Sawfly and gall wasp chromosomes reveal ancestral rDNA blueprint for Hymenoptera

Deep in the genome of every eukaryotic cell sits a set of instructions for building ribosomes, the molecular machines that translate genes into proteins. The genes encoding the ribosomal RNA are not scattered randomly across chromosomes. Instead, they cluster together at specific chromosomal addresses known as nucleolus organizing regions, or NORs, where the densest structure inside the nucleus, the nucleolus, assembles around them. Mapping these clusters of 45S ribosomal DNA across the tree of life has become one of the most powerful tools in comparative cytogenetics, because the number and position of rDNA sites can be tracked and compared between species to reconstruct what the chromosomes of long-dead ancestors probably looked like. Now, a new study of sawflies and gall wasps has delivered a surprisingly clean answer to a question that has vexed hymenopteran cytogeneticists for decades: a single rDNA cluster per haploid chromosome set appears to be the ancestral condition for the entire order Hymenoptera, an insect lineage whose described species exceed 150,000.

The research, published in the journal Comparative Cytogenetics, was led by Vladimir Gokhman of the Botanical Garden of Moscow State University together with colleagues who collected adult sawflies near Ozhigovo, roughly 60 kilometers southwest of Moscow, and galls containing immature gall wasps near Saratov, about 730 kilometers to the southeast. The team examined three species: Tenthredo campestris, a common sawfly of the family Tenthredinidae; Arge ciliaris, a sawfly of the family Argidae; and Aulacidea hieracii, a gall wasp of the family Cynipidae. Two of these belong to the superfamily Tenthredinoidea within the sawfly suborder Symphyta, while the third represents the superfamily Cynipoidea among the higher wasps of the suborder Apocrita. The choice was deliberate. Symphyta, the so-called lower Hymenoptera, harbor fewer than nine thousand described species, a fraction of the order’s total diversity, but they retain the least advanced body plans and chromosome sets. Studying them is therefore essential for pinning down ancestral character states for the order as a whole, a logic that has already paid off with the confirmation of the canonical insect-type telomeric repeat TTAGG in sawflies.

Previous work on rDNA clusters in Tenthredinoidea had produced a confusing picture. FISH and other techniques had shown that Rhogogaster viridis, Diprion pini and Neodiprion abietis each carry a single 45S rDNA cluster per haploid karyotype, but two species of the genus Tenthredo told a different story: Tenthredo velox has one rDNA site, while Tenthredo arcuata has two. To make matters worse, Athalia rosae, now placed in its own family Athaliidae, carries four clusters, the highest number recorded in any sawfly. With such variation, the ancestral number and location of rDNA sites in the Symphyta remained ambiguous. The new results cut through this uncertainty. Using fluorescence in situ hybridization with an 18S rDNA probe, the researchers found that both Tenthredo campestris and Arge ciliaris harbor exactly one pericentromeric rDNA cluster per haploid set of ten biarmed chromosomes. In Tenthredo campestris, the cluster sits on a particular medium-sized chromosome, while in Arge ciliaris it co-localizes with an obvious pericentromeric secondary constriction on the longer arm of the second metacentric chromosome, a structure long suspected to mark the NOR.

The technique behind these observations is as elegant as it is technically demanding. Fluorescence in situ hybridization relies on a labeled DNA probe that binds specifically to complementary sequences on prepared chromosomes. The team isolated genomic DNA from the firebug Pyrrhocoris apterus, an unrelated insect chosen as a convenient probe source, PCR-amplified the 18S ribosomal RNA gene from it, and labeled the product with biotin. Chromosome preparations were made from haploid embryos dissected from unfertilized mature eggs of female sawflies and from cerebral ganglia of gall wasp prepupae, following classic protocols developed by Naito and by Imai and colleagues with modern modifications. The preparations were treated with RNase A and pepsin to remove unwanted nucleic acids and proteins, fixed with paraformaldehyde, denatured at 75 degrees Celsius, and incubated with the hybridization mixture for 42 to 44 hours at 37 degrees Celsius. After stringent washes in formamide and SSC buffer, the probe was detected with fluorescent avidin, and the chromosomes were counterstained with DAPI and mounted in antifade medium for microscopy.

For Aulacidea hieracii, the gall wasp of the study, the diploid chromosome set contains twenty chromosomes that gradually decrease in size, with a single pericentromeric band highlighted by chromomycin A3 staining on the largest metacentric. Earlier work had suggested that this GC-enriched band might represent the NOR, but chromomycin A3-positive segments do not always correspond to rDNA clusters, so the finding needed independent confirmation. The new FISH results delivered exactly that: the 18S rDNA probe revealed a single paired rDNA site precisely where the chromomycin A3 band had been visualized, demonstrating that both techniques were looking at the same rDNA cluster. This corroboration matters because it validates chromomycin A3 staining as a reliable, if indirect, proxy for NORs in this group, allowing cytogeneticists to screen more species quickly before committing to the more laborious FISH procedure.

Putting the new data together with everything already known about rDNA clusters in Hymenoptera produces a coherent evolutionary narrative. Within Tenthredinoidea, the single rDNA cluster per haploid set predominates, and the new species reinforce this pattern. Arge ciliaris is particularly informative because it remains the only examined member of the Argidae plus Pergidae clade, which is sister to the remaining Tenthredinoidea except Blasticotomidae, the most basal family whose chromosomes are entirely unknown. If the single-cluster condition holds across the superfamily, then the higher numbers of rDNA sites found in Tenthredo arcuata and Athalia rosae represent apomorphic, or derived, conditions that evolved independently in those lineages. The authors caution that studying rDNA clusters in other sawfly superfamilies could either confirm or reject this assumption, but the weight of evidence now clearly favors a single ancestral site.

The story extends beyond the sawflies. Tenthredinoidea is sister to all other Hymenoptera except the family Xyelidae, in which the structure and location of rDNA sites remain unknown. Among the higher wasps of the suborder Apocrita, a single 45S rDNA cluster per haploid karyotype is widespread, particularly among parasitoid lineages. All known members of Cynipoidea, including Diplolepis rosae and four species of Figitidae examined by FISH in earlier studies, as well as the three Cynipidae species screened with chromomycin A3, carry a single cluster. The same condition is considered ancestral for Aculeata, the stinging wasps, ants and bees, despite dramatic variation within the group, where chromosome sets can harbor up to fifteen NORs. Taken together, the evidence points to a single rDNA site per haploid genome as the ancestral state for the entire order Hymenoptera, and, perhaps unsurprisingly, a similar pattern appears to characterize the class Insecta in general.

What makes this finding scientifically satisfying is the way it resolves a long-standing ambiguity with a modest but well-chosen sample. The number and location of NORs vary substantially across parasitoids and aculeate Hymenoptera, so reconstructing the ancestral condition for Apocrita alone had been far from straightforward. By adding data from the least advanced sawflies and from a gall wasp whose earlier chromomycin A3 results needed confirmation, the study anchors the ancestral state at the base of the order and shows that the widespread single-cluster pattern is not a coincidence of sampling but a genuine evolutionary signal. The confirmation that a single pericentromeric rDNA cluster per haploid karyotype is ancestral for Hymenoptera also aligns the order with the broader insect pattern, suggesting that the machinery of ribosomal RNA gene organization has remained remarkably stable across hundreds of millions of years of insect evolution, even as chromosome numbers, genome sizes and life histories have diversified spectacularly.

For the field of comparative cytogenetics, the update is a reminder that ancestral states are best reconstructed from the least derived taxa, and that techniques as different as FISH and chromomycin A3 staining can be combined to build a robust picture of chromosome architecture. The remaining gaps, most notably the unknown chromosomes of Blasticotomidae and Xyelidae, now stand out as the obvious next targets. Until those basal lineages are examined, the single rDNA cluster per haploid genome stands as the best-supported ancestral blueprint for the chromosomes of wasps, bees, ants and sawflies, a quiet but fundamental constant in one of the most species-rich orders of life on Earth.

Subject of Research: Comparative cytogenetics of ribosomal DNA clusters in Hymenoptera

Article Title: Number and location of rDNA clusters in the superfamilies Tenthredinoidea and Cynipoidea ( Hymenoptera ): an update

Article References: Gokhman, V. E., Kuznetsova, V. G., & Anokhin, B. A. (2024). Number and location of rDNA clusters in the superfamilies Tenthredinoidea and Cynipoidea (Hymenoptera): an update. Comparative Cytogenetics, 18, 239-246. https://doi.org/10.3897/compcytogen.18.142301

Image Credits: AI Generated

DOI: 10.3897/compcytogen.18.142301

Keywords: Hymenoptera, rDNA clusters, FISH, nucleolus organizing regions, Tenthredinoidea, Cynipoidea, comparative cytogenetics, sawflies, gall wasps, chromosome evolution, Number, location