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RESEARCH ARTICLE
DNA Barcoding Evaluation and Its
Taxonomic Implications in the Species-Rich
Genus Primula L. in China
Hai-Fei Yan
1
, Yun-Jiao Liu
2
, Xiu-Feng Xie
3
, Cai-Yun Zhang
2
, Chi-Ming Hu
1
, Gang Hao
2
*,
Xue-Jun Ge
1
*
1Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical
Garden, Chinese Academy of Sciences, Guangzhou, China, 2College of Life Sciences, South China
Agricultural University, Guangzhou, China, 3Tropical Agriculture department, Guangdong Agriculture
Industry Business Polytechnic College, Guangzhou, China
*haogang@scau.edu.cn (GH); xjge@scbg.ac.cn (XJG)
Abstract
The genus Primula is extremely diverse in the east Himalaya-Hengduan Mountains (HHM)
in China as a result of rapid radiation. In order to overcome the difficulty of morphological
classification of this genus, we surveyed three plastid regions (rbcL, matK, and trnH-psbA)
and two nuclear markers (ITS and ITS2) from 227 accessions representing 66 Primula spe-
cies across 18 sections, to assess their discriminatory power as barcodes. We found that
ITS alone or combined with plastid regions showed the best discrimination across different
infrageneric ranks and at species level. We suggest rbcL+ matK + ITS as the first choice at
present to barcode Primula plants. Although the present barcoding combination performed
poorly in many closely related species of Primula, it still provided many new insights into cur-
rent Primula taxonomy, such as the underlying presence of cryptic species, and several po-
tential improper taxonomic treatments. DNA barcoding is one useful technique in the
integrative taxonomy of the genus Primula, but it still requires further efforts to improve its ef-
fectiveness in some taxonomically challenging groups.
Introduction
There is a critical need for rigorously delineated species for many theoretical studies and practi-
cal applications [1]. However, using traditional morphology-based taxonomy is difficult to dis-
cover morphologically cryptic taxa [2]. Species that are the product of rapid radiations within
single genera can represent suites of morphologically similar taxa that are difficult to distin-
guish both in the field and the herbarium [3]. DNA barcoding is a valuable addition to the tax-
onomic tool box. After 10 years development of DNA barcoding, it has been found that large
genera with rapid evolutionary radiations still pose a significant challenge for a universal bar-
coding system [4,5,6]. In order to understand better the overall discriminatory power of the
PLOS ONE | DOI:10.1371/journal.pone.0122903 April 13, 2015 1/15
a11111
OPEN ACCESS
Citation: Yan H-F, Liu Y-J, Xie X-F, Zhang C-Y, Hu C-
M, Hao G, et al. (2015) DNA Barcoding Evaluation
and Its Taxonomic Implications in the Species-Rich
Genus Primula L. in China. PLoS ONE 10(4):
e0122903. doi:10.1371/journal.pone.0122903
Academic Editor: Shilin Chen, Chinese Academy of
Medical Sciences, Peking Union Medical College,
CHINA
Received: November 27, 2014
Accepted: February 24, 2015
Published: April 13, 2015
Copyright: © 2015 Yan et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This study was financially supported by the
National Natural Science Foundation of China (grant
nos. 31270009, 31170205) [http://www.nsfc.gov.cn/].
Competing Interests: The authors have declared
that no competing interests exist.
plant barcoding loci, future work should focus on groups that experienced rapid evolutionary
radiations, for example, the closely related species within a single genus.
Primula L. is an extraordinarily species-rich group within the east Himalaya-Hengduan
Mountains (HHM) in China. The genus consists of about 500 species with over 300 of these
found in China, and most of them (approximately 200 species) are restricted to populations in
Southwest China, and are mainly confined to the HHM [7]. The HHM and its adjacent regions
have been considered to represent the modern diversification centre of the genus [8]. The ex-
ceptionally high Primula species and/or lineage diversity in China occurred no more than 10
Mya [9], and may have been triggered by the extensive uplifts of the Qinghai-Tibet Plateau
(QTP) since the early Miocene and strengthened by topographical complexity of the QTP and
climate oscillations during the Quaternary [10]. Like other large plant groups co-occurring on
the QTP (such as Pedicularis,Rhododendron,Gentiana and Saussurea), Primula is a taxonomi-
cally challenging group because: 1) many key diagnostic features are tiny and empirical, and
cannot be determined correctly by non-specialists, these features include the shape of calyx
and bracts [7]; 2) many dwarf species (such as Primula section Minutissimae) are too small in
size to separate; and 3) frequent hybridization or introgression can confuse the Primula species
boundaries. Primula species, even distantly related ones, can be hybridized readily in green-
house conditions [11] and in the wild, as reported recently [12–14]. In addition, new Primula
species in the HHM and adjacent area have been described a number of times in recent years
[15–20]. This suggests that the species diversity of Primula is still underestimated. Although
monographs describing Primula do exist [7,11,21,22], the use of keys for the genus requires a
high level of specialized expertise. A more efficient approach to facilitate delimitating Primula
species and discovering cryptic species or lineages in the genus is urgently required. Despite the
promise of DNA barcoding, only a few studies have used it in plant groups that have a high di-
versity in the HHM or in neighboring regions [23–27].
Although the limited ability of DNA barcoding to discriminate species in large genera is
well known, the following questions are still unclear: 1) to what extent could DNA barcoding
discriminate infrageneric levels (i.e., subgenus, section, and series) within large genera? 2)
Could DNA barcodes discriminate between certain closely related species pairs? 3) In rapidly
evolved genera, could DNA barcoding reveal cryptic species? As a typical rapidly evolved plant
taxon in the HHM, the genus Primula provides a good opportunity to answer these questions.
In the current study, we sampled 66 species representing 18 sections of Primula in China; these
contained many closely related groups. The discriminatory ability of three common plastid
barcoding candidates (rbcL, matK, and trnH-psbA) and nuclear regions (ITS and ITS2)
were evaluated.
Materials and Methods
Ethics statement
All samples employed in this study are not endangered nor protected in the sampled area, and
none of the sampled locations are privately owned or protected by any law. No specific permits
were required for the described field studies.
Taxon sampling, DNA extraction and sequencing
During this study we examined a total of 227 accessions of 66 Primula species from 18 of the
24 sections of the genus in China recognized by Hu [21]. We used Omphalogramma delavayi
Franch. as an outgroup [28,29]. In order to explore the pattern of genetic variability in mor-
phological species, more than two individuals of each species were collected. Taking account of
the effect of geographical sampling scale on DNA barcoding [30], more individuals (>10)
DNA Barcodes of Primula Species
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were sampled from widespread species, such as P.secundiflora Franch. and P.Poissonii
Franch., across their ranges to allow for their intraspecific variability.
To test the effectiveness of DNA barcoding in more closely related groups, section Proliferae
was exhaustively sampled in this study. There are approximately 23 species in this section [11].
In China, nineteen species have been described [7,8,21], and a new record species, P.burma-
nica Balf. f. et Ward, in the section was recently discovered on the south side of Ailao Mountain
in Simao (Szemao) region, China (Yan et al., unpublished data). We collected 84 accessions
representing all species of the section in China except P.stenodonta Balf. f. ex W. W. Smith et
Fletcher. In addition, we selected several of the most closely related species groups in the genus,
such as P.chungensis Balf. f. et Ward vs. P.cockburinana Hemsl., P.ovalifolia Franch. vs. P.tar-
diflora C. M. Hu, P.prattii Hemsl. vs. P.pulchella Franch., P.fasciculata Balf. f. et Ward vs. P.
munroi ssp. yagongensis (Petitm) W. W. Smith et Forr., and the P.poissonii complex. Collec-
tion details, voucher numbers, taxonomy, and GenBank accession numbers are listed in S1
Table.
Genomic DNA was extracted from silica gel-dried leaf material following a modified version
of the cetyltrimethyl ammonium bromide (CTAB) protocol of Doyle & Doyle [31]. Five candi-
date DNA barcodes, containing two coding plastid genes (rbcL and matK), one intergenic plas-
tid spacer (trnH-psbA), the nuclear ribosomal internal transcribed spacer (ITS, including ITS1,
5.8s and ITS2) and the internal transcribed spacer2 (ITS2), were evaluated in this study. RbcL
was amplified using the primer combination (rbcLa_f and 724R) as suggested by Fay et al.[
32]
and Kress & Erickson [33], respectively. The amplification of matK was achieved using the
primer pair 3F-KIM and XF ([34]; Kim unpublished data). For trnH-psbA, the primers trnH05
and psbA3 were used [35,36]. ITS was amplified with the primers proposed by White et al.[37].
PCR amplification and sequencing conditions followed Yan et al.[24]. ITS2 was retrieved from
the ITS data in this study.
Data analyses
Sequences for each marker were aligned with Muscle 3.8 [38] and then manually adjusted
using Se-Al 2.0a11 [39]. We focused on evaluating five single markers and their combinations
(rbcL+ matK, rbcL+ matK+ trnH-psbA,rbcL+ matK+ITS, rbcL+ matK+ITS2, rbcL
+ matK+ trnH-psbA + ITS, and rbcL+ matK+ trnH-psbA + ITS2). For the pair-wise genetic
distance (PWG-distance) method, the genetic pairwise distance was determined by MEGA6
using the Kimura two-parameter distance model (K2P) with pairwise deletion of missing sites
[40]. Three parameters (average intraspecific distance, average theta (ө), and coalescent depth)
were calculated for all markers. In order to evaluate the ‘local’barcoding gap for each species
[41,42], we plotted the maximum intraspecific divergences against the smallest interspecific
distances for each species [41,43].
To test whether accurate species assignments can be made among the samples using a single
marker or combinations of markers, we used another two distance-based methods the ‘best
match’(BM) and ‘best close match’(BCM) using the TaxonDNA/Species Identifier 1.7.7-dev3
program [44]. BM assigns the query to the species with the smallest distance sequence, whereas
BCM only identifies the query when the closest sequence is within a distance threshold. The
threshold value is determined by using the distance less than 95% of all intraspecific distances,
which was calculated by the pairwise summary function [44].
For the tree-building method, we calculated the proportion of monophyletic groups using a
Neighbor-Joining (NJ) tree. The test was performed using PAUPv4b10 with the K2P model
[45]. If all individuals of a species cluster together with a bootstrap value above 70%, then the
species was considered as having been successfully identified.
DNA Barcodes of Primula Species
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Results
Sequence characteristics and genetic divergence
All plastid markers (rbcL, matK, trnH-psbA) were successfully amplified across all individuals,
but amplification of ITS failed in two Primula species (P.virginis Lévl. and P.duclouxii Petitm.)
and one accession of P.gemmifera Batal. (GXJ253, voucher: Hao940) in this study (S1 Table).
The characteristics of the five DNA markers are presented in Table 1. Overall, the aligned
length of the five markers ranged from 241 bp (ITS2) to 857 bp (trnH-psbA). The proportion
of variable sites were the lowest for rbcL and highest for ITS2. RbcL exhibited the lowest intra-
specific and/or inter-specific divergence as well, whilst trnH-psbA showed the highest intra-
specific divergence (0.87%), followed by ITS2 (0.80%). However, the greatest interspecific dis-
tance was found in ITS2 (12.73%), followed by trnH-psbA (11.69%). The box-and-whisker
plots (Fig 1) indicate the distance distribution of inter- and intra-specific distances for all
single markers.
The mean intra and inter-specific genetic divergence for the main combinations varied in
the ranges 0.24% to 0.47% and 3.71% to 6.70%, respectively (Table 1). The combination of
rbcL+ matK+ trnH-psbA + ITS exhibited the highest mean intra- and inter-specific distance,
followed by rbcL+ matK + ITS. The core barcode rbcL+ matK exhibited the smallest intra-and
inter-specific genetic difference (Table 1).
Discrimination success of candidate barcodes
The local barcoding gap, with an interspecific distance larger than the intraspecific distance for
a species, directly reveals the species discrimination ability of barcodes. The proportion of the
local barcoding gap varied between the regions tested (Figs 2and 3,S2 Table). ITS showed the
best discriminatory power (54.69%) among the five single candidate barcodes, followed by
trnH-psbA (48.40%). In contrast, rbcL provided the lowest discrimination rate (24.24%). Of all
the combinations tested, the proportion of the barcoding gap of the core barcode combination
(rbcL+ matK) was the lowest (42.42%) (Fig 3,S2 Table), while rbcL+ matK+ trnH-psbA+
ITS exhibited the highest local barcoding gap (68.75%) followed by rbcL+ matK + ITS and
rbcL+ trnH-psbA + ITS (65.63%). TrnH-psbA and ITS2 individually and/or combined with
other plastid markers did not perform well enough to discriminate Primula species in this
study (Fig 3,S2 Table). For example, rbcL+ matK+ trnH-psbA and rbcL+ matK + ITS2 could
Table 1. Summary of genetic variability and sequence characteristics of the candidate barcodes and their main combinations in this study.
rbcLmatKtrnH-
psbA
ITS ITS2 R + M R + M
+T
R+M
+I
R+M
+I2
R+M+T
+I
Aligned length (bp) 614 718 857 680 241 1333 2191 2015 1575 2872
Average intra-distance 0.14% 0.33% 0.87% 0.75% 0.80% 0.24% 0.36% 0.41% 0.32% 0.47%
Average inter-distance 2.14% 5.12% 11.69% 11.10% 12.73% 3.71% 5.06% 6.04% 4.92% 6.70%
Average theta (ө)0.17% 0.24% 0.25% 0.48% 0.58% 0.21% 0.21% 0.29% 0.26% 0.29%
Coalescent Depth 5.57% 2.04% 4.15% 5.30% 6.36% 2.58% 1.91% 2.39% 2.19% 2.31%
Proportion of variable sites 15.79% 33.43% 47.37% 50.88% 51.19% 25.36% 32.63% 32.90% 29.21% 37.05%
Proportion of parsimony sites 12.38% 27.72% 32.56% 43.09% 48.13% 20.63% 25.33% 27.84% 24.57% 29.18%
Rate of PCR and sequencing
success
100% 100% 100% 97.80% 97.80% N/a N/a N/a N/a N/a
R, rbcL; M, matK; T, trnH-psbA; I, ITS; I2, ITS2.
doi:10.1371/journal.pone.0122903.t001
DNA Barcodes of Primula Species
PLOS ONE | DOI:10.1371/journal.pone.0122903 April 13, 2015 4/15
identify 36 Primula species (56.65%), while rbcL+ matK + ITS performed better and identified
65.63% of the Primula species.
Compared with the PWG-distance method, the BM and BCM analyses all showed better
discrimination success. BCM always had a lower identification rate than BM analysis (S2
Table). Based on the BM model, ITS performed best among the five single DNA regions, and
successfully assigned 81.98% sequences to the correct species (Fig 3). The identification rate of
the two-locus combinations ranged from 71.36% to 89.63%. Among them, the core barcode
combination rbcL+ matK correctly identified 72.24% of specimens, which was only slightly
better than rbcL+ trnH-psbA (71.36%). For three-locus combinations, matK+ trnH-psbA+
ITS, rbcL+trnH-psbA + ITS, and rbcL+ matK + ITS provided similar discrimination rates
(90.99%, 90.54%, and 89.18%), followed by rbcL+ matK+ trnH-psbA (78.41%). In addition,
combinations with ITS2 always produced a lower identification rate compared to combinations
with ITS (Fig 3,S2 Table).
The tree-building method provided a similar result to the distance-based method. In this
analysis, we found that ITS was the best of all single markers, successfully identifying 53.13% of
species. Of the combinations, rbcL+ matK showed the poorest discriminatory power (37.88%),
while rbcL+ matK+ trnH-psbA + ITS was the best one with a 64.06% discrimination rate, fol-
lowed by rbcL+ matK + ITS and matK+ trnH-psbA + ITS (60.94%) (Fig 3,S2 Table).
When we considered the previously recognized infrageneric taxa (the twenty four sections,
[21]), rbcL and trnH-psbA each only identified four sections (S1 Fig). The discrimination rate
of ITS was the best among all single barcodes, distinguishing eight sections (section Pycnoloba,
Fig 1. Comparisons of the distribution ranges of inter- and intraspecific distances using boxplots.
doi:10.1371/journal.pone.0122903.g001
DNA Barcodes of Primula Species
PLOS ONE | DOI:10.1371/journal.pone.0122903 April 13, 2015 5/15
section Auganthus, section Souliei, section Soldanelloides, section Sikkimensis, section Amethys-
tina, section Muscarioides, and section Petiolares)(S1 Fig). Among the main combinations, the
core barcode (rbcL+matK) only successfully identified five sections (section Pycnoloba, section
Fig 2. Relationships between inter- and intraspecific distance indicating the local gaps for species.
doi:10.1371/journal.pone.0122903.g002
Fig 3. Species discrimination rates of several main barcodes in Primula.R,rbcL; M, matK; T, trnH-psbA; I, ITS; I2, ITS2.
doi:10.1371/journal.pone.0122903.g003
DNA Barcodes of Primula Species
PLOS ONE | DOI:10.1371/journal.pone.0122903 April 13, 2015 6/15
Auganthus, section Souliei, section Soldanelloides, and section Sikkimensis), followed by the
combinations rbcL+ matK+trnH-psbA + ITS2, rbcL+ matK+ trnH-psbA + ITS and rbcL+
matK+ trnH-psbA, which all identified the same eight same sections as ITS. In contrast, rbcL+
matK + ITS was the best combination, and was able to discriminate nine sections (including
section Proliferae)(Fig 4). Our sampling represented four subgenera (subgenus Auriculastrum,
subgenus Auganthus, subgenus Carolinella and subgenus Aleuritia) according to the revised
classification of Primula [11], nevertheless, the majority of DNA barcodes singly or jointly could
only separate out subgenus Auriculastrum correctly.
Fig 4. Neighbor-joining tree based on the combination rbcL+ matK + ITS with the K2P distance model. (A) The whole tree of Primula except
section Proliferae. (B) The tree of section Proliferae.Asterisks along branches indicate monophyletic species with bootstrap values above 70%.
Accessions are suffixed by sample ID. Monophyletic sections are highlighted with grey shading.
doi:10.1371/journal.pone.0122903.g004
DNA Barcodes of Primula Species
PLOS ONE | DOI:10.1371/journal.pone.0122903 April 13, 2015 7/15
Discrimination ability of DNA barcoding in closely related groups
Section Proliferae is an example containing closely related taxa that is suitable for testing the
discriminatory performance of DNA barcoding. Using the tree-building method, the core bar-
code (rbcL+ matK) could only correctly identify P.smithiana Craib with a relatively high boot-
strap value (i.e. over 70%), whereas ITS alone could distinguish five species (S1 Fig). rbcL+
matK + ITS was the most efficient and precise combination in this study, as stated above, but it
only discriminated 10 species correctly (52.63%) in this section (Fig 4). Section Proliferae con-
tained three taxonomically challenging groups (or species complexes). Although the three
groups could be easily distinguished by rbcL+ matK + ITS, the species within each group were
difficult to discriminate using the current barcodes singly and/or in combination. For example,
the P.poissonii complex is resolved as monophyletic with high support by rbcL+ matK + ITS,
however, only two narrowly distributed species (P.anisodora Balf. f. et. Forr. and P.miyabeana
Ito et Kawakami) could be readily distinguished (Fig 4).
Compared with section Proliferae, the discrimination performance of DNA barcoding
in other Primula species was much better (64.44%, based on the tree-building result of
rbcL+matK + ITS). However, we found that a failure often occurred in the most closely re-
lated species groups (Table 2), such as P.chungensis vs. P.cockburinana,P.ovalifolia vs. P.
tardiflora,P.prattii vs. P.pulchella,andP.fasciculatavs. P.munroi ssp. yagongensis. In addi-
tion, some species showed extremely high intraspecific divergence (>1%); these included P.
moupingensis Franch., P.bella Franch., P.fasciculata,P.malvacea Franch. and P.yunnanen-
sis Franch. (Table 2). Most of the species with extremely high intraspecific divergence cannot
Table 2. Summary of the candidate barcode rbcL+ matK + ITS divergence pattern for unidentified species.
Taxon The nearest
relative
Mean intraspecific divergence
(%)
Maximum intraspecific distance
(%)
Minimum interspecific distance
(%)
P.prattii P pulchella 00 0
P.pulchella P.prattii 0.1 0.15 0
P.burmanica P.mallophylla 0.19 0.21 0.16
P.chungensis P.cockburniana 0.17 0.21 0.11
P.bulleyana P.aurantiaca 0.32 0.21 0.11
P.aurantiaca P.bulleyana 0.13 0.22 0.11
P.chrysochlora P.helodoxa 0.13 0.26 0.26
P.poissonii P.anidosora 0.12 0.38 0.38
P.chionantha P.melanops 0.18 0.41 0.1
P.beesiana P.bulleyana 0.02 0.43 0.21
P.wilsonii P.miyabeana 0.25 0.48 0.37
P.septemloba P.heucherifolia 0.32 0.56 0.46
P.prenantha P.helodoxa 0.21 0.58 0.26
P.ovalifolia P.tardiflora 0.42 0.61 0.36
P.alpicola P.sikkimensis 0.26 0.77 0.48
P.
blattariformis
P.malvacea 0.6 0.93 0.83
P.moupinensis P.epilosa 0.54 1.24 0.61
P.denticulata P.kialensis 0.48 1.6 1.3
P.bella P.yunnanensis 0.9 1.7 1.03
P.fasciculata P.munroi 0.99 1.72 1.55
P.malvacea P.blattariformis 0.85 1.84 0.83
P.yunnanensis P.bella 1.82 2.39 1.03
doi:10.1371/journal.pone.0122903.t002
DNA Barcodes of Primula Species
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be correctly distinguished by any of the three methods, which probably indicates their status
should be further examined.
Discussion
The resolution of the tested DNA markers in Primula
In this study, all the three plastid regions tested individually showed a relatively low discrimi-
natory efficacy ranging from 15.16% to 31.82% (based on monophyletic analysis) in Primula
species (S2 Table). The core barcode rbcL+ matK also provided low discrimination at a rate of
37.88% (S2 Table). One of the most promising supplementary plastid barcodes, trnH-psbA,
varied in size from 154 bp (P.poissonii) to 523 bp (P.polynuera Franch.), so there were a large
number of gaps in the alignment matrix. Based on tree-building analysis, trnH-psbA identified
42.19% of species; this was the best among the plastid regions but lower than the nuclear mark-
ers (ITS) (S2 Table). The combination of trnH-psbA with rbcLormatK did not result in higher
resolution (S2 Table), which demonstrated that trnH-psbA is not a preferred barcode in
Primula.
The strong identification ability of ITS has been verified based on a comprehensive study
[46], even in some complex plant groups, such as Panassia [25], Ficus [47], Lysimachia [27],
and Sisyrinchium [48]. In this study, ITS exhibited the highest discriminatory power among
all five markers, and any combinations with ITS were able to discriminate more species than
combinations without ITS (Fig 3,S2 Table). Of the three-locus combinations, rbcL+ matK+
ITS and matK+trnH-psbA + ITS all distinguished 60.94% of monophyletic species, which was
the best discrimination performance (Appendix S2). Therefore, as suggested by Yan et al.[24],
rbcL+matK + ITS should be the first choice to barcode Primula plants. Compared with primer
problems associated with ITS, ITS2 has conserved regions for designing universal primers, and
can be readily amplified in various groups [49]. However, ITS2 itself or combined with plastid
markers did not produce better results than ITS and/or corresponding combinations (S2 Table).
We suggest that ITS2 may be an ideal supplementary barcode when ITS amplification fails.
Discrimination performance on section rank in Primula
DNA barcoding should be able to help identify some groups within large genera, thus reducing
the time required for morphological studies to produce definitive species lists. Although it is
well known that DNA barcoding has difficulties in resolving closely related species, it is not
clear whether such barcoding could identify samples correctly to section level within large gen-
era. There are more than 200 Primula species concentrated in the HHM region in China [11].
Primula has always been divided into subgroups, usually with the rank of section [22,50]. In a
well-accepted infrageneric system, Smith and Fletcher divided the genus into a total of 31 sec-
tions [22]. Twenty-four sections of the Chinese Primula were adopted by Hu [21].
In this study, DNA barcoding performed well for distinguishing sections, and could resolve
nine of the current 18 sections [21]. However, of the resolved sections, three (namely section
Auganthus,sectionSouliei, and section Soldanelloides) together with the monotypic section Pyc-
noloba were each represented by one species in the current study. Considering the fact that the
phylogeny of many sections and their close relatives, such as section Soldanelloides,Minutissimae,
and Souliei, still lack detailed studies [28,29], the discrimination rate of DNA barcoding would
probably drop further if we expanded the number of members in these sections. These results
demonstrated that DNA barcoding is useful in some sections of Primula. In addition to barcoding
discriminatory ability, the infragenetric classification system will also influence the results. A reli-
able and well-recognized infrageneric rank in a large genus is a prerequisite for applying DNA
DNA Barcodes of Primula Species
PLOS ONE | DOI:10.1371/journal.pone.0122903 April 13, 2015 9/15
barcoding. Therefore, a number of new sectional delimitations will be necessary in the genus
Primula [11,28].
Resolving ability of DNA barcoding in section Proliferae
The Primula section Proliferae is a well-delimited and natural group characterized by numer-
ous whorls of flowers resembling candelabra [11]. It is mainly concentrated in the HHM [7]. In
China, 19 species have been described and, with the exception of P.miyabeana (endemic to
Taiwan), they are narrowly distributed in southwest China [7]. This section contains several
taxonomically challenging groups, such as the P.poissonii complex, which consists of P.aniso-
dora,P.wilsonii Dunn and P.poissonii, and the P.beesiana group with P.beesiana Forr., P.bul-
leyana Forr., P.burmanica and P.pulverulenta Duthie. This complex section provides a good
example to test the discriminatory ability of candidate barcodes in closely related species, espe-
cially those formed through rapid evolutionary radiation.
Although the discriminatory power of DNA barcoding is limited in section Proliferae (dis-
crimination rate of 52.63%), in the current study it confirmed the monophyly of section Prolif-
erae (tree-building method) (Fig 4), and divided the section into three clades with high support
(over 85%), which agree well with the study based on their morphology [51]. It is convenient
for us to assign unknown Primula specimens to a rough position in the section. This could
help to narrow the scope of identification. Within each complex or clade, DNA barcoding
could still provide some clues for identification and taxonomic treatment. For example, P.pois-
sonii and P.anisodora have the closest relationship and they were confirmed by the current
barcodes (Fig 4), but only P.anisodora exhibited monophyly. DNA barcoding could also help
to solve several classification disputes in this section. For example, barcoding supported treat-
ing P.wilsonii and P.anisodora,P.burmarica and P.beesiana as separate species [7,11,21,51]
(Fig 4). Therefore, even for very closely related species, DNA barcoding may still provide help
to some extent, and narrow the identification range.
It is well known that using the universal DNA barcode (two core barcodes and two alterna-
tive barcodes, trnH-psbA, ITS) it is almost impossible to separate very closely related species
formed through rapid radiation. Therefore, species-specific barcodes need to be developed for
difficult taxa [6]. These markers may be based on other rapidly evolved molecular markers such
as low or single copy nuclear genes (e.g. waxy and leafy)[52] or even using high-throughput se-
quencing methods (such as RAD and GBS).
Biological implications of DNA barcoding in Primula
Traditional taxonomy mainly depends on morphological diagnosis, and it should be corrobo-
rated by other sources of data, such as geographical, ecological, reproductive and DNA se-
quence information [53]. However, constructing a robust taxonomy for recently diverged plant
taxa is more difficult, because they often show little difference in their morphological and ge-
netic profiles. In addition, many other aspects could also cause the failure of DNA barcoding,
such as imperfect taxonomy, interspecific hybridization, paralogy, and incomplete lineage sort-
ing [42,52,54,55]. For many such taxa, DNA barcoding provides an opportunity to solve some
of the taxonomic problems through discovering the underlying biological issues.
By surveying the non-monophyletic taxa at species level and examining genetic distance (Fig
4,Table 2), we filtered out barcoding failures in several species probably caused by incomplete
lineage sorting. For example, narrowly distributed P.tardiflora,P.prattii, and P.cockburiana
each experienced peripheral isolated speciation from their widely distributed relatives (putative
parents) (P.ovalifolia,P.pulchella, and P.chungensis)[55]. The barcoding results were partial-
ly supported by a complementary phylogeographic study [56]. It is a question for taxonomy to
DNA Barcodes of Primula Species
PLOS ONE | DOI:10.1371/journal.pone.0122903 April 13, 2015 10 / 15
reflect on these incomplete speciation processes by synonymizing the nested and parent species
or elevating lineages in the paraphyletic lineage to species status [55]. In the context, we prefer
to treat the nested and parent species as one species because of their similarity in morphology
[7,21], but of course additional research is necessary.
Imperfect taxonomy in several plant and animal taxa has been detected by DNA barcoding
(e.g. [27,42,57–59]), providing significant support for the taxonomic value of the technique. P.
bella examined in this study is an excellent example of over-lumping in traditional taxonomy,
as the species appeared polyphyletic and exhibited unexpectedly large intraspecific divergence
(Table 2,Fig 4). Given the variable morphological characters (such as shape of bracts and the
stem length), there are classification disputes about the delimitation of P.bella [7,11,21,60].
DNA barcoding supported the suggestion that the anomalous individual P.bella GXJ096
(voucher: Hao & Yan 956) should be raised to species status (P.cyclostegia Hand.-Mazz.) on
the basis of its genetic profile, although additional work is essential to validate this as a robust
species. A similar situation is also probably the case for P.denticulata Smith.
Discovering the potential presence of cryptic species and/or lineages is an important appli-
cation of DNA barcoding, and this remains within the domain of taxonomy [53]. The taxo-
nomic usefulness of DNA barcoding has been validated in a wide range of animals (see, for
example [61–67]), but there are few studies of large plant groups that have recently experienced
evolutionary radiation. It is plausible that the frequent occurrence of cryptic species in Chinese
Primula represents adaptation to the variable habitats on the HHM and rapid radiation evolu-
tion in a relatively short time [7,9,11,21]. By iteratively reexamining peculiar specimens de-
tected by DNA barcoding (such as P.yunnanensis GXJ099, P.fasciculata GXJ249, and P.
moupinensis GXJ259) (Table 2,Fig 4), several tiny morphological or geographical divergences
may be identified in these taxa, which indicate the possibility of cryptic species; however, fur-
ther taxonomic scrutiny is required.
Another great challenge for barcoding plant species is linked to hybridization events
[23,52,54,68]. Natural or artificial hybrids in Primula have been reported recently [11–14], and
these may cause a failure in barcoding Primula species. In the current study, underlying hy-
bridization might occur in P.anisodora and its most close relative P.poissonii. They were
found in the same populations, and a putative hybrid (P.poissonii Y640) was also discovered
(S2 Fig). Additional research is needed to resolve the biological situation (e.g. [69,70]).
Conclusion
Primula species examined in the present study are difficult to distinguish using the core bar-
code (rbcL+ matK). Another plastid marker, trnH-psbA, varied in size and exhibited lower dis-
crimination compared to ITS, suggesting that it is not a suitable barcode for studies of Primula.
In contrast, ITS showed the best discriminatory ability of all the single markers tested, discrimi-
nating 65.63% and 60.94% of species (according to the PWG-distance method and tree-build-
ing method) when combined with rbcL+ matK, which performed best among all three-locus
combinations. We propose that rbcL+ matK+ITS should be treated as the first local barcode
in the genus Primula at present, although its discrimination rates with respect to infrageneric
rank and separating closely related Primula species are limited.
Despite the limited discrimination for closely related pairs, DNA barcoding provided many
new insights into the current Primula taxonomy, such as detecting potential cryptic species, and
revealing several probably improper taxonomic treatments. Obviously, it is difficult to resolve
all closely related groups based on the current limited and relatively conserved molecular mark-
ers, especially in taxa such as Primula, which have experienced recent rapid radiation. Other
more rapidly evolved molecular markers should be incorporated into future DNA barcoding
DNA Barcodes of Primula Species
PLOS ONE | DOI:10.1371/journal.pone.0122903 April 13, 2015 11 / 15
projects, for example low or single copy nuclear genes, nuclear SNPs, nuclear SSRs [23,52], and
the complete chloroplast genome [71–74]. As proposed by Twyford [6], we are building a robust
phylogeny framework for the Primula section Proliferae using RAD (restriction-site-associated
DNA, [75]), and expect to resolve the true evolutionary relationships; these may be necessary to
develop robust species-specific barcodes in the future [6]. Overall, DNA barcoding is a useful
technique for the integrative taxonomy of the genus, but it still requires further work to improve
its value for studying taxonomically challenging groups.
Supporting Information
S1 Fig. Neighbor-joining trees based on candidate barcodes and their main combinations
with K2P distance model. Asterisks along branches indicate monophyletic species with boot-
strap values above 70%. Accessions are suffixed by sample ID. Monophyletic sections are
highlighted with grey shading.
(PDF)
S2 Fig. Three individuals of Primula poissonii complex and their flowers.
(DOCX)
S1 Table. Taxon, voucher, collection information, and Genbank accession numbers.
(XLSX)
S2 Table. Discrimination success based on different analysis methods.
(DOCX)
Acknowledgments
We are grateful to Drs. Lian-Ming Gao, Zhi-Kun Wu, Xun Gong, Yu-Ming Shui, Zhong-Lai
Luo, Yuan Xu, Bing-Qiang Xu, and Xin Wu for their help in collecting plant material. We also
thank Dr. Juan Liu and Ms. Yu-Ying Zhou for help in data analysis and laboratory work.
Author Contributions
Conceived and designed the experiments: HFY GH XJG. Performed the experiments: HFY YJL
CYZ. Analyzed the data: HFY YJL CYZ. Contributed reagents/materials/analysis tools: CMH
GH XFX XJG. Wrote the paper: HFY XJG GH.
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