Journal of Threatened
Taxa | www.threatenedtaxa.org | 26 February 2025 | 17(2): 26547–26552
ISSN 0974-7907 (Online)
| ISSN 0974-7893 (Print)
https://doi.org/10.11609/jott.9486.17.2.26547-26552
#9486 | Received 11
November 2024 | Final received 23 December 2024 | Finally accepted 06 February
2025
New distribution
record and DNA barcoding of the steno-endemic plant Cordia diffusa (Boraginaceae)
M. Haritha 1,
D. Leena Lavanya 2 & H. Abinaya 3
1,2,3 Department of
Botany, Avinashilingam Institute of Home Science and
Higher Education for Women, Coimbatore,
Tamil Nadu 641043,
India.
1 harithamuthu0111@gmail.com,
2 leenalavanyad@gmail.com (corresponding author), 3 abisiva238@gmail.com
Editor: Mandar Paingankar, Government Science College Gadchiroli,
Maharashtra, India. Date of publication: 26 February
2025 (online & print)
Citation: Haritha, M., D.L. Lavanya & H. Abinaya (2025). New distribution record and
DNA barcoding of the steno-endemic plant Cordia diffusa
(Boraginaceae). Journal of Threatened Taxa 17(2): 26547–26552. https://doi.org/10.11609/jott.9486.17.2.26547-26552
Copyright: © Haritha et al. 2025. Creative Commons Attribution 4.0 International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: The author declares that no funds, grants, or other support were received during the preparation of this manuscript.
Competing interests: The authors declare no competing interests.
Author details: Haritha, M., is a research scholar in the Department of Botany at Avinashilingam Institute for Home Science and Higher Education for Women. Her research focuses on plant anatomy, DNA barcoding, and phytochemistry. Dr. D. Leena Lavanya is an assistant professor in the Department of Botany at Avinashilingam Institute for Home Science and Higher Education for Women, holds a doctoral degree in Botany. Her research expertise includes molecular biology, phytochemistry, and mushroom cultivation. Abinaya, H., is a research scholar in the Department of Botany at Avinashilingam Institute for Home Science and Higher Education for Women. Her research focuses on the biofortification of mushrooms.
Author contributions: MH–data collection, data analysis, and writing-original draft. DLL–supervision, guidance in research methodology, and writing-review & editing. HA–assistance in data collection and visualization.
Acknowledgements: The author thanks Dr. R. Sathishkumar, Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India, for carrying out the DNA barcoding work.
Abstract: The research marks
the first documented occurrence of Cordia diffusa
in Kurumbapalayam, a location where it had not been
reported previously. In this study, genomic markers were also utilized to
document the endemic plant species Cordia diffusa
K.C.Jacob of the Boraginaceae family. This study employed chloroplast
markers such as matK, rbcL,
and the psbA-tnH region to determine their efficiency
in identifying the species. For the first time, C. diffusa
has been analyzed and documented using these genetic markers. Sequences
obtained from three different primer sets for each marker were submitted to
GenBank. This research highlights the first genetic documentation and
identification of C. diffusa using chloroplast
markers, contributing valuable genetic data to GenBank and advancing the
understanding of this endangered species distribution.
Keywords: Boraginaceae,
chloroplast markers, Endangered species, Kurumbapalayam,
matk, psbA-tnH, rbcL, species identification.
Introduction
The Cordia
plant genus is part of the Boraginaceae family often
referred to as the forget me-not family and includes herbs and trees, among its
2,700 species spread across almost 125 genera historically categorized into
five subfamilies named Boraginoideae, Cordioideae, Ehretioideae, Heliotropioideae, and Wellstedioideae
(De Candolle in 1845; Bentham & Hooker in 1876; Engler in 1898; Pilger
& Krause in 1915). However, according to evolutionary studies, physical
traits, and consistent naming conventions, these have now been expanded to 11
separate and related plant families. These include Boraginaceae
sensu stricto, Codonaceae, the established Coldeniaceae
family, Cordiaceae, Ehretiacaea,
Heliotropceae, Hoplestigmataceae,
Hydrophylacaea, Lennoaceae,
Namaceae, and Wellstediaceae
as detailed by Luebert et al. (2016). The genus name Cordia
pays tribute to the botanist and pharmacist Valerus Cordus as referenced by Quattrocchi
(2012).
Henry et al. (1978)
reported Cordia diffusa K.C.Jacob, a shrub commonly known
as Kovai Manjack or Sirunaruvilli, is a rare and threatened flowering plant of
southern India. While it is believed to be endemic to this region (Arumugam et
al. 2019), reports suggest its presence in other areas like the Gulf of Mannar (Daniel & Umamaheshwari
2001) and Chengalpattu District (Ramamurthy 1987; Nayar
1996; Singh et al. 2015). In the region of Kurumbapalayam,
in Sarkarsamakulam, within Coimbatore C. diffusa has been recently discovered. Highlighted by
its traits the identification of plant species has been a common practice.
Spotting endangered plants that resemble species based on their vegetative
features poses a challenge. A solution to this dilemma could lie in DNA
barcoding, introduced by Hebert et al. (2003) for the purpose of species
identification or discovery. The ideal DNA barcode should be a consistent short
DNA sequence (400–800 bp) that can be produced and
characterized efficiently for all living species (Savolainen
et al. 2005). CBOL Plant Working Group (2009) evaluated the performance of psbK–psbI, atpF–atpH, and trnH–psbA spacers, and matK, rbcL, rpoB, and rpoC1 and among
the seven plastid barcode loci, recommends that rbcL
and matk as standard barcoding region, ITS and trnH-psbA as supplementary barcode loci (China plant BOL
group et al. 2011; Pang et al. 2012) these four loci were widely accepted as
plant DNA barcodes (Yu et al. 2011; Li et al. 2012; Parveen et al. 2012; Aubriot et al. 2013; Saarela et
al. 2013; Yan et al. 2015). Plastome sequences have
also been used widely to study the phylogenetic connections of numerous plant
taxa (Lv et al. 2022; Su et al. 2022; Xu et al. 2022;
Hu et al. 2023).
It should be
remembered that DNA barcoding is not a substitute for taxonomy, but rather an
important tool that generates information on undiscovered species (Ebach et al. 2005). Rapid economic development and its
impact on the flora and fauna of numerous nations, particularly in the tropical
and subtropical regions, molecular identification can speed up identification
and improve the accuracy of species separation in complex species and tree
plants and estimate the biodiversity of these regions to preserve the rare
endemic and endangered species. In the present study, DNA barcoding of the
endangered species Cordia diffusa, which has
not been done so far, is done, and its phylogenetic relationship with other
species was determined based on BLAST results.
Materials and Methods
Plant collection
Plant materials were
collected in 2022 in Kurumbapalayam, Coimbatore
(Figure 1, Image 1). The collected specimens were authenticated, and a herbarium voucher was submitted to the Botanical Survey of
India, Southern Circle, Coimbatore, Tamil Nadu.
DNA isolation, PCR
amplification and DNA sequencing
DNA from C. diffusa leaf was obtained using the Macherey
Nagel Nucleospin Plant II mini spin DNA extraction
kit from 100 mg of tissue. The amount of DNA extracted was measured using a Thermo Scientific Nanodrop 2000 spectrophotometer. To
barcode the DNA samples, for identification purposes the specific genes matK, rbcL and trnH-psbA were used for PCR amplification using a thermal
cycler (Bio-Rad, USA). PCR reactions were performed with a total volume of 30µl
reaction mixture containing 2µl (100 ng) of DNA, 15 µl of EmeraldAmp
GT PCR mix (TaKaRa, Bio USA, Inc.), 0.5 µl (10 pM) of each forward and reverse prime, and 12 µl of
nuclease-free water. PCR cycling conditions were: one cycle of 5 min at 95°C,
followed by 30 cycles at 95°C for 1 min. Annealing at different temperatures
for different genes: 58°C for the matK gene, 55°C for
the rbcL gene, and 57°C for the trnH
psba gene for 30 seconds and extension at 72°C for a
minute and the final extension step at 72°C for 10 minutes. The reaction
mixture consists of 2µl (100 ng) of DNA, 15µl of EmeraldAmp
GT PCR mix (TaKaRa, Bio USA, Inc.), 0.5µl (10 pM) of each forward and reverses primer, and 12µl of
nuclease-free water made up to 30µl. The primer set descriptions are given in
Table 1. The amplified PCR products were loaded in a 1% TAE agarose gel and
observed using a UV-Transilluminator. The amplified DNA was sequenced at Biokart (Karnataka, India). Sequencing was performed in
both primers to obtain the full length of the targeted region.
Sequence submission
and data analysis
The DNA sequences
were assembled and edited using codon code Aligner V 2.06 (Codon Code Co, USA)
to remove unclear base calls, and both sequences were assembled. The sequences
were modified according to the NCBI criteria (https://www.ncbi.nlm.nih.gov/)
and compared to the global sequence in the GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi)
using the nucleotide BLAST algorithm. Both BLASTn and
BLASTx were performed sequentially. The sequences
were submitted to NCBI through the Bankit submission
channel (https://www.ncbi.nlm.nih.gov/WebSub/). To determine the percentage of
sequence similarity, the FASTA format of Cordia diffusa
DNA sequences was compared with the NCBI database using BLAST + 2.14.).
Results
A detailed literature
survey on the occurrence of C. diffusa was
done, and the species previously reported in Vaalankulam,
Ramanathapuram (Chandrabose
1968), and IFGTB (Prabu et al. 2019) were identified in Kurumbapalayam,
Coimbatore. The herbarium of the plant was submitted to the Botanical Survey of
India, Coimbatore, with Voucher number MH178154 (Image 2).
PCR Results and DNA
Barcode Sequence Analysis
Partial DNA
sequencing was performed using matK, rbcL, and trnH-psbA gene regions.
The accession number of the GenBank submitted sequences with their respective
base lengths and nucleotide composition percentages are summarized in Table 2.
Among the genes used for the study, the highest G+C content (42%) was observed
in the rbcL gene compared to matK
and trnH-psbA.
Discussion
Cordia diffusa, a rare, endemic shrub species geographically
restricted to Tamil Nadu and Sri Lanka, was first identified by Dr. K. Cherian
Jacob in 1938 and later described in 1944. Henry et al. (1978) listed C. diffusa as a rare and threatened plant of southern
India. Later, Chandrabose & Nair (1988) expressed
concerns over its potential extinction, classifying it as indeterminate on the
IUCN Red List of Threatened Plants (Rao et al. 2003). Although C. diffusa has been reported at several locations in
Coimbatore, recent observations by Prabhu et al.
(2019) indicated the removal of the species from the IFGTB region where it had
previously been found. This study adds new findings by documenting the presence
of C. diffusa in Kurumbapalayam,
Coimbatore.
Plant species
identification primarily relies on sequencing variation from reference
sequences and reconstructing phylogenetic relationships (Altschul
et al. 1997). But as Smith et al. (2008) pointed out, in order to comprehend
the detected species, it is crucial to combine plant DNA data with other
traits, including morphology, ecology, and development. Because the chloroplast
genomes of angiosperms are so well conserved, the chloroplast regions matK, rbcL, and trnH-psbA were used in this study to barcode C. diffusa (Chevenet et al.
2006; Amiryousefi et al. 2018; Mehmood et al. 2020).
Reliable molecular markers for species identification are provided by these
conserved areas.
Across the plant
taxa, rbcL is highly conserved, enabling the
development of universal primers for PCR amplification. However, the variation
within the rbcL region allows for species
identification and phylogenetic studies (Kress & Erickson 2007;
Hollingsworth et al. 2009). C. diffusa
(OR096230.1) has a base pair length of 548. The main purpose of DNA barcoding
is to tell species apart effectively due to its significant variability, which
helps in identifying plant species, especially when dealing with diverse groups
where other markers might struggle with distinguishing closely related taxa as
mentioned by Kress et al. (2005) and Shaw et al. (2007).
Conclusion
The study serves a
significant part in documenting the molecular identification of Cordia diffusa, an endangered and steno-endemic plant species.
In addition to enriching molecular databases, DNA sequences submitted to
GenBank are a useful resource for upcoming studies in conservation biology and
plant taxonomy. The new occurrence of the plant in Kurumbapalayam,
Coimbatore, extends its known distribution and shows the need for continuous
research and observation of regional plants. Further studies will concentrate
on creating plans for the plant’s conservation and sustainable management.
Table 1. List
of primers used for DNA amplification.
|
Primer name |
Sequence |
|
F |
CTTCCGATATGAGGAATTCTTCC |
|
R |
CTTCCGATATGAGGAATTCTTCC |
|
F |
ATGTCACCACAAACAGAGACTAAAGC |
|
R |
TCGCATGTACCTGCAGTAGC |
|
F |
CGCGCATGGTGGATTCACAATCC |
|
R |
GTTATGCATGAACGTAATGCTC |
Table 2.
Average AT and GC nucleotide composition of Cordia diffusa
species in three primers.
|
Primers |
Accession number |
Total base pairs
obtained after sequencing |
A (%) |
T (%) |
G (%) |
C (%) |
AT (%) |
GC (%) |
|
matK |
OQ852800.1 |
727 bp |
36 |
32 |
17 |
15 |
68 |
32 |
|
rbcL |
OR096230.1 |
548 bp |
28 |
30 |
21 |
21 |
58 |
42 |
|
trnH-psbA |
OR096231.1 |
315 bp |
28 |
45 |
17 |
10 |
73 |
27 |
For
figure & images - - click here for full PDF
References
Arumugam, S., K.S. Kumar, B. Karthik & V.
Ravichandran (2019). Cordia diffusa
K.C. Jacob, the Kovai Manjack
(Boraginaceae): a highly threatened steno-endemic
species from Coimbatore City, Tamil Nadu, India. Journal of Threatened Taxa
11(14): 14870–14875. https://doi.org/10.11609/jott.5292.11.14.14870-14875
Mehmood, F., I. Shahzadi, S.
Waseem, B. Mirza, I. Ahmed & M.T. Waheed (2020). Chloroplast genome
of Hibiscus rosa-sinensis (Malvaceae):
Comparative analyses and identification of mutational hotspots. Genomics
112(1): 581–591. https://doi.org/10.1016/j.ygeno.2019.04.010
Altschul, S., T.L. Madden,
A.A. Schäffer, J. Zhang, Z. Zhang, W. Miller &
D.J. Lipman (1997). Gapped BLAST and PSI-BLAST: A new generation of
protein database search programs. Nucleic Acids Research 25(17):
3389–3402. https://doi.org/10.1093/nar/25.17.3389
Amiryousefi, A., J. Hyvonen & P. Poczai (2018). The chloroplast
genome sequence of bittersweet (Solanum dulcamara):
Plastid genome structure evolution in Solanaceae. PLoS
One 13(4): e0196069. https://doi.org/10.1371/journal.pone.0196069
Aubriot, X., P.P. Lowry, C. Cruaud, A. Couloux & T. Haevermans (2013). DNA barcoding in a
biodiversity hot spot: potential value for the identification of Malagasy
Euphorbia L. listed in CITES Appendices I and II. Molecular Ecology
Resources 13(1): 57–65. https://doi.org/10.1111/1755-0998.12028
PMID: 23095939 17
Bentham, G. & J.D. Hooker (1876). Genera Plantarum, Vol. 1.
Reeve, London, 57–102.
CBOL Plant Working Group, P.M. Hollingsworth, L.L.
Forrest, J.L. Spouge, M. Hajibabaei, S. Ratnasingham, M. Van der Bank, M. Chase, W. Cowan, R.S.
Erickson, D.L. Fazekas, A.J. Graham, S.W. James, K.E. Kim, K.J. Kress, W.J.
Schneider, H. van AlphenStahl, J. Barrett, S.C.H. van
den Berg & C. Little (2009). A DNA barcode for land plants. Proceeding
of the National Academy of Science 106(31): 12794–12797. https://doi.org/10.1073/pnas.0905845106
Chandrabose, M. (1968). A New Species of
Polygala from South India. Bulletin of the Botanical Survey of India
9(1–4): 288–290.
Chandrabose., M. & N.C. Nair
(1988). Flora of Coimbatore. Bishen Singh Mahendra Pal Singh, Dehra Dun, 398 pp.
Chevenet, F., C. Brun, A.L. Bañuls, B. Jacq & R. Christen (2006). TreeDyn:
Towards dynamic graphics and annotations for analyses of trees. BMC
Bioinformatics 7: 439. https://doi.org/10.1186/1471-2105-7-439
China Plant BOL Group; D.Z. Li, L.M. Gao, H.T. Li, H.
Wang, X.J. Ge, J.Q. Liu, Z.D. Chen, S.L. Zhou, S.L. Chen, J.B. Yang, C.X. Fu,
C.X. Zeng, H.F. Yan, Y.J. Zhu, Y.S. Sun, S.Y. Chen, L. Zhao, K. Wang, T. Yang
& G.W. Duan (2011). Comparative
analysis of a large dataset indicates that internal transcribed spacer (ITS)
should be incorporated into the core barcode for seed plants. Proceeding of
the National Academy of Science 108(49): 19641–19646. https://doi.org/10.1073/pnas.1104551108
PMID: 2210073
Daniel, P. & P. Umamaheshwari
(2001). Flora of Gulf of Mannar Southern India.
Botanical Survey of India, Calcutta, 688 pp.
De Candolle, A.P. (1845). Prodromus
Systematis Naturalis Regni Vegetabilis, Vol. 9. Paris, 573 pp. https://doi.org/10.5962/bhl.title.286
Ebach, M.C. & C. Holrege (2005). DNA barcoding is no substitute
for taxonomy. Nature 434(7034): 697–697. https://doi.org/10.1038/434697b
Engler, A. (1898). Syllabus der Pflanzenfamilien, second edition. Berlin: Gebrüder Borntraeger. https://archive.org/details/syllabusderpfla00eng
Hebert, P.D., S. Ratnasingham
& J.R. De Waard (2003). Barcoding animal
life: cytochrome c oxidase subunit 1 divergences among closely related species.
Proceedings of the Royal Society of London. Series B: Biological Sciences
270: S96–S99. https://doi.org/10.1098/rsbl.2003.0025
Henry, A.N., K. Vivekananthan
& N.C. Nair (1978). Rare and threatened flowering plants of South
India. Journal of the Bombay Natural History Society 75(3): 684–697.
Hollingsworth, M.L., A. Clark, L.L. Forrest, J.
Richardson, R.T. Pennington, D.G. Long & P.M. Hollingsworth (2009). Selecting barcoding
loci for plants: evaluation of seven candidate loci with species-level sampling
in three divergent groups of land plants. Molecular Ecology Resources 9(2):
439–457. https://doi.org/10.1111/j.1755-0998.2008.02439.x
Hu, H.S., J.Y. Mao, X. Wang, Y. Liang, B. Jiang &
D.Q. Zhang (2023). Plastid phylogenomics
and species discrimination in the “Chinese” clade of Roscoea
(Zingiberaceae). Plant Diversity 45: 523–534. https://doi.org/10.1016/j.pld.2023.03.012
Kress, W.J. & D.L. Erickson (2007). A two-locus global
DNA barcode for land plants: the coding rbcL gene
complements the non-coding trnH-psbA spacer region. PLOS
one 2(6): e508. https://doi.org/10.1371/journal.pone.0000508
Kress, W.J., K.J. Wurdack,
E.A. Zimmer, L.A. Weigt & D.H. Janzen (2005). Use of DNA barcodes
to identify flowering plants. Proceedings of the National Academy of
Sciences 102(23): 8369–8374. https://doi.org/10.1073/pnas.0503123102
Li, H.Q., J.Y. Chen, S. Wang & S.Z. Xiong (2012). Evaluation of six candidate DNA barcoding loci
in Ficus (Moraceae) of
China. Molecular Ecology Resources 12(5): 783–790. https://doi.org/10.1111/j.1755-0998.2012.03147.x
Luebert, F., L. Cecchi, M.W.
Frohlich, M. Gottschling, C.M. Guilliams,
K.E. Hasenstab-Lehman, H.H. Hilger,
J.S. Miller, M. Mittelbach, M. Nazaire, M. Nepi, D. Nocentini, D. Ober, R.G.
Olmstead, F. Selvi, M.G. Simpson, K. Sutory, B. Valdes, G.K. Walden & M. Weigend
(2016). Familial classification of the Boraginales. Taxon
65(3): 502–522. https://doi.org/10.12705/653.5
Lv, S.Y., X.Y. Ye, Z.H.
Li, P.F. Ma & D.Z. Li (2022). Testing complete plastomes
and nuclear ribosomal DNA sequences for species identification in a
taxonomically difficult bamboo genus. Fargesia. Plant
Diversity 45: 147–155. https://doi.org/10.1016/j.pld.2022.04.002
Nayar, M.P. (1996). Hotspots of
Endemic Plants of India, Nepal, and Bhutan.
Tropical Botanic Garden and Research Institute, Thiruvananthapuram, 254
pp.
Pang, X., C. Liu, L. Shi, R. Liu, D. Liang, H. Li,
S.S. Cherny & S. Chen (2012). Utility of the trnH–psbA intergenic spacer
region and its combinations as plant DNA barcodes: a meta-analysis. PLOS One
7(11): e48833. https://doi.org/10.1371/journal.pone.0048833
Parveen, I., H.K. Singh, S. Raghuvanshi, U.C. Pradhan
& S.B. Babbar (2012). DNA barcoding of
endangered Indian Paphiopedilum species. Molecular Ecology Resources
12(1): 82–90. https://doi.org/10.1111/j.1755-0998
Pilger, R. & K. Krause (1915). Die Natürlichen Pflanzenfamilien, Ergänzungsheft 3. Leipzig: W. Engelmann.
Prabu, K., A. Rajasekaran,
D. Bharathi & S. Ramalakshmi (2019). Anti-oxidant
activity, phytochemical screening and HPLC profile of rare endemic Cordia diffusa. Journal of King Saud University - Science
31(4): 724–727. https://doi.org/10.1016/j.jksus.2018.04.025
Quattrocchi, U. (2012). CRC World
Dictionary of Medicinal and Poisonous Plants: Common Names, Scientific Names,
Eponyms, Synonyms, and Etymology. CRC Press. https://doi.org/10.1201/b16504
Ramamurthy, K. (1987). Boraginaceae. Flora
of Tamil Nadu, India, 97 pp.
Rao, C.K., B.L. Geetha & S. Geetha (2003). Red list of
threatened vascular plant species in India. Botanical Survey of India,
Kolkata.
Saarela, J.M., P.C.
Sokoloff, L.J. Gillespie, L.L. Consaul & R.D.
Bull (2013). DNA barcoding the Canadian Arctic flora: core plastid
barcodes (rbcL + matK) for
490 vascular plant species. PLOS One 8(10): e77982. https://doi.org/10.1371/journal.pone.0077982
Savolainen, V., R.S. Cowan,
A.P. Vogler, G.K. Roderick & R. Lane (2005). Towards writing the
encyclopedia of life: an introduction to DNA barcoding. Philosophical
Transactions of the Royal Society B 360: 1805–1811. https://doi.org/10.1098/rstb.2005.1730
Shaw, J., E.B. Lickey, E.E.
Schilling & R.L. Small (2007). Comparison of whole chloroplast genome
sequences to choose noncoding regions for phylogenetic studies in angiosperms:
the tortoise and the hare III. American Journal of Botany 94(3):
275–288. https://doi.org/10.3732/ajb.94.3.275
Singh, P., K. Karthikeyan, P. Lakshminarasimhan
& S.S. Dash (2015). Endemic Vascular Plants of India.
Botanical Survey of India, Kolkata, 339 pp.
Smith, M.A., J.J. Rodriguez, J.B. Whitfield, A.R.
Deans, D.H. Janzen, W. Hallwachs & P.D. Hebert
(2008). Extreme diversity of tropical parasitoid wasps exposed by iterative
integration of natural history, DNA barcoding, morphology, and collections. Proceedings
of the National Academy of Sciences 105(34): 12359–12364. https://doi.org/10.1073/pnas.0805319105
Su, T., Y.F. Geng, C.L.
Xiang, F. Zhao, M. Wang, L. Gu & G.X. Hu (2022). Chloroplast genome
of salvia sect. Drymosphace: comparative and
phylogenetic analysis. Diversity 14: 324. https://doi.org/10.3390/d14050324
Xu, Y.L., H.H. Shen, X.Y. Du & L. Lu (2022). Plastome
characteristics and species identification of Chinese medicinal wintergreens (Gaultheria,
Ericaceae). Plant Diversity 44: 519–529. https://doi.org/10.1016/j.pld.2022.06.002
Yan, H.F., Y.J. Liu, X.F. Xie,
C.Y. Zhang, C.M. Hu, G. Hao & X.J. Ge (2015). DNA barcoding
evaluation and its taxonomic implications in the species-rich genus Primula
L. in China. PLOS One 10(4): e0122903. https://doi.org/10.1371/journal.pone.0122903
Yu, W.B., P.H. Huang,
R.H. Ree, M.L. Liu, D.Z. Li & H. Wang (2011). DNA barcoding of Pedicularis L. (Orobanchaceae):
evaluating four universal barcode loci in a large and hemiparasitic
genus. Journal of Systematics and Evolution 49(5): 425–437. https://doi.org/10.1111/j.1759-6831.2011.00154.x
12