Insights into chloroplast genome evolution in Rutaceae through population genomics

Chloroplast genomes, pivotal for understanding plant evolution, remain unexplored in Rutaceae, a family with key perennial crops like citrus. Leveraging next-generation sequencing data from 509 Rutaceae accessions across 15 species, we conducted a de novo assembly of 343 chloroplast genomes, unveiling a chloroplast variation map highlighting the heterogeneous evolution rates across genome regions. Notably, differences in chloroplast genome size primarily originate from large single-copy and small single-copy regions. Structural variants predominantly occurred in the single-copy region, with two insertions located at the single-copy and inverted repeat region boundary. Phylogenetic analysis, principal component analysis, and population genetic statistics confirmed the cohesive clustering of different Citrus species, reflecting evolutionary dynamics in Citrus diversification. Furthermore, a close chloroplast genetic affinity was revealed among Atalantia (previously regarded as primitive citrus), Clausena, and Murraya. Zanthoxylum formed a distinct group with heightened genetic diversity. Through expanding our analysis to include 34 published chloroplast genomes, we explored chloroplast gene selection, revealing divergent evolutionary trends in photosynthetic pathways. While Photosystem I and Photosystem II exhibited robust negative selection, indicating stability, the Nicotinamide adenine dinucleotide (NADH) dehydrogenase pathway demonstrated rapid evolution, which was indicative of environmental adaptation. Finally, we discussed the effects of gene length and GC content on chloroplast gene evolution. In conclusion, our study reveals the genetic characterization of chloroplast genomes during Rutaceae diversification, providing insights into the evolutionary history of this family.