Characteristics and Phylogenetic Analysis of the Complete Plastomes of Anthogonium gracile and Eleorchis japonica (Epidendroideae, Orchidaceae)

Phylogenetic relationships within the subtribe Arethusinae (Arethuseae: Epidendroideae: Orchidaceae) remain unresolved, with particular uncertainty surrounding the phylogenetic positions of Anthogonium gracile and Eleorchis japonica. The monophyly of this subtribe remains contentious, making it one of the challenging taxa in Orchidaceae phylogenetics. In this study, we sequenced and analyzed the complete plastome sequences of A. gracile and E. japonica for the first time, aiming to elucidate their plastome characteristics and phylogenetic relationships. Both plastomes exhibited a conserved quadripartite structure, with 158,358 bp in A. gracile and 152,432 bp in E. japonica, and GC contents of 37.1% and 37.3%, respectively. Comparative analyses revealed strong structural conservation, but notable gene losses: E. japonica lacked seven ndh genes (ndhC/D/F/G/H/I/K), whereas A. gracile retained a complete ndh gene set. Repetitive sequence analysis identified an abundance of simple sequence repeats (68 and 77), tandem repeats (43 and 30), and long repeats (35 and 40). Codon usage displayed a bias toward the A/U termination, with leucine and isoleucine being the most frequent. Selection pressure analysis indicated that 68 protein-coding genes underwent purifying selection (Ka/Ks < 1), suggesting evolutionary conservation of plastome protein-coding genes. Nucleotide diversity analysis highlighted six hypervariable regions (rps8-rpl14, rps16-trnQUUG, psbB-psbT, trnTUGU-trnLUAA, trnFGAA-ndhJ, and ycf1), suggesting their potential as molecular markers. Phylogenomic reconstruction, using complete plastome sequences, (ML, MP, and BI) indicated that Arethusinae was non-monophyletic. A. gracile formed a sister relationship with Mengzia foliosa and E. japonica, whereas Arundina graminifolia exhibited a sister relationship with Coelogyninae members. These results shed new light on the plastome characteristics and phylogenetic relationships of Arethusinae.