Spore morphometric analysis in Sino-Himalayan and W-Himalayan Athyriaceae taxa: taxonomic insights and molecular phylogenetic comparison

The application of advanced microscopic technique like scanning electron microscopy (SEM) has been pivotal in offering phylogenetically informative and taxonomically significant insights into the spore characteristics of various pteridophyte groups. The spore morphology of seven Pakistani Athyriaceae taxa, phytogeographically Sino-Himalayan and W-Himalayan elements; Athyrium attenuatum, Athyrium mackinnoniorum, Athyrium wallichianum, Deparia allantodioides, Deparia japonica, Deparia macdonellii, and Diplazium maximumwas investigated using light microscopy and scanning electron microscopy (SEM). The study aimed to explore detailed spore morphological characteristics for taxonomic differentiation. Notably, A. mackinnoniorum and D. macdonellii were identified as W-Himalayan endemics, and the spore morphology of D. maximum and D. macdonellii was documented for the first time. The spores exhibited a monolete structure, bilateral symmetry, medium size, and displayed diverse shapes in polar and equatorial views. Variations in spore size, shape, color, and perispore characteristics provided essential data for taxonomic differentiation within the studied Athyriaceae taxa. D. maximum had the smallest polar diameter (26.3 ± 1 µm), while D. japonica exhibited the largest polar spore diameter (40.4 ± 3 µm). Athyrium attenuatum displayed the smallest equatorial diameter (25.3 ± 1 µm), and A. wallichianum featured the largest equatorial spores (39.5 ± 10 µm). The perispore types varied among rugate, cristate-retate, baculate, and rugate-retate, with surface ornamentation, including granulate, folded, tuberculate/lamellate, and muriform obscure folds, contributing to distinct morphological characteristics. The study utilized classical clustering and principal component analysis to evaluate both qualitative and quantitative spore traits. These analyses identified distinct groups, underscoring taxonomic significance. The study-highlighted effectiveness of spore morphological characteristics for distinguishing closely related taxa, improving the taxonomic framework for Pakistan’s fern flora. Additionally, the study provides a spore morphological-based key for accurate species identification through perispore type and surface ornamentation.