Caladenia drummondii is pollinated by males of a single species of pompilid wasp, making this the... more Caladenia drummondii is pollinated by males of a single species of pompilid wasp, making this the first well-documented case of a pollination system specialised on pompilids outside of Africa.
1. DNA sequences in Fasta format for each of sequence Sets 1 to 5, as provided to Roche NimbleGen... more 1. DNA sequences in Fasta format for each of sequence Sets 1 to 5, as provided to Roche NimbleGen for the probe design.
Caladenia is a diverse Australian genus that is exceptional among orchids in having both specie... more 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 for using GC-MS, and reproductive success was quantified for two 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, though the sexually deceptive species had a dull-red labellum. Reproductive success was generally low, and highly variable between sites and years.
For organisms with limited vagility and/or occupying patchy habitats, we often encounter nonrando... more For organisms with limited vagility and/or occupying patchy habitats, we often encounter nonrandom patterns of genetic affinity over relatively small spatial scales, labelled fine-scale genetic structure. Both the extent and decay rate of that pattern can be expected to depend on numerous interesting demographic, ecological, historical, and mating system factors, and it would be useful to be able to compare different situations. There is, however, no heterogeneity test currently available for fine-scale genetic structure that would provide us with any guidance on whether the differences we encounter are statistically credible. Here, we develop a general nonparametric heterogeneity test, elaborating on standard autocorrelation methods for pairs of individuals. We first develop a 'pooled within-population' correlogram, where the distance classes (lags) can be defined as functions of distance. Using that pooled correlogram as our null-hypothesis reference frame, we then develop a heterogeneity test of the autocorrelations among different populations, lag-by-lag. From these single-lag tests, we construct an analogous test of heterogeneity for multilag correlograms. We illustrate with a pair of biological examples, one involving the Australian bush rat, the other involving toadshade trillium. The Australian bush rat has limited vagility, and sometimes occupies patchy habitat. We show that the autocorrelation pattern diverges somewhat between continuous and patchy habitat types. For toadshade trillium, clonal replication in Piedmont populations substantially increases autocorrelation for short lags, but clonal replication is less pronounced in mountain populations. Removal of clonal replicates reduces the autocorrelation for short lags and reverses the sign of the difference between mountain and Piedmont correlograms.
Sexually deceptive orchids attract specific pollinators by mimicking insect sex pheromones. Norma... more 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 structureactivity 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)-βcitronellol 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-6methylacetophenone, has the strongest intramolecular hydrogen bond of all regioisomers, which at least in part may explain the specific activity.
† Background The remarkable diversity of mating patterns and sexual systems in flowering plants h... more † Background The remarkable diversity of mating patterns and sexual systems in flowering plants has fascinated evolutionary biologists for more than a century. Enduring questions about this topic include why sexual polymorphisms have evolved independently in over 100 plant families, and why proportions of self-and crossfertilization often vary dramatically within and among populations. Important new insights concerning the evolutionary dynamics of plant mating systems have built upon a strong foundation of theoretical models and innovative field and laboratory experiments. However, as the pace of advancement in this field has accelerated, it has become increasingly difficult for researchers to follow developments outside their primary area of research expertise. † Scope In this Viewpoint paper we highlight three important themes that span and integrate different subdisciplines: the changes in morphology, phenology, and physiology that accompany the transition to selfing; the evolutionary consequences of pollen pool diversity in flowering plants; and the evolutionary dynamics of sexual polymorphisms. We also highlight recent developments in molecular techniques that will facilitate more efficient and cost-effective study of mating patterns in large natural populations, research on the dynamics of pollen transport, and investigations on the genetic basis of sexual polymorphisms. This Viewpoint also serves as the introduction to a Special Issue on the Evolution of Plant Mating Systems. The 15 papers in this special issue provide inspiring examples of recent discoveries, and glimpses of exciting developments yet to come.
Sexually deceptive plants achieve pollination by enticing specific male insects as pollinators us... more 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.
Orchids are renowned not only for their diversity of floral forms, but also for their many and of... more Orchids 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.
The Orchidaceae is rivaled only by the Asteraceae as the largest plant family, with the estimated... more 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...
With over 25,000 species, the drivers of diversity in the Orchidaceae remain to be fully understo... more 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 numbe...
The Pompilidae is a cosmopolitan and diverse group of wasps, which commonly feed on nectar. Howev... more The Pompilidae is a cosmopolitan and diverse group of wasps, which commonly feed on nectar. However, pollination systems specialized 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 reve...
Bioactive natural products underpin the intriguing pollination strategy used by sexually deceptiv... more 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.
Hundreds of orchid species secure pollination by sexually luring specific male insects as pollina... more Hundreds of orchid species secure pollination by sexually luring specific male insects as pollinators by chemical and morphological mimicry. Yet, the biochemical pathways involved in the synthesis of the insect sex pheromone-mimicking volatiles in these sexually deceptive plants remain poorly understood. Here, we explore the biochemical pathways linked to the chemical mimicry of female sex pheromones (chiloglottones) employed by the Australian sexually deceptive Chiloglottis orchids to lure their male pollinator. By strategically exploiting the transcriptomes of chiloglottone 1-producing Chiloglottis trapeziformis at distinct floral tissues and at key floral developmental stages, we identified two key transcriptional trends linked to the stage-and tissue-dependent distribution profiles of chiloglottone in the flower: (i) developmental upregulation of fatty acid biosynthesis and β-oxidation genes such as KETOACYL-ACP SYNTHASE, FATTY ACYL-ACP THIOESTERASE, and ACYL-COA OXIDASE during the transition from young to mature buds and flowers and (ii) the tissue-specific induction of fatty acid pathway genes in the callus (the insectiform odor-producing structure on the labellum of the flower) compared to the labellum remains (non-odor-producing) regardless of development stage of the flower. Enzyme inhibition experiments targeting KETOACYL-ACP SYNTHASE activity alone in three chiloglottone-producing species (C. trapeziformis, C. valida, and C. aff. valida) significantly inhibited chiloglottone biosynthesis up to 88.4% compared to the controls. These findings highlight the role of coordinated (developmental stage-and tissue-dependent) fatty acid gene expression and enzyme activities for chiloglottone production in Chiloglottis orchids.
Selection exerted by pollinators on flowers is predicted to occur along two distinct axes. While ... more 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. 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.
Mobile species pose major challenges for conservation because of their unpredictable, large scale... more 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.
Context Dispersal is essential for species persistence and landscape genetic studies are valuable... more Context 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. Objectives 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. Methods We studied scarlet macaws (Ara macao) over a 13,000 km 2 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). Results 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. Conclusions 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 Electronic supplementary material The online version of this article (
The majority of flowering plants rely on animals as pollen vectors. Thus, plant mating systems an... more The majority of flowering plants rely on animals as pollen vectors. Thus, plant mating systems and pollen dispersal are strongly influenced by pollinator behaviour. In Australian sexually deceptive orchids pollinated by male thynnine wasps, outcrossing and extensive pollen flow is predicted due to floral deception, which minimizes multiple flower visitations within patches, and the movement of pollinators under mate‐search rather than foraging behaviours. This hypothesis was tested using microsatellite markers to reconstruct and infer paternity in two clonal, self‐compatible orchids. Offspring from naturally pollinated Chiloglottis valida and C. aff. jeanesii were acquired through symbiotic culture of seeds collected over three seasons. In both species, outcrossing was extensive (tm = 0.924–1.00) despite clone sizes up to 11 m wide. The median pollen flow distance based on paternity for both taxa combined was 14.5 m (n = 18, range 0–69 m), being larger than typically found by patern...
Caladenia drummondii is pollinated by males of a single species of pompilid wasp, making this the... more Caladenia drummondii is pollinated by males of a single species of pompilid wasp, making this the first well-documented case of a pollination system specialised on pompilids outside of Africa.
1. DNA sequences in Fasta format for each of sequence Sets 1 to 5, as provided to Roche NimbleGen... more 1. DNA sequences in Fasta format for each of sequence Sets 1 to 5, as provided to Roche NimbleGen for the probe design.
Caladenia is a diverse Australian genus that is exceptional among orchids in having both specie... more 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 for using GC-MS, and reproductive success was quantified for two 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, though the sexually deceptive species had a dull-red labellum. Reproductive success was generally low, and highly variable between sites and years.
For organisms with limited vagility and/or occupying patchy habitats, we often encounter nonrando... more For organisms with limited vagility and/or occupying patchy habitats, we often encounter nonrandom patterns of genetic affinity over relatively small spatial scales, labelled fine-scale genetic structure. Both the extent and decay rate of that pattern can be expected to depend on numerous interesting demographic, ecological, historical, and mating system factors, and it would be useful to be able to compare different situations. There is, however, no heterogeneity test currently available for fine-scale genetic structure that would provide us with any guidance on whether the differences we encounter are statistically credible. Here, we develop a general nonparametric heterogeneity test, elaborating on standard autocorrelation methods for pairs of individuals. We first develop a 'pooled within-population' correlogram, where the distance classes (lags) can be defined as functions of distance. Using that pooled correlogram as our null-hypothesis reference frame, we then develop a heterogeneity test of the autocorrelations among different populations, lag-by-lag. From these single-lag tests, we construct an analogous test of heterogeneity for multilag correlograms. We illustrate with a pair of biological examples, one involving the Australian bush rat, the other involving toadshade trillium. The Australian bush rat has limited vagility, and sometimes occupies patchy habitat. We show that the autocorrelation pattern diverges somewhat between continuous and patchy habitat types. For toadshade trillium, clonal replication in Piedmont populations substantially increases autocorrelation for short lags, but clonal replication is less pronounced in mountain populations. Removal of clonal replicates reduces the autocorrelation for short lags and reverses the sign of the difference between mountain and Piedmont correlograms.
Sexually deceptive orchids attract specific pollinators by mimicking insect sex pheromones. Norma... more 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 structureactivity 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)-βcitronellol 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-6methylacetophenone, has the strongest intramolecular hydrogen bond of all regioisomers, which at least in part may explain the specific activity.
† Background The remarkable diversity of mating patterns and sexual systems in flowering plants h... more † Background The remarkable diversity of mating patterns and sexual systems in flowering plants has fascinated evolutionary biologists for more than a century. Enduring questions about this topic include why sexual polymorphisms have evolved independently in over 100 plant families, and why proportions of self-and crossfertilization often vary dramatically within and among populations. Important new insights concerning the evolutionary dynamics of plant mating systems have built upon a strong foundation of theoretical models and innovative field and laboratory experiments. However, as the pace of advancement in this field has accelerated, it has become increasingly difficult for researchers to follow developments outside their primary area of research expertise. † Scope In this Viewpoint paper we highlight three important themes that span and integrate different subdisciplines: the changes in morphology, phenology, and physiology that accompany the transition to selfing; the evolutionary consequences of pollen pool diversity in flowering plants; and the evolutionary dynamics of sexual polymorphisms. We also highlight recent developments in molecular techniques that will facilitate more efficient and cost-effective study of mating patterns in large natural populations, research on the dynamics of pollen transport, and investigations on the genetic basis of sexual polymorphisms. This Viewpoint also serves as the introduction to a Special Issue on the Evolution of Plant Mating Systems. The 15 papers in this special issue provide inspiring examples of recent discoveries, and glimpses of exciting developments yet to come.
Sexually deceptive plants achieve pollination by enticing specific male insects as pollinators us... more 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.
Orchids are renowned not only for their diversity of floral forms, but also for their many and of... more Orchids 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.
The Orchidaceae is rivaled only by the Asteraceae as the largest plant family, with the estimated... more 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...
With over 25,000 species, the drivers of diversity in the Orchidaceae remain to be fully understo... more 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 numbe...
The Pompilidae is a cosmopolitan and diverse group of wasps, which commonly feed on nectar. Howev... more The Pompilidae is a cosmopolitan and diverse group of wasps, which commonly feed on nectar. However, pollination systems specialized 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 reve...
Bioactive natural products underpin the intriguing pollination strategy used by sexually deceptiv... more 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.
Hundreds of orchid species secure pollination by sexually luring specific male insects as pollina... more Hundreds of orchid species secure pollination by sexually luring specific male insects as pollinators by chemical and morphological mimicry. Yet, the biochemical pathways involved in the synthesis of the insect sex pheromone-mimicking volatiles in these sexually deceptive plants remain poorly understood. Here, we explore the biochemical pathways linked to the chemical mimicry of female sex pheromones (chiloglottones) employed by the Australian sexually deceptive Chiloglottis orchids to lure their male pollinator. By strategically exploiting the transcriptomes of chiloglottone 1-producing Chiloglottis trapeziformis at distinct floral tissues and at key floral developmental stages, we identified two key transcriptional trends linked to the stage-and tissue-dependent distribution profiles of chiloglottone in the flower: (i) developmental upregulation of fatty acid biosynthesis and β-oxidation genes such as KETOACYL-ACP SYNTHASE, FATTY ACYL-ACP THIOESTERASE, and ACYL-COA OXIDASE during the transition from young to mature buds and flowers and (ii) the tissue-specific induction of fatty acid pathway genes in the callus (the insectiform odor-producing structure on the labellum of the flower) compared to the labellum remains (non-odor-producing) regardless of development stage of the flower. Enzyme inhibition experiments targeting KETOACYL-ACP SYNTHASE activity alone in three chiloglottone-producing species (C. trapeziformis, C. valida, and C. aff. valida) significantly inhibited chiloglottone biosynthesis up to 88.4% compared to the controls. These findings highlight the role of coordinated (developmental stage-and tissue-dependent) fatty acid gene expression and enzyme activities for chiloglottone production in Chiloglottis orchids.
Selection exerted by pollinators on flowers is predicted to occur along two distinct axes. While ... more 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. 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.
Mobile species pose major challenges for conservation because of their unpredictable, large scale... more 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.
Context Dispersal is essential for species persistence and landscape genetic studies are valuable... more Context 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. Objectives 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. Methods We studied scarlet macaws (Ara macao) over a 13,000 km 2 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). Results 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. Conclusions 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 Electronic supplementary material The online version of this article (
The majority of flowering plants rely on animals as pollen vectors. Thus, plant mating systems an... more The majority of flowering plants rely on animals as pollen vectors. Thus, plant mating systems and pollen dispersal are strongly influenced by pollinator behaviour. In Australian sexually deceptive orchids pollinated by male thynnine wasps, outcrossing and extensive pollen flow is predicted due to floral deception, which minimizes multiple flower visitations within patches, and the movement of pollinators under mate‐search rather than foraging behaviours. This hypothesis was tested using microsatellite markers to reconstruct and infer paternity in two clonal, self‐compatible orchids. Offspring from naturally pollinated Chiloglottis valida and C. aff. jeanesii were acquired through symbiotic culture of seeds collected over three seasons. In both species, outcrossing was extensive (tm = 0.924–1.00) despite clone sizes up to 11 m wide. The median pollen flow distance based on paternity for both taxa combined was 14.5 m (n = 18, range 0–69 m), being larger than typically found by patern...
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