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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.

The use of diversity metrics has a long history in population ecology, while population genetic work has been dominated by variance-derived metrics instead, a technical gap that has slowed cross-communication between the fields. Interestingly, Rao's Quadratic Entropy (RQE), comparing elements for 'degrees of divergence', was originally developed for population ecology, but has recently been deployed for evolutionary studies. We here translate RQE into a continuous diversity analogue, and then construct a multiply nested diversity partition for alleles, individuals, populations, and species, each component of which exhibits the behavior of proper diversity metrics, and then translate these components into [0,1]-scaled form. We also deploy non-parametric statistical tests of the among-stratum components and novel tests of the homogeneity of within-stratum diversity components at any hierarchical level. We then illustrate this new analysis with eight nSSR loci and a pair of close Australian marsupial (Antechinus) congeners, using both 'different is different' and 'degree of difference' distance metrics. The total diversity in the collection is larger than that within either species, but most of the within-species diversity is resident within single populations. The combined A. agilis collection exhibits more diversity than does the combined A. stuartii collection, possibly attributable to localized differences in either local ecological disturbance regimes or differential levels of population isolation. Beyond exhibiting different allelic compositions, the two congeners are becoming more divergent for the arrays of allele sizes they possess.

Sexually deceptive plants achieve pollination by enticing specific male insects as pollinators using a combination of olfactory, visual, and morphological mimicry. The sexually deceptive orchid genus Chiloglottis is comprised of some 30 species with predominantly dull green-red flowers except for the dark insectiform calli/callus structure from the labellum lamina. This unique structure mimics the female of the pollinator and potentially enhances the visibility of the mimic. However, the chemical and genetic basis for the color of these structures remains poorly understood across the genus. The goal of this study was to investigate the flower color biochemistry and patterns of gene expression across the anthocyanin and flavonol glycoside biosynthetic pathway within the calli structures across the three distinct clades of Chiloglottis (Formicifera, Reflexa, and Valida) using chemical and transcriptome analysis. Our phylogenomic analysis confirmed the close sister relationship between the Reflexa/Formicifera clades and reaffirms the basal position of the Valida clade. Additionally, the biochemical basis of the dark calli/callus structures is conserved across the genus. Nonetheless, the proportion of methoxylated anthocyanin and flavonol glycoside derivatives and the mean gene expression levels appear to differentiate the Reflexa and Formicifera clades from the Valida clade. In future studies, it will be of interest to tease apart the role of phylogeny, environment, pollinators, and other factors as potential drivers of the observed biochemistry and gene expression differences. It will also be important to characterize the function of candidate genes such as DFR , LDOX, and FLS in this fascinating case of flower color mimicry.

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.

Sexually deceptive orchids employ floral volatiles to sexually lure their specific pollinators. How and why this pollination system has evolved independently on multiple continents remains unknown, although preadaptation is considered to have been important. Understanding the chemistry of sexual deception is a crucial first step towards solving this mystery. The combination of gas chromatography‐electroantennographic detection (GC‐EAD), GC‐MS, synthesis and field bioassays allowed us to identify the volatiles involved in the interaction between the orchid Drakaea glyptodon and its sexually attracted male thynnine wasp pollinator, Zaspilothynnus trilobatus. Three alkylpyrazines and one novel hydroxymethyl pyrazine were identified as the sex pheromone of Z. trilobatus and are also used by D. glyptodon for pollinator attraction. Given that our findings revealed a new chemical system for plants, we surveyed widely across representative orchid taxa for the presence of these compounds. With one exception, our chemical survey failed to detect pyrazines in related genera. Collectively, no evidence for preadaptation was found. The chemistry of sexual deception is more diverse than previously known. Our results suggest that evolutionary novelty may have played a key role in the evolution of sexual deception and highlight the value of investigating unusual pollination systems for advancing our understanding of the role of chemistry in evolution.

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.

Background and Aims Pterostylisis an Australasian terrestrial orchid genus of more than 400 species, most of which use a motile, touch-sensitive labellum to trap dipteran pollinators. Despite studies dating back to 1872, the mechanism of pollinator attraction has remained elusive. This study tested whether the fungus gnat-pollinated Pterostylis sanguinea secures pollination by sexual deception.MethodsThe literature was used to establish criteria for confirming sexual deception as a pollination strategy. Observations and video recordings allowed quantification of each step of the pollination process. Each floral visitor was sexed and DNA barcoding was used to evaluate the degree of pollinator specificity. Following observations that attraction to the flowers is by chemical cues, experimental dissection of flowers was used to determine the source of the sexual attractant and the effect of labellum orientation on sexual attraction. Fruit set was quantified for 19 populations to test for a relationship with plant density and population size.Key ResultsA single species of male gnat (Mycetophilidae) visited and pollinated the rewardless flowers. The gnats often showed probing copulatory behaviour on the labellum, leading to its triggering and the temporary entrapment of the gnat in the flower. Pollen deposition and removal occurred as the gnat escaped from the flower via the reproductive structures. The labellum was the sole source of the chemical attractant. Gnats always alighted on the labellum facing upwards, but when it was rotated 180 ° they attempted copulation less frequently. Pollination rate showed no relationship with orchid population size or plant density.ConclusionsThis study confirms for the first time that highly specific pollination by fungus gnats is achieved by sexual deception in Pterostylis. It is predicted that sexual deception will be widespread in the genus, although the diversity of floral forms suggests that other mechanisms may also operate.

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.

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.

The \"sexually deceptive\" orchid Chiloglottis trapeziformis attracts males of its pollinator species, the thynnine wasp Neozeleboria cryptoides, by emitting a unique volatile compound, 2-ethyl-5-propylcyclohexan-1,3-dione, which is also produced by female wasps as a male-attracting sex pheromone.