Nettle Family Genomes Reveal Rare Mitochondrial DNA Invasion of Chloroplasts

Hidden in the shaded understories of tropical forests, on limestone karst cliffs and beside cave entrances, grows one of botany’s most species-rich but least understood plant lineages. The tribe Elatostemateae, part of the nettle family Urticaceae, encompasses roughly 1,300 species ranging from delicate herbs to woody shrubs, and includes the popular Chinese money plant, Pilea peperomioides, that graces countless windowsills. Now a large-scale genomic study has peeled back the chloroplast genomes of this enigmatic group and uncovered a startling degree of structural chaos, including a rare event in which mitochondrial DNA appears to have invaded the chloroplast genome, something documented in only a handful of flowering plant lineages.

The research, published in the journal Ecology and Evolution, assembled and compared complete plastid genomes from 40 species spanning the recognized genera of Elatostemateae, together with nuclear ribosomal DNA sequences from the same samples. The team, led by Long-Fei Fu of the Guangxi Institute of Botany along with Alexandre K. Monro of the Natural History Museum in London and colleagues, generated dozens of new plastome sequences and combined them with existing data to build one of the most comprehensive phylogenomic frameworks yet attempted for the tribe. Their goal was threefold: to resolve relationships among the tribe’s genera, to map structural variation across the chloroplast genome, and to identify fast-evolving DNA regions that could serve as genetic barcodes for identifying species.

Elatostemateae has long been a taxonomic headache. Classical classifications relied on combinations of leaf arrangement, stipule position, inflorescence architecture and achene morphology, but these characters turn out to be prone to convergent evolution, meaning unrelated species can look deceptively similar. Earlier molecular studies relying on a handful of genes confirmed that the tribe as a whole is monophyletic, descended from a single common ancestor, but also revealed that the genus Pellionia, as traditionally defined, was polyphyletic, its species scattered across the family tree. Those same studies elevated Elatostematoides and Procris to full generic rank, resurrected Achudemia and Lecanthus as separate lineages within Pilea, and established a modern classification recognizing eight genera: Achudemia, Elatostema, Elatostematoides, Lecanthus, Metapilea, Pilea, Polychroa and Procris.

The new study sequenced 40 accessions using Illumina paired-end technology, extracting total genomic DNA from silica-dried leaves and assembling the chloroplast genomes de novo with the software GetOrganelle, without using a reference genome. The researchers also fished out the nuclear ribosomal DNA region spanning the 18S, 5.8S and 26S genes plus the internal transcribed spacers from the same sequencing reads, a technique known as genome skimming that squeezes phylogenetic information from low-depth whole-genome data. Phylogenetic trees were then built from four separate data partitions, complete plastomes, plastid protein-coding sequences, nuclear ribosomal DNA, and a concatenation of plastome plus ribosomal DNA, using both maximum-likelihood and Bayesian inference, with the latter run for 100 million generations until independent analyses converged.

The resulting trees recovered the same three major clades regardless of which dataset was used. Clade A corresponds to Elatostema in the broad sense, containing four subclades that map neatly onto the genera Polychroa, Procris, Elatostematoides and Elatostema itself, with Elatostema further subdivided into four lineages. Clade B contains the Caribbean genus Gyrotaenia, and Clade C holds Pilea in the broad sense alongside Achudemia and Lecanthus, with Pilea itself splitting into eight subclades. Notably, Urticeae, the tribe containing the stinging nettles, emerged as the sister lineage to Elatostemateae in this family-level framework. The agreement between chloroplast and nuclear ribosomal DNA at the level of major clades lends confidence to these backbone relationships, even though some internal branches, particularly within Elatostema and Pilea, showed weaker statistical support in the ribosomal analysis.

But the headline-grabbing results came from the structural comparisons. Plastome size ranged from 143,738 base pairs in Pilea micromeriifolia to 163,969 base pairs in Elatostema oblongifolium, a spread of more than 20,000 base pairs driven almost entirely by shifts in the boundaries of the inverted repeat, a pair of large duplicated regions that bracket the chloroplast genome’s small single-copy region. In Pilea tetraphylla, the researchers documented an unusually asymmetric combination of inverted repeat contraction and expansion: one boundary contracted, moving genes such as ycf2 and rpl2 out of the repeat into the single-copy region where they became single-copy, while the opposite boundary expanded, pulling ycf1 and rps15 into the repeat so that they became duplicated. Pilea micromeriifolia showed the opposite extreme, with pronounced inverted repeat contraction shrinking the repeat by roughly seven kilobases and stripping out five genes.

Even more dramatic were the findings in two species of Elatostema. Elatostema parvum and Elatostema pycnodontum carry an approximately 20-kilobase inversion in the large single-copy region, spanning from rpoB to trnQ-UUG and encompassing about 14 genes. These are the first large plastome inversions documented in Elatostemateae, and their breakpoints lie near tRNA genes, a pattern consistent with recombination-prone regions reported in other plant chloroplast genomes, though the authors caution that proximity alone does not prove the tRNA loci caused the inversion. Crucially, the inversion-bearing species form a single recognized subclade, meaning the structural variant is phylogenetically clustered rather than scattered randomly across the tree, hinting that it arose once in a common ancestor of that lineage.

The most remarkable structural find, however, was a chunk of foreign DNA wedged between the ycf15 and trnV-GAC genes within the inverted repeat of Elatostema nanchuanense and Elatostema oblongifolium, adding between 3,941 and 7,033 base pairs and producing inverted repeats far longer than those of their relatives. When the team searched this inserted sequence against public databases, the best matches were to mitochondrial genomes, not chloroplasts. The researchers verified the insertion was genuine rather than an assembly artifact by rebuilding the plastomes with two independent assembly pipelines and checking that sequencing read coverage was continuous across the insertion boundaries. The verdict: a plastid-to-mitochondrial DNA transfer in reverse, a so-called PTMT insertion. While the transfer of organellar DNA to the nucleus is commonplace in plant evolution, movement of mitochondrial DNA into the plastid genome is exceedingly rare, with only a few documented examples in distantly related angiosperms. The authors suggest the insertion likely arose when a double-strand break in the chloroplast genome was repaired using mitochondrial DNA as raw material, with subsequent sequence loss leaving behind the remnant visible today. Because the insertion is restricted to a subset of Elatostema, it may prove phylogenetically informative, though its precise age and mechanism cannot yet be pinned down.

Beyond the structural fireworks, the study catalogued 5,477 repeat elements across the 40 plastomes, including 3,042 simple sequence repeats, 1,379 dispersed long repeats and 1,056 tandem repeats, with mononucleotide repeats dominated by poly-A and poly-T motifs making up two-thirds of the microsatellites. Nucleotide diversity scans pinpointed seven hypervariable regions, including the genes ycf1, rpl22 and rps16 and the intergenic spacers rps15-ycf1, trnH-GUG-psbA, rpl32-trnL-UAG and ndhF-rpl32, which show elevated mutation rates and stand out as prime candidates for future DNA barcoding of this morphologically slippery tribe.

Perhaps the most tantalizing thread running through the work is the discordance surrounding Gyrotaenia. The plastid and ribosomal DNA data place this Caribbean genus inside Elatostemateae as sister to Pilea, consistent with earlier multi-locus studies. Yet recent analyses using hundreds of low-copy nuclear genes recovered with the Angiosperms353 probe set place Gyrotaenia together with Myriocarpa as sister to the core of the tribe instead. The authors point out that incomplete lineage sorting during rapid ancient divergence and historical introgression, sometimes called chloroplast capture, are both plausible and non-exclusive explanations, but that the current data cannot distinguish between them. Disentangling that conflict will require denser sampling of independent nuclear loci or whole genomes. Meanwhile, the observation that the tribe’s major plastome structural variants, the inversion, the mitochondrial insertion, the extreme repeat contraction and the asymmetric boundary shift, each map to different subclades occupying different geographic regions, from the Indomalayan realm to the Neotropics and Africa, offers an intriguing hypothesis linking phylogeny, geography and genome architecture that population-level studies are now poised to test. For a tribe that has dodged tidy classification for decades, the chloroplast genome is finally starting to give up its secrets.

Subject of Research: Plastid genome evolution and phylogenetic relationships in the nettle tribe Elatostemateae

Article Title: Plastid Phylogenomics of Elatostemateae (Urticaceae): Insights Into Structural Variation and Phylogenetic Relationships

Article References: Fu, L.-F., Xin, Z.-B., Xiong, C., Wen, F., Wei, Y.-G., & Monro, A. K. (2026). Plastid Phylogenomics of Elatostemateae (Urticaceae): Insights Into Structural Variation and Phylogenetic Relationships. Ecology and Evolution, 16(10), Article e74416. https://doi.org/10.1002/ece3.74416

Image Credits: AI Generated

DOI: 10.1002/ece3.74416

Keywords: Elatostemateae, Urticaceae, plastid phylogenomics, chloroplast genome, inverted repeat, mitochondrial DNA transfer, phylogeny, Pilea, Elatostema, DNA barcoding, genome rearrangement, cytonuclear discordance