Hidden Chromosomal Diversity Uncovered in a Midge Once Thought Genetically Uniform

The non-biting midge Chironomus agilis has long been regarded as one of the least chromosomally variable members of its species group, but a sweeping survey of natural populations across Eurasia tells a very different story. Researchers examined the giant polytene chromosomes of larvae collected from 19 populations spanning Eastern Europe, Siberia and the Russian Far East, and found that the species carries considerably more chromosomal polymorphism than earlier records suggested. The study, which revises the species’ banding sequence pool and adds three banding sequences never before described, shows that populations in Western Siberia in particular harbor a striking level of inversion diversity, while populations at the edges of the range remain almost entirely monomorphic.

Chironomus agilis belongs to the Ch. plumosus group of sibling species, a cluster of closely related midges that has become a classic model for studying how karyotypes evolve during speciation and how chromosomal polymorphism is maintained in natural populations. Before the new work, chromosomal polymorphism data existed for only two populations, one from the Rybinskoe reservoir in Eastern Europe and one from Berdsky pond in Siberia, in which a total of just nine banding sequences had been recorded. Two other published studies could not be relied upon because the karyotypes shown in their photographs, labeled as Ch. agilis, actually belong to different species. On that limited evidence, the species had been judged to have a low level of polymorphism. A later monograph brought the total number of known banding sequences to 13, but published no new quantitative data on their frequencies. The new survey changes that picture substantially.

The researchers prepared squashes of polytene chromosomes from sixth-instar larvae collected at 19 sites, ranging from the Rybinsk Reservoir in Yaroslavl region through a dense network of water bodies in the Novosibirsk region and the Altai territory, to the Amur River, Evoron Lake in Khabarovsk territory and Solyonoe Lake near Yakutsk. Sample sizes varied widely, from single larvae at some sites to 259 larvae collected across multiple years from a pond on the Shadrikha River. Larvae were fixed in a 3:1 mixture of 96% ethanol and glacial acetic acid and stored at minus 20 degrees Celsius, and chromosome squashes were prepared by a routine aceto-orcein method. When a site was sampled in several different years, the data were pooled after statistical testing showed no significant differences in banding sequence frequencies between probes.

Chironomus agilis belongs to the so-called thummi cytocomplex, with a haploid chromosome number of four and an arm combination of AB, CD, EF and G. Chromosomes I and II are metacentric, chromosome III is submetacentric and chromosome IV is telocentric. The species’ karyotype carries a distinctive signature: two nucleoli, both located on arm G, one at the centromeric end and the other near the telomere at the opposite end. This arrangement makes Ch. agilis easy to distinguish from other Chironomus species. The karyotype also contains three Balbiani rings; two sit on arm G, and the third, unlike in many related species, is usually well developed and easily identifiable on arm B. The researchers also presented revised mapping of the main banding sequences in arms A through F, including two alternative versions of the arm E map depending on whether the older mapping of the reference species Ch. plumosus by Keyl or the revised mapping by Golygina and Kiknadze is followed. In total, 16 banding sequences were present in the studied populations, and inversion polymorphisms were detected in every chromosome arm except G. Yet the distribution of that variation is highly uneven.

Arms B and F show a noticeably high level of polymorphism across populations, while arms A, C, D and E contain only rare or unique inversion sequences. Arm A was monomorphic everywhere except in a single larva from the Eltsovka River in Novosibirsk, which carried a heterozygous sequence differing from the standard by a short paracentric inversion. Arm C was monomorphic except for one larva from Travianoe Lake in the Altai territory carrying a sequence that, notably, is identical to the main banding sequence of the sibling species Ch. sp. prope agilis; the researchers present its mapping for the first time. Arm D harbors three sequences, two of which are rare or unique, and arm E was monomorphic except for one heterozygous larva from the Shadrikha River pond.

The most dramatic variation lies in arm B. The standard sequence p’agiB1 reaches high frequency only in the western part of the range, in the Rybinsk Reservoir population, where it is fixed. In every population studied from Siberia and the Far East, the alternative sequence p’agiB2, which differs from p’agiB1 by a large simple inversion, is dominant instead, reaching a frequency of 100 percent in the Evoron Lake population. A third sequence in this arm, p’agiB3, a short simple inversion in the middle of the arm derived from p’agiB2, was found only once, in the Shadrikha River population, and is described for the first time. Arm F likewise carries three sequences: the main sequence p’agiF1 occurs in all larvae from European and Far Eastern populations and dominates in Siberia, while p’agiF2, differing by a complex paracentric inversion on the distal part of the arm, appears in most Siberian populations mostly in heterozygous form, and a newly described p’agiF3 was found once in the Shadrikha River pond. Arm G was monomorphic in all populations and was not mapped, because its two nucleoli and two Balbiani rings make banding patterns difficult to compare.

Quantitatively, the geographic contrast is stark. Among populations suitable for analysis, the highest levels of chromosomal polymorphism were found in Siberia, in the Novosibirsk and Altai regions, where between 44.4 and 73.1 percent of larvae were heterozygous for inversions and the number of heterozygous inversions per larva ranged from 0.467 to 0.937. By contrast, the European population from the Rybinsk Reservoir and the Far Eastern populations were almost monomorphic, with heterozygote percentages of zero in the European sample and just 2.8 percent in the Evoron Lake sample. The Shadrikha River population was the richest of all, with 12 banding sequences and 14 genotypic combinations, while most other populations carried 9 to 10 sequences. Beyond inversions, the researchers also observed genomic polymorphism in the form of supernumerary B-chromosomes in two Siberian populations, from ponds on the Ora and Shadrikha rivers.

Tests against Hardy-Weinberg expectations revealed deviations in two Siberian populations, those from the Shadrikha and Koynikha river ponds, where heterozygotes in arm B exceeded expected values, with observed frequencies of the p’agiB1.2 genotype at 49.8 percent versus 41.1 percent expected in the former, and 65.6 percent versus 46.0 percent expected in the latter. The authors note that water quality data for the studied sites are not available, so the causes of these deviations cannot presently be assessed. Cytogenetic distances between populations, calculated by Nei’s criteria, ranged from zero to 0.155, well below the threshold of roughly 1 that is considered diagnostic of clearly differentiated chironomid species. The smallest distances connected Western Siberian populations, while the largest separated the border populations from the Rybinsk Reservoir and Evoron Lake. A neighbor-joining tree built from these distances reflects the same pattern: the European population belongs to cytogenetic type 0, in which all main banding sequences are dominant, whereas all Siberian and Far Eastern populations belong to type B, in which the alternative arm B sequence p’agiB2 predominates.

Compared with its better-studied relatives, Ch. agilis emerges as moderately polymorphic rather than depauperate. Palearctic populations of Ch. plumosus average 63.2 percent heterozygotes and 0.95 inversions per larva, against 45.1 percent and 0.52 for Ch. agilis, and the average number of banding sequences per population is 9.2 for Ch. agilis versus 12.5 for Ch. plumosus, 15.3 for Ch. entis and 14.3 for Ch. balatonicus. The pattern of arm-specific variation also resembles Ch. balatonicus, in which only two arms are highly polymorphic, although the affected arms differ between the two species: A and D in Ch. balatonicus but B and F in Ch. agilis, while in Ch. plumosus and Ch. entis inversions occur at high frequency in most or all arms. The authors caution that it is too early to draw final conclusions, since karyological data remain scarce for Europe, Eastern Siberia and the Far East. Still, the evidence points to Western Siberia as the center of the species’ range, where populations hold both higher levels and greater diversity of chromosomal polymorphism than those at the margins.

Subject of Research: Chromosomal polymorphism and banding sequence revision in natural populations of the midge Chironomus agilis

Article Title: Revision of the banding sequence pool and new data on chromosomal polymorphism in natural populations of Chironomus agilis Shobanov et Djomin, 1988 ( Diptera , Chironomidae )

Article References: Golygina, V. V., & Ermolaeva, O. V. (2021). Revision of the banding sequence pool and new data on chromosomal polymorphism in natural populations of Chironomus agilis Shobanov et Djomin, 1988 (Diptera, Chironomidae). Comparative Cytogenetics, 15(4), 527-541. https://doi.org/10.3897/compcytogen.v15.i4.76761

Image Credits: AI Generated

DOI: 10.3897/CompCytogen.v15.i4.76761

Keywords: Chironomus agilis, polytene chromosomes, chromosomal polymorphism, banding sequences, inversions, Chironomidae, karyotype, B-chromosomes, Siberia, population genetics, cytogenetics, Revision