The research team, led by Xing-Xing Shen of Zhejiang University together with co-first authors Xianfeng Mi and Guo-Zheng Ou and colleague Yixiao Zhu, leveraged a large-scale dataset originally assembled by Tao and colleagues comprising 472 insect species spanning 19 orders. From each species they extracted amino acid sequences of 13 mitochondrial protein-coding genes and 1,367 single-copy nuclear protein-coding genes. Using concatenation-based maximum likelihood inference with the IQ-TREE software and 1,000 ultrafast bootstrap replicates, they reconstructed two genome-scale phylogenies, one from the mitochondrial data under the best-fitting mtInv+F+G4 model and one from the nuclear data under the LG+G4 model. Both trees were exceptionally well supported: 98.30 percent of nodes in the nuclear phylogeny and 88.11 percent of nodes in the mitochondrial phylogeny carried bootstrap values of 90 percent or higher, and within the five largest insect orders, Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera, highly supported nodes remained above 95 percent in nuclear trees and above 80 percent in mitochondrial trees.

That high support made the conflict all the more striking. When the researchers compared the two topologies using the normalized Robinson-Foulds distance, a metric ranging from 0, indicating identical trees, to 1, indicating maximal disagreement, they obtained a value of 0.306 for the full dataset, a considerable level of topological discordance that could not be dismissed as an artifact of weak phylogenetic signal. The degree of conflict also varied substantially among the major orders. Lepidoptera, the order containing butterflies and moths, showed the greatest incongruence with an nRF distance of 0.446, while Hymenoptera, the bees, wasps, and ants, showed the least at 0.158. Because both trees were statistically robust, the discordance had to reflect genuine differences in the evolutionary histories or compositional properties of the two genomes rather than simple failure to resolve branching order.

One obvious suspect was model choice. Mitochondrial genomes evolve faster than nuclear genomes and display strong compositional heterogeneity, so critics might argue that standard models simply fail to capture the true substitution process in mitochondrial sequences, generating misleading trees. To test this, the team rebuilt the mitochondrial phylogeny from concatenated amino acid sequences using a site-heterogeneous mixture model, mtInv+F+G4+C60, which allows substitution patterns to vary across sites in far more flexible ways. The result was unambiguous: the more complex model did not eliminate the mito-nuclear discordance. This finding aligns with a growing body of work suggesting that increasing evolutionary model complexity improves fit to the data but does not necessarily resolve gene tree conflict, meaning the source of the disagreement had to lie elsewhere.

The researchers then cast a wide net, systematically quantifying nine gene properties for every mitochondrial and nuclear gene: alignment length, GC content, amino acid substitution saturation, effective number of amino acids, proportion of constant sites, proportion of parsimony-informative sites, external branch length, average bootstrap support value, and treeness, defined as the proportion of internal branch lengths over all branch lengths. Their central analytical innovation was a property-matching strategy. For each property, they selected 13 nuclear genes whose values closely matched those of the 13 mitochondrial genes, for example nuclear genes whose alignment lengths fell within plus or minus 5 percent of the corresponding mitochondrial genes, concatenated these mito-like nuclear genes, built a phylogeny, and measured its topological distance to the mitochondrial reference tree. This sampling and tree-building procedure was repeated 20 times for each property and compared against a background in which 13 nuclear genes were chosen at random, which yielded a median nRF of 0.334.

Against that background, only one property made a difference. When nuclear genes were selected to match the GC content of mitochondrial genes, the resulting trees became significantly more similar to the mitochondrial phylogeny, while matching on alignment length, saturation, amino acid diversity, or any of the other properties failed to reduce the discordance. The compositional gulf between the two genomes is dramatic: mitochondrial genes exhibited significantly lower GC content than nuclear genes, with mitochondrial codons showing a strong bias against codons ending in G or C and a marked preference for A/T-ending codons, whereas nuclear codons displayed a more balanced pattern across the first, second, and third codon positions. This compositional bias cascades into protein composition, since the mitochondrial genome favors amino acids encoded by low-GC codons such as leucine, isoleucine, phenylalanine, methionine, asparagine, and tyrosine, while the nuclear genome favors amino acids encoded by high-GC codons such as alanine, arginine, glutamate, and aspartate.

The GC effect extends beyond the boundary between the two genomes and into the structure of the nuclear genome itself. When the researchers compared pairwise topological differences among mitochondrial genes, they found that mitochondrial genes were more topologically similar to one another than nuclear genes were to each other, consistent with their shared maternal inheritance. Yet even within the mitochondrial genome, low-GC genes produced trees more similar to each other than high-GC genes did. To probe the nuclear side, the team divided 1,367 nuclear genes into three non-overlapping groups by GC content, a low-GC group at 38 to 40 percent, a medium-GC group at 42 to 44 percent, and a high-GC group at 46 to 48 percent, with each group spanning exactly a 2 percent range to ensure comparable variation. Pairwise comparisons of gene trees within each group revealed a clear trend: topological differences increased steadily with GC content, meaning low-GC nuclear genes are more topologically consistent with one another than high-GC nuclear genes are.

Intriguingly, GC content also tracks functional differentiation within the nuclear genome. Using Gene Ontology analysis, in which detailed GO terms were consolidated into 18 broader biological process categories, the researchers found that high-GC nuclear genes were disproportionately involved in complex biological processes including biological regulation, developmental processes, response to stimulus, multicellular organismal processes, growth, and homeostatic processes, while low-GC nuclear genes represented the greatest proportion of genes involved in metabolic processes. This functional stratification suggests that GC content is not merely a technical nuisance for phylogenetic inference but a biologically meaningful axis of genome organization, one that correlates with gene expression patterns and evolutionary dynamics documented in prior studies of codon usage bias and compositional evolution across insect genomes.

What mechanism might link nucleotide composition to tree topology? The authors point to GC-biased gene conversion, a process in which recombination preferentially fixes G and C alleles over A and T during DNA repair, as a plausible driver. GC-rich genomic regions are thought to experience higher recombination rates, and biased conversion can distort substitution patterns in ways that mislead tree-building algorithms, potentially generating greater topological discordance among high-GC genes. The study’s conclusions are notably conservative in what they rule out: gene alignment length, inheritance pattern, and evolutionary model complexity all failed to explain the observed conflicts, overturning several common assumptions in the field. Because mitochondrial genomes are shorter than nuclear genomes, differences in alignment length have long been considered a contributing factor, yet matching on length had no effect, and the persistence of discordance among mitochondrial genes grouped by GC content shows that maternal inheritance alone cannot guarantee topological agreement.

The authors are careful to acknowledge the limits of their analysis. Multiple biological processes are known to generate nuclear phylogenomic incongruence, including incomplete lineage sorting, introgression, hybridization, sex-biased dispersal, and horizontal gene transfer, and future work integrating these factors will be essential to test whether they also contribute to mito-nuclear discordance. The team emphasizes that their aim was to elucidate the cause of discordance rather than to declare which genome tells the truer evolutionary story. Even so, the practical implications are immediate for anyone reconstructing insect phylogenies: sampling nuclear genes whose GC content resembles that of mitochondrial genes, so-called mito-like nuclear genes, can measurably reduce topological conflict, and researchers interpreting mitochondrial barcodes or mitogenomic trees should now weigh compositional bias as a first-order consideration. By bridging molecular composition and evolutionary inference, the study transforms GC content from a footnote in methods sections into a central character in one of phylogenetics’ most persistent mysteries.

Subject of Research: Mito-nuclear phylogenetic discordance and the role of GC content in insect genome evolution

Article Title: Dissecting discordance of mitochondrial and nuclear phylogenetic trees in insects

Article References: Mi, X., Ou, G.-Z., Zhu, Y., & Shen, X.-X. (2025). Dissecting discordance of mitochondrial and nuclear phylogenetic trees in insects. Crop Health, 3(1), Article 23. https://doi.org/10.1007/s44297-025-00062-3

Image Credits: AI Generated

DOI: 10.1007/s44297-025-00062-3

Keywords: phylogenetics, mito-nuclear discordance, insects, GC content, mitochondrial genome, nuclear genome, insect genomics, GC-biased gene conversion, Gene Ontology, Robinson-Foulds distance, codon usage bias, evolutionary models

The investigation was led by Márcio Galvão Pavan of the Oswaldo Cruz Institute (Fiocruz) in Rio de Janeiro, together with Rodrigo Gurgel-Gonçalves of the University of Brasília and colleagues, including Fernando Abad-Franch and Fernando Araujo Monteiro, who contributed equally to the work. The team set out to answer a deceptively simple question: when light-colored bugs turn up inside carnaúba palms, are they R. nasutus, the species whose primary habitat these palms represent in northeastern Brazil, or could they actually be R. neglectus wearing, in effect, a different species’ colors? The answer, according to the combined genetic and morphological evidence, is frequently the latter.

The study’s foundation lies in an ecological and biogeographical puzzle. Rhodnius nasutus is typical of the Caatinga, Brazil’s seasonally dry, thorny scrubland biome, where it dwells chiefly in the crowns of Copernicia prunifera palms and is characteristically light brown in color. Rhodnius neglectus, by contrast, is a core-Cerrado species associated with Mauritia flexuosa palms and typically shows a much darker brown body. Because the Caatinga and Cerrado intergrade across a broad transition zone, the two palm species, and the bugs that inhabit them, occasionally come into geographic contact. That overlap set the stage for the color confusion the researchers document.

To untangle the situation, the team sampled triatomines from C. prunifera palms across the semiarid Caatinga and the Caatinga-Cerrado transition, comparing them with specimens collected from core-Cerrado M. flexuosa palms. Every insect was first identified by phenotype, meaning its visible color pattern and morphology. The researchers then turned to genetics, sequencing clones of the mitochondrial cytochrome b gene, a standard barcoding marker, and the nuclear ribosomal DNA internal transcribed spacer 2, or ITS2. This two-marker strategy allowed the team to compare signals from the maternally inherited mitochondrial genome with those from the nuclear genome, a contrast that proved decisive.

The morphological results were clear at the extremes but murky in the middle. Adult specimens from core-Cerrado Mauritia palms consistently displayed the dark brown coloration typical of R. neglectus, while adults from core-Caatinga localities showed the light brown hue typical of R. nasutus. But in bugs captured in carnaúba palms from the Caatinga-Cerrado transition zone and parts of the central-western Caatinga, color patterns were dubious, matching neither type reliably. Specimens that looked like typical light brown R. nasutus could not confidently be assigned on appearance alone, raising the possibility that field identifications based on color might have been systematically wrong in exactly these regions.

Genetics began to resolve the ambiguity. Bayesian genealogies built from cytochrome b sequences placed the dubious-phenotype specimens that resembled typical R. nasutus in a sub-clade that is sister to typical R. neglectus from Mauritia flexuosa palms, with roughly 2% mean cytb divergence separating the two sub-clades. In other words, the bugs that looked like R. nasutus carried mitochondrial genomes pointing to R. neglectus ancestry. This mitochondrial signal effectively dissolved the taxonomic uncertainty created by the shared coloration, demonstrating that insects occupying carnaúba palms in the transition zone belong, at least by maternal lineage, with R. neglectus.

The nuclear ITS2 marker told a less tidy story. ITS2 genealogies recovered both R. neglectus and R. nasutus as paraphyletic, meaning individuals of each nominal species were scattered across the gene tree rather than clustering neatly by species. Such patterns often arise when recently diverged species still share ancestral genetic variation. When the researchers applied a multispecies-coalescent analysis, a statistical framework designed to account for this shared ancestry, the dubious-phenotype bugs from C. prunifera clustered with typical R. neglectus. The analysis suggested that the messy ITS2 picture reflects incomplete lineage sorting, the retention of ancestral polymorphisms, rather than hybridization or misassigned species limits. The mito-nuclear discordance observed in the study is thus itself scientifically informative, illustrating how different genomes can carry different historical signals during the early stages of species divergence.

Why would two species converge on the same color? The authors propose that predator-driven natural selection is the most plausible mechanism. Triatomine bugs live in palm crowns where the substrate color is set by the palm’s dried leaf bases and fibers. In the core Cerrado, dark Mauritia crowns favor dark R. neglectus; in the core Caatinga, the lighter carnaúba substrate favors pale R. nasutus. But when R. neglectus colonizes carnaúba palms, individuals whose coloration better matches the light palm-crown substrate presumably enjoy better camouflage against visual predators such as birds and lizards. Over time, this selection pressure could produce R. neglectus populations whose light brown bodies are nearly indistinguishable from typical R. nasutus. This is a textbook scenario of adaptive phenotypic convergence, conceptually related to classic camouflage and mimicry systems in evolutionary biology, but documented here in disease vectors, where species identity has public health consequences.

The practical implications are significant. Chagas disease remains a major neglected tropical disease in Latin America, and surveillance programs depend on correctly identifying which vector species are present in a given area, because species differ in habitat preferences, domestic invasion behavior, and epidemiological relevance. If R. neglectus can masquerade as R. nasutus, field records based on color alone may misattribute vectors to the wrong species and thereby misguide control strategies. The study’s findings, the authors argue, call into question the widespread use of overall body color as a key phenotypic character in triatomine taxonomy. As molecular tools become more accessible, integrating DNA-based confirmation into routine vector surveillance may be essential wherever palm-dwelling Rhodnius species co-occur or share similar microhabitats.

Beyond the immediate applied concerns, the work contributes to a broader understanding of how color variation evolves in triatomines and how convergence can erode the morphological signals taxonomists depend on. The researchers received support from Brazil’s CAPES and CNPq funding agencies, and sampling was conducted under permit from the country’s biodiversity authority, ICMBio. By combining careful field collection across two biomes with mitochondrial and nuclear sequence analysis and modern coalescent modeling, the team has shown that even a character as apparently straightforward as body color can be shaped powerfully by ecology, and that species identities written in DNA may diverge sharply from those written on the insect’s back. For the bugs of the carnaúba palms, looking like the neighbor may be good for survival, but it makes life considerably harder for the scientists trying to tell them apart.

Subject of Research: Phenotypic convergence in the Chagas disease vector bugs Rhodnius neglectus and Rhodnius nasutus inhabiting Copernicia prunifera palms in Brazil

Article Title: Phenotypic convergence in Chagas disease vectors: Rhodnius neglectus from Copernicia prunifera palms may express the typical Rhodnius nasutus color phenotype

Article References: Pavan, M. G., Gurgel-Gonçalves, R., Corrêa-Antônio, J., Morelli, K. A., Bahia, A. C., Abad-Franch, F., & Monteiro, F. A. (2026). Phenotypic convergence in Chagas disease vectors: Rhodnius neglectus from Copernicia prunifera palms may express the typical Rhodnius nasutus color phenotype. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07651-3

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07651-3

Keywords: Triatominae, Rhodnius, Chagas disease, phenotypic convergence, convergent evolution, camouflage, Copernicia prunifera, cytochrome b, ITS2, incomplete lineage sorting, mito-nuclear discordance, Brazil