High mountains of Central Europe as a refuge of surprising cytotype diversity of Huperzia selago (Lycopodiaceae)

Abstract Polyploidization is pivotal in plant speciation, enhancing adaptability, ecological tolerance and specific geographical distribution pattern. While cytotype diversity is extensively studied in angiosperms and ferns, knowledge in homosporous lycophytes remains very limited. This study addresses this gap, focusing on the homosporous lycophyte Huperzia selago in Central Europe. Employing flow cytometry, we assessed genome size in 1330 H. selago individuals from 208 populations identifying five distinctive cytotypes (2x, 3x, 4x, 5x, 6x). Challenging chromosome counting on somatic gemmae roots was applied for the three lowest cytotypes yielded average counts of 140, 204, and 262 chromosomes, respectively. Geographical separation of cytotypes was not observed. Tetraploids were most widespread (72.7%), while triploids (21.3%) were rare, and extremely rare are cytotypes 2x, 5x, 6x constituted less than 5% of the dataset. Uncommon cytotypes were associated with the Alps and triploids occurs also in the highest parts of Western Carpathians. Hexaploid genome size (29 pg) approached upper limits reported in lycophytes. Around 27.3% of populations exhibited mixed cytotypes. Except for atypical diploids, spore abortion differed between even and odd ploidies, approximately 21.8% and 65.2%, respectively. Microcharacter sizes (stoma, spore) did not linearly correspond with increasing ploidy levels. The high ploidy-level diversity and cytotype coexistence in Central European Huperzia selago match the well documented patterns in ferns and angiosperms. These findings provide valuable insights into lycophyte polyploidy, underscoring the necessity for broader geographical sampling and applicance of molecular studies to elucidate phylogenetic relationships and taxonomic classifications within the genus Huperzia.