Introduction
Bird’s-foot trefoil (Lotus corniculatus L.; Persian: Yonjeh pa’kalaghi) is a salt-tolerant, nitrogen-fixing forage legume from the Fabaceae family, valued for its adaptability to diverse environments and its role in sustainable agriculture. The species exhibits considerable polymorphism and morphological variation across its natural range, with the Mediterranean region recognized as a center of genetic diversity. Despite its ecological and agronomic significance, morphological diversity in natural habitats of Iran remains understudied, especially in the southwest, where no comprehensive evaluation has been conducted. This study addresses this gap by assessing morphological traits in accessions collected from multiple areas of the region to better understand variation under natural conditions.
 Material and Methods
This study investigated the morphological diversity of bird’s-foot trefoil by collecting 126 genotypes from nine distinct accessions across Khuzestan province. The accessions included Ahvaz, Sheyban, Mollasani, Hamidiyeh, Susangerd, Karun, Minoo Island, Hendijan, and Shavoor. Sample collection was conducted from March 2023 to January 2024 in natural habitats to capture the full range of phenotypic variation. A total of 18 morphological traits were evaluated-14 qualitative and 4 quantitative-selected based on the guidelines of the International Board for Plant Genetic Resources (IBPGR) and previous research findings. The qualitative traits included growth habit, vegetative form, relative size of the three leaflets, leaflet shape, leaflet indumentum, leaflets thickness, leaflet apex, stem firmness, stem cross-sectional shape, stem color, stem indumentum, corolla color, calyx teeth shape, and calyx indumentum. The quantitative traits measured were central leaflet length, central leaflet width, the ratio of calyx teeth to calyx tube length, and plant height. Data were analyzed using multivariate statistical methods. Cluster analysis was performed using Ward’s method and the Euclidean distance coefficient to group accessions. The accuracy of clustering was validated through discriminant function analysis using SPSS software. In addition, principal component analysis (PCA) was conducted in R software to identify the key traits contributing to morphological variability and to visualize the relationships among genotypes.
Results and Discussion
Analysis of vegetative form among the studied genotypes revealed that 88.10% of the genotypes were herbaceous, while 11.90% exhibited woody structure. Regarding growth habit, 65.87% of genotypes were decumbent, 22.22% were erect or ascending, and 11.90% were like procumbent or prostrate. The Minoo Island accession was distinct in both vegetative form (woody) and growth habit (similar to procumbent or prostrate plants), setting it apart from the other accessions. Considerable variation was observed in several qualitative traits, including leaflet size (three leaflets per leaf, same or different sizes), leaflet apex, leaflet shape, and corolla color within the accessions, demonstrating high variation even on a single plant. Conversely, certain traits, such as stem color and cross-sectional shape, exhibited no variation. Analysis of variance (ANOVA) indicated that the three of the four quantitative traits were statistically significant at the 5% level. Duncan's multiple range test effectively distinguished homogeneous groups and identified similarities and differences among accessions. Cluster analysis grouped the accessions based on morphological traits, with Minoo Island forming a distinct cluster due to its unique vegetative form and growth habit. These results were further supported by principal component analysis (PCA), which helped visualize trait contributions and genotype relationships. Discriminant function analysis confirmed the accuracy of the cluster groupings. Interestingly, some accessions from geographically distant locations clustered together, suggesting that similar ecological conditions may influence morphological traits. To isolate genetic variation from environmental effects, future evaluations should be conducted under uniform cultivation conditions.
 Conclusion
The observed morphological diversity among bird’s-foot trefoil accessions highlights the species’ adaptability to varying environmental conditions. This variability is particularly valuable for conservation and breeding programs. The unique characteristics of the Minoo Island accession, especially its vegetative form and growth habit, make it a promising candidate for targeted germplasm conservation and further agronomic evaluation.