The larvae were collected on 28 June 2022 from a small, nearly circular lake about 1.5 kilometers west of the settlement of Kurush in the Dokuzparinsky district. The lake reaches a maximum depth of about 3 meters, and its water carries roughly 310 parts per million of dissolved minerals. Fourth-instar larvae were preserved for two parallel investigations: ten individuals were slide-mounted in Fora-Berlese solution and deposited at the Tembotov Institute of Ecology of Mountain Territories in Nalchik, while 24 larvae were fixed in ethanol-glacial acetic acid for karyological work. Twelve of those karyologically studied larvae were then used for DNA extraction.

The morphological evidence pointed clearly to Ch. degelenus. The larvae are red, 14 to 18 millimeters long, and belong to the plumosus-type, bearing lateral tubuli on the seventh abdominal segment and two pairs of strongly twisted ventral tubuli on the eighth that extend beyond the posterior parapods. The head capsule is yellow with a darkened occipital sclerite, and the height of the fourth lateral tooth of the mentum roughly equals that of the fifth. Only one small difference emerged: in the original Kazakh description, the antennal blade does not reach the fifth antennal segment, whereas in the Caucasian population it extends beyond it. Everything else matched the 2001 diagnosis.

The karyotype told the same story with even greater precision. Chironomus degelenus has a diploid chromosome number of eight, with arm combinations AB, CD, EF, and G that place it firmly in the so-called thummi cytocomplex. Arm AB is metacentric, CD and EF are submetacentric, and G is telocentric, with prominent centromeric bands. Two nucleoli are visible, one at the centromeric end of arm G and another in the middle of arm B, and two Balbiani rings develop on arm G. The Caucasian population carries eight banding sequences in total: all seven previously described by Seisebaev and colleagues, plus one entirely new sequence, degC2, discovered for the first time in this study.

Chromosomal polymorphism proved remarkably low. Only arm C showed inversion variation, with the new sequence degC2 differing from degC1 by a simple inversion in the region 8a-11c. The sequence degC2 occurred at a frequency of 0.708 and the homozygous combination degC2.2 at 0.542, making it the dominant arrangement, while degC1 appeared at just 0.292. About a third of the larvae, 33.3 percent, were heterozygous for the inversion, and the average number of heterozygous inversions per larva was only 0.333. Arms A, B, D, E, F, and G were all monomorphic in the sample.

Then came the surprise that elevates the study beyond a regional record. When the team ran their newly obtained COI sequences through the BLAST algorithm against the NCBI database, the closest match was not any Palaearctic species but Chironomus atrella, a Nearctic midge described in 1945 and found predominantly in North America. A comparison of polytene chromosome banding patterns, using photomaps of Ch. atrella published by Martin and colleagues in 2006, revealed that the banding sequences of the two species are almost identical in every arm except G. In arm A, the sequence degA1.1 is completely identical to atrA1.1 except for a small feature near the centromere, where Ch. atrella carries a region of at least three pale bands between the band group 19ef and the thick centromeric band K. That pale region is present in Kazakh Ch. degelenus but absent in the Caucasian samples. In arm C, degC1 is identical to atrC2 and degC2 is identical to atrC1. Arms D, E, and F show equally striking matches, and arm E is the only one where the chromosome mappings of the two species correspond directly.

On the strength of this cytological kinship, the authors propose including Ch. degelenus in the Ch. atrella group of closely related species. Yet the DNA data show the two are unmistakably distinct. The team obtained eight COI sequences from the 12 Caucasian larvae, representing six haplotypes, and deposited them in GenBank under accession numbers OQ873429 through OQ873436. These are the first DNA data ever generated for the species, which had been absent from both GenBank and BOLD. Phylogenetic analysis of 51 nucleotide sequences, run in BEAST with a GTR+G+I substitution model, a strict clock, and the Yule speciation process, placed the Caucasian Ch. degelenus sequences in their own cluster, with the nearest neighbor being a cluster of Ch. atrella sequences from the first Barcode Index Number, or BIN, in BOLD.

The genetic distances expose a deeper problem. Within the Ch. degelenus cluster, K2P distances range from 0 to 1.65 percent, averaging 0.9 percent, and within the two Ch. atrella BIN clusters they are even smaller, at most 0.36 and 0.55 percent. But between clusters the distances leap far above the 3 percent threshold that earlier work on Canadian Chironomus suggested as a rough species boundary. The distance between the Ch. degelenus cluster and the first Ch. atrella BIN is 5.1 percent; between Ch. degelenus and the second BIN it is 6.6 percent; and between the two Ch. atrella BINs themselves it is 6.2 percent. In other words, the name Ch. atrella has been applied to two genetically separate entities that last shared a common ancestor millions of years ago. The authors note that Martin had already observed in 2006 that the name has been ascribed to two cytologically distinct species, one from his own work and one reported by Hitchcock and Anderson in 1968 from a tidal cove in Old Saybrook, Connecticut.

This confusion is a textbook case of what biologists call dark taxa: genetic sequences in public databases like GenBank and BOLD that come from unknown or taxonomically challenging species. Almost all of the 559 Ch. atrella COI sequences in BOLD were identified by the automated BOLD Identification System, without karyological verification. The scale of the problem is sobering; one 2019 estimate found that about 65 percent of German Chironomidae sequences in BOLD lack species-level assignment. For specialists working on Chironomus, where correct larval identification by karyotype must precede any DNA study, dark taxa can quietly poison downstream analyses, and the two-entity problem within Ch. atrella is a vivid demonstration of the cost.

The study also reaches back into deep time for an explanation. Using molecular clock calibrations anchored to a 36-million-year split between the outgroup Pagastiella orophila and all Chironomus, and to a 5.75 to 3.43 million-year divergence within the Ch. plumosus group, the authors estimate that the split between the Ch. degelenus cluster and the Ch. atrella clusters occurred between 3.72 and 2.67 million years ago, within the Piacenzian stage of the Pliocene. The common ancestor of Ch. degelenus and the first Ch. atrella BIN lived between 2.05 and 1.47 million years ago, in the Calabrian Pleistocene, while the Ch. degelenus cluster itself coalesced only 0.504 to 0.356 million years ago. The authors propose that the Pliocene spread of the first true grasslands, which replaced retreating forests and scattered stagnant waterbodies across newly open landscapes, created a mosaic of isolated aquatic habitats that drove populations apart. Because chironomids are poor fliers whose active flight is limited to less than about 10 kilometers, wind currents sweeping across those open grasslands may have carried their ancestors, likely native to the Nearctic, across the Beringia land bridge into the Palaearctic, where the relatively young Ch. degelenus lineage eventually arrived in the mountains of Kazakhstan and, as now revealed, the Eastern Caucasus.

Subject of Research: Karyotype and COI barcoding of the non-biting midge Chironomus degelenus and its relationship with the Nearctic species Chironomus atrella

Article Title: Karyotype characteristics and COI gene data of Chironomus degelenus Seisebaev, Bakhtin & Siirin, 2001 ( Diptera , Chironomidae ) from the Eastern Caucasus (Dagestan) and its relationship with the Nearctic species Chironomus atrella (Townes, 1945)

Article References: Karmokov, M., & Akkizov, A. (2025). Karyotype characteristics and COI gene data of Chironomus degelenus Seisebaev, Bakhtin & Siirin, 2001 (Diptera, Chironomidae) from the Eastern Caucasus (Dagestan) and its relationship with the Nearctic species Chironomus atrella (Townes, 1945). Caucasiana, 4, 173-194. https://doi.org/10.3897/caucasiana.4.e165329

Image Credits: AI Generated

DOI: 10.3897/caucasiana.4.e165329

Keywords: Chironomus degelenus, Chironomus atrella, Chironomidae, karyotype, polytene chromosomes, COI barcode, dark taxa, Dagestan, Pliocene, biogeography, Beringia, inversion polymorphism