Phylogenomics Clarifies Plastome Reduction and Phylogenetic Relationships in Monotropoideae

Plastome evolution and biogeographic history remain poorly understood in mycoheterotrophic lineages. Here, we sequenced and assembled the complete plastome of Monotropa callistoma (Ericaceae), a fully mycoheterotrophic species, and compared it with other mycoheterotrophic plastomes. The M. callistoma plastome is 29,784 bp long, lacks the canonical quadripartite structure, and contains 40 genes, representing an intermediate level of reduction within Monotropoideae. Comparative analyses revealed extreme contraction in some lineages, including Sciaphila (12.8-21.5 kb), and a weak positive correlation between GC content and genome size. Within Monotropoideae, plastomes become smaller from the early-branching Pterospora lineage to the more recently diverged Monotropa lineage. This reduction is accompanied by progressively poorer synteny and lineage-specific loss of rps23 in Monotropa. Phylogenomic analyses of 30 Ericaceae plastomes strongly supported Monotropoideae (bootstrap = 100) and clarified relationships among its 10 sampled genera. Divergence-time estimation and ancestral-range reconstruction placed the origin of Clade 1 (Pyroloideae, Arbutoideae and Monotropoideae) in Asia during the late Cretaceous to early Cenozoic. The lineage subsequently dispersed to North America via Beringia, whereas the Monotropsis + Monotropastrum + Monotropa clade later dispersed back to Asia during the late Eocene to early Oligocene. This history gave rise to East Asian endemics, including Monotropa humile and M. callistoma. These findings illuminate plastome reduction in mycoheterotrophs, resolve relationships within Monotropoideae, and support a complex history of bidirectional intercontinental dispersal.