High-quality nuclear genome resources remain scarce for most orchids, particularly Australia's diverse terrestrial lineages. Chiloglottis trapeziformis is a well-studied sexually deceptive orchid that provides a valuable system for investigating orchid genome evolution, structural variation, and the molecular basis of specialized pollination. Here, we integrated PacBio HiFi, Oxford Nanopore ultra-long reads, and Hi-C chromatin-contact data to generate the first chromosome-scale, haplotype-resolved nuclear genome assembly for any Australian terrestrial orchid. Hi-C guided scaffolding resolved two haplotypes into 20 chromosomes each, consistent with the reported karyotype and genome size (2n = 40, haplotype sizes of 1.58 and 1.91 Gb). Genome completeness was high for both haplotypes, recovering 94.67% and 94.82% single-copy BUSCO genes for Haplotype 1 and Haplotype 2, respectively. De novo repeat annotation revealed a repeat-rich genome (85.79% to 88.25% repetitive sequence), dominated by LTR retrotransposons. Evidence-guided annotation identified 16,287 and 16,548 protein-coding genes in Haplotype 1 and Haplotype 2, respectively. Phylogenetically informed comparisons placed C. trapeziformis as sister to Anoectochilus roxburghii among sampled Orchidoideae and showed broad gene-order conservation. Comparing the two haplotypes for structural variation, we identified large interhaplotype inversions containing functionally annotated genes with detectable RNA expression, with focal examples further supported by local Hi-C contact patterns and breakpoint-level inspection. Inversion-overlapping genes did not show elevated dS relative to collinear background. This assembly and annotation resource provides a foundation for population and conservation genomics, structural and comparative analyses, and genome-enabled hypothesis testing of molecular traits underlying sexual deception in orchids.

A new study shows that a single unusual apocarotenoid floral volatile compound, (+) dihydroedulan I, attracts an obligate gall midge pollinator within a complex brood-site pollination mutualism.

Abstract Chiloglottis trapeziformis is a sexually deceptive Australian orchid that provides a valuable system for studying orchid genome evolution, structural variation, and the molecular basis of specialized pollination. However, high-quality nuclear genome resources remain scarce for most orchids, particularly Australia’s diverse terrestrial lineages. To address this gap, we integrated PacBio HiFi, Oxford Nanopore ultra-long reads, and Hi-C chromatin-contact data to generate the first chromosome-scale, haplotype-resolved nuclear genome assembly for an Australian terrestrial orchid, Chiloglottis trapeziformis . Hi-C guided scaffolding resolved two haplotypes into 20 chromosomes each, consistent with the reported karyotype and genome size (2n=40, haplotype sizes of 1.58 Gb and 1.91 Gb). Genome completeness was high for both haplotypes, recovering 95.1% and 95.5% complete BUSCO genes for haplotype 1 and haplotype 2, respectively. De novo repeat annotation revealed a repeat-rich genome (85.79– 88.25% repetitive sequence), dominated by LTR retrotransposons. Evidence-guided annotation identified 16,287 and 16,548 protein-coding genes in Haplotype 1 and Haplotype 2, respectively. Phylogenetically informed comparisons placed C. trapeziformis as sister to Anoectochilus roxburghii among sampled Orchidoideae and showed broad gene-order conservation. Comparing the two haplotypes for structural variation, we identified large inter-haplotype inversions containing functionally annotated genes with detectable RNA expression, with focal examples further supported by local Hi-C contact patterns and breakpoint-level inspection. Inversion-overlapping genes did not show elevated dS relative to collinear background. This assembly and annotation resource provides a foundation for population and conservation genomics, structural and comparative analyses, and genome-enabled hypothesis testing of molecular traits underlying sexual deception in orchids. Significance statement Sexually deceptive orchids use remarkable chemical, visual, and tactile mimicry to attract specific pollinators, but the genome resources needed to understand how these complex traits evolved remain scarce. By generating a chromosome-scale, haplotype-resolved genome for Chiloglottis trapeziformis , we provide the first genomic framework for the large and unique Australasian tribe Diurideae. We show extensive structural variation between haplotypes in an otherwise highly collinear genome, with several large inversions potentially impacting expressed genes. The absence of elevated coding divergence in these regions highlights structural variation as a potentially underappreciated driver of orchid genome evolution. This resource fills a major gap for Australian orchids and provides a foundation for linking genome structure with orchid diversification, conservation, and the evolution of sexual deception.

Orchid mycorrhizal fungi (OMF) associations in the Orchidaceae are thought to have been a major driver of diversification in the family. In the terrestrial orchid tribe Diurideae, it has long been hypothesized that OMF symbiont associations may reflect evolutionary relationships among orchid hosts. Given that recent phylogenomic efforts have been unable to fully resolve relationships among subtribes in the Diurideae, we sought to ascertain whether orchid OMF preferences may lend support to certain phylogenetic hypotheses. First, we used phylogenomic methods and Bayesian divergence time estimation to produce a genus-level tree for the Diurideae. Next, we synthesized decades of published fungal sequences and morphological/germination data to identify dominant fungal partners at the genus scale and perform ancestral state reconstruction to estimate the evolutionary trajectory of fungal symbiont shifts. Across the tribe, we found phylogenomic discordance stemming from incomplete lineage sorting. However, our results also revealed unprecedented phylogenetic niche conservatism of fungal symbionts within the tribe: entire genera, subtribes, and even groups of related subtribes associate with only a single fungal family, suggesting that fungal symbiont preferences in the Diurideae do indeed reflect phylogenetic relationships among orchid hosts. Moreover, we show that these relationships have evolved directionally from generalist associations with multiple fungal families towards more specific partnerships with only one fungal family. Orchid symbiont preferences here provide new insights into the placement of several groups with longstanding phylogenetic uncertainty. In spite of complex evolutionary histories, host-symbiont relationships can be used to help detangle alternative phylogenetic hypotheses.

Pollination by sexual deception has evolved multiple times in the Orchidaceae, with most known cases involving male Hymenoptera as pollinators. The diverse Australasian orchid genus Pterostylis, characterized by elaborate trap flowers, contains some species pollinated by sexual deception of fungus gnats (Diptera). However, there is considerable variation in floral morphology, suggesting that additional pollination strategies or pollen vectors may be involved. Here, we test the hypothesis that sexual deception of male Diptera is taxonomically widespread by investigating the pollination systems across a representative subset spanning nine out of 10 sections and 18 Pterostylis species. We confirm four new cases of pollination by sexual deception of male fungus gnats (families Mycetophilidae, Keroplatidae, and Sciaridae) and accrued evidence for three further cases. Each of these orchids was pollinated by a single species of fungus gnat, with two species exploiting the same pollinator. Unexpectedly, we observed insect feeding behaviour on two species pollinated by sciarid gnats and phorid flies, respectively, with trace levels of sucrose detected where feeding was observed. Our results show that the sexual deception of male fungus gnats is likely to be the dominant mode of pollination in Pterostylis, although other poorly understood pollination strategies are also present.

Chemically mediated floral volatile signals are crucial for pollinator attraction across angiosperms. However, beyond model plant systems, the molecular mechanisms underpinning their tissue-specific biosynthesis, regulation, and emission are still poorly understood. In this study of a food-deceptive insect-pollinated orchid (Caladenia denticulata), we elucidated the molecular basis of α-pinene biosynthesis-the major floral volatile emitted by this species and diverse lower abundance monoterpenes and sesquiterpenes. To achieve this, we combined comparative transcriptomics between active glandular trichome-rich sepal tips and labellum and non-active remaining flower tissues, floral volatile headspace profiling, phylogenetic analysis of a multigene family, and protein functional assays. We found (i) multiple branch points of the terpene synthase (TPS) biosynthetic pathway were highly expressed and coordinately upregulated in the active floral tissues compared to non-active ones, (ii) the monoterpene synthase CdTPS-b3 underpinning α-pinene biosynthesis and a bona fide promiscuous TPS CdTPS-b4 that may contribute to the diverse array of low-abundance mono- and sesquiterpenes found in its flowers, and (iii) dual localization (plastid and cytosol) of CdTPS-b3 and CdTPS-b4. Our findings highlight metabolic pathway specialization at multiple TPS pathway branch points supporting the biosynthesis and emission of α-pinene in C. denticulata flowers that are implicated in its generalist pollinator attraction. Furthermore, the complexity of diverse floral terpenes in Caladenia is likely mediated by finely tuned TPS gene expression, functional promiscuity, and subcellular localization. We predict that the combination of these three mechanisms underpin the evolution of multiple deceptive pollination strategies in Caladenia.

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.

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.

In understanding the impact of commercial whaling, it is important to estimate the mixing of low latitude breeding populations on Antarctic feeding grounds, particularly the endangered humpback whale populations of Oceania. This paper estimates the degree of genetic differentiation among the putative populations of Oceania (New Caledonia, Tonga, the Cook Islands and French Polynesia) and Australia (western Australia and eastern Australia) using ten microsatellite loci and mtDNA, assesses the power of the data for a mixed-stock analysis, determines ways to improve statistical power for future studies and estimates the population composition of Antarctic samples collected in 2010 south of New Zealand and eastern Australia. A large proportion of individuals could not be assigned to a population of origin (> 52%) using a posterior probability threshold of > 0.90. The mixed-stock analysis simulations however, produced accurate results with humpback whales reapportioned to their population of origin above the 90% threshold for western Australia, New Caledonia and Oceania grouped using a combined mtDNA and microsatellite dataset. Removing the Cook Islands, considered a transient region for humpback whales, from the simulation analysis increased the ability to reapportion Tonga from 86% to 89% and French Polynesia from 89% to 92%. Breeding ground sample size was found to be a factor influencing the accuracy of population reapportionment whereas increasing the mixture or feeding ground sample size improved the precision of results. The mixed-stock analysis of our Antarctic samples revealed substantial contributions from both eastern Australia (53.2%, 6.8% SE) and New Caledonia (43.7%, 5.5% SE) [with Oceania contributing 46.8% (5.9% SE)] but not western Australia. Despite the need for more samples to improve estimates of population allocation, our study strengthens the emerging genetic and non-genetic evidence that Antarctic waters south of New Zealand and eastern Australia are used by humpback whales from both eastern Australia and the more vulnerable breeding population of New Caledonia, representing Oceania.

The flower is arguably the centrepiece of angiosperm evolution. Its primary function is to secure pollination - the transfer of pollen from the anther (male) to the stigma (female). As plants are sessile organisms, the extraordinary diversity of flowers in large part reflects countless alternative evolutionary solutions to achieve this critical step in the flowering plant life cycle. The majority of flowering plants, some 87% by one estimate, depend on animals for pollination, with most of these paying for the service of pollination via food rewards of nectar or pollen. As in human economic systems, however, some cheating and deception occurs, with the pollination strategy of sexual deception being one such example.

Flowering plants have evolved extraordinarily diverse metabolites that underpin the floral visual and olfactory signals enabling plant-pollinator interactions. In some cases, these metabolites also provide unusual rewards that specific pollinators depend on. While some metabolites are shared by most flowering plants, many have evolved in restricted lineages in response to the specific selection pressures encountered within different niches. The latter are designated as specialized metabolites. Recent investigations continue to uncover a growing repertoire of unusual specialized metabolites. Increased accessibility to cutting-edge multi-omics technologies (e.g. genome, transcriptome, proteome, metabolome) is now opening new doors to simultaneously uncover the molecular basis of their synthesis and their evolution across diverse plant lineages. Drawing upon the recent literature, this perspective discusses these aspects and, where known, their ecological and evolutionary relevance. A primer on omics-guided approaches to discover the genetic and biochemical basis of functional specialized metabolites is also provided.

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.

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.

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.

Sexually deceptive plants secure pollination by luring specific male insects as pollinators using a combination of olfactory, visual, and morphological mimicry. Flower color is a key component to this attraction, but its chemical and genetic basis remains poorly understood. Chiloglottis trapeziformis is a sexually deceptive orchid which has predominantly dull green-red flowers except for the central black callus projecting from the labellum lamina. The callus mimics the female of the pollinator and the stark color contrast between the black callus and dull green or red lamina is thought to enhance the visibility of the mimic. The goal of this study was to investigate the chemical composition and genetic regulation of temporal and spatial color patterns leading to visual mimicry, by integrating targeted metabolite profiling and transcriptomic analysis. Even at the very young bud stage, high levels of anthocyanins were detected in the dark callus, with peak accumulation by the mature bud stage. In contrast, anthocyanin levels in the lamina peaked as the buds opened and became reddish-green. Coordinated upregulation of multiple genes, including dihydroflavonol reductase and leucoanthocyanidin dioxygenase, and the downregulation of flavonol synthase genes (FLS) in the callus at the very young bud stage underpins the initial high anthocyanin levels. Conversely, within the lamina, upregulated FLS genes promote flavonol glycoside over anthocyanin production, with the downstream upregulation of flavonoid O-methyltransferase genes further contributing to the accumulation of methylated flavonol glycosides, whose levels peaked in the mature bud stage. Finally, the peak anthocyanin content of the reddish-green lamina of the open flower is underpinned by small increases in gene expression levels and/or differential upregulation in the lamina in select anthocyanin genes while FLS patterns showed little change. Differential expression of candidate genes involved in specific transport, vacuolar acidification, and photosynthetic pathways may also assist in maintaining the distinct callus and contrasting lamina color from the earliest bud stage through to the mature flower. Our findings highlight that flower color in this sexually deceptive orchid is achieved by complex tissue-specific coordinated regulation of genes and biochemical pathways across multiple developmental stages.

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

Biology and economics are surprisingly similar disciplines. At their core, both fields are the study of competitive interactions for scarce resources and the consequences of those interactions over time. Perhaps the first person to notice this similarity was Charles Darwin, who credited his reading of the influential economist Thomas Robert Malthus with catalysing his understanding of natural selection as the driving force of evolution. While it may not have been recognised at the time, this was not the only area of Darwin's thinking to parallel economic concepts.

The Orchidaceae is rivaled only by the Asteraceae as the largest plant family, with the estimated number of species exceeding 25,000 and encompassing more than 700 genera. To gain insights into the mechanisms driving species diversity across both global and local scales, well-supported phylogenies targeting different taxonomic groups and/or geographical regions will be crucial. High-throughput sequencing technologies have revolutionized the field of molecular phylogenetics by simplifying the process of obtaining genome-scale sequence data. Consequently, there has been an explosive growth of such data in public repositories. Here we took advantage of this unprecedented access to transcriptome data from predominantly non-phylogenetic studies to assess if it can be repurposed to gain rapid and accurate phylogenetic insights across the orchids. Exhaustive searches revealed transcriptomic data for more than 100 orchid species spanning 5 subfamilies, 13 tribes, 21 subtribes, and 50 genera that were amendable for exploratory phylotranscriptomic analysis. Next, we performed re-assembly of the transcriptomes before strategic selection of the final samples based on a gene completeness evaluation. Drawing on these data, we report phylogenetic analyses at both deep and shallow evolutionary scales via maximum likelihood and shortcut coalescent species tree methods. In this perspective, we discuss some key outcomes of this study and conclude by highlighting other complementary, albeit rarely explored, insights beyond phylogenetic analysis that repurposed multi-tissue transcriptome can offer.

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.