Plant Biology ISSN 1435-8603
RESEARCH PAPER
Pollen transfer efficiency and its effect on inflorescence size in deceptive pollination strategies G. Scopece1,2, F. P. Schiestl3 & S. Cozzolino1 1 Department of Biology, University Federico II, Complesso Universitario MSA, Naples, Italy 2 Institute for Plant Protection, Consiglio Nazionale delle Ricerche, Sesto Fiorentino (FI), Italy 3 Institute of Systematic Botany, University of Zurich, Zurich, Switzerland
Keywords Flower number; food deception; inflorescence size; orchids; pollen loss; resource allocation; sexual deception. Correspondence G. Scopece, Department of Biology, University of Naples Federico II, Complesso Universitario MSA, via Cinthia, I-80126 Naples, Italy. E-mail: [email protected] Editor A. Dafni Received: 4 February 2014; Accepted: 16 May 2014 doi:10.1111/plb.12224
ABSTRACT Pollination systems differ in pollen transfer efficiency, a variable that may influence the evolution of flower number. Here we apply a comparative approach to examine the link between pollen transfer efficiency and the evolution of inflorescence size in food and sexually deceptive orchids. We examined pollination performance in nine food-deceptive, and eight sexually deceptive orchids by recording pollen removal and deposition in the field. We calculated correlations between reproductive success and flower number (as a proxy for resources allocated during reproductive process), and directional selection differentials were estimated on flower number for four species. Results indicate that sexually deceptive species experience decreased pollen loss compared to food-deceptive species. Despite producing fewer flowers, sexually deceptive species attained levels of overall pollination success (through male and female function) similar to food-deceptive species. Furthermore, a positive correlation between flower number and pollination success was observed in food-deceptive species, but this correlation was not detected in sexually deceptive species. Directional selection differentials for flower number were significantly higher in food compared to sexually deceptive species. We suggest that pollination systems with more efficient pollen transfer and no correlation between pollination success and number of flowers produced, such as sexual deception, may allow the production of inflorescences with fewer flowers that permit the plant to allocate fewer resources to floral displays and, at the same time, limit transpiration. This strategy can be particularly important for ecological success in Mediterranean water-deprived habitats, and might explain the high frequency of sexually deceptive species in these specialised ecosystems.
INTRODUCTION The early evolution and the extraordinary flowering plant radiation have been strongly linked to animal exploitation as pollen vectors (Crane et al. 1995; Hu et al. 2008; Van der Niet & Johnson 2012). This key innovation that characterises most basal angiosperm groups conferred the advantage of reducing pollen loss by more direct pollen transfer than provided through wind pollination, leading to more efficient reproduction (Pellmyr 2002; Endress 2010). Among flowering plants, the surprising variety of pollination systems has yet to be exhaustively described, and new adaptations continue to be discovered. The adaptations of floral traits to pollinators result in a wide range of variability, and under some conditions, can confer strong and immediate reproductive isolation (Grant 1949). As a consequence, transitions between pollination systems are often associated with speciation events (Grant 1949). In this context, understanding the mechanisms responsible for transitions in pollination systems is a primary goal for plant evolutionary biologists, and can assist in providing new insights into the vast radiation of several groups of flowering plants (Armbruster & Muchhala 2009). Entomophilous plants entice pollinators by producing a set of floral traits that pollen vectors associate with the presence of a reward (Dukas & Real 1993). However, production of an
attractive floral display is predicted to be costly (Pyke 1991), and available resources may limit the evolutionary trajectory towards increased pollination efficiency (Levins 1968). Life-history traits, including growth, survival and reproduction, are linked via trade-offs, which indicates an investment in one trait may come at the expense of another (Obeso 2002). In hermaphroditic plants, overall reproductive success is the combination of two elements, a male siring seeds and a female producing seeds. The allocation of resources to produce larger inflorescences can have different effects on each of these processes (Campbell 1989; Karron & Mitchell 2012). Male and female reproductive success can be a decelerating or accelerating function of inflorescence size (De Jong & Klinkhamer 1994). In the former case, evolution of pollination strategies that increases the efficiency of one or both reproductive functions facilitates a reduction in the inflorescence size investment. This resourcesaving strategy may represent an important improvement in the allocation strategy because it can release energy for other lifehistory traits and can be particularly relevant in resource-limited habitats where a reduction of inflorescence size also directly limits transpiration through the flower surface. Among the angiosperms, orchids represent one of the most astonishing examples of complex interactions with animal pollinators (Darwin 1862; Van der Pijl & Dodson 1966). In this
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plant group, an unusually high number of species do not produce a reward, and therefore attract pollinators using deceptive strategies (Dafni 1984; Ackerman 1986; Schiestl 2005; Jersakova et al. 2006). The mechanisms favouring evolution of a deceptive strategy remain poorly understood (Nilsson 1992; Jersakova et al. 2006). One popular hypothesis, with some empirical evidence, is that deception increases outcrossing by limiting pollinator-mediated self-pollination (Dressler 1981; Peakall & Beattie 1996; Johnson & Nilsson 1999; Johnson et al. 2004; Peakall & Schiestl 2004; Cozzolino & Widmer 2005; Jersakova & Johnson 2006). Several different deceptive pollination mechanisms have been described in orchids, however food-based deception is by far the most common strategy (Dafni 1984; Ackerman 1986). Sexual deception, with multiple origins and occurring on at least four different continents (Dafni & Bernhardt 1990; Pridgeon et al. 1997; Steiner 1998; Blanco & Barboza 2005), is successfully employed in several hundred species. Food deception operates on generalised mimicry of floral traits typical of nectariferous species and attracts na€ıve nectar-searching insects (Dafni 1984). Sexual deception is derived from visual, tactile and olfactory mimicry cues from female insects to attract males to copulate on the flowers (Schiestl 2005). The two pollination strategies differ substantially in floral specialisation and pollinator sharing (Schiestl & Schl€ uter 2009; Peakall et al. 2010). Sexual deception is a highly specific pollination strategy (Paulus & Gack 1990; Bower 1996), and sympatric species are reproductively isolated through strong floral isolation (Xu et al. 2011); food-deceptive species typically attract a wide range of pollinators that frequently overlap among sympatric species (Nilsson 1992; Cozzolino et al. 2005). In the Mediterranean subtribe Orchidinae, pollination strategy mapping based on molecular phylogenies shows that sexually deceptive species are nested in food-deceptive clades, suggesting that floral exploitation of insect sexual behaviour evolved from food-deceptive ancestors (Cozzolino et al. 2001; Cozzolino & Widmer 2005; Inda et al. 2012). A similar pattern was reported in the Australian subtribe Caladeniinae, where the transition from food to sexual deception occurred multiple times (Kores et al. 2001; Cozzolino & Widmer 2005), even within a single genus (as in Caladenia: Phillips et al. 2009). Interestingly, Schiestl & Cozzolino (2008) reported in the Orchidinae that several food-deceptive species produce floral compounds similar to those produced by sexually deceptive species involved in triggering male sexual responses, suggesting pre-adaptations for the evolution of sexual deception in fooddeceptive species (see also Vereecken et al. 2012). For some food-deceptive species, males and females have been shown to contribute to pollination equally, which may be mediated by putative sex pheromone compounds (Bino et al. 1982; Scopece et al. 2009), leading to pollination systems intermediate between food and sexual deception. While a trend towards specialisation is considered unlikely by some authors (e.g. Waser et al. 1996; but see Tremblay 1992), in the Orchidaceae it appears that the multiple independent transitions from food to sexual deception always represent a trend towards specialisation. Specialisation can be advantageous for male function, because it reduces pollen loss mediated by promiscuous or inefficient pollinators (Lankinen & Larsson 2009). Concordantly, it has been reported that sexual deception confers an increase in pollen transfer efficiency com2
pared to food deception, which has been interpreted as a potential factor selecting for the evolution of sexual deception, a highly specific pollination mechanism (Scopece et al. 2010; see also Ellis & Johnson 2010a). These results elucidate the food to sexual deception transition and the importance of pollen transfer efficiency along an evolutionary trajectory, from generalised food deception to specialised sexual deception. In this study we suggest the hypothesis that the reduction in pollen loss and the consequent increase of efficiency in pollen transfer may have an effect on inflorescence size in deceptive pollination systems. Specifically, we suggest the hypothesis that a more efficient pollen transfer in sexually deceptive species may allow the production of fewer flowers, which could in turn allow the allocation of resources to other life-history traits, and thus contributes to the success of this pollination strategy. We comparatively investigated the reproductive performances of two groups of Mediterranean orchids pollinated either through food or sexual deception, and counted flower number as a proxy for resources allocated to the floral display. We also analysed correlations between flower number and reproductive performance in the field. MATERIAL AND METHODS Data collection Sampling was conducted in 17 European orchid populations belonging to the orchid subtribe Orchidinae, selected with the aim of acquiring a representative sample of food-deceptive and sexually deceptive species. The orchid populations analysed represent four genera, with nine food-deceptive and eight sexually deceptive species (Table 1). Field observations were conducted on a total of 754 individuals (>4900 inspected flowers). Pollinators influence the fitness of hermaphroditic plants through male and female functions; therefore, we collected data on pollen removal and deposition. When flowers were open, we estimated number of flowers with pollinia removed (hereafter referred to as visited flowers) and number of flowers with at least one pollen massula on the stigma (hereafter referred to as pollinated flowers). For each sampled individual, we also tallied flower number on the inflorescence and number of flowers open at the time of sampling. In each population, data were collected from a random sample of individuals (44.35 6.68 on average; see Table 1). Flower number was also estimated from a selection of foodand sexually deceptive species belonging to the Australian subtribe Caladeniinae using data reported in Scopece et al. (2010) and unpublished observation. We included in this analysis 11 species (seven sexually deceptive and four food-deceptive), all belonging to the genus Caladenia (see Table S1). Reproductive parameters and data analyses The efficiency of the two pollination strategies was evaluated by estimating the proportion of exported pollen that did not reach intraspecific stigmas. The methods of Scopece et al. (2010) were applied with minor modifications. We calculated an index as follows: pollen loss = 1 (pollinated flowers/visited flowers). Pollen loss indices were compared between fooddeceptive and sexually deceptive species using a non-parametric Mann–Whitney U test.
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Pollen transfer efficiency and flower number
Table 1. The location and strategy of the analysed orchid populations: four genera containing nine food-deceptive and eight sexually deceptive species.
species
pollination strategya
locality
sampled individuals
mean no. flowers
mean no. open flowers
APS
pollen loss
Anacamptis pyramidalis Anacamptis papilionacea ssp. papilionacea Anacamptis papilionacea ssp. grandiflora Dactylorhiza incarnata Dactylorhiza saccifera Orchis italica Orchis mascula Orchis pauciflora Orchis simia Ophrys bertolonii Ophrys bilunulata Ophrys exaltata Ophrys fusca Ophrys insectifera Ophrys lutea Ophrys sphegodes Ophrys tenthredinifera
FD FD FD FD FD FD FD FD FD SD SD SD SD SD SD SD SD
Monte Catalfano (Sicily) Monte San Giacomo (Campania) Monte San Giacomo (Campania) Monte Matese (Campania) Ruvo del Monte (Basilicata) Novara di Sicilia (Sicily) Monte San Giacomo (Campania) Monte San Giacomo (Campania) Monte San Giacomo (Campania) Monte Genuardo (Sicily) Marina di Lesina (Puglia) Marina di Lesina (Puglia) Montebello (Calabria) Vigo di Fassa (Trentino A. Adige) Monte Catalfano (Sicily) Marina di Lesina (Puglia) Monte San Giacomo (Campania)
48 71 9 10 20 20 29 53 25 60 28 98 63 28 28 79 85
4.19 4.66 13.11 13.5 35.55 22.32 23.72 4.85 17.28 3.17 2.96 3.69 3,70 4.00 2.39 3.61 3.77
3.71 4.66 13.11 13.5 30.2 20.16 23.72 4.85 17.28 1.48 2.96 2.59 3.21 4.00 2.21 2.52 2.49
1.58 1.52 0.89 5.56 8.3 3.67 2.56 1.87 2.56 1.75 3.44 3.19 2.23 1.5 3.27 1.81 1.12
0.55 0.46 0.84 0.49 0.58 0.63 0.41 0.61 0.55 0.07 0.20 0.16 0.24 0.4 0.18 0.17 0.50
SD, sexual deception. a FD, food deception.
The flower number produced by individuals of each species was estimated on all sampled individuals and averaged in each sampled population. Overall variation in flower dimensions between food-deceptive and sexually deceptive species is generally small (Delforge 2006), therefore flower number was considered a proxy for resources allocated in the reproductive process. This parameter was compared both between fooddeceptive and sexually deceptive Mediterranean Orchidinae and between Australian food-deceptive and sexually deceptive Caladeniinae using a non-parametric Mann–Whitney U test. For both pollination strategies, basing on individuals with at least one visited flower, we calculated absolute pollination success (APS) as follows: APS = male function + female function. For logistical reasons, male function was not estimated following pollen fate, but by estimating the number of flowers that showed pollen removal. Interpretation of male function derived only from pollen removal can result in erroneous conclusions (Johnson et al. 2005); therefore we multiplied it by the probability that exported pollen reached conspecific stigmas (i.e. the ratio between pollinated and visited flowers; Ellis & Johnson 2010b). Female function was estimated as the number of pollinated flowers in each sampled individual. Therefore, the APS index estimated the expected fruit number that individuals should produce through male and female functions. The link between flower number and pollination success for fooddeceptive and sexually deceptive strategies was assessed with a non-parametric Kendall’s tau correlation between APS and the number of open flowers. For four species (two sexually deceptive, i.e. Ophrys exaltata and Ophrys sphegodes, and two food-deceptive, i.e. Orchis mascula and Orchis pauciflora) we estimated directional selection differentials (Lande & Arnold 1983; Brodie et al. 1995) as the covariance between relative reproductive success and flower number produced. We calculated relative reproductive success of an individual as fruit set divided by mean population fruit set (Lande & Arnold 1983). Bootstrap bias-corrected confidence intervals (CI) were computed to compare selection dif-
ferentials among different species (Maad & Alexandersson 2004). Non-overlapping CIs indicated significantly different mean trait values among populations. RESULTS Pollen loss was significantly higher in food-deceptive compared to sexually deceptive species (U = 3.0, Z = 3.177, P < 0.001; Fig. 1). Food-deceptive species produced more flowers than sexually deceptive species (U = 0.0, Z = 3.464, P < 0.001; Fig. 2a). The same pattern was also found in the Australian subtribe Caladeniinae (U = 3.0, Z = 2.088, P < 0.05; Fig. 2b). Absolute pollination success (APS) was not significantly different between the two pollination systems (U = 30.0, Z = 0.578, P = 0.606) and was significantly correlated with open flower number in food-deceptive species (Kendall tau = 0.535, P < 0.05; Fig. 3a), but not in sexually deceptive species (Kendall tau = 0.071, P = 0.805; Fig. 3b). A trend towards a positive correlation between pollen loss and flower number in sexually deceptive species (Kendall tau = 0.429, P = 0.138) was observed, but not in food-deceptive species (Kendall tau = 0.028, P = 0.917).
Fig. 1. Comparison of pollen loss between food- and sexually deceptive species. Circles represent outliers.
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A
Scopece, Schiestl & Cozzolino
B
Fig. 2. Comparisons of flower number between foodand sexually deceptive species of Orchidinae (A) and Caladeniinae (B). Circles represent outliers.
A
B
Fig. 3. Correlation between mean number of open flowers and absolute pollination success (APS) in fooddeceptive (A) and sexually deceptive (B) species. Black solid lines represent regression lines; dashed lines represent mean confidence intervals (CI).
Food-deceptive species showed higher directional selection differential values compared to sexually deceptive species (Fig. 4; food-deceptive species: Orchis mascula = 0.023, Orchis pauciflora = 0.043; sexually deceptive species: Ophrys exaltata = 0.001, Ophrys sphegodes = 0.001). DISCUSSION Floral display is a key investment in plants, and often comprises a significant part of an individual plant’s total biomass. Our results suggest that efficiency in pollen transfer might have a strong impact on floral display evolution, and therefore on plant resource allocation strategies. In orchids, many key adaptations are associated with resource allocation, including the lack of seed endosperm (Arditti 1987), the absence of female gametophyte development until pollination (Zhang & O’Neill 1993) and the exploitation of symbiotic fungi for germination (Waterman & Bidartondo 2008). Moreover, deceptively pollinated species conserve the resources that nectariferous species
Fig. 4. Directional selection differentials for flower number in sexually deceptive Ophrys sphegodes and O. exaltata, and food-deceptive Orchis mascula and O. pauciflora. Different letters indicate significant differences.
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allocate in potentially expensive nectar production (Pyke 1991). The present study emphasises an important advantage in resource allocation for sexually deceptive orchids relative to food-deceptive species: comparable reproductive success can be achieved with fewer flowers. This advantage may contribute to the success of sexually deceptive orchids, which commonly grow in nutrient-deprived soils (Delforge 2006). When comparing pollen loss between food-deceptive and sexually deceptive species we found significant differences. That is, as already shown for pollen transfer in Scopece et al. (2010), sexually deceptive species proved to have more efficient pollination, with a higher fraction of removed pollen that does reach conspecific stigmas (Fig. 1). Different proportions of pollen loss are likely due to different specialisation levels of the two strategies, and the behaviour of insects visiting food- or sexually deceptive species. Food-deceptive species attract a wide range of pollinators that associate floral traits with the presence or absence of a nectar reward (Dukas & Real 1993). Following a few unsuccessful visits to deceptive flowers, the pollinators shift to other flower types. This inconsistent flower visitation behaviour likely leads to high pollen loss (Cozzolino et al. 2005). In contrast, it has been reported that sexually deceptive species attract males of only a few pollinator species (Paulus & Gack 1990; Schiestl & Schl€ uter 2009), which show high fidelity in flower visitation (Xu et al. 2011). Furthermore, male bees spend less time foraging on other flowers than female bees (Ne’eman et al. 2006; Schiestl & Schl€ uter 2009). Inflorescence size has often been linked to selection for the male component of reproductive success (Campbell 1989). The advantage in the male component can thus explain the lower number of flowers produced in sexually deceptive versus fooddeceptive species found in our study (Fig. 2a). Reduced pollen loss in sexually deceptive species (with smaller inflorescence size compared to food-deceptive species) suggests this pollination system, by increased pollen dissemination efficiency, releases the selection for larger inflorescences resulting in a
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Pollen transfer efficiency and flower number
reduction in flower production. This is also supported by the finding of a positive correlation trend between pollen loss and flower number in sexually deceptive species. Comparative studies can be influenced by phylogenetic constraints that can overestimate differences due to the tendency of closely related taxa to resemble each other (Felsenstein 1985). In the present study, an appropriate test to exclude phylogenetic constraints could not be performed on the dataset of Mediterranean orchids because all sexually deceptive species belong to the same genus (and thus represent a single transition). However, we found that within the Australian subtribe Caladeniinae, congruent with results from Mediterranean Orchidinae, sexually deceptive species produce fewer flowers than food-deceptive species (Fig. 2b). The subtribe Caladeniinae is comprised of sister species with alternative pollination strategy (Phillips et al. 2009), suggesting transitions from food to sexual deception occurred multiple times. Therefore, in contrast with Mediterranean orchids, the Australian Caladeniinae is a viable model to test pattern independence from phylogenetic constraints. In this context, the finding of similar patterns in phylogenetically independent groups such as Orchidinae and Caladeniinae may suggest wider applicability of our results. In all species, independent of pollination strategy, selection should lead to an optimisation of reproductive success. However, although a positive correlation between absolute pollination success and number of open flowers in food-deceptive species (Fig. 3a) was observed, in sexually deceptive species the absence of a correlation was detected (Fig. 3b), which suggests selection towards increased flower number primarily affects food-deceptive species. Similarly, our directional selection differential analysis in four orchid species showed that selection towards an increase in flower production is higher in fooddeceptive than in sexually deceptive species (Fig. 4). Food-deceptive flowers primarily exploit the pollinator’s visual perception by producing showy flowers, nectar guides and long spurs (Dafni 1984). In such a pollination strategy, the enhanced visibility due to increased flower number (i.e. in floral display) can be important for overall pollination success. In contrast, the lack of a correlation between APS and flower number in sexually deceptive species can be related to the rapid individual learning of male insects that, after pseudocopulation, may visit one or two more flowers but then never come back to the same flowers (Ayasse et al. 2000). This behaviour, together with the limited number of individuals attracted (only males of only one or a few species), suggests that increased flower production might not result in higher pollination success under this pollination system. In terms of individual pollination performance, we found that APS for food-deceptive and sexually deceptive individuals was similar, which may suggest that sexually deceptive individuals can produce a comparable number of fruits (via male or female function) despite decreased flower production. This may relieve the selection towards increasing resource allocation in the proREFERENCES Ackerman J.D. (1986) Mechanisms and evolution of food-deceptive pollination systems in orchids. Lindleyana, 1, 108–113. Arditti J. (1987) Orchid biology, reviews and perspectives. Cornell University Press, New York, USA.
duction of more flower, thus releasing a higher proportion of energy for other life-history traits, such as those involved in plant growth or longevity. This improved resource allocation strategy may be an important advantage for reducing transpiration in species that live in water-deprived habitats such as Mediterranean regions where sexually deceptive species typically occur, and where this pollination system has evolved multiple times in different continents (e.g. Dafni 1984; Cozzolino & Widmer 2005). Our results thus suggest that sexual deception, through more efficient pollen transfer, confers an important advantage in terms of resource allocation, favouring the exploitation of resource-limited habitats. Future studies are needed to understand whether even food-deceptive species that occupy strictly Mediterranean habitats show adaptations to reduce the investment in floral display or to increase pollen transfer efficiency as may be the case, for e.g. O. pauciflora, a food-deceptive species with small inflorescences that produces male bumblebee pheromone components that have been shown to increase pollen removal (Valterova et al. 2008) and of Anacamptis papilionacea that likely exploits a mixed pollination strategy also involving sexual attraction (Vogel 1972; Scopece et al. 2009). CONCLUSIONS In this study, we show that enhanced pollen transfer efficiency in sexually deceptive orchids could confer an important advantage in resource allocation. We suggest this advantage contributed to the evolutionary success of sexually deceptive species in resource-limited habitats by facilitating re-allocation from flower number to other life-history traits or to defence. This may render an important ecological advantage, which can explain the absence of reversals from sexual to food deception. Further studies are necessary to elucidate whether sexual deception efficiency has implications on other life-history traits, such as longevity and growth. Future studies should also investigate the link between pollen transfer efficiency, floral display and resource allocation in other pollination systems and plant taxa to better understand the impact on plant evolution. ACKNOWLEDGEMENTS The authors thank V. Tranchida-Lombardo, N. Juillet and S. Q. Xu for help in field data collection and data analyses. The authors also thank Steve Johnson and R. Mitchell for helpful comments on a previous version of the manuscript. The Progetto di Rilevante Interesse Nazionale (PRIN) funded this study. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Caladenia species included in the analysis.
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sexual deception for pollinator specificity, fruit set, population genetics and conservation of Caladenia (Orchidaceae). Australian Journal of Botany, 57, 287–306. Pridgeon A.M., Bateman R.M., Cox A.V., Hapemann J.R., Chase M.W. (1997) Phylogenetics of subtribe Orchidinae (Orchidoideae, Orchidaceae) based on nuclear ITS sequences. 1. Intergeneric relationships and polyphyly of Orchis sensu lato. Lindleyana, 12, 89–109. Pyke G. (1991) What does it cost a plant to produce floral nectar? Nature, 350, 58–59. Schiestl F.P. (2005) On the success of a swindle: pollination by deception in orchids. Naturwissenschaften, 92, 255–264. Schiestl F.P., Cozzolino S. (2008) Evolution of sexual mimicry in the Orchidinae: the role of preadaptations in the attraction of male bees as pollinators. BMC Evolutionary Biology, 8, 27. Schiestl F.P., Schl€ uter P.M. (2009) Floral isolation, specialized pollination, and pollinator behavior in orchids. Annual Review of Entomology, 54, 425–446. Scopece G., Juillet N., Muller A., Schiestl F.P., Cozzolino S. (2009) Pollinator attraction in Anacamptis papilionacea (Orchidaceae): a food or a sex promise? Plant Species Biology, 24, 109–114. Scopece G., Cozzolino S., Johnson S.D., Schiestl F.P. (2010) Pollination efficiency and the evolution of specialized deceptive pollination systems. American Naturalist, 175, 98–105. Steiner K.E. (1998) The evolution of beetle pollination in a South African orchid. American Journal of Botany, 85, 1180–1193. Tremblay R.L. (1992) Trends in the pollination ecology of the Orchidaceae: evolution and systematics. Canadian Journal of Botany, 70, 642–650. Valterova I., Kunze J., Gumbert A., Luxova A., Liblikas I., Kalinova B., Borg-Karlson A.-B. (2008) Male bumble bee pheromonal components in the scent of deceit pollinated orchids; unrecognized pollinator cues? Arthropod-Plant Interactions, 1, 137–145. Van der Niet T., Johnson S.D. (2012) Phylogenetic evidence for pollinator-driven diversification of angiosperms. Trends in Ecology & Evolution, 27, 353–361. Van der Pijl L., Dodson C.H. (1966) Orchid flowers: their pollination and evolution. University of Miami Press, Coral Gables, FL, USA. Vereecken N.J., Wilson C.A., H€ otling S., Schulz S., Banketov S.A., Mardulyn P. (2012) Pre-adaptations and the evolution of pollination by sexual deception: Cope’s rule of specialization revisited. Proceedings of the Royal Society B: Biological Sciences, 279, 4786–4794. Vogel S. (1972) Pollination von Orchis papilionacea L. in den Schwarmbahnen von Eucera tuberculata F. Jahresberichte des Naturwissenschaftlichen Vereins in Wuppertal, 85, 67–74. Waser N.M., Chittka L., Price M.V., Williams N.M., Ollerton J. (1996) Generalization in pollination systems, and why it matters. Ecology, 77, 1043–1060. Waterman R.J., Bidartondo M.I. (2008) Deception above, deception below: linking pollination and mycorrhizal biology of orchids. Journal of Experimental Botany, 59, 1085–1096. Xu S.Q., Schl€ uter P.M., Scopece G., Breitkopf H., Gross K., Cozzolino S., Schiestl F.P. (2011) Floral isolation is the main reproductive barrier among closely related sexually deceptive orchids. Evolution, 65, 2606–2620. Zhang X.S., O’Neill S.D. (1993) Ovary and gametophyte development are coordinately regulated by auxin and ethylene following pollination. Plant Cell, 5, 403–418.
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RESEARCH PAPER
Pollen transfer efficiency and its effect on inflorescence size in deceptive pollination strategies G. Scopece1,2, F. P. Schiestl3 & S. Cozzolino1 1 Department of Biology, University Federico II, Complesso Universitario MSA, Naples, Italy 2 Institute for Plant Protection, Consiglio Nazionale delle Ricerche, Sesto Fiorentino (FI), Italy 3 Institute of Systematic Botany, University of Zurich, Zurich, Switzerland
Keywords Flower number; food deception; inflorescence size; orchids; pollen loss; resource allocation; sexual deception. Correspondence G. Scopece, Department of Biology, University of Naples Federico II, Complesso Universitario MSA, via Cinthia, I-80126 Naples, Italy. E-mail: [email protected] Editor A. Dafni Received: 4 February 2014; Accepted: 16 May 2014 doi:10.1111/plb.12224
ABSTRACT Pollination systems differ in pollen transfer efficiency, a variable that may influence the evolution of flower number. Here we apply a comparative approach to examine the link between pollen transfer efficiency and the evolution of inflorescence size in food and sexually deceptive orchids. We examined pollination performance in nine food-deceptive, and eight sexually deceptive orchids by recording pollen removal and deposition in the field. We calculated correlations between reproductive success and flower number (as a proxy for resources allocated during reproductive process), and directional selection differentials were estimated on flower number for four species. Results indicate that sexually deceptive species experience decreased pollen loss compared to food-deceptive species. Despite producing fewer flowers, sexually deceptive species attained levels of overall pollination success (through male and female function) similar to food-deceptive species. Furthermore, a positive correlation between flower number and pollination success was observed in food-deceptive species, but this correlation was not detected in sexually deceptive species. Directional selection differentials for flower number were significantly higher in food compared to sexually deceptive species. We suggest that pollination systems with more efficient pollen transfer and no correlation between pollination success and number of flowers produced, such as sexual deception, may allow the production of inflorescences with fewer flowers that permit the plant to allocate fewer resources to floral displays and, at the same time, limit transpiration. This strategy can be particularly important for ecological success in Mediterranean water-deprived habitats, and might explain the high frequency of sexually deceptive species in these specialised ecosystems.
INTRODUCTION The early evolution and the extraordinary flowering plant radiation have been strongly linked to animal exploitation as pollen vectors (Crane et al. 1995; Hu et al. 2008; Van der Niet & Johnson 2012). This key innovation that characterises most basal angiosperm groups conferred the advantage of reducing pollen loss by more direct pollen transfer than provided through wind pollination, leading to more efficient reproduction (Pellmyr 2002; Endress 2010). Among flowering plants, the surprising variety of pollination systems has yet to be exhaustively described, and new adaptations continue to be discovered. The adaptations of floral traits to pollinators result in a wide range of variability, and under some conditions, can confer strong and immediate reproductive isolation (Grant 1949). As a consequence, transitions between pollination systems are often associated with speciation events (Grant 1949). In this context, understanding the mechanisms responsible for transitions in pollination systems is a primary goal for plant evolutionary biologists, and can assist in providing new insights into the vast radiation of several groups of flowering plants (Armbruster & Muchhala 2009). Entomophilous plants entice pollinators by producing a set of floral traits that pollen vectors associate with the presence of a reward (Dukas & Real 1993). However, production of an
attractive floral display is predicted to be costly (Pyke 1991), and available resources may limit the evolutionary trajectory towards increased pollination efficiency (Levins 1968). Life-history traits, including growth, survival and reproduction, are linked via trade-offs, which indicates an investment in one trait may come at the expense of another (Obeso 2002). In hermaphroditic plants, overall reproductive success is the combination of two elements, a male siring seeds and a female producing seeds. The allocation of resources to produce larger inflorescences can have different effects on each of these processes (Campbell 1989; Karron & Mitchell 2012). Male and female reproductive success can be a decelerating or accelerating function of inflorescence size (De Jong & Klinkhamer 1994). In the former case, evolution of pollination strategies that increases the efficiency of one or both reproductive functions facilitates a reduction in the inflorescence size investment. This resourcesaving strategy may represent an important improvement in the allocation strategy because it can release energy for other lifehistory traits and can be particularly relevant in resource-limited habitats where a reduction of inflorescence size also directly limits transpiration through the flower surface. Among the angiosperms, orchids represent one of the most astonishing examples of complex interactions with animal pollinators (Darwin 1862; Van der Pijl & Dodson 1966). In this
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plant group, an unusually high number of species do not produce a reward, and therefore attract pollinators using deceptive strategies (Dafni 1984; Ackerman 1986; Schiestl 2005; Jersakova et al. 2006). The mechanisms favouring evolution of a deceptive strategy remain poorly understood (Nilsson 1992; Jersakova et al. 2006). One popular hypothesis, with some empirical evidence, is that deception increases outcrossing by limiting pollinator-mediated self-pollination (Dressler 1981; Peakall & Beattie 1996; Johnson & Nilsson 1999; Johnson et al. 2004; Peakall & Schiestl 2004; Cozzolino & Widmer 2005; Jersakova & Johnson 2006). Several different deceptive pollination mechanisms have been described in orchids, however food-based deception is by far the most common strategy (Dafni 1984; Ackerman 1986). Sexual deception, with multiple origins and occurring on at least four different continents (Dafni & Bernhardt 1990; Pridgeon et al. 1997; Steiner 1998; Blanco & Barboza 2005), is successfully employed in several hundred species. Food deception operates on generalised mimicry of floral traits typical of nectariferous species and attracts na€ıve nectar-searching insects (Dafni 1984). Sexual deception is derived from visual, tactile and olfactory mimicry cues from female insects to attract males to copulate on the flowers (Schiestl 2005). The two pollination strategies differ substantially in floral specialisation and pollinator sharing (Schiestl & Schl€ uter 2009; Peakall et al. 2010). Sexual deception is a highly specific pollination strategy (Paulus & Gack 1990; Bower 1996), and sympatric species are reproductively isolated through strong floral isolation (Xu et al. 2011); food-deceptive species typically attract a wide range of pollinators that frequently overlap among sympatric species (Nilsson 1992; Cozzolino et al. 2005). In the Mediterranean subtribe Orchidinae, pollination strategy mapping based on molecular phylogenies shows that sexually deceptive species are nested in food-deceptive clades, suggesting that floral exploitation of insect sexual behaviour evolved from food-deceptive ancestors (Cozzolino et al. 2001; Cozzolino & Widmer 2005; Inda et al. 2012). A similar pattern was reported in the Australian subtribe Caladeniinae, where the transition from food to sexual deception occurred multiple times (Kores et al. 2001; Cozzolino & Widmer 2005), even within a single genus (as in Caladenia: Phillips et al. 2009). Interestingly, Schiestl & Cozzolino (2008) reported in the Orchidinae that several food-deceptive species produce floral compounds similar to those produced by sexually deceptive species involved in triggering male sexual responses, suggesting pre-adaptations for the evolution of sexual deception in fooddeceptive species (see also Vereecken et al. 2012). For some food-deceptive species, males and females have been shown to contribute to pollination equally, which may be mediated by putative sex pheromone compounds (Bino et al. 1982; Scopece et al. 2009), leading to pollination systems intermediate between food and sexual deception. While a trend towards specialisation is considered unlikely by some authors (e.g. Waser et al. 1996; but see Tremblay 1992), in the Orchidaceae it appears that the multiple independent transitions from food to sexual deception always represent a trend towards specialisation. Specialisation can be advantageous for male function, because it reduces pollen loss mediated by promiscuous or inefficient pollinators (Lankinen & Larsson 2009). Concordantly, it has been reported that sexual deception confers an increase in pollen transfer efficiency com2
pared to food deception, which has been interpreted as a potential factor selecting for the evolution of sexual deception, a highly specific pollination mechanism (Scopece et al. 2010; see also Ellis & Johnson 2010a). These results elucidate the food to sexual deception transition and the importance of pollen transfer efficiency along an evolutionary trajectory, from generalised food deception to specialised sexual deception. In this study we suggest the hypothesis that the reduction in pollen loss and the consequent increase of efficiency in pollen transfer may have an effect on inflorescence size in deceptive pollination systems. Specifically, we suggest the hypothesis that a more efficient pollen transfer in sexually deceptive species may allow the production of fewer flowers, which could in turn allow the allocation of resources to other life-history traits, and thus contributes to the success of this pollination strategy. We comparatively investigated the reproductive performances of two groups of Mediterranean orchids pollinated either through food or sexual deception, and counted flower number as a proxy for resources allocated to the floral display. We also analysed correlations between flower number and reproductive performance in the field. MATERIAL AND METHODS Data collection Sampling was conducted in 17 European orchid populations belonging to the orchid subtribe Orchidinae, selected with the aim of acquiring a representative sample of food-deceptive and sexually deceptive species. The orchid populations analysed represent four genera, with nine food-deceptive and eight sexually deceptive species (Table 1). Field observations were conducted on a total of 754 individuals (>4900 inspected flowers). Pollinators influence the fitness of hermaphroditic plants through male and female functions; therefore, we collected data on pollen removal and deposition. When flowers were open, we estimated number of flowers with pollinia removed (hereafter referred to as visited flowers) and number of flowers with at least one pollen massula on the stigma (hereafter referred to as pollinated flowers). For each sampled individual, we also tallied flower number on the inflorescence and number of flowers open at the time of sampling. In each population, data were collected from a random sample of individuals (44.35 6.68 on average; see Table 1). Flower number was also estimated from a selection of foodand sexually deceptive species belonging to the Australian subtribe Caladeniinae using data reported in Scopece et al. (2010) and unpublished observation. We included in this analysis 11 species (seven sexually deceptive and four food-deceptive), all belonging to the genus Caladenia (see Table S1). Reproductive parameters and data analyses The efficiency of the two pollination strategies was evaluated by estimating the proportion of exported pollen that did not reach intraspecific stigmas. The methods of Scopece et al. (2010) were applied with minor modifications. We calculated an index as follows: pollen loss = 1 (pollinated flowers/visited flowers). Pollen loss indices were compared between fooddeceptive and sexually deceptive species using a non-parametric Mann–Whitney U test.
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Table 1. The location and strategy of the analysed orchid populations: four genera containing nine food-deceptive and eight sexually deceptive species.
species
pollination strategya
locality
sampled individuals
mean no. flowers
mean no. open flowers
APS
pollen loss
Anacamptis pyramidalis Anacamptis papilionacea ssp. papilionacea Anacamptis papilionacea ssp. grandiflora Dactylorhiza incarnata Dactylorhiza saccifera Orchis italica Orchis mascula Orchis pauciflora Orchis simia Ophrys bertolonii Ophrys bilunulata Ophrys exaltata Ophrys fusca Ophrys insectifera Ophrys lutea Ophrys sphegodes Ophrys tenthredinifera
FD FD FD FD FD FD FD FD FD SD SD SD SD SD SD SD SD
Monte Catalfano (Sicily) Monte San Giacomo (Campania) Monte San Giacomo (Campania) Monte Matese (Campania) Ruvo del Monte (Basilicata) Novara di Sicilia (Sicily) Monte San Giacomo (Campania) Monte San Giacomo (Campania) Monte San Giacomo (Campania) Monte Genuardo (Sicily) Marina di Lesina (Puglia) Marina di Lesina (Puglia) Montebello (Calabria) Vigo di Fassa (Trentino A. Adige) Monte Catalfano (Sicily) Marina di Lesina (Puglia) Monte San Giacomo (Campania)
48 71 9 10 20 20 29 53 25 60 28 98 63 28 28 79 85
4.19 4.66 13.11 13.5 35.55 22.32 23.72 4.85 17.28 3.17 2.96 3.69 3,70 4.00 2.39 3.61 3.77
3.71 4.66 13.11 13.5 30.2 20.16 23.72 4.85 17.28 1.48 2.96 2.59 3.21 4.00 2.21 2.52 2.49
1.58 1.52 0.89 5.56 8.3 3.67 2.56 1.87 2.56 1.75 3.44 3.19 2.23 1.5 3.27 1.81 1.12
0.55 0.46 0.84 0.49 0.58 0.63 0.41 0.61 0.55 0.07 0.20 0.16 0.24 0.4 0.18 0.17 0.50
SD, sexual deception. a FD, food deception.
The flower number produced by individuals of each species was estimated on all sampled individuals and averaged in each sampled population. Overall variation in flower dimensions between food-deceptive and sexually deceptive species is generally small (Delforge 2006), therefore flower number was considered a proxy for resources allocated in the reproductive process. This parameter was compared both between fooddeceptive and sexually deceptive Mediterranean Orchidinae and between Australian food-deceptive and sexually deceptive Caladeniinae using a non-parametric Mann–Whitney U test. For both pollination strategies, basing on individuals with at least one visited flower, we calculated absolute pollination success (APS) as follows: APS = male function + female function. For logistical reasons, male function was not estimated following pollen fate, but by estimating the number of flowers that showed pollen removal. Interpretation of male function derived only from pollen removal can result in erroneous conclusions (Johnson et al. 2005); therefore we multiplied it by the probability that exported pollen reached conspecific stigmas (i.e. the ratio between pollinated and visited flowers; Ellis & Johnson 2010b). Female function was estimated as the number of pollinated flowers in each sampled individual. Therefore, the APS index estimated the expected fruit number that individuals should produce through male and female functions. The link between flower number and pollination success for fooddeceptive and sexually deceptive strategies was assessed with a non-parametric Kendall’s tau correlation between APS and the number of open flowers. For four species (two sexually deceptive, i.e. Ophrys exaltata and Ophrys sphegodes, and two food-deceptive, i.e. Orchis mascula and Orchis pauciflora) we estimated directional selection differentials (Lande & Arnold 1983; Brodie et al. 1995) as the covariance between relative reproductive success and flower number produced. We calculated relative reproductive success of an individual as fruit set divided by mean population fruit set (Lande & Arnold 1983). Bootstrap bias-corrected confidence intervals (CI) were computed to compare selection dif-
ferentials among different species (Maad & Alexandersson 2004). Non-overlapping CIs indicated significantly different mean trait values among populations. RESULTS Pollen loss was significantly higher in food-deceptive compared to sexually deceptive species (U = 3.0, Z = 3.177, P < 0.001; Fig. 1). Food-deceptive species produced more flowers than sexually deceptive species (U = 0.0, Z = 3.464, P < 0.001; Fig. 2a). The same pattern was also found in the Australian subtribe Caladeniinae (U = 3.0, Z = 2.088, P < 0.05; Fig. 2b). Absolute pollination success (APS) was not significantly different between the two pollination systems (U = 30.0, Z = 0.578, P = 0.606) and was significantly correlated with open flower number in food-deceptive species (Kendall tau = 0.535, P < 0.05; Fig. 3a), but not in sexually deceptive species (Kendall tau = 0.071, P = 0.805; Fig. 3b). A trend towards a positive correlation between pollen loss and flower number in sexually deceptive species (Kendall tau = 0.429, P = 0.138) was observed, but not in food-deceptive species (Kendall tau = 0.028, P = 0.917).
Fig. 1. Comparison of pollen loss between food- and sexually deceptive species. Circles represent outliers.
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A
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B
Fig. 2. Comparisons of flower number between foodand sexually deceptive species of Orchidinae (A) and Caladeniinae (B). Circles represent outliers.
A
B
Fig. 3. Correlation between mean number of open flowers and absolute pollination success (APS) in fooddeceptive (A) and sexually deceptive (B) species. Black solid lines represent regression lines; dashed lines represent mean confidence intervals (CI).
Food-deceptive species showed higher directional selection differential values compared to sexually deceptive species (Fig. 4; food-deceptive species: Orchis mascula = 0.023, Orchis pauciflora = 0.043; sexually deceptive species: Ophrys exaltata = 0.001, Ophrys sphegodes = 0.001). DISCUSSION Floral display is a key investment in plants, and often comprises a significant part of an individual plant’s total biomass. Our results suggest that efficiency in pollen transfer might have a strong impact on floral display evolution, and therefore on plant resource allocation strategies. In orchids, many key adaptations are associated with resource allocation, including the lack of seed endosperm (Arditti 1987), the absence of female gametophyte development until pollination (Zhang & O’Neill 1993) and the exploitation of symbiotic fungi for germination (Waterman & Bidartondo 2008). Moreover, deceptively pollinated species conserve the resources that nectariferous species
Fig. 4. Directional selection differentials for flower number in sexually deceptive Ophrys sphegodes and O. exaltata, and food-deceptive Orchis mascula and O. pauciflora. Different letters indicate significant differences.
4
allocate in potentially expensive nectar production (Pyke 1991). The present study emphasises an important advantage in resource allocation for sexually deceptive orchids relative to food-deceptive species: comparable reproductive success can be achieved with fewer flowers. This advantage may contribute to the success of sexually deceptive orchids, which commonly grow in nutrient-deprived soils (Delforge 2006). When comparing pollen loss between food-deceptive and sexually deceptive species we found significant differences. That is, as already shown for pollen transfer in Scopece et al. (2010), sexually deceptive species proved to have more efficient pollination, with a higher fraction of removed pollen that does reach conspecific stigmas (Fig. 1). Different proportions of pollen loss are likely due to different specialisation levels of the two strategies, and the behaviour of insects visiting food- or sexually deceptive species. Food-deceptive species attract a wide range of pollinators that associate floral traits with the presence or absence of a nectar reward (Dukas & Real 1993). Following a few unsuccessful visits to deceptive flowers, the pollinators shift to other flower types. This inconsistent flower visitation behaviour likely leads to high pollen loss (Cozzolino et al. 2005). In contrast, it has been reported that sexually deceptive species attract males of only a few pollinator species (Paulus & Gack 1990; Schiestl & Schl€ uter 2009), which show high fidelity in flower visitation (Xu et al. 2011). Furthermore, male bees spend less time foraging on other flowers than female bees (Ne’eman et al. 2006; Schiestl & Schl€ uter 2009). Inflorescence size has often been linked to selection for the male component of reproductive success (Campbell 1989). The advantage in the male component can thus explain the lower number of flowers produced in sexually deceptive versus fooddeceptive species found in our study (Fig. 2a). Reduced pollen loss in sexually deceptive species (with smaller inflorescence size compared to food-deceptive species) suggests this pollination system, by increased pollen dissemination efficiency, releases the selection for larger inflorescences resulting in a
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reduction in flower production. This is also supported by the finding of a positive correlation trend between pollen loss and flower number in sexually deceptive species. Comparative studies can be influenced by phylogenetic constraints that can overestimate differences due to the tendency of closely related taxa to resemble each other (Felsenstein 1985). In the present study, an appropriate test to exclude phylogenetic constraints could not be performed on the dataset of Mediterranean orchids because all sexually deceptive species belong to the same genus (and thus represent a single transition). However, we found that within the Australian subtribe Caladeniinae, congruent with results from Mediterranean Orchidinae, sexually deceptive species produce fewer flowers than food-deceptive species (Fig. 2b). The subtribe Caladeniinae is comprised of sister species with alternative pollination strategy (Phillips et al. 2009), suggesting transitions from food to sexual deception occurred multiple times. Therefore, in contrast with Mediterranean orchids, the Australian Caladeniinae is a viable model to test pattern independence from phylogenetic constraints. In this context, the finding of similar patterns in phylogenetically independent groups such as Orchidinae and Caladeniinae may suggest wider applicability of our results. In all species, independent of pollination strategy, selection should lead to an optimisation of reproductive success. However, although a positive correlation between absolute pollination success and number of open flowers in food-deceptive species (Fig. 3a) was observed, in sexually deceptive species the absence of a correlation was detected (Fig. 3b), which suggests selection towards increased flower number primarily affects food-deceptive species. Similarly, our directional selection differential analysis in four orchid species showed that selection towards an increase in flower production is higher in fooddeceptive than in sexually deceptive species (Fig. 4). Food-deceptive flowers primarily exploit the pollinator’s visual perception by producing showy flowers, nectar guides and long spurs (Dafni 1984). In such a pollination strategy, the enhanced visibility due to increased flower number (i.e. in floral display) can be important for overall pollination success. In contrast, the lack of a correlation between APS and flower number in sexually deceptive species can be related to the rapid individual learning of male insects that, after pseudocopulation, may visit one or two more flowers but then never come back to the same flowers (Ayasse et al. 2000). This behaviour, together with the limited number of individuals attracted (only males of only one or a few species), suggests that increased flower production might not result in higher pollination success under this pollination system. In terms of individual pollination performance, we found that APS for food-deceptive and sexually deceptive individuals was similar, which may suggest that sexually deceptive individuals can produce a comparable number of fruits (via male or female function) despite decreased flower production. This may relieve the selection towards increasing resource allocation in the proREFERENCES Ackerman J.D. (1986) Mechanisms and evolution of food-deceptive pollination systems in orchids. Lindleyana, 1, 108–113. Arditti J. (1987) Orchid biology, reviews and perspectives. Cornell University Press, New York, USA.
duction of more flower, thus releasing a higher proportion of energy for other life-history traits, such as those involved in plant growth or longevity. This improved resource allocation strategy may be an important advantage for reducing transpiration in species that live in water-deprived habitats such as Mediterranean regions where sexually deceptive species typically occur, and where this pollination system has evolved multiple times in different continents (e.g. Dafni 1984; Cozzolino & Widmer 2005). Our results thus suggest that sexual deception, through more efficient pollen transfer, confers an important advantage in terms of resource allocation, favouring the exploitation of resource-limited habitats. Future studies are needed to understand whether even food-deceptive species that occupy strictly Mediterranean habitats show adaptations to reduce the investment in floral display or to increase pollen transfer efficiency as may be the case, for e.g. O. pauciflora, a food-deceptive species with small inflorescences that produces male bumblebee pheromone components that have been shown to increase pollen removal (Valterova et al. 2008) and of Anacamptis papilionacea that likely exploits a mixed pollination strategy also involving sexual attraction (Vogel 1972; Scopece et al. 2009). CONCLUSIONS In this study, we show that enhanced pollen transfer efficiency in sexually deceptive orchids could confer an important advantage in resource allocation. We suggest this advantage contributed to the evolutionary success of sexually deceptive species in resource-limited habitats by facilitating re-allocation from flower number to other life-history traits or to defence. This may render an important ecological advantage, which can explain the absence of reversals from sexual to food deception. Further studies are necessary to elucidate whether sexual deception efficiency has implications on other life-history traits, such as longevity and growth. Future studies should also investigate the link between pollen transfer efficiency, floral display and resource allocation in other pollination systems and plant taxa to better understand the impact on plant evolution. ACKNOWLEDGEMENTS The authors thank V. Tranchida-Lombardo, N. Juillet and S. Q. Xu for help in field data collection and data analyses. The authors also thank Steve Johnson and R. Mitchell for helpful comments on a previous version of the manuscript. The Progetto di Rilevante Interesse Nazionale (PRIN) funded this study. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Caladenia species included in the analysis.
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