Taxonomic and molecular characterization of 15 wild-growing tulip species of Greece using the internal transcribed spacer (ITS) nuclear marker in combination with the psbA-trnH and trnL/trnF plastid markers

Abstract

Due to the absence or scarcity of DNA sequence data in GenBank regarding Mediterranean, Balkan or Greek native tulip species, we performed molecular characterization of the 15 wild-growing Greek tulip species through analysis of the internal transcribed spacer nrDNA and the psbA/trnH and trnL/trnF cpDNA regions. To facilitate conservation and sustainable utilization efforts, the aim was to identify and complement the genetic distinctiveness of six Greek endemic and five Balkan or Aegean endemic and/or subendemic wild-growing Greek native tulips and two naturalized ones of Asiatic origin, among which seven are threatened with extinction. Molecular phylogenetic analysis of 148 DNA sequences was conducted, and 85 Tulipa taxa (species and subspecies) were aligned and compared to 40 new DNA sequences generated herein for the 15 wild-growing Greek tulips. These three molecular markers resulted in 354 single-nucleotide polymorphisms (SNPs) and 20 distinct indels for all Tulipa taxa, of which 100 SNPs and 12 indels were related to the Greek tulips. Phylogenetic dendrograms were constructed for all the above-mentioned molecular markers, and the DNA sequences were analyzed separately and jointly, leading to three distinct clusters for the 15 wild-growing Greek tulips. Group 1 included Greek members of subgenus Tulipa, that is, T. scardica, T. undulatifolia, T. rhodopea, T. agenensis and T. raddii; Group 2 included only T. clusiana (subgenus Clusianae) and Group 3 included Greek members of subgenus Eriostemones, that is, *T. bakeri, T. saxatilis, T. australis, *T. cretica, *T. goulimyi, *T. orphanidea T. bithynica, *T. hageri and *T. doerfleri (asterisks indicate single-country endemics).

Keywords:

• Barcoding
• PCR
• botanical collections
• tulip bulbs
• SNPs
• indel
• identification
• phylogenetic analysis

Acknowledgements

The authors are thankful to V. Papiomytoglou and M. Avramakis (Natural History Museum of Crete) for assistance in field work and the permission to reproduce photographs of Tulipa cretica and T. saxatilis, respectively. We are also thankful to the staff of the Balkan Botanic Garden of Kroussia and the Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization Demeter for hosting the ex-situ conservation of the tulip accessions of TULIPS.GR. We would also like to thank Stina Weststrand and Mats Havström of Gothenburg Botanical Garden (Sweden) for the Tulipa scardica samples analyzed in this investigation.

Authors’ contribution

Conceptualization: G.T., N.K.; methodology: G.T., N.K., M.S., I.S.; validation: G.T., N.K., I.S., A.T., M.S., I.T., P.M.; formal analysis: G.T., N.K., I.S., A.T., M.S.; investigation: G.T., N.K., I.S., A.T., P.M.; resources: G.T., N.K.; data curation: G.T., N.K., A.T.; writing – original draft preparation: G.T., N.K., I.S., A.T.; writing – review and editing: G.T., N.K., I.S., A.T.; visualization: G.T., N.K., I.S.; supervision: G.T., N.K., I.T.; project administration: G.T., N.K.; funding acquisition: G.T., N.K.; All authors read and approved the final submitted manuscript.

Disclosure statement

The authors declare that they have no conflict interests.

Ethical standards

The authors declare that this study complies with the current laws of E.U.

Data availability statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Funding

This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH-CREATE-INNOVATE (project code: T2EDK-05115; acronym: TULIPS.GR), entitled ‘Value chain for Greek native tulips: development of documented propagation material and integrated conservation for sustainable exploitation’.