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...

The ndhF sequences of 99 taxa, representing all sections in extant Magnoliaceae, were analyzed to address phylogenetic questions in the family. Magnolia macrophylla and M. dealbata, North American species of Magnolia section Rytidospermum, are placed at the base in the subfamily Magnolioideae although its supporting value is low. In the remaining taxa, several distinctive lineages are recognized: (1) Magnolia, the biggest genus in the family, is not monophyletic; (2) Michelia, including section Maingola of Magnolia subgenus Magnolia, is closely related with Elmerrillia and sections Alcimandra and Aromadendron of Magnolia subgenus Magnolia; (3) the associates of Michelia are grouped with Magnolia subgenus Yulania and section Gynopodium of Magnolia subgenus Magnolia; (4) Pachylarnax forms a clade with sections Manglietiastrum and Gynopodium of Magnolia; (5) a well-supported Manglietia clade is recognized; (6) Caribbean species of section Theorhodon of Magnolia subgenus Magnolia, which are section Splendentes sensu Vázquez-Garcia, are closely allied with New World members of Magnolia subgenus Talauma; and (7) section Rytidospermum of Magnolia subgenus Magnolia and subgenus Talauma are polyphyletic. The separated clades in the molecular tree are considerably different from traditional taxonomic dispositions in the family. The molecular data strongly suggest that a taxonomic realignment of infrafamilial delimitations and compositions should be considered.

Species of Orchidaceae are under severe threat of extinction mainly due to overcollection and habitat destruction; accurate identification of orchid species is critical in conservation biology and sustainable utilization of orchids as plant resources. We examined 647 sequences of the cpDNA regions rbcL, matK, atpF-atpH IGS, psbK-psbI IGS and trnH-psbA IGS from 89 orchid species (95 taxa) and four outgroup taxa to develop an efficient DNA barcode for Orchidaceae in Korea. The five cpDNA barcode regions were successfully amplified and sequenced for all chlorophyllous taxa, but the amplification and sequencing of the same regions in achlorophyllous taxa produced variable results. psbK-psbI IGS showed the highest mean interspecific K2P distance (0.1192), followed by matK (0.0803), atpF-atpH IGS (0.0648), trnH-psbA IGS (0.0460) and rbcL (0.0248). The degree of species resolution for individual barcode regions ranged from 60.5% (rbcL) to 83.5% (trnH-psbA IGS). The degree of species resolution was significantly enhanced in multiregion combinations of the five barcode regions. Of the 26 possible combinations of the five regions, six provided the highest degree of species resolution (98.8%). Among these, a combination of atpF-atpH IGS, psbK-psbI IGS and trnH-psbA IGS, which comprises the least number of DNA regions, is the best option for barcoding of the Korean orchid species.

Sequences from internal transcribed spacers (ITS) of nuclear ribosomal DNA were determined to examine phylogenetic relationships in the genus Acer. ITS 1 sequences in twenty-eight species of Acer and a species of Diptemnia in the family Aceraceae ranged from 220 to 242 bp and ITS 2 sequences from 215 to 251 bp. The size of the 5.8s coding region was 164 bp for all species examined in the family. Phylogenetic analysis of TTS sequences placed a very robust clade of section Palmata at the base of the tree. Three species of section Parvifora sensu de Jong (1994), A. spkatum, A. distylum and A. nipponicum, did not form a monophyletic clade. Acer spimfum was separated from the robust clade of A. disty/um and A. nipponicum. Molecular tree strongly supports the close relationship among section Plafanoidea, Glabra series Arguta, and section Macrantha. The close relationship between sections Penfaphyla and Trifoliata was also strongly suggested in ITS tree. Sections Rubra and Hyptiocarpa appeared to be closely allied with each other. The average rate of nucleotide substitution was estimated as (8.0f1.9)~10-" substitutions per site per year for ITS 1 and (9.0t-1.6)~10-~ for ITS 2.

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Invasive giant knotweed (Fallopia sachalinensis) is native to northeastern Asia. In Korea, F. sachalinensis is confined to two volcanic islands, Ullung and Dok islands, where it occurs as dodecaploids (2n = 132). We investigated the molecular variation in 104 accessions from 94 populations of F. sachalinensis and its relatives throughout their native range to elucidate the origin of these island populations. All F. sachalinensis plants on Ullung and Dok islands were uniquely dodecaploid, whereas other populations were tetraploid (2n = 44). Among the 39 cpDNA haplotypes identified, the accessions from these islands shared two unique haplotypes, and were resolved as a well-supported monophyletic clade. However, this clade was sister to a clade comprising F. japonica accessions from southwestern Japan and separated from the clade comprising F. sachalinensis from other areas; this relationship is inconsistent with morphological evidence. The monophyly of the F. sachalinensis populations on Ullung and Dok islands suggests a single colonization event. The progenitor was likely from Japan, where it possibly captured F. japonica var. japonica cpDNA via introgression. The Ullung Island populations subsequently differentiated through polyploidization and mutations post-introduction. Our results also indicate that giant knotweed in Europe and North America likely originated from northern Japan and/or Sakhalin Island.

The strong FSGS from genets is consistent with two biological and ecological traits of E. koreensis: bee-pollination and limited seed dispersal. Furthermore, potential clone mates over repeated generations would contribute to the observed high F(ij) values among genets at short distance. To ensure long-term ex situ genetic variability of the endangered E. koreensis, individuals located at distances of 10-12 m should be collected across entire populations of E. koreensis.

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