While Darwin (1862Darwin ( , 1877 showed that reproductive success in orchid populations depended on adaptive floral morphology coupled with pollinator visitation a more recent review of the literature (Tremblay et al., 2005) confirmed that many out-breeding species are pollinator-limited because most orchid species showing low fecundity also lack rewards. The absence of rewards depresses both pollinator fidelity and the frequency of pollinator visits to an orchid population even though orchid flowers that lack rewards retain the same interlocking floral structures for precise pollinia removal and deposition found in related species that offer rewards. Using the genus, Cypripedium, as a model lineage of non-rewarding flowers this study also shows that the correlation between low fruit set in a Cypripedium sp. and its specific pollinator(s) is insufficient to predict specific frequencies of low fecundity. Annual rates of fruit set often vary broadly between populations of the same species and within the same population over several seasons. We speculate that fruit-set rates also decline when orchid demography and additional biotic and abiotic factors interrupt rates of pollinator activity (pre-zygotic) and fertilization/fruit maturation (post-zygotic). We suggest that that traditional field studies on pollination ecology and breeding systems be combined with data sets recording genetic variation and orchid flower demography in relation to seasonal variation in climate. We also propose that the same information be collected in regard to genetic variation, demography and phenology of populations of known orchid pollinators and co-blooming angiosperm species native to orchid habitats.

As in most pollinator-limited orchids lacking edible rewards, a population of C. reginae in southern Missouri showed a low conversion ratio of flowers into fruits (0.046-0.23) over two seasons. There was no relationship between the length of the secondary flowering stem, the number of foliage leaves on the same stem and the number of flowers (one or two) produced at the terminus of the stem. However, the size mattered based on the physical dimensions of pollinia-carrying insects vs. parameters of floral architecture. While a diverse range of floral visitors (Coleoptera, Diptera and Hymenoptera) to C. reginae were observed over three seasons, only six medium-sized bees (Anthophora, Apis and Megachile spp.) carried segments of massulate pollinia after three seasons of observation and collection. Pollinia were always deposited dorsally on the thorax. These bees had a mean width of 4.44 mm and depth of 3.41 mm whereas the rear exit length and width of the orchid measured 6.53 mm and 3.41 mm. respectively. In contrast, the more numerous but smaller bees (2.66 mm width and 2.16 mm depth) in the genera Augochlorella, Augochlora, Ceratina, Lasioglossum spp. etc., exited the flower via the same rear orifices without pressing against the dehiscent anthers. Larger bees (gynes of Bombus spp.) measuring 9.06 mm in width and 6.25 mm in depth, were too large to escape via the rear exits so they left the flower via the large, dorsal entrance (through which they first entered the labellum) never contacting either anther. As in the small-flowered C. plectrochilum, the larger flowered C. reginae receives many floral visitors but selects for pollinia-vectors of a discrete body size.

Fluorescence microscopy is used to compare frequencies of pollen tube penetration in in situ populations of Cypripedium 1.iardo/phicinum W.\X/. Smith et Farrer, Crpnpc'dzum flavum W.W. Smith, Cyprzpedtum iitofltafluJ)1 Dougl. ex !.indl., Cypripedium pari'iflorum Salisbury var. pubescens (Wildenow) ().'X. Knight, Cypripedtwn regincie Walter, and Cypripedium tibeticum Schltr. The average natural (insect-mediated) pollination rates measured over five seasons are wide ranging among the six species (0.08-0.74). However, the pollination rate of hand-manipulated populations (self and/or cross) is significantly greater than the rate of insect-mediated polimations in all species studied. A few pollen tubes in both self-and cross-pollinations display aberrant growth in the styles and/or ovaries, but their numbers are too small to suggest evidence of self-incompatibility. Pollen tubes germinate and grow up to the bases of styles within 48 h in C. bardolphianum, C. flavum, and C. tibeiicuni. Pollen tubes remain at the bases of the styles in C. rnonlanum for 5 d after pollination. In C. parviflorum, pollen tubes penetrate ovaries at 7 d. Pollen tube penetration of ovaries is observed within 15 d after hand pollination in all six species but remains incomplete at this time, with the greatest number of ovule penetrations observed in C. reginae (which has the shortest floral life span). Therefore, we suggest there are additional fsct()rs aside from low pollinator visitation for low conversion rates of flowers into fruits in these Cvpripediunz species. These include inadequate pollen loads deposited on receptive stigmas (pollen limitation), coupled with environmental stress and/or predation disrupting or destroying the slow processes of fertilization and/or fruit maturation.

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Historically, only a few large flowered species in the genus Thelymitra were identified as obligate out-breeders. We compared floral presentation, pollen-pistil interactions, pollination ecology, and interspecific hybridization in two populations of T. macrophylla where its flowering periods overlapped with T. antennifera (Tenterden) and T. crinita (Lesmurdie) respectively. Pollen-pistil interactions were studied using glasshouse collections of T. crinita and T. macrophylla at KPBG. The number of flowers per inflorescence in T. macrophylla varied significantly between sites. Climatic conditions influenced flower opening and closing regimes differently in T. crinita vs. T. macrophylla. While all three Thelymitra species opened on warm, sunny mornings and closed by late afternoon, T. crinita at Lesmurdie was significantly more likely to open its perianth segments on cool days compared to the co-blooming, sympatric flowers of T. macrophylla. The floral lifespans of individual flowers of T. macrophylla and T. crinita were reduced significantly following application of Thelymitra pollen onto the stigmatic surface but were not reduced by pollinarium removal. Flowers of both species were self-compatible but neither species self-pollinated mechanically (autogamy). Fluorescence microscopy also showed that both species were inter-compatible. Natural rates of pollinarium removal by insects were low in all three species at both sites. Natural rates of pollen deposition on receptive stigmas were significantly higher in T. crinita vs. T. macrophylla but pollen deposition was less than 12% in both species. Observations and collections of pollinators were infrequent and pollinaria vectors were restricted to a few polylectic, female bees in the families Apidae, Colletidae, and Halictidae. We found a large number of hybrids between T. antennifera and T. macrophylla at Tenterden but few obvious hybrids between T. crinita and T. macrophylla at Lesmurdie. As expected, all hybrids showed characteristics intermediate between their two putative parent species, including pollen configuration in the T. crinita × T. macrophylla specimens. Due to malformations of the column, the majority of T. antennifera × T. macrophylla flowers appeared unable to attach their pollinia to their respective rostella.