Research Article |
Corresponding author: Mukhamed Kh. Karmokov ( lacedemon@rambler.ru ) Academic editor: Veronika Golygina
© 2018 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 MKh (2018) Karyotype characteristics and chromosomal polymorphism of Chironomus “annularius” sensu Strenzke (1959) (Diptera, Chironomidae) from the Caucasus region. Comparative Cytogenetics 12(3): 267-284. https://doi.org/10.3897/CompCytogen.v12i3.25832
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The study presents data on the karyotype characteristics and features of chromosomal polymorphism of Chironomus “annularius” sensu
Diptera , Chironomidae , Chironomus annularius , polytene chromosomes, chromosome polymorphism, Central Caucasus, South Caucasus, Eastern Ciscaucasia
There are a great number of publications that mention the name of Chironomus annularius from the 18th century (
Keyl & Keyl (1959) described the karyotype of Ch. “annularius” sensu
The aim of the work was to present the description of karyotype characteristics and chromosomal polymorphism of Ch. “annularius” from three Caucasian populations. In addition, it was also very important to compare the chromosomal polymorphism characteristics of Ch. “annularius” from the Caucasus with earlier studies.
We used fourth instar larvae of Chironomus in the karyological study. We provide the collection sites and abbreviations of earlier studied populations (
Collections sites of Ch. “annularius” in Caucasus region. Collection sites are marked with black dots.
Collection sites and number of analyzed Ch. “annularius” larvae from the European, Siberian, Kazakhstan and Nearctic populations per
Localities | Population abbreviation | Collection sites | Collection date | Number of specimens |
---|---|---|---|---|
European population | NL-NT-NT | The Netherlands | 07.1998 | 16 |
Siberian populations | RU-OMS-IR | Omskaya Oblast’: former riverbed or river Irtysh near Omsk | 08.1996 | 39 |
RU-NSK-EP | Reservoir near river Nizhnyaya Eltsovka | 07.2006 | 26 | |
RU-NSK-BE | Pond in Berdsk | 06.1998 | 52 | |
Kazakhstan population | KZ-SIP-UB | Alma Ata, pond in the Botanical garden | 09.1989 | 17 |
Nearctic populations | US-ND-WA | USA, Warsing Dam | 09.05.96 | 16 |
US-ND-IS | USA, Isabel Lake | 02.1995 | 33 |
Collection sites and number of analyzed Ch. “annularius” larvae from the Caucasus region.
Localities | Population abbreviation | Collection sites | Collection date | Number of specimens |
---|---|---|---|---|
Central Caucasus | CC-OS-ZM | 43°19.9067’ N; 44°11.1333’ E, Republic of North-Ossetia-Alania, puddle in the bed of drained pond, beside the Zmeiskaya settlement, altitude ca 310 m a.s.l. | 05.05.10 | 32 |
Eastern Ciscaucasia | ECS-BK-ART |
44°45.965’ N; 46°48.2037’ E, Republic of Dagestan, Tarumovsky District, ca 8 km southwest of “Biriuziak” holyday base, a puddle beside the artesian well, altitude ca -25 m b.s.l. |
26.05.17 | 47 |
South Caucasus | SC-SJ-PA | 41°19.3018’ N; 43°45.5577’ E, Republic of Georgia, Samtskhe-Javakheti region, ca 1 km north to the Sagamo settlement, one of branches of the Paravani river, altitude ca 2010 m a.s.l. | 18.07.17 | 36 |
Consequently, the site from Republic of North-Ossetia-Alania belongs to the Central Caucasus, the site from the Republic of Dagestan belongs to the Eastern Ciscaucasia and the site from the Republic of Georgia belongs to South Caucasus or Transcaucasia. Regarding vertical zonation (
The head capsule and body of 25 larvae were slide mounted in Fora-Berlese solution. The specimens have been deposited in the Tembotov Institute of Ecology of Mountain territories RAS in Nalchik, Russia. We studied the karyotype and chromosomal polymorphism in 115 larvae from the Caucasus region.
We fixed the larvae for karyological study in ethanol-glacial acetic acid solution (3:1). The slides of the chromosomes were prepared 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 the photomaps of
We studied the chromosome slides using a Carl Zeiss Axio Imager A2 microscope and performed the statistical data processing using software packages PAST 3.18 (
We used the following parameters of chromosomal polymorphism characteristics for comparison: percentage of heterozygous larvae, number of heterozygous inversions per larvae, the number of banding sequences in a population and a number of genotypic combinations per population. We calculated the genetic distances between populations according to Nei criteria (
We used the software package GenALEx 6.503 (
We performed a principal component analysis (PCA) of all the studied populations using original and previous data of
We measured the genetic distances (Table
We attributed the larvae of Chironomus in the studied sites to Ch. “annularius” by both morphological and chromosomal characteristics. The morphological larval characters of Ch. “annularius” from the Caucasian sites are similar to those previously described for this species by
The diploid number of chromosomes in Ch. “annularius” karyotype is 2n = 8, chromosome arm combination is AB, CD, EF, and G (the “thummi” cytocomplex) (Fig.
Previously,
Arm A has four banding sequences: annA1, annA2, annA3, and annA5 (Figs
annA5 1a-2c 10a-12a 13ba 4a-c 2g-d 9e-4d 2h-3i 12cb 13c-16c 19d-16d 19ef C
The banding sequence annA5 was found only in the population of the South Caucasus with relatively low frequency (annA5 – 0.069) and only in the heterozygous state (annA1.5 – 0.139) (Tables
Arm B has three banding sequences: annB1, annB2, and annB4 (Fig.
Arm C has two banding sequences: annC1 and annC2 (Fig.
Arm D has three banding sequences: annD1, annD2, and annD4 (Fig.
annD4 1a-3a-c 12ba 11c-a 3g-d 12dc 13a 10a 7a-4a 10e-b 13b-15e 20b-18e 17f-a 8a 18d-a 7g-b 9e-8b 16e-a 20c-24g C
The banding sequence annD4 was found only in the population of the South Caucasus with very low frequency (annD4 – 0.014) and only in the heterozygous state (annD1.4 – 0.028) (Tables
Arm E has two banding sequences: annE1 and annE2 (Fig.
Arm F has two banding sequences: annF1 and annF2 (Fig.
Arm G was monomorphic with banding sequence annG1.1 (Fig.
The data for European (Netherlands), Siberian, Kazakhstan and Nearctic (USA) populations are available due to
Arm A. Most earlier studied populations (
Arm B is polymorphic in most parts of studied populations, except for the Nearctic population of Warsing Dam where only banding sequence annB2 was present and the population of the Central Caucasus where also only sequence annB4 was found (Table
Arm C of Ch. “annularius” is polymorphic in two Caucasian populations (Eastern Ciscaucasia and Central Caucasus), Kazakhstan population and one Siberian population (Pond in Berdsk). The arm is monomorphic in populations of Europe, South Caucasus and rest of the Siberian populations, where only genotypic combinations annC1.1 was present. In addition, the arm is monomorphic in both Nearctic populations where the other genotypic combination annC3.3 was found (Table
Arm D of Ch. “annularius” is polymorphic in most of the studied populations, except for both Nearctic populations, where only banding sequence annD3 was present and population of the Europe where only sequence annD1 was found (Table
Arm E of Ch. “annularius” is polymorphic in most part of the studied populations, except for the Nearctic population of Warsing Dam and the population of the Europe where only banding sequence annE1 was present (Table
Frequency of banding sequences in different populations of Ch. “annularius”. N – the number of individuals, * – original data.
Banding sequences | European population | Caucasian populations | Siberian populations | Kazakhstan population | Nearctic populations | |||||
---|---|---|---|---|---|---|---|---|---|---|
NL-NT-NT N=16 |
ECS-BK-ART N=47* |
CC-OS-ZM N=32* |
SC-SJ- PA N=36* |
RU-OMS-IR N=39 |
RU-NSK-EP N=26 |
RU-NSK-BE N=52 |
KZ-AA-BG N=17 |
US-ND-WA N=16 |
US-ND-IS N=33 |
|
A1 | 0.438 | 0.766 | 0.313 | 0.708 | 0.910 | 0.769 | 0.865 | 0.736 | 0 | 0 |
A2 | 0.562 | 0.074 | 0.687 | 0.181 | 0.052 | 0.212 | 0.096 | 0.235 | 1 | 0.985 |
A3 | 0 | 0.160 | 0 | 0.042 | 0.038 | 0.019 | 0.039 | 0.029 | 0 | 0 |
A4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.015 |
A5 | 0 | 0 | 0 | 0.069 | 0 | 0 | 0 | 0 | 0 | 0 |
B1 | 0.844 | 0 | 0 | 0.778 | 0.051 | 0.173 | 0.106 | 0 | 0 | 0 |
B2 | 0.156 | 0.596 | 0 | 0 | 0.949 | 0.827 | 0.894 | 0.706 | 1 | 0.985 |
B4 | 0 | 0.404 | 1 | 0.222 | 0 | 0 | 0 | 0.234 | 0 | 0 |
B5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.015 |
C1 | 1 | 0.394 | 0.969 | 1 | 1 | 1 | 0.981 | 0.029 | 0 | 0 |
C2 | 0 | 0.606 | 0.031 | 0 | 0 | 0 | 0.019 | 0.971 | 0 | 0 |
C3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
D1 | 1 | 0.085 | 0.156 | 0.944 | 0.538 | 0.788 | 0.673 | 0.588 | 0 | 0 |
D2 | 0 | 0.915 | 0.844 | 0.042 | 0.462 | 0.212 | 0.327 | 0.412 | 0 | 0 |
D3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
D4 | 0 | 0 | 0 | 0.014 | 0 | 0 | 0 | 0 | 0 | 0 |
E1 | 1 | 0.170 | 0.875 | 0.806 | 0.500 | 0.538 | 0.462 | 0.794 | 1 | 0.970 |
E2 | 0 | 0.830 | 0.125 | 0.194 | 0.500 | 0.462 | 0.538 | 0.206 | 0 | 0.030 |
F1 | 0.156 | 0.723 | 0.141 | 0.153 | 0.243 | 0.173 | 0.163 | 0.206 | 0 | 0 |
F2 | 0.844 | 0.277 | 0.859 | 0.847 | 0.757 | 0.827 | 0.837 | 0.794 | 0.906 | 0.742 |
F3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.094 | 0.258 |
G1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 |
G3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
Frequency of genotypic combinations in different populations of Ch. “annularius”. N – the number of individuals, * – original data.
Genotypic combinations | European population | Caucasian populations | Siberian populations | Kazakhstan population | Nearctic populations | |||||
---|---|---|---|---|---|---|---|---|---|---|
NL-NT-NT N=16 |
ECS-BK-ART N=47* |
CC-OS-ZM N=32* |
SC-SJ- PA N=36* |
RU-OMS-IR N=39 |
RU-NSK-EP N=26 |
RU-NSK-BE N=52 |
KZ-AA-BG N=17 |
US-ND-WA N=16 |
US-ND-IS N=33 |
|
A1.1 | 0.187 | 0.574 | 0.218 | 0.500 | 0.820 | 0.654 | 0.750 | 0.529 | 0 | 0 |
A1.2 | 0.500 | 0.064 | 0.188 | 0.250 | 0.103 | 0.193 | 0.153 | 0.353 | 0 | 0 |
A2.2 | 0.313 | 0.043 | 0.594 | 0.028 | 0 | 0.115 | 0.030 | 0.059 | 1 | 0.970 |
A1.3 | 0 | 0.319 | 0 | 0.028 | 0.077 | 0.038 | 0.077 | 0.059 | 0 | 0 |
A1.5 | 0 | 0 | 0 | 0.139 | 0 | 0 | 0 | 0 | 0 | 0 |
A2.3 | 0 | 0 | 0 | 0.055 | 0 | 0 | 0 | 0 | 0 | 0 |
A2.4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.030 |
B1.1 | 0.687 | 0 | 0 | 0.611 | 0.103 | 0.115 | 0.038 | 0 | 0 | 0 |
B1.2 | 0.313 | 0 | 0 | 0 | 0 | 0.115 | 0.135 | 0 | 0 | 0 |
B1.4 | 0 | 0 | 0 | 0.333 | 0 | 0 | 0 | 0 | 0 | 0 |
B2.2 | 0 | 0.532 | 0 | 0 | 0.897 | 0.770 | 0.827 | 0.412 | 1 | 0.970 |
B2.4 | 0 | 0.128 | 0 | 0 | 0 | 0 | 0 | 0.588 | 0 | 0 |
B4.4 | 0 | 0.340 | 1 | 0.056 | 0 | 0 | 0 | 0 | 0 | 0 |
B2.5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.030 |
C1.1 | 1 | 0.234 | 0.937 | 1 | 1 | 1 | 0.961 | 0 | 0 | 0 |
C1.2 | 0 | 0.319 | 0.063 | 0 | 0 | 0 | 0.039 | 0.059 | 0 | 0 |
C2.2 | 0 | 0.447 | 0 | 0 | 0 | 0 | 0 | 0.941 | 0 | 0 |
C3.3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
D1.1 | 1 | 0 | 0 | 0.889 | 0.359 | 0.616 | 0.481 | 0.353 | 0 | 0 |
D1.2 | 0 | 0.17 | 0.313 | 0.083 | 0.359 | 0.346 | 0.385 | 0.470 | 0 | 0 |
D1.4 | 0 | 0 | 0 | 0.028 | 0 | 0 | 0 | 0 | 0 | 0 |
D2.2 | 0 | 0.83 | 0.687 | 0 | 0.282 | 0.038 | 0.134 | 0.177 | 0 | 0 |
D3.3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
E1.1 | 1 | 0.043 | 0.750 | 0.639 | 0.256 | 0.308 | 0.250 | 0.706 | 1 | 0.940 |
E1.2 | 0 | 0.255 | 0.250 | 0.333 | 0.488 | 0.461 | 0.423 | 0.176 | 0 | 0.060 |
E2.2 | 0 | 0.702 | 0 | 0.028 | 0.256 | 0.231 | 0.327 | 0.118 | 0 | 0 |
F1.1 | 0 | 0.532 | 0 | 0 | 0 | 0 | 0 | 0.059 | 0 | 0 |
F1.2 | 0.313 | 0.383 | 0,281 | 0.306 | 0.487 | 0.346 | 0.327 | 0.294 | 0 | 0 |
F2.2 | 0.687 | 0.085 | 0.719 | 0.694 | 0.513 | 0.654 | 0.673 | 0.647 | 0.813 | 0.546 |
F2.3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.187 | 0.394 |
F3.3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.060 |
G1.1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 |
G3.3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
Arm F of Ch. “annularius” is polymorphic in all the studied populations. In most of them, with the exception of the population from Eastern Ciscaucasia, genotypic combination annF2.2 was predominant (Table
Arm G is monomorphic in all the studied populations. However, there is an important difference. In Holarctic populations genotypic combination annG1.1 was dominant, while in Nearctic populations another combination annG3.3 was dominant.
The level of inversion polymorphism of Caucasian Ch. “annularius” populations is quite similar to those of previously studied Holarctic populations (Table. 5). The populations of the South Caucasus and Eastern Ciscaucasia are generally close to Asian populations (Siberia and Kazakhstan) by all the parameters of chromosomal polymorphism. The population of Central Caucasus is close to the European population by the average number of heterozygous inversions per larvae, number of banding sequences per population and number of genotypic combinations per population. The percentage of heterozygous larvae in population of Central Caucasus is lowest (72%) among all the Holarctic populations (81-90%) (Table
Cytogenetical characteristics of chromosomal polymorphism in different populations of Ch. “annularius”. N – the number of individuals, * – original data.
Cytogenetical characteristics | European population | Caucasian populations | Siberian populations | Kazakhstan population | Nearctic populations | |||||
---|---|---|---|---|---|---|---|---|---|---|
NL-NT-NT N=16 |
ECS-BK-ART N=47* |
CC-OS-ZM N=32* |
SC-SJ- PA N=36* |
RU-OMS-IR N=39 |
RU-NSK-EP N=26 |
RU-NSK-BE N=52 |
KZ-AA-BG N=17 |
US-ND-WA N=16 |
US-ND-IS N=33 |
|
Heterozygous larvae, % | 81 | 83 | 72 | 89 | 90 | 85 | 89 | 88 | 18 | 48 |
Average number of heterozygous inversions per larvae | 1.1 | 1.6 | 1.2 | 1.6 | 1.6 | 1.5 | 1.5 | 1.0 | 0.2 | 0.5 |
Number of banding sequences per population |
10 | 14 | 12 | 15 | 13 | 13 | 14 | 14 | 8 | 11 |
Number of genotypic combinations per population |
11 | 19 | 13 | 19 | 15 | 17 | 18 | 18 | 8 | 12 |
On the dendrogram of genetic distances, there are four clear clusters that we conditionally assigned as European, Asian, Siberian and Nearctic clusters (Fig.
Principal component analysis (PCA) of genotypic combination frequencies in 10 Ch. “annularius” populations. For abbreviations of the populations, see Tables
Values of genetic distances between the different populations of Ch. “annularius”.
Population | NL-NT-NT | ECS-BK-ART | CC-OS-ZM | SC-SJ- PA | RU-OMS-IR | RU-NSK-EP | RU-NSK-BE | KZ-AA-BG | US-ND-WA | US-ND-IS |
---|---|---|---|---|---|---|---|---|---|---|
NL-NT-NT | 0 | |||||||||
ECS-BK-ART | 1.0628 | 0 | ||||||||
CC-OS-ZM | 0.3919 | 0.5318 | 0 | |||||||
SC-SJ-PA | 0.0724 | 0.8232 | 0.3853 | 0 | ||||||
RU-OMS-IR | 0.4069 | 0.3421 | 0.4084 | 0.2454 | 0 | |||||
RU-NSK-EP | 0.2589 | 0.4732 | 0.3952 | 0.1566 | 0.0272 | 0 | ||||
RU-NSK-BE | 0.3422 | 0.3909 | 0.409 | 0.2124 | 0.0147 | 0.0084 | 0 | |||
KZ-AA-BG | 0.5162 | 0.4259 | 0.5787 | 0.4828 | 0.4121 | 0.3845 | 0.3807 | 0 | ||
US-ND-WA | 1.1784 | 2.0094 | 1.1094 | 1.5183 | 1.2745 | 1.1585 | 1.2025 | 1.1412 | 0 | |
US-ND-IS | 1.2873 | 2.0136 | 1.2059 | 1.6637 | 1.3387 | 1.2404 | 1.2917 | 1.2424 | 0.0093 | 0 |
The principal component analysis shows almost the same picture as the dendrogram of genetic distances (Fig.
Among Caucasian populations, the frequencies of genotypic combinations in all arms of Ch. “annularius” follow Hardy-Weinberg expectation only in the population of South Caucasus. In population of Central Caucasus, the frequencies of genotypic combinations in arm A do not follow Hardy-Weinberg expectation (χ2 = 10.166, p – 0.001). The homozygotes annA1.1 were observed 2.22 times more frequently than it was expected and heterozygotes annA1.2 should be occurred 2.29 times more frequently than they were observed. One can observe an even more complex picture in the population of Eastern Ciscaucasia where the frequencies of genotypic combinations do not follow Hardy-Weinberg expectation across three arms: arm A (χ2 = 16.046, p – 0.001), arm B (χ2 = 25.388, p – 0.000), and arm C (χ2 = 5.163, p – 0.023). In the arm A the heterozygotes annA1.2 should be occurred 1.78 times more frequently than they were observed, homozygotes annA2.2 were observed 7.17 times more frequently than it was expected, also expected combinations annA2.3 and annA3.3 were not found at all. In arm B the homozygotes annB2.2 and annB4.4 were observed 1.5/2.1 times more frequently than it was expected and heterozygotes annB2.4 should be occurred 3.77 times more frequently than they were observed. Finally, in arm C the homozygotes annC1.1 and annC2.2 were observed 1.5/1.2 times more frequently than it was expected and heterozygotes annC1.2 should be occurred 1.5 times more frequently than they were observed.
We found the species Ch. “annularius” in the South Caucasus for the first time. Earlier (
Overall, the Caucasian populations of the species can be characterized as relatively polymorphic. We found two new banding sequences annA5 and annD4 in the banding sequences pool of Ch. “annularius”. We observed inversion polymorphism almost in all chromosome arms except for arm G, which was monomorphic in Caucasian populations.
Observed picture with Hardy-Weinberg expectation in the site from Eastern Ciscaucasia can be explained in several ways. First, it can be a negative selection of heterozygotes due to some adaptive processes that are still ongoing. Another possibility is that it is due to short time of existence of this population and founder effect.
The climate of Terek-Kuma lowland is much hotter and drier than in both other collection sites. We collected the larvae here from the puddle beside an active artesian well. This habitat is stable because it is constantly fed by water from the well. There are about 3 000 of such kind of wells (most of them still active), within the radius of ca 100 km. Most of them were drilled in the 50–60s of the 20th century for the aims of animal husbandry. Considering this, we can expect a lot of new records of this species from habitats situated beside those wells. The puddle that served as collection site is quite small (3×5m of water surface, max. depth about 0.5m) and thus the total size of the population is not so big. Possibly this population is relatively young and just over 50–60 years old. It can be presumed that initially a very small number of individuals from some nearby habitats established this population and the influx of new migrants is not so large. It is quite possible that most part of the larvae here could be relatives and so the inbreeding could occur quite often. Possibly, there was not enough time for the population to come to the equilibrium. Perhaps we see the founder effect that can also explain the observed picture with Hardy-Weinberg expectation.
All the obtained data are indicative of the complex genetic structure of Caucasian populations of Ch. “annularius” and total complexity of microevolution processes occurring in the Caucasus region. In spite of geographic proximity, one Caucasian population is separated from other populations of the Caucasus at the level of subspecies.