Research Article |
Corresponding author: Mukhamed Kh. Karmokov ( lacedemon@rambler.ru ) Academic editor: Vladimir Gokhman
© 2022 Mukhamed Kh. Karmokov.
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:
Karmokov MK (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
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The study presents data on the karyotype characteristics and the mitochondrial gene COI sequences of the non-biting midge Chironomus bonus Shilova et sDzhvarsheishvili 1974 (Diptera Chironomidae) from the South Caucasus. The species belongs to the Ch. plumosus group of sibling species one of the most widespread and successful groups in the genus Chironomus Meigen 1803. The karyotype of the studied population is monomorphic. The morphological and chromosomal characteristics of Ch. bonus from the Caucasus are similar to those previously described for this species (Kiknadze et al. 1991a). In the phylogenetic tree based on the COI gene sequences one can observe several clear clusters. We named them Palearctic Ch. plumosus Far Eastern Ch. borokensis-Ch. suwai and Nearctic Ch. entis-Ch. plumosus clusters. The calculated K2P genetic distances within each cluster have not exceeded the 3% threshold for the genus Chironomus. Contrary to this the distances between the clusters exceed this range and correspond to separate species. The Ch. bonus sequences belong to the cluster consisting of Ch. plumosus (Linnaeus 1758) sequences from European populations and do not form a separate clade of the phylogenetic tree. One can suppose that the origin of the Ch. plumosus group of sibling species dates back to 5.75–3.43 million years ago (Mya) the epochs of Late Miocene (7,3–5,3 Mya) and early Pliocene (5,3–2,58 Mya). On the other hand Palearctic Ch. plumosus Far Eastern Ch. borokensis-Ch. suwai and Nearctic Ch. entis-Ch. plumosus clusters appeared relatively recently in the Middle Pleistocene 1.288–0.307 Mya. The possible relationship between the climate changes in the Pliocene and the origin of the Ch. plumosus group are discussed. Chironomidae
Chironomus bonus, COI gene, Diptera, mitochondrial DNA, phylogeny, polytene chromosomes, South Caucasus
Shilova and Dzhvarsheishvili first described Chironomus bonus Shilova et Dzhvarsheishvili, 1974 from Paravani Lake in the Republic of Georgia (
The species Ch. bonus belongs to the Ch. plumosus group of sibling species, one of the most widespread and successful groups in the genus Chironomus Meigen, 1803. According to
According to
The majority of the species in the Ch. plumosus group have a Palearctic distribution. Only two of them, Ch. plumosus and Ch. entis, are also found in the Nearctic, and they can therefore be considered as Holarctic species (
The karyotype of Ch. bonus has been described by
The aim of the work is to present the description of the karyotype and gene COI sequences of Ch. bonus from the South Caucasus, as well as to compare the karyotype characteristics and DNA data of Ch. bonus with the corresponding information available for other species of the Ch. plumosus group.
For both DNA and karyological studies, we used fourth-instar larvae of Ch. bonus. We collected larvae from a particular site in the Republic of Georgia (South Caucasus): 18.07.17, 41°19.305'N, 43°45.563'E, Ninotsminda district in the region of Samtskhe-Javakheti, one of the branches of the Paravani river, just 0.6 km north of Saghamo settlement, altitude of ca. 2000 m a.s.l. The maximum depth of the river is about 1 m, and the salinity of the water is about 40 ppm. The collection site is marked on the map with a dark circle (Fig.
Collection site of Ch. bonus in South Caucasus. The collection site is marked with dark circle.
The head capsules and bodies of six larvae were slide-mounted in Faure-Berlese medium. The specimens have been deposited at the Tembotov Institute of Ecology of the Mountain Territories RAS in Nalchik, Russia. We studied the karyotype of all six larvae from the Caucasus region.
For karyological study, we fixed the larvae in an ethanol-glacial acetic acid solution (3:1). The preparations of the chromosomes were made using the ethanol-orcein technique (see Dyomin and Ilyinskaya 1988;
We performed the identification of chromosome banding sequences for arms A, E, and F using photomaps by
We used four karyologically studied larvae of Ch. bonus for further DNA extraction. DNA was extracted from the larvae and preserved in 96% ethanol using a Diatom DNA Prep 100 kit (Izogen Laboratory Ltd, Moscow, Russia) according to the manufacturer’s protocol. DNA extraction was performed on vacuum-dried samples without prior homogenization. Samples were incubated in a lysis buffer at a temperature of 55.5 °C for 16 h. After the extraction, the head capsules were retrieved for dry mounting. The resulting DNA solutions were stored at -18 °C. The amplification of the mitochondrial COI gene was conducted using the MasterMix X5 kit (Dialat Ltd, Moscow).
To amplify the mitochondrial COI gene’s barcoding region, primers 911 (5´-TTTCTACAAATCATAAAGATATTGG-3´) and 912 (5´- TAAACTTCAGGGTGACCAAAAAATCA-3´) (
Purified PCR products were sequenced in both directions. DNA sequencing of the COI gene was performed according to Sanger using the BigDye Terminator v3.1 commercial kit (ThermoFisher) and the ABI 3130×l genetic analyzer (ThermoFisher) at Syntol JSC (Moscow, Russia). The GenBank accession numbers of the three sequences obtained in this study are MZ014021, MZ014022, and MZ014023.
For the phylogenetic comparison, we used DNA data (sixty-one COI gene sequences) from both the GenBank and BOLD (
We found some COI gene data in both the GenBank and BOLD databases only for seven species of the Ch. plumosus group out of 14. We used in our study COI gene sequences from both the aforementioned databases for Ch. balatonicus, Ch. muratensis, Chironomus prope agilis, Ch. borokensis, Ch. usenicus, Ch. entis, and Ch. plumosus, with available data for species with Holarctic and Nearctic distributions. The most abundant data on the COI gene are available for Ch. plumosus (GenBank and BOLD – 66 and 138 sequences, respectively) and Ch. entis (GenBank and BOLD – 339 and 13 sequences, respectively). DNA sequences of Ch. plumosus obtained from material collected from both Western and Eastern Europe, the Middle East, the Far East, and Northern America were included into the analysis. Concerning Ch. entis, available DNA sequences are more uniform and were obtained from material collected almost exclusively from Northern America (Canada). In cases when a large number of sequences were available from the same region, we used no more than 5–6 sequences with different haplotypes to avoid overloading the phylogenetic tree.
We conducted the alignment of COI sequences with MUSCLE with a genetic code of “invertebrate mitochondrial” packaged in MEGA 6 (
Estimates of evolutionary divergence between sequences of Palearctic Ch. plumosus cluster. The number of base substitutions per site (%) from between sequences are shown. Analyses were conducted using the Kimura 2-parameter model (
№ | Sequences | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | JN016807.1 Ch. usenicus Russia, Saratov terr. | 0 | ||||||||||||||||||||||||
2 | JN016809.1 Ch. usenicus Russia, Saratov terr. | 0.364 | 0 | |||||||||||||||||||||||
3 | JN016808.1 Ch. usenicus Russia, Saratov terr. | 0.182 | 0.182 | 0 | ||||||||||||||||||||||
4 | MZ014023 Ch. bonus Georgia, S. Caucasus | 0.547 | 0.547 | 0.364 | 0 | |||||||||||||||||||||
5 | MZ014022 Ch. bonus Georgia, S. Caucasus | 0.364 | 0.364 | 0.182 | 0.182 | 0 | ||||||||||||||||||||
6 | MZ014021 Ch. bonus Georgia, S. Caucasus | 0.547 | 0.547 | 0.364 | 0.364 | 0.182 | 0 | |||||||||||||||||||
7 | JN016830.1 Ch. plumosus Russia, Saratov Terr. | 0.547 | 0.547 | 0.364 | 0.730 | 0.547 | 0.731 | 0 | ||||||||||||||||||
8 | JN016829.1 Ch. plumosus Russia Saratov Terr. | 0.182 | 0.182 | 0 | 0.364 | 0.182 | 0.364 | 0.364 | 0 | |||||||||||||||||
9 | AB740263.1 Ch. plumosus Russia | 0.547 | 0.547 | 0.364 | 0.730 | 0.547 | 0.730 | 0.730 | 0.364 | 0 | ||||||||||||||||
10 | AB740262.1 Ch. plumosus Russia | 0.182 | 0.182 | 0 | 0.364 | 0.182 | 0.364 | 0.364 | 0 | 0.364 | 0 | |||||||||||||||
11 | CHBAL014-20 Ch. plumosus Montenegro | 0.364 | 0.364 | 0.182 | 0.547 | 0.364 | 0.547 | 0.182 | 0.182 | 0.547 | 0.182 | 0 | ||||||||||||||
12 | PGBAL006-19 Ch. plumosus Montenegro | 0.364 | 0.364 | 0.182 | 0.547 | 0.364 | 0.547 | 0.182 | 0.182 | 0.547 | 0.182 | 0 | 0 | |||||||||||||
13 | PGBAL007-19 Ch. plumosus Montenegro | 0.547 | 0.547 | 0.364 | 0.730 | 0.547 | 0.731 | 0.364 | 0.364 | 0.730 | 0.364 | 0.182 | 0.182 | 0 | ||||||||||||
14 | PGBAL009-19 Ch. plumosus Montenegro | 0.547 | 0.547 | 0.364 | 0.730 | 0.547 | 0.731 | 0.364 | 0.364 | 0.730 | 0.364 | 0.182 | 0.182 | 0 | 0 | |||||||||||
15 | PGCBG089-20 Ch. plumosus Montenegro | 0.364 | 0.364 | 0.182 | 0.547 | 0.364 | 0.547 | 0.182 | 0.182 | 0.547 | 0.182 | 0 | 0 | 0.182 | 0.182 | 0 | ||||||||||
16 | BSCHI661-17 Ch. plumosus Poland | 0.547 | 0.547 | 0.364 | 0.730 | 0.547 | 0.731 | 0.731 | 0.364 | 0.730 | 0.364 | 0.547 | 0.547 | 0.731 | 0.731 | 0.547 | 0 | |||||||||
17 | BSCHI063-11 Ch. plumosus Sweden | 0.731 | 0.731 | 0.547 | 0.914 | 0.731 | 0.916 | 0.916 | 0.547 | 0.914 | 0.547 | 0.731 | 0.731 | 0.916 | 0.916 | 0.731 | 0.182 | 0 | ||||||||
18 | BSCHI115-17 Ch. plumosus Sweden | 0.364 | 0.364 | 0.182 | 0.547 | 0.364 | 0.547 | 0.547 | 0.182 | 0.547 | 0.182 | 0.364 | 0.364 | 0.547 | 0.547 | 0.364 | 0.547 | 0.731 | 0 | |||||||
19 | BSCHI219-17 Ch. plumosus Sweden | 0.182 | 0.182 | 0 | 0.364 | 0.182 | 0.364 | 0.364 | 0 | 0.364 | 0 | 0.182 | 0.182 | 0.364 | 0.364 | 0.182 | 0.364 | 0.547 | 0.182 | 0 | ||||||
20 | BSCHI284-17 Ch. plumosus Sweden | 0.182 | 0.182 | 0 | 0.364 | 0.182 | 0.364 | 0.364 | 0 | 0.364 | 0 | 0.182 | 0.182 | 0.364 | 0.364 | 0.182 | 0.364 | 0.547 | 0.182 | 0 | 0 | |||||
21 | BSCHI350-17 Ch. plumosus Sweden | 0.364 | 0.364 | 0.182 | 0.547 | 0.364 | 0.547 | 0.547 | 0.182 | 0.547 | 0.182 | 0.364 | 0.364 | 0.547 | 0.547 | 0.364 | 0.547 | 0.731 | 0 | 0.182 | 0.182 | 0 | ||||
22 | BSCHI517-17 Ch. plumosus Sweden | 0.547 | 0.547 | 0.364 | 0.730 | 0.547 | 0.731 | 0.731 | 0.364 | 0.730 | 0.364 | 0.547 | 0.547 | 0.731 | 0.731 | 0.547 | 0 | 0.182 | 0.547 | 0.364 | 0.364 | 0.547 | 0 | |||
23 | BSCHI644-17 Ch. plumosus Sweden | 0.182 | 0.182 | 0 | 0.364 | 0.182 | 0.364 | 0.364 | 0 | 0.364 | 0 | 0.182 | 0.182 | 0.364 | 0.364 | 0.182 | 0.364 | 0.547 | 0.182 | 0 | 0 | 0.182 | 0.364 | 0 | ||
24 | GBDP44143-19 Ch. plumosus UK | 0.547 | 0.547 | 0.364 | 0.730 | 0.547 | 0.731 | 0.731 | 0.364 | 0.730 | 0.364 | 0.547 | 0.547 | 0.731 | 0.731 | 0.547 | 0 | 0.182 | 0.547 | 0.364 | 0.364 | 0.547 | 0 | 0.364 | 0 | |
25 | GBDP44180-19 Ch. plumosus Iran | 2.028 | 2.406 | 2.217 | 2.592 | 2.406 | 2.595 | 2.217 | 2.217 | 2.219 | 2.217 | 2.028 | 2.028 | 2.217 | 2.217 | 2.028 | 2.217 | 2.406 | 2.406 | 2.217 | 2.217 | 2.406 | 2.217 | 2.217 | 2.217 | 0 |
We conducted the estimation of phylogenetic relationships in BEAST V1.10.4 (
Estimates of evolutionary divergence between sequences of Nearctic Ch. entis-Ch. plumosus cluster. The number of base substitutions per site (%) from between sequences are shown. Analyses were conducted using the Kimura 2-parameter model (
№ | Sequences | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | GBDPC429-14 Ch. entis US, Michigan | 0 | ||||||||||||
2 | KF278213.1 Ch. entis Canada, Quebec | 0.914 | 0 | |||||||||||
3 | KF278212.1 Ch. entis Canada, Quebec | 1.099 | 0.182 | 0 | ||||||||||
4 | MGOCF102-16 Ch. entis US, New York | 0.914 | 0 | 0.182 | 0 | |||||||||
5 | KJ085531.1 Ch. entis Canada, Ontario | 0.547 | 0.730 | 0.914 | 0.730 | 0 | ||||||||
6 | KJ087284.1 Ch. entis Canada, Ontario | 0.547 | 0.730 | 0.914 | 0.730 | 0 | 0 | |||||||
7 | KJ089893.1 Ch. entis Canada, Ontario | 0.547 | 0.730 | 0.914 | 0.730 | 0 | 0 | 0 | ||||||
8 | GBDPC430-14 Ch. plumosus US, Michigan | 0.730 | 0.730 | 0.913 | 0.730 | 0.547 | 0.547 | 0.547 | 0 | |||||
9 | SDP408034-15 Ch. plumosus US, Minnisota | 0.730 | 0.730 | 0.913 | 0.730 | 0.547 | 0.547 | 0.547 | 0 | 0 | ||||
10 | GBDPC133-14 Ch. plumosus Canada | 1.285 | 0.364 | 0.547 | 0.364 | 1.099 | 1.099 | 1.099 | 1.098 | 1.098 | 0 | |||
11 | GBDPC138-14 Ch. plumosus Canada | 1.285 | 0.364 | 0.547 | 0.364 | 1.099 | 1.099 | 1.099 | 1.098 | 1.098 | 0 | 0 | ||
12 | GBDPC144-14 Ch. plumosus Canada | 0.730 | 0.730 | 0.913 | 0.730 | 0.547 | 0.547 | 0.547 | 0 | 0 | 1.098 | 1.098 | 0 | |
13 | GBDPC166-14 Ch. plumosus Canada | 1.099 | 0.547 | 0.731 | 0.547 | 0.914 | 0.914 | 0.914 | 1.098 | 1.098 | 0.916 | 0.916 | 1.098 | 0 |
We also tried to get average estimates of divergence time between different branches and clusters that appear on the obtained phylogenetic tree (Figs
Estimates of evolutionary divergence between sequences of Far Eastern Ch. borokensis-Ch. suwai cluster. The number of base substitutions per site (%) from between sequences are shown. Analyses were conducted using the Kimura 2-parameter model (
№ | Sequences | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | AB740261.1 Ch. borokensis Russia | 0 | |||||||||||||
2 | GBDP17582-15 Ch. plumosus Japan | 1.283 | 0 | ||||||||||||
3 | GBDP17583-15 Ch. plumosus Japan | 1.468 | 0.547 | 0 | |||||||||||
4 | JCDB364-15 Ch. plumosus Japan | 1.469 | 0.182 | 0.364 | 0 | ||||||||||
5 | JCDB363-15 Ch. plumosus Japan | 1.469 | 0.182 | 0.364 | 0.000 | 0 | |||||||||
6 | GBDP11685-12 Ch. plumosus South Korea | 0.364 | 1.654 | 1.839 | 1.841 | 1.841 | 0 | ||||||||
7 | GBDP11686-12 Ch. plumosus South Korea | 0.182 | 1.469 | 1.654 | 1.656 | 1.656 | 0.182 | 0 | |||||||
8 | GBDP11687-12 Ch. plumosus South Korea | 0.182 | 1.469 | 1.654 | 1.656 | 1.656 | 0.182 | 0 | 0 | ||||||
9 | GBDP12282-12 Ch. plumosus South Korea | 0 | 1.283 | 1.468 | 1.469 | 1.469 | 0.364 | 0.182 | 0.182 | 0 | |||||
10 | XJDQD1039-18 Ch. plumosus China | 1.468 | 0.913 | 1.467 | 1.097 | 1.097 | 1.839 | 1.654 | 1.654 | 1.468 | 0 | ||||
11 | XJDQD1037-18 Ch. plumosus China | 1.468 | 0.913 | 1.467 | 1.097 | 1.097 | 1.839 | 1.654 | 1.654 | 1.468 | 0 | 0 | |||
12 | XJDQD1038-18 Ch. plumosus China | 1.468 | 0.913 | 1.467 | 1.097 | 1.097 | 1.839 | 1.654 | 1.654 | 1.468 | 0 | 0 | 0 | ||
13 | XJDQD1036-18 Ch. plumosus China | 1.468 | 0.913 | 1.467 | 1.097 | 1.097 | 1.839 | 1.654 | 1.654 | 1.468 | 0 | 0 | 0 | 0 | |
14 | MN750315.1 Ch. plumosus China | 1.845 | 2.028 | 2.592 | 2.217 | 2.217 | 2.219 | 2.034 | 2.034 | 1.845 | 2.214 | 2.214 | 2.214 | 2.214 | 0 |
Estimates of evolutionary divergence between sequences of Ch. plumosus from Finland and sequences of Ch. balatonicus, Ch. muratensis and Chironomus prope agilis. The number of base substitutions per site (%) from between sequences are shown. Analyses were conducted using the Kimura 2-parameter model (
№ | Sequences | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
---|---|---|---|---|---|---|---|---|
1 | CHIFI299-16 Ch. plumosus Finland, Satakunta | 0 | ||||||
2 | CHIFI298-16 Ch. plumosus Finland, Satakunta | 1.099 | 0 | |||||
3 | LEFIJ3947-16 Ch. plumosus Finland, Regio aboensis | 3.939 | 3.555 | 0 | ||||
4 | LEFIJ3948-16 Ch. plumosus Finland, Regio aboensis | 3.566 | 3.573 | 3.362 | 0 | |||
5 | AF192190.1 Chironomus prope agilis Russia | 6.306 | 6.315 | 8.357 | 6.930 | 0 | ||
6 | JN016826.1 Ch. balatonicus Russia, Saratov_terr. | 3.372 | 3.378 | 3.555 | 0.547 | 6.315 | 0 | |
7 | AF192194.1 Ch. muratensis Russia | 4.119 | 4.123 | 3.156 | 4.123 | 8.115 | 3.929 | 0 |
Recent research demonstrates that the range of divergence rates of the COI gene sequence in insects varies from 1.5% to 2.3% per 1 Mya (
Substitutions that distinguish Ch. bonus sequence from sequences in the Palearctic Ch. plumosus cluster; nonsyn. and syn. - nonsynonymous and synonymous substitutions respectively.
№ | Substitution type | Position in the sequence | Codon | Position in codon | Ch. bonus sequence |
---|---|---|---|---|---|
1 | nonsyn. | 2 | 1 | 1st | MZ014021.1 |
2 | syn. | 212 | 71 | 1st | MZ014021.1 |
3 | syn. | 340 | 113 | 3rd | MZ014021.1 MZ014022.1 MZ014023.1 |
4 | nonsyn. | 609 | 203 | 2nd | MZ014023.1 |
5 | nonsyn. | 642 | 214 | 2nd | MZ014023.1 |
6 | nonsyn. | 644 | 215 | 1st | MZ014023.1 |
Based on morphological and chromosomal characters, we identified the larvae belonging to the genus Chironomus at the studied site as Ch. bonus. The morphology of Ch. bonus larvae from the South Caucasus is similar to that previously described for this species by
The diploid number of chromosomes in the Ch. bonus karyotype is 2n = 8 plus the B-chromosome. Such a picture for the C. bonus karyotype is based on the almost constant presence of an additional B-chromosome in the karyotype of each larva. The chromosome arm combinations are AB, CD, EF, and G (the “thummi” cytocomplex) (Fig.
The karyotype of Ch. bonus from the South Caucasus is monomorphic. The banding sequences of all the chromosome arms of Ch. bonus are identical to those of Ch. plumosus. The difference between the karyotypes of both species is the presence of one additional B-chromosome in almost all studied Ch. bonus larvae. In total, there are 7 banding sequences in the Ch. bonus banding sequences pool (Fig.
h’bonA1 1a-2c 10a-12a 13ba 4a-c 2g-d 9e-4d 2h-3i 12cb 13c-14f 15a-14g 15b-19f C*
h’bonB1 25s-a 24s-a 23z-a 22u-a 21t-a 20n-a 19p-a 18o-a 17m-a 16m-a 15r-a 14r-a 13z-a 12y-v C**
p’bonC1 1a-2c 6c-f 7a-d 16a-17a 6hg 11d-12d 4a-6b 11c-8a 15e-13a 3c-2d 17b-22g C
p’bonD1 1a-3g 11a-13a 10a-8a 18d-a 7g-4a 10e-b 13b-17f 18e-24g C
h’bonE1 1a-3e 5a-10b 4h-3f 10c-13g C***
h’bonE1 1a-3a 4c-10b 3e-b 4b-3f 10c-13g C*
h’bonF1 1a-d 6e-1e 7a-10b 18ed 17d-11a 18a-c 10dc 19a-23f C
h’bonG1 not mapped
* revised mapping by
** mapped according to system of Maximova-Shobanov (
*** mapped according to
Overall, we successfully obtained three complete COI gene sequences of Ch. bonus from six larvae from the South Caucasus. (MZ014021.1: 627 bp, base composition is 25.99% A, 36.84% T, 16.91% G, and 20.26% C; MZ014022.1: 658 bp, base composition is 26.59% A, 36.17% T, 16.57% G, and 20.67% C; MZ014023.1: 650 bp, base composition is 27.08% A, 35.38% T, 16.77% G, and 20.77% C). Each of the three sequences had a different haplotype. This is the first DNA data obtained for Ch. bonus.
The resulting phylogenetic tree (Fig.
Phylogenetic tree of Chironomus species estimated by the Bayesian inference (BA). Support values are given if they exceed 0.5. The numbers at the nodes indicate posterior probabilities; Node 1, Node 2 etc. – nodes of the tree for which TMRCAs were calculated.
The Palearctic Ch. plumosus cluster (Figs
Phylogenetic tree of Palearctic Ch. plumosus cluster estimated by the Bayesian inference (BA). Support values are given if they exceed 0.5. The numbers at the nodes indicate posterior probabilities.
The Far Eastern Ch. borokensis-Ch. suwai cluster mostly formed by Ch. plumosus sequences from the Far East (China, South Korea, and Japan) and a sequence of Ch. borokensis from Russia. We named this branch as the Far Eastern Ch. borokensis-Ch. suwai cluster because this particular Ch. borokensis sequence (AB740261.1) was obtained from the material identified through karyological analysis (
Almost the same pattern is observed in the Nearctic Ch. entis-Ch. plumosus cluster, consisting equally of Ch. plumosus and Ch. entis sequences. According to the data from
In addition to the above-mentioned obvious clusters of the tree (Fig.
Calculated pairwise sequence distances (Tables
Almost the same pattern is observed in the Nearctic Ch. entis-Ch. plumosus cluster, where the distances between the sequences are also lower than the 3% range, varying from 0 to 1.285% (Table
In the Far Eastern Ch. borokensis-Ch. suwai cluster, the distances between the sequences are also lower than the 3% range, varying from 0 to 2.217% (Table
At the same time, the average distances between the various clusters exceed the 3% threshold. The distance between Palearctic Ch. plumosus and Far Eastern Ch. borokensis-Ch. suwai clusters is 3.55%. The distance between Palearctic Ch. plumosus and Nearctic Ch. entis-Ch. plumosus clusters is 3.75%. Finally, the distance between Far Eastern Ch. borokensis-Ch. suwai and Nearctic Ch. entis-Ch. plumosus clusters is 5.98%.
In the fourth cluster, which contains Ch. plumosus sequences from Finland, the distances between the sequences are generally higher than the 3% range (Table
Some sequences in the Palearctic Ch. plumosus cluster initially were not complete, and it was hard to make a good comparison. But still, we found a small number of substitutions that distinguish the sequences of Ch. bonus from other sequences in the Palearctic Ch. plumosus cluster. Overall, we found six substitutions of that kind (Table
According to the obtained results, the earliest split of the Ch. plumosus group of sibling species occurred during the Late Miocene (7,3–5,3 Mya) and early Pliocene (5,3–2,58 Mya) epoch (Fig.
Estimations of the age of the most recent common ancestors (TMRCAs) for DNA clades.
Node number | Mean value (Mya) | Stdev. | 95% HPD interval | ESS |
---|---|---|---|---|
Divergence rate 1.5% | ||||
Node 0 | 29.177 | 5.499 | 19.403, 40.569 | 6368 |
Node 1 | 17.288 | 3.874 | 10.391, 25.051 | 5221 |
Node 2 | 5.746 | 1.293 | 3.321, 8.280 | 4724 |
Node 3 | 2.883 | 0.698 | 1.689, 4.333 | 4562 |
Node 4 | 3.895 | 0.995 | 2.147, 5.844 | 5323 |
Node 5 | 0.638 | 0.212 | 0.284, 1.057 | 3230 |
Node 6 | 0.906 | 0.26 | 0.447, 1.427 | 3866 |
Node 7 | 1.971 | 0.505 | 1.027, 2.927 | 4836 |
Node 8 | 1.288 | 0.395 | 0.612, 2.077 | 5470 |
Node 9 | 0.517 | 0.169 | 0.229, 0.852 | 3570 |
Divergence rate 2.3% | ||||
Node 0 | 24.538 | 4.519 | 15.765, 33.035 | 7072 |
Node 1 | 13.716 | 2.992 | 8.518, 20.073 | 5990 |
Node 2 | 4.380 | 0.933 | 2.639, 6.220 | 5168 |
Node 3 | 2.204 | 0.511 | 1.288, 3.228 | 5429 |
Node 4 | 2.962 | 0.727 | 1.683, 4.467 | 5722 |
Node 5 | 0.481 | 0.155 | 0.217, 1.378 | 3759 |
Node 6 | 0.692 | 0.197 | 0.335, 1.071 | 4840 |
Node 7 | 1.503 | 0.375 | 0.820, 2.230 | 5210 |
Node 8 | 0.979 | 0.296 | 0.466, 1.570 | 5899 |
Node 9 | 0.395 | 0.129 | 0.177, 0.649 | 3576 |
Divergence rate 3.54% | ||||
Node 0 | 21.017 | 3.923 | 13.900, 28.841 | 6753 |
Node 1 | 11.123 | 2.492 | 6.596, 16.182 | 5914 |
Node 2 | 3.431 | 0.763 | 2.043, 4.944 | 4731 |
Node 3 | 1.715 | 0.410 | 1.013, 2.548 | 4676 |
Node 4 | 2.317 | 0.586 | 1.257, 3.468 | 5414 |
Node 5 | 0.378 | 0.124 | 0.164, 0.624 | 3784 |
Node 6 | 0.539 | 0.151 | 0.271, 0.835 | 4660 |
Node 7 | 1.170 | 0.295 | 0.634, 1.743 | 4902 |
Node 8 | 0.759 | 0.229 | 0.349, 1.206 | 6108 |
Node 9 | 0.307 | 0.101 | 0.137, 0.509 | 3832 |
Studied larvae of Ch. bonus have a monomorphic karyotype, with its details similar to those previously described for this species by
We can propose two possible explanations for the observed pattern within the Palearctic Ch. plumosus cluster (Fig.
The observed pattern also can be explained by a relatively recent separation of the two species, with Ch. plumosus being a parental species to Ch. bonus. The COI gene sequences of these species are therefore very similar, with a very low number of new substitutions in the Ch. bonus lineage. However, we discovered a number of substitutions that clearly distinguish Ch. bonus from Ch. usenicus and Ch. plumosus from European populations (Table
We can assume that the Ch. plumosus group originated from the common ancestor during the Pliocene of 5.75–3.43 Mya. However, since we have certain DNA data only for seven species of the Ch. plumosus group out of 14, this temporary estimate could change in the future in favor of the older age. At the same time, the obtained age of the most recent common ancestor of the Ch. plumosus group corresponds rather well to the estimations by
We can be more confident about the age of the most recent common ancestors of species constituting the Palearctic Ch. plumosus, Far Eastern Ch. borokensis-Ch. suwai, and Nearctic Ch. entis-Ch. plumosus clusters. It is possible that the age of the Palearctic Ch. plumosus, Far Eastern Ch. borokensis-Ch. suwai, and Nearctic Ch. entis-Ch. plumosus clusters is 0.638–0.378, 0.906–0.539 and 1.288–0.759 million years (Myr) respectively. The age of European populations of Ch. plumosus is approximately 0.517–0.307 Myr. We therefore suggest that observed clusters have arisen relatively recently in the Middle Pleistocene sub-epoch.
We concluded that the most recent common ancestor of the Ch. plumosus group originated in the Pliocene epoch (5.3–2.58 Mya). It is known that this epoch is characterized by the appearance of a new type of biome, the first true grasslands, due to the retreat of the forests associated with the gradual cooling of the climate that began in the previous epochs. True grasslands and Serengeti-like communities of grazing animals probably did not appear until the Late Miocene in the New World and the Pliocene in the Old World (ca. 5 Mya) (Pärtel 2005).
Due to the heterogeneity of the landscapes, new stagnant water bodies became increasingly abundant. In contrast to lowland rivers, which usually have similar environmental parameters, each of these stagnant water bodies was often characterized by a unique combination of size, shape, depth, temperature profile, mineralization level etc. This variation in environmental parameters could easily lead to differences in breeding time between various populations or individuals that can potentially lead to reproductive isolation and the emergence of new species. We suggest that the species divergence in this group could have been caused by invasion of their common ancestor into newly originated water bodies.
The data (Figs and Tables) that support this study are available in FigShare at https://doi.org/10.6084/m9.figshare.17060912.v1 and https://doi.org/10.6084/m9.figshare.17060666.v1.
We are sincerely grateful to E.A. Kuchinova, a researcher of the Laboratory of Ecology and Evolution of Vertebrates, Tembotov Institute of Ecology of Mountain territories RAS, for her big help in lab work.
This study was partially supported by a grant No. 18-04-00961 from the Russian Foundation for Basic Research (RFBR).
Mukhamed Kh. Karmokov https://orcid.org/0000-0002-3797-2511