Palynological diversity and plastid phylogenetic placement of Papilionaceae in the Kurram Valley, Pakistan, a Himalayan floristic crossroads

Abstract

The family Papilionaceae remains taxonomically challenging due to high phenotypic plasticity and convergent floral traits. Pollen morphology offers more stable diagnostic signatures. The Kurram Valley, a biogeographic crossroads of the Irano-Turanian, Sino-Japanese, and Saharo-Sindian regions along the Pak-Afghan border, has remained palynologically unexplored. We examined pollen traits of 15 Papilionaceous taxa using light and scanning electron microscopy, coupled with multivariate ordination (PCA, NMDS, PCoA), heatmap analysis, and a phylogenetic framework inferred from rbcL and matK markers. In total, seven quantitative and eleven qualitative pollen characters were recorded; the quantitative characters were used in multivariate analyses, whereas the qualitative characters were used for taxonomic comparison and identification. The ordination analyses consistently separated Alhagi maurorum Medik. from the remaining 14 species, with the former characterized by negative PC1 values, scabrate-perforate exine, and the largest mesocolpium (29.90 µm). Cluster 2 was further subdivided into three subgroups: Subgroup 2a (high PC2 values > 0.8, mainly associated with equatorial diameter and P/E ratio), Subgroup 2b1, and Subgroup 2b2. Subgroups 2b1 and 2b2 both showed low PC2 values associated with colpus width, colpus length, and exine thickness. Pollen fertility ranged from 69% (Medicago sativa L.) to 90% (Leobordea platycarpa (Viv.) B.-E.van Wyk & Boatwr.). The plastid phylogeny recovered eight tribes, with strong support for most nodes (BS ≥ 80%, PP ≥ 0.90), although discordant support values (Medicago pair 47/0.97, Astragaleae 61/0.82, Phaseolus lunatus L. 58/0.96) indicated limited phylogenetic resolution at some nodes, particularly for relationships among closely related taxa. Phylogeny-based tribal affiliations were partially consistent with pollen‑based groupings. A diagnostic key is provided for laboratory-based microscopic identification. The palynological dataset generated in this study provides valuable reference information for taxonomic identification and has broader relevance to melissopalynology and forensic palynology.