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Orchids are amongst the most diverse of all plant families, and produce a wide array of volatile compounds for pollinator attraction.

Sexually deceptive orchids exploit the innate sexual preferences of their male insect pollinators to achieve pollen transfer. Although floral volatiles are critical for pollinator attraction in sexually deceptive systems, floral morphology is also expected to exploit the sexual preferences of the pollinator. Here, we investigate the pollination of the Australian orchid Pterostylis cycnocephala. We confirm that male fungus gnats of a single undescribed Bradysia sp. (Diptera, Sciaridae) act as pollinators and display sexual behaviour on flowers, including wing fanning, abdomen curling and genitalic clasping of the prominent labellum appendage. Gnats only triggered the labellum and became trapped in the flower after attempting pseudocopulation with the appendage, a process necessary for pollen removal and deposition. Male gnats located flowers hidden from view, suggesting long-range attraction is primarily due to floral odour. However, male gnats displayed reduced copulatory behaviour when the labellum was absent and when the labellum appendage was inaccessible, suggesting that appropriate morphology may be required to elicit the copulatory behaviour needed for pollination. Our study is the first detailed investigation of sexual deception of male Sciaridae in Australian orchids and represents an example of convergent evolution with some Neotropical Lepanthes orchids, which also sexually deceive male Bradysia.

A new study finds that, in the forests of tropical China, hungry hornets are lured to the fruits of Aquilaria sinensis by highly volatile compounds structurally similar to volatiles from herbivore-damaged leaves. The hornets disperse the short-lived seeds rapidly to optimal new habitats.

Fire drives animal population dynamics across many ecosystems. Yet, we still lack an understanding of how most species recover from fire and the effects of fire severity and patchiness on recovery processes. This information is crucial for fire‐mediated biodiversity conservation, particularly as fire regimes change globally. We conducted an experiment to test whether post‐fire recovery is driven by in situ survival or recolonisation, and to determine whether this varies with fires of increasing percentage area burnt (burn cover) and severity. We used the pale field rat Rattus tunneyi as a model, because it represents the extinction process for a suite of mammal species suffering population collapse across Australia's northern savannas. Our treatments spanned a gradient from patchy, low severity fires (simulating early dry season management burns) to thorough, high severity fires (simulating wildfires). We performed capture–mark–recapture, vegetation and aerial surveys before, 6 weeks after and 1 year after fire. Six weeks after fire, pale field rats were only captured in unburnt patches of vegetation, and capture rates were proportional to the amount of unburnt habitat. One year later, both vegetation and pale field rat populations recovered across all sites. However, population recovery after low severity fires was likely achieved through in situ survival and reproduction in unburnt micro‐refuges, compared to recolonisation driving recovery after high severity fires. Synthesis and applications. Pale field rat persistence is strongly dependent on the retention of unburnt habitat patches within fire‐affected areas. Management strategies that increase micro‐refugia within burnt areas may facilitate pale field rat population recovery. Globally, building recovery mechanisms into fire management will be vital for supporting the long‐term persistence of fire‐affected species.

Interview with Rod Peakall, who studies the evolution of specialised pollinator interactions at the Australian National University.

With over 25,000 species, the drivers of diversity in the Orchidaceae remain to be fully understood. Here, we outline a multitiered sequence capture strategy aimed at capturing hundreds of loci to enable phylogenetic resolution from subtribe to subspecific levels in orchids of the tribe Diurideae. For the probe design, we mined subsets of 18 transcriptomes, to give five target sequence sets aimed at the tribe (Sets 1 & 2), subtribe (Set 3), and within subtribe levels (Sets 4 & 5). Analysis included alternative de novo and reference-guided assembly, before target sequence extraction, annotation and alignment, and application of a homology-aware k-mer block phylogenomic approach, prior to maximum likelihood and coalescence-based phylogenetic inference. Our evaluation considered 87 taxa in two test data sets: 67 samples spanning the tribe, and 72 samples involving 24 closely related Caladenia species. The tiered design achieved high target loci recovery (>89%), with the median number of recovered loci in Sets 1-5 as follows: 212, 219, 816, 1024, and 1009, respectively. Interestingly, as a first test of the homologous k-mer approach for targeted sequence capture data, our study revealed its potential for enabling robust phylogenetic species tree inferences. Specifically, we found matching, and in one case improved phylogenetic resolution within species complexes, compared to conventional phylogenetic analysis involving target gene extraction. Our findings indicate that a customized multitiered sequence capture strategy, in combination with promising yet underutilized phylogenomic approaches, will be effective for groups where interspecific divergence is recent, but information on deeper phylogenetic relationships is also required.

Natural disturbance regimes in forest ecosystems are being rapidly modified by anthropogenic pressures, including silvicultural practices and climate change. Australian forests dominated by mountain ash (Eucalyptus regnans) are critically endangered, with wildfires and clearfell logging predicted to cause ecosystem collapse within the next 50 years. To investigate the influence of disturbance on patterns and extent of genetic diversity in mountain ash, we compare replicated sites with three different disturbance histories (undisturbed, burnt, and logged). We employ genetic analysis at five chloroplast microsatellite loci and 2866 nuclear single-nucleotide polymorphisms (SNPs) to estimate within- and among- population genetic diversity, and assess the extent of fine-scale spatial genetic structure among individuals, for the three disturbance treatments. Consistent with the expectation of extensive pollen dispersal but limited seed dispersal, we detected low levels of genetic differentiation at nuclear SNPs (FST = 0.067), and very high levels of differentiation at cpDNA microsatellites (FST = 0.751). While differences among treatments at nuclear SNPs were small, we found stronger spatial genetic structure in the undisturbed treatment, higher levels of genetic differentiation in the logged treatment and greater partitioning of genetic diversity among logged sites. Analysis of cpDNA revealed significantly higher levels of total and within-site genetic diversity in the logged treatment than the burnt or undisturbed treatments, with haplotypes entering the system via the use of non-local seed in the regeneration process. We suggest that artificial regeneration activities should utilise a greater number of maternal parents, which could be achieved via variable retention harvesting or utilising a regional admixture provenancing approach.

Caladenia is a diverse Australian genus that is exceptional among orchids in having both species pollinated by food-seeking and sexually deceived insects. Here, we investigated the pollination of Caladenia nobilis, a species predicted to be food-deceptive due to its large, cream-coloured and apparently nectarless flowers. Pollinator observations were made using experimental clumps of flowers. Measurements of floral colour were undertaken with a spectrometer, nectar was tested using GC-MS, and reproductive success was quantified for 2 years. While C. nobilis attracted nine species of insect, only males of the thynnine wasp Rhagigaster discrepans exhibited the correct size and behaviour to remove and deposit pollen. Male R. discrepans attempted to feed from the surface of the labellum, often crawling to multiple flowers, but showed no evidence of sexual attraction. Most flowers produced little or no nectar, although some may provide enough sucrose to act as a meagre reward to pollinators. Floral colouration was similar to a related Caladenia species pollinated by sexual deception, although the sexually deceptive species had a dull-red labellum. Reproductive success was generally low and highly variable between sites and years. In addition to most visitors being of inappropriate size for pollinia removal, the lack of response to the orchid by several co-occurring species of thynnine wasp suggests filtering of potential pollinators at the attraction phase. Our discovery of a pollination strategy that may be intermediate between food deception and food reward raises the question, how many putatively rewardless orchids actually produce meagre amounts of nectar?

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 β-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.

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 β-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.

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.

The Pompilidae is a cosmopolitan and diverse group of wasps, which commonly feed on nectar. However, pollination systems specialised on pompilids have not been documented in detail outside of southern Africa. Here, we studied Caladenia drummondii (Orchidaceae), where based on floral traits and preliminary field observations we predicted pollination by sexual deception of male pompilid wasps. Detailed pollinator observations were undertaken using floral baiting experiments at sites spanning 375 km. Following evidence for nectar on some flowers of C. drummondii, the sugar content on the labellum was analysed by GC-MS. Floral spectral reflectance was measured and compared with Caladenia using other pollination strategies. Males of a single species of pompilid wasp (Calopompilus sp.) were the only visitors capable of pollinating C. drummondii. Attempts to feed from the surface of the labellum were frequent, and were associated with removal and deposition of pollinia. GC-MS analysis revealed larger quantities of sugar on the labellum than reported in other Caladenia species. While no sexual or courtship behaviour was observed, the zig-zag and circling flight on approach to the flower is suggestive of odour-based attraction. Floral spectral reflectance was similar to sexually deceptive Caladenia. This study represents the first confirmation of a specialised pompilid pollination system outside of Africa. Although pollination occurs during nectar-foraging, long-distance sexual attraction cannot be ruled out as an explanation for the exclusive male visitation. The similarity in floral spectral reflectance to other Caladenia indicates colour may not impose a constraint on the evolution of pollination by pompilids.

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 β-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.

BackgroundGiven 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.ScopeWe 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.ConclusionsMajor 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.

Methods for 3D‐imaging of biological samples are experiencing unprecedented development, with tools such as X‐ray micro‐computed tomography (μCT) becoming more accessible to biologists. These techniques are inherently suited to small subjects and can simultaneously image both external and internal morphology, thus offering considerable benefits for invertebrate research. However, methods for visualising 3D‐data are trailing behind the development of tools for generating such data. Our aim in this article is to make the processing, visualisation and presentation of 3D‐data easier, thereby encouraging more researchers to utilise 3D‐imaging. Here, we present a comprehensive workflow for manipulating and visualising 3D‐data, including basic and advanced options for producing images, videos and interactive 3D‐PDFs, from both volume and surface‐mesh renderings. We discuss the importance of visualisation for quantitative analysis of invertebrate morphology from 3D‐data, and provide example figures illustrating the different options for generating 3D‐figures for publication. As more biology journals adopt 3D‐PDFs as a standard option, research on microscopic invertebrates and other organisms can be presented in high‐resolution 3D‐figures, enhancing the way we communicate science.

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)-β-citronellol and 2-hydroxy-6-methylacetophenone, to lure its Zeleboria sp. thynnine wasp pollinator. We show that the blend is barely attractive when (S)-β-citronellol is substituted with its enantiomer, (R)-β-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.

Mobile species pose major challenges for conservation because of their unpredictable, large scale movements in response to fluctuating resources. If locations with critical resources overlap with threats, large proportions of a mobile population may be exposed to threats. Critically endangered and nomadic swift parrots Lathamus discolor nest wherever food is most abundant in their breeding range, but concern exists that nest predation from an introduced predator may severely affect their population. Although swift parrots nest on predator-free offshore islands, population viability analysis indicates that is land nesting alone may be insufficient to offset extinction risk from high mainland predation rates assuming that the species is a single panmictic population. We test the assumption that swift parrots act as a single conservation unit. We undertook a population genetic analysis using seven microsatellite loci and samples obtained over 6 years from across the breeding range of swift parrots. We found no evidence of departure from Hardy–Weinberg expectations across the species and both Analysis of Molecular Variance and Bayesian Structure analyses failed to detect any evidence for genetic differentiation across the samples both spatially and temporally. These results, supported by simulations, indicate panmixia and a lack of population genetic structure in swift parrots. Unlike a sedentary or site philopatric species, the majority of the swift parrot population may be at risk of exposure to predation when unpredictable resources draw individuals away from islands. These findings support a key assumption of population viability models that predict an extreme reduction in population size for swift parrots, and address a major gap in knowledge of the species’ ecology. Our study has implications both for the development of effective conservation management strategies and for the longer-term evolution of avoidance of predator-infested habitat in swift parrots.

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.

One of the most intriguing natural observations is the pollination of orchids by sexual deception. Here, we identify floral semiochemicals from Caladenia (spider orchids) for the first time. We demonstrate that C. crebra, attracts its single pollinator species with a unique system of (methylthio)phenols, three of which are new natural products. Furthermore, 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.

Genetic tagging, the unique identification of individuals by their DNA profile, has proven to be an effective method for research on several animal species. In this study we apply non-invasive genetic tagging from feather samples to reveal the genetic structure and estimate local population size of red-and-green macaws (Ara chloropterus) without the need to capture these animals. The study was centered in the Tambopata region of the Peruvian Amazon. Here macaws frequently visit clay licks and their naturally molted feathers provide a unique source of non-invasively sampled DNA. We analyzed 249 feathers using nine microsatellite loci and identified 221 unique genotypes. The remainder revealed 21 individuals which were ‘recaptured’ one or more times. Using a capture-mark-recapture model the average number of different individuals visiting clay licks within one breeding season was estimated to fall between 84 and 316 individuals per clay lick. Analysis of population genetic structure revealed only small genetic differences among regions and clay licks, suggesting a single red-and-green macaw genetic population. Our study confirms the utility of non-invasive genetic tagging in harsh tropical environment to obtain crucial population parameters about an abundant parrot species that is very difficult to capture in the wild.

The increasing availability of DNA sequence data enables exciting new opportunities for fungal ecology. However, it amplifies the challenge of how to objectively classify the diversity of fungal sequences into meaningful units, often in the absence of morphological characters. Here, we test the utility of modern multilocus Bayesian coalescent-based methods for delimiting cryptic fungal diversity in the orchid mycorrhiza morphospecies Serendipita vermifera. We obtained 147 fungal isolates from Caladenia, a speciose clade of Australian orchids known to associate with Serendipita fungi. DNA sequence data for 7 nuclear and mtDNA loci were used to erect competing species hypotheses by clustering isolates based on: (a) ITS sequence divergence, (b) Bayesian admixture analysis, and (c) mtDNA variation. We implemented two coalescent-based Bayesian methods to determine which species hypothesis best fitted our data. Both methods found strong support for eight species of Serendipita among our isolates, supporting species boundaries reflected in ITS divergence. Patterns of host plant association showed evidence for both generalist and specialist associations within the host genus Caladenia. Our findings demonstrate the utility of Bayesian species delimitation methods and suggest that wider application of these techniques will readily uncover new species in other cryptic fungal lineages.

Recent studies using sequence data from eight sequence loci and coalescent-based species delimitation methods have revealed several species-level lineages of Tulasnella associated with the orchid genera Arthrochilus, Caleana, Chiloglottis, and Drakaea in Australia. Here we formally describe three of those species, Tulasnella prima, T. secunda, and T. warcupii spp. nov, as well as an additional Tulasnella species associated with Chiloglottis growing in Sphagnum, T sphagneti sp. nov. Species were identified by phylogenetic analyses of the ITS with up to 1.3 % sequence divergence within taxa and a minimum of 7.6 % intraspecific divergence. These new Tulasnella (Tulasnellaceae, Cantharellales) species are currently only known from orchid hosts, with each fungal species showing a strong relationship with an orchid genus. In this study, T. prima and T. sphagneti associate with Chiloglottis, while T. secunda associates with Drakaea and Caleana, and T. warcupii associates with Arthrochilus oreophilus.

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.

Cross-kingdom mimicry of female insect sex pheromones by sexually deceptive orchids has evolved multiple times.1 Fungus gnats (Diptera) are predicted to be pollinators of hundreds of sexually deceptive orchids,2-4 yet unlike orchids that sexually attract bees and wasps (Hymenoptera),5-11 the chemistry of fungus gnat-pollinated orchids remains unknown. Furthermore, despite the importance of fungus gnats as pollinators, pests, and decomposers of organic material, and evidence for sex pheromones since 1971,12-17 no structure of any fungus gnat sex pheromone has to date been confirmed. In this study, we found a mixture of five hydrocarbons shared between Pterostylis orbiculata orchids and female Mycomya sp. (Mycetophilidae) fungus gnats, which included three alkanes, a C23 diene, and a C23 triene. The triene was an undescribed natural product, which we synthesized and confirmed to be (6Z,9Z)-1,6,9-tricosatriene. Field bioassays with a synthetic blend of the five hydrocarbons elicited attraction and sexual behavior from male gnats. The triene alone elicited attraction and low levels of sexual behavior, but the blend without it was unattractive, suggesting that this compound is a key component of orchid pollinator attraction and the female fungus gnat sex pheromone. In two closely related Pterostylis species, we found related C23 trienes, but not (6Z,9Z)-1,6,9-tricosatriene. These results suggest that unusual long-chain unsaturated hydrocarbons hold the key to sexual deception in Pterostylis orchids, and are an important step toward deciphering female fungus gnat sex pheromones.

Dispersal is essential for species persistence and landscape genetic studies are valuable tools for identifying potential barriers to dispersal. Macaws have been studied for decades in their natural habitat, but we still have no knowledge of how natural landscape features influence their dispersal. We tested for correlations between landscape resistance models and the current population genetic structure of macaws in continuous rainforest to explore natural barriers to their dispersal. We studied scarlet macaws (Ara macao) over a 13,000 km2 area of continuous primary Amazon rainforest in south-eastern Peru. Using remote sensing imagery from the Carnegie Airborne Observatory, we constructed landscape resistance surfaces in CIRCUITSCAPE based on elevation, canopy height and above-ground carbon distribution. We then used individual- and population-level genetic analyses to examine which landscape features influenced gene flow (genetic distance between individuals and populations). Across the lowland rainforest we found limited population genetic differentiation. However, a population from an intermountain valley of the Andes (Candamo) showed detectable genetic differentiation from two other populations (Tambopata) located 20–60 km away (F ST = 0.008, P = 0.001–0.003). Landscape resistance models revealed that genetic distance between individuals was significantly positively related to elevation. Our landscape resistance analysis suggests that mountain ridges between Candamo and Tambopata may limit gene flow in scarlet macaws. These results serve as baseline data for continued landscape studies of parrots, and will be useful for understanding the impacts of anthropogenic dispersal barriers in the future.

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.

Where dispersal distances are restricted or generations overlap, kin may remain spatially clustered, leading to positive spatial genetic structure and the potential for inbreeding. In such circumstances, post-dispersal behavioral mechanisms may be required if individuals are to avoid mating with kin. Here, we conducted an empirical investigation of mate choice in the presence of fine-scale genetic structure. We assessed the potential for mating among relatives using genetic spatial autocorrelation analysis among adult mountain brushtail possums (Trichosurus cunninghami). There was significant positive spatial genetic structure among opposite-sexed adults (on a scale of 200 m), suggesting that kin remained spatially clustered after dispersal. Despite this, no genetic evidence of inbreeding was found. We assessed whether females may potentially avoid inbreeding: (1) by seeking distant mates and/or (2) by the active avoidance of kin in mate choice. Individuals did not choose distant mates, as 97 % of pairs that mated were separated by

Summary Sexually deceptive orchids are renowned for their olfactory mimicry of female insect sex pheromones to lure male pollinators, but the role of floral morphology remains unknown. Here, we reveal compelling new experimental evidence that morphology also matters in sexual deception. Our study exploited two morphologically distinct Chiloglottis orchids that both employ the same semiochemical (chiloglottone 1) to attract their respective primary pollinator. In these experiments, we monitored attempted copulation of pollinators with orchid labella as this likely impacts plant fitness. Reciprocal pollinator choice tests revealed significant reductions in the frequency and duration of attempted copulation when pollinators were presented with alternate orchid species that differ in floral morphology, but nevertheless exhibit identical semiochemicals. Experimentally shortening the labellum also reduced the duration of attempted copulation in one of the species. Pollinators exhibited contrasting orientations during attempted copulation and pollination, which seem to be correlated with fundamental differences in the morphological adaptations for both mechanical fit and female mimicry in these orchids. Our findings confirm the overlooked importance of floral morphology for sexually deceptive orchid pollination and indicate that pollinator behaviour could impose strong selection on specific floral traits.

Plants are predicted to show floral adaptation to geographic variation in the most effective pollinator, potentially leading to reproductive isolation and genetic divergence. Many sexually deceptive orchids attract just a single pollinator species, limiting opportunities to experimentally investigate pollinator switching. Here, we investigate Drakaea concolor, which attracts two pollinator species. Using pollinator choice tests, we detected two morphologically similar ecotypes within D. concolor. The common ecotype only attracted Zaspilothynnus gilesi, whereas the rare ecotype also attracted an undescribed species of Pogonothynnus. The rare ecotype occurred at populations nested within the distribution of the common ecotype, with no evidence of ecotypes occurring sympatrically. Surveying for pollinators at over 100 sites revealed that ecotype identity was not correlated with wasp availability, with most orchid populations only attracting the rare Z. gilesi. Using microsatellite markers, genetic differentiation among populations was very low (GST = 0.011) regardless of ecotype, suggestive of frequent gene flow. Taken together, these results may indicate that the ability to attract Pogonothynnus has evolved recently, but this ecotype is yet to spread. The nested distribution of ecotypes, rather than the more typical formation of ecotypes in allopatry, illustrates that in sexually deceptive orchids, pollinator switching could occur throughout a species' range, resulting from multiple potentially suitable but unexploited pollinators occurring in sympatry. This unusual case of sympatric pollinators highlights D. concolor as a promising study system for further understanding the process of pollinator switching from ecological, chemical and genetic perspectives.

Flowers have evolved diverse strategies to attract animal pollinators, with visual and olfactory floral cues often crucial for pollinator attraction. While most plants provide reward (e.g., nectar, pollen) in return for the service of pollination, 1000s of plant species, particularly in the orchid family, offer no apparent reward. Instead, they exploit their often specific pollinators (one or few) by mimicking signals of female insects, food source, and oviposition sites, among others. A full understanding of how these deceptive pollination strategies evolve and persist remains an open question. Nonetheless, there is growing evidence that unique blends that often contain unusual compounds in floral volatile constituents are often employed to secure pollination by deception. Thus, the ability of plants to rapidly evolve new pathways for synthesizing floral volatiles may hold the key to the widespread evolution of deceptive pollination. Yet, until now the biosynthesis of these volatile compounds has been largely neglected. While elucidating the biosynthesis in non-model systems is challenging, nonetheless, these cases may also offer untapped potential for biosynthetic breakthroughs given that some of the compounds can be exclusive or dominant components of the floral scent and production is often tissue-specific. In this perspective article, we first highlight the chemical diversity underpinning some of the more widespread deceptive orchid pollination strategies. Next, we explore the potential metabolic pathways and biosynthetic steps that might be involved. Finally, we offer recommendations to accelerate the discovery of the biochemical pathways in these challenging but intriguing systems.