Deciphering <i>Plantago</i>'s Evolutionary Tree: The Role of Nuclear ITS1 Data to Resolve Phylogenetic Links and Cross-Species Comparisons

Abstract

Background: Plantago is an important genus in the Plantaginaceae that has been studied for its genetic diversity, medicinal value, and evolutionary significance. Objectives: This study aimed to assess the genetic diversity and phylogenetic relationships of Plantago using the ITS1 region. Methods: ITS1 sequences were retrieved from the NCBI database and aligned using ClustalW in MEGA. Genetic diversity parameters, including GC content, haplotype number, nucleotide diversity, and mutation count, were calculated. Neutrality tests, including Tajima's D and Fu's Fs, were performed to assess neutrality. A phylogenetic tree was constructed using the Neighbor-Joining method to analyze evolutionary relationships and divergence patterns among ITS1 sequences from different Plantago species. Results: The ITS1 region exhibited a GC content of 51.7% and a balanced nucleotide composition, supporting its suitability as a molecular marker. A total of 123 polymorphic sites and 100 distinct haplotypes were identified, indicating high genetic variation among the studied populations. The mean nucleotide diversity was 0.02929, with 138 total mutations detected, confirming substantial genetic polymorphism. Neutrality tests yielded negative but non-significant values, suggesting possible effects of natural selection or recent population expansion. Phylogenetic analysis using the Neighbor-Joining method revealed clear clustering among sequences, reflecting consistent evolutionary relationships. Short branch lengths at the basal nodes of the phylogenetic tree indicated relatively recent divergence events within the genus. Overall, ITS1 demonstrated strong resolving power for distinguishing genetic variation and evolutionary relationships among Plantago taxa. Conclusions: These findings demonstrate that ITS1 is a reliable molecular marker for studying genetic diversity and evolution in the genus Plantago.