Taxonomic and molecular characterization of 15 wild-growing tulip species of Greece using the internal transcribed spacer (ITS) nuclear marker in combination with the <i>psb</i>A-<i>trn</i>H and <i>trn</i>L/<i>trn</i>F plastid markers

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 <i>psb</i>A<i>/trn</i>H and <i>trn</i>L/<i>trn</i>F 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 <i>Tulipa</i> 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 <i>Tulipa</i> 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 <i>Tulipa</i>, that is, <i>T. scardica</i>, <i>T. undulatifolia</i>, <i>T. rhodopea</i>, <i>T. agenensis</i> and <i>T. raddii</i>; Group 2 included only <i>T. clusiana</i> (subgenus <i>Clusianae</i>) and Group 3 included Greek members of subgenus <i>Eriostemones</i>, that is, *<i>T. bakeri</i>, <i>T. saxatilis</i>, <i>T. australis</i>, *<i>T. cretica</i>, *<i>T. goulimyi</i>, *<i>T. orphanidea T. bithynica</i>, *<i>T. hageri</i> and *<i>T. doerfleri</i> (asterisks indicate single-country endemics).