A DNA barcode for land plants
ABSTRACT 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.
- SourceAvailable from: Elizabeth A Zimmer[show abstract] [hide abstract]
ABSTRACT: Methods for identifying species by using short orthologous DNA sequences, known as "DNA barcodes," have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short ( approximately 450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes.Proceedings of the National Academy of Sciences 07/2005; 102(23):8369-74. · 9.74 Impact Factor
- Science 11/2007; 318(5848):190-1. · 31.20 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination.PLoS ONE 02/2007; 2(6):e508. · 3.73 Impact Factor
A DNA barcode for land plants
CBOL Plant Working Group1
Communicated by Daniel H. Janzen, University of Pennsylvania, Philadelphia, PA, May 27, 2009 (received for review March 18, 2009)
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
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 investiga-
tions 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 vari-
ous 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 labo-
ratories 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
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
arge-scale standardized sequencing of the mitochondrial
gene CO1 has made DNA barcoding an efficient species
Universality. 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 ?50% success in gymno-
sperms and/or cryptogams based on data compiled from several
laboratories (Fig. 1A).
Sequence Quality. Evaluation of sequence quality and coverage
from the candidate loci demonstrated that high quality bidirec-
tional sequences were routinely obtained from rbcL, rpoC1, and
rpoB (Fig. 1B, x axis). The remaining 4 loci required more
manual editing and produced fewer bidirectional reads. matK
performed best of this group, although it showed discordance
between forward and reverse reads more frequently than other
coding regions. The greatest problems in obtaining bidirectional
sequences with few ambiguous bases were encountered with the
intergenic spacers trnH–psbA and psbK–psbI, in part attributable
to a high frequency of mononucleotide repeats disrupting indi-
vidual sequencing reads.
Species Discrimination. Among 397 samples successfully se-
quenced for all 7 loci, species discrimination for single-locus
barcodes ranged from 43% (rpoC1) to 68%–69% (psbK–psbI
and trnH–psbA), with rbcL and matK providing 61% and 66%
discrimination respectively (rank order: rpoC1?rpoB?atpF–
Author contributions: P.M.H., L.L.F., J.L.S., M.H., S.R., M.v.d.B., M.W.C., R.S.C., D.L.E., A.J.F.,
V.S., O.S., M.J.W., and D.P.L. designed research; D.L.E., A.J.F., K.E.J., J.v.A.S., D.B., K.S.B.,
K.M.C., J.C., A.C., J.J.C., F.C., D.S.D., C.S.F., M.L.H., L.J.K., P.R.K., J.S.K., Y.D.K., R.L., H.-L.L.,
M.H., S.R., and D.P.L. analyzed data; and P.M.H., S.W.G., S.C.H.B., and D.P.L. wrote the
Conflict of interest statement: Following the publication of Lahaye et al. (PNAS 105:2923,
2008), the process of filing a patent on DNA barcoding of land plants using matK was
initiated by V.S., M.v.d.B., R.L., and D.B., but because of the lack of commercial interest the
patent application was subsequently dropped.
Freely available online through the PNAS open access option.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database. For a list of accession numbers, see SI Table 1. FASTA files of sequences are
available on request.
See Commentary on page 12569.
1CBOL Plant Working Group: Peter M. Hollingswortha,2, Laura L. Forresta, John L. Spougeb,
Mehrdad Hajibabaeic, Sujeevan Ratnasinghamc, Michelle van der Bankd, Mark W. Chasee,
Robyn S. Cowane, David L. Ericksonf, Aron J. Fazekasg, Sean W. Grahamh, Karen E. Jamesi,
Mark Carinei, Juliana Chaco ´np, Alexandra Clarka, James J. Clarksone, Ferozah Conradq,
Husbandg, Laura J. Kellya,e, Prasad R. Kesanakurtig, Jung Sung Kimj, Young-Dong Kimt,
Renaud Lahayed, Hae-Lim Leej, David G. Longa, Santiago Madrin ˜a ´np, Olivier Maurind,
Isabelle Meusnierc, Steven G. Newmasterg, Chong-Wook Parku, Diana M. Percyh, Gitte
Petersenv, James E. Richardsona, Gerardo A. Salazarw, Vincent Savolainene,x, Ole Sebergv,
Michael J. Wilkinsonr, Dong-Keun Yij, and Damon P. Littley
aRoyal Botanic Garden Edinburgh, Edinburgh EH3 5LR, United Kingdom;bNational Center
Computational Biology Branch, Bethesda, MD 20894;cBiodiversity Institute of Ontario,
Department of Integrative Biology, University of Guelph, Guelph, ON, Canada N1G 2W1;
Auckland Park, Johannesburg 2006, South Africa;eRoyal Botanic Gardens, Kew, Richmond
TW9 3DS, United Kingdom;fDepartment of Botany, Smithsonian Institution, Washington
DC, 20013-7012;gDepartment of Integrative Biology, University of Guelph, Guelph, ON,
Canada N1G 2W1;hUBC Botanical Garden and Centre for Plant Research, Faculty of Land
and Food Systems, and Department of Botany, University of British Columbia, Vancouver,
BC, Canada V6T 1Z4;iBotany Department, Natural History Museum, London SW7 5BD,
United Kingdom;jSchool of Life Sciences and Biotechnology, Korea University, Seoul
136-701, Korea;kDepartment of Ecology and Evolutionary Biology, University of Toronto,
Toronto, ON, Canada M5S 3B2;lLaborato ´rio de Sistema ´tica Molecular de Plantas, Univer-
sidade Estadual de Feira de Santana, Departamento de Cie ˆncias Biolo ´gicas, 44031-460,
Feira de Santana, Bahia, Brazil;mJardín Bota ´nico Lankester, Universidad de Costa Rica,
Cartago, Costa Rica;nDepartment of Biology, Columbus State University, Columbus, GA
31907-5645;oDepartment of Botany, University of Wisconsin, Madison, WI 53508;pUni-
versidaddelosAndes,ApartadoAe ´reo4976,Bogota ´,D.C.,Colombia;qLeslieHillMolecular
South Africa;rInstitute of Biological, Environmental and Rural Sciences, Aberystwyth
University, Ceredigion SY23 3DA, United Kingdom;sDepartment of Botany, University of
Cape Town, Rondebosch 7700, South Africa;tDepartment of Life Sciences, Hallym Univer-
sity, Chuncheon 200-702, Korea;uSchool of Biological Sciences, Seoul National University,
Seoul 151-742, Korea;vNatural History Museum of Denmark, University of Copenhagen,
1307 Copenhagen K, Denmark;wInstituto de Biología, Universidad Nacional Auto ´noma de
Me ´xico,04510Me ´xico,D.F.,Mexico;xImperialCollegeLondon,SilwoodParkCampus,Ascot
SL5 7PY, United Kingdom; andyCullman Program for Molecular Systematics, New York
Botanical Garden, Bronx, NY, 10458-5126
2To whom correspondence should be addressed. E-mail: P.Hollingsworth@rbge.org.uk.
This article contains supporting information online at www.pnas.org/cgi/content/full/
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no. 31 www.pnas.org?cgi?doi?10.1073?pnas.0905845106
atpH?rbcL?matK?psbK–psbI?trnH–psbA; Fig. 1B, y axis).
Two-locus combinations gave 59%–75% resolution, and 3-locus
combinations 65%–76% (Fig. 1C). Ten of the 2-locus combina-
tions gave 70%–75% discrimination. The top 5 of these involved
various combinations of rbcL, psbK–psbI, matK, and trnH–psbA.
Using all 7 loci, 73% of species were discriminated. When the
species discrimination analyses are extended to the full sample,
which includes those that failed to sequence for 1 or more loci,
the rank order among single-locus comparisons is rpoC1 (38%),
rpoB (40%), atpF–atpH (50%), matK (57%), rbcL (58%), trnH–
psbA (58%), and psbK–psbI (64%). The rise in relative perfor-
mance of rbcL is associated with its strong (87%) discriminatory
power in the cryptogam samples. These were excluded from the
preceding analyses as all had missing data from 1 or more loci.
An ideal DNA barcode should be routinely retrievable with a
single primer pair, be amenable to bidirectional sequencing
with little requirement for manual editing of sequence traces,
and provide maximal discrimination among species. Based on
these criteria, 4 of the candidate loci can be excluded (Fig. 1
A and B). Both rpoC1 and rpoB performed well in terms of
universality and/or sequence quality, but had low discrimina-
tory power; atpF–atpH fell below the median for species
resolution in single and multilocus barcodes and for recovery
of high-quality bidirectional sequences; whereas psbK–psbI
showed good discriminatory power, but had the lowest se-
quencing success in these trials, and substantial problems
generating bidirectional reads.
Choosing a plant barcode from the 3 remaining candidate
loci was more difficult. Individually, trnH–psbA, rbcL, and
matK possess attributes that are highly desirable in a plant
DNA barcoding system, although none of the 3 loci fits all 3
criteria perfectly. As reported elsewhere (7), trnH–psbA dem-
onstrated good amplification across land plants with a single
pair of primers (93% for angiosperms; Fig. 1A) and high levels
of species discrimination. However, problems obtaining high
quality bidirectional sequences are the primary limitation
for this locus. In addition, trnH–psbA has a median length of
418 bp (IQR ? 296–500 bp) in the dataset examined here,
which is well-suited for DNA barcoding, but its upper length
of ?1,000 bp in some monocot (3) and conifer (11) species can
lead to problems obtaining bidirectional sequences without
using taxon-specific internal sequencing primers.
Among plastid regions, rbcL is the best characterized gene.
Improvements in primer design make it easily retrievable
across land plants (8) and it is well suited for recovery of
high-quality bidirectional sequences. Although not the most
variable region (Fig. 1B), it is a frequent component of the best
performing multi-locus combinations for species discrimina-
tion (Fig. 1C).
matK is one of the most rapidly evolving plastid coding
regions and it consistently showed high levels of discrimination
among angiosperm species (Fig. 1C) (8, 9). Mixed reports have
been published regarding the universality of matK primers,
ranging from routine success (9) to more patchy recovery (7,
8), which has led to reservations about this locus by some
researchers. In the current study, 90% of the angiosperm
samples tested were successfully amplified and sequenced
using a single primer pair (Fig. 1A). Success in gymnosperms
(83%) and particularly cryptogams (10%) was more limited,
even when multiple primer sets were used.
In summary, rbcL offers high universality and good, but not
outstanding discriminating power, whereas matK and
trnH–psbA offer higher resolution, but each requires further
development work. Primer universality needs improvement
for matK in some clades, and trnH–psbA does not consis-
tently provide bidirectional unambiguous sequences, often
requiring manual editing of sequence traces. Thus, no single
locus meets CBOL’s data standards and guidelines for locus
selection, and as a result a synergistic combination of loci is
One option preferred by some researchers in the CBOL Plant
Working Group was a 3-locus barcode of matK?rbcL?trnH-psbA,
to allow further testing of these loci. Based on the relative perfor-
mance of the 3 loci, the best 2-locus barcode could be selected at
a later date. The majority preference, however, was to select a
rather than 2 in very large sample sets, and (b) prevent further
locus codes at head of Fig. 1A). (A) Universality success based on 170 angio-
for up to 81 gymnosperm and 156 cryptogam samples. (B) Assessment of
which high quality bidirectional sequences (contigs) could be assembled (see
are indicated. Colors reflect sequence quality (red, worse; green, better). (C)
Discrimination success for 1–3 and 7 locus barcodes for species for which
multiple individuals from multiple congeneric species were sampled, and all 7
loci were recovered. Outer error bars (thin lines) demarcate 95% confidence
intervals. Inner error bars (thick lines) indicate the relative magnitude of
discrimination failure as measured by the interquartile range (IQR) for the
number of species that are indistinguishable from a given query sequence.
Discrimination success from all 7 loci is shown with a white line, with the
associated 95% confidence interval in light gray, and the magnitude of
discrimination failure in dark gray. Colors indicate the average percentage of
finished bidirectional sequences expected for each locus combination. The
arrow indicates the recommended standard 2-locus barcode.
Comparison of the performance of 7 candidate barcoding loci (see
CBOL Plant Working GroupPNAS ?
August 4, 2009 ?
vol. 106 ?
no. 31 ?
delays in implementing a standard barcode for land plants. In the
datasets examined here, sequencing 3 loci did not improve discrim-
ination beyond the best performing 2-locus barcodes.
Among the 2-locus barcode combinations, rbcL?matK was
the majority choice for several reasons. High-quality sequences
of rbcL are easily retrievable across phylogenetically divergent
lineages, and it performs well in discrimination tests in combi-
nation with other loci. Developing amplification strategies for
matK was considered an investment with better prospects for
return than solving the problem of sequence quality in trnH–
psbA caused by mononucleotide repeats (13). Recent primer
development for matK has improved its recovery from angio-
sperms, and so prospects for further improvement in angio-
sperms and other land plant groups seem reasonable, analogous
to the extensive improvements made to primer sets for CO1 for
animal DNA barcoding (14).
We therefore propose rbcL?matK as the standard barcode for
land plants. This combination represents a pragmatic solution to
a complex trade-off between universality, sequence quality,
discrimination, and cost. Using rbcL?matK in the sample set
examined here, species discrimination was successful in 72% of
cases, with the remaining species being matched to groups of
congeneric species with 100% success. Given the logistical
difficulties of undertaking identifications with some ?400,000
species of land plant, this 2-locus barcode offers the opportunity
to harness high-throughput automated sequencing technologies
to establish a powerful universal framework for DNA-based
identification of plants.
The unique identification to species level in 72% of cases
and to ‘species groups’ in the remainder will be useful for many
applications of DNA barcoding such as studies of plant-animal
interactions (15), establishing whether plant products in in-
ternational trade belong to protected species (9, 16, 17),
discriminating among seedlings to establish forest regenera-
tion dynamics, or undertaking large-scale biodiversity surveys
with limited access to taxonomic expertise. A particular
strength of the barcoding approach is that these identifications
can be made with small amounts of tissue from sterile, juvenile
or fragmentary materials from which morphological identifi-
cations are difficult or impossible (18). In addition, it is
important to emphasize that the discriminatory power of this
standard barcode will be higher in situations that involve
geographically restricted sample sets, such as studies focusing
on the plant biodiversity of a given region or local area (19, 20).
A future challenge for DNA barcoding in plants is to
increase the proportion of cases in which unique species
identifications are achieved. In the short term, where further
resolution and universality are required, we envisage that the
core rbcL?matK barcode will be augmented in individual
projects from a flexible short-list of supplementary loci in-
cluding the noncoding plastid regions examined here (trnH–
psbA, atpF–atpH, and psbK–psbI), and the trnL intron which
has been advocated for situations involving highly degraded
tissue (19). The rapidly evolving internal transcribed spacers of
nuclear ribosomal DNA also represent a useful supplementary
barcode in taxonomic groups in which direct sequencing of this
locus is possible (21). Moving beyond these currently available
supplementary barcodes, ongoing advances in sequencing
technologies and the concomitant accumulation of genomic
and transcriptomic sequence data from plants will greatly
increase opportunities for targeting the nuclear genome as a
source of informative characters.
There is little doubt that the approaches used in plant DNA
barcoding will be refined in future (22). However, the key
foundation step for plant barcoding is in reaching agreement on
a standard set of loci to enable large-scale sequencing and the
development of a global plant barcoding infrastructure. The
broad community agreement presented here, to sequence rbcL
and matK as a standard 2-locus barcode, is thus an important
for taxonomy, conservation, and the multitude of other appli-
cations (23) that require identification of plant material.
Materials and Methods
Plant Materials. We used a total of 907 samples from 550 species representing
the major lineages of land plants (including 670/445 angiosperm, 81/38 gym-
nosperm, and 156/67 cryptogam samples/species) to evaluate the candidate
barcoding loci (Fig. S1, Fig. S2, and Table S1; cryptogams are defined here as
all non–seed bearing embryophytes).
Universality. To provide directly comparable information on universality
and trace quality (see below), we generated de novo sequence data from
190 samples (including 170 angiosperms) at the Canadian Centre for DNA
Barcoding (CCDB), University of Guelph, using a single primer pair per locus
(Table S1). We used this dataset to quantify universality in angiosperms. As
amplification and sequencing success is typically lower in nonangiosperm
data on amplification and sequencing success from different laboratories
for cryptogams; Table S1). Our assessments of universality simply record
whether sequence data were obtained, regardless of the amount of man-
ual trace editing required or the extent of read bidirectionality. Full details
of molecular methods are available from the corresponding author on request.
with minimal requirement for manual editing of sequences, we examined the
quality of the de novo generated sequence traces via the CCDB automated
defined such that both the forward and reverse reads should have a minimum
be ?50% of the original read length; the assembled contig should have ?50%
bases (?20QV) and ?1% internal gaps and substitutions when aligning the
forward and reverse reads. These quality control criteria were selected as a
quality sequences. Various permutations of the parameters resulted in the same
general conclusions (rbcL, rpoC1, and rpoB performed well, matK was interme-
psbA, psbK–psbI, and atpF–atpH).
which all 7 loci were successfully sequenced (397 samples, all seed plants). We
restricted assessment of discrimination success to species where multiple
species from 34 genera). Although not counted in the discrimination success
statistics, a further 104 singleton-sampled species congeneric with the above,
as potential sources of discrimination failure. Using the same samples for all 7
loci allowed us to directly compare the relative discriminatory power of the
different loci. We considered discrimination as successful if the minimum
uncorrected interspecific p-distance involving a species was larger than its
maximum intraspecific distance [all distances were calculated from pairwise
global alignments counting unambiguous base substitutions only (24)]. We
of the distance measure for all possible 2–7 locus combinations and recording
the success of each multi-locus combination. We used the binomial distribu-
tion to calculate 95% confidence intervals to establish whether performance
Species discrimination assessments were then repeated on a dataset of 907
but not all loci. Multi-locus combinations were not evaluated in this dataset
because of large numbers of zero-distances introduced by individuals being
represented by mutually exclusive loci.
for data formatting, and George Weiblen for plant material. This work was
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Foundation, Genome Canada, Scottish Government’s Rural and Environment
Research and Analysis Directorate, Royal Society, South African National
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