Variation in sexual reproduction in orchids and its evolutionary consequences: a spasmodic journey to diversification

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1 Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society The Linnean Society of London, 2005? Original Article EVOLUTIONARY PROCESSES IN ORCHIDS R. L. TREMBLAY ET AL. Biological Journal of the Linnean Society, 2005, 84, With 5 figures Variation in sexual reproduction in orchids and its evolutionary consequences: a spasmodic journey to diversification RAYMOND L. TREMBLAY 1 *, JAMES D. ACKERMAN 2, JESS K. ZIMMERMAN 3 and RICARDO N. CALVO 4 1 Department of Biology, 100 carr. 908, University of Puerto Rico-Humacao, Humacao, Puerto Rico Department of Biology, PO Box 23360, University of Puerto Rico-Rio Piedras, San Juan, Puerto Rico Institute for Tropical Ecosystem Studies, University of Puerto Rico, PO Box 21910, San Juan, Puerto Rico Department of Biology, University of Miami, Coral Gables, FL 33124, USA Received 8 April 2003; accepted for publication 1 April 2004 The great taxonomic diversity of the Orchidaceae is often attributed to adaptive radiation for specific pollinators driven by selection for outcrossing. However, when one looks beyond the product to the process, the evidence for selection is less than overwhelming. We explore this problem by discussing relevant aspects of orchid biology and asking which aspects of reproduction explain the intricate pollination mechanisms and diversification of this family. We reaffirm that orchids are primarily pollination limited, the severity of which is affected by resource constraints. Fruit set is higher in temperate than in tropical species, and in species which offer pollinator rewards than those that do not. Reproductive success is skewed towards few individuals in a population and effective population sizes are often small. Population structure, reproductive success and gene flow among populations suggest that in many situations genetic drift may be as important as selection in fostering genetic and morphological variation in this family. Although there is some evidence for a gradualist model of evolutionary change, we believe that the great diversity in this family is largely a consequence of sequential and rapid interplay between drift and natural selection The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, ADDITIONAL KEYWORDS: cost of reproduction fruit set gene flow genetic drift natural selection Orchidaceae pollinator limitations resource limitation speciation. INTRODUCTION Early in the history of evolutionary biology, orchids had a prominent role in providing evidence for natural selection. Their unusual pollination mechanisms attracted the attention of Darwin (1877), who argued that they offer strong evidence both for natural selection and for the advantages of cross-pollination. Since then, much effort has been devoted to describing *Corresponding author. raymond@hpcf.upr.edu Current address: HDR Engineering Inc., 2202 N. West Shore Blvd. Suite 250, Tampa, FL 33607, USA orchid pollination mechanisms (e.g. van der Pijl & Dodson, 1966; van der Cingel, 1995). These reports contribute to Darwin s arguments, although it is not often stated explicitly. Most agree that there is a link between orchid pollination systems and orchid diversity, the distinction between cause and effect is often not clear. In this review we argue that the predominance of pollination limitation has had a significant effect on the evolution of the family and propose mechanisms by which orchids may have diversified. Natural selection should favour levels of reproductive effort that yield optimal fruit and seed set. Many hermaphroditic plants produce far more flowers than 1

2 2 R. L. TREMBLAY ET AL. fruits; orchids are superlative examples of this phenomenon. Low fruit-to-flower ratios in many plants are believed to be the result of a paucity of resources available for fruit development. This assumption forms the basis for the resource-limitation hypothesis whereby consistently more flowers are pollinated than fruits are matured (Stephenson, 1981; Lee, 1988). Regulation of maternal investment occurs through abortion of flowers and immature fruits (Lloyd, 1980; Stephenson, 1981) which may also be a mechanism for regulating seed quality (e.g. Lee & Bazzaz, 1982; Bookman, 1984; Stephenson & Winsor, 1986). The hypothesis that resources are the ultimate limiting factor in angiosperm reproduction has gained widespread acceptance because: (a) levels of fruit maturation remain unchanged following supplementary pollination, and (b) experimental reduction of resource availability causes elevated levels of fruit abortion (Stephenson, 1980, 1981; Bawa & Beach, 1981; Willson & Burley, 1983). Thus, according to this view, variation in reproductive success should be closely tied to the severity of resource constraints. Flowers that fail to become fruits are not always wasted as they may function to enhance plant fitness through pollen donation (Willson & Rathcke, 1974). For example, in many milkweeds (Asclepias) fruit production is poorly correlated with the number of flowers in an inflorescence, but the amount of pollen removed by pollinators, an index of male fitness, is strongly correlated with inflorescence size (Willson & Price, 1977; Bell, 1985; Queller, 1985). In fact, some researchers regard the corolla as primarily a male organ (Bell, 1985) because pollinator attractants influence fitness through pollen donation to a much greater extent than through seed production (Stanton, Snow & Handel, 1986). However, some evidence suggests that larger inflorescences do not always result in proportionally higher male fitness (Campbell, 1989). A common thread in these arguments is Bateman s Principle (Bateman, 1948), which assigns the two aspects of sexual selection (Darwin, 1871), intrasexual competition and mate choice, to the individual sexes. Noting the asymmetry in resource investment in offspring between males and females, this principle states that: (1) for males, reproduction is limited by access to mates, so that they must compete for opportunities to mate with females, and (2) for females, reproduction is limited by resources and they should therefore exercise a choice of mates to sire their relatively costly offspring. Although Bateman (1948) and others mentioned the possibility that sexual selection operated in plants as well as in animals, it was much later before patterns of pollination and fruit maturation in plants were interpreted in this context (Janzen, 1977; Charnov, 1979, 1982; Willson, 1979; Stephenson & Bertin, 1983). Despite the theoretical neatness and evidence for resource constraints, variation in reproductive success in a number of species was found instead to be caused by low levels of pollination (Bierzychudek, 1981a; Garwood & Horvitz, 1985; Hainsworth, Wolf & Mercier, 1985; Burd, 1994, and references therein), from which the pollinator limitation hypothesis emerged. The evolutionary and ecological consequences of pollinator limitation are likely to differ from those of resource limitation. If reproduction is pollen limited, Bateman s Principle is inapplicable (Stephenson & Bertin, 1983). Pollen-limited female reproduction is equivalent to saying that females are limited by access to mates and therefore the potential for selective mate choice is reduced under these conditions (Willson & Burley, 1983). In fact, it would seem that any degree of selectivity, not just that related to sexual selection, is of dubious value when the probability of a flower receiving pollen becomes small. While males may still compete amongst themselves for mates under pollenlimited reproduction (as do females), the intensity of male-male competition is restricted (Stephenson & Bertin, 1983; Tremblay, 1994). The dichotomy of resource vs. pollination limitation may be an oversimplification. Pollination limited plants often show effects of resource constraints, so it may be more realistic to say that such plants are affected by both (Montalvo & Ackerman, 1987). In fact, the theoretical model of Calvo & Horvitz (1990), often cited as demonstrating pollination limitation, showed that the degree of limitation is affected by the severity of resource constraints. Furthermore, there can be substantial variations from year to year (Schemske & Horvitz, 1988; Vaughton, 1991). The theoretical consequences of resource limitation, such as female choice, are not likely to be manifested in combination with pollination constraints. Ackerman & Montalvo (1990) and Meléndez-Ackerman et al. (2000) noted that some plants are pollen limited within a season but resource limited over their lifetimes. Under such conditions, the evolutionary consequences would be an optimization between increased investment in pollinator attraction (and a reduction in allocation to ovules) and various aspects of life history, primarily longevity, age to first reproduction and reproductive effort within and across years (Ackerman & Montalvo, 1990). Orchidaceae comprise c. one fifteenth of all angiosperms. While the intricate relationships between orchid flowers and their pollinators have long received a great deal of attention (Darwin, 1877; van der Pijl & Dodson, 1966; van der Cingel, 1995), relatively little consideration has been given to the fact that orchids often exhibit low fruit-to-flower ratios (Darwin, 1877; Ackerman, 1986a; Gill, 1989; Neiland

3 EVOLUTIONARY PROCESSES IN ORCHIDS 3 & Wilcock, 1998). Many studies in which researchers have performed supplemental pollination clearly indicate that these low ratios are best explained by pollinator limitation (see Darwin, 1877; Ackerman, 1989; Zimmerman & Aide, 1989; Calvo & Horvitz, 1990). Thus, orchids provide an excellent illustration of the evolution of reproductive strategies under pollenlimitation. In this review we discuss the ecology and evolution of reproduction in orchids, pursuing our principal argument that the predominance of pollination limitation explains both their intricate pollination mechanisms as well as the diversification of the family. Following an overview of the essential details of orchid reproduction, we discuss the evidence that reproductive success (both male and female) is pollination limited in orchids. We then summarize global patterns in orchid fruit reproduction, assembling data from almost 200 species of orchids. We then look at the causes of pollinator limitation, resource constraints and other ecological factors that have been shown to limit orchid reproduction. We conclude with a discussion of the relationship between variation in reproductive success, evolutionary processes, and the apparent high rates of speciation in the Orchidaceae. OVERVIEW OF ORCHID FLORAL BIOLOGY Darwin (1877) produced the first treatise on orchid pollination in order to corroborate his thesis that sexual reproduction (and cross-pollination in particular) is fundamental to organic evolution. He thoroughly described the functional floral morphology of a number of orchids. These essays provide indirect evidence of selection for floral characteristics that enhance the probability of cross-pollination. Much of the subsequent literature has followed suit. Three primary features, in combination, distinguish the flowers of orchids from those of other families: (1) The column, the fusion of male and female organs within a single structure located at the centre of the flower. (2) Pollinia, tightly packed masses of pollen found in most orchids, transported as a unit by pollinators (Freudenstein & Rasmussen, 1997; Pacini & Hesse, 2002); a single visit is potentially sufficient to produce a full seed complement (e.g. Montalvo & Ackerman, 1987; Proctor & Harder, 1994; Nazarov & Gerlach, 1997). (3) Zygomorphy, whereby a labellum is often highly modified to serve different functions (reviewed in van der Pijl & Dodson, 1966). The diversity of floral shapes and functional modifications found across the family are largely the result of variation in these three features. Most orchids require an external pollinating agent (Dressler, 1981). Among non-autogamous species, we find a wide variety of pollination systems: only abiotic and mammalian pollination are absent. Animal groups that pollinate orchids include birds, moths, butterflies, a wide variety of flies, numerous bees and, to a lesser extent, wasps. Hymenopterans alone account for the pollination of around 60% of the family (van der Pijl & Dodson, 1966). There is also a wide range of levels of specificity in plant pollinator interactions in the family (Tremblay, 1992). For example, the European Herminium monorchis was visited and presumably pollinated by 69 insect species, including members of four different orders: Lepidoptera, Coleoptera, Diptera and Hymenoptera (Nilsson, 1979a). Epipactis palustris has as many as 103 species of potentially effective pollinators (Nilsson, 1978a; Tremblay, 1992). Nevertheless, high pollinator specificity in orchid species is much more common: about 60% of orchids have only one recorded pollinator (Tremblay, 1992). This relationship has been well documented in a number of tropical orchids that are visited by one or a few species of euglossine bee (Ackerman, 1983; Williams & Whitten, 1983; Roubik & Ackerman, 1987). There are perhaps three kinds of floral rewards among orchids. The most common type is nutritional, consumed by the pollinators or their larvae. Nearly all such species are nectariferous, although some produce oils (Vogel, 1974; Steiner, 1989) and a handful offer pollen (Gregg, 1991b; Koryan & Endress, 2001) or pseudopollen (Dodson & Frymire, 1961; Goss, 1977; Davies, Winters & Turner, 2000). The second type is peculiar to orchids pollinated by male euglossine bees. The bees are attracted by floral fragrances that they collect for some as yet unknown aspect of mate attraction (Dressler, 1981, 1982; Williams & Whitten, 1983; Eltz et al., 1999). A third type has not been well documented. Some Maxillaria species produce waxes and resins (Dondon et al., 2002) that are collected by wasps and bees (Braga, 1977; M. Whitten, pers. comm. 2002) and presumably used for nest construction. Although most orchids offer some type of reward, an unusually high number of species offer no reward whatsoever. About a third of all Orchidaceae are deceptive (van der Pijl & Dodson, 1966; Ackerman, 1986a; Nilsson, 1992). Deception may be achieved by the flowers resemblance to larval food, or to the nectariferous flowers of other families, or even to female individuals of the insect pollinator (reviewed in Dafni, 1984; Ackerman, 1986a; Nilsson, 1992). Other distinguishing features of orchid reproductive biology include delayed ovule development (Wirth & Withner, 1959), the large number (20 up to as many as 4 million) of dust-like seeds contained in a single capsule (Arditti & Ghani, 2000), and their dependence on

4 4 R. L. TREMBLAY ET AL. fungal associations for germination and seedling establishment (Batty et al., 2001; McKendrick et al., 2002; Rasmussen & Whigham, 2002; Selosse et al., 2002). POLLINATION LIMITATION IN ORCHIDS Pollination limitation of sexual reproduction in plants may be detected experimentally when enhanced pollination elevates seed or fruit set above natural levels (Burd, 1994). Here, we consider three aspects of pollen limitation: (1) frequency - when pollinator abundance is low, some flowers or individual plants may never be visited; (2) quantity - even when pollinator visits are frequent, the amount of pollen actually reaching the stigmas or ovules may be low; (3) quality - the source of pollen (whether it arrives from the same plant or a close relative, or from an unrelated donor) can influence fruit and seed set and even the vigour of offspring (Charlesworth & Charlesworth, 1987). To test for pollen limitation on fruit and seed set one must increase pollen availability to flowers in naturally occurring populations. Supplemental pollination is best done by using all the flowers on an individual. This reveals whether or not the plant becomes stressed by eliminating the possibility that it shunts resources away from less intensely pollinated flowers in favour of the experimentally pollinated ones [Stephenson, 1981; but see Zimmerman & Pyke (1988)]. The source of pollen used in supplemental pollination can be problematic because of the potential for inbreeding and outbreeding depression of fruit and seed set (Waser & Price, 1991). We distinguish between cross- and self-pollination because researchers rarely note how supplementary crossed pollen was collected (e.g. at what distance from target plants). EVIDENCE FROM THE LITERATURE Data for this analysis and that presented in subsequent sections were gleaned from an exhaustive review of the literature (including Biological Abstracts and the Science Citation Index). Data were available for 15 species of non-autogamous orchids in which researchers tested for pollination limitation of fruit set by cross-pollinating all the flowers on individual plants growing in field populations (Table 1), comparing natural levels of fruit set with those obtained from supplemental cross-pollination. As researchers rarely stated which plants were used for pollen sources, these data do not control for variation in pollen quality other than that self-pollen was not used. However, the Table 1. Natural fruit set and hand cross-pollination of non-autogamous orchids. In all studies included here, the experiments were performed by cross-pollinating all flowers on individual plants in field populations. Differences between natural and experimental groups are statistically significant (Wilcoxon signed rank test, tied Z-value 3.180, P = 0.002) Natural fruit Cross-pollination Species set (%) fruit set (%) References Aspasia principissa Rchb. f Zimmerman & Aide, 1989 Calopogon tuberosus (L.) Britton, Firmage & Cole, 1988 Sterns & Poggenb. Calypso bulbosa var. occidentalis (L.) Ackerman, 1981 Oakes Alexandersson & Ågren, 1996 Cyclopogon cranichoides (Griseb.) Schltr Calvo, 1990a Cypripedium acaule Ait Davis, 1986; Gill, 1989; Primack & Hall, 1990; O Connell & Johnston, 1998 Dendrobium toressae (Bailey) Dockrill Bartareau, 1994 Encyclia cordigera (Humb., Bonpl. & Janzen et al., 1980 Kunth) Dressler Epidendrum ciliare L Ackerman & Montalvo, 1990 Isotria vertcilata Muhl. ex Willd Mehrhoff, 1983 Orchis boryi Rchb. f Gumbert & Kunze, 2001 Platanthera blephariglottis (Willd.) Lindl Cole & Firmage, 1984 Stelis argentata Lindl Christensen, 1992 Tipularia discolor (Pursh) Nutt Snow & Whigham, 1989 Tolumnia variegata (Sw.) Braem Ackerman & Montero Oliver, 1985 Mean (SE) 23.1 (6.6) 71.2 (8.5)

5 EVOLUTIONARY PROCESSES IN ORCHIDS 5 Table 2. Additional studies of non-autogamous orchids that suggest pollen limitation of fruit set. These studies include those in which not all flowers on an individual were experimentally pollinated or in which hand-pollination was not performed on field populations Species Reference Aspidogyne argentea (Vell.) Garay Singer & Sazima, 2001b Aspidogyne longicornu (Cogn.) Garay Singer & Sazima, 2001b Aerangis ellisii (Rchb. f.) Schltr. Nilsson & Rabakonandrianina, 1988 Brassavola nodosa (L.) Lindl. Schemske, 1980 Catasetum viridiflavum Hook. Zimmerman, Roubik & Ackerman,1989 Cleistes divaricata (L.) Ames Gregg, 1989 Comparettia falcata Poepp. & Endl. Rodríguez-Robles, Meléndez & Ackerman, 1992 Dactylorhiza sambucina (L.) Soó Nilsson, 1980 D. incarnata (L.) Soó M. T. Kuitunen, pers. comm. Disa uniflora Berg Johnson & Bond, 1992 Diuris maculata R. Br. Beardsell et al., 1986 Goodyera oblongifolia Raf. Ackerman, 1975; Kallunki, 1976 Ionopsis utricularioides (Sw.) Lindl. Montalvo & Ackerman, 1987 Isotria verticillata Muhl. ex Willd. Mehrhoff, 1983 Liparis lilifolia (L.) Rich. ex Lindl. Whigham & O Neil, 1991 Listera cordata (L.) R. Br. Ackerman & Mesler, 1979 L. ovata (L.) R. Br. Nilsson, 1981 Malaxis massonii Ridl. Aragón & Ackerman, 2001 Myrosmodes cochleare Garay Berry & Calvo, 1991 Orchis collina Sol. ex Russ Dafni & Ivri, 1979 O. coriophora L. Dafni & Ivri, 1979 O. laxifolia Lam. A. Fritz, pers. comm. O. mascula L. Nilsson, 1983b O. morio L. Nilsson, 1984 O. spectabilis (L.) Raf. Dieringer, 1982 O. spitzelli Saut. ex Koch Fritz, 1990 Paphiopedilum virens (Rchb. f.) Pfitz. Atwood, 1985 P. volonteanum (Sand.) Stein Atwood, 1985 Platanthera bifolia (L.) Rich. Nilsson, 1983a P. chlorantha (Custer) Rchb. Nilsson, 1983a P. ciliaris (L.) Lindl. Robertson & Wyatt, 1990 P. okuboi Makino Inoue, 1985 P. stricta Lindl. Patt et al., 1989 Prescottia densiflora Lindl. Singer & Sazima, 2001a P. plantaginea Lindl. Singer & Sazima, 2001a P. stachyodes Lindl. Singer & Sazima, 2001a Pogonia japonica Rchb. f. Matsui, Ushimaru & Fujita, 2001 Prosthechea cochleata (L.) W. E. Higgins J. K. Zimmerman & J. D. Ackerman, unpubl. data Psychilis krugii (Bello) Sauleda Ackerman, 1989 Schomburgkia tibicinis Bateman Rico-Gray & Thien, 1987 Thelymitra epipactoides F. Muell. Cropper & Calder, 1990 Thelymitra antennifera (Lindl.) Hook. f. Dafni & Calder, 1987 evidence for pollination limitation of fruit set is unequivocal. In all cases studied, orchids given supplemental pollination produced higher levels of fruit set than those pollinated naturally (Wilcoxon signrank test, Z = 3.180, P = 0.002). These differences were often quite substantial. Levels of fruit set between two and ten times those seen with natural pollination were common when plants were hand pollinated. In the case of Tolumnia variegata, hand pollinations produced a 50-fold increase compared with natural pollination (Ackerman & Montero Oliver, 1985; Calvo, 1993). In studies on 42 additional orchid species (Table 2), researchers performed experimental hand pollination

6 6 R. L. TREMBLAY ET AL. without specifying whether: (1) it was applied to all the flowers on an individual; (2) they had used plants not growing under normal field conditions, or (3) they had used pollen from the same plant. While these studies are not unequivocal demonstrations of pollination limitation, all but one indicate that fruit set in these orchids may be pollen-limited in the field. The single exception is Inoue s (1985) study of Platanthera mandarinorum spp. hachijoensis, in which natural levels of fruit set often approached 100% and could not therefore be raised through supplemental pollination. While representing only a tiny proportion of the or so species, these results were obtained from taxa across the taxonomic spectrum of the family and strongly suggest that pollination limitation of fruit set is a common characteristic of non-autogamous orchids. GLOBAL PATTERNS OF FRUIT SET In addition to summarizing data on pollination limitation, we compiled a larger data set on patterns of fruit set (Table 3). This was done in order to compare levels of fruit set among temperate and tropical localities, compare different pollinator types, explore the contrasts between deceptive and rewarding plants and to contrast different inflorescence sizes. The global patterns of fruit set provide a framework for discussion of the ecological and evolutionary implications of orchid reproduction. Data on natural levels of fruit set and results of hand pollination were compiled from the literature for 216 non-autogamous species (92 genera). These species are representative of the diversity of geographical distribution, habitat and pollination systems in the Orchidaceae. There are species from all continents where orchids are known, including 123 temperate and 93 tropical species. Both terrestrial and epiphytic species, as well as rewarding (N = 84) and deceptive (N = 132) species were considered. Most pollinator groups are also included. In compiling the data, we had to accommodate differences among studies in scope, sample size, number of populations and years, and ways of reporting fruit set. For each species, one value of natural and experimental fruit set was sought. For studies that included more than one population and/or year, and for species that have been studied by more than one author, the median of all reported fruit set values was used. Fruit set was reported either as the ratio between the total number of fruit and the total number of flowers in the sample or as the average of all individuals in the sample. For hand pollination, the largest value reported was included in order to denote the highest fruit set achievable. Because data on inflorescence size are not consistently reported, we assigned each species a qualitative index according to the range of sizes most frequently represented. Species that had an average of less than ten flowers per inflorescence were assigned a value of 0, and species with more than ten flowers had a value of 1. In this paper, inflorescence size is broadly defined as the total number of flowers produced throughout the reproductive season. To facilitate comparison, pollinators were grouped in broad categories: bees, wasps, moths, butterflies, ants, flies, birds, beetles and generalist. A three-way non-parametric ANOVA with interaction (Iman, 1974; Zar, 1996) was used to determine whether latitude (tropical and temperate), presence or absence of pollinator reward, and inflorescence size (less than vs. more than ten flowers/inflorescence), had a significant effect on median fruit set. Values were ranked before analysis. Pollinator reward and latitude had a significant effect on median fruit set (Table 4). Median fruit set in temperate species (34.6 ± 2.3; N = 123) is more than twice that of tropical species (17.0 ± 2.1; N = 91); deceptively pollinated species have a per cent fruit set (20.7 ± 1.7; N = 130) half that of rewarding species (37.1 ± 3.2; N = 84). Inflorescence size and none of the interactions were significant (Table 4). In general, most species have low fruit set (Fig. 1). FRUITING FAILURE OF INDIVIDUAL PLANTS Species vary in the percentage of flowering plants that fail to set fruit (Table 3). Sample size for this analysis is limited because few surveys have reported fruiting failure (temperate N = 18, tropical N = 18; deceptive N = 28, rewarding N = 8). A two-way non-parametric ANOVA with interaction of geographical area and pollination mechanism was performed on the ranked Frequency Per cent fruit set Figure 1. Frequency distribution of median fruit set of 216 orchids using data from Table 3.

7 EVOLUTIONARY PROCESSES IN ORCHIDS 7 Table 3. Reproductive success of non-autogamous orchids. PR, per cent pollinaria removed. PFS, per cent fruit set (open and hand pollination). IS, inflorescence size: 0, less than ten flowers; 1, more than ten flowers. FF, per cent number of plants which do not set fruits (fruiting failure). PG, pollinator groups: 0, moths; 1, bees; 2, wasps; 3, flies; 4, generalist; 5, butterflies; 6, ants; 7, birds; 8, beetles. PFS Species PR open hand IS FF PG References TEMPERATE, DECEPTIVE Anacamptis pyramidalis (L.) Rich Neiland & Wilcock, 1998 Anoectochilus formosanus Hayata 86.7 Shiau et al., 2002 Aplectrum hyemale (Mulh. ex Willd.) Torr Hogan, 1983 Arethusa bulbosa L Thien & Marcks, 1972 Bletilla striata (Thunb.) Rchb. f Sugiura, 1995 Caladenia tentaculata Schltdl Peakall & Beattie, 1996 Calopogon tuberosus (L.) Britton, Sterns Firmage & Cole, 1988; Thien & Marcks, 1972 & Poggenb. Calypso bulbosa (L.) Oakes var. americana (R. Br.) Luer Calypso bulbosa (L.) Oakes var. occidentalis (Holz.) Calder & Taylor Boyden, Ackerman, 1981 Cephalanthera longifolia (L.) Fritsch Dafni & Ivri, 1981b Cephalantera rubra (L.) Rich Nilsson, 1983c Ceratandra grandiflora Lindl Steiner, 1998 Chloraea lamellate Lindl Lehnebach & Riveros, 2003 Cleistes divaricata (L.) Ames Gregg, 1989, 1991a, b Corysanthes triloba Hook. f Fitzgerald, (cited in Darwin, 1877) Cyclopogon cranichoides (Griseb.) Schltr Calvo, 1990b Cypripedium acaule Ait Primack & Hall, 1990; Gill, 1989; Davis, 1986; O Connell & Johnston, 1998 Cypripedium calceolus L Nilsson, 1979b; Kull & Kull, 1991; Kull, Blivona, 2002 Cypripedium californicum A. Gray Kipping, 1971 Cypripedium candidum Muhl. ex Willd Carroll, Miller & Whitson, 1984; Curtis, 1954 Cypripedium fasciculatum Kellogg ex S. Watson Kipping, Lipow, Bernhardt & Vance, 2002 Cypripedium macranthos Sw. var. rebunense (Kudo) Miyabe & Kudo Site Site Sugiura et al., 2001

8 8 R. L. TREMBLAY ET AL. Table 3. Continued PFS Species PR open hand IS FF PG References Cypripedium montanum Douglas ex Lindl Coleman, 1995 Cypripedium reginae Walter Proctor, K. B. Gregg, unpubl. data Dactylorhiza fuschii (Druce) Verm Dafni & Woodell, 1986; Neiland & Wilcock, 1998; Waite, Hopkins & Hitchings, 1991 Dactylorhiza incarnata (L.) Soó Lammi & Kuitunen, 1995 Mattila & Kuitunen, 2000 Dactylorhiza lapponica (Laest. ex Hartman) Soó Neiland & Wilcock, 1998 Dactylorhiza maculata (L.) Soó Neiland & Wilcock, 1998 Dactylorhiza purpurella (T. & T. A. Stephenson) Soó Neiland & Wilcock, 1998 Dactylorhiza sambucina (L.) Soó Nilsson, 1980; Pettersson & Nilsson, 1983 Disa atricapilla (Thunb.) Sw Steiner, Whitehead & Johnson, 1994 Disa bivalvata (L. f.) Durand & Schinzl Steiner, Whitehead & Johnson, 1994 Disa ferruginea (Thunb.) Sw Johnson, 1994 Disa grandiflora L Darwin, 1877 Disa racemosa L. f Johnson et al., 1998 Disa tenuifolia (Thunb.) Linder Johnson & Steiner, 1994 Disa venosa Sw Johnson et al., 1998 Diuris maculata R. Br Beardsell et al., 1986 Drakaea glyptodon Fitz Peakall, 1990 Galearis spectabilis (L.) Raf. 2.8 Dieringer, 1982 Herschelianthes graminifolia (Spreng.) Durand & Schinzl Johnson, 1993 Isotria verticillata Muhl. ex Willd Mehrhoff, 1983 Leporella fimbriata (Lindl.) A. S. George Peakall, 1989a; Peakall, Beattie & James, 1987 Liparis lilifolia (L.) Rich. ex Lindl Whigham & O Neill, 1991 Ophrys aranifera Huds Delphino in Darwin, 1877 Ophrys bombyliflora Link Neiland & Wilcock, 1998 Ophrys insectifera L Neiland & Wilcock, 1998 Ophrys sphegodes Mill Neiland & Wilcock, 1998 Ophrys tenthredinifera Willd Neiland & Wilcock, 1998

9 EVOLUTIONARY PROCESSES IN ORCHIDS 9 PFS Species PR open hand IS FF PG References Ophrys vernixia Brot Neiland & Wilcock, 1998 Orchis boryi Rchb. f Gumbert & Kunze, 2001 Orchis caspia Trautv Dafni, 1983 Orchis collina Sol. ex Russ Dafni & Ivri, 1979 Orchis galilaea (Bornm. & M. Schulze) Bino, Dafni & Meeuse, 1982 Schltr. Orchis israelitica H. Baumann & Dafni Dafni & Ivri, 1981a Orchis italica Poir Neiland & Wilcock, 1998 Orchis laxiflora Lam. ssp. palustris (Jacq.) Asch. & Graebn A.-L. Fritz, pers. comm. Orchis mascula L Nilsson 1983b, Johnson & Nilsson, 1999 Orchis militaris L Farrell 1985, Sprengel (cited in Darwin, 1877) Kisseleva & Timonin, 2001 Orchis morio L Nilsson, 1984 Orchis pallens L Vöth, 1982 (cited in van der Cingel, 1995) Orchis papilionacea L Vogel, 1972 (cited in Dafni, 1987) Orchis purpurea Huds Neiland & Wilcock, 1998 Orchis spitzelii Saut. ex Koch Fritz, 1990 Pogonia ophioglossoides (L.) Ker Boland & Scott, 1991; Proctor, 1998 Sepapias cordigera L Neiland & Wilcock, 1998 Serapias parviflora Parl Neiland & Wilcock, 1998 Serapias vomeracea Briq Dafni, Ivri & Brantjes, 1981 Steveniella satyroides (Steven) Schltr Nazarov, 1995 Thelymitra epipactoides F. Muell Cropper, Calder & Tomkinson, 1989 Thelymitra ixioides Sw Sydes & Calder, 1993 Triphora trianthophora (Sw.) Rydb Williams, 1994 TEMPERATE, REWARD Acianthus sinclairii Hook. f Cheeseman, cited in Darwin, 1877 Arethusa bulbosa L Thien & Marcks, 1972 Cremastra appendiculata D. Don Chung & Chung, 2003 var. variabilis Blume Sugiura, 1996a Cymbidium goeringii (Rchb. f.) Rchb. f ? Chung & Chung, 2003 Dactylorhiza fuchsii (Druce) Soó Dafni & Woodell, 1986 Disa uniflora Berg Johnson & Bond, 1992 Valley Gorge Epipactis consimilis D. Don Ivri & Dafni,1977 Epipactis helleborine (L.) Crantz Piper & Waite, 1988

10 10 R. L. TREMBLAY ET AL. Table 3. Continued PFS Species PR open hand IS FF PG References Epipactis palustris (L.) Crantz Nilsson, 1978a Epipactis thunbergii A. Gray Sugiura, 1996b Goodyera foliosa (Kuntze) Benth. ex Sugiura & Yamaguchi, 1997 Hook. f. var. maximowicziana Makino Goodyera oblongifolia Raf Ackerman 1975; Kallunki, 1976 Goodyera procera Ker-Gawl Wong & Sun, 1999 Goodyera repens (L.) R. Br Neiland & Wilcock, 1998 Goodyera repens (L.) R. Br. var. ophioides Fernald Kallunki, 1981 Goodyera tesselata Lodd Kallunki, 1981 Gymnadenia conopsea (L.) R. Br Neiland & Wilcock, 1998 Herminium monorchis (L.) R. Br Nilsson, 1979a Liparis kumokiri Maek Oh et al., 2001 Liparis makinoama Schltr Oh et al., 2001 Liparis reflexa (R. Br.) Lindl Wallace, 1974 Listera cordata (L.) R. Br Ackerman & Mesler, Meléndez-Ackerman & Ackerman, 2001 Listera ovata (L.) R. Br Nilsson, 1981 Microtis parviflora R. Br Peakall & Beattie, 1989 Monadenia ophrydea Lindl Johnson, 1995 Orchis coriophora L Dafni & Ivri, 1979 Orchis spectabilis L Dieringer, 1982 Oreorchis patens Lindl Sugiura, Okajima & Maeta, 1997 Platanthera bifolia (L.) Rich Nilsson, 1983a; Mattila, 2000 Platanthera blephariglottis (Willd.) Smith & Snow 1976; Cole & Firmage, 1984 Lindl. Platanthera chlorantha (Cust.) Rchb Nilsson 1978b, 1983a Platanthera ciliaris (L.) Lindl Smith & Snow 1976; Robertson & Wyatt, 1990; Gregg, 1990 Platanthera integrilabia (Correll) Luer Zettler & Fairey, 1990 Platanthera lacera (Michx.) G. Don Gregg, 1990 Platanthera mandarinorum Rchb. f. ssp. hachijoensis (Honda) Murata Inoue, 1986b Platanthera metabifolia F. Maek Inoue, 1986a Platanthera obtusata (Banks ex Pursh) Thien & Utech, 1970 Lindl.

11 EVOLUTIONARY PROCESSES IN ORCHIDS 11 PFS Species PR open hand IS FF PG References Platanthera okuboi Makino Inoue, 1985 Platanthera stricta Lindl Patt et al., 1989 Pogonia japonica Rchb. f Matsui, Ushimaru & Fujita, Ushimaru & Nakata, 2001 Pogonia ophioglossoides (L.) Ker-Gawl Thien & Marcks 1972; Proctor, 1998 Prasophylum odoratum R. Br Bernhart & Burns-Balogh, 1986 Prasophyllum romanzoffia Cham. > Larson & Larson, 1987 Pteroglossapsis ruwenzoriensis (Rendle) Rolfe Singer & Cocucci, 1997a Satyrium bicorne Thunb Ellis & Johnson, 1999 Satyrium coriifolium Sw Ellis & Johnson, 1999 Satyrium erectum Sw Ellis & Johnson, 1999 Spiranthes lacera (Raf.) Raf. var. lacera Catling, 1982 Spiranthes lucida (H. H. Eaton) Ames Catling, 1982 Spiranthes ochroleuca (Rydb.) Rydb Catling, 1982 Spiranthes romanzoffiana Cham Catling, 1982 Spiranthes vernalis Engelm. & Gray Catling, 1982 Tipularia discolor (Pursh) Nutt Whigham & McWethy, 1980; Snow & Whigham, 1989 TROPICAL, DECEPTIVE Aspasia principissa Rchb. f Zimmerman & Aide, 1989 Bletia patula Graham Ackerman 1995; J. D. Ackerman & Carromero, unpubl. data Brassavola nodosa (L.) Lindl Schemske, 1980; Murren & Ellisson 1996 Bulbophyllum involutum Borba, Semir & Borba & Semir, 1998; Borba & Semir 1999b; F. Barros Borba, Sheppard & Semir, 1999 Bulbophyllum ipanemense Hoehne Borba & Semir, 1998; Borba & Semir 1999b; Borba, Sheppard & Semir, 1999 Bulbophyllum warmingianum Cogn Sazima, 1978 Bulbophyllum weddellii (Lindl.) Rchb. f Borba & Semir, 1998; Borba & Semir 1999b; Borba, Sheppard & Semir, 1999 Cochleanthes lipscombiae (Rolfe) Gray Ackerman, 1983 Coryanthes elegantium Linden & Rchb. f Dodson, 1965 Coryanthes leucocorys Rolfe Dodson, 1965 Coryanthes macrantha (Hook.) Hook Dodson, 1965 Coryanthes rodriguesii Hoehne Dodson, 1965 Coryanthes trifoliata C. Schweinf Dodson, 1965 Corymborkis forcipigera L. O. Williams Ackerman, 1995 Cyclopogon cranichoides (Griseb.) Schltr Calvo, 1990a Cymbidiella flabellata (Thou.) Rolfe Nilsson et al., 1986

12 12 R. L. TREMBLAY ET AL. Table 3. Continued PFS Species PR open hand IS FF PG References Cyrtopodium broadwayi Ames Quesnel et al., 1982 Dendrobium infundibulum Lindl Kjellsson, Rasmussen & Dupuy, 1985 Dendrobium monophyllum F. Muell Bartareau, 1995 Dendrobium speciosum Sm Calder, Adams & Slater, 1982 Dendrobium toressae (Bailey) Dockrill Bartareau, 1994 Dilomilis montana (Sw.) Summerh. I. Rodríguez-Colón & J. D. Ackerman, unpubl. data Site ? Site ? Elleanthus cf. brenesii B. Grabowski, pers comm. Encyclia cordigera (Humb., Bonpl. & Kunth) Dressler Janzen et al., 1980 Epidendrum ciliare L Ackerman & Montalvo, 1990 Epidendrum exasperatum Rchb. f Calvo, 1990b Ionopsis utricularioides (Sw.) Lindl Montalvo & Ackerman, 1987 Laelia speciosa (Humb., Bonpl. & Kunth.) Schltr. Lepanthes caritensis Tremblay & Ackerman Hernández-Apolinar, Tremblay, 1997b; Tremblay et al., 1998 Lepanthes eltoroensis Stimson Tremblay, 1996 Lepanthes rubripetala Stimson Tremblay, 1996 Lepanthes rupestris Stimson Tremblay, 1996 Lepanthes sanguinea Hook Ackerman & Zimmerman (cited in Christensen, 1992) Lepanthes wendlandii Rchb. f Calvo, 1990b Lepanthes woodburyana Stimson J. D. Ackerman & J. K. Zimmerman, unpubl. data Malaxis massonii (Ridl.) Kuntze Aragón & Ackerman, 2001 Mormodes tuxtlensis Salazar Sosa & Rodríguez-Angulo, 2000 Myrmecophila tibicinis (Bateman) Rolfe Rico-Gray & Thien, 1987 Nervilia bicarinata (Bl.) Schltr Pettersson, 1989 Nervilia humilis Schltr Pettersson, 1989 Nervilia shirensis (Rolfe) Schltr Pettersson, 1989 Nervilia stolziana Schltr Pettersson, 1989

13 EVOLUTIONARY PROCESSES IN ORCHIDS 13 PFS Species PR open hand IS FF PG References Oncidium altissimum (Jacq.) Sw Ackerman, 1995 Oncidium ascendens Lindl. Forest Parra-Tabla et al., 2000 Pastoral field Parra-Tabla et al., 2000 Oncidium stipitatum Lindl J. K. Zimmerman, unpubl. data Paphiopedilum villosum (Lindl.) Stein Bänziger, 1996 Polystachya concreta (Jacq.) Garay & Sweet Goss, 1977 Pleurothallis adamantinensis Brade Borba, Semir & Shepherd, 2001; Borba & Semir 2001 Pleurothallis fabiobarrosii Borba & Borba, Semir & Shepherd, 2001; Borba & Semir Semir 2001 Pleurothallis johannensis Barb. Rodr Borba, Semir & Shepherd, 2001; Borba & Semir 2001 Prosthechea cochleata (L.) W. E. Higgins J. D. Ackerman & J. K. Zimmerman, unpubl. data Psychilis krugii (Bello) Sauleda Ackerman, 1989 Stelis argentata Lindl Christensen, 1992 Stelis sp Christensen, 1992 Stelis sp Christensen, 1992 Stelis sp Christensen, 1992 Stelis sp Christensen, 1992 Tetramicra canaliculata (Aubl.) Urb Pagán, Martínez & Ackerman (cited in Ackerman, 1995) Tolumnia variegata (Sw.) Braem Ackerman & Montero Oliver, 1985; Ackerman, Meléndez-Ackerman & Salguero-Faría, 1997; Calvo, 1990b Vanilla barbellata Rchb. f I. Panetto & J. D. Ackerman, unpubl. data; L. R. Nielsen & J. D. Ackerman, unpubl. data Vanilla claviculata (W. Wright) Sw I. Panetto & J. D. Ackerman, unpubl. data; L. R. Nielsen & J. D. Ackerman, unpubl. data Vanilla dilloniana Correll I. Panetto & J. D. Ackerman, unpubl. data; L. R. Nielsen & J. D. Ackerman, unpubl. data Vanilla planifolia Andrews Ackerman, 1995 Vanilla poitaei Rchb. f I. Panetto & J. D. Ackerman, unpubl. data TROPICAL, REWARD Aerangis ellisii (Rchb. f.) Schltr Nilsson & Rabakonandrianina, 1988 Angraecum arachnites Schltr. Aspidogyne argentea (Vell.) Garay Nilsson, 1985; Nilsson et al., 1985, Singer & Sazima, 2001b

14 14 R. L. TREMBLAY ET AL. Table 3. Continued PFS Species PR open hand IS FF PG References Aspidogyne longicornu (Cogn.) Garay Singer & Sazima, 2001b Catasetum macrocarpum Rich. ex Kunth Carvalho & Machado, 2002 Catasetum viridiflavum Hook Zimmerman, Roubik & Ackerman, 1989; Zimmerman, 1991 Comparettia falcata Poepp. & Endl Rodríguez-Robles, Meléndez & Ackerman, 1992; Ackerman,Rodríguez-Robles & Meléndez, 1994; Salguero-Faría & Ackerman, 1999 Cyclopogon congestus (Vell.) Hoehne Singer & Sazima, 1999 Dendrochilum longbracteatum Pfitzer Pedersen, 1995 Erythrodes arietina (Rchb. f. & Warm.) Ames Singer & Sazima, 2001b Habenaria gourlieana Lindl Singer & Coccuci, 1997b Habenaria hieronymi Kraenzl Singer & Coccuci, 1997b Habenaria montevidensis Spreng Singer & Coccuci, 1997b Habenaria parviflora Lindl Singer, 2001 Habenaria rupicola Barb. Rodr Singer & Coccuci, 1997b Leochilus scriptus (Scheidw.) Rchb. f Chase, 1986 Myrosmodes cochleare Garay Berry & Calvo, 1991 Mystacidium venosum Lindl Luyt & Johnson, 2001 Notylia nemorosa Barb. Rodr Singer & Koehler, 2002 Pelexia oestrifera (Rchb. f. & Warm.) Schltr Singer & Sazima, 1999 Pleurothallis ochreata Lindl Borba, Semir & Shepherd, 2001; Borba & Semir, 2001 Pleurothallis racemiflora Lindl. ex Hook Ackerman, 1995 Pleurothallis teres Lindl Borba, Semir & Shepherd, 2001; Borba & Semir, 2001 Prescottia densiflora Lindl Singer & Sazima, 2001a Prescottia plantaginea Lindl Singer & Sazima, 2001a Prescottia stachyodes (Sw.) Lindl Singer & Sazima, 2001a Sarcoglottis faciculata (Vell.) Schltr Singer & Sazima, 1999 Sauroglossum elatum Lindl Singer, 2002 Stenorrhychos lanceolatus (Aubl.) L. C. Rich Singer & Sazima, 2000 Xylobium squalens Lindl Pintaúdi, Stort & Marin-Morales, 1990

15 EVOLUTIONARY PROCESSES IN ORCHIDS 15 Table 4. Results of three-way non-parametric ANOVA with interactions for ranked median fruit set. Data of open pollination only, from Table 3. Distribution: temperate vs. tropical. Reward: deceit or reward. Inflorescence size: Inflorescence smaller than ten flowers or equal to or larger than ten flowers Source d.f. MS F P Distribution < Reward Inflorescence size Distribution reward Distribution inflorescence Reward inflorescence Three-way interaction Residual Per cent fruiting failure Deceptive Reward Temperate Tropical Figure 2. The frequency of deceptive and rewarding plants that fail to set fruits. Data are from Table 3. Means + SE; N = 44. Table 5. Median per cent fruit set and standard error (SE) of naturally pollinated orchids by pollinator types. Median fruit set is significantly distinct among pollinator groups (Kruskal-Wallis H, corrected for ties = 24.82, P = ). Beetles and ants excluded because of low sample size Pollinator N Fruit set SE Bees Birds Butterflies Generalist Flies Moths Wasps Moreover, median fruit set and per cent fruiting failure were significantly negatively correlated (Spearman s rank correlation, corrected for ties, rho = ; P < ; N = 44). Thus, deceptive species with low overall fruit set and small inflorescence size are most likely to exhibit high levels of fruiting failure. POLLINATOR GROUP EFFECT Per cent median fruit set varies according to pollinator group (Kruskal Wallis H, corrected for ties = 24.82, P = ; Table 5). We tested the difference between the median fruit set of orchids pollinated by moths, bees, wasps, flies, butterflies and birds (beetles and ants were excluded because of small sample size) as well as generalist orchid species. Fruit set was lowest for species pollinated by flies and highest for those pollinated by birds and for generalists. However, the pattern may be confused by the fact that all the generalist species with fruit set higher than 50% are temperate, nectariferous species, while of the species pollinated by bees, many with fruit set below 10% are tropical. data set. No difference was detected between tropical and temperate species; mean fruiting failure was in the range of 50 60% of plants from both areas (F 1,32 = 0.18, P = 0.67; Fig. 2). However, failure depended on the pollination mechanism: in deceptive species, most plants do not set fruit (66.5%); rewarding species have much lower failure rates (29.1%; F 1,32 = 8.28, P = 0.007). Per cent fruiting failure was significantly higher for species with less than ten flowers per inflorescence (68.0% ± 6.4; mean ± SE) than for those with more than ten (42.8% ± 8.2; mean ± SE; Mann Whitney U- test, Z = 2.353; P < 0.02; N = 46). GENERAL PATTERNS OF POLLINATOR LIMITATION Our data demonstrate that fruit production in nonautogamous orchids is pollen-limited. For almost all species where data are available for natural and hand pollination, the minimum fruit set difference of the latter was 10% larger (mean and SE; open pollination, 26.6 ± 1.7 (N = 210); cross hand-pollination, 80.0 ± 2.6 (N = 98); Table 3). Median natural fruit set in temperate species is approximately twice that of tropical species (mean and SE: tropical 17.0 ± 2.1 (N = 91); temperate 34.6 ± 2.3 (N = 123). It is not evident why the former should be more efficient at setting fruit. A possible explanation (further research is required) is that it may be a result of population size or population

16 16 R. L. TREMBLAY ET AL. dispersion. Tropical species are frequently organized in small groups of individuals separated by large distances (Ackerman, 1986b; Tremblay, 1997c). Hyperdispersed populations are perhaps more common among tropical epiphytes because they are immersed in a tangled canopy. Data are generally lacking on population dispersion in orchids. However, an alternative explanation for differences among temperate and tropical orchids might be that they are due to taxonomic differences and phylogenetic constraints. An approach to testing this hypothesis would be to investigate fruit set in species of the same genus that are in both regions. If fruit production is phylogenetically constrained among regions, then we would expect fruit set within a genus to be more similar than among genera. Our results are similar to those of Neiland & Wilcock (1998), who observed that fruit set in temperate species is about three times as successful as that of tropical species (38.2% vs. 13.6%). We found just a twofold difference, which may simply be a consequence of different sample sizes: Neiland & Wilcock (1998) included 96 species while this survey has 216. CAUSES OF POLLINATION LIMITATION Pollinator effectiveness Orchids are visited by a number of different insects or birds, but not all visitors are pollinators (Ackerman & Mesler, 1979; Nilsson, 1979a, b). The relative frequency of effective visits is not often quantified but variation in visitor performance can be substantial. For example, the most frequent visitors to Herminium monorchis were rarely effective pollinators and even the best carried pollinaria only about 70% of the time (Nilsson, 1979a). Pollinarium removal and deposition The frequency of effective visits, quantified as pollinarium removals and pollinations, is often very low. Although the data show substantial variation among populations and species, usually just under half the flowers fail to export their pollen (Table 3). Pollinaria removals usually exceed pollinations, so one can express efficiency as a ratio of the former to the latter and this can be used as an index of efficiency. Mean ratio is 1.7 : 1 in Tolumnia variegata (Ackerman, Meléndez-Ackerman & Salguero-Faría, 1997), 1.35 : 1 in Comparettia falcata (Salguero-Faría & Ackerman, 1999), 2.5 : 1 in Epidendrum ciliare (Ackerman & Montalvo, 1990), and 1.96 : 1 in Ionopsis utricularioides (Montalvo & Ackerman, The range of per cent removals to per cent fruit set is large (0.24 : : 1). The most effective systems are those of Satyrium bicorne, Vanilla barbellata (< 0.30 : 1), while the most inefficient are those of Stelis argentata and Bulbophyllum ipanemense (> 24.0; Table 3; ratios not shown). Pollinarium deposition efficiency among species is approximately divided equally between efficient (< 1 : 1) and inefficient (> 1 : 1; Fig. 3). We expect pollinaria removals to depositions to be greater than 1 unless the pollen masses can be broken unto subunits. For example, in Herminium monorchis the number of removals per deposition ranged from 0.74 to 0.94 (Nilsson, 1979a). We tested the hypothesis that removal to fruit set ratio is more efficient in mealy pollen orchid species and found it to be true (Mann Whitney U-test, Z = 5.443, P = 0.001; Table 3). Orchids with mealy pollen have a mean ratio of 1.1 ± 0.19 : 1 (N = 36) whereas those with hard pollinia have a mean of 6.97 ± 1.56 : 1 (N = 21). Pollinator abundance and diversity Fruit set within and among populations may be influenced by pollinator activity and diversity. In several Swedish populations of Listera ovata it varied between 13 and 70% and was positively correlated with visitation rates (Nilsson, 1981). Visitation frequencies to two populations of the epiphytic Comparettia falcata were related to the abundance of its hummingbird pollinator (Rodríguez-Robles et al., 1992). Ackerman et al. (1997) observed the effect of pollinator abundance and floral fragrance on fruit set in the deceptive orchid Tolumnia variegata and found that the main cause of fruit production among populations could be attributed to pollinator abundance. Moreover, flower production often needs to be synchro- Frequency < <20 <30 Pollinaria removal per fruit set Figure 3. Frequency distribution of pollinaria removal to fruit set ratio. Values below 1 represent species that set more then one fruit per pollinarium removal (mainly orchids with mealy pollen) while values larger than 1 represent species that remove more than one pollinarium per fruit set.