Research Article |
Corresponding author: Viktor V. Bolshakov ( victorb@ibiw.ru ) Academic editor: Paraskeva Michailova
© 2022 Viktor V. Bolshakov, Ekaterina A. Movergoz.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Bolshakov VV, Movergoz EA (2022) Karyotype and COI gene sequences of Chironomus melanotus Keyl, 1961 from the Yaroslavl region, Russia, and the difficulties with its identification using GenBank and BOLD systems. Comparative Cytogenetics 16(3): 161-172. https://doi.org/10.3897/compcytogen.v16.i3.90336
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Karyotype and COI gene sequences of Chironomus melanotus Keyl, 1961 from the Yaroslavl region (Russia) were analyzed. A low level of chromosomal polymorphism has been confirmed, eventually eight banding sequences were found: melA1, melB1, melC1, melD1, melE1, melF1, and melG1; only melD2 was found in two larvae from the Sunoga river. Analysis of phylogenetic tree and estimated genetic distances has shown not all COI gene sequences of Ch. melanotus in GenBank and BOLD to belong to this species. The lower distance of 0.4% was observed between two sequences from the Yaroslavl region and Finland, apparently these are true Ch. melanotus sequences. The distances between true Ch. melanotus and other sequences from Finland were 9.5% and 12.4%, and from Sweden it was 11%. The average genetic distance between studied sequences of 9.1% is out of the range of the 3% threshold previously determined for chironomids. According to our estimates, there are two sequences with a distance of 2.9% that may belong to Ch. annularius Meigen, 1818, and one sequence with a genetic distance of 2.1%, may belonging to Ch. cingulatus Meigen, 1830, which has been confirmed karyologically. Another two sequences form a separate cluster. We suggest that they either belong to a known species, but are not present in the databases, or belong to a distinct, undescribed species.
Chironomidae, Chironomus melanotus, COI, dark taxa, DNA-barcode, karyotype
Chironomus melanotus Keyl, 1961 is one of the most widespread and well-known species. It does not belong to any sibling species group (
We could not find any studies of Ch. melanotus involving approaches of morphology, cytogenetics and DNA barcoding published in one article. In the GenBank and BOLD databases were found five and one COI gene sequences, respectively. These sequences were obtained from individuals collected in Finland and Sweden, and deposited during the preparation of this paper (
The present study aims to calculate and compare the genetic distances between COI gene sequences of Ch. melanotus from Yaroslavl region identified by morphology and cytogenetics and the sequences obtained from GenBank and BOLD of Ch. melanotus from different populations identified by morphology or molecular-genetics (barcode), and additional sequences from GenBank and BOLD of several Chironomus identified by cytogenetics.
Fourth instar larvae of Ch. melanotus were collected from a few places in the Yaroslavl region, Russia. Thirty-one larvae were found in a puddle on the Shumarovka river shore (58°02'25.5"N, 38°15'33.2"E) in October 2018. The depth is 0.5 m, and the bottom is black silt. Seven larvae were collected in the Sunoga river (58°03'20.3"N, 38°14'04.2"E) in August 2018. The depth is 0.1 – 0.2 m, and the bottom is gray silt with sand. Four larvae were collected in a small stream (brook) in the shore zone of the Kotorosl’ river (57°22'41.6"N, 39°50'08.5"E) in June 2016. The depth is 0.5 m, and the bottom is black silt and rotting wood.
The age was determined by the standard method (Ilyinskaya, 1983). All larvae were taken for karyotype analysis using the ethanol-orcein technique (
One larva from a small stream (brook) in the shore zone of Kotorosl river studied karyologically was taken for the total DNA extraction using “M-sorb-OOM” (Sintol, Moscow) kit with magnet particles according to manufacturer’s protocol. For amplification of COI (cytochrome oxidase subunit I) we used primers LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA -3’) (Eurogen, Moscow) (
For alignment of COI nucleotide sequences, we used MUSCLE in the MEGA6 software (
In addition, thirty-four COI gene sequences of the genus Chironomus from “GenBank” and “Barcode of Life Data Systems” (BOLD) were analyzed. Accession numbers of used sequences in GenBank and BOLD: Ch. acutiventris Wülker et al., 1983 (AF192200.1), Ch. annularius Meigen, 1818 (AF192189.1), Ch. anthracinus Zetterstedt, 1860 (KF278222), Ch. balatonicus Devai et al., 1983 (JN016826.1), Ch. bernensis Wülker et Klötzli, 1973 (AF192188.1), Ch. borokensis Kerkis et al., 1988 (AB740261), Ch. cingulatus Meigen, 1830 (AF192191.1), Ch. commutatus Keyl, 1960 (AF192187.1), Ch. curabilis et al., 1990 (JN016810.1), Ch. dilutus et al., 1999 (KF278335.1), Ch. entis Shobanov, 1989 (KM571024.1), Ch. heterodentatus Konstantinov, 1956 (AF192199.1), Ch. heteropilicornis Wülker, 1996 (MK795770.1), Ch. maturus Johannsen, 1908 (DQ648204.1), Ch. melanotus (MZ659620, MZ657748, MZ658877, MZ657558, MZ658420, BSCHI737-17), Ch. nipponensis Tokunaga, 1940 (DQ648206), Ch. novosibiricus Kiknadze et al., 1993 (AF192197.1), Ch. nuditarsis Keyl, 1961 (KY225345.1), Ch. obtusidens Goetghebuer, 1921 (CHMNO207-15*), Ch. piger Strenzke, 1959 (AF192202.1), Ch. pilicornis Fabricius, 1787 (HM860166.1), Ch. plumosus Linnaeus, 1758 (KF278217.1), Ch. riparius Meigen, 1804 (KR756187.1), Ch. sokolovae Istomina et al., 1999 (MW471100), Ch. sororius Wulker, 1973 (MZ324811), Ch. tentans Fabricius, 1805 (AF110157.1), Ch. tuvanicus Kiknadze et al., 1993 (AF192196.1), Ch. usenicus Loginova et Belyanina, 1994 (JN016820.1), Ch. whitseli Sublette et Sublette, 1974 (KR683438.1). The COI sequence of Drosophila melanogaster Meigen, 1830 (HQ551913) was used as outgroup in phylogenetic analysis.
The species has a 2n = 8 set of chromosomes. By the chromosome arm combination – AB, CD, EF and G, the species belongs to Chironomus “thummi” cytocomplex. The chromosomes AB and CD are metacentric, EF is submetacentric, and G is telocentric. The nucleus and Balbiani ring were found in arm G. The peculiarity of the karyotype of Ch. melanotus is a heterochromatinized centromeric region that forms an unstable chromocenter (Fig.
Karyotype of Chironomus melanotus from the Sunoga river, Yaroslavl, Russia. Arrows indicate centromeric bands, melA1, melB1 and etc. – genotypic combinations of banding sequences in chromosome arms, BR – Balbiani rings, N – nucleous.
We found two zygotic combinations: melA1.1. B1.1. C1.1. D1.1. E1.1. F1.1. G1.1, and melA1.1. B1.1. C1.1. D1.2. E1.1. F1.1. G1.1, which was found only in two larvae from the Sunoga river.
All eight banding sequences coincide with banding sequences in Keyl et al. (1961, 1962),
Arm A. One banding sequence: melA1 1a-2c 10a-12c 3i-2h 4d-9e 2g-d 4c-a 13a-19f C.
Arm B. One banding sequence: melB1 28-27-26-25-24-23-22-21-20-19 C (mapped according to Hirvenoja, Michailova 1991). Different from Ch. plumosus by four inversion steps.
Arm C. One banding sequence: melC1 1a-2e 15c-e 11c-8a 6b-2f 15b-11d 6gh 17a-16a 7d-a 6f-c 17b-22g C.
Arm D. Two banding sequences: melD1 1a-3g 11a-13a 16a-e 8a-10a 13b-15e 10e-b 4a-7g 17a-24g C in heterozygous state with melD2 1a-3g 11a-13a 16a-e 8a-9e 7g-4a 10b-e 15e-13b 10a 17a-24g C.
Arm E. One banding sequence: melE1 1a-3e 5a-10b 4h-3f 10c-13g C.
Arm F. One banding sequence: melF1 1a-8f 9a-11i 12a-15i 16a-23f C.
Arm G. One banding sequence: melG1. Not mapped.
The level of polymorphism in Ch. melanotus is known to be very low (
The obtained COI gene sequence for Ch. melanotus from the Yaroslavl region was deposited in the GenBank with accession number OL546775; the length of the sequence is 658 bp (percentage of nucleotides A: 25; T: 38; G: 17; C: 19).
More interesting was the analysis of COI gene sequences. As was said previously, for the species name Ch. melanotus in the databases match six sequences of the COI gene from Finland (MZ659620, MZ657748, MZ658877, MZ657558, MZ658420) identified by molecular-genetics and Sweden (BSCHI737-17) identified by imago characters, and the average genetic distance between them of 9.1% is out of the range of 3% distances previously determined for chironomids (
Pairwise genetic distances (p-distances, %) between COI gene sequences of Ch. melanotus and closest sequences of Chironomus from GenBank and BOLD.
Ch. melanotus OL546775 Yaroslavl, RUS | Ch. melanotus MZ659620 Finland | Ch. melanotus MZ657748 Finland | Ch. melanotus MZ658877 Finland | Ch. melanotus BSCHI737-17 Sweden | Ch. melanotus MZ657558 Finland | Ch. melanotus MZ658420 Finland | Ch. anthracinus KF278222 | Ch. annularius AF192189.1 | Ch. cingulatus AF192191.1 | |
---|---|---|---|---|---|---|---|---|---|---|
Ch. melanotus MZ659620 Finland | 0,4 | |||||||||
Ch. melanotus MZ657748 Finland | 9,5 | 9,7 | ||||||||
Ch. melanotus MZ658877 Finland | 9,5 | 9,7 | 0,0 | |||||||
Ch. melanotus BSCHI737-17 Sweden | 11,0 | 11,4 | 7,0 | 7,0 | ||||||
Ch. melanotus MZ657558 Finland | 12,4 | 12,6 | 10,7 | 10,7 | 11,2 | |||||
Ch. melanotus MZ658420 Finland | 12,4 | 12,6 | 10,7 | 10,7 | 11,2 | 0,4 | ||||
Ch. anthracinus KF278222 | 4,0 | 3,6 | 10,5 | 10,5 | 11,6 | 12,0 | 12,4 | |||
Ch. annularius AF192189.1 | 12,6 | 12,8 | 9,7 | 9,7 | 10,1 | 2,9 | 2,9 | 12,6 | ||
Ch. cingulatus AF192191.1 | 10,9 | 11,2 | 7,2 | 7,2 | 2,1 | 11,0 | 11,0 | 11,8 | 10,1 | |
Ch. nipponensis DQ648206 | 10,3 | 10,7 | 6,5 | 6,5 | 4,2 | 11,4 | 11,4 | 11,2 | 9,9 | 4,8 |
The distance between the two similar sequences (MZ657558 and MZ658420) from Finland and Ch. annularius (AF192189.1) confirmed karyologically (
On the phylogenetic tree constructed by Bayesian inference (Fig.
Bayesian tree of the analyzed samples of Chironomus spp. inferred from COI sequences. Species name, GenBank accession numbers and group name are shown to the right of the branches. Support values are given if they exceed 0.3. The numbers at the nodes indicate posterior probabilities.
All the obtained data show that several species are hidden in GenBank and BOLD under the name “Chironomus melanotus”. First, there is a true Ch. melanotus cluster (Fig.
Two similar sequences (MZ657748 and MZ658877) need special attention. The samples of Ch. melanotus from Finland were investigated during the project of FinBOL (Finnish Barcode Of Life), in the framework of which the authors tested the system FinPROTAX (Probabilistic Taxonomic Assignment Tool) (
On the example of Ch. melanotus, we confirmed that in Chironomus species identification we must use all available comprehensive approaches, involving morphological, cytogenetic and molecular genetic studies (mitochondrial and nuclear genes) (
At least four species of Chironomus could be in the databases under the name “Ch. melanotus” from Finland and Sweden. This suggests that at the present stage of the collection of genetic data, it is impossible to trust only a computer algorithm. We agree with
The work was realized according to the Russia state projects 121050500046-8 and 121051100099-5. The authors are grateful to B.A. Levin, A.A. Bobrov (IBIW RAS) and M.Kh. Karmokov (IEMT RAS) for their help and consultations during all stages of the investigation and manuscript preparation; P. Michailova (IBER BAS), J. Martin (The University of Melbourne, Australia) and V.G. Kuznetsova (ZIN RAS) for the opportunity to see the work in a new light; D.D. Pavlov (IBIW RAS) for the linguistic corrections of the text.
Viktor V. Bolshakov https://orcid.org/0000-0002-8028-3818
Ekaterina A. Movergoz https://orcid.org/0000-0002-3677-6631