Comparative genomics of chloroplast structural evolution in cactus

Most angiosperm chloroplast genomes (cpDNAs) exhibit a conserved quadripartite architecture consisting of two single-copy (SC) regions separated by two inverted repeats (IRs). Most of these cpDNAs conserve gene composition and arrangement. In this study, we compared 102 taxa and their cpDNAs, of which 95 were de novo assembled and annotated, to identify structural patterns associated with cpDNAs structure. A phylogeny was obtained using 64 coding sequences and phylogenetic signal was estimated for 20 traits, including length, GC and gene content of four cpDNA regions, and correlations between variables were assessed using PGLS. Additionally, clustering and stochastic mapping analyses were performed for IR structures. In total, 40 distinct IR structures were identified, and structure-14 grouped 15 taxa from three genera. Strong phylogenetic signal was detected for cpDNA-GC content (λ = 0.95) and SC-length (λ = 1), whereas PGLS indicated a significant negative correlation between SC-GC and IR-GC content (R 2 = 0.49). Clustering of IR structures showed two groups mainly differing in the presence of rRNAs, whereas stochastic mapping indicated that the absence of such genes represents the ancestral state. Overall, these results reveal the evolutionary structural diversity of cpDNA across the Mammilloid clade, highlighting the compensatory role of IR, as well as evolutionary tendencies through the gain and loss of rRNA genes.