Comparative chloroplast genomics and phylogenomic insights into the genus Zehneria (Cucurbitaceae).

The genus Zehneria (Cucurbitaceae) presents significant taxonomic challenges due to morphological similarities among species and a lack of resolution in molecular studies. In this study, we sequenced and assembled the chloroplast genomes of seven African Zehneria species collected from Kenya, complementing a previously published accession (Zehneria sp. MZ427944.1), to establish a comprehensive plastome-based framework for the genus. The plastomes ranged from 157,150 to 157,365 bp and exhibited a conserved quadripartite structure, with 130-132 annotated genes, including 86-87 protein-coding genes, 36-37 tRNAs, and 8 rRNAs. Structural variation, particularly in non-coding regions and inverted repeat boundaries, was observed, with eight hypervariable loci providing potential targets for molecular marker development. Simple sequence repeat (SSR) analysis revealed a predominance of A/T mononucleotides across species. Codon usage analysis revealed a strong AT-bias across all species, with codons ending in A or U accounting for 72.2% of synonymous codon preferences. Selective pressure analysis identified eight protein-coding genes with Ka/Ks ratios exceeding 1.0, including psaJ and rps8, proposed as candidate molecular barcodes pending further validation. Phylogenomic analysis of 79 protein-coding genes strongly supported the monophyly of Zehneria (ML bootstrap = 96-100%; PP = 1.0), with one node showing slightly reduced support (BS = 96%), and enhanced resolution of interspecific relationships compared to prior studies using fewer loci. This work offers novel insights into genomic diversity and evolutionary history of sampled Zehneria species, providing a critical molecular resource for future taxonomic and phylogenetic studies within Cucurbitaceae. Broader taxon sampling, particularly from Asian lineages, will be necessary to fully resolve interspecific relationships across the genus.