DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbI spacer, and trnH-psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL؉matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.matK ͉ rbcL ͉ species identification L arge-scale standardized sequencing of the mitochondrial gene CO1 has made DNA barcoding an efficient species identification tool in many animal groups (1). In plants, however, low substitution rates of mitochondrial DNA have led to the search for alternative barcoding regions. From initial investigations of plastid regions (2-4), 7 leading candidates have emerged (5, 6). Four are portions of coding genes (matK, rbcL, rpoB, and rpoC1), and 3 are noncoding spacers (atpF-atpH, trnH-psbA, and psbK-psbI). Different research groups have proposed various combinations of these loci as their preferred plant barcodes, but no consensus has emerged (5-12). This lack of an agreed standard has impeded progress in plant barcoding.Our aim here is to identify a standard DNA barcode for land plants. To achieve this goal, we have pooled data across laboratories including sequence data from 907 samples, representing 445 angiosperm, 38 gymnosperm, and 67 cryptogam species. Using various subsets of these data, we evaluated the 7 candidate loci using criteria in the Consortium for the Barcode of Life's (CBOL) data standards and guidelines for locus selection (http:// www.barcoding.si.edu/protocols.html). Universality: Which loci can be routinely sequenced across the land plants? Sequence quality and coverage: Which loci are most amenable to the production of bidirectional sequences with few or no ambiguous base calls? Discrimination: Which loci enable most species to be distinguished? ResultsUniversality. Direct universality assessments using a single primer pair for each locus in angiosperms resulted in 90%-98% PCR and sequencing success for 6/7 regions. Success for the seventh region, psbK-psbI, was 77% (Fig. 1A). Greater problems were encountered in other land plant groups, with rpoB, matK, atpF-atpH, and psbK-psbI all showing Ͻ50% success in gymnosperms and/or cryptogams based on data compiled from several laboratories (Fig. 1 A).Sequence Quality. Evaluation of sequence quality and coverage from the candidate loci demonstrated that high quality bidirectional sequences were routinely obtained from rbcL, rpoC1, and rpoB (Fig. 1B, x axis). The remaining 4 loci required more manual editing and produced f...

Since the last classification of Orchidaceae in 2003, there has been major progress in the determination of relationships, and we present here a revised classification including a list of all 736 currently recognized genera. A number of generic changes have occurred in Orchideae (Orchidoideae), but the majority of changes have occurred in Epidendroideae. In the latter, almost all of the problematic placements recognized in the previous classification 11 years ago have now been resolved. In Epidendroideae, we have recognized three new tribes (relative to the last classification): Thaieae (monogeneric) for Thaia, which was previously considered to be the only taxon incertae sedis; Xerorchideae (monogeneric) for Xerorchis; and Wullschlaegelieae for achlorophyllous Wullschlaegelia, which had tentatively been placed in Calypsoeae. Another genus, Devogelia, takes the place of Thaia as incertae sedis in Epidendroideae. Gastrodieae are clearly placed among the tribes in the neottioid grade, with Neottieae sister to the remainder of Epidendroideae. Arethuseae are sister to the rest of the higher Epidendroideae, which is unsurprising given their mostly soft pollinia. Tribal relationships within Epidendroideae have been much clarified by analyses of multiple plastid DNA regions and the low-copy nuclear gene Xdh. Four major clades within the remainder of Epidendroideae are recognized: Vandeae/Podochileae/Collabieae, Cymbidieae, Malaxideae and Epidendreae, the last now including Calypsoinae (previously recognized as a tribe on its own) and Agrostophyllinae s.s. Agrostophyllinae and Collabiinae were unplaced subtribes in the 2003 classification. The former are now split between two subtribes, Agrostophyllinae s.s. and Adrorhizinae, the first now included in Epidendreae and the second in Vandeae. Collabiinae, also probably related to Vandeae, are now elevated to a tribe along with Podochileae. Malaxis and relatives are placed in Malaxidinae and included with Dendrobiinae in Malaxideae. The increased resolution and content of larger clades, recognized here as tribes, do not support the 'phylads' in Epidendroideae proposed 22 years ago by Dressler.

We propose in this paper to use three regions of plastid DNA as a standard protocol for barcoding all land plants. We review the other markers that have been proposed and discuss their advantages and disadvantages. The low levels of variation in plastid DNA make three regions necessary; there are no plastid regions, coding or non‐coding, that evolve as rapidly as mitochondrial DNA generally does in animals. We outline two, three‐region options, (1) rpoC1, rpoB and 1matK or (2) rpoC1, matK and psbA‐trnH as viable markers for land plant barcoding.

Recent debates on the number of plant species in the vast lowland rain forests of the Amazon have been based largely on model estimates, neglecting published checklists based on verified voucher data. Here we collate taxonomically verified checklists to present a list of seed plant species from lowland Amazon rain forests. Our list comprises 14,003 species, of which 6,727 are trees. These figures are similar to estimates derived from nonparametric ecological models, but they contrast strongly with predictions of much higher tree diversity derived from parametric models. Based on the known proportion of tree species in neotropical lowland rain forest communities as measured in complete plot censuses, and on overall estimates of seed plant diversity in Brazil and in the neotropics in general, it is more likely that tree diversity in the Amazon is closer to the lower estimates derived from nonparametric models. Much remains unknown about Amazonian plant diversity, but this taxonomically verified dataset provides a valid starting point for macroecological and evolutionary studies aimed at understanding the origin, evolution, and ecology of the exceptional biodiversity of Amazonian forests.Amazonia | floristics | rain forests | seed plants | species diversity

With more than 56,000 species (excluding fungi), Brazil has one of the richest floras in the worldnearly 19% of the world flora. Our knowledge of the diversity and status of nonvascular plants in Brazil is still fragmentary, although localized studies on algae have revealed loss of species resulting from environmental pollution. Emphasis on local floral surveys, rather than wider taxonomic studies, has obscured estimates of national totals for most taxonomic groups. Knowledge of angiosperms, especially monocotyledons (of which 45% of the species are endemic), is more complete than most. For this group figures are more reliable, with some distribution patterns, endemism levels, and centers of diversity identified. Much, however, still awaits discovery. Coordinated efforts to catalog Brazil's flora are in progress and include projects such as the conservation priority-setting workshops of the Brazilian Ministry of the Environment, which have identified key conservation areas in the major biomes; development of threatened species lists for plants; and the assembly of type data on species of northeastern Brazil through the Darwin Initiative-all of which greatly assist in increasing our knowledge. These initiatives also underline the urgent need to expand the numbers and geographic spread of projects on plant systematics and taxonomy in Brazil, a measure that demands adequate provision of funding and training programs for plant specialists. Finally, Brazil's environmental agency (IBAMA) could play a proactive role in opening protected areas under its jurisdiction, thereby facilitating botanical research by university departments and research institutes. Biodiversidad y Conservación de Plantas en BrasilResumen: Con más de 56,00 especies (excluyendo hongos), Brasil tiene una de las floras más ricas del mundo-casi 19% de la flora mundial. Nuestro conocimiento de la diversidad y del estatus de plantas no vasculares aun es muy incompleto, aunque estudios sobre algas han revelado la pérdida de especies debido a la contaminación ambiental. Elénfasis en estudios florísticos locales, en lugar de estudio taxonómicos más amplios, ha enmascarado a las estimaciones de totales nacionales en casi todos los grupos taxonómicos. El conocimiento sobre angiospermas, especialmente monocotiledóneas (de las cuales 45% de las especies son endémicas), es el más completo. Las cifras para este grupo son más confiables, y se han identificado algunos patrones de distribución, niveles de endemismo y centros de diversidad. Sin embargo, falta mucho por descubrir. Están en progreso esfuerzos coordinados para catalogar la flora de Brasil e incluyen proyectos-que contribuyen enormemente al incremento de nuestro conocimiento -como talleres de definición de prioridades organizados por el Ministerio del Ambiente, que ha identificadoáreas clave para la conservación en los principales biomas; desarrollo de listas de especies de plantas amenazadas; y la organización de datos sobre especies tipo del noreste de Brasil por medio de la Iniciativa Darwin. Sin emb...

An expanded plastid DNA phylogeny for Orchidaceae was generated from sequences of rbcL and matK for representatives of all five subfamilies. The data were analyzed using equally weighted parsimony, and branch support was assessed with jackknifing. The analysis supports recognition of five subfamilies with the following relationships: (Apostasioideae (Vanilloideae (Cypripedioideae (Orchidoideae (Epidendroideae))))). Support for many tribal-level groups within Epidendroideae is evident, but relationships among these groups remain uncertain, probably due to a rapid radiation in the subfamily that resulted in short branches along the spine of the tree. A series of experiments examined jackknife parameters and strategies to determine a reasonable balance between computational effort and results. We found that support values plateau rapidly with increased search effort. Tree bisection-reconnection swapping in a single search replicate per jackknife replicate and saving only two trees resulted in values that were close to those obtained in the most extensive searches. Although this approach uses considerably more computational effort than less extensive (or no) swapping, the results were also distinctly better. The effect of saving a maximal number of trees in each jackknife replicate can also be pronounced and is important for representing support accurately.

Phylogenetic relationships within the epidendroid orchids with emphasis on tribes Epidendreae and Arethuseae were assessed with parsimony and model-based analyses of individual and combined DNA sequence data from ITS nuclear ribosomal DNA and plastid trnL intron, the trnL-F spacer, matK (gene and spacers), and rbcL regions. Despite the absence of boostrap support for some of the relationships, a well-resolved and supported consensus was found, for which most clades were present in more than one individual analysis. Most clades of this consensus attained high posterior probabilities with a Bayesian approach. Circumscription of Arethuseae and Epidendreae are different from most orchid systems based on morphology, but they correspond to a combination of patterns from several less comprehensive orchid phylogenetic analyses previously published. A new circumscription of Epidendreae includes only Neotropical subtribes (Bletiinae, Chysiinae, Laeliinae, Ponerinae, and Pleurothallidinae), whereas Arethuseae include Coelogyninae (all Old World) and Arethusinae (pantropical). Many previously included genera will need to be moved to other tribes. Taxa previously assigned to be Old World Epidendreae are related to different groups of Old World orchids, and this study can serve as a guide for sampling strategies in future studies to resolve troublesome epidendroid orchid clades.

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