Comparative chloroplast genomics of Gnaphalieae (Asteraceae)

The tribe Gnaphalieae (Asteroideae, Asteraceae) comprises approximately 2,100 species worldwide; however, its chloroplast genomic resources remain limited. Here, we present the most comprehensive comparative chloroplast genomic study of Gnaphalieae to date, achieved through the de novo assembly of 25 species from 13 genera, including the first reported chloroplast genomes for seven of these genera. These newly assembled genomes were combined with 36 previously reported chloroplast genomes retrieved from the NCBI database, yielding a dataset of 61 species representing 26 genera that span both subtribes and all major informal clades of Gnaphalieae. Chloroplast genome sizes ranged from 151,073 bp to 154,259 bp, exhibiting a typical quadripartite structure with highly conserved gene content (112 unique genes), organization, guanine–cytosine content, and intron composition. Inverted repeat boundaries, amino acid composition, and codon usage demonstrated high similarity across species, whereas a moderate level of variation was detected in simple sequence repeats. Maximum likelihood phylogenetic analyses supported the two-subtribe classification of Relhaniinae and Gnaphaliinae and resolved the Relhania clade as the earliest-diverging lineage. Furthermore, several large genera, including Gnaphalium , Anaphalis , Pseudognaphalium , and Helichrysum , were resolved as non-monophyletic in the chloroplast tree. The backbone of the Filago–Leontopodium–Antennaria–Gamochaeta (FLAG) clade received only weak support, contrasting with previous nuclear ITS/ETS topologies. This study provides the most comprehensive chloroplast genomic framework for Gnaphalieae to date, establishing essential genetic resources for future phylogenetic and conservation research. The observed cytonuclear discordance highlights the need to integrate nuclear and chloroplast data for accurate taxonomic inference in rapidly radiating lineages.