The origin and speciation of orchids.

Orchids constitute one of the most spectacular radiations of flowering plants. However, their origin, spread across the globe, and hotspots of speciation remain uncertain due to the lack of an up-to-date phylogeographic analysis. We present a new Orchidaceae phylogeny based on combined high-throughput and Sanger sequencing data, covering all five subfamilies, 17/22 tribes, 40/49 subtribes, 285/736 genera, and c. 7% (1921) of the 29 524 accepted species, and use it to infer geographic range evolution, diversity, and speciation patterns by adding curated geographical distributions from the World Checklist of Vascular Plants. The orchids' most recent common ancestor is inferred to have lived in Late Cretaceous Laurasia. The modern range of Apostasioideae, which comprises two genera with 16 species from India to northern Australia, is interpreted as relictual, similar to that of numerous other groups that went extinct at higher latitudes following the global climate cooling during the Oligocene. Despite their ancient origin, modern orchid species diversity mainly originated over the last 5 Ma, with the highest speciation rates in Panama and Costa Rica. These results alter our understanding of the geographic origin of orchids, previously proposed as Australian, and pinpoint Central America as a region of recent, explosive speciation.

Genomic, spatial and morphometric data for discrimination of four species in the Mediterranean Tamus clade of yams (Dioscorea, Dioscoreaceae).

BACKGROUND AND AIMS: Among the numerous pantropical species of the yam genus, Dioscorea, only a small group occurs in the Mediterranean basin, including two narrow Pyrenean endemics (Borderea clade) and two Mediterranean-wide species (D. communis and D. orientalis, Tamus clade). However, several currently unrecognized species and infraspecific taxa have been described in the Tamus clade due to significant morphological variation associated with D. communis. Our overarching aim was to investigate taxon delimitation in the Tamus clade using an integrative approach combining phylogenomic, spatial and morphological data. METHODS: We analysed 76 herbarium samples using Hyb-Seq genomic capture to sequence 260 low-copy nuclear genes and plastomes, together with morphometric and environmental modelling approaches. KEY RESULTS: Phylogenomic reconstructions confirmed that the two previously accepted species of the Tamus clade, D. communis and D. orientalis, are monophyletic and form sister clades. Three subclades showing distinctive geographic patterns were identified within D. communis. These subclades were also identifiable from morphometric and climatic data, and introgression patterns were inferred between subclades in the eastern part of the distribution of D. communis. CONCLUSIONS: We propose a taxonomy that maintains D. orientalis, endemic to the eastern Mediterranean region, and splits D. communis sensu lato into three species: D. edulis, endemic to Macaronesia (Canary Islands and Madeira); D. cretica, endemic to the eastern Mediterranean region; and D. communis sensu stricto, widespread across western and central Europe. Introgression inferred between D. communis s.s. and D. cretica is likely to be explained by their relatively recent speciation at the end of the Miocene, disjunct isolation in eastern and western Mediterranean glacial refugia and a subsequent westward recolonization of D. communis s.s. Our study shows that the use of integrated genomic, spatial and morphological approaches allows a more robust definition of species boundaries and the identification of species that previous systematic studies failed to uncover.

Molecular Clocks and Archeogenomics of a Late Period Egyptian Date Palm Leaf Reveal Introgression from Wild Relatives and Add Timestamps on the Domestication.

The date palm, Phoenix dactylifera, has been a cornerstone of Middle Eastern and North African agriculture for millennia. It was first domesticated in the Persian Gulf, and its evolution appears to have been influenced by gene flow from two wild relatives, P. theophrasti, currently restricted to Crete and Turkey, and P. sylvestris, widespread from Bangladesh to the West Himalayas. Genomes of ancient date palm seeds show that gene flow from P. theophrasti to P. dactylifera may have occurred by ∼2,200 years ago, but traces of P. sylvestris could not be detected. We here integrate archeogenomics of a ∼2,100-year-old P. dactylifera leaf from Saqqara (Egypt), molecular-clock dating, and coalescence approaches with population genomic tests, to probe the hybridization between the date palm and its two closest relatives and provide minimum and maximum timestamps for its reticulated evolution. The Saqqara date palm shares a close genetic affinity with North African date palm populations, and we find clear genomic admixture from both P. theophrasti, and P. sylvestris, indicating that both had contributed to the date palm genome by 2,100 years ago. Molecular-clocks placed the divergence of P. theophrasti from P. dactylifera/P. sylvestris and that of P. dactylifera from P. sylvestris in the Upper Miocene, but strongly supported, conflicting topologies point to older gene flow between P. theophrasti and P. dactylifera, and P. sylvestris and P. dactylifera. Our work highlights the ancient hybrid origin of the date palms, and prompts the investigation of the functional significance of genetic material introgressed from both close relatives, which in turn could prove useful for modern date palm breeding.

Phylotranscriptomics reveals the reticulate evolutionary history of a widespread diatom species complex.

In contrast to surveys based on a few genes that often provide limited taxonomic resolution, transcriptomes provide a wealth of genomic loci that can resolve relationships among taxonomically challenging lineages. Diatoms are a diverse group of aquatic microalgae that includes important bioindicator species and many such lineages. One example is Nitzschia palea, a widespread species complex with several morphologically defined taxonomic varieties, some of which are critical pollution indicators. Morphological differences among the varieties are subtle and phylogenetic studies based on a few genes fail to resolve their evolutionary relationships. We conducted morphometric and transcriptome analyses of 10 Nitzschia palea strains to resolve the relationships among strains and taxonomic varieties. Nitzschia palea was resolved into three clades, one of which corresponds to a group of strains with narrow linear-lanceolate valves. The other morphological group recovered in the shape outline analysis was not monophyletic and consisted of two clades. Gene-tree concordance analyses and phylogenetic network estimations revealed patterns of incomplete lineage sorting and gene flow between intraspecific lineages. We detected reticulated evolutionary patterns among lineages with different morphologies, resulting in a putative recent hybrid. Our study shows that phylogenomic analyses of unlinked nuclear loci, complemented with morphometrics, can resolve complex evolutionary histories of recently diverged species complexes.

Asymmetric contributions of seed and pollen to gene dispersal in the marsh orchid Dactylorhiza umbrosa in Asia Minor.

Orchids differ from other plants in their extremely small and partly air-filled seeds that can be transported long distances by wind. Seed dispersal in orchids is expected to contribute strongly to overall gene flow, and orchids generally express low levels of genetic differentiation between populations and low pollen to seed flow ratios. However, studies in orchids distributed in northern Europe have often found a poor geographic structuring of genetic variation. Here, we studied geographic differentiation in the marsh orchid Dactylorhiza umbrosa, which is widely distributed in upland regions from Asia Minor to Central Asia. These areas were less affected by Pleistocene ice ages than northern Europe and the orchid should have been able to survive the last ice age in local refugia. In the plastid genome, which is dispersed by seeds, populations at close distance were clearly divergent, but the differentiation still increased with geographic distance, and a significant phylogeographic structure had developed. In the nuclear genome, which is dispersed by both seeds and pollen, populations showed an even stronger correlation between genetic and geographic distance, but average levels of differentiation were lower than in the plastid genome, and no phylogeographic structure was evident. Combining plastid and nuclear data, we found that the ratio of pollen to seed dispersal (mp/ms) decreases with physical distance. Comparison with orchids that grow in parts of Europe that were glaciated during the last ice suggests that a balanced structure of genetic diversity develops only slowly in many terrestrial orchids, despite efficient seed dispersal.

Hundreds of nuclear and plastid loci yield novel insights into orchid relationships.

PREMISE: The inference of evolutionary relationships in the species-rich family Orchidaceae has hitherto relied heavily on plastid DNA sequences and limited taxon sampling. Previous studies have provided a robust plastid phylogenetic framework, which was used to classify orchids and investigate the drivers of orchid diversification. However, the extent to which phylogenetic inference based on the plastid genome is congruent with the nuclear genome has been only poorly assessed. METHODS: We inferred higher-level phylogenetic relationships of orchids based on likelihood and ASTRAL analyses of 294 low-copy nuclear genes sequenced using the Angiosperms353 universal probe set for 75 species (representing 69 genera, 16 tribes, 24 subtribes) and a concatenated analysis of 78 plastid genes for 264 species (117 genera, 18 tribes, 28 subtribes). We compared phylogenetic informativeness and support for the nuclear and plastid phylogenetic hypotheses. RESULTS: Phylogenetic inference using nuclear data sets provides well-supported orchid relationships that are highly congruent between analyses. Comparisons of nuclear gene trees and a plastid supermatrix tree showed that the trees are mostly congruent, but revealed instances of strongly supported phylogenetic incongruence in both shallow and deep time. The phylogenetic informativeness of individual Angiosperms353 genes is in general better than that of most plastid genes. CONCLUSIONS: Our study provides the first robust nuclear phylogenomic framework for Orchidaceae and an assessment of intragenomic nuclear discordance, plastid-nuclear tree incongruence, and phylogenetic informativeness across the family. Our results also demonstrate what has long been known but rarely thoroughly documented: nuclear and plastid phylogenetic trees can contain strongly supported discordances, and this incongruence must be reconciled prior to interpretation in evolutionary studies, such as taxonomy, biogeography, and character evolution.

Evolution of endemism on a young tropical mountain.

Tropical mountains are hot spots of biodiversity and endemism, but the evolutionary origins of their unique biotas are poorly understood. In varying degrees, local and regional extinction, long-distance colonization, and local recruitment may all contribute to the exceptional character of these communities. Also, it is debated whether mountain endemics mostly originate from local lowland taxa, or from lineages that reach the mountain by long-range dispersal from cool localities elsewhere. Here we investigate the evolutionary routes to endemism by sampling an entire tropical mountain biota on the 4,095-metre-high Mount Kinabalu in Sabah, East Malaysia. We discover that most of its unique biodiversity is younger than the mountain itself (6 million years), and comprises a mix of immigrant pre-adapted lineages and descendants from local lowland ancestors, although substantial shifts from lower to higher vegetation zones in this latter group were rare. These insights could improve forecasts of the likelihood of extinction and 'evolutionary rescue' in montane biodiversity hot spots under climate change scenarios.

Satellite DNA in Paphiopedilum subgenus Parvisepalum as revealed by high-throughput sequencing and fluorescent in situ hybridization.

BACKGROUND: Satellite DNA is a rapidly diverging, largely repetitive DNA component of many eukaryotic genomes. Here we analyse the evolutionary dynamics of a satellite DNA repeat in the genomes of a group of Asian subtropical lady slipper orchids (Paphiopedilum subgenus Parvisepalum and representative species in the other subgenera/sections across the genus). A new satellite repeat in Paphiopedilum subgenus Parvisepalum, SatA, was identified and characterized using the RepeatExplorer pipeline in HiSeq Illumina reads from P. armeniacum (2n = 26). Reconstructed monomers were used to design a satellite-specific fluorescent in situ hybridization (FISH) probe. The data were also analysed within a phylogenetic framework built using the internal transcribed spacer (ITS) sequences of 45S nuclear ribosomal DNA. RESULTS: SatA comprises c. 14.5% of the P. armeniacum genome and is specific to subgenus Parvisepalum. It is composed of four primary monomers that range from 230 to 359 bp and contains multiple inverted repeat regions with hairpin-loop motifs. A new karyotype of P. vietnamense (2n = 28) is presented and shows that the chromosome number in subgenus Parvisepalum is not conserved at 2n = 26, as previously reported. The physical locations of SatA sequences were visualised on the chromosomes of all seven Paphiopedilum species of subgenus Parvisepalum (2n = 26-28), together with the 5S and 45S rDNA loci using FISH. The SatA repeats were predominantly localisedin the centromeric, peri-centromeric and sub-telocentric chromosome regions, but the exact distribution pattern was species-specific. CONCLUSIONS: We conclude that the newly discovered, highly abundant and rapidly evolving satellite sequence SatA is specific to Paphiopedilum subgenus Parvisepalum. SatA and rDNA chromosomal distributions are characteristic of species, and comparisons between species reveal that the distribution patterns generate a strong phylogenetic signal. We also conclude that the ancestral chromosome number of subgenus Parvisepalum and indeed of all Paphiopedilum could be either 2n = 26 or 28, if P. vietnamense is sister to all species in the subgenus as suggested by the ITS data.

Anchored hybrid enrichment generated nuclear, plastid and mitochondrial markers resolve the Lepanthes horrida (Orchidaceae: Pleurothallidinae) species complex.

Phylogenetic relationships in species complexes and lineages derived from rapid diversifications are often challenging to resolve using morphology or standard DNA barcoding markers. The hyper-diverse genus Lepanthes from Neotropical cloud forest includes over 1200 species and many recent, explosive diversifications that have resulted in poorly supported nodes and morphological convergence across clades. Here, we assess the performance of 446 nuclear-plastid-mitochondrial markers derived from an anchored hybrid enrichment approach (AHE) coupled with coalescence- and species network-based inferences to resolve phylogenetic relationships and improve species recognition in the Lepanthes horrida species group. In addition to using orchid-specific probes to increase enrichment efficiency, we improved gene tree resolution by extending standard angiosperm targets into adjacent exons. We found high topological discordance among individual gene trees, suggesting that hybridization/polyploidy may have promoted speciation in the lineage via formation of new hybrid taxa. In addition, we identified ten loci with the highest phylogenetic informativeness values from these genomes. Most previous phylogenetic sampling in the Pleurothallidinae relies on two regions (ITS and matK), therefore, the evaluation of other markers such as those shown here may be useful in future phylogenetic studies in the orchid family. Coalescent-based species tree estimation methods resolved the phylogenetic relationships of the L. horrida species group. The resolution of the phylogenetic estimations was improved with the inclusion of extended anchor targets. This approach produced longer loci with higher discriminative power. These analyses also disclosed two undescribed species, L. amicitiae and L. genetoapophantica, formally described here, which are also supported by morphology. Our study demonstrates the utility of combined genomic evidence to disentangle phylogenetic relationships at very shallow levels of the tree of life, and in clades showing convergent trait evolution. With a fully resolved phylogeny, is it possible to disentangle traits evolving in parallel or convergently across these orchid lineages such as flower color and size from diagnostic traits such as the shape and orientation of the lobes of the petals and lip.

Exploring the evolutionary origin of floral organs of Erycina pusilla, an emerging orchid model system.

BACKGROUND: Thousands of flowering plant species attract pollinators without offering rewards, but the evolution of this deceit is poorly understood. Rewardless flowers of the orchid Erycina pusilla have an enlarged median sepal and incised median petal ('lip') to attract oil-collecting bees. These bees also forage on similar looking but rewarding Malpighiaceae flowers that have five unequally sized petals and gland-carrying sepals. The lip of E. pusilla has a 'callus' that, together with winged 'stelidia', mimics these glands. Different hypotheses exist about the evolutionary origin of the median sepal, callus and stelidia of orchid flowers. RESULTS: The evolutionary origin of these organs was investigated using a combination of morphological, molecular and phylogenetic techniques to a developmental series of floral buds of E. pusilla. The vascular bundle of the median sepal indicates it is a first whorl organ but its convex epidermal cells reflect convergence of petaloid features. Expression of AGL6 EpMADS4 and APETALA3 EpMADS14 is low in the median sepal, possibly correlating with its petaloid appearance. A vascular bundle indicating second whorl derivation leads to the lip. AGL6 EpMADS5 and APETALA3 EpMADS13 are most highly expressed in lip and callus, consistent with current models for lip identity. Six vascular bundles, indicating a stamen-derived origin, lead to the callus, stelidia and stamen. AGAMOUS is not expressed in the callus, consistent with its sterilization. Out of three copies of AGAMOUS and four copies of SEPALLATA, EpMADS22 and EpMADS6 are most highly expressed in the stamen. Another copy of AGAMOUS, EpMADS20, and the single copy of SEEDSTICK, EpMADS23, are most highly expressed in the stelidia, suggesting EpMADS22 may be required for fertile stamens. CONCLUSIONS: The median sepal, callus and stelidia of E. pusilla appear to be derived from a sepal, a stamen that gained petal identity, and stamens, respectively. Duplications, diversifying selection and changes in spatial expression of different MADS-box genes shaped these organs, enabling the rewardless flowers of E. pusilla to mimic an unrelated rewarding flower for pollinator attraction. These genetic changes are not incorporated in current models and urge for a rethinking of the evolution of deceptive flowers.

DNA metabarcoding of orchid-derived products reveals widespread illegal orchid trade.

In eastern Mediterranean countries orchids continue to be collected from the wild for the production of salep, a beverage made of dried orchid tubers. In this study we used nrITS1 and nrITS2 DNA metabarcoding to identify orchid and other plant species present in 55 commercial salep products purchased in Iran, Turkey, Greece and Germany. Thirty samples yielded a total of 161 plant taxa, and 13 products (43%) contained orchid species and these belonged to 10 terrestrial species with tuberous roots. Another 70% contained the substitute ingredient Cyamopsis tetraganoloba (Guar). DNA metabarcoding using the barcoding markers nrITS1 and nrITS2 shows the potential of these markers and approach for identification of species used in salep products. The analysis of interspecific genetic distances between sequences of these markers for the most common salep orchid genera shows that species level identifications can be made with a high level of confidence. Understanding the species diversity and provenance of salep orchid tubers will enable the chain of commercialization of endangered species to be traced back to the harvesters and their natural habitats, and thus allow for targeted efforts to protect or sustainably use wild populations of these orchids.

You are what you get from your fungi: nitrogen stable isotope patterns in Epipactis species.

Background and Aims: Partially mycoheterotrophic plants are enriched in 13 C and 15 N compared to autotrophic plants. Here, it is hypothesized that the type of mycorrhizal fungi found in orchid roots is responsible for variation in 15 N enrichment of leaf tissue in partially mycoheterotrophic orchids. Methods: The genus Epipactis was used as a case study and carbon and nitrogen isotope abundances of eight Epipactis species, fungal sporocarps of four Tuber species and autotrophic references were measured. Mycorrhizal fungi were identified using molecular methods. Stable isotope data of six additional Epipactis taxa and ectomycorrhizal and saprotrophic basidiomycetes were compiled from the literature. Key Results: The 15 N enrichment of Epipactis species varied between 3·2 ± 0·8 ‰ ( E. gigantea ; rhizoctonia-associated) and 24·6 ± 1·6 ‰ ( E. neglecta ; associated with ectomycorrhizal ascomycetes). Sporocarps of ectomycorrhizal ascomycetes (10·7 ± 2·2 ‰) were significantly more enriched in 15 N than ectomycorrhizal (5·2 ± 4·0 ‰) and saprotrophic basidiomycetes (3·3 ± 2·1 ‰). Conclusions: As hypothesized, it is suggested that the observed gradient in 15 N enrichment of Epipactis species is strongly driven by 15 N abundance of their mycorrhizal fungi; i.e. ɛ 15 N in Epipactis spp. associated with rhizoctonias < ɛ 15 N in Epipactis spp. with ectomycorrhizal basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes and basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes.

Evolution of wood anatomical characters in Nepenthes and close relatives of Caryophyllales.

Background and Aims: Nepenthes attracts wide attention with its spectacularly shaped carnivorous pitchers, cultural value and horticultural curiosity. Despite the plant's iconic fascination, surprisingly little anatomical detail is known about the genus beyond its modified leaf tip traps. Here, the wood anatomical diversity of Nepenthes is explored. This diversity is further assessed with a phylogenetic framework to investigate whether the wood characters within the genus are relevant from an evolutionary or ecological perspective, or rather depend on differences in developmental stages, growth habits, substrates or precipitation. Methods: Observations were performed using light microscopy and scanning electron microscopy. Ancestral states of selected wood and pith characters were reconstructed using an existing molecular phylogeny for Nepenthes and a broader Caryophyllales framework. Pairwise comparisons were assessed for possible relationships between wood anatomy and developmental stages, growth habits, substrates and ecology. Key Results: Wood anatomy of Nepenthes is diffuse porous, with mainly solitary vessels showing simple, bordered perforation plates and alternate intervessel pits, fibres with distinctly bordered pits (occasionally septate), apotracheal axial parenchyma and co-occurring uni- and multiseriate rays often including silica bodies. Precipitation and growth habit (stem length) are linked with vessel density and multiseriate ray height, while soil type correlates with vessel diameter, vessel element length and maximum ray width. For Caryophyllales as a whole, silica grains, successive cambia and bordered perforation plates are the result of convergent evolution. Peculiar helical sculpturing patterns within various cell types occur uniquely within the insectivorous clade of non-core Caryophyllales. Conclusions: The wood anatomical variation in Nepenthes displays variation for some characters dependent on soil type, precipitation and stem length, but is largely conservative. The helical-banded fibre-sclereids that mainly occur idioblastically in pith and cortex are synapomorphic for Nepenthes , while other typical Nepenthes characters evolved convergently in different Caryophyllales lineages.

Advances in DNA metabarcoding for food and wildlife forensic species identification.

Species identification using DNA barcodes has been widely adopted by forensic scientists as an effective molecular tool for tracking adulterations in food and for analysing samples from alleged wildlife crime incidents. DNA barcoding is an approach that involves sequencing of short DNA sequences from standardized regions and comparison to a reference database as a molecular diagnostic tool in species identification. In recent years, remarkable progress has been made towards developing DNA metabarcoding strategies, which involves next-generation sequencing of DNA barcodes for the simultaneous detection of multiple species in complex samples. Metabarcoding strategies can be used in processed materials containing highly degraded DNA e.g. for the identification of endangered and hazardous species in traditional medicine. This review aims to provide insight into advances of plant and animal DNA barcoding and highlights current practices and recent developments for DNA metabarcoding of food and wildlife forensic samples from a practical point of view. Special emphasis is placed on new developments for identifying species listed in the Convention on International Trade of Endangered Species (CITES) appendices for which reliable methods for species identification may signal and/or prevent illegal trade. Current technological developments and challenges of DNA metabarcoding for forensic scientists will be assessed in the light of stakeholders' needs.

Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing.

We explored genetic variation by sequencing a selection of 84 tomato accessions and related wild species representative of the Lycopersicon, Arcanum, Eriopersicon and Neolycopersicon groups, which has yielded a huge amount of precious data on sequence diversity in the tomato clade. Three new reference genomes were reconstructed to support our comparative genome analyses. Comparative sequence alignment revealed group-, species- and accession-specific polymorphisms, explaining characteristic fruit traits and growth habits in the various cultivars. Using gene models from the annotated Heinz 1706 reference genome, we observed differences in the ratio between non-synonymous and synonymous SNPs (dN/dS) in fruit diversification and plant growth genes compared to a random set of genes, indicating positive selection and differences in selection pressure between crop accessions and wild species. In wild species, the number of single-nucleotide polymorphisms (SNPs) exceeds 10 million, i.e. 20-fold higher than found in most of the crop accessions, indicating dramatic genetic erosion of crop and heirloom tomatoes. In addition, the highest levels of heterozygosity were found for allogamous self-incompatible wild species, while facultative and autogamous self-compatible species display a lower heterozygosity level. Using whole-genome SNP information for maximum-likelihood analysis, we achieved complete tree resolution, whereas maximum-likelihood trees based on SNPs from ten fruit and growth genes show incomplete resolution for the crop accessions, partly due to the effect of heterozygous SNPs. Finally, results suggest that phylogenetic relationships are correlated with habitat, indicating the occurrence of geographical races within these groups, which is of practical importance for Solanum genome evolution studies.

Pollination of Specklinia by nectar-feeding Drosophila: the first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae.

BACKGROUND AND AIMS: The first documented observation of pollination in Pleurothallidinae was that of Endrés, who noticed that the 'viscid sepals' of Specklinia endotrachys were visited by a 'small fly'. Chase would later identify the visiting flies as being members of the genus Drosophila. This study documents and describes how species of the S. endotrachys complex are pollinated by different Drosophila species. METHODS: Specimens of Specklinia and Drosophila were collected in the field in Costa Rica and preserved in the JBL and L herbaria. Flies were photographed, filmed and observed for several days during a 2-year period and were identified by a combination of non-invasive DNA barcoding and anatomical surveys. Tissue samples of the sepals, petals and labellum of Specklinia species were observed and documented by SEM, LM and TEM. Electroantennogram experiments were carried out on Drosophila hydei using the known aggregation pheromones ethyl tiglate, methyl tiglate and isopropyl tiglate. Floral compounds were analysed by gas chromatography-mass spectometry using those same pheromones as standards. KEY RESULTS: Flowers of S. endotrachys, S. pfavii, S. remotiflora and S. spectabilis are visited and pollinated by several different but closely related Drosophila species. The flies are arrested by aggregation pheromones, including ethyl tiglate, methyl tiglate and isopropyl tiglate, released by the flowers, and to which at least D. hydei is very sensitive. Visible nectar drops on the adaxial surface of sepals are secreted by nectar-secreting stomata, encouraging male and female Drosophila to linger on the flowers for several hours at a time. The flies frequently show courtship behaviour, occasionally copulating. Several different Drosophila species can be found on a single Specklinia species. CONCLUSIONS: Species of the S. endotrachys group share a similar pollination syndrome. There seem to be no species-specific relationships between the orchids and the flies. It is not expected that Specklinia species will hybridize naturally as their populations do not overlap geographically. The combination of pheromone attraction and nectar feeding is likely to be a generalized pollination syndrome in Pleurothallidinae.

The HTS barcode checker pipeline, a tool for automated detection of illegally traded species from high-throughput sequencing data.

BACKGROUND: Mixtures of internationally traded organic substances can contain parts of species protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). These mixtures often raise the suspicion of border control and customs offices, which can lead to confiscation, for example in the case of Traditional Chinese medicines (TCMs). High-throughput sequencing of DNA barcoding markers obtained from such samples provides insight into species constituents of mixtures, but manual cross-referencing of results against the CITES appendices is labor intensive. Matching DNA barcodes against NCBI GenBank using BLAST may yield misleading results both as false positives, due to incorrectly annotated sequences, and false negatives, due to spurious taxonomic re-assignment. Incongruence between the taxonomies of CITES and NCBI GenBank can result in erroneous estimates of illegal trade. RESULTS: The HTS barcode checker pipeline is an application for automated processing of sets of 'next generation' barcode sequences to determine whether these contain DNA barcodes obtained from species listed on the CITES appendices. This analytical pipeline builds upon and extends existing open-source applications for BLAST matching against the NCBI GenBank reference database and for taxonomic name reconciliation. In a single operation, reads are converted into taxonomic identifications matched with names on the CITES appendices. By inclusion of a blacklist and additional names databases, the HTS barcode checker pipeline prevents false positives and resolves taxonomic heterogeneity. CONCLUSIONS: The HTS barcode checker pipeline can detect and correctly identify DNA barcodes of CITES-protected species from reads obtained from TCM samples in just a few minutes. The pipeline facilitates and improves molecular monitoring of trade in endangered species, and can aid in safeguarding these species from extinction in the wild. The HTS barcode checker pipeline is available at https://github.com/naturalis/HTS-barcode-checker.

Orchid fruit and root movement analyzed using 2D photographs and a bioinformatics pipeline for processing sequential 3D scans.

Premise: Most studies of the movement of orchid fruits and roots during plant development have focused on morphological observations; however, further genetic analysis is required to understand the molecular mechanisms underlying this phenomenon. A precise tool is required to observe these movements and harvest tissue at the correct position and time for transcriptomics research. Methods: We utilized three-dimensional (3D) micro-computed tomography (CT) scans to capture the movement of fast-growing Erycina pusilla roots, and built an integrated bioinformatics pipeline to process 3D images into 3D time-lapse videos. To record the movement of slowly developing E. pusilla and Phalaenopsis equestris fruits, two-dimensional (2D) photographs were used. Results: The E. pusilla roots twisted and resupinated multiple times from early development. The first period occurred in the early developmental stage (77-84 days after germination [DAG]) and the subsequent period occurred later in development (140-154 DAG). While E. pusilla fruits twisted 45° from 56-63 days after pollination (DAP), the fruits of P. equestris only began to resupinate a week before dehiscence (133 DAP) and ended a week after dehiscence (161 DAP). Discussion: Our methods revealed that each orchid root and fruit had an independent direction and degree of torsion from the initial to the final position. Our innovative approaches produced detailed spatial and temporal information on the resupination of roots and fruits during orchid development.

Orchidinae-205: A new genome-wide custom bait set for studying the evolution, systematics, and trade of terrestrial orchids.

Terrestrial orchids are a group of genetically understudied, yet culturally and economically important plants. The Orchidinae tribe contains many species that produce edible tubers that are used for the production of traditional delicacies collectively called 'salep'. Overexploitation of wild orchids in the Eastern Mediterranean and Western Asia threatens to drive many of these species to extinction, but cost-effective tools for monitoring their trade are currently lacking. Here we present a custom bait kit for target enrichment and sequencing of 205 novel genetic markers that are tailored to phylogenomic applications in Orchidinae s.l. A subset of 31 markers capture genes putatively involved in the production of glucomannan, a water-soluble polysaccharide that gives salep its distinctive properties. We tested the kit on 73 taxa native to the area, demonstrating universally high locus recovery irrespective of species identity, that exceeds the total sequence length obtained with alternative kits currently available. Phylogenetic inference with concatenation and coalescent approaches was robust and showed high levels of support for most clades, including some which were previously unresolved. Resolution for hybridizing and recently radiated lineages remains difficult, but could be further improved by analysing multiple haplotypes and the non-exonic sequences captured by our kit, with the promise to shed new light on the evolution of enigmatic taxa with a complex speciation history. Offering a step-up from traditional barcoding and universal markers, the genome-wide custom loci targeted by Orchidinae-205 are a valuable new resource to study the evolution, systematics and trade of terrestrial orchids.

Dating the origin of the Orchidaceae from a fossil orchid with its pollinator.

Since the time of Darwin, evolutionary biologists have been fascinated by the spectacular adaptations to insect pollination exhibited by orchids. However, despite being the most diverse plant family on Earth, the Orchidaceae lack a definitive fossil record and thus many aspects of their evolutionary history remain obscure. Here we report an exquisitely preserved orchid pollinarium (of Meliorchis caribea gen. et sp. nov.) attached to the mesoscutellum of an extinct stingless bee, Proplebeia dominicana, recovered from Miocene amber in the Dominican Republic, that is 15-20 million years (Myr) old. This discovery constitutes both the first unambiguous fossil of Orchidaceae and an unprecedented direct fossil observation of a plant-pollinator interaction. By applying cladistic methods to a morphological character matrix, we resolve the phylogenetic position of M. caribea within the extant subtribe Goodyerinae (subfamily Orchidoideae). We use the ages of other fossil monocots and M. caribea to calibrate a molecular phylogenetic tree of the Orchidaceae. Our results indicate that the most recent common ancestor of extant orchids lived in the Late Cretaceous (76-84 Myr ago), and also suggest that the dramatic radiation of orchids began shortly after the mass extinctions at the K/T boundary. These results further support the hypothesis of an ancient origin for Orchidaceae.