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Planta Med 2021; 87(07): 528-537
DOI: 10.1055/a-1379-3249
Natural Product Chemistry and Analytical Studies
Original Papers

DNA Barcoding of St. Johnʼs wort (Hypericum spp.) Growing Wild in North-Eastern Greece

Ioanna Pyrka
1   Postgraduate Studies Program, Conservation of Biodiversity and Sustainable Exploitation of Native Plants (BNP), School of Biology, Faculty of Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
,
Anastasia Stefanaki
2   Biosystematics Group, Wageningen University, Wageningen, Netherlands
,
Konstantinos E. Vlachonasios 
1   Postgraduate Studies Program, Conservation of Biodiversity and Sustainable Exploitation of Native Plants (BNP), School of Biology, Faculty of Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
3   Department of Botany, School of Biology, Faculty of Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
4   Natural Products Research Centre of Excellence (NatPro-AUTh), Center of Interdisciplinary Research and Innovation of Aristotle University of Thessaloniki (CIRI-AUTh), Thessaloniki, Greece
› Author AffiliationsSupported by: European Regional Development Fund MIS 5002803
Supported by: National Strategic Reference Framework (NSRF), Research Funding Programme of the Action RESEARCH – CREATE – INNOVATE AROMADISTIL – 95783
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Abstract

Plants of the genus Hypericum, commonly known as “St. Johnʼs wort” (“spathohorto” or “valsamo” in Greek), have been used since antiquity for their therapeutic properties. Wild-harvested Hypericum plants are still popular today in herbal medicines, commercially exploited due to their bioactive compounds, hypericin and hyperforin, which have antidepressant, antimicrobial and antiviral activity. Species identification of commercial products is therefore important and DNA barcoding, a molecular method that uses small sequences of organismsʼ genome as barcodes, can be useful in this direction. In this study, we collected plants of the genus Hypericum that grow wild in North-Eastern Greece and explored the efficiency of matK, and trnH-psbA regions as DNA barcodes for their identification. We focused on 5 taxa, namely H. aucheri, H. montbretii, H. olympicum, H. perforatum subsp. perforatum, and H. thasium, the latter a rare Balkan endemic species collected for the first time from mainland Greece. matK (using the genus-specific primers designed herein), trnH-psbA, and their combination were effectively used for the identification of the 5 Hypericum taxa and the discrimination of different H. perforatum subsp. perforatum populations. These barcodes were also able to discriminate Greek populations of H. perforatum, H. aucheri, H. montbretii, and H. olympicum from populations of the same species growing in other countries.

Key words

Hypericaceae - Hypericum perforatum - Hypericum thasium - matK - Greek flora - trnH-psbA

Supporting Information

    NJ tree for (a) matK, (b) rbcL, and (c) trnH-psbA sequences retrieved from GenBank based on K2P model of nucleotide substitution (Fig. 1S), matK and trnH-psbA sequence length (Fig. 2S), PCR products of (a) matK and (b) trnH-psbA as shown in agarose gel 1% (Fig. 3S), nucleotide sequence alignment of (a) matK, (b) trnH-psbA, and (c) matK + trnH-psbA of Hypericum taxa collected from NE Greece (Fig. 4S). Percentage of variable sites in matK, rbcL and trnH-psbA sequences retrieved from GenBank (Table 1S), percentage of PCR and sequence success (Table 2S), percentage of variable characters in matK and trnH-psbA sequences (Table 3S), K2P genetic distances in matK (Table 4S), trnH-psbA (Table 5S) and matK + trnH-psbA sequences (Table 6S), nucleotide primers used in this study (Table 7S).

  • Supporting Information


Publication History

Received: 10 October 2020

Accepted after revision: 28 January 2021

Article published online:
22 February 2021

© 2021. Thieme. All rights reserved.

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  • References

  • 1 Robson NKB. Studies in the genus Hypericum L. (Guttiferae). 1. Infrageneric classification. Bull Brit Mus Nat Hist (Botany) 1977; 5: 293-355
    PubMedGoogle Scholar
  • 2 Beck L. Pedanius Dioscorides of Anazarbus; De materia medica. 4th enlarged edition. Altertumwissenschaftliche Texte und Studien. Hildesheim: Olms; 2020
    Google Scholar
  • 3 Barnes J, Anderson LA, Phillipson JD. St Johnʼs wort (Hypericum perforatum L.): a review of its chemistry, pharmacology and clinical properties. J Pharm Pharmacol 2001; 53: 583-600
    CrossrefPubMedGoogle Scholar
  • 4 Kladar N, Anačkov G, Srđenović B, Gavarić N, Hitl M, Salaj N, Jeremić K, Babović S, Božin B. St. Johnʼs wort herbal teas–biological potential and chemometric approach to quality control. Plant Foods Hum Nutr 2020; 75: 390-395
    CrossrefPubMedGoogle Scholar
  • 5 Sarris J. Herbal medicines in the treatment of psychiatric disorders: 10-year updated review. Phytother Res 2018; 32: 1147-1162
    CrossrefPubMedGoogle Scholar
  • 6 Robson NKB. And then came molecular phylogenetics – reactions to a monographic study of Hypericum (Hypericaceae). Phytotaxa 2016; 255: 181-198
    CrossrefPubMedGoogle Scholar
  • 7 Dimopoulos P, Raus T, Bergmeier E, Constantinidis T, Iatrou G, Kokkini S, Strid A, Tzanoudakis D. Vascular Plants of Greece: an annotated Checklist. Berlin: Botanic Garden and Botanical Museum Berlin-Dahlem; Athens: Hellenic Botanical Society [Englera 31]; 2013
    Google Scholar
  • 8 Dimopoulos P, Raus T, Bergmeier E, Constantinidis T, Iatrou G, Kokkini S, Strid A, Tzanoudakis D. Vascular plants of Greece: an annotated checklist. Supplement. Willdenowia 2016; 46: 301-347
    CrossrefPubMedGoogle Scholar
  • 9 Greuter W, Karl R. Ein neues Johanniskraut von den Kykladen (Ägäis, Griechenland): Hypericum (sect. Drosocarpium) perfoliatum subsp. phitosianum (Hypericaceae). Bot Chron 2019; 22: 39-47
    PubMedGoogle Scholar
  • 10 Robson NKB. Studies in the genus Hypericum L. (Hypericaceae). 9. addenda, corrigenda, keys, lists and general discussion. Phytotaxa 2012; 72: 1-111
    CrossrefPubMedGoogle Scholar
  • 11 Trigas P. A new Hypericum (sect. Drosocarpium, Hypericaceae) from the Cyclades Islands (Greece). Nord J Bot 2018; 36: e02205
    CrossrefPubMedGoogle Scholar
  • 12 Vladimirov V, Mehmet A, Tan K. New floristic records in the Balkans: 35. Phytol Balcan 2018; 24: 155-174
    PubMedGoogle Scholar
  • 13 Hanlidou E, Karousou R, Kleftoyanni V, Kokkini S. The herbal market of Thessaloniki (N Greece) and its relation to the ethnobotanical tradition. J Ethnopharmacol 2004; 91: 281-299
    CrossrefPubMedGoogle Scholar
  • 14 Hebert PDN, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Royal Soc B 2003; 270: 313-321
    CrossrefPubMedGoogle Scholar
  • 15 Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH. Use of DNA barcodes to identify flowering plants. Proc Natl Acad Sci U S A 2005; 102: 8369-8374
    CrossrefPubMedGoogle Scholar
  • 16 CBOL Plant Working Group. A DNA barcode for land plants. Proc Natl Acad Sci U S A 2009; 106: 12794-12797
    CrossrefPubMedGoogle Scholar
  • 17 China Plant BOL Group. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proc Natl Acad Sci U S A 2011; 108: 19641-19646
    CrossrefPubMedGoogle Scholar
  • 18 Li X, Yang Y, Henry RJ, Rossetto M, Wang Y, Chen S. Plant DNA barcoding: from gene to genome. Biol Rev 2015; 90: 157-166
    CrossrefPubMedGoogle Scholar
  • 19 Kress WJ. Plant DNA barcodes: applications today and in the future. J Syst Evol 2017; 55: 291-307
    CrossrefPubMedGoogle Scholar
  • 20 Theodoridis S, Stefanaki A, Tezcan M, Aki C, Kokkini S, Vlachonasios KE. DNA barcoding in native plants of the Labiatae (Lamiaceae) family from Chios Island (Greece) and the adjacent Çeşme-Karaburun Peninsula (Turkey). Mol Ecol Resour 2012; 12: 620-633
    CrossrefPubMedGoogle Scholar
  • 21 Tezcan M, Vlachonasios K, Aki C. DNA barcoding study on Sideritis trojana Bornm. An endemic medicinal plant of Ida mountain Turkiye. Fresenius Environ Bull 2010; 19: 1352-1355
    PubMedGoogle Scholar
  • 22 Crockett SL, Douglas AW, Scheffler BE, Khan IA. Genetic profiling of Hypericum (St. Johnʼs Wort) species by nuclear ribosomal ITS sequence analysis. Planta Med 2004; 70: 929-935
    Article in Thieme ConnectPubMedGoogle Scholar
  • 23 Meseguer AS, Aldasoro JJ, Sanmartín I. Bayesian inference of phylogeny, morphology and range evolution reveals a complex evolutionary history in St. Johnʼs wort (Hypericum). Mol Phylogenet Evol 2013; 67: 379-403
    CrossrefPubMedGoogle Scholar
  • 24 Nürk NM, Madriñán S, Carine MA, Chase MW, Blattner FR. Molecular phylogenetics and morphological evolution of St. Johnʼs wort (Hypericum; Hypericaceae). Mol Phylogenet Evol 2013; 66: 1-16
    CrossrefPubMedGoogle Scholar
  • 25 Nürk NM, Scheriau C, Madriñán S. Explosive radiation in high Andean Hypericum – rates of diversification among New World lineages. Front Genet 2013; 4: 175
    PubMedGoogle Scholar
  • 26 Park SJ, Kim KJ. Molecular phylogeny of the genus Hypericum (Hypericaceae) from Korea and Japan: evidence from nuclear rDNA ITS sequence data. J Plant Biol 2004; 47: 366-374
    CrossrefPubMedGoogle Scholar
  • 27 Costa J, Campos B, Amaral JS, Nunes ME, Oliveira MBPP, Mafra I. HRM analysis targeting ITS1 and matK loci as potential DNA mini-barcodes for the authentication of Hypericum perforatum and Hypericum androsaemum in herbal infusions. Food Control 2016; 61: 105-114
    CrossrefPubMedGoogle Scholar
  • 28 Howard C, Hill E, Kreuzer M, Mali P, Masiero E, Slater A, Sgamma T. DNA authentication of St Johnʼs Wort (Hypericum perforatum L.) commercial products targeting the ITS region. Genes (Basel) 2019; 10: 286
    CrossrefPubMedGoogle Scholar
  • 29 Newmaster SG, Grguric M, Shanmughanandhan D, Ramalingam S, Ragupathy S. DNA barcoding detects contamination and substitution in North American herbal products. BMC Med 2013; 11: 122
    CrossrefPubMedGoogle Scholar
  • 30 Pawar RS, Handy SM, Cheng R, Shyong N, Grundel E. Assessment of the authenticity of herbal dietary supplements: comparison of chemical and DNA barcoding methods. Planta Med 2017; 83: 921-936
    Article in Thieme ConnectPubMedGoogle Scholar
  • 31 Raclariu AC, Paltinean R, Vlase L, Labarre A, Manzanilla V, Ichim MC, Crisan G, Brysting AK, de Boer H. Comparative authentication of Hypericum perforatum herbal products using DNA metabarcoding, TLC and HPLC-MS. Sci Rep 2017; 7: 1-12
    Google Scholar
  • 32 Howard C, Bremner PD, Fowler MR, Isodo B, Scott NW, Slater A. Molecular identification of Hypericum perforatum by PCR amplification of the ITS and 5.8S rDNA region. Planta Med 2009; 75: 864-869
    Article in Thieme ConnectPubMedGoogle Scholar
  • 33 Howard C, Lockie-Williams C, Slater A. Applied Barcoding: The practicalities of DNA testing for herbals. Plants 2020; 9: 1150
    CrossrefPubMedGoogle Scholar
  • 34 Zeliou K, Koui EM, Papaioannou C, Koulakiotis NS, Iatrou G, Tsarbopoulos A, Papasotiropoulos V, Lamari FN. Metabolomic fingerprinting and genetic discrimination of four Hypericum taxa from Greece. Phytochemistry 2020; 174: 112290
    CrossrefPubMedGoogle Scholar
  • 35 Robson NKB. Studies in the genus Hypericum L. (Hypericaceae) 5 (1). Sections 10. Olympia to 15/16. Crossophyllum . Phytotaxa 2010; 4: 5-126
    CrossrefPubMedGoogle Scholar
  • 36 Hilu KW, Liang H. The matK gene: sequence variation and application in plant systematics. Am J Bot 1997; 84: 830-839
    CrossrefPubMedGoogle Scholar
  • 37 DeVere N, Rich TCG, Ford CR, Trinder SA, Long C, Moore CW, Satterthwaite D, Davies H, Allainguillaume J, Ronca S, Tatarinova T, Garbett H, Walker K, Wilkinson MJ. DNA barcoding the native flowering plants and conifers of Wales. PLoS One 2012; 7: e37945
    CrossrefPubMedGoogle Scholar
  • 38 Storchova H, Olson MS. The architecture of the chloroplast psbA-trnH noncoding region in angiosperms. Plant Syst Evol 2007; 268: 235-256
    CrossrefPubMedGoogle Scholar
  • 39 Graham SW, Reeves PA, Burns ACE, Olmstead RG. Microstructural changes in noncoding chloroplast DNA: interpretation, evolution, and utility of indels and inversions in basal angiosperm phylogenetic inference. Int J of Plant Sci 2000; 161: 83-96
    CrossrefPubMedGoogle Scholar
  • 40 Blair C, Murphy RW. Recent trends in molecular phylogenetic analysis: where to next?. J Hered 2011; 102: 130-138
    CrossrefPubMedGoogle Scholar
  • 41 Boissier E. Flora Orientalis, Vol. 1. Basel: H. Georg; 1867
    Google Scholar
  • 42 Edwards K, Johnstone C, Thompson C. A simple method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 1991; 19: 1349
    CrossrefPubMedGoogle Scholar
  • 43 Hall TA. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999; 41: 95-98
    PubMedGoogle Scholar
  • 44 Larsson A. AliView: a fast and lightweight alignment viewer and editor for large data sets. Bioinformatics 2014; 30: 3276-3278
    CrossrefPubMedGoogle Scholar
  • 45 R Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2018. Accessed January 10, 2020 at: https://www.R-project.org/
    Google Scholar