Comparative Phylogenomic Study of Malaxidinae (Orchidaceae) Sheds Light on Plastome Evolution and Gene Divergence

Malaxidinae is one of the most confusing groups in the Orchidaceae classification. Previous phylogenetic analyses have revealed that the relationships between the taxa in Malaxidinae have not yet been reliably established, using only a few plastome regions and nuclear ribosomal internal transcribed spacer (nrITS). In the present study, the complete plastomes of <i>Oberonia integerrima</i> and <i>Crepidium purpureum</i> were assembled using high-throughput sequencing. Combined with publicly available complete plastome data, this resulted in a dataset of 19 plastomes, including 17 species of Malaxidinae. The plastome features and phylogenetic relationships were compared and analyzed. The results showed the following: (1) Malaxidinae species plastomes possess the quadripartite structure of typical angiosperms, with sizes ranging from 142,996 to 158,787 bp and encoding from 125 to 133 genes. The <i>ndh</i> genes were lost or pseudogenized to varying degrees in six species. An unusual inversion was detected in the large single-copy region (LSC) of <i>Oberonioides microtatantha</i>. (2) Eight regions, including <i>ycf1</i>, <i>matK</i>, <i>rps16</i>, <i>rpl32</i>, <i>ccsA</i>-<i>ndhD</i>, <i>clpP</i>-<i>psbB</i>, <i>trnF^GAA</i>-<i>ndhJ</i>, and <i>trnS^GCU</i>-<i>trnG^UCC</i>, were identified as mutational hotspots. (3) Based on complete plastomes, 68 protein-coding genes, and 51 intergenic regions, respectively, our phylogenetic analyses revealed the genus-level relationships in this subtribe with strong support. The <i>Liparis</i> was supported as non-monophyletic.