The road less taken: Dihydroflavonol 4-reductase inactivation and delphinidin anthocyanin loss underpins a natural intraspecific flower colour variation.

Visual cues are of critical importance for the attraction of animal pollinators, however, little is known about the molecular mechanisms underpinning intraspecific floral colour variation. Here, we combined comparative spectral analysis, targeted metabolite profiling, multi-tissue transcriptomics, differential gene expression, sequence analysis and functional analysis to investigate a bee-pollinated orchid species, Glossodia major with common purple- and infrequent white-flowered morphs. We found uncommon and previously unreported delphinidin-based anthocyanins responsible for the conspicuous and pollinator-perceivable colour of the purple morph and three genetic changes underpinning the loss of colour in the white morph - (1) a loss-of-function (LOF; frameshift) mutation affecting dihydroflavonol 4-reductase (DFR1) coding sequence due to a unique 4-bp insertion, (2) specific downregulation of functional DFR1 expression and (3) the unexpected discovery of chimeric Gypsy transposable element (TE)-gene (DFR) transcripts with potential consequences to the genomic stability and post-transcriptional or epigenetic regulation of DFR. This is one of few known cases where regulatory changes and LOF mutation in an anthocyanin structural gene, rather than transcription factors, are important. Furthermore, if TEs prove to be a frequent source of mutation, the interplay between environmental stress-induced TE evolution and pollinator-mediated selection for adaptive colour variation may be an overlooked mechanism maintaining floral colour polymorphism in nature.

Sexual attraction with pollination during feeding behaviour: implications for transitions between specialized strategies.

BACKGROUND AND AIMS: Understanding the origin of pollination by sexual deception has proven challenging, as sexually deceptive flowers are often highly modified, making it hard to resolve how any intermediate forms between sexual deception and an ancestral strategy might have functioned. Here, we report the discovery in Caladenia (Orchidaceae) of sexual attraction with pollination during feeding behaviour, which may offer important clues for understanding shifts in pollination strategy. METHODS: For Caladenia robinsonii, we observed the behaviour of its male wasp pollinator, Phymatothynnus aff. nitidus (Thynnidae), determined the site of release of the sexual attractant, and experimentally evaluated if the position of the attractant influences rates of attempted copulation and feeding behaviour. We applied GC-MS to test for surface sugar on the labellum. To establish if this pollination strategy is widespread in Caladenia, we conducted similar observations and experiments for four other Caladenia species. KEY RESULTS: In C. robinsonii, long-range sexual attraction of the pollinator is via semiochemicals emitted from the glandular sepal tips. Of the wasps landing on the flower, 57 % attempted copulation with the sepal tips, while 27 % attempted to feed from the base of the labellum, the behaviour associated with pollen transfer. A similar proportion of wasps exhibited feeding behaviour when the site of odour release was manipulated. A comparable pollination strategy occurs in another phylogenetically distinct clade of Caladenia. CONCLUSIONS: We document a previously overlooked type of sexual deception for orchids involving long-distance sexual attraction, but with pollination occurring during feeding behaviour at the labellum. We show this type of sexual deception operates in other Caladenia species and predict that it is widespread across the genus. Our findings may offer clues about how an intermediate transitional strategy from a food-rewarding or food-deceptive ancestor operated during the evolution of sexual deception.

The volatile chemistry of orchid pollination.

Covering: up to September 2022Orchids are renowned not only for their diversity of floral forms, but also for their many and often highly specialised pollination strategies. Volatile semiochemicals play a crucial role in the attraction of a wide variety of insect pollinators of orchids. The compounds produced by orchid flowers are as diverse as the pollinators they attract, and here we summarise some of the chemical diversity found across orchid taxa and pollination strategies. We focus on compounds that have been experimentally demonstrated to underpin pollinator attraction. We also highlight the structural elucidation and synthesis of a select subset of important orchid pollinator attractants, and discuss the ecological significance of the discoveries, the gaps in our current knowledge of orchid pollination chemistry, and some opportunities for future research in this field.

Drakolide Structure-activity Relationships for Sexual Attraction of Zeleboria Wasp Pollinator.

Orchids pollinated by sexual deception lure their specific male pollinators by sex pheromone mimicry. Despite the growing list of chemically diverse semiochemicals known to be involved, the chemical basis and flexibility of this extreme pollinator specificity are not fully understood. One promising but rarely applied tool is the synthesis and field testing of chemically related variants for investigating the structural specificity of the pheromone mimics. Here, we build on the discovery of the unusual semiochemical blend used by Drakaea micrantha to sexually lure its male Zeleboria thynnine wasp pollinator. This blend consists of a ß-ketolactone (drakolide) and two specific hydroxymethylpyrazines, presumably drawn from two distinct biosynthetic pathways. Here, we synthesized and tested the activity of various stereo- and structural isomers of the naturally occurring drakolide. Our study confirmed that in blends with the two pyrazines, both a mixture of stereoisomers, and the specific stereoisomer of the natural drakolide, elicit high rates of landings and attempted copulations. However, in the absence of pyrazines, both the number of responses and the level of sexual attraction were significantly reduced. When structural analogs were substituted for the natural drakolide, attractiveness and degree of sexual behaviour varied but were generally reduced. Based on our findings, and prior knowledge that related hydroxymethylpyrazines are active in other Drakaea spp., we conclude that the dual sex pheromone mimicry of D. micrantha likely evolved via initial changes in just one of the two biosynthetic pathways. Most plausibly, this involved modifications in the drakolides, with the pyrazines as a 'pre-adaption' enhancing the sexual response.

Evolution of reproductive structures for in-flight mating in thynnine wasps (Hymenoptera: Thynnidae: Thynninae).

Thynnine wasps have an unusual mating system that involves concurrent in-flight copulation and nuptial feeding of wingless females by alate males. Consequently, thynnine genitalia play a multifunctional role and have likely been subject to various different selective pressures for both reproductive success and food provisioning. Here, we present a new molecular phylogeny for the Australian Thynninae and use 3D-geometric morphometrics and comparative methods to investigate the morphological evolution of select genital structures across the group. We found significant morphological integration between all male and female structures analysed, which is likely influenced by sexual selection, but also reproductive isolation requirements and mechanical constraints. The morphology of the primary male and female coupling structures was correlated with female body size, and female genitalia exhibited strong negative size allometry. Those male and female coupling structures have evolved at similar evolutionary rates, whereas female structures appear to have evolved a higher degree of morphological novelty over time. We conclude that the unique reproductive strategies of thynnine wasps have resulted in complex evolutionary patterns in their genital morphology, which has likely played a central role in the extensive diversification of the subfamily across Australasia and South America. Our study reinforces the need to treat composite characters such as genitalia by their component parts, and to consider the roles of both male and female reproductive structures in evolutionary studies.

Orchid conservation: from theory to practice.

BACKGROUND: Given the exceptional diversity of orchids (26 000+ species), improving strategies for the conservation of orchids will benefit a vast number of taxa. Furthermore, with rapidly increasing numbers of endangered orchids and low success rates in orchid conservation translocation programmes worldwide, it is evident that our progress in understanding the biology of orchids is not yet translating into widespread effective conservation. SCOPE: We highlight unusual aspects of the reproductive biology of orchids that can have important consequences for conservation programmes, such as specialization of pollination systems, low fruit set but high seed production, and the potential for long-distance seed dispersal. Further, we discuss the importance of their reliance on mycorrhizal fungi for germination, including quantifying the incidence of specialized versus generalized mycorrhizal associations in orchids. In light of leading conservation theory and the biology of orchids, we provide recommendations for improving population management and translocation programmes. CONCLUSIONS: Major gains in orchid conservation can be achieved by incorporating knowledge of ecological interactions, for both generalist and specialist species. For example, habitat management can be tailored to maintain pollinator populations and conservation translocation sites selected based on confirmed availability of pollinators. Similarly, use of efficacious mycorrhizal fungi in propagation will increase the value of ex situ collections and likely increase the success of conservation translocations. Given the low genetic differentiation between populations of many orchids, experimental genetic mixing is an option to increase fitness of small populations, although caution is needed where cytotypes or floral ecotypes are present. Combining demographic data and field experiments will provide knowledge to enhance management and translocation success. Finally, high per-fruit fecundity means that orchids offer powerful but overlooked opportunities to propagate plants for experiments aimed at improving conservation outcomes. Given the predictions of ongoing environmental change, experimental approaches also offer effective ways to build more resilient populations.

A Specific Blend of Drakolide and Hydroxymethylpyrazines: An Unusual Pollinator Sexual Attractant Used by the Endangered Orchid Drakaea micrantha.

Bioactive natural products underpin the intriguing pollination strategy used by sexually deceptive orchids. These compounds, which mimic the sex pheromones of the female insect, are emitted in particular blends to lure male insect pollinators of specific species. By combining methods from field biology, analytical chemistry, electrophysiology, crystallography, and organic synthesis, we report that an undescribed ß-hydroxylactone, in combination with two specific hydroxymethylpyrazines, act as pollinator attractants in the rare hammer orchid Drakaea micrantha. This discovery represents an unusual case of chemically unrelated compounds being used together as a sexual attractant. Furthermore, this is the first example of the identification of pollinator attractants in an endangered orchid, enabling the use of chemistry in orchid conservation. Our synthetic blend is now available to be used in pollinator surveys to locate suitable sites for plant conservation translocations.

Experimental examination of pollinator-mediated selection in a sexually deceptive orchid.

Background and Aims: Selection exerted by pollinators on flowers is predicted to occur along two distinct axes. While pollinator attraction to flowers is governed by pollinator preferences, pollen transfer efficiency is mediated by the mechanical fit of pollinators to flower morphology. Although pollinator attraction in sexually deceptive orchids is typically underpinned by floral odour, morphological traits are expected to play a vital role in mechanical fit during floral contact with pollinators. Methods: Here we utilize a comprehensive and novel procedure to test for pollinator-mediated selection through mechanical fit with the flower labellum in the orchid Chiloglottis trapeziformis. This approach combines detailed pollinator observations related to plant reproductive fitness with complementary experimental manipulation and phenotypic selection analysis. Key Results: Experiments with virgin flowers revealed that pollen removal occurs only during vigorous pseudocopulation. This behaviour involves male wasps that grasp the insectiform callus structure on the labellum while probing the labellum tip in a forward orientation. Both orientation and duration of pseudocopulation were significant predictors of pollen removal, confirming a direct relationship between pollinator behaviour and plant fitness. Controlled floral manipulation that either shortened or elongated the distance between the callus and the labellum tip detected no change in pollinator attraction. The duration of pseudocopulation, however, was significantly reduced on flowers with shortened or elongated callus-tip distances, consistent with stabilizing selection. Phenotypic selection analysis confirmed this prediction in natural populations by uncovering evidence for stabilizing selection on the distance between the callus and the labellum tip. Conclusions: Our experimental manipulations and selection analysis in natural populations thus demonstrate stabilizing selection on the distance from the callus to the labellum tip, and illustrate the utility of employing multiple approaches to confirm selection exerted by pollinators on floral form.

Duplication and selection in ß-ketoacyl-ACP synthase gene lineages in the sexually deceptive Chiloglottis (Orchidaceace).

BACKGROUND AND AIMS: The processes of gene duplication, followed by divergence and selection, probably underpin the evolution of floral volatiles crucial to plant-insect interactions. The Australian sexually deceptive Chiloglottis orchids use a class of 2,5-dialkylcyclohexan-1,3-dione volatiles or 'chiloglottones' to attract specific male wasp pollinators. Here, we explore the expression and evolution of fatty acid pathway genes implicated in chiloglottone biosynthesis. METHODS: Both Chiloglottis seminuda and C. trapeziformis produce chiloglottone 1, but only the phylogenetically distinct C. seminuda produces this volatile from both the labellum callus and glandular sepal tips. Transcriptome sequencing and tissue-specific contrasts of the active and non-active floral tissues was performed. The effects of the fatty acid synthase inhibitor cerulenin on chiloglottone production were tested. Patterns of selection and gene evolution were investigated for fatty acid pathway genes. KEY RESULTS: Tissue-specific differential expression of fatty acid pathway transcripts was evident between active and non-active floral tissues. Cerulenin significantly inhibits chiloglottone 1 production in the active tissues of C. seminuda. Phylogenetic analysis of plant ß-ketoacyl synthase I (KASI), a protein involved in fatty acid biosynthesis, revealed two distinct clades, one of which is unique to the Orchidaceae (KASI-2B). Selection analysis indicated a strong signal of positive selection at the split of KASI-2B followed by relaxed purifying selection in the Chiloglottis clade. CONCLUSIONS: By capitalizing on a phylogenetically distinct Chiloglottis from earlier studies, we show that the transcriptional and biochemical dynamics linked to chiloglottone biosynthesis in active tissues are conserved across Chiloglottis. A combination of tissue-specific expression and relaxed purifying selection operating at specific fatty acid pathway genes may hold the key to the evolution of chiloglottones.

Sex ratio bias and shared paternity reduce individual fitness and population viability in a critically endangered parrot.

Sex-biased mortality can lead to altered adult sex ratios (ASRs), which may in turn lead to harassment and lower fitness of the rarer sex and changes in the mating system. Female critically endangered swift parrots (Lathamus discolor) suffer high predation while nesting due to an introduced mammalian predator, the sugar glider (Petaurus breviceps). High predation on females is causing severe population decline alongside strongly biased adult sex ratios (≥73% male). Our 6-year study showed that 50.5% of critically endangered swift parrot nests had shared paternity although the birds remained socially monogamous. Shared paternity increased significantly with the local rate of predation on breeding females, suggesting that rates of shared paternity increased when the ASR became more biased. Nests that were not predated produced fewer fledglings as the local ASR became more male-biased possibly due to higher interference during nesting from unpaired males. Population viability analyses showed that part of the predicted decline in the swift parrot population is due to reduced reproductive success when paternity is shared. The models predicted that the population would decline by 89.4% over three generations if the birds maintained the lowest observed rate of shared paternity. This compares with predicted population reductions of 92.1-94.9% under higher rates of shared paternity. We conclude that biases in the ASR, in this case caused by sex-specific predation from an introduced predator, can lead to changes in the mating system and negative impacts on both individual fitness and long-term population viability.

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.

The impact of mating systems and dispersal on fine-scale genetic structure at maternally, paternally and biparentally inherited markers.

For decades, studies have focused on how dispersal and mating systems influence genetic structure across populations or social groups. However, we still lack a thorough understanding of how these processes and their interaction shape spatial genetic patterns over a finer scale (tens-hundreds of metres). Using uniparentally inherited markers may help answer these questions, yet their potential has not been fully explored. Here, we use individual-level simulations to investigate the effects of dispersal and mating system on fine-scale genetic structure at autosomal, mitochondrial and Y chromosome markers. Using genetic spatial autocorrelation analysis, we found that dispersal was the major driver of fine-scale genetic structure across maternally, paternally and biparentally inherited markers. However, when dispersal was restricted (mean distance = 100 m), variation in mating behaviour created strong differences in the comparative level of structure detected at maternally and paternally inherited markers. Promiscuity reduced spatial genetic structure at Y chromosome loci (relative to monogamy), whereas structure increased under polygyny. In contrast, mitochondrial and autosomal markers were robust to differences in the specific mating system, although genetic structure increased across all markers when reproductive success was skewed towards fewer individuals. Comparing males and females at Y chromosome vs. mitochondrial markers, respectively, revealed that some mating systems can generate similar patterns to those expected under sex-biased dispersal. This demonstrates the need for caution when inferring ecological and behavioural processes from genetic results. Comparing patterns between the sexes, across a range of marker types, may help us tease apart the processes shaping fine-scale genetic structure.

An experimental evaluation of traits that influence the sexual behaviour of pollinators in sexually deceptive orchids.

Pollination by sexual deception of male insects is perhaps one of the most remarkable cases of mimicry in the plant kingdom. However, understanding the influence of floral traits on pollinator behaviour in sexually deceptive plants is challenging, due to the risk of confounding changes in floral odour when manipulating morphology. Here, we investigated the floral traits influencing the sexual response of male Zaspilothynnus nigripes (Tiphiidae) wasps, a pollinator of two distantly related sexually deceptive orchids with contrasting floral architecture, Caladenia pectinata and Drakaea livida. In D. livida, the chemical sexual attractant is emitted from the labellum, whereas in C. pectinata, it is produced from the distal sepal tips, allowing manipulative experiments. When controlling for visual cues, there was no difference in long-distance attraction, although the floral odour of D. livida induced copulation more frequently than that of C. pectinata. The role of colour in pollinator sexual attraction was equivocal, indicating that colour may not be a strong constraint on the initial evolution of sexual deception. The frequency of wasp visitors landing on C. pectinata decreased when the amount of floral odour was reduced, but attempted copulation rates were enhanced when the source of floral odour was associated with the labellum. These latter variables may represent axes of selection that operate across many sexually deceptive species. Nonetheless, the observed variation in floral traits suggests flexibility among species in how sexual deception can be achieved.

Structure-Activity Studies of Semiochemicals from the Spider Orchid Caladenia plicata for Sexual Deception.

Sexually deceptive orchids attract specific pollinators by mimicking insect sex pheromones. Normally this mimicry is very specific and identical compounds have been identified from orchids and matching females of the pollinators. In this study, we conduct a detailed structure-activity investigation on isomers of the semiochemicals involved in the sexual attraction of the male pollinator of the spider orchid Caladenia plicata. This orchid employs an unusual blend of two biosynthetically unrelated compounds, (S)-ß-citronellol and 2-hydroxy-6-methylacetophenone, to lure its Zeleboria sp. thynnine wasp pollinator. We show that the blend is barely attractive when (S)-ß-citronellol is substituted with its enantiomer, (R)-ß-citronellol. Furthermore, none of the nine-possible alternative hydroxy-methylacetophenone regioisomers of the natural semiochemical are active when substituted for the natural 2-hydroxy-6-methylacetophenone. Our results were surprising given the structural similarity between the active compound and some of the analogues tested, and results from previous studies in other sexually deceptive orchid/wasp systems where substitution with analogues was possible. Interestingly, high-level ab initio and density functional theory calculations of the hydroxy-methylacetophenones revealed that the active natural isomer, 2-hydroxy-6-methylacetophenone, has the strongest intramolecular hydrogen bond of all regioisomers, which at least in part may explain the specific activity.

The Spider Orchid Caladenia crebra Produces Sulfurous Pheromone Mimics to Attract its Male Wasp Pollinator.

One of the most intriguing natural observations is the pollination of orchids by sexual deception. Chemicals underpin this interaction between the orchid and its sexually attracted male insect pollinator, with the signaling compounds involved, called semiochemicals, predicted to mimic the chemical composition of the sex pheromone. We identified floral semiochemicals from Caladenia (spider orchids) for the first time. We further demonstrate that C. crebra attracts its single pollinator species with a unique system of (methylthio)phenols, three of which are new natural products. Furthermore, as predicted, the same compounds constitute the sex pheromone of the pollinator, the thynnine wasp Campylothynnus flavopictus, representing the first occurrence of sulfurous sex pheromones in Hymenoptera.

Weeds, as ancillary hosts, pose disproportionate risk for virulent pathogen transfer to crops.

BACKGROUND: The outcome of the arms race between hosts and pathogens depends heavily on the interactions between their genetic diversity, population size and transmission ability. Theory predicts that genetically diverse hosts will select for higher virulence and more diverse pathogens than hosts with low genetic diversity. Cultivated hosts typically have lower genetic diversity and thus small effective population sizes, but can potentially harbour large pathogen population sizes. On the other hand, hosts, such as weeds, which are genetically more diverse and thus have larger effective population sizes, usually harbour smaller pathogen population sizes. Large pathogen population sizes may lead to more opportunities for mutation and hence more diverse pathogens. Here we test the predictions that pathogen neutral genetic diversity will increase with large pathogen population sizes and host diversity, whereas diversity under selection will increase with host diversity. We assessed and compared the diversity of a fungal pathogen, Rhynchosporium commune, on weedy barley grass (which have a large effective population size) and cultivated barley (low genetic diversity) using microsatellites, effector locus nip1 diversity and pathogen aggressiveness in order to assess the importance of weeds in the evolution of the neutral and selected diversity of pathogens. RESULTS: The findings indicated that the large barley acreage and low host diversity maintains higher pathogen neutral genetic diversity and lower linkage disequilibrium, while the weed maintains more pathotypes and higher virulence diversity at nip1. Strong evidence for more pathogen migration from barley grass to barley suggests transmission of virulence from barley grass to barley is common. CONCLUSIONS: Pathogen census population size is a better predictor for neutral genetic diversity than host diversity. Despite maintaining a smaller pathogen census population size, barley grass acts as an important ancillary host to R. commune, harbouring highly virulent pathogen types capable of transmission to barley. Management of disease on crops must therefore include management of weedy ancillary hosts, which may harbour disproportionate supplies of virulent pathogen strains.

Parapheromones for Thynnine Wasps.

Sexually deceptive orchids produce floral volatiles that attract male insect pollinators. This interaction between flower and pollinator normally is highly specific. In the few cases where the chemical composition of the volatiles is known, the compounds have been found to be identical to those that comprise the sex pheromone of the female wasp. In this study, we investigated whether there is potential for flexibility in the molecular structure of the chemical cues used to mediate these specific interactions. Specifically, we asked whether strong sexual attraction can be maintained with structural modifications of sex pheromone components. Our study focused on the orchid, Drakaea glyptodon, which is pollinated by males of the thynnine wasp, Zaspilothynnus trilobatus. Three alkylpyrazines and a unique hydroxymethylpyrazine are components of the female produced sex pheromone of Z. trilobatus, and also the semiochemicals produced by the orchid that lures the males as pollinators. A blend of 2-butyl-3,5-dimethylpyrazine and 2-hydroxymethyl-3,5-diethyl-6-methylpyrazine (3:1) is as attractive as the full blend of four compounds. Therefore, in this study we substituted 2-hydroxymethyl-3,5-diethyl-6-methylpyrazine with one of five structurally related parapheromones in a blend with 2-butyl-3,5-dimethylpyrazine. All blends tested stimulated approaches by male wasps, with some also eliciting landing and attempted copulation. High-level calculations (G4(MP2)) showed the energy differences between the structural isomers were small, although the degree of sexual attraction varied, indicating the importance of structural factors for activity. One of the parapheromones, 2-hydroxymethyl-3,5-dimethyl-6-ethylpyrazine, elicited similar proportions of approaches, landings, and attempted copulations as the sex pheromone at the ratio and dose tested. The findings suggest that there is potential for chemical flexibility in the evolution of sexual deception.

The effect of sex-biased dispersal on opposite-sexed spatial genetic structure and inbreeding risk.

Natal sex-biased dispersal has long been thought to reduce the risk of inbreeding by spatially separating opposite-sexed kin. Yet, comprehensive and quantitative evaluations of this hypothesis are lacking. In this study, we quantified the effectiveness of sex-biased dispersal as an inbreeding avoidance strategy by combining spatially explicit simulations and empirical data. We quantified the extent of kin clustering by measuring the degree of spatial autocorrelation among opposite-sexed individuals (FM structure). This allowed us to systematically explore how the extent of sex-biased dispersal, generational overlap, and mate searching distance, influenced both kin clustering, and the resulting inbreeding in the absence of complementary inbreeding avoidance strategies. Simulations revealed that when sex-biased dispersal was limited, positive FM genetic structure developed quickly and increased as the mate searching distance decreased or as generational overlap increased. Interestingly, complete long-range sex-biased dispersal did not prevent the development of FM genetic structure when generations overlapped. We found a very strong correlation between FM genetic structure and both FIS under random mating, and pedigree-based measures of inbreeding. Thus, we show that the detection of FM genetic structure can be a strong indicator of inbreeding risk. Empirical data for two species with different life history strategies yielded patterns congruent with our simulations. Our study illustrates a new application of spatial genetic autocorrelation analysis that offers a framework for quantifying the risk of inbreeding that is easily extendable to other species. Furthermore, our findings provide other researchers with a context for interpreting observed patterns of opposite-sexed spatial genetic structure.

UV-B light contributes directly to the synthesis of chiloglottone floral volatiles.

BACKGROUND AND AIMS: Australian sexually deceptive Chiloglottis orchids attract their specific male wasp pollinators by means of 2,5-dialkylcyclohexane-1,3-diones or 'chiloglottones', representing a newly discovered class of volatiles with unique structures. This study investigated the hypothesis that UV-B light at low intensities is directly required for chiloglottone biosynthesis in Chiloglottis trapeziformis. METHODS: Chiloglottone production occurs only in specific tissue (the callus) of the labellum. Cut buds and flowers, and whole plants with buds and flowers, sourced from the field, were kept in a growth chamber and interactions between growth stage of the flowers and duration and intensity of UV-B exposure on chiloglottone production were studied. The effects of the protein synthesis inhibitor cycloheximide were also examined. KEY RESULTS: Chiloglottone was not present in buds, but was detected in buds that were manually opened and then exposed to sunlight, or artificial UV-B light for ≥5 min. Spectrophotometry revealed that the sepals and petals blocked UV-B light from reaching the labellum inside the bud. Rates of chiloglottone production increased with developmental stage, increasing exposure time and increasing UV-B irradiance intensity. Cycloheximide did not inhibit the initial production of chiloglottone within 5 min of UV-B exposure. However, inhibition of chiloglottone production by cycloheximide occurred over 2 h of UV-B exposure, indicating a requirement for de novo protein synthesis to sustain chiloglottone production under UV-B. CONCLUSIONS: The sepals and petals of Chiloglottis orchids strongly block UV-B wavelengths of light, preventing chiloglottone production inside the bud. While initiation of chiloglottone biosynthesis requires only UV-B light, sustained chiloglottone biosynthesis requires both UV-B and de novo protein biosynthesis. The internal amounts of chiloglottone in a flower reflect the interplay between developmental stage, duration and intensity of UV-B exposure, de novo protein synthesis, and feedback loops linked to the starting amount of chiloglottone. It is concluded that UV-B light contributes directly to chiloglottone biosynthesis. These findings suggest an entirely new and unexpected biochemical reaction that might also occur in taxa other than these orchids.

Not all types of host contacts are equal when it comes to E. coli transmission.

The specific processes that facilitate pathogen transmission are poorly understood, particularly for wild animal populations. A major impediment for investigating transmission pathways is the need for simultaneous information on host contacts and pathogen transfer. In this study, we used commensal Escherichia coli strains as a model system for gastrointestinal pathogens. We combined strain-sharing information with detailed host contact data to investigate transmission routes in mountain brushtail possums. Despite E. coli being transmitted via the faecal-oral route, we revealed that, strain-sharing among possums was better explained by host contacts than spatial proximity. Furthermore, and unexpectedly, strain-sharing was more strongly associated with the duration of brief nocturnal associations than day-long den-sharing. Thus, the most cryptic and difficult associations to measure were the most relevant connections for the transmission of this symbiont. We predict that future studies that employ similar approaches will reveal the importance of previously overlooked associations as key transmission pathways.