant species, but also within species among populations. tra among populations with most abundant flower visitors However, there is a big gap in our knowledge of whether being bees and syrphid flies at low and high altitudes, such variability is the result of divergent selective pres- respectively. Pollinator climate differed also among popu- sures exerted by a variable pollinator climate or alternative lations; however, independent of altitude, most pollinators scenarios (e.g., genetic drift). Cypripedium calceolus is a were bees of Lasioglossum, Andrena, and Nomada. Only Eurasian deceptive lady’s-slipper orchid pollinated by bees. few syrphids acted as pollinators and this is the first record It is found from near sea level to altitudes of 2500 m. We of flies as pollinators in C. calceolus. The electrophysiolog- asked whether pollinator climate and floral scents vary in ical tests showed that bees and syrphid flies sensed many a concerted manner among different altitudes. Floral scents of the compounds released by the flowers, among them of four populations in the Limestone Alps were collected linalool and octyl acetate. Overall, we found that both flo- by dynamic headspace and analyzed by gas chromatog- ral scent and visitor/pollinator climate differ among popu- raphy coupled to mass spectrometry (GC/MS). Flower lations. We discuss whether interpopulation variation in visitors and pollinators (the subset of visitors with pollen scent is a result of pollinator-mediated selection. loads) were collected and identified. Preliminary coupled gas chromatographic and electroantennographic measure- Keywords Deceptive pollination · ments with floral scents and pollinators revealed biologi- Electroantennography · Dynamic headspace · Pollinator cally active components. More than 70 compounds were climate detected in the scent samples, mainly aliphatics, terpenoids, and aromatics. Although several compounds were found in all samples, and all samples were dominated by linalool Introduction The majority of angiosperms is pollinated by animals, Handling editors: Isabel Alves dos Santos and Isabel Machado. mostly insects (Ollerton et al. 2011). The pollinator cli- * Stefan Dötterl mate (composition, abundance, and efficiency of pol- [email protected] linators; Grant and Grant 1965) of a plant species often 1 varies among populations and potentially contributes to Department of Ecology and Evolution, Plant Ecology, the evolution of pollination ecotypes, i.e., forms adapted University of Salzburg, Hellbrunnerstr. 34, 5020 Salzburg, Austria to the local pollinator fauna, especially if differences in the pollinator climate among populations are temporally 2 Department of Biological Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Palermo, and spatially stable in an evolutionary timescale (Johnson Italy 2006). Such ecotypes often differ in floral characteristics 3 Centro Nazionale per lo Studio e la Conservazione della which allow recognizing subspecies or other intra-spe- Biodiversità Forestale “Bosco Fontana”, Marmirolo, Italy cific taxa (e.g., Johnson 1997). Such variations in floral Vol.:(0123456789)1 3 H. Braunschmid et al. traits are not only caused by qualitative shifts in the polli- Here, we examined four populations of C. calceolus nator spectrum, but more often by a gradual, quantitative between 600 and 1450 m a.s.l. and asked whether polli- variation in the availability of pollinator species (Aigner nators and floral scents differ among populations. In pre- 2001; Waser 2001). liminary analyses, we also determined biologically active The flowers of animal-pollinated plants typically adver- compounds in flower visitors/pollinators. In detail, we (i) tise their presence by visual and olfactory cues (Chittka and collected scent from flowers by dynamic headspace and Raine 2006). Olfactory cues are most important for plants analyzed samples by thermal desorption (TD) gas chroma- pollinated at night (Cordeiro et al. 2017; Dobson 2006; tography and mass spectrometry (GC/MS), (ii) recorded Dötterl et al. 2012), and for highly specialized pollination insects trapped in the flowers and those being potential pol- systems (Heiduk et al. 2016; Milet-Pinheiro et al. 2013; len vectors, and (iii) used gas chromatography coupled to Schäffler et al. 2015; Schiestl et al. 1999). electroantennographic detection (GC/EAD) to determine Several thousand scent compounds are known so far electrophysiologically active floral scent compounds in sev- (Knudsen et al. 2006). As with other floral traits, floral eral flower visitors/pollinators. As shown in other species, scents are known to vary within species, both within and we predict that there are differences in scent and flower among populations (Dötterl et al. 2005; Chartier et al. visitors/pollinators among populations (see above) of C. 2013; Mant et al. 2005; Stökl et al. 2009; Sun et al. 2014), calceolus, and that antennae of different insects respond to and this variability is strongly suggested to have a genetic a different set of compounds (compare with Jürgens et al. basis (e.g., Andargie et al. 2014; Friberg et al. 2013; Zu 2014). We discuss whether results point to local pollinator et al. 2016). However, little is known whether such genetic adaptation in olfactory cues in C. calceolus. variability is the result of divergent selection or other sce- narios (e.g., drift). The effectiveness of an olfactory signal depends on how well it matches the pollinator’s percep- Materials and methods tive abilities, and these olfactory traits are thus expected to diverge between plant populations experiencing differ- Plant species ent pollinator environments. Indeed, plants experiencing different pollinators among population were found to pro- Cypripedium calceolus L. (C. calceolus, ssp. calceolus in duce population-specific scents and likely be adapted to the Bergström et al. 1992) is a pollen-limited perennial orchid locally available pollinator climate (Mant et al. 2005; Stökl (Kull 1998; Blinova 2002) distributed across the boreal and et al. 2009; Sun et al. 2014). temperate zones of Europe and Asia. It is found in a vari- Although typically involved in mutualistic interactions ety of habitats including open to medium-shaded decidu- with their pollinators, 4–6% of plants are pollinated by ous and coniferous forests, and alpine meadows and rubble, deceit (Renner 2006). The flowers of such plants signal the predominantly on calcareous soil (Cribb 1997). It is one of presence of a reward by visual, olfactory, thermal, and/or the biggest European orchids with a stem height ranging tactile cues without providing it. Populations of deceptive from 20 to 60 cm, and moreover, with one of the biggest plants are believed to be more variable than populations of and conspicuous flowers of all European orchids. One or rewarding species in traits associated with pollinator attrac- two flowers per stem consist of three purple-brown sepals, tion. This is because deceived pollinators then have more two similarly colored petals, and a petal called labellum, difficulties in learning to avoid the deceptive plant and which is yellow and shoe shaped to form a trap. The pollen discriminate it from rewarding resources (Ackerman et al. consists of single pollen grains contained in a sticky smear. 2011). Seeds are with a size of 1.2 × 0.3 mm among the biggest A widespread deceptive plant in Eurasia is Cypripedium of temperate orchids and are produced in high numbers calceolus L. (Orchidaceae). It occurs in central and north- (6000–16,000; Kull 1999). The plant propagates also veg- ern parts of Europe and Asia, where it spans an altitudinal etatively with horizontal rhizomes, building patches with gradient from near sea level to 2500 m a.s.l. (Cribb 1997). many ramets belonging to one or several clones (Brzosko It is known from previous studies that the pollinators of et al. 2002). The successful pollination of C. calceolus the trap flowers of C. calceolus are predominantly various depends on small insects temporarily trapped in the label- solitary bees from the genera Andrena, Lasioglossum, and lum and leaving the slippery cavern through a posterior Halictus (Kull 1999; Nilsson 1979 and references therein) exit opening, thereby depositing pollen imported from which are attracted by visual and olfactory floral cues other flowers on the stigma and collecting pollen from the (Daumann 1968; Nilsson 1979). However, it is unknown anthers (Nilsson 1979, and references therein). whether the pollinator climate of C. calceolus varies among Cypripedium calceolus is regarded as a rare plant and is altitude as shown for other plants (e.g., Pellmyr 1986; Sun protected by Annex II of the Habitats Directive of the Euro- et al. 2014). pean Commission. In the IUCN Red List, however, it has 1 3 Interpopulation variation in pollinators and floral scent of