Identification of Indigenous Thai Phlegmariurus Genotypic Population by Integrating Morphological and Molecular Studies

<i>Phlegmariurus</i>, a diverse genus within the Lycopodiaceae family, has wide diversity in tropical regions, including Thailand. Accurate species delimitation in the tropical clubmoss genus <i>Phlegmariurus</i> is challenged by high morphological plasticity and genetic complexity. This study applied an integrative multilocus approach combining morphometric analysis of 27 complete specimens, 35 <i>Phlegmariurus</i> and <i>one Lycopodiella</i> accessions for AFLP genotyping (926 loci; PIC 0.32), SSR profiling (44 loci; PIC 0.57; expected heterozygosity 0.35), and chloroplast barcoding using <i>rbcL</i> (1308 bp; bootstrap 89-99%) and the <i>psbA</i>-<i>trnH</i> intergenic spacer (308 bp; bootstrap ≥ 94%). A total of 13 were identified as belonging to seven known species, including <i>P. nummulariifolius</i> (NST01, NST15, NST36), <i>P. goebelii</i> (JP04), <i>P. phlegmaria</i> (NST13), <i>P. verticillatus</i> (PHI16), <i>P. squarrosus</i> (NST21, NST22, MY31), <i>P. tetrastichus</i> (NST30), and <i>P. carinatus</i> (MY32, MY33, NST34). Morphological clustering and molecular markers consistently distinguished <i>Phlegmariurus</i> accessions from the <i>Lycopodiella</i> outgroup. Additionally, 19 previously unclassified <i>Phlegmariurus</i> accessions were successfully identified as belonging to the species <i>P. nummulariifolius</i> (NST23), <i>P. goebelii</i> (NST03, JP05, STN12, PNA14, SKA25, CPN26, KRB27, PNA28), <i>P. phlegmaria</i> (NWT07, STN08, NST09, NST10, PHI29), <i>P. squarrosus</i> (NST17), and <i>P. carinatus</i> (PNA06, STN18, CPN19, JP24). Moreover, this study identified three novel lineages (NST02, STN11, NST20) with strong support across datasets. The combination of broad genomic coverage (AFLP), fine-scale allelic resolution (SSR), deep-branch backbone (<i>rbcL</i>), and terminal-branch discrimination (<i>psb</i>A-<i>trn</i>H) yields a robust framework for species identification. These results define clear operational units for conservation prioritization and establish a foundation for marker-assisted development of ornamental <i>Phlegmariurus</i> cultivars.