Botanical Journal of the Linnean Society, 2009, 159, 1–11.

Selection of candidate coding DNA barcoding regions

for use on land plants
CAROLINE S. FORD1, KAREN L. AYRES2, NICOLA TOOMEY3, NADIA HAIDER4,
JONATHAN VAN ALPHEN STAHL5, LAURA J. KELLY5, NIKLAS WIKSTRÖM6,
PETER M. HOLLINGSWORTH7, R. JOEL DUFF8, SARAH B. HOOT9,
ROBYN S. COWAN5, MARK W. CHASE5 and MIKE J. WILKINSON1*
1
Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth
SY23 3DA, UK
2
School of Biological Sciences, University of Reading, Reading RG6 6FN, UK
3
Department of Biological Sciences, Imperial College, Silwood Park, Ascot SL5 7PY, UK
4
Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS),
Damascus, PO Box 6091, Syria
5
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond TW9 3DS, UK
6
Department of Systematic Botany, Uppsala University, Norbyvägen 18D, 752 36 Uppsala, Sweden
7
Royal Botanic Garden Edinburgh, 20a Inverleith Row, Edinburgh EH3 5LR, UK
8
Department of Plant Biology, Southern Illinois University, Carbondale, IL 62901-6509, USA
9
Department of Biological Sciences, University of Wisconsin, Lapham Hall 181, 3209 N. Maryland
Ave., Milwaukee, WI 53211, USA

Received 13 April 2008; accepted for publication 25 September 2008

An in silico screen of 41 of the 81 coding regions of the Nicotiana plastid genome generated a shortlist of 12
candidates as DNA barcoding loci for land plants. These loci were evaluated for amplification and sequence
variation against a reference set of 98 land plant taxa. The deployment of multiple primers and a modified
multiplexed tandem polymerase chain reaction yielded 85–94% amplification across taxa, and mean sequence
differences between sister taxa of 6.1 from 156 bases of accD to 22 from 493 bases of matK. We conclude that loci
should be combined for effective diagnosis, and recommend further investigation of the following six loci: matK,
rpoB, rpoC1, ndhJ, ycf5 and accD. © 2009 The Linnean Society of London, Botanical Journal of the Linnean
Society, 2009, 159, 1–11.

ADDITIONAL KEYWORDS: genetic barcodes – matK – MT-PCR – multi-locus barcode – plant identification
– plastid genes.

INTRODUCTION divergent selection and efficacy of genome repair. The

absence of routine protocols for rapid, low-cost and
The proliferation of genome sequence data has
reliable genome sequencing of all species means that
allowed paradigm shifts in our understanding of cell
there is a growing desire to target shared genomic
biology and gene function, but has largely failed to
regions as a common platform for DNA-based species
enhance species diagnosis. Absolute genome sequence
diagnosis. However, some incongruity is inescapable
divergence between sister species is unknown, but
if identification is based on limited DNA information,
will inevitably be highly variable between species
because there can only be a correlative link between
pairs, and will depend on a range of factors, including
the sequences used and true species identity. To con-
time since speciation, mutation rate, extent of
found matters, there is variability in the mechanisms
of speciation and contexts in which speciation occurs
*Corresponding author. E-mail: jjw@aber.ac.uk and inconsistency in the taxonomic treatment of

© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 1–11 1
2 C. S. FORD ET AL.
analogous situations across divergent taxa. Despite
such limitations, the strategy of using limited DNA
information to aid species identification has consider-
able attraction for many practical, commercial and
scientific applications (Newmaster, Fazekas &
Ragupathy, 2006). This is known as DNA barcoding.
Barcoding efforts are proceeding for many animal
groups using the mitochondrial cytochrome oxidase C
gene (CO1). DNA sequences of this gene contain suf-
ficient variability to distinguish species of birds
(Hebert et al., 2004a), fish (Ward et al., 2005), spiders
(Greenstone et al., 2005) and some butterflies (Hebert
et al., 2004b; Hajibabaei et al., 2006), and the gene
has been designated as the current barcoding region
for the animal kingdom (Hebert, Ratnasingham &
deWaard, 2003). Moreover, pilot studies are now
assessing the utility of CO1 for barcoding fungi
(Seifert et al., 2007), diatoms (Evans, Wortley &
Mann, 2007) and red algae (Robba et al., 2006).
The use of this mitochondrial region largely avoids
problems of locus and genome duplication that are
commonplace in the nuclear genome, as they only
occasionally occur for mitochondrial genes through
horizontal transfer (Timmis et al., 2004). However, in
some groups, CO1 is an unsuitable barcoding marker.
Barcoding efforts are less well developed for these
taxa, primarily because an equivalent diagnostic
region has yet to be identified. Land plants are the
most prominent of these ‘orphan groups’, in which
relative sequence uniformity of the mitochondrial
genome precludes the use of CO1 as a barcoding
marker (Cho et al., 1998, 2004; Adams & Palmer,
2003; Chase et al., 2005). The difficulty in developing
barcoding markers for land plants is exacerbated by
the extensive evolutionary divergence between taxa
since they first appeared c. 475 million years ago
(Wellman, Osterloff & Mohiuddin, 2003), their
propensity to undergo both large- and small-scale
nuclear genome rearrangements, the widespread
appearance of interspecific and intergeneric hybrids,
and the huge diversity in their life history and breed-
ing behaviour. This complexity has long been recog-
nized and has even complicated the establishment of
universal species concepts across the group. Thus, the
‘land plant genome’ has great depth and breadth in its
variability and presents a particularly difficult chal-
lenge in the search for suitable DNA barcoding loci.
KEY CRITERIA FOR BARCODING LOCI
As an underlying principle of DNA barcoding is the
high-throughput standardized identification of bio-
logical samples, the DNA region(s) used for plant
barcoding must be: (1) routinely amplifiable; (2) easily
sequenced via single-pass sequencing; (3) sufficiently
variable to separate most sister species pairs and yet
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 1–11
also show sufficient sequence consistency to ensure
that infraspecific variation does not confound species
assignation; (4) unsusceptible to the amplification
of loci other than the targeted plastid region; and
(5) easily annotated for the critical evaluation of
sequence quality and error detection.
The search for a parallel region to CO1 in land plants
that matches these criteria has focused on the plastid
genome, which benefits from a high copy number per
cell, enabling amplification from degraded samples,
and has a known gene order and function for an
increasing number of land plants. However, it seems
likely that the modest levels of sequence divergence of
plastid DNA when compared with CO1 in animals
(Chase et al., 2007) will dictate the use of multiple loci
to allow species diagnosis. Several plastid regions have
been proposed (Chase et al., 2007), with greatest inter-
est shown in the protein-encoding rbcL gene (Chase
et al., 2005; Newmaster et al., 2006; Kress & Erickson,
2007) and the predominantly non-coding psbA-trnH
intergenic spacer (Kress et al., 2005; Kress & Erickson,
2007; Shaw et al., 2007). In addition, K. J. Kim (http://
www.barcoding.si.edu/plant_working_group.html) has
suggested the combined use of coding and non-coding
loci (either matK + atpF-H + psbK-I or matK + atpF-
H + trnH-psbA) as multi-locus barcodes.
The suggestion of rbcL as a barcoding region relates
to its historical popularity as a gene for deep-level
phylogenetic studies, rather than being based on a
systematic evaluation of plastid coding regions for
DNA barcoding. There are thousands of rbcL
sequences from phylogenetic studies already depos-
ited in public databases, and routine protocols exist
for sequence generation. However, the absence of
vouchers or electropherograms for the great majority
of sequences precludes their use for barcoding,
although they may still have utility as an aid to
identification. The length of the rbcL gene (c. 1400
bases) also dictates the use of multiple-pass sequenc-
ing, which further compromises its value as a barcode
region if used in its entirety. Its application therefore
depends on the identification of suitable diagnostic
sections of the gene (Kress & Erickson, 2007). In silico
tests of a 550–600-base subset of rbcL have shown
that the section can generally resolve samples to the
family and genus level (Kress & Erickson, 2007).
Despite efforts, hopes of producing universal primers
to amplify short, diagnostic regions of rbcL suitable
for barcoding (allowing single-pass sequencing) have
proved elusive (Chase et al., 2007).
The psbA-trnH intergenic spacer is one of the most
variable regions of the plastid genome (Shaw et al.,
2007) and, in this respect, has potential as a candi-
date barcoding region for land plants (Kress et al.,
2005; Kress & Erickson, 2007). However, much of its
variability occurs as indels, with amplicons targeting
 DNA BARCODING REGIONS FOR LAND PLANTS
the region typically exhibiting considerable variation
in size and leading to alignment difficulties even
between closely related taxa (Chase et al., 2007).
There are also problems associated with duplications
and deletions in this locus (Sass et al., 2007). The
degree to which sequence alignment is necessary for
barcoding purposes is a matter of debate (Chase et al.,
2005, 2007; Kress et al., 2005; Newmaster et al., 2006;
Kress & Erickson, 2007). Little & Stevenson (2007)
contend that alignment-free methods can perform
well in barcoding studies. However, a more practical
issue arising from the use of a non-coding region is
that the absence of a reading frame means that it is
impossible for end-users to exploit the translated
(amino acid) sequence as an independent assessment
of sequence quality. Also lost is the capacity to use the
amino acid sequence as a strong supporting criterion
to infer orthology of genome location and function
between amplicon and target locus, i.e. to confirm
whether the amplicon derives from the plastid or from
a pseudogene within the nuclear genome that shares
primer binding sites and possibly a common ancestral
origin via horizontal gene transfer.
Although the debate regarding the use of non-
coding regions in DNA barcoding is ongoing based on
the high levels of sequence variability observed in
such loci, there are clear benefits of using easily
alignable barcode data from coding loci, as these
allow independent checks of sequence quality (via in
silico translation) and thereby provide some protec-
tion against the inadvertent use of nuclear pseudo-
genes. An added benefit of coding regions is that these
data are also more amenable for re-use in phyloge-
netic studies. Given these potential benefits, a thor-
ough systematic investigation of the potential plastid
coding regions for DNA barcoding in land plants is
important. In this paper, we examine the genic
regions of the plastid genome with barcoding poten-
tial. Our search has focused on regions containing
areas of conserved amino acid sequence and hence
assumed functional importance to act as targets for
primer binding; these flank exons of 200–800 bases
and do not contain introns.
MATERIAL AND METHODS
IDENTIFICATION OF GENE REGIONS
We examined 41 of the 81 functionally characterized
loci from the 95 protein coding regions of the Nicotiana
tabacum plastid genome (GenBank accession
NC_001879) for suitability as barcoding candidates
(Table S1, see Supporting Information). Each coding
region, as listed on the Entrez Genome page of the
National Center for Biotechnology Information (NCBI)
website (http://www.ncbi.nlm.nih.gov/genomes/altvik.
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 1–11
3
cgi?gi=13349&db=g&from=0), was individually ‘blast
searched’ using the search function on this page to
identify related sequences in the database. All plant
sequences returned were aligned together as amino
acids using default settings of ClustalW (http://
www.ebi.ac.uk/clustalw/). These alignments were then
employed as the basis for the elimination of genes from
further assessment using the following categories: (1)
genes not conserved across > 98% of species for which
complete plastid sequences are available; (2) genes
lacking conserved regions for the development of
universal primers; and (3) genes containing introns
or showing extensive size variation (such as those
showing > 1.3-fold difference in length). The remaining
genes were examined further to identify regions of the
sequence of suitable amplicon size (180–800 bases) in
which variation could be observed between species and
that were flanked by conserved regions that could be
utilized as target sites for polymerase chain reaction
(PCR) primers. The remaining 40 loci from the
Nicotiana plastid were evaluated in a separate study
(Haider, 2003; N. Haider, pers. comm.), and are not
presented here.
UNIVERSAL PRIMER DEVELOPMENT
Primer binding sites were selected in regions of con-
served amino acid sequence where the nucleotide
sequence contained 40–60% GC content in a region
flanking a 180–800-bp amplicon. Primers were
designed: (1) to be 20–24 bp in length with similar
melting temperature (Tm); (2) to lack internal nucle-
otide complementarity and redundancy; (3) to be
exonic and terminate the 5′ end of the primer on
codon position 2 (forward primers) or 1 (reverse
primers); and (4) to have any existing variation
between sequences only at the third position nucle-
otide in the triplet. Four primers were designed for
each of the 12 loci, two forward and two reverse
(Table S2, see Supporting Information). An additional
forward primer was designed for matK to address
some of the problems experienced with universality
during testing.
DNA SAMPLES
The ability of primers to generate amplicons was
tested empirically using 98 taxa comprising 46 con-
generic species pairs and two species triplets from the
same genus. The test series comprised four liver-
worts, six pteridophytes, six gymnosperms, 28 mono-
cotyledons and 54 other angiosperms (Table S3, see
Supporting Information). An initial screen for
sequence polymorphisms between ‘sister species’ was
performed, with ten diverse pairs of angiosperm
species selected for reliable amplification in the initial
4 C. S. FORD ET AL.
PCR screen (Table S3). The second round of screening
for sequence variability was performed using all 98
taxa. Nearly all DNA was supplied pre-extracted from
the Royal Botanic Gardens, Kew DNA Bank (http://
www.kew.org/data/dnabank/homepage.html). Excep-
tions were those samples for bryophytes and some
fern allies.
STANDARD PCR AND SEQUENCING
PCR was performed using Bioline BIOTAQTM DNA
polymerase or Bioline BioMixTM in a 20-mL reaction
according to the manufacturer’s instructions. Ther-
mocycling conditions were optimized for all primer
pairs at 94 °C for 1 min, followed by 40 cycles of 94 °C
for 30 s, 53 °C for 40 s and 72 °C for 40 s, with a final
extension step of 72 °C for 5 min. Products were sub-
mitted for sequence analysis to either The BioCentre,
University of Reading (http://www.biocentre.reading.
ac.uk) or Macrogen (http://www.macrogen.com). Cycle
sequencing reactions were carried out in both cases
according to Sanger, Nicklen & Coulson (1977) using
BigDye Terminator 3.1 (Applied Biosystems). Manual
editing of raw traces and subsequent alignments of
forward and reverse sequences enabled us to assign
edited sequences for most species. The 3′ and 5′
termini were clipped to generate consensus sequences
for each taxon. Nucleotide sequences were then
translated into amino acid sequences using ExPASY
(http://www.expasy.ch/tools/dna.html).
MODIFIED MULTIPLEXED TANDEM PCR (MT-PCR)
Taxa showing repeated null amplification for specific
loci were subjected to a modified MT-PCR technique.
This method was originally developed to ensure
the detection of rare mRNAs from mixed samples
(Stanley & Szewczuk, 2005), and generates high-
quality sequences from low levels of cDNA template
(Nolan, Hands & Bustin, 2006; Jex et al., 2008). The
same approach was used here to increase the capacity
to detect potentially low-concentration (or quality)
template DNA. The nested approach used by MT-PCR
utilizes the two primer pairs designed for each loci,
with the outer pair yielding first-round pre-
amplification and the inner pair using the first-round
product as template for second-round amplification.
First-round pre-amplification was performed employ-
ing the outer forward primer (usually designated 1,
but see Table S2) and the outer reverse primer
(usually designated 4, but see Table S2) in a PCR
using Bioline BioMixTM in a 20-mL reaction compris-
ing: 94 °C for 1 min, followed by 15 cycles of 94 °C for
30 s, 51 °C for 40 s and 72 °C for 40 s, with a final
extension step of 72 °C for 5 min. The use of only 15
cycles ensures that the amplification remains in the
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 1–11
exponential phase during pre-amplification. This
reduces the incidence of de novo mutations arising
from mispriming during first-round amplification,
and so increases the fidelity of the template for
second-round amplification. The resulting products
were diluted 1 : 12.5, and 2.5 mL of this product was
used as template for the second amplification employ-
ing the inner forward primer (Table S2) and the inner
reverse primer (Table S2) in a PCR using Bioline
BioMix(TM) in a 20-mL reaction as follows: 94 °C for
1 min, followed by 40 cycles of 94 °C for 30 s, 51 °C for
40 s and 72 °C for 40 s, with a final extension step of
72 °C for 5 min. The resultant amplicons were sub-
mitted to Macrogen for purification and single exten-
sion sequencing in the forward and reverse directions
using the inner primer pair.
SEQUENCE ANALYSIS AND VERIFICATION
Consensus sequences were produced for each taxon at
each locus by alignment of the forward and reverse
sequences using ClustalW (http://www.ebi.ac.uk/
clustalw/). All sequences were blastn searched (http://
www.ncbi.nlm.nih.gov/BLAST/) to verify taxon (or
close taxonomic group) and locus. The sequences were
then translated into amino acids using ExPASY to
yield sequence and frame-shift errors identified by the
presence of stop codons. Errors were removed if pos-
sible via comparison with the sister taxon amino acid
sequence and with the original trace data. If ambi-
guities remained, sequences were either discarded
or re-sequenced. Corrected amino acid sequences
were then verified using a blastp search to known
sequences. Sequences that failed to blast (either as a
nucleotide or amino acid sequence) to the expected
target were excluded from further analysis.
Sequences remaining after verification were aligned
in species pairs using ClustalW and clipped to equal
length. Verified sequences for selected loci were sub-
mitted to the European Molecular Biology Laboratory
(EMBL) (Table S4, see Supporting Information).
SEQUENCE VARIABILITY
Comparison of species pairs to determine numbers of
polymorphisms and indels for each marker was per-
formed using a program written in C (Program S1).
Variation in size and sequence meant that global
alignments resulted in short read lengths for most of
the loci used. We therefore aligned the sequences in
congeneric pairs separately, so that we maximized the
read length for each pair. For species triplets (Erica,
Protea), one sequence was discarded to leave a pair.
Polymorphisms were identified whenever a site dif-
fered between the two aligned sequences for a species
pair. Each individual indel was treated as a dummy
 DNA BARCODING REGIONS FOR LAND PLANTS 5

Table 1. Polymerase chain reaction (PCR) amplification for 12 target loci (one primer pair each) across 98 taxa and
sequence polymorphisms between ten species pairs for 11 loci

PCR amplification Sequence polymorphisms

Species Percentage Combined amplicon Variable Percentage of

Gene amplified N/98 success Rank length (bp) bases variable bases Rank (%)

accD 65 66.3 6 2155 84 3.90 1

matK 40 41.8 11 4548 52 1.14 6
ndhA 63 64.3 8 3171 22 0.69 8
ndhJ 78 79.6 2 3946 63 1.60 4
ndhK 88 89.8 1 1710 5 0.29 10
rpl22 27 27.6 12 – – – –
rpoB 74 75.5 4 4129 38 0.92 7
rpoC1 78 79.6 2 4690 70 1.49 5
rpoC2 64 65.3 7 4867 32 0.66 9
ycf2 67 68.4 5 3504 9 0.26 11
ycf5 51 52.0 10 2927 66 2.25 3
ycf9 60 61.2 9 1263 29 2.30 2

The amplification of products with the expected size was expressed and ranked by the number of species (N/98) and
percentage success. For each marker, sequence variability is expressed as the number and percentage of variable bases
of total length. Markers ranked (highest to lowest) by percentage of variable bases.

base, and therefore usually resulted in a single nucle-
otide polymorphism (SNP). Sequence length was
recorded as zero for species pairs that did not produce
clean sequences. A further C program (Program S2)
took the resulting number of SNPs and sequence
length per species pair, and summed these over all
possible combinations of markers. To maximize the
use of the available data, all species pairs yielding
adequate sequences for a given subset of markers
were used to obtain results for that subset, resulting
in different numbers of species pairs being used for
different marker subsets. The results were expressed
as the mean number and percentage of polymorphic
sites over available pairs.
RESULTS
LOCUS SELECTION
ClustalW alignments of the amino acid sequences of
41 of the 81 functionally characterized coding regions
in the Nicotiana plastid genome (GenBank accession
NC-001879) yielded a candidate shortlist of 12 loci
meeting the criteria given in ‘Materials and methods’.
These top 12 plastid gene regions were selected for
further primer design and empirical evaluation
(Tables 1, S1, S2).
INITIAL SCREEN OF THE POTENTIAL FOR
WIDESPREAD AMPLIFICATION
The initial screen tested the ability of one primer pair
for each locus to yield a single strongly amplified
product of appropriate size across 98 divergent taxa.
All loci yielded individual products of appropriate size
in > 40% of species trialled, except for rpl22 (27.6%),
with ndhK (89.8%) amplifying products most consis-
tently (Table 1). Accordingly, 11 loci (all except rpl22)
were carried forward to a second screen to assess the
fidelity of amplification and to provide preliminary
information on sequence variability.
AMPLIFICATION FIDELITY AND PRELIMINARY
COMPARISON OF DNA SEQUENCES
A second screen was performed to test amplicon orthol-
ogy and to provide an initial evaluation of sequence
variation. In this case, we used ten congeneric species
pairs selected for consistency of amplification across
most loci in the initial screen (Table S3). In this way,
we sought to rank loci on the basis of sequence
variability rather than on the ability to amplify.
Representative sequences of all coding regions were
obtained from four species pairs (Begonia, Coffea,
Hieracium and Paeonia), whereas four species pairs
produced good sequences for all except one locus
(Aglaia and Sophora missing matK; Betula missing
rpoC2; Dioscorea missing ycf9). Acorus failed to yield
sequenced amplicons for two loci (matK and ndhA) and
Carex failed to amplify for six loci (rpoC1, matK, ycf2,
ycf5, ndhK and ndhA) (Table S5, see Supporting Infor-
mation). Thus, data for all species pairs were obtained
for accD, ndhJ and rpoB.
In a few cases, in silico translation of raw trace data
revealed sequences that included one or more stop

© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 1–11
6 C. S. FORD ET AL.
Table 2. Amplification and sequence success of six plastid markers
Region accD matK
Species pairs tested 48 48
Species pair sequences 35 32
Percentage amplification 73 67
Mean amplicon length (bp) 212 724
The first two species in each of the triplets are counted as a ‘pair’ in this analysis.
codons. There were often modest quality traces around
these codons and their appearance was frequently
associated with mononucleotide repeats. We therefore
adopted the conservative approach of deeming these
sites to contain possible artefactual, frame-shifting
indels. It was often possible to confirm such artefacts
by direct comparison with the equivalent sequence
from the sister species, but only sometimes by careful
re-examination of all trace data. For this screen,
manual correction of such instances generally involved
a simple frame-shift correction that removed the stop
codon and increased the stretch of amplicon sequence
over which there was clear amino acid and nucleotide
sequence concordance with orthologous regions from
other species. There was no obvious pattern to the
appearance of these indels, with the abundance of
frame-shift indels ranging from none in ndhA, ndhJ
and rpoB to nine in accD (Table S6, see Supporting
Information). Corrected amino acid sequences were
then screened for putative homology with known
sequences using protein blast (blastp) searches on the
NCBI database. The highest ranking blastp similarity
scores matched the target locus for all genes.
There was considerable variability between the
edited sequences of selected loci in the abundance of
SNPs. By far the highest proportion of polymorphic
sites (83/2155, 3.8%) was generated in accD, with a
marked drop to ycf9 and ycf5 (2.29% and 2.25%,
respectively), ranked second and third (Tables S5, 2).
The least variable locus proved to be ycf2, exhibiting
only nine polymorphisms among the 3500 bases
amplified across all species (0.25%). Thus, viewed
purely from the perspective of the percentage of
variable sites, the six highest ranking loci were
accD, ycf9, ycf5, ndhJ, rpoC1 and matK (Table S5).
However, although ycf9 yielded the second most vari-
able sequences (proportion of variable sites), the
amplicons it generated were both small and highly
variable in size, with the largest amplicon (Coffea,
168 bp) being almost 1.5 times longer than the short-
est (Acorus, 115 bp; Table S5). These features would
profoundly complicate the process of demonstrating
that the amplicon derived from the intended plastid
locus, as short, variable sequences with multiple
indels provide little scope for establishing sufficient
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 1–11
ndhJ rpoB rpoC1 ycf5
48 48 48 48
45 42 45 32
94 87 94 67
361 388 498 297
alignment with reference sets to have confidence in
true amplicon origin. Accordingly, ycf9 was replaced
by the proportionately less variable but longer rpoB
(ranked 7, Table S5) and taken forward for more
detailed sequence comparisons.
SEQUENCE VARIABILITY ACROSS 98
DIVERGENT SPECIES
Primers targeting the selected six loci (accD, ycf5,
ndhJ, rpoB, rpoC1 and matK) were used for a more
detailed examination of sequence variability between
sister species across 98 divergent taxa. All four pos-
sible primer combinations for all six loci were used in
this screen. This action enabled sequences to be
secured across the vast majority of taxa for ndhJ,
rpoC1, rpoB and ycf5 (98%, 98%, 95% and 95% respec-
tively; Table S7, see Supporting Information). The
consistency of amplification was more problematic for
matK (70% of species amplified) and accD (86% of
species). Some species were also markedly more prone
to consistent failure across several loci than others;
Equisetum telmateia failed to amplify the intended
target for any locus, whereas no amplification was
obtained for Diosporos bejaudii and Erica anguliger
for three loci (accD, matK and rpoB, and rpoC1, rpoB
and matK, respectively; Table S7). Thus, given that
every species, except E. telmateia, amplified products
using at least one primer pair, the quality of DNA
extraction was not considered to be a significant
factor in determining PCR amplification. The use of
four primer combinations nevertheless dramatically
improved the overall frequency of loci amplified and
sequenced in most species. This is perhaps best illus-
trated by Alcantarea regina, for which amplification
was recovered from just five of 24 primer combina-
tions tested across all loci, but nevertheless secured
sequence from all loci except matK. The limited cov-
erage for problematic loci was addressed by the intro-
duction of a new forward primer for matK (matKX)
and the generation of amplicons using modified
MT-PCR (improving amplification to 85%; Table S7).
The intended locus invariably generated the highest
amino acid and nucleotide hits by blastp and blastn,
respectively.
 DNA BARCODING REGIONS FOR LAND PLANTS
No locus successfully produced unambiguous elec-
tropherograms across all species pairs, although the
two most consistently amplified loci, ndhJ and rpoC1,
only failed to provide sequence for three pairs (Anas-
trophyllum, Pinus and Stephanodaphne, and Anastro-
phyllum, Carex and Equisetum, respectively) (i.e.
45/48 pairs, 94%; Table S8, see Supporting Informa-
tion). The rpoB primers universally produced ampli-
cons for all angiosperms (i.e. 88% of all species
groups), but consistently failed in all other groups,
namely Araucaria, Ephedra, Equisetum, Isoetes, Lyco-
podium and Mannia. The remaining three loci were
notably less consistent and yielded robust sequence
in both/all sister species on just 50% (matK), 67%
(ycf5) or 73% (accD) of occasions (Table S8). As with
rpoB, these loci performed markedly better among
angiosperms, with the majority of failed reactions
deriving from gymnosperm and spore-producing land
plants (Table S8). The adoption of the MT-PCR tech-
nique greatly improved the proportion of taxa from
which sequences were secured, primarily from matK,
but also from ndhJ and rpoB. Sequence data for an
additional 13 taxa were obtained via this method for
matK (Table S9, see Supporting Information). The use
of this technique, combined with the inclusion of an
additional forward primer (matKX), improved the
amplification of the matK primers to 32/48 (67%,
Table S8) of sister species examined. The only robust
sequence obtained for E. telmateia from all primer
combinations and loci was acquired using the
MT-PCR technique with the ndhJ primers, thereby
confirming the utility of the technique for difficult
taxa or DNA.
When all species pair comparisons were made,
matK and ndhJ generated the highest mean number
of differences between species pairs (14.0 and 12.3,
respectively), followed by rpoC1, rpoB, ycf5 and accD
in that order (Table S8). However, it should be
remembered that this ranking is based on different
species arrays for each locus. To compare sequence
variability between loci directly, it is better to con-
sider only the 19 species pairs that amplified across
all loci. When this was performed, matK again gen-
erated the highest number of mean sequence differ-
ences (20.5 per species pair), followed by ndhJ (7.0),
ycf5 (6.4), rpoC1 (4.5), rpoB (4.0) and accD (3.4)
(Table S10, see Supporting Information). Signifi-
cantly, no single locus was able to distinguish
between all species pairs included in the study, with
some variability between loci in the number and
selection of species pairs that they were unable to
diagnose. Loci differed in their ability to discriminate
between species pairs, with ycf5 (33/33 pairs) and
matK (31/33) proving to be the best performers, fol-
lowed by ndhJ (38/45), rpoC1 (37/46), accD (28/36)
and rpoB (32/43).
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 1–11
7
The combined use of two or more markers therefore
provides the opportunity to increase the variance
between species pairs and the overall frequency of
diagnostic sequence differences. Overall, this measure
increased the mean number of sequence differences
between species pairs across all individual loci from
7.6 to 15.3 bases when pairs of loci were used, and
30.5 when four loci were used (Table S11, see Sup-
porting Information). The combined use of all loci
yielded a mean of 45.8 sequence differences between
sister species, but required a combined mean
sequence length of over 2500 bases per species.
Among the combinations excluding one locus, there
was a notable and sharp drop in mean sequence
divergence generated from the combination that
lacked matK (25.3 bases compared with 38.8–42.4 for
the remaining combinations). This pattern was main-
tained for all shorter combinations that lacked matK.
For instance, performance was distinctly bimodal for
the triplet combinations, with the ten combinations
including matK yielding 27.9–33.9 mean differences
between species pairs, whereas the ten combinations
lacking this locus fell in the range 11.9–17.9
(Table S11).
DISCUSSION
The search for genomic regions to provide appropriate
targets for genetic barcoding is a task that extends
beyond a simple quest for sequence variability.
Although there is a need for sufficient polymorphism
to allow species-level diagnosis, there is an equal need
to consider the requirements of users and the
demands placed upon them when seeking to match
experimental data to reference barcode sequences. A
greater level of technical rigour is required to gener-
ate reference sequences, and it is important that
appropriate procedural mechanisms are adopted by
database providers to ensure that such data are
robust and reliable. If the emergent barcoding
resource is to prove popular, however, it is also vital
that the same constraints are not imposed on end-
users. One must balance the desire for universality of
amplification, sequence divergence and the provision
of internal procedures to correct sequencing errors.
The volume and taxonomic breadth of plastid
genome sequences held in public databases is impres-
sive and expanding, with 121 eukaryote plastid
genomes sequenced in their entirety (http://www.
ncbi.nlm.nih.gov/genomes/ORGANELLES/plastids_
tax.html), and information relating to several loci
known for over 1000 species (e.g. http://www.ncbi.
nlm.nih.gov). This resource provides a valuable plat-
form from which candidate barcoding loci can be
sought, although it must be remembered that the
nucleotide sequence coverage is taxonomically skewed
8 C. S. FORD ET AL.
and heavily biased towards crop and model species.
One key issue is that these data are inevitably
insufficient to cover all natural sequence variation
(particularly for poorly described loci), and this com-
plicates primer design. We ameliorated this problem
by targeting conserved amino acid regions likely to be
under functional constraint, such that variation in
the corresponding nucleotide sequence should be
restricted to synonymous mutations. Our strategy to
identify candidate loci was to seek two such regions of
sufficient size to allow nested primer design, and in
sufficiently close proximity to allow reliable amplifi-
cation and single-pass sequencing. Experimentally,
these precautions appeared to yield reasonable levels
of amplification across 98 test taxa, with all loci,
except rpl22, producing amplicons in > 40% of taxa
with single primer pairs and without PCR optimiza-
tion. The marked improvement when all possible
primer combinations and MT-PCR were applied seem-
ingly validated the approach (success among > 85% of
species, > 67% of species pairs).
Data quality is an important determinant of the
experimental utility of molecular barcodes and, for
this reason, it is widely acknowledged that submis-
sions to public databases as DNA barcodes (i.e.
barcode reference sequences) will be subject to several
additional requirements, most notably that the raw
trace data are used to provide a post-hoc means of
sequence validation. Exposing trace data to indepen-
dent scrutiny will certainly reduce instances of
erroneous base calling among reference barcoding
sequences, and will permit statistical approaches to
characterize base peak and signal baseline fluctua-
tions (for example, Izmailov et al., 2002; Andrade &
Manolakos, 2003). Likewise, the users may them-
selves opt to employ standard quality assurance
approaches, such as Phred (Ewing et al., 1998), to
verify the quality of electropherogram traces (for
example, Lewers et al., 2008). Such measures do not
provide protection against the sequencing of heterolo-
gous amplicons, although the use of nucleotide simi-
larity searches provides one means of indicating
whether the amplicon is likely to originate from the
intended plastid locus. In silico translation of nucle-
otide sequences therefore provides an attractive
additional test for the orthology of genic regions, with
comparison of amino acid alignments across taxa also
revealing those coding regions within the amplicon
likely to be under strong selection. We found that all
nucleotide and translated sequences of our genic
candidates exhibited greatest sequence similarity to
their intended targets, although in the preliminary
screen the use of translated sequences also uncovered
several instances where frame shifts led to the intro-
duction of stop codons in all reading frames. Here,
manual correction was relatively simple for the vast
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 1–11
majority of instances, implying that most arose from
sequencing, annotation or transcriptional artefacts,
despite our initial editing of the trace data, utilization
of experienced DNA sequence service providers and
sequencing in both directions (a small number of
sequencing reactions repeatedly failed in one direc-
tion). These errors could not be detected or corrected
using non-coding sequences as DNA barcodes.
There is now a growing acceptance that the genetic
barcoding of land plants requires a multi-locus
approach (Chase et al., 2005, 2007; Newmaster et al.,
2006). The selection of loci to feature in a multi-locus
barcode requires a balance between conflicting
factors. Cognizance should be taken of the universal-
ity of amplification (with the more variable loci
tending also to be the more difficult to amplify across
groups), but also whether there is sufficient sequence
variation to distinguish between species (with the
more variable loci generating greater capacity to
effect diagnosis). Newmaster et al. (2006) also consid-
ered familiarity and the value of considerable existing
sequence resources when suggesting the combination
of rbcL with other plastid loci as part of a multi-locus
barcode. More recently, Kress & Erickson (2007)
evaluated a subset of the loci and primers described
here with several other loci on the basis of amplifi-
cation success and sequence variability. They pro-
posed that psbA-trnH should be combined with part of
the rbcL gene (rbcL-a), with the former providing the
sequence variation for species identification and the
latter providing a less variable taxonomic ‘anchor’.
However, inclusion of psbA-trnH exposes the compos-
ite barcode to problems of excessive size variation and
an absence of corroborative amino acid sequence for
end-user sequence annotation. It is the potential cor-
roborative value of the translated sequence that led
us to focus on coding regions as potential barcode
regions, although reduced sequence variability may
necessitate the inclusion of multiple loci.
Previously, Chase et al. (2007) cited preliminary
data from the present study to suggest that two
triplet loci combinations may be suitable for use as
universal barcodes from land plants: rpoC1, rpoB and
matK, or rpoC1, matK and psbA-trnH. Comprehen-
sive examination of the data generated provides some
support for this combination, but also highlights
alternative combinations that may perform a simi-
larly effective role as a multi-locus barcode. When loci
were compared individually, matK yielded by far the
greatest level of variability. Indeed, the performance
of matK was such that there was clear division
between combinations that included this locus and
those that did not. This feature clearly favoured the
inclusion of matK in any locus combination being
proposed as the multi-locus barcode, although
thought must also be given to its modest performance
 DNA BARCODING REGIONS FOR LAND PLANTS
when individual matK primers were used and that
the large size of the amplicon generated also favours
its inclusion. The use of nested MT-PCR greatly
improved the amplification rates for this locus (85% of
species, 67% of species pairs), but still left the pros-
pect of a proportion of taxa for which no sequence was
obtained. Although it is possible that a reasonable
number of such cases may be resolved by PCR opti-
mization or through the targeted design of taxon-
specific primers (Hebert et al., 2004a, b; Ward et al.,
2005; Seifert et al., 2007), it is questionable whether
this option would be appropriate for certain barcoding
applications. For instance, workers interested in
establishing community species audits will require
broad coverage across all members of the communi-
ties being surveyed, and so may be less concerned
with resolving problematic groups or with distin-
guishing between the comparatively infrequent
instances of sympatric sister species. Under such cir-
cumstances, the additional effort needed to develop
new primers or protocols to ‘fill data gaps’ may be
unwarranted. Thus, selection of the remaining loci in
the multi-locus barcode should ideally aim to comple-
ment the deficiency in universality of matK, and yet
also maximize the probability of realizing a species
diagnosis in the absence of a matK product. Combi-
nations including ndhJ, ycf5 and accD yielded the
highest proportion of variable sites, and so appear to
be the most natural complements to matK. However,
difficulty here resides in taxonomic coverage, with
accD known to be systematically absent from grasses
(Katayama & Ogihara, 1996), ycf5 from some mosses
(Sugiura et al., 2003) and ndhJ from the commer-
cially important genus Pinus (Wakasugi et al., 1994).
Given the relatively minor difference in the combined
performance of these loci with matK, when compared
with the more universally present (and amplifiable)
rpoC1 and rpoB, we therefore propose that
rpoC1 + rpoB + matK currently represents the most
prudent choice for an interim multi-locus coding
barcode combination. Although increasing the
number of loci can only increase the number of
species pairs that can be distinguished (from 18 to
19), the expansion in the mean number of differences
(20.5 for matK to 29.1 for rpoB + rpoC1 + matK)
(Table S11) enhances the capacity to allow for
infraspecific variation. Furthermore, given the higher
consistency of amplification from rpoB and rpoC1, the
use of these markers also provides useful ‘insurance’
for instances in which matK fails to amplify. As
supplementary barcoding loci, ndhJ and, to a slightly
lesser extent, ycf5 and accD may perform a valuable
function for some groups as supplementary barcoding
loci. Although our study encompasses a broad taxo-
nomic spectrum of species pairs, the comparatively
limited sampling used restricts our ability to make
© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 1–11
9
broad generalizations. This requires a more extensive
survey, in which the utility of markers is assessed in
greater detail for a number of taxonomically diverse
groups. There have only been a small number of such
studies to date. In the first, Sass et al. (2007) com-
pared all six loci identified above across 21 species
from ten genera of cycads, and achieved greatest
sequencing success with rpoC1, which enabled iden-
tification to the generic level, but not to species. In
concordance with the results presented here, matK
offered greater species-level variation in sequence,
but performed less well in terms of amplification
(Sass et al., 2007). Lahaye et al. (2008) compared the
same loci across the taxonomically problematic
orchids, and found that, for this group, matK per-
formed best. Similarly, Newmaster et al. (2008) found
matK to provide greater resolution at the species
level, compared with the other five loci, when applied
to a set of 40 taxa (composed of 38 species from one
genus) from Myristicaceae known to have a low level
of genetic divergence. We await the outcome of
similar studies across a wider selection of taxa before
finally advocating the optimal composition of a multi-
locus barcode for all land plants. In the longer term,
we expect that wider access to complete plastid
genome sequences will enable finer focusing of loci for
use as part of multi-locus barcodes and, as sequencing
technology advances for end-users, more complex
multi-locus barcodes may become an increasingly
attractive proposition, including appropriate loci from
the nuclear genome.
ACKNOWLEDGEMENTS
We thank the Alfred P. Sloan Foundation and the
Gordon and Betty Moore Foundation for funding.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Table S1. Gene regions of the Nicotiana plastid genome assessed for their potential as barcoding markers.
Markers in bold selected for primer development and screening
Table S2. Candidate regions selected for development as potential barcoding markers and their primer pairs.
*Primer added after initial screen to improve universality of matK
Table S3. Ninety-eight taxa (46 species pairs and two species triplets) selected for the assessment of candidates
for universal primers for barcoding. All taxa were screened for polymerase chain reaction (PCR) amplification
across 12 candidate loci. *Ten species pairs used in the first round of sequencing to screen polymorphisms across
11 candidate loci. All taxa were screened in the second round of sequencing to identify sequence polymorphisms
across six candidate loci
Table S4. European Molecular Biology Laboratory (EMBL) sequence accession numbers
Table S5. Results of the initial screen for sequence polymorphisms across ten species pairs. Percentage of
variable sites (ranked from most to least variable): calculated as the percentage of single nucleotide polymor-
phisms (SNPs) and indel sites of the total number of bases for each marker. P (ranked from the highest to the
lowest) represents the proportion of species pairs discriminated by each marker
Table S6. Distribution of stop codons in translated sequences of target loci. Stop codons detected in translated
nucleotide sequences during the initial screen for polymorphisms of 11 loci against ten species pairs
Table S7. Primer universality. Results of the application of all four possible primer pairs for the six primary
loci to the full set of 98 taxa. 1, amplification; 0, no amplification; dark shade, failed after both standard and
multiplexed tandem polymerase chain reaction (MT-PCR)
Table S8. Number of single nucleotide polymorphisms (SNPs) and indels per marker across 48 species pairs
(first two species in the two species triplets analysed as pairs). Additional sequences for each locus from Pinus
koraiensis and P. thunbergii were downloaded from GenBank and added to this dataset (accessions NC004677,
NC001631). Only sequence data corresponding to the section amplified by the primer pairs presented here were
aligned and submitted to SNP analysis protocols. The results appear in this table as Pinus2. Num diffs, number
of SNPs and indels between species pair. Seq len, length of sequence generated. *Extra species pair comparisons
secured by multiplexed tandem polymerase chain reaction (MT-PCR) or additional primer (MatKX) (see also
Table S9)
Table S9. Sequences generated using multiplexed tandem polymerase chain reaction (MT-PCR)
Table S10. Numbers of single nucleotide polymorphisms (SNPs) and indels per marker across the 19 species
pairs for which sequence data were obtained across all loci. Num diffs, number of SNPs and indels between
species pair. Seq len, length of sequence generated
Table S11. Marker combination analysis for the 19 species pairs for which sequence data were obtained for all
loci
Program S1. barcode.c.
Program S2. barcode2.c.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing material) should be directed to the corresponding
author for the article.

© 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 1–11