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.

Plastome phylogenomics reveals an early Pliocene North- and Central America colonization by long-distance dispersal from South America of a highly diverse bromeliad lineage.

Understanding the spatial and temporal frameworks of species diversification is fundamental in evolutionary biology. Assessing the geographic origin and dispersal history of highly diverse lineages of rapid diversification can be hindered by the lack of appropriately sampled, resolved, and strongly supported phylogenetic contexts. The use of currently available cost-efficient sequencing strategies allows for the generation of a substantial amount of sequence data for dense taxonomic samplings, which together with well-curated geographic information and biogeographic models allow us to formally test the mode and tempo of dispersal events occurring in quick succession. Here, we assess the spatial and temporal frameworks for the origin and dispersal history of the expanded clade K, a highly diverse Tillandsia subgenus Tillandsia (Bromeliaceae, Poales) lineage hypothesized to have undergone a rapid radiation across the Neotropics. We assembled full plastomes from Hyb-Seq data for a dense taxon sampling of the expanded clade K plus a careful selection of outgroup species and used them to estimate a time- calibrated phylogenetic framework. This dated phylogenetic hypothesis was then used to perform biogeographic model tests and ancestral area reconstructions based on a comprehensive compilation of geographic information. The expanded clade K colonized North and Central America, specifically the Mexican transition zone and the Mesoamerican dominion, by long-distance dispersal from South America at least 4.86 Mya, when most of the Mexican highlands were already formed. Several dispersal events occurred subsequently northward to the southern Nearctic region, eastward to the Caribbean, and southward to the Pacific dominion during the last 2.8 Mya, a period characterized by pronounced climate fluctuations, derived from glacial-interglacial climate oscillations, and substantial volcanic activity, mainly in the Trans-Mexican Volcanic Belt. Our taxon sampling design allowed us to calibrate for the first time several nodes, not only within the expanded clade K focal group but also in other Tillandsioideae lineages. We expect that this dated phylogenetic framework will facilitate future macroevolutionary studies and provide reference age estimates to perform secondary calibrations for other Tillandsioideae lineages.

New and noteworthy species of the genus Epidendrum (Orchidaceae, Laeliinae) from the Área de Conservación Privada La Pampa del Burro, Amazonas, Peru.

Fourteen species of the genus Epidendrum, recently collected in the Área de Conservación Privada La Pampa del Burro (ACPPB), five of them new to science (Epidendrumechinatiantherumsp. nov., E.imazaensesp. nov., E.parvireflexilobumsp. nov., E.rosulatumsp. nov., and E.ochrostachyumsp. nov.), are described and illustrated. The other species include a new record for Peru (E.acrobatesii) and four for the department of Amazonas (E.brachyblastum, E.forcipatum, E.mavrodactylon, and E.tridens). Epidendrumenantilobum is here considered a synonym of Epidendrumbrachyblastum. The type locality of Epidendrumcryptorhachis, originally stated as Ecuador, Guayabamba, is corrected to indicate that it refers to the valley of Guayabamba, Rodríguez de Mendoza, Amazonas, Peru. Our results show the need to continue conducting botanical exploration in the ACPPB as a baseline for subsequent studies, including a full inventory of the orchid diversity.

A new species of Liparis (Orchidaceae, Epidendroideae, Malaxidinae) from the Bosque de Protección Alto Mayo, San Martín, Peru.

Liparisaltomayoënsissp. nov. is described, illustrated, and tentatively assigned to the Neotropical section Decumbentes on the basis of its branching, prostrate rhizomes and upright stems bearing several leaves. Vegetatively, the new species is distinguished by its short, upward stems bearing 3-6 leaves, these with undulate, translucent margins and reticulate, prominent veining on the upper surface. Florally, it is distinctive in the labellum with fleshy basal one-half provided with a central, rounded cavity limited on each side by a prominent, bilobulate ridge and apically by a lunate ridge, and membranaceous, trilobulate apical one-half deflexed ca. 90°. In contrast with other species of section Decumbentes, in which fruit formation is infrequent, in L.altomayoënsis a high proportion (⁓50-100%) of flowers develop into a fruit; in some flowers the pollinaria rotate and contact the stigma, apparently resulting in at least facultative self-pollination. The main differences among the six species of L.sectionDecumbentes hitherto known are contrasted in a dichotomous key. The new species is known only from three populations located in the Bosque de Protección Alto Mayo, on the Amazonian slope of the Andes in northeastern Peru but appears to be under no foreseeable threats.

ResumenLiparisaltomayoënsissp. nov. es descrita, ilustrada y asignada tentativamente a la sección neotropical Decumbentes con base en sus rizomas postrados, ramificados y tallos ascendentes portando varias hojas. Vegetativamente, la nueva especie se distingue por sus tallos ascendentes cortos con 3­6 hojas, éstas con márgenes undulados, translúcidos y venación reticulada prominente en la superficie superior. Floralmente, es distintivo su labelo con la mitad basal carnosa, provista de una cavidad central redondeada que está limitada a cada lado por una cresta bilobulada prominente y apicalmente por una cresta lunada, y la mitad apical del labelo membranácea, trilobulada y deflexa ca. 90°. En contraste con otras especies de la sección Decumbentes, en las que la producción de frutos es infrecuente, en L.altomayoënsis una alta proporción (⁓50­100%) de las flores produce fruto; en algunas flores los polinarios rotan y contactan el estigma, aparentemente resultando en autopolinización al menos facultativa. Las diferencias principales entre las seis especies de L. sección Decumbentes conocidas hasta ahora se contrastan en una clave dicotómica. La nueva especie se conoce solamente de tres poblaciones localizadas en el Bosque de Protección Alto Mayo, en la vertiente amazónica de los Andes en el noreste de Perú, pero no parece estar sujeta a amenazas previsibles.

New plastome structural rearrangements discovered in core Tillandsioideae (Bromeliaceae) support recently adopted taxonomy.

Full plastome sequences for land plants have become readily accessible thanks to the development of Next Generation Sequencing (NGS) techniques and powerful bioinformatic tools. Despite this vast amount of genomic data, some lineages remain understudied. Full plastome sequences from the highly diverse (>1,500 spp.) subfamily Tillandsioideae (Bromeliaceae, Poales) have been published for only three (i.e., Guzmania, Tillandsia, and Vriesea) out of 22 currently recognized genera. Here, we focus on core Tillandsioideae, a clade within subfamily Tillandsioideae, and explore the contribution of individual plastid markers and data categories to inform deep divergences of a plastome phylogeny. We generated 37 high quality plastome assemblies and performed a comparative analysis in terms of plastome structure, size, gene content and order, GC content, as well as number and type of repeat motifs. Using the obtained phylogenetic context, we reconstructed the evolution of these plastome attributes and assessed if significant shifts on the evolutionary traits' rates have occurred in the evolution of the core Tillandsioideae. Our results agree with previously published phylogenetic hypotheses based on plastid data, providing stronger statistical support for some recalcitrant nodes. However, phylogenetic discordance with previously published nuclear marker-based hypotheses was found. Several plastid markers that have been consistently used to address phylogenetic relationships within Tillandsioideae were highly informative for the retrieved plastome phylogeny and further loci are here identified as promising additional markers for future studies. New lineage-specific plastome rearrangements were found to support recently adopted taxonomic groups, including large inversions, as well as expansions and contractions of the inverted repeats. Evolutionary trait rate shifts associated with changes in size and GC content of the plastome regions were found across the phylogeny of core Tillandsioideae.

A new species and a new record of Liparis sect. Decumbentes (Malaxidinae, Orchidaceae) from Peru.

A new species of Liparis sect. Decumbentes, Liparis sessilis Damián, Salazar & Rimarachín, sp. nov is described and illustrated from Amazonas (Perú), including color photographs, a detailed comparison and an identification key to all the species of Liparis sect. Decumbentes. In addition, we report Liparis brachystalix Rchb.f. for the first time for the orchid flora of Peru, and select a lectotype for its synonym, L. commelinoides Schltr.

ResumenUna nueva especie de Liparis sect. Decumbentes, Liparis sessilis Damián, Salazar & Rimarachín, sp. nov se describe e ilustra de Amazonas (Perú), incluyendo fotografías a color, una comparasion detallada y una clave de identificación de todas las especies de Liparis sect. Decumbentes. Ademas, reportamos Liparis brachystalix Rchb.f. por primera vez para la orquiflora del Perú, y seleccionamos un lectótipo para uno de sus sinónimos, L. commelinoides Schltr.

Target Nuclear and Off-Target Plastid Hybrid Enrichment Data Inform a Range of Evolutionary Depths in the Orchid Genus Epidendrum.

Universal angiosperm enrichment probe sets designed to enrich hundreds of putatively orthologous nuclear single-copy loci are increasingly being applied to infer phylogenetic relationships of different lineages of angiosperms at a range of evolutionary depths. Studies applying such probe sets have focused on testing the universality and performance of the target nuclear loci, but they have not taken advantage of off-target data from other genome compartments generated alongside the nuclear loci. Here we do so to infer phylogenetic relationships in the orchid genus Epidendrum and closely related genera of subtribe Laeliinae. Our aims are to: 1) test the technical viability of applying the plant anchored hybrid enrichment (AHE) method (Angiosperm v.1 probe kit) to our focal group, 2) mine plastid protein coding genes from off-target reads; and 3) evaluate the performance of the target nuclear and off-target plastid loci in resolving and supporting phylogenetic relationships along a range of taxonomical depths. Phylogenetic relationships were inferred from the nuclear data set through coalescent summary and site-based methods, whereas plastid loci were analyzed in a concatenated partitioned matrix under maximum likelihood. The usefulness of target and flanking non-target nuclear regions and plastid loci was assessed through the estimation of their phylogenetic informativeness. Our study successfully applied the plant AHE probe kit to Epidendrum, supporting the universality of this kit in angiosperms. Moreover, it demonstrated the feasibility of mining plastome loci from off-target reads generated with the Angiosperm v.1 probe kit to obtain additional, uniparentally inherited sequence data at no extra sequencing cost. Our analyses detected some strongly supported incongruences between nuclear and plastid data sets at shallow divergences, an indication of potential lineage sorting, hybridization, or introgression events in the group. Lastly, we found that the per site phylogenetic informativeness of the ycf1 plastid gene surpasses that of all other plastid genes and several nuclear loci, making it an excellent candidate for assessing phylogenetic relationships at medium to low taxonomic levels in orchids.

A pilot study applying the plant Anchored Hybrid Enrichment method to New World sages (Salvia subgenus Calosphace; Lamiaceae).

We conducted a pilot study using Anchored Hybrid Enrichment to resolve relationships among a mostly Neotropical sage lineage that may have undergone a recent evolutionary radiation. Conventional markers (ITS, trnL-trnF and trnH-psbA) have not been able to resolve the relationships among species nor within portions of the backbone of the lineage. We sampled 12 representative species of subgenus Calosphace and included one species of Salvia's s.l. closest relative, Lepechinia, as outgroup. Hybrid enrichment and sequencing were successful, yielding 448 alignments of individual loci with an average length of 704bp. The performance of the phylogenomic data in phylogenetic reconstruction was superior to that of conventional markers, increasing both support and resolution. Because the captured loci vary in the amount of net phylogenetic informativeness at different phylogenetic depths, these data are promising in phylogenetic reconstruction of this group and likely other lineages within Lamiales. However, special attention should be placed on the amount of phylogenetic noise that the data could potentially contain. A prior exploration step using phylogenetic informativeness profiles to detect loci with sites with disproportionately high substitution rates (showing "phantom" spikes) and, if required, the ensuing filtering of the problematic data is recommended. In our dataset, filtering resulted in increased support and resolution for the shallow nodes in maximum likelihood phylogenetic trees resulting from concatenated analyses of all the loci. Additionally, it is expected that an increase in sampling (loci and taxa) will aid in resolving weakly supported, short deep internal branches.

Molecular phylogenetics of the species-rich genus Habenaria (Orchidaceae) in the New World based on nuclear and plastid DNA sequences.

Habenaria is a large genus of terrestrial orchids distributed throughout the tropical and subtropical regions of the world. The integrity and monophyly of this genus have been under discussion for many years, and at one time or another, several genera have been either included in a broadly defined Habenaria or segregated from it. In this study, the phylogenetic relationships of the Neotropical members of the genus and selected groups of African Habenaria were investigated using DNA sequences from the nuclear internal transcribed spacer (ITS) region and the plastid matK gene sampled from 151 taxa of Habenaria from the Neotropics (ca. 51% of the total) as well as 20 species of Habenaria and Bonatea from the Old World. Bayesian and parsimony trees were congruent with each other, and in all analyses, the Neotropical species formed a highly supported group. African species of Habenaria in sections Dolichostachyae, Podandria, Diphyllae, Ceratopetalae and Bilabrellae, and the Neotropical clade formed a highly supported "core Habenaria clade", which includes the type species of the genus from the New World. The topology of the trees indicates an African origin for the Neotropical clade and the low sequence divergence among the Neotropical species suggests a recent radiation of the genus in the New World. Species of Bonatea and Habenaria sections Chlorinae and Multipartitae formed a well-supported clade that was sister to the "core Habenaria clade". The Neotropical clade consists of at least 21 well-supported subgroups, but all Neotropical sections of the current sectional classification are paraphyletic or polyphyletic and will need extensive revision and recircumscription. Most of the Neotropical subgroups formed morphologically uniform assemblage of species, but some cases of morphological divergence within subgroups and convergence between subgroups indicated that morphology alone can be misleading for inferring relationships within the genus. The genera Bertauxia, Kusibabella and Habenella, segregated from New World Habenaria, are not monophyletic and a revision of the sectional classification rather than a generic division seems most appropriate. Our results do not support an extensive generic fragmentation of Habenaria as previously suggested and will provide a framework for revising the infrageneric classification and investigating the patterns of morphological evolution and geographical distribution of the genus in the New World.

Stelis zootrophionoides (Orchidaceae: Pleurothallidinae), a new species from Mexico.

BACKGROUND: Stelis (Orchidaceae) encompasses approximately 1100 species of epiphytic orchids distributed throughout the Neotropics, with the highest diversity in Andean South America. Sixty-two species were recorded previously in Mexico. METHODS: We formally describe here Stelis zootrophionoides as a new species from Chiapas, Mexico. To determine its systematic position, we conducted a morphological comparison with other members of Pleurothallidinae and a phylogenetic analysis of nucleotide sequences from the plastid matK/trnK and trnL/trnF regions, as well as the nuclear ribosomal ITS region for 52 species of Pleurothallidinae. Sequences of 49 species were downloaded from GenBank and those of three species, including the new taxon, were newly generated for this work. The new species is described and illustrated; notes on its ecological preferences and a comparison with closely related species are presented. CONCLUSIONS: The new species, known only from one location and apparently restricted to the cloud forest in the central highlands of Chiapas, Mexico, is considered a rare species. This small epiphyte is unique among the Mexican species of Stelis by the combination of dark purple flowers with the distal third of the dorsal sepal adhered to the apices of the lateral sepals, which are partially united into a bifid synsepal, leaving two lateral window-like openings, and sagittate labellum. Stelis jalapensis, known from southern Mexico and Guatemala, also has the apices of the sepals adhered to each other, but it is distinguished by its larger flowers with lanceolate, acute dorsal sepal, completely fused lateral sepals (i.e. the synsepal is not bifid), and oblong-elliptic labellum. The phylogenetic analysis shows that S. zootrophionoides is closely related to other Mexican Stelis and corroborates previous suggestions that fused sepal apices have arisen independently in different lineages of Pleurothallidinae.

Molecular phylogeny of Cypripedium (Orchidaceae: Cypripedioideae) inferred from multiple nuclear and chloroplast regions.

A molecular analysis was performed on 56 taxa in the orchid genus Cypripedium using nrDNA ITS and five chloroplast regions (trnH-psbA, atpI-atpH, trnS-trnfM, trnL-F spacer, and the trnL intron). The genus Cypripedium was confirmed as monophyletic. Our data provided strong support for monophyletic grouping of eight infrageneric sections (Subtropica, Obtusipetala, Trigonopedia, Sinopedilum, Bifolia, Flabelinervia, Arietinum, and Cypripedium) defined in earlier taxonomic treatments, and paraphyletic grouping of two sections (Irapeana and Retinervi). Within the genus Cypripedium, the first divergent lineage consisted of two Mesomaerican species, and subsequently the Cypripedium debile lineage from eastern Asia was split. Our study did not support the notion that two Asian species (Cypripedium subtropicum and Cypripedium singchii) were closely related to either Mesoamerican Cypripedium irapeanum or North American Cypripedium californicum, as indicated by previous interpretations based on morphological evidences. In addition, one pair of vicariant species, Cypripedium plectrochilum (eastern Asia) and Cypripedium arietinum (North America), unique to section Arietinum, was confirmed. Furthermore, within the monophyletic section Cypripedium two previously recognized subsections, Cypripedium and Macrantha, were shown to be paraphyletic. Our results suggested that this section split into two groups based on distribution (North America vs. Eurasia) instead of such previously used, morphological traits as flower color, and the shape of the lips (labellum) and lateral petals.

Phylogenetic relationships of Cranichidinae and Prescottiinae (Orchidaceae, Cranichideae) inferred from plastid and nuclear DNA sequences.

BACKGROUND AND AIMS: Phylogenetic relationships of subtribes Cranichidinae and Prescottiinae, two diverse groups of neotropical terrestrial orchids, are not satisfactorily understood. A previous molecular phylogenetic study supported monophyly for Cranichidinae, but Prescottiinae consisted of two clades not sister to one another. However, that analysis included only 11 species and eight genera of these subtribes. Here, plastid and nuclear DNA sequences are analysed for an enlarged sample of genera and species of Cranichidinae and Prescottiinae with the aim of clarifying their relationships, evaluating the phylogenetic position of the monospecific genera Exalaria, Ocampoa and Pseudocranichis and examining the value of various structural traits as taxonomic markers. METHODS: Approx. 6000 bp of nucleotide sequences from nuclear ribosomal (ITS) and plastid DNA (rbcL, matK-trnK and trnL-trnF) were analysed with cladistic parsimony and Bayesian inference for 45 species/14 genera of Cranichidinae and Prescottiinae (plus suitable outgroups). The utility of flower orientation, thickenings of velamen cell walls, hamular viscidium and pseudolabellum to mark clades recovered by the molecular analysis was assessed by tracing these characters on the molecular trees. KEY RESULTS: Spiranthinae, Cranichidinae, paraphyletic Prescottia (with Pseudocranichis embedded), and a group of mainly Andean 'prescottioid' genera (the 'Stenoptera clade') were strongly supported. Relationships among these clades were unresolved by parsimony but the Bayesian tree provided moderately strong support for the resolution (Spiranthinae-(Stenoptera clade-(Prescottia/Pseudocranichis-Cranichidinae))). Three of the four structural characters mark clades on the molecular trees, but the possession of a pseudolabellum is variable in the polyphyletic Ponthieva. CONCLUSIONS: No evidence was found for monophyly of Prescottiinae and the reinstatement of Cranichidinae s.l. (including the genera of 'Prescottiinae') is favoured. Cranichidinae s.l. are diagnosed by non-resupinate flowers. Lack of support from parsimony for relationships among the major clades of core spiranthids is suggestive of a rapid morphological radiation or a slow rate of molecular evolution.

Root character evolution and systematics in Cranichidinae, Prescottiinae and Spiranthinae (Orchidaceae, Cranichideae).

BACKGROUND AND AIMS: Previous studies have suggested that velamen characteristics are useful as taxonomic markers in Orchidaceae. Members of tribe Cranichideae have been assigned to two velamen types constructed based on combinations of characters such as the presence of secondary cell-wall thickenings and pores. However, such characters have not been analysed on an individual basis in explicit cladistic analyses. METHODS: The micromorphology of roots of 26 species of Cranichideae was examined through scanning electron microscopy and light microscopy, scoring the variation and distribution of four characters: number of velamen cell layers, velamen cell-wall thickenings, presence and type of tilosomes, and supraendodermal spaces. The last three characters were analysed cladistically in combination with DNA sequence data of plastid trnK/matK and nuclear ribosomal internal transcribed spacer (ITS) regions and optimized on the resulting phylogenetic tree. KEY RESULTS: Thickenings of velamen cell walls group Prescottiinae with Spiranthinae, whereas tilosomes, documented here for the first time in Cranichideae, provide an unambiguous synapomorphy for subtribe Spiranthinae. Supraendodermal spaces occur mostly in species dwelling in seasonally dry habitats and appear to have evolved three times. CONCLUSIONS: Three of the four structural characters assessed are phylogenetically informative, marking monophyletic groups recovered in the combined molecular-morphological analysis. This study highlights the need for conducting character-based structural studies to overcome analytical shortcomings of the typological approach.

Phylogenetic relationships of aroids and duckweeds (Araceae) inferred from coding and noncoding plastid DNA.

Familial, subfamilial, and tribal monophyly and relationships of aroids and duckweeds were assessed by parsimony and Bayesian phylogenetic analyses of five regions of coding (rbcL, matK) and noncoding plastid DNA (partial trnK intron, trnL intron, trnL-trnF spacer) for exemplars of nearly all aroid and duckweed genera. Our analyses confirm the position of Lemna and its allies (formerly Lemnaceae) within Araceae as the well-supported sister group of all aroids except Gymnostachydoideae and Orontioideae. The last two subfamilies form the sister clade of the rest of the family. Monophyly of subfamilies Orontioideae, Pothoideae, Monsteroideae, and Lasioideae is supported, but Aroideae are paraphyletic if Calla is maintained in its own subfamily (Calloideae). Our results suggest expansion of the recently proposed subfamily Zamioculcadoideae (Zamioculcas, Gonatopus) to include Stylochaeton and identify problems in the current delimitation of tribes Anadendreae, Heteropsideae, and Monstereae (Monsteroideae), Caladieae/Zomicarpeae, and Colocasieae (Aroideae). Canalization of traits of the spathe and spadix considered typical of Araceae evolved after the split of Gymnostachydoideae, Orontioideae, and Lemnoideae. An association with aquatic habitats is a plesiomorphic attribute in Araceae, occurring in the helophytic Orontioideae and free-floating Lemnoideae, but evolving independently in various derived aroid lineages including free-floating Pistia (Aroideae).

Phylogenetics of Cranichideae with emphasis on Spiranthinae (Orchidaceae, Orchidoideae): evidence from plastid and nuclear DNA sequences.

DNA sequences from plastid rbcL and matK genes and the trnL-F region, as well as the nuclear ribosomal ITS region, were used to evaluate monophyly and subtribal delimitation of Cranichideae and generic relationships in Spiranthinae. Cranichideae are moderately supported as monophyletic, with Chloraeinae and Pterostylis-Megastylis indicated as their collective sisters. Within Cranichideae, Pachyplectroninae and Goodyerinae form a well-supported monophyletic group sister to a "core spiranthid" clade that includes, according to their branching order, Galeottiellinae, Manniellinae, and a Prescottiinae-Cranichidinae-Spiranthinae subclade. Inclusion of Galeottiella in Spiranthinae, as in previous classifications, renders the latter paraphyletic to all other spiranthid subtribes. Cranichidinae and Spiranthinae (minus Galeottiella) are monophyletic and strongly supported, but Prescottiinae form a grade that includes a strongly supported prescottioid Andean clade and a weakly supported Prescottia-Cranichidinae clade sister to Spiranthinae. Well-supported major clades in Spiranthinae identified in this study do not correspond to previous alliances or the narrowly defined subtribes in which they have been divided recently. Morphological characters, especially those that have been used for taxonomic delimitation in Cranichideae, are discussed against the framework of the molecular trees, emphasizing putative synapomorphies and problems derived from lack of information or inadequate interpretation of the characters.