Morphological and Morphometric Characterization of Lycopodiaceae Spores from the Białowieża Primeval Forest Ecosystem (NE Poland)

Spores offer the most accessible diagnostic characters for the early-divergent Lycopodiaceae. We quantified eight morphometric traits—equivalent diameter, polar length, equatorial width, projected area, perimeter, and aspect ratio—in a balanced sample of 50 spores from each of six Central European taxa (Diphasiastrum alpinum, D. tristachyum, D. complanatum, Lycopodium annotinum, L. clavatum, and Huperzia selago) collected in the Białowieża Primeval Forest. Integrated light-microscope and scanning-electron-microscope imaging revealed three discrete wall-ornamentation syndromes (reticulate, verrucate, and granulose) that parallel the quantitative gradients. Principal component analysis showed that a single, collinear size axis accounts for 79% of variance, situating H. selago at the large-diameter extreme (mean: 37μm) and the three Diphasiastrum species at the small-diameter pole (mean: 32–33μm). One-way ANOVA (p<10−31) and PERMANOVA (R2=0.52) confirmed decisive interspecific separation that mirrors published molecular phylogenies, underscoring a strong phylogenetic signal in spore form. While trait baselines are taxonomically stable, moderate microhabitat-driven shifts indicate limited ecophenotypic plasticity. The resulting high-resolution benchmark refines palynological identification, enables rapid spore-based bioindication of demographic stress, and strengthens conservation monitoring in relic temperate forest ecosystems.