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.

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 Gongora gibba genome assembly provides new insights into the evolution of floral scent in male euglossine bee-pollinated orchids.

Orchidaceae is one of the most prominent flowering plant families, with many species exhibiting highly specialized reproductive and ecological adaptations. An estimated 10% of orchid species in the American tropics are pollinated by scent-collecting male euglossine bees; however, to date, there are no published genomes of species within this pollination syndrome. Here we present the first draft genome of an epiphytic orchid from the genus Gongora, a representative of the male euglossine bee-pollinated subtribe Stanhopeinae. The 1.83 Gb de novo genome with a scaffold N50 of 1.7Mb was assembled using short- and long-read sequencing and chromosome capture (Hi-C) information. Over 17,000 genes were annotated, and 82.95% of the genome was identified as repetitive content. Furthermore, we identified and manually annotated 26 terpene synthase (TPS) genes linked to floral scent biosynthesis and performed a phylogenetic analysis with other published orchid TPS genes. The Gongora gibba genome assembly will serve as the foundation for future research to understand the genetic basis of floral scent biosynthesis and diversification in orchids. Las orquídeas (Orchidaceae) son una de las familias de plantas con mayor riqueza de especies y exhiben adaptaciones reproductivas altamente especializadas. Se estima que el 10% de las especies de orquídeas en los trópicos americanos son polinizadas por abejas euglosinas; sin embargo, hasta la fecha no existen genomas publicados de especies con este síndrome de polinización. Aquí presentamos el primer genoma de una orquídea epífita del género Gongora, un representante de la subtribu Stanhopeinae, que es polinizada exclusivamente por abejas euglosinas macho. El genoma de 1,83 Gb se ensambló de novo utilizando secuenciación e información de captura de cromosomas (Hi-C), logrando un N50 de 1,7 Mb. Se anotaron más de 17.000 genes y se identificó que el 82,95% del genoma presenta elementos repetitivos. Además, identificamos y anotamos manualmente 26 genes de la familia de genes terpeno sintasa (TPS) y realizamos un análisis filogenético con otros genes TPS de orquídeas publicados. El ensamblaje del genoma de Gongora gibba servirá como base para futuras investigaciones para comprender la base genética de la biosíntesis y la diversificación de los aromas florales en las orquídeas.

Convergent evolution of floral signals underlies the success of Neotropical orchids.

The great majority of plant species in the tropics require animals to achieve pollination, but the exact role of floral signals in attraction of animal pollinators is often debated. Many plants provide a floral reward to attract a guild of pollinators, and it has been proposed that floral signals of non-rewarding species may converge on those of rewarding species to exploit the relationship of the latter with their pollinators. In the orchid family (Orchidaceae), pollination is almost universally animal-mediated, but a third of species provide no floral reward, which suggests that deceptive pollination mechanisms are prevalent. Here, we examine floral colour and shape convergence in Neotropical plant communities, focusing on certain food-deceptive Oncidiinae orchids (e.g. Trichocentrum ascendens and Oncidium nebulosum) and rewarding species of Malpighiaceae. We show that the species from these two distantly related families are often more similar in floral colour and shape than expected by chance and propose that a system of multifarious floral mimicry--a form of Batesian mimicry that involves multiple models and is more complex than a simple one model-one mimic system--operates in these orchids. The same mimetic pollination system has evolved at least 14 times within the species-rich Oncidiinae throughout the Neotropics. These results help explain the extraordinary diversification of Neotropical orchids and highlight the complexity of plant-animal interactions.

DNA barcoding the floras of biodiversity hotspots.

DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. DNA barcoding is well established in animals, but there is not yet any universally accepted barcode for plants. Here, we undertook intensive field collections in two biodiversity hotspots (Mesoamerica and southern Africa). Using >1,600 samples, we compared eight potential barcodes. Going beyond previous plant studies, we assessed to what extent a "DNA barcoding gap" is present between intra- and interspecific variations, using multiple accessions per species. Given its adequate rate of variation, easy amplification, and alignment, we identified a portion of the plastid matK gene as a universal DNA barcode for flowering plants. Critically, we further demonstrate the applicability of DNA barcoding for biodiversity inventories. In addition, analyzing >1,000 species of Mesoamerican orchids, DNA barcoding with matK alone reveals cryptic species and proves useful in identifying species listed in Convention on International Trade of Endangered Species (CITES) appendixes.

Molecular phylogenetics and the evolution of fruit and leaf morphology of Dichaea (Orchidaceae: Zygopetalinae).

BACKGROUND AND AIMS: The orchid genus Dichaea, with over 100 species found throughout the neotropics, is easily recognized by distichous leaves on long stems without pseudobulbs and flowers with infrastigmatic ligules. The genus has previously been divided into four sections based primarily on presence of ovary bristles and a foliar abscission layer. The aim of this work is to use DNA sequence data to estimate phylogenetic relationships within Dichaea and map the distribution of major morphological characters that have been used to delimit subgenera/sections. METHODS: Sequence data for the nuclear ribosomal internal transcribed spacers and plastid matK, trnL intron, trnL-F spacer and ycf1 for 67 ingroup and seven outgroup operational taxonomic units were used to estimate phylogenetic relationships within Dichaea. Taxa from each of the four sections were sampled, with the greatest representation from section Dichaea, the most diverse and taxonomically puzzling group. KEY RESULTS: Molecular data and morphology support monophyly of Dichaea. Results indicate that section Dichaeopsis is polyphyletic and based on symplesiomorphies, including deciduous leaves and smooth ovaries that are widespread in Zygopetalinae. There are at least three well-supported clades within section Dichaeopsis. Section Pseudodichaea is monophyletic and defined by setose ovaries and leaves with an abscission layer. Sections Dichaea and Dichaeastrum are monophyletic and defined by pendent habit and persistent leaves. Section Dichaeastrum, distinguished from section Dichaea primarily by a glabrous ovary, is potentially polyphyletic. CONCLUSIONS: The leaf abscission layer was lost once, occurring only in the derived sections Dichaea and Dichaeastrum. The setose fruit is a more homoplasious character with several losses and gains within the genus. We propose an informal division of the genus based upon five well-supported clades.

Phylogenetic comparative methods improve the selection of characters for generic delimitations in a hyperdiverse Neotropical orchid clade.

Taxonomic delimitations are challenging because of the convergent and variable nature of phenotypic traits. This is evident in species-rich lineages, where the ancestral and derived states and their gains and losses are difficult to assess. Phylogenetic comparative methods help to evaluate the convergent evolution of a given morphological character, thus enabling the discovery of traits useful for classifications. In this study, we investigate the evolution of selected traits to test for their suitability for generic delimitations in the clade Lepanthes, one of the Neotropical species-richest groups. We evaluated every generic name proposed in the Lepanthes clade producing densely sampled phylogenies with Maximum Parsimony, Maximum Likelihood, and Bayesian approaches. Using Ancestral State Reconstructions, we then assessed 18 phenotypic characters that have been traditionally employed to diagnose genera. We propose the recognition of 14 genera based on solid morphological delimitations. Among the characters assessed, we identified 16 plesiomorphies, 12 homoplastic characters, and seven synapomorphies, the latter of which are reproductive features mostly related to the pollination by pseudocopulation and possibly correlated with rapid diversifications in Lepanthes. Furthermore, the ancestral states of some reproductive characters suggest that these traits are associated with pollination mechanisms alike promoting homoplasy. Our methodological approach enables the discovery of useful traits for generic delimitations in the Lepanthes clade and offers various other testable hypotheses on trait evolution for future research on Pleurothallidinae orchids because the phenotypic variation of some characters evaluated here also occurs in other diverse genera.

Specklinia dunstervillei, a New Species Long Confused with Specklinia endotrachys (Orchidaceae: Pleurothallidinae).

Specklinia dunstervillei is described as a new species in recognition of the distinctness of a Venezuelan species related to and confused with Specklinia endotrachys. It was illustrated for the first time by G. C. K. Dunsterville in 1963 from a plant collected in Trujillo on the Cordillera de Merida. The newly named species can be easily recognized by its small habit, short leaves and small reddish-orange flowers, the non-ascending dorsal sepal and the obtuse petals that are shortly apiculate. Specklinia dunstervillei is formally described and illustrated once again and compared morphologically and genetically with its closest relatives.