Comparative complete chloroplast genome analysis of Cucurbita (Cucurbitaceae) species revealed insights into phylogenetic evolution, adaptive pressure, and lineage diversification

Cucurbita is an economically and nutritionally important genus within the Cucurbitaceae. However, its taxonomy and evolutionary relationships remain difficult to resolve due to extensive phenotypic diversity, domestication history, and frequent hybridization. In this study, we generated ten complete chloroplast genomes (CPGs), including newly sequenced genomes of Cucurbita moschata and C. maxima and de novo assemblies of eight additional species. We conducted a comprehensive comparative phylogenomic analysis across twelve Cucurbita species by integrating the two previously published genomes. The obtained results showed that the CPGs of these Cucurbita species were 157,309 bp ± 568 bp in size and encoded 130~131 annotated genes, including 86 protein-coding genes, 36~37 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. All these CPGs presented a conserved quadripartite circular structure, containing an LSC, an SSC and two IR regions. Analysis of codon usage bias indicated leucine (Leu) was the most abundant amino acid, and the relative synonymous codon usage (RSCU) values of thirty codons were greater than 1.00. A total of nine simple sequence repeat (SSR) primers were validated as highly polymorphic among Cucurbita species, and the trnL - trnF region was identified as the most suitable DNA barcode in Cucurbita . The chloroplast phylogenomic analysis of 61 representative Cucurbitaceae species confirmed Cucurbita as a strongly supported monophyletic core group, with C. cordata and C. digitata in a basal position. Furthermore, Cucurbita was found to be sister to a group of Benincaseae containing Citrullus , Zehneria , and Cucumis . A total of seven genes ( atpE , atpF , matK , ndh A, rpoC2 , ycf1 , and ycf2 ) harbored significant selective sites under positive selection and promoted the plant development and environmental adaptation of Cucurbita . This study substantially enriches chloroplast genomic resources for Cucurbita and provides an integrated framework for resolving its phylogenetic relationships, evolutionary history, taxonomic classification, and adaptive diversification, with important implications for taxonomy, breeding, and conservation of cucurbit germplasm.