The research team collected scale-leaf tissue from M. callistoma in Shiyan, Hubei Province, and sequenced it on an Illumina HiSeq 4000 platform at a mean depth of over 900-fold. Assembly with GetOrganelle and polishing with Pilon produced a single circular plastome of 29,784 base pairs with a GC content of 30.8 percent. That figure might sound unremarkable until compared with a typical photosynthetic plant, whose plastome runs roughly 150,000 base pairs and carries more than a hundred genes. The M. callistoma plastome contains just 40 genes: 20 protein-coding genes, 16 transfer RNAs, and four ribosomal RNAs. Strikingly, the canonical quadripartite structure found in almost all land plant plastids, with two inverted repeats separating large and small single-copy regions, is entirely absent. No inverted-repeat boundaries could be detected at all.

The gene inventory tells the story of a genome in terminal decline. Every photosynthetic function has been erased. There are no photosystem subunits, no NADH dehydrogenase genes, no cytochrome b6f complex components, no ATP synthase subunits, and no rubisco large subunit. What remains is essentially a minimal housekeeping kit: ten genes for small ribosomal subunit proteins, seven for large subunit proteins, and just three other coding genes, infA, matK, and accD. Four of the retained genes carry a single intron. This is the genomic signature of an organelle whose core reason for existence, photosynthesis, has been outsourced to fungal partners, leaving relaxed selection to strip away everything the plant no longer needs.

To place this genome in context, the researchers compiled published plastomes from 30 mycoheterotrophic species across six families, ranging from orchids such as Gastrodia and Lecanorchis to the bizarre holoparasites Sciaphila and Mitrastemon. The comparison revealed a spectrum of decay. Sciaphila thaidanica and Mitrastemon yamamotoi carry plastomes of only about 20 kilobases, among the smallest known in flowering plants, while at the other extreme some Burmannia species retain genomes approaching 100 kilobases. GC content showed a weak but statistically significant positive correlation with genome size, suggesting that as these genomes shrink, their nucleotide composition shifts in parallel. Within Monotropoideae itself, Pterospora andromedea holds the largest plastome at roughly 40 kilobases, while the more recently diverged Monotropa uniflora has the smallest at about 27 kilobases.

Whole-genome alignments exposed another dimension of decay: the collapse of synteny, the conserved order of genes along the chromosome. Among Monotropoideae species, gene order is scrambled, particularly in Hypopitys, Monotropsis, Monotropastrum, and Monotropa. Fragment loss has accumulated in more recently diverged lineages, so that even within the genus Monotropa, where synteny is relatively well preserved, the overall picture across the subfamily is one of progressive disintegration. Ancestral-state reconstruction across the 30 species showed that losses of the large genes ycf1 and ycf2 occurred convergently among eudicot lineages, while all sampled Monotropa species lacked rps23, pointing to a lineage-specific loss within that genus.

The phylogenomic analysis went beyond genome description to resolve long-standing questions about relationships within Ericaceae. Using a concatenated alignment of plastid protein-coding genes nearly 48,000 base pairs long, with Actinidia chinensis as an outgroup, the team recovered a well-supported split into two major clades. Clade 1 united Pyroloideae, Arbutoideae, and Monotropoideae, with Pyroloideae branching earliest and Monotropoideae emerging as the youngest lineage. Within Monotropoideae, the sampling covered 10 of the 12 recognized genera and achieved maximum bootstrap support of 100 for the backbone. Pterospora and Sarcodes occupied successive early-branching positions, followed by Pleuricospora, while a clade of Monotropsis, Monotropastrum, and Monotropa was sister to a group containing Allotropa, Hemitomes, Pityopus, and Hypopitys. Clade 2 comprised the core Ericaceae, subdivided into Vaccinioideae and Ericoideae.

Divergence dating with fossil calibrations placed the split between Pyroloideae and the remaining subfamilies at approximately 67.77 million years ago, in the latest Cretaceous. Arbutoideae and Monotropoideae diverged around 66.57 million years ago, and diversification within Monotropoideae began about 63.35 million years ago, immediately after the end-Cretaceous extinction. The genus-level splits came much later: Sarcodes and Pterospora separated roughly 45.79 million years ago, Monotropsis split from the lineage leading to Monotropa and Monotropastrum about 39.73 million years ago, and those two genera diverged about 26.74 million years ago. The rapid radiation of Rhododendron and Vaccinium, by contrast, occurred only around 1 to 2 million years ago.

Perhaps the most striking finding concerns geography. Ancestral-range estimation using the DIVALIKE+J model in RASP placed the most recent common ancestor of Clade 1 in Asia during the late Cretaceous to early Cenozoic. From there, the lineage dispersed to North America, most plausibly across the Bering Land Bridge, which connected the continents from the latest Cretaceous into the early Paleocene, when a warm, humid belt of deciduous broadleaf forest stretched across the region. Then came a remarkable reversal: the ancestor of the Monotropsis, Monotropastrum, and Monotropa lineage appears to have dispersed back to Asia during the late Eocene to early Oligocene, around 39.70 million years ago, giving rise to the East Asian endemics Monotropastrum humile and Monotropa callistoma. The ghost plants, in other words, crossed the world twice.

The comparative analysis also yielded a testable hypothesis about the order in which plastid genes are lost as plants abandon photosynthesis. Across the 30 species examined, the ndh genes were absent everywhere, followed by photosystem, cytochrome, rubisco, and ycf3/4 genes, then the RNA polymerase genes, then atp genes, and finally the ribosomal, transfer RNA, and housekeeping genes such as accD, clpP, and matK. The sole exception was Burmannia cryptopetala, which retains atp genes, a pattern associated with mixotrophic or partially parasitic lifestyles. The team proposes that the degree of a plant’s absolute dependence on fungal hosts correlates with the breadth of plastid gene loss, with Monotropa, whose plastid genes evolve roughly 13 times faster than its nuclear genes, representing an advanced stage of degradation in which even clpP has been discarded.

Yet the decay is not random, and that is the deeper lesson of the study. Housekeeping genes persist with remarkable consistency: accD, which catalyzes the first committed step of fatty acid biosynthesis and is essential for building the plastid’s own membranes, was retained in every single mycoheterotrophic species examined. The retention of matK across all Monotropoideae and Burmanniaceae, despite its patchy record in orchids, and the lineage-specific survival of ycf1 and ycf2 in some orchids and Burmanniaceae but not others, show that different lineages face different residual functional constraints. The endpoints of plastid genome reduction form a spectrum rather than a single destination, ranging from the streamlined but intact genomes of Monotropoideae to the complete loss of the plastid genome in Balanophora, which has transferred even accD to the nucleus. When an organelle’s core function is outsourced, its genetic system converges, again and again across independent lineages, toward the same extreme simplification. The ghost plants of Asia and North America, silent and white beneath the forest canopy, are carrying out that experiment in real time, and their genomes are the record.

Subject of Research: Plastome evolution and phylogenomics of the mycoheterotrophic subfamily Monotropoideae (Ericaceae)

Article Title: Phylogenomics Clarifies Plastome Reduction and Phylogenetic Relationships in Monotropoideae

Article References: Deng, G., Duan, K., Xiang, N., Gao, F., Zhan, P., Shu, R., Lin, Q., Zuo, Q., & Yuan, T. (2026). Phylogenomics Clarifies Plastome Reduction and Phylogenetic Relationships in Monotropoideae. Ecology and Evolution, 16(10), Article e74449. https://doi.org/10.1002/ece3.74449

Image Credits: AI Generated

DOI: 10.1002/ece3.74449

Keywords: Monotropoideae, mycoheterotrophy, plastome reduction, Ericaceae, Monotropa callistoma, phylogenomics, Bering Land Bridge, biogeography, gene loss, relaxed selection, ghost plants, divergence dating