Evolutionary histories determine DNA barcoding success in vascular plants: Seven case studies using intraspecific broad sampling of closely related species

Abstract

Background:
Four plastid regions, rpoB, rpoC1, matK, and trnH-psbA, have been recommended as DNA barcodes for plants. Their success in delimiting species boundaries depends on the existence of a clear-cut difference between inter- and intraspecific variability. We tested the ability of these regions to discriminate among closely related species in seven genera of flowering plants with different generation times (trees, perennials, and annuals). To ensure a maximum coverage of intraspecific diversity, and therefore to better evaluate the resolution power of each barcode, we applied a population genetics approach by sampling three to 45 individuals per species over a wide geographical range.

Results:
All possible combinations between loci were analysed, which showed that using more than one locus does not always improve the resolution power. The trnH-psbA locus was most effective at discriminating among closely related species (Acer, Lonicera, Geranium, and Veronica), singly or in combination. For Salix, Adenostyles, and Gentiana, the best results were obtained with the combination of matK, rpoB, and trnH-psbA. No barcoding gap was found within six genera analysed, excepting Lonicera. This is due to shared polymorphisms among species, combined with very divergent sequences within species. These genetic patterns reflect incomplete lineage sorting and hybridization events followed by chloroplast capture.

Conclusions:
Our results strongly suggest that adding trnH-psbA to the two obligate DNA barcodes proposed by the CBOL plant-working group (matK and rbcL) should be mandatory for closely related species. In our sampling, generation time had no influence on DNA barcoding success, as the best and worst identification successes were found for the two tree genera (Acer, 64 % success and Salix, 86 % failure). Evolutionary histories are the main factor influencing DNA barcoding success in the studied genera.

Full text

R E S E A R C H A R T I C L E Open Access
Evolutionary histories determine DNA
barcoding success in vascular plants: seven
case studies using intraspecific broad
sampling of closely related species
Sofia Caetano Wyler
1,2
and Yamama Naciri
1*
Abstract
Background: Four plastid regions, rpoB, rpoC1, matK, and trnH-psbA, have been recommended as DNA barcodes
for plants. Their success in delimiting species boundaries depends on the existence of a clear-cut difference between
inter- and intraspecific variability. We tested the ability of these regions to discriminate among closely related species in
seven genera of flowering plants with different generation times (trees, perennials, and annuals). To ensure a maximum
coverage of intraspecific diversity, and therefore to better evaluate the resolution power of each barcode, we applied a
population genetics approach by sampling three to 45 individuals per species over a wide geographical range.
Results: All possible combinations between loci were analysed, which showed that using more than one locus does
not always improve the resolution power. The trnH-psbA locus was most effective at discriminating among closely
related species (Acer,Lonicera,Geranium,andVeronica), singly or in combination. For Salix,Adenostyles, and Gentiana,
the best results were obtained with the combination of matK, rpoB, and trnH-psbA. No barcoding gap was found
within six genera analysed, excepting Lonicera. This is due to shared polymorphisms among species, combined with
very divergent sequences within species. These genetic patterns reflect incomplete lineage sorting and hybridization
events followed by chloroplast capture.
Conclusions: Our results strongly suggest that adding trnH-psbA to the two obligate DNA barcodes proposed by the
CBOL plant-working group (matKandrbcL) should be mandatory for closely related species. In our sampling, generation
time had no influence on DNA barcoding success, as the best and worst identification successes were found for the
two tree genera (Acer,64%successandSalix, 86 % failure). Evolutionary histories are the main factor influencing DNA
barcoding success in the studied genera.
Keywords: Acer,Adenostyles, Chloroplast capture, Incomplete lineage sorting, Interspecific hybridization, Gentiana,
Geranium,Lonicera,Salix,Veronica
Background
DNA barcoding uses a short DNA sequence from a
standard locus to identify the species to which a particu-
lar specimen belongs [1]. Since DNA barcoding was first
used in plants, several regions have been recommended
as universal barcodes [2–7]. Primarily located in the
chloroplast genome, these regions focus on coding and
non-coding loci. Kress and Erickson [2] proposed the
combined use of rbcL and trnH-psbA, but other combi-
nations have been suggested as well ([8–10]; among
others). More recently, the Plant Working Group of the
Consortium for the Barcode of Life adopted rbcLand
matK as the core DNA barcodes for plants [11], with
trnH-psbA as an additional marker. Other studies have
suggested the use of the nuclear ribosomal locus ITS
[4, 12, 13], but the aim of the present study was to test
for the accuracy of the chloroplast barcodes per se and
we therefore selected matK, rpoC1, rpoB, and trnH-
* Correspondence: Yamama.Naciri@ville-ge.ch
1
Laboratoire de Systématique Végétale et Biodiversité, Conservatoire et
Jardin botaniques & University of Geneva, Chemin de l’Impératrice, 1, 1292,
Chambésy, Geneva, Switzerland
Full list of author information is available at the end of the article
© 2016 Caetano Wyler and Naciri. Open Access This article is distributed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution,
and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link
to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication
waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise
stated.
Caetano Wyler and Naciri BMC Evolutionary Biology (2016) 16:103
DOI 10.1186/s12862-016-0678-0

psbA. The barcode studies published so far agree that
matKandtrnH-psbA are the two most promising
chloroplast regions for discriminating among closely re-
lated species, whereas other regions, such as rbcL, are
more suitable for identifications at the family and/or the
genus level [14]. This is the main reason why we decided
to discard rbcL, although it is one of the official barcodes.
Because the debate has long focused on which
marker(s) should be used to obtain the best assignment
to species [2, 4, 7, 10, 11, 13–20], other fundamental is-
sues have received less attention, although they are of
high relevance for barcoding success. One of these issues
is how many individuals should be analysed within a
species to generate a reliable reference for an accurate
identification. Early studies that analysed the success of
DNA barcoding [2, 14, 16] did not use the closest spe-
cies when pairs of species were selected. Accordingly, a
higher identification success was usually obtained when
barcoding floras, for which closely related taxa are not
always included, versus taxonomical groups for which it
is usually the case [7, 8]. Meyer and Paulay [21] raised
the sample size concern but it has rarely been addressed
in barcoding studies (but see [7, 22]), although it is par-
ticularly critical when working with closely related spe-
cies, for which intra- and interspecific genetic variation
may overlap quite frequently. Therefore the methods by
which intraspecific variability is documented has a direct
influence on the accuracy with which a given DNA
sequence identifies species.
DNA barcoding success depends on the existence of a
clear cut-off between intraspecific variation and inter-
specific divergence, the so-called “barcoding gap”.The
barcoding gap is largely dependent on the studied
groups and species, which constitutes a second issue
that has hardly ever been addressed (but see [7]). Many
plant species evolved recently through adaptive radia-
tions and rapid speciation [3, 23–26]. Recent speciation
with consecutive incomplete lineage sorting often results
in reduced sequence divergence between the newly spe-
ciating taxa [27–29]. In the worst case, i.e. retention of
ancestral polymorphism(s) among species, the identifica-
tion of specimens is impossible [30]. Problematic identi-
fication of specimens also arises from hybridization
between species, which is very frequent [31, 32], and
polyploidization [29, 33, 34]. Therefore, the success of
DNA barcoding is expected to vary among groups
depending on their evolutionary history.
Still, a general prediction about DNA barcoding suc-
cess can be made based on life traits such as the gener-
ation time. The short generation times that characterize
annual plants are expected to lead to a rapid accumula-
tion of mutations and to prompt species differentiation.
Significant barcoding gaps are expected for such plants,
leading to high DNA barcoding assignment success. The
longer life spans and slower accumulation of mutations
in woody plants are expected to result in poorer species
delimitations [35, 36].
In this study, we analysed the impact of generation
times and large sample sizes on DNA barcoding success.
We addressed this question using four chloroplast loci
(matK, rpoB, rpoC1, and trnH-psbA) that have been pro-
posed as barcodes [14]. These markers were evaluated
for closely related species within seven genera that dis-
play different generation times: Acer and Salix (trees);
Adenostyles,Gentiana, and Lonicera (perennials); and
Geranium and Veronica (annuals). Within genera, we se-
lected species that have clear taxonomical status with
overlapping geographical distributions. We then sampled
as many populations as possible in order to assess intra-
specific and interspecific variation in the barcoding loci
to infer how well specimens could be assigned to species
with the selected chloroplast barcodes.
Results and discussion
Sampling
A total of 485 individuals were sampled for the 27 spe-
cies used in this study (Additional file 1). Differences in
sampling sizes per genus are explained by the relative
abundance of some species (Acer—103 individuals) com-
pared to others (Geranium—16 individuals) and by the
effort put into sampling Gentiana (137 individuals) for a
detailed study on the phylogeography of the Ciminalis
group [37]. Samples were collected in Austria, the Czech
Republic, France, Italy, Norway, Portugal, Switzerland,
the United Kingdom, Spain, and Sweden from 37.05° to
69.30° in latitude and from −8.38° to 22.48° in longitude.
Primer universality and amplification success
A DNA barcode must fulfil several requirements and
should optimally be universal (present in all taxa), easily
amplified (i.e., without species-specific PCR primers),
short enough (so that it can be easily sequenced, even
on degraded samples), informative at the species level
(with enough variation insuring a satisfactory identifica-
tion of species), and conserved or slightly polymorphic
at the intraspecific level (so that a barcode gap can be
observed).
Four candidate chloroplast regions were targeted in
the present study: matK, rpoC1, rpoB, and trnH-psbA.
Only 440 specimens were amplified and sequenced suc-
cessfully for the four loci (91 %). Loci were sequenced
with 100 % success, except for rpoC1 and rpoB in one
individual of Gentiana, and matKinAcer,Gentiana,
Lonicera, and Veronica (Table 1). We used four combi-
nations of five matK primers (one of them newly de-
signed in this study) to improve the results (Additional
file 2). Still, we were not able to obtain matK sequences
from 13 individuals of Veronica hederifolia (sequencing
Caetano Wyler and Naciri BMC Evolutionary Biology (2016) 16:103 Page 2 of 11

success: 58.6 %; Table 1). This marker is known to have
a lower success rate of PCR amplification and sequen-
cing [11, 13] and our results emphasize the lack of
primer universality for this DNA barcode, even at the
genus level (Acer and Veronica; Additional file 2). More-
over, generating fully bidirectional sequences for matK
was sometimes challenging, a problem that has also been
reported in many families, including Asteraceae [27] and
Lemnaceae [10].
Sequence variation and discriminating power
Alignments, sequence variation analyses, and identifica-
tion of unique sequences were performed within each
genus separately. The alignment lengths for rpoC1 and
rpoB were conserved for all genera, while those of matK
and trnH-psbA ranged from 761 to 1228 bp and from
325 to 525 bp, respectively (Table 1). For the trnH-psbA
spacer, the differences in length are not surprising and
are easily explained by a high number of insertion/dele-
tion events. The use of different primer pairs for differ-
ent genera explains the range in matK product size.
Sequence variation was quantified using the number
of conserved and parsimony informative sites. The
percentage of conserved sites was high for each genus,
ranging from 80 % in Gentiana for trnH-psbA to 100 %
in Salix and Adenostyles for rpoC1 and rpoB, respectively
(Table 1). The percentage of congeneric species resolved
as monophyletic was accordingly very low for rpoC1 and
rpoB. This is not a surprising result given the slow evo-
lutionary rate of these two coding regions. These loci are
Table 1 Diversity measures for matK, rpoC1, rpoB, and trnH-psbA, given separately for the seven genera (n is the number of sampled
individuals)
matKrpoC1 rpoBtrnH-psbA
Acer (n= 103) Aligned length (bp) 849 508 349 512
Sequencing success (%) 95.1 100 100 100
Conserved sites (%) 98 99.2 99.1 93.8
Parsimony informative sites (%) 1.3 0.8 0.9 3.7
Salix (n= 69) Aligned length (bp) 855 508 349 325
Sequencing success (%) 100 100 100 100
Conserved sites (%) 96.4 100 99.7 91.4
Parsimony informative sites (%) 0 0 0.3 0.9
Adenostyles (n= 37) Aligned length (bp) 798 508 349 508
Sequencing success (%) 100 100 100 100
Conserved sites (%) 98.2 99.8 100 99
Parsimony informative sites (%) 0.1 0.2 0 0.8
Gentiana (n= 135) Aligned length (bp) 761 508 349 460
Sequencing success (%) 98.5 99.3 99.3 100
Conserved sites (%) 92.6 96.7 98.3 80
Parsimony informative sites (%) 6.6 1.6 1.7 19.6
Lonicera (n= 67) Aligned length (bp) 1190 508 340 525
Sequencing success (%) 85.1 100 100 100
Conserved sites (%) 96.7 99.8 98.8 97.1
Parsimony informative sites (%) 1.7 0.2 1.2 2.7
Geranium (n= 16) Aligned length (bp) 769 508 349 356
Sequencing success (%) 87.5 100 100 100
Conserved sites (%) 94.1 96.7 99.4 89
Parsimony informative sites (%) 5.6 1.6 0 2.5
Veronica (n= 58) Aligned length (bp) 1228 508 349 393
Sequencing success (%) 58.6 100 100 100
Conserved sites (%) 91.5 94.5 94.3 81.7
Parsimony informative sites (%) 5.7 5.3 5.2 15.5
Total aligned sequence length (bp), percentage of individuals successfully amplified and sequenced, percentage of conserved and parsimony informative
characters in the aligned sequences
Caetano Wyler and Naciri BMC Evolutionary Biology (2016) 16:103 Page 3 of 11

therefore not suitable to distinguish closely related spe-
cies, as also reported in other studies (e.g., [10]). We
highlight that both loci have slightly lower resolution
powers compared to that of the recommended DNA
barcode rbcL [11]. Therefore the use of the latter region
would not have dramatically changed our results in the
present study.
The percentage of parsimony informative sites was low
for most markers in all genera, especially rpoC1 and
rpoB (mean = 1.4 and 1.3 %, respectively). The locus
trnH-psbA harbours the highest percentage of parsi-
mony informative sites, except in Geranium, for which
the highest value is found with matK (5.6 % instead of
2.5 % with trnH-psbA).
When considered separately, the locus with the highest
number of sequences private to a single species was found
with trnH-psbA (Table 2). Accordingly, the highest identifi-
cation success at the species level was also observed using
this locus. The ability of trnH-psbA to distinguish species is
generally well accepted [16]. Many studies have recom-
mended using this marker as a DNA barcode on a regular
basis [2, 38–40]. Moreover, its use in intraspecific popula-
tion studies [41, 42] highlights its utility for discriminating
closely related species, which agrees with the results
obtained here. Intergenic spacers are generally difficult to
align across genera [43], but performing the analyses inde-
pendently within each genus can surpass this obstacle.
Monophyly tested by phylogenetic trees
For each barcode, we estimated the recovered species
monophyly using multiple individuals per species and
phylogenetic NJ trees (Additional file 3). It should be noted
that the main purpose of the trees was not to study evolu-
tionary relationships, but rather species identification.
The 103 Acer individuals were divided into three clades
for matK, rpoC1, and trnH-psbA: 1, A. campestre L. and A.
platanoides L., 2, A. opalus Mill. and A. monspessulanum
L., and 3, A. pseudoplatanus Falk. With rpoB, A. campestre,
A. platanoides,A. opalus,andA. monspessulanum grouped
together in a single clade. Adenostyles species did not clus-
ter into distinguishable clades with the four markers. For
Gentiana, the four loci separated the four species into two
main clades: 1, G. alpina Vill. and G. clusii E.P.Perrier &
Songeon, and 2, G. acaulis L. and G. angustifolia Vill. Still,
three G. alpina individuals were clustered in the second
clade. For Geranium,rpoC1 and trnH-psbAweretheonly
markers able to distinguish G. columbinum L. from the
other two species that clustered together. For Lonicera,only
rpoC1 failed to distinguish the four species into monophy-
letic clades (L. caerulea L., L. nigra L., and L. alpigena L.
clustered in a single clade). Salix species were indiscernible
with the four DNA barcodes. Veronica hederifolia L. indi-
viduals formed a monophyletic clade with three loci (matK
failed to amplify this species). With matK, two clades could
be observed, the first one comprising almost all V. arvensis
Table 2 Assignment success for matK, rpoC1, rpoB, and trnH-psbA given alone and arranged according to all possible combinations
Opt. for Option. Option1: matK+rpoC1; Option2: matK+rpoB; Option3: matK+ trnH-psbA; Option4: rpoC1 + rpoB; Option5: rpoC1 + trnH-psbA; Option6: rpoB+trnH-
psbA; Option7: matK+rpoC1 + rpoB; Option8: matK+rpoC1 + trnH-psbA; Option9: matK+rpoB+trnH-psbA; Option10: rpoC1 + rpoB+trnH-psbA; Option11: matK+
rpoC1 + rpoB+trnH-psbA. Number of individuals successfully amplified and sequenced, number of total different sequences within the genus and the ones that
are private to a single species, and number of individuals harbouring species’private sequences. The statistics are given for the seven genera separately (n is the
number of sampled individuals). The best DNA barcode(s) are highlighted in grey for each genus
Caetano Wyler and Naciri BMC Evolutionary Biology (2016) 16:103 Page 4 of 11

L. individuals and the second one grouping V. persica Poir.
and V. polita Fr. together. The four loci also agreed in clus-
tering two V. polita individuals within the V. arvensis clade
and two V. arvensis individuals in the persica-polita clade
(Additional file 3).
Therefore, monophyletic clades grouping conspecific
individuals were only observed in Lonicera with matK,
rpoB, and trnH-psbA. For the six remaining genera,
none of the chloroplast regions was successful in recon-
structing monophyletic species clades.
Locus combination and barcode gaps
Combining markers improves the rate of correct species
identification [20, 27]. In the present study, all possible
combinations between loci were analysed and are re-
ported in Table 2. Our results clearly showed that com-
bining loci is not always an advantage. For instance,
option 11, which combines all four loci, did not result in
the highest identification rate, as one might expect if
each locus was informative. The highest success in dis-
criminating closely related species was always attained
with a combination involving trnH-psbA. We stress,
however, that it is not always the same combination of
loci that gave the best results. With two loci (options 1
to 6), option 6 (rpoB+trnH-psbA) performed well for
most genera in terms of private intraspecific diversity
and number of individuals unambiguously identified.
The exceptions were Salix,Adenostyles, and Gentiana,
for whom identical or better results were obtained with
option 3 (matK+trnH-psbA). For the combinations with
three loci (options 7–10), the same pattern was ob-
served: whenever the number of individuals sequenced
was the same among options, the combination of matK
and trnH-psbA performed slightly better in discriminat-
ing species. The barcoding success was enhanced when
these two loci were combined, but the lower sequencing
success of matK limited its utility in this dataset.
None of the loci or combinations of loci performed
equally for the seven genera in terms of sequencing
and identification successes and no locus or combin-
ation of loci proved to be ideal for DNA barcoding.
We selected trnH-psbA alone as the DNA barcode
for Acer,Lonicera,Geranium,andVeronica,asthe
addition of other loci did not improve discrimination
of species in these four genera. This is in line with
the original concept of DNA barcoding, which advo-
cates the use of a single sequence. For Adenostyles,
combining matKandtrnH-psbA (option 3) performed
equally or better than other options while minimizing
the number of loci involved. For Salix and Gentiana,
option 9, which combined matK, rpoB, and trnH-
psbA, gave the best discriminatory results.
Barcoding gaps were evaluated by comparing the
intra- and interspecific divergences within each genus
[21]. The Kimura 2-parameter (K2P) distances were
computed for the chosen locus/combination according
to the above chosen options: trnH-psbA for Acer,Loni-
cera,Geranium, and Veronica, option 3 for Adenostyles,
and option 9 for Salix and Gentiana. Lonicera was the
only genus with a clear barcoding gap (Fig. 1). The
expected cut-off between intra- and interspecific K2P
distances was not observed in all other genera. Acer,
Geranium,Veronica, and Gentiana also tend to have
higher inter- than intraspecific distances though there is
some overlap at frequencies ranging between 9 and
20 %. Conversely, intra- and interspecific distances over-
lap completely in Veronica and Geranium.
Analyses were performed separately in each genus, so
overlaps between intra- and interspecific variation are
expected when closely related taxa are included. In our
study, the overlap between the two distributions indi-
cated that DNA barcoding with the studied chloroplast
loci is not effective for the studied genera, except Loni-
cera. Indeed, the nearest-neighbour distance (minimum
average interspecific distance) was, with the exception of
Lonicera, lower than the maximum intraspecific distance
(Fig. 2). This type of result is associated to two main
population genetic factors, incomplete lineage sorting
and interspecific hybridization [21, 28, 44]. Recently
diverged species are likely to have a null or very low
average sequence distance to the most closely related
species. Moreover, hybridization events associated with
chloroplast captures tend to maximize the intraspecific
divergence, as divergent chloroplasts can be exchanged
and shared among species [28]. This seems to be the
case in Geranium,Gentiana, and Veronica.
Influence of sampling size
The extent to which large sample sizes influenced the cap-
ture of intraspecific variability was analysed. The correl-
ation between sampling sizes and number of unique
sequences was only found for Adenostyles (r=0.99, n=3,
P< 0.05). The lack of correlation was observed for the ma-
jority of the genera, within genera (n=3—5) and overall
(r=0.21, n=27, P> 0.31). We employed the rarefaction
method to quantify the average number of different se-
quences that would be recovered using a small sampling
size within species. For a sampling size of three individ-
uals, the sequence richness (Rs) ranged between one for
species with no intraspecific diversity for the studied loci
(Acer monspessulanum, A. platanoides, Adenostyles leuco-
phylla DC., Gentiana acaulis, Geranium columbinum,
Lonicera nigra,andVeronica persica)and2.5(Gentiana
clusii,Salix herbacea Schrenk, and S. reticulata L.). Inter-
estingly, the most variable species never reached Rs =3,
despite having six to nine sequences. Similarly, other spe-
cies that displayed moderate variation (two to three unique
sequences) had very low Rs values (Acer pseudoplatanus Rs
Caetano Wyler and Naciri BMC Evolutionary Biology (2016) 16:103 Page 5 of 11

Fig. 1 Relative distribution of intra- (light grey) and interspecific (dark grey) divergence, as measured by the K2P distance, of the defined DNA
barcode alone or combined within each genus
Caetano Wyler and Naciri BMC Evolutionary Biology (2016) 16:103 Page 6 of 11

=1.2 and Gentiana angustifolia Rs =1.1). Rs was calculated
using observed sequence frequencies, emphasizing the fact
that small samples will often miss rare sequences.
Median joining networks and life histories
Median joining networks were drawn with the selected
barcode for each genus separately (Fig. 3) and illustrate
why barcoding gaps were seldom observed. Sister species
shared the same sequences in six out of the seven genera.
Lonicera was the only genus for which complete lineage
sorting was observed. According to the most recent phyl-
ogeny of the genus, the four species analysed here belong
to separate subclades of the Lonicera clade [45]. However,
these four sections were poorly supported, so it would be
interesting to analyse the DNA barcoding performance if
one had considered species from the same subclade.
Among the other genera, three different processes can
explain the sharing of chloroplast sequences between spe-
cies. The first is incomplete lineage sorting among sister
species, which is observed in four genera. 1) Within Acer
this pattern occurs twice: between A. campestre and A.
platanoides (sequence B) and between A. monspessula-
num and A. opalus (sequence F). The latest Acer phyl-
ogeny [46] confirms that these two pairs are sister species.
2) Within Gentiana, the AAA sequences are shared be-
tween G. angustifolia and G. acaulis. According to Christe
et al. [37], who studied the phylogeographic patterns
within the Ciminalis group, these species have diverged
recently. 3) Within Veronica,V. persica and V. polita share
sequence A. The latest phylogeny, based on ITS, reported
that these are sister species within subgenus Pocilla [47].
4) Adenostyles alliariae Kern and A. leucophylla share se-
quence A, which reflects their status as sister species [48].
The second process that explains sequence sharing is pu-
tative hybridization between species. This is observed
within both genera of annual plants: Veronica polita is
characterized by sequences A, G, and C. Sequence C, which
is distinct by 66 mutations from the two others, is
shared with V. arvensis. Hybridization is recognized as
an important evolutionary force for some subgenera of
Veronica [9]. In published phylogenies, the species for
which hybridization is suspected are grouped together
in the ITS consensus tree and the cladogram based on
the ITS sequences, chromosome numbers, and iridoid
composition [47, 49]. Geranium pusillum L. harbours two
different sequences that are separated by 23 mutations;
one is shared with G. dissectum L. (sequence A). A third
case of hybridization was also observed within Gentiana.
Indeed, G. alpina possesses two sequences that are dis-
tinct at 63 positions, one of which (BAJ) is closely related
to the most frequent sequence (CBC) in G. clusii. Hybrid-
izations between Gentiana species have often been re-
ported [50–52], and distinct events of chloroplast capture
involving these species have also been suggested [37].
The complete lack of structure observed within Salix
was surprising, but not new. The three most common se-
quences were shared among the four species analysed in
this study, and only 14.5 % of the specimens had private
sequences. Our results agreed with a recent study that
documented little variation in chloroplast loci among
Salix species, with most taxa sharing the same barcode se-
quence. Complex processes involving “recent repeated
plastid capture events, aided by widespread hybridization
and long-range seed dispersal, but primarily propelled by
one or more trans-species selective sweeps”were sug-
gested to explain the observed pattern [53].
In summary, our results illustrate the effect of species’
evolutionary histories on DNA barcoding success. In this
study, evolutionary history refers to recent speciation
events with incomplete lineage sorting and retention of
ancestral sequences, interspecific hybridization events
Fig. 2 Minimum average interspecific distance (light grey) against the maximum intraspecific divergence (dark grey), as measured by the K2P
distance, of the defined DNA barcode within each genus
Caetano Wyler and Naciri BMC Evolutionary Biology (2016) 16:103 Page 7 of 11

Fig. 3 Sequence networks drawn for selected DNA barcode(s) within each genus. Each unique sequence combination is represented by a circle,
with size proportional to the number of individuals sharing the sequence. Each branch segment represents a single mutation; substitutions are
coded as full lines and indels as double lines
Caetano Wyler and Naciri BMC Evolutionary Biology (2016) 16:103 Page 8 of 11

with chloroplast capture, and spatial expansions with se-
quence surfing [54]. It is commonly acknowledged that
several processes underlying the evolutionary patterns in
plants cause a partial failure of DNA barcodes to track
species boundaries [13, 28, 29, 43], but this study shows
that the absence of a barcoding gap among closely re-
lated species is quite common, with extensive sharing of
diversity among species (49 %).
Conclusions
The main factor that impacts DNA barcoding success is a
species’evolutionary history. Sampling many specimens
from a wide geographical distribution within species was
shown to be important as it increases the likelihood of cap-
turing the intraspecific genetic variation. However, sam-
pling sizes were not correlated to the number of different
sequences found within a species, because variability is
mostly influenced by the species’evolutionary history. Our
study shows that within the same genus, and even within
the same section, sequence variation can range from low to
high, depending on the species (for instance, Gentiana clu-
sii and G. acaulis –11 and 2 different sequences, respect-
ively, with similar sampling sizes collected from the whole
distribution range). Such diverse patterns were obtained
through different demographic regimes (bottlenecks, spatial
or demographic expansions) that shaped the diversity and
its structuring.
Life traits, such as generation time, do not influence
the DNA barcode success in our study. The best and
worst identification successes were indeed found for
the two tree genera (Acer, 64 % success and Salix,86%
failure). The annual plants analysed here showed, on
average, a higher number of mutations between se-
quences than was observed in perennials. This should,
theoretically, be an advantage for DNA barcoding suc-
cess, but the incidence of interspecific hybridization
within these genera highly shapes the observed genetic
pattern and results in specimen identification failures.
Therefore, our results underline the impact of species’
evolutionary histories on the ability to successfully
identify a given specimen.
We found that the most useful combination of loci for
discriminating closely related species can differ from one
genus to another, and this agrees with other papers that
discussed the interest of different loci as DNA barcodes.
However, our results demonstrated that trnH-psbAisal-
most always the best DNA barcode locus. This supports
the proposal for trnH-psbA to be added to the two core
DNA chloroplast barcodes proposed by the CBOL plant
working group. Moreover, our results show that the K2P
metric is not the most appropriate, as it does not take into
account invertion/deletion events that are of high interest,
especially for trnH-psbA, to distinguish and document
sequence variation.
Methods
Sampling strategy
Genera and species were selected for the present study
based on the following criteria: generation times, geo-
graphic distribution ranges, clear taxonomical status,
and ease of recognition. In each case, all possible closely
related species were sampled except any rare or endan-
gered ones. Species of two genera are trees (Acer and
Salix), three genera include perennial herbaceous or
woody species (Adenostyles,Gentiana, and Lonicera),
whereas two genera include annual species (Geranium
and Veronica). For each species, as many localities as
possible were sampled, over the largest possible geo-
graphical range, to gather as much intraspecific variation
as possible (Additional file 1). For each individual, an
herbarium voucher was collected, identified by an ex-
pert, and deposited at the Geneva herbarium (G). For
protected Gentiana species, high-quality photos were
taken in lieu of herbarium specimens.
DNA extraction, amplification and sequencing
Total genomic DNA was extracted using the NucleoS-
pin© Plant II kit (Macherey-Nagel, GmbH & Co. KG,
Düren, Germany) following the supplier’s instructions.
Three cpDNA coding regions (matK, rpoC1, and rpoB)
and one cpDNA spacer (trnH-psbA) were amplified and
sequenced. PCR was performed in 20 μL total volume
with 0.60 U Taq (Roche, Mannheim, Germany), 2 μLof
10X buffer containing 20 mM MgCl
2
, 0.8 μL of each pri-
mer (10 mM), 0.4 μl of a mix containing 10 mM of each
dNTP (Roche), and 0.85 μL of template DNA of un-
known concentration. The PCR program had an initial
heating step at 95 °C for 6 min, followed by 35 cycles of
denaturation at 95 °C for 30 s, annealing for 30 s at a
locus-specific temperature, elongation at 72 °C for 45 s,
and a final elongation step at 72 °C for 10 min. Anneal-
ing temperatures varied between 45 and 52 °C depend-
ing on locus and species (see Additional file 2 for
details). The primers used are also listed in Additional
file 2. PCR products were cleaned and bidirectionally
sequenced using the PCR primers on an ABI 377 auto-
mated sequencer (Applied Biosystems, Foster City, CA,
USA) following the manufacturer’s protocols.
Sequence alignment and data analyses
Contig assembly and sequence consensus were gener-
ated using Sequencher (GeneCodes Corporation, Ann
Arbor, Michigan, USA). Barcode sequences were aligned
in BIOEDIT 7.0.3.5 [55] and edited manually. Sequence
variation was then characterized using the percentage of
conserved sites, the percentage of parsimony informative
sites, and the number of unique sequences per species.
This last measure is the only one that takes into account
insertion/deletion and inversion events. Both events
Caetano Wyler and Naciri BMC Evolutionary Biology (2016) 16:103 Page 9 of 11

were manually coded as single mutation steps [56]. Se-
quence variation analyses were then performed in MEGA
version 6 [57]. All sequences were deposited in GenBank
under accession numbers KU672731—KU674305 and
KU672731—KU674305 (Additional file 4).
In order to investigate how well the different markers
performed individually in identifying species within a
genus, the number of sequences that were private to a sin-
gle species was checked and the number of individuals
unambiguously identified was reported. We also per-
formed a comparison of all possible locus-combinations.
Species discrimination was evaluated using tree-
based analyses. The Neighbour-Joining tree recon-
struction recommended as the standard barcoding
method [1] was adopted and performed with SeaView
4.4.0, based on the K2P model and 100 replicates for
bootstrap analyses [58].
The presence of barcoding gaps was analysed by
graphing the distributions of intra- and interspecific gen-
etic distances for each genus. Sequence divergences were
calculated using pairwise distances with the Kimura 2-
parameter in MEGA [57].
The correlation between sampling size and the
number of unique sequences was computed overall
species and within genera for trnH-psbA, which was
the most diverse barcode within species and the only
one common to all genera. The sequence richness
(Rs) was computed for a sample size of three individ-
uals, using the rarefaction methods that takes into ac-
count sequence frequencies in each species [59]. Rs
was used to quantify the average number of different
sequences that would be recovered using a sampling
size of three individuals within species. Correlations
and Rs were computed in Excel and confidence inter-
vals for correlation coefficients were assessed in the
online program VassarStat (http://vassarstats.net/)
using the Fisher r-to-z transformation.
Median joining networks of the sequences were drawn
using the program Network [60]. These analyses were
performed, within each genus, on the defined DNA
barcode alone or combined: trnH-psbA alone for Acer,
Lonicera,Geranium,andVeronica; option 3 (matK and
trnH-psbA) for Adenostyles, and option 9 (matK, rpoB,
and trnH-psbA) for Salix and Gentiana. Site mutations
and indels were equally weighted and all the structural
mutations (inversions and insertions/deletions of more
than 1 bp) were treated as single-step events.
Availability of data and materials
The datasets supporting the conclusions of this article
are available in the Genbank repository, [accession num-
bers KU672731—KU674305 and KU672731—KU674305
at http://www.ncbi.nlm.nih.gov/genbank/].
Additional files
Additional file 1: List of individuals. For each individual, the country,
district, and absolute coordinates in decimal degrees are given.
(PDF 29 kb)
Additional file 2: Amplification conditions. For each species, annealing
temperature and the primer combination are given for matK, rpoC1, rpoB,
and trnH-psbA. (PDF 27 kb)
Additional file 3: Phylogenetic trees. For each genus and locus, a
neighbour joining tree is presented. Bootstrap values above 80 % are
shown above branches. Codes following species names are individual
numbers (see Additional file 1). (PDF 572 kb)
Additional file 4: Genbank accession numbers. For each sample,
accession numbers for the four loci are given. (PDF 71 kb)
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
Both authors conceived the experiment, conducted field surveys and wrote
the manuscript, SCW performed molecular and statistical analyses. Both
authors read and approved the final manuscript.
Acknowledgements
We wish to thank David Aeschimann, Beat Baumler, Ariane Cailliau, Camille
Christe, Daniel Jeanmonod, Philippe Juillierat, Catherine Lambelet, and Pierre-
André Loizeau for their help in sampling; David Aeschimann for specimen
identifications; Régine Niba and Camille Christe for their help with lab work,
and Slim Chraiti for his help with figures. We are extremely grateful for the
assistance provided by one of the reviewers to improve this manuscript. The
Swiss National Foundation supported this project with grant n°3100A0-
120390 to YN. We are also grateful to the Société Académique de Genève
and to SwissBOL (Swiss Barcode of Life) for their financial support to YN and
SCW, respectively.
Author details
1
Laboratoire de Systématique Végétale et Biodiversité, Conservatoire et
Jardin botaniques & University of Geneva, Chemin de l’Impératrice, 1, 1292,
Chambésy, Geneva, Switzerland.
2
SwissBOL, University of Geneva,
Department of Genetics and Evolution, Quai Ernest Ansermet 30, 1211
Geneva, Switzerland.
Received: 8 January 2016 Accepted: 5 May 2016
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