Phylogenetic exploration, codon usage bias, and genomic divergence in Hydrocotyle: a comparative plastome study across different geographical locations.

Hydrocotyle himalaica from Bhutan, a perennial herb that thrives from 1500 to 2600 m, possesses both ecological importance and medicinal properties. The plastome analysis revealed a length of 153,383 bp, showing variation from conspecific taxa in China. Its standard structure comprises two IR regions (18,336 bp IRa and 18,336 bp IRb), an LSC region of 97,944 bp, and an SSC region of 18,767 bp, with a GC content of 37.63%. Non-coding regions showed higher mutation susceptibility, with Pi values from 0.006 to 0.107. An AT-rich codon bias was consistent across all 18 Hydrocotyle species. Nucleotide composition and GC% in coding sequences differed among the species. The codon preference in Hydrocotyle is shaped by multiple factors, with natural selection being the primary influence, as indicated by the ENC-plot, PR2-plot, and Neutrality-plot. Codon usage patterns varied, with RSCU values from 0 to 2.23. Codons ending in A or U had RSCU > 1, while those ending in C or G had RSCU < 1. GC2 content surpassed GC3 and GC1 in most genes. The phylogenetic analysis placed H. himalaica, sourced from Kanglung, Bhutan, within the monophyly of the Hydrocotyloideae subfamily. However, the species showed weaker bootstrap support (BS < 50) with H. javanica and H. hookeri subsp., a deviation from a prior report on the same species from Jiangkou, Guizhou, China. This analysis highlighted the genomic characteristics and evolutionary relationships of H. himalaica from Bhutan, underscoring the need for a comprehensive phylogenetic, ecological, and botanical characterization to confirm intra-specific variation within Hydrocotyle species.