Pollination ecology of Australian sexually deceptive orchids with contrasting patterns of pollinator exploitation

Sexual deception, entailing the pollination of flowers through mimicry of female insects, is one of the most remarkable pollination strategies to have evolved. This thesis explores two Australian sexually deceptive orchid systems with contrasting patterns of pollinator exploitation. The first three chapters focus on the genus Cryptostylis, a system with a unique case of pollinator sharing - five Australian species, four of which are largely sympatric, all deceive the same male ichneumonid pollinator. In Chapter One, mark-recapture experiments were used to investigate the consequences of ichneumonid pollination on pollen movement in C. ovata. A high pollinator revisitation rate indicated some potential for self-pollination. In Chapter Two, reproductive barriers contributing to the absence of hybrids between Cryptostylis species were investigated. Pre-pollination barriers, assessed in field experiments, did not prevent hybridisation. Hand cross-pollinations conducted among the four common Cryptostylis species in a greenhouse all produced fruits, however seed mass and the percentage of formed embryos were reduced in hybrids. Major differences in ploidy and chromosome number likely explain this post-pollination fitness reduction. Two Cryptostylis species were found to be self-incompatible, marking the first case of self-incompatibility in the Diurideae. The unique reproductive biology of Australian Cryptostylis, encompassing pollinator sharing, self-incompatibility, and post-pollination reproductive isolation driven by large ploidy differences, may indicate that its mode of diversification may differ greatly to those in other sexually deceptive genera. Chapter Three presents the first phylogeny to encompass both Australian and Asiatic Cryptostylis. An Australian origin of Cryptostylis is supported, with a likely single dispersal event to Asia. Ploidy variation and geographic barriers appear to have played a role in diversification across Cryptostylis. In Chapter Four, the potential presence of pollination ecotypes in Drakaea livida was tested for. Patterns of chemical diversity and pollinator availability across the distribution of the species are investigated. Pollinator choice trials revealed the presence of three discrete ecotypes each attracting its own pollinator species. Patterns of pollinator availability did not correlate with ecotype distribution. Each ecotype possessed a significantly different floral volatile composition. Using Partial Least Squares Discriminant Analysis (PLS-DA), the presence-absence of a subset of taxonomically informative compounds could be used to accurately predict the ecotype of a flower. Different classes of electrophysiologically active compounds were present in different ecotypes. These marked differences in chemical composition between the ecotypes suggest either a long time since their divergence and may hint at a scenario of convergent evolution of floral morphology. In Chapter Five, the ecotype geographic ranges and methods of identifying the ecotypes were investigated. Species distribution modelling predicted each ecotype to have a different core range. Two ecotypes were widespread, while one had a limited distribution within extensively cleared agricultural land, raising conservation concerns. PLS-DA correctly identified the ecotype of a flower when labella extracts were made from pollinated flowers, thereby providing a non-destructive identification technique. The pollinator specificity, morphology, floral chemistry, and ranges of the ecotypes supported them as Evolutionary Significant Units. In conclusion, the ecological and evolutionary consequences of pollination by sexual deception may vary extensively between plant taxa in accordance with their different patterns of pollinator exploitation. The taxonomy, species richness of the pollinator group, and the plant species to pollinator species ratio all influence the evolution and diversification of sexually deceptive orchids

Three Chemically Distinct Floral Ecotypes in Drakaea livida, an Orchid Pollinated by Sexual Deception of Thynnine Wasps

Sexually deceptive orchids are unusual among plants in that closely related species typically attract different pollinator species using contrasting blends of floral volatiles. Therefore, intraspecific variation in pollinator attraction may also be underpinned by differences in floral volatiles. Here, we tested for the presence of floral ecotypes in the sexually deceptive orchid Drakaea livida and investigated if the geographic range of floral ecotypes corresponded to variation in pollinator availability. Pollinator choice trials revealed the presence of three floral ecotypes within D. livida that each attracts a different species of thynnine wasp as a pollinator. Surveys of pollinator distribution revealed that the distribution of one of the ecotypes was strongly correlated with that of its pollinator, while another pollinator species was present throughout the range of all three ecotypes, demonstrating that pollinator availability does not always correlate with ecotype distribution. Floral ecotypes differed in chemical volatile composition, with a high degree of separation evident in principal coordinate analysis. Some compounds that differed between ecotypes, including pyrazines and (methylthio)phenols, are known to be electrophysiologically active in thynnine wasp antennae. Based on differences in pollinator response and floral volatile profile, the ecotypes represent distinct entities and should be treated as such in conservation management

Conservation assessment of the Drakaea livida (Orchidaceae) ecotypes and an evaluation of methods for their identification

Morphologically cryptic taxa must be accounted for when quantifying biodiversity and implementing effective conservation measures. Some orchids pollinated by sexual deception of male insects contain morphologically cryptic ecotypes, such as the warty hammer orchid Drakaea livida (Orchidaceae). This species is comprised of three cryptic pollination ecotypes, which can be distinguished based on differences in pollinator species and floral volatiles. The present study aims were: (a) to investigate the geographic range of the three D. livida ecotypes, enabling assessment of their conservation status; and (b) to test the efficacy of different methods of identifying the D. livida ecotypes. Three methods of ecotype identification were assessed: morphometric analysis, genome size comparison, and analysis of chemical volatile composition of labellum extracts from pollinated flowers. MaxEnt species distribution models revealed that each ecotype has a different predicted geographic range, with small areas of overlap at the range margins. One ecotype is known from just ten populations over a limited geographic area, the majority of which has been cleared for agriculture, and urban development. While there was broad overlap between the ecotypes in individual morphological traits, multivariate analysis of morphological traits provided correct assignment to ecotype in 87% of individuals. Using the labellum of pollinated flowers, screening for volatile chemical compounds associated with particular ecotypes returned an even higher correct assignment rate, of 96.5%. As such, we advocate that the use of volatiles from the labellum of recently pollinated flowers is an effective way to determine the ecotype of unknown individuals of D. livida, with minimal impact on the flowering plant

Three Chemically Distinct Floral Ecotypes in Drakaea livida, an Orchid Pollinated by Sexual Deception of Thynnine Wasps

Sexually deceptive orchids are unusual among plants in that closely related species typically attract different pollinator species using contrasting blends of floral volatiles. Therefore, intraspecific variation in pollinator attraction may also be underpinned by differences in floral volatiles. Here, we tested for the presence of floral ecotypes in the sexually deceptive orchid Drakaea livida and investigated if the geographic range of floral ecotypes corresponded to variation in pollinator availability. Pollinator choice trials revealed the presence of three floral ecotypes within D. livida that each attracts a different species of thynnine wasp as a pollinator. Surveys of pollinator distribution revealed that the distribution of one of the ecotypes was strongly correlated with that of its pollinator, while another pollinator species was present throughout the range of all three ecotypes, demonstrating that pollinator availability does not always correlate with ecotype distribution. Floral ecotypes differed in chemical volatile composition, with a high degree of separation evident in principal coordinate analysis. Some compounds that differed between ecotypes, including pyrazines and (methylthio)phenols, are known to be electrophysiologically active in thynnine wasp antennae. Based on differences in pollinator response and floral volatile profile, the ecotypes represent distinct entities and should be treated as such in conservation management.The Holsworth Wildlife Research Endowment and the Australian Systematic Botany Society are thanked for their provision of research funding. AMW was supported by an Australian Government Research Training Program (RTP), and BB and RD were supported by Australian Research Council (ARC) Discovery Early Career Researcher Awards (DE 160101313 and DE150101720)

2-(Tetrahydrofuran-2-yl)acetic Acid and Ester Derivatives as Long-Range Pollinator Attractants in the Sexually Deceptive Orchid Cryptostylis ovata

Sexually deceptive orchids achieve pollination by luring male insects to flowers through chemical and sometimes visual mimicry of females. An extreme example of this deception occurs in Cryptostylis, one of only two genera where sexual deception is known to induce pollinator ejaculation. In the present study, bioassay-guided fractionations of Cryptostylis solvent extracts in combination with field bioassays were implemented to isolate and identify floral volatiles attractive to the pollinator Lissopimpla excelsa. ( S)-2-(Tetrahydrofuran-2-yl)acetic acid [( S)-1] and the ester derivatives methyl ( S)-2-(tetrahydrofuran-2-yl)acetate [( S)-2] and ethyl ( S)-2-(tetrahydrofuran-2-yl)acetate [( S)-3], all previously unknown semiochemicals, were confirmed to attract L. excelsa males in field bioassays. Chiral-phase GC and HPLC showed that the natural product 1 comprised a single enantiomer, its S-configuration being confirmed by synthesis of the two enantiomers from known enantiomers of tetrahydrofuran-2-carboxylic acid.B.B. and R.D.P.: Australian Research Council (ARC) Discovery Early Career Researcher Awards (DE 160101313 and DE150101720), A.M.W.: Australian Government Research Training Program, and Australian Orchid Foundation Grant 319.17, R.P. and G.R.F.: ARC Linkage Program Award (LP130100162). D. Bainbridge is gratefully acknowledged for designing and fabricating the preparative GC collector used in this stud

The US Program in Ground-Based Gravitational Wave Science: Contribution from the LIGO Laboratory

Recent gravitational-wave observations from the LIGO and Virgo observatories have brought a sense of great excitement to scientists and citizens the world over. Since September 2015,10 binary black hole coalescences and one binary neutron star coalescence have been observed. They have provided remarkable, revolutionary insight into the "gravitational Universe" and have greatly extended the field of multi-messenger astronomy. At present, Advanced LIGO can see binary black hole coalescences out to redshift 0.6 and binary neutron star coalescences to redshift 0.05. This probes only a very small fraction of the volume of the observable Universe. However, current technologies can be extended to construct "3rd Generation" (3G) gravitational-wave observatories that would extend our reach to the very edge of the observable Universe. The event rates over such a large volume would be in the hundreds of thousands per year (i.e. tens per hour). Such 3G detectors would have a 10-fold improvement in strain sensitivity over the current generation of instruments, yielding signal-to-noise ratios of 1000 for events like those already seen. Several concepts are being studied for which engineering studies and reliable cost estimates will be developed in the next 5 years