An experimental evaluation of traits that influence the sexual behaviour of pollinators in sexually deceptive orchids Ryan D. Phillips1,2,3* & Rod Peakall1 5 1 Ecology and Evolution, Research School of Biology, The Australian National University, Canberra, ACT, 2601, Australia 2 Kings Park and Botanic Garden, The Botanic Garden and Parks Authority, West Perth, 6005, Western Australia, Australia 10 3 Department of Ecology, Environment & Evolution, La Trobe University, Victoria, 3086, Australia *Corresponding author: email: [email protected] Ph: 61 2 61252866 Fax: 61 2 61255573 15 Running head: floral traits and sexual behaviour Abstract 20 Pollination by sexual deception of male insects is perhaps one of the most remarkable cases of mimicry in the plant kingdom. However, understanding the influence of floral traits on pollinator behaviour in sexually deceptive orchids is challenging, due to the risk of confounding changes in floral odour when manipulating morphology. Here, we investigated the floral traits influencing the sexual response of male Zaspilothynnus nigripes (Tiphiidae) 25 wasps, a pollinator of two distantly related sexually deceptive orchids with contrasting floral architecture, Caladenia pectinata and Drakaea livida. In D. livida the chemical sexual attractant is emitted from the labellum, while in C. pectinata it is produced from the distal sepal tips, allowing manipulative experiments. When controlling for visual cues there was no difference in long distance attraction, though the floral odour of D. livida induced copulation 30 more frequently than that of C. pectinata. The role of colour in pollinator sexual attraction was equivocal, indicating that colour may not be a strong constraint on the initial evolution of sexual deception. The frequency of wasp visitors landing on C. pectinata decreased when the amount of floral odour was reduced, but attempted copulation rates were enhanced when the source of floral odour was associated with the labellum. These latter variables may represent 1 35 axes of selection that operate across many sexually deceptive species. Nonetheless, the observed variation in floral traits suggests flexibility in how sexual deception can be achieved. Keywords: sexual deception, mimicry, floral traits, pollinator behaviour 40 Introduction Pollination by sexual deception, where male insects are attracted to the flower by chemical 45 and/or physical mimicry of a female insect (Ellis & Johnson, 2010; Schiestl et al., 1999, Schiestl et al., 2003; de Jager & Peakall, 2016), is perhaps one of the most remarkable of all pollination strategies. The investigation of plants with unconventional floral forms is revealing an increasing diversity of plants pollinated via this strategy (Vereecken et al., 2012; Phillips et al., 2014b; Arakaki et al., 2016). Consequently, sexual deception is now known to 50 be geographically widespread, and to involve at least several hundred species, with examples from Europe, Australia, South and central America, southern Africa and Asia (Singer, 2002; Blanco & Barboza, 2005; Ellis & Johnson, 2010; Vereecken et al., 2012; Phillips et al., 2014b; Arakaki et al., 2016; Bohman et al., 2016a). Despite recently discoveries in the Asteraceae (Ellis & Johnson, 2010) and Iridaceae (Vereecken et al., 2012), the vast majority 55 of species using this strategy are in the Orchidaceae, where at least 20 genera are known to contain sexually deceptive species (Johnson & Schiestl, 2016; Bohman et al., 2016a). While there is a strong trend towards reduced floral size, dull colouration and insectiform structure of the labellum (Johnson & Schiestl, 2016), there are also sexually deceptive species with large, sometimes colourful flowers that lack insectiform structures (Phillips et al., 2009, 60 2017; Phillips & Peakall 2018) raising the important question of which traits are needed to achieve the sexual attraction of pollinators. In sexually deceptive pollination systems, one intuitively expects selection to favour traits that increase sexual behavior at the flower, since more time on the flower, or more vigorous 65 sexual behavior, may increase the frequency of pollen removal and pollination (de Jager & Peakall, 2018). However, teasing apart the effect of various floral traits on sexual behavior raises some important methodological complexities. For example, in many sexually deceptive orchids, pollinator attraction is achieved via chemical cues (Kullenberg, 1961; Stoutamire, 2 1983; Schiestl et al., 2003; Bohman et al., 2016a). Therefore, when experimentally 70 disentangling the role of the chemical, visual and morphological cues, any manipulation must avoid confounding floral odour effects with other cues (de Jager & Ellis 2012; de Jager & Peakall, 2016, 2018). This is not so easy to achieve in most genera of sexually deceptive orchids where odour release occurs from the labellum (Kullenberg, 1961; Phillips et al., 2013; Phillips et al., 2014b; de Jager & Peakall, 2016), meaning that labellum shape or size 75 cannot be manipulated without the need to control for odour. Furthermore, interspecific comparison of the effect of floral traits on pollinator behaviour is also confounded when plants use different pollinator species. This is a particularly acute challenge for sexually deceptive orchids where pollinator specificity is extreme, and pollinator sharing rare or absent in most genera (but see Gaskett & Herberstein, 2010; Gogler et al., 2009; Phillips et 80 al., 2017 for some exceptions). In order to understand the floral traits that affect the attraction of sexually deceived pollinators, in this study we take advantage of an exceptional case of pollinator sharing where the thynnine wasp Zaspilothynnus nigripes (Guérin, 1842) pollinates two distantly related 85 orchids, Caladenia pectinata R.S. Rogers and Drakaea livida J.Drumm., by sexual deception (Phillips et al., 2013; Fig. 1). Unlike D. livida, C. pectinata produces the sexual attractant entirely from glands on the distal sepal tips rather than the labellum (hereafter called clubs), meaning that other floral traits can be manipulated without altering the amount of sexual attractant produced. Interestingly, the copulation rate is over three times higher in D. livida 90 than C. pectinata (Phillips et al., 2013), providing the opportunity to explore traits that affect levels of sexual attraction. Research on other sexually deceptive systems has provided some clues about the factors that are likely to be important for enhancing sexual attraction. For example, experiments using 95 synthesized semio-chemicals have demonstrated that both changes in the blend of chemicals produced, and the quantity of compounds released, affect the number of pollinators responding and the frequency of attempted copulation (Ayasse et al., 2003; Schiestl, 2004; Vereecken & Schiestl, 2008; Peakall et al., 2010; Bohman et al., 2014; Bohman et al., 2017; Xu et al., 2017). In some sexually deceptive orchids, colour also appears to be important for 100 locating the flower, either through visual mimicry or colour contrast to maximize detectability (Streinzer et al., 2009; Gaskett & Herberstein, 2010; Gaskett et al., 2016), though the effect of colour on copulation rate is unknown in any orchid. Once pollinators 3 have landed on the flower, morphological traits are known to enhance copulation rates and duration at least in some systems (de Jager & Ellis, 2012; Phillips et al., 2014b; de Jager & 105 Peakall, 2016, 2018). For example, experimental reduction in labellum size of Chiloglottis, while avoiding removal of the odour producing callus, showed that a labellum size corresponding to the length of the mimicked female elicited a longer duration of attempted copulation than smaller labella (de Jager & Peakall, 2016). 110 Here, we apply a series of innovative floral manipulations to test if three floral traits (amount of floral odour, site of odour release, and colour), which are predicted to affect reproductive success in sexually deceptive orchids, influence the extent of pollinator response and sexual behaviour with the flower. Further, we seek to understand the basis of pronounced variation in pollinator sexual behaviour with different species of sexually deceptive orchid (see Fig. 1). 115 Due to the large size of Z. nigipres (approximately 20 mm body length) and its vigorous copulatory behaviour with the flower, in this system pollinator responses can be readily classified and into three discrete steps, long-distance attraction, landing on the flower, and attempted copulation. Such a classification is not always possible in other sexually deceptive species where the pollinator and flower is small and/or the sexual behavior is more subtle 120 (e.g. Phillips et al., 2014b). Further, we can subdivide ‘sexual attraction’ into two quantifiable components. We define ‘enhanced sexual response’ as a significant increase in the total number of wasps responding to the flower. We define ‘enhanced sexual behavior’ as a significant increase in the proportion of wasps landing on the flower, and/or the proportion of landing wasps that attempt copulation with the flower. In this context, we address four 125 specific questions for Z. nigripes: (i) Is there interspecific variation in the attractiveness of the floral odour? (ii) Does a decrease in the amount of floral odour reduce sexual attraction? (iii) Does sexual attraction increase when the attractant is associated with the labellum? (iv) Does a dull-coloured labellum enhance sexual attraction? 130 Materials and