Research report: Karyotype characteristics and gene COI sequences of Chironomus bonus Shilova et Dzhvarsheishvili, 1974 ( Diptera , Chironomidae ) from the South Caucasus (Republic of Georgia, Paravani river)

A population of the non-biting midge Chironomus bonus collected beside a branch of Georgia’s Paravani River has provided the first mitochondrial COI sequences for the species, alongside a detailed chromosomal description. The researchers examined fourth-instar larvae collected on 18 July 2017 in the Ninotsminda district of Samtskhe-Javakheti, about 0.6 kilometres north of Saghamo at an altitude of approximately 2,000 metres. The site was a river reach with a maximum depth of about 1 metre and water salinity of about 40 ppm.

Chironomus bonus was first described from Paravani Lake in 1974 and has also been recorded in Armenia, France, Switzerland and Bulgaria, but neither GenBank nor BOLD previously contained DNA data for it. The study combined chromosome analysis with mitochondrial DNA sequencing to characterize the Georgian specimens and compare them with other members of the Chironomus plumosus species group. Six larvae were examined cytologically.

Following fixation in an ethanol–glacial acetic acid solution, chromosome preparations were made with the ethanol-orcein technique and examined under a Carl Zeiss Axio Imager A2 microscope. Banding sequences were identified for the chromosome arms using established photomaps and mapping systems, allowing the authors to compare the Georgian material directly with previously published karyotypes. All six larvae had the same basic chromosome complement: a diploid number of 2n = 8 plus an additional B chromosome.

Their chromosomes formed the AB, CD, EF and G arm combinations associated with the thummi cytocomplex. The AB and CD chromosomes were metacentric, EF was submetacentric and G was telocentric. Clearly identifiable centromeric bands, one nucleolus, two Balbiani rings on the G arm and a further Balbiani ring on arm B completed the observed profile.

The principal visible distinction from C. plumosus was the additional B chromosome, which was present in almost all examined C. bonus larvae. Chromosome examination was central to species identification because the C. plumosus group comprises 14 recognized species that can be similar in external form and often occur together in the same body of water. The source review describes karyological analysis as the reliable method for identifying species in the group.

DNA extraction was therefore performed on four larvae that had already been studied by chromosome analysis. Their DNA was extracted with a commercial preparation kit, and the mitochondrial COI barcoding region was amplified with primers 911 and 912 in 25-µL PCR reactions. Products were purified, checked by agarose gel electrophoresis and sequenced in both directions by Sanger sequencing.

The analysis produced three complete COI sequences, deposited in GenBank as MZ014021, MZ014022 and MZ014023. Their lengths were 627, 658 and 650 base pairs, respectively, and each represented a different haplotype within the small Georgian sample. For phylogenetic comparison, the researchers assembled 61 nucleotide sequences from GenBank and BOLD, including members of the C. plumosus group and the outgroup Pagastiella orophila.

The alignment was performed in MUSCLE with the invertebrate mitochondrial genetic code in MEGA 6, leaving 579 positions after gaps and missing data were removed. Pairwise evolutionary distances were calculated with the Kimura two-parameter model. Bayesian phylogenetic analysis was performed in BEAST v1.10.4 using the HKY+G substitution model, a strict clock and a Yule speciation process.

The resulting tree contained several strongly supported but internally complex groupings. The three Georgian C. bonus sequences joined C. plumosus sequences from western and eastern Europe in a Palearctic cluster that also included available C. usenicus sequences from Russia. A separate Far Eastern cluster combined C. borokensis with sequences labelled C. plumosus from China, South Korea and Japan, while a Nearctic cluster mixed C. entis and C. plumosus sequences from North America.

The C. bonus sequences did not form an independent branch in this analysis. Genetic distances were low within the three principal clusters but higher between them. Within the Palearctic cluster, Kimura two-parameter distances ranged from 0 to 2.595%; excluding an Iranian C. plumosus sequence reduced the range to 0–0.914%.

The three C. bonus sequences differed from one another by 0.182–0.364%. The Nearctic C. entis–C. plumosus cluster contained distances of 0–1.285%, while the Far Eastern C. borokensis–C. suwai cluster ranged from 0 to 2.217%, or 0–1.839% when one Chinese sequence was omitted. Average distances between clusters were 3.55% for the Palearctic and Far Eastern groups, 3.75% for the Palearctic and Nearctic groups, and 5.98% for the Far Eastern and Nearctic groups.

The authors compared those between-cluster values with a reference range of up to 3% for intraspecific COI distances in Chironomus, while emphasizing that COI results require support from other forms of identification. The 3.75% separation between Palearctic and Nearctic sequences labelled C. plumosus was interpreted as evidence that Nearctic populations assigned to that name may represent a separate species. The authors also cautioned that several database sequences had been identified through morphology alone.

Their clustering patterns suggested that some records labelled C. plumosus in the Far East or North America could belong to C. borokensis, C. suwai or C. entis, although the study did not directly resolve the identity of every database specimen. Comparison of the Georgian sequences with other members of the Palearctic cluster identified six substitutions that could distinguish C. bonus. Four were nonsynonymous and two were synonymous.

A substitution at position 340 was present in all three Georgian C. bonus sequences and was the only change found in the complete dataset that clearly distinguished them from the other species. The remaining substitutions occurred in individual sequences and were also detected elsewhere in the comparison. The authors discussed two possible explanations for the close mitochondrial similarity between C. bonus and European C. plumosus: a relatively recent separation with few accumulated differences, or an older separation followed by mitochondrial introgression through interspecific hybridisation.

The available results did not distinguish between those scenarios. Model-based divergence estimates depended on assumed mitochondrial substitution rates of 1.5%, 2.3% or 3.54% per million years. The earliest split among the three principal clusters was estimated at 5.75–3.43 million years ago, placing it in the Late Miocene to early Pliocene.

The common ancestor of all three clusters was dated to 2.88–1.72 million years ago. Estimated separation between the Palearctic and Far Eastern clusters was 1.97–1.17 million years, while common ancestors for the Palearctic, Far Eastern and Nearctic clusters were estimated at 1.288–0.759, 0.906–0.539 and 0.638–0.378 million years ago, respectively. The Palearctic cluster excluding the Iranian sequence had an estimated common ancestor at 0.517–0.307 million years ago.

The authors proposed that diversification within the group may have been associated with the expansion of separate stagnant-water habitats during the Pliocene. Lakes and ponds can differ in depth, temperature, mineralisation, size and other environmental conditions, potentially producing differences in breeding timing and reproductive isolation. This remains an evolutionary interpretation rather than a directly observed outcome of the Georgian study.

The timing estimates were also provisional because COI data were available for only seven of the 14 species in the C. plumosus group, and the analysis contained ambiguous clusters. Nevertheless, the study established a distinctive karyological identity for the Georgian C. bonus population and supplied the species’ first COI reference sequences, showing why chromosome and mitochondrial evidence were evaluated together rather than treating either marker as sufficient on its own.

Subject of Research: Karyotypic and mitochondrial COI characterization of Chironomus bonus from the South Caucasus

Article Title: Karyotype characteristics and gene COI sequences of Chironomus bonus Shilova et Dzhvarsheishvili, 1974 ( Diptera , Chironomidae ) from the South Caucasus (Republic of Georgia, Paravani river)

Article References: Karmokov, M. K. (2022). Karyotype characteristics and gene COI sequences of Chironomus bonus Shilova et Dzhvarsheishvili, 1974 (Diptera, Chironomidae) from the South Caucasus (Republic of Georgia, Paravani river). Comparative Cytogenetics, 16(1), 19-38. https://doi.org/10.3897/compcytogen.v16.i1.79182

Image Credits: AI Generated

DOI: 10.3897/CompCytogen.v16.i1.79182

Keywords: Chironomus bonus, Chironomus plumosus group, mitochondrial COI, karyotype, B chromosome, South Caucasus, Phylogeography, Mitochondrial divergence, Non-biting midges, characteristics, gene, sequences