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 the Kings Park and Botanic Garden (Perth, WA). The number of flowers/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 spp. 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 X T. macrophylla specimens. Due to malformations of the column, the majority of T. antennifera X T. macrophylla flowers appeared unable to attach their pollinia to their respective rostella.
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