Abstract
Chrysosplenium, a perennial herb in the family Saxifragaceae, prefers to grow in low light and moist environments and is divided into two sections of Alternifolia and Oppositifolia based on phyllotaxy. Although there has been some progress in the phylogeny of Chrysosplenium over the years, the phylogenetic position of some species is still controversial. In this study, we assembled chloroplast genomes (cp genomes) of 34 Chrysosplenium species and performed comparative genomic and phylogenetic analyses in combination with other cp genomes of previously known Chrysosplenium species, for a total of 44 Chrysosplenium species. The comparative analyses revealed that cp genomes of Chrysosplenium species were more conserved in terms of genome structure, gene content and arrangement, SSRs, and codon preference, but differ in genome size and SC/IR boundaries. Phylogenetic analysis showed that cp genomes effectively improved the phylogenetic support and resolution of Chrysosplenium species and strongly supported Chrysosplenium species as a monophyletic taxon and divided into three branches. The results also showed that the sections of Alternifolia and Oppositifolia were not monophyletic with each other, and that C. microspermum was not clustered with other Chrysosplenium species with alternate leaves, but with C. sedakowii into separate branches. In addition, we identified 10 mutational hotspot regions that could serve as potential DNA barcodes for Chrysosplenium species identification. In contrast to Peltoboykinia, the clpP and ycf2 genes of Chrysosplenium were subjected to positive selection and had multiple significant positive selection sites. We further detected a significant positive selection site on the petG gene between the two sections of Chrysosplenium. These evolutionary characteristics may be related to the growth environment of Chrysosplenium species. This study enriches the cp genomes of Chrysosplenium species and provides a reference for future studies on its evolution and origin.