Cytogenetic variability in species of genus Chironomus (Dip-tera, Chironomidae) from Poland

CARYOLOGIA Vol. 58, no. 4: 345-358, 2005

Cytogenetic variability in species of genus Chironomus (Dip-tera, Chironomidae) from Poland

Paraskeva Michailova1*

, Elzbieta Warchałowska-Sliwa2, Boris Krastanov

1and

Andrzej Kownacki3

1 Institute of Zoology, Bulgarian Academy of Sciences, blv. Tzar Osvoboditel 1, 1000 Sofia, Bulgaria.2 Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, str. Slawkowska 17, 31-016 Kra-kow, Poland.3 Karol Starmach Institute of Freshwater Biology, Polish Academy of Sciences, str. Slawkowska 17, 31 − 016 Kra-kow, Poland.

Abstract — Chromosome variability was analyzed in three Chironomus species: Chironomus plumosus L., Chi-ronomus muratensis Ryser, Scholl & Wulker, and Chironomus annularius Meigen, from the natural populationsin Poland. A comparative analysis of band sequences with other Palearctic populations was done. Chromosomeband sequences of C. plumosus and C. muratensis did not differ from other Palearctic populations. A new ho-mozygous band sequence was discovered in C. annularius. The ten sequences observed in the Polish populations ofC. plumosus were represented in all cytogenetically studied European populations of this species. However, these se-quences showed substantial interpopulational variation and they didn’t demonstrate any geographical gradient.Some of the inversions in C. plumosus and C. annularius were also involved in the function of the so-called polymor-phic system. The sensitivity of important chromosome structures (BRs and NOR) in the polytene chromosomes of C.annularius to organic contaminants in the studied area was presented. The morphology of the additional “B” chro-mosome found in C.plumosus can vary in different salivary gland cells. The existence of the “B” chromosome in theC. plumosus genome was considered in the light of its selective value in the studied area.

Key words: aberrations, additional “B” chromosome, Chironomus, polymorphic system, polytene chromosomes.

INTRODUCTION

The karyotype and chromosome variability ofwidely distributed, Palearctic Chironomid specieshave been studied extensively (Keyl 1962;

Michailova 1989; Kiknadze et al. 1991a, 1996;Butler et al. 1999; Golygina and Kiknadze

2001). A detailed cytogenetic analysis of salivarygland chromosomes revealed the existence of sib-ling species in different Palearctic populations ofthe genus Chironomus (Keyl 1962; Ryser et al.1983; Kerkis et al. 1988, 1989; Michailova 1989;Kiknadze et al. 1991a, 1996). Also, the evaluationof chromosome polymorphism in natural popula-tions of Chironomid species has contributed dataon adaptation, cytogenetic differentiation andkaryotype divergence of this genus (Petrova

1991; Kiknadze et al. 1998; Butler et al. 1999;Gunderina et al.1999). That’s way the knowledge

of chromosome variability among the Chironomidpopulations is essential. However, information onthe karyotype and chromosome polymorphism ofthe Chironomus species in Poland is very scarce.Only one study, reporting the presence of severalcytotaxonomic species of the genus Chironomus(Michailova et al. 2002), is available in spite ofthe fact that this genus is very common in differ-ent aquatic biotopes (Kownacki 1999).

The present paper contributes information onpolytene chromosomes and their cytogenetic vari-ability in three Chironomus species: Chironomusplumosus L., Chironomus muratensis Ryser,

Scholl & Wulker and Chironomus annulariusMeigen. These species belong to the cytocomplex“thummi” with chromosome arm combinations:AB CD EF G (Michailova 1989). Two of them(C. plumosus and C. muratensis) are closely relatedand are incorporated in the chromosome group“plumosus”, while the third (C. annularius) isa single representative of the cytocomplex“thummi”. Cytogenetic data concerning C. plumo-sus and C. annularius provide information on

* Corresponding author: phone: ++359 2 988 51 15; fax:++ 359 988 28 97; e-mail: [email protected]

chromosome polymorphism and the process ofmicrodifferentiation in C. plumosus. Genomepolymorphism of C. plumosus is also discussed.Additionally, we compared our results of chromo-some variability of C.plumosus with informationof its cytogenetic variability in different Palearcticpopulations (Gunderina et al. 1999; Golygina

and Kiknadze 2001).

MATERIALS AND METHODS

Twenty three specimens of Chironomus plu-mosus and three specimens of C. muratensis werecollected during May/June 2002 from a post-ex-ploitation gravel pit in Rybnik, located in the Vis-tula Valley, 15 km south from Torun (northernPoland). A lake was formed after the pit was filledwith subterranean water. It is characterized by amaximum depth of more than 5 m, an area of 8ha,sand-mud bottom, a water volume of 240 000m3,high alkaline reaction (pH=8,9), moderate con-ductivity (400 µS/cm), and biochemical oxygendemand (BOD5) − 4.79 mg/dm3. The lake is sur-rounded by forest with the exception of its south-ern bank, which is farmland.

Twenty one specimens of C. annularius wereobtained during June 2002 from a retention pondof a sugar factory in Chybie (southern Poland).Michailova et al. (2001) gave information aboutthe water quality of this pond. Oxidation was el-evated in this water body, and NH2, PO4, and N-NO3 ions were highly concentrated.

Larvae were fixed in a solution of 960C etha-nol and glacial acetic acid (3:1). Cytogenetic andmorphological preparations were obtained fromeach larva. Isolated salivary glands weresquashed for polytene chromosome preparationsusing a routine aceto — orcein method(Michailova 1989). Freezing in liquid nitrogenfor removal of cover slips did permanent slides.After several steps in alcohol and xylene, theywere remounted in Euparal. Larval head cap-sules and bodies were mounted in Euparal formorphological analysis. Slides of polytene chro-mosomes and larval morphology were depositedat the Institute of Zoology, Sofia — BulgarianAcademy of Sciences.

Polytene chromosomes of C. plumosus wereidentified by applying the maps of Butler et al.(1999), Gunderina et al. (1999), Golygina andKiknadze (2001). The polytene chromosomes ofC. muratensis were identified by comparing withthe literature (Kiknadze et al. 1991a; Michailova

et al. 2002). For a subsequent, detailed karyotype

analysis of C. annularius, standard chromosomemaps done by Keyl and Keyl (1959); Keyl

(1962); Petrova and Michailova (1986), andKiknadze et al. (1991a, 1996) were used. Se-quences occurring in our species were symbolizedaccording to Kiknadze et al. (1991a, 1996) andGolygina and Kiknadze (2001) − for instance,homozygous states of arms: A - A1.1 or A2.2; B-B1.1or B2.2 etc.; heterozygous states: A1.2, B1.2and etc.

Inverted and standard band sequences wereconsidered as alleles and their frequencies wereestimated according to Butler et al. (1999).This permits the calculation of cytogenetic dis-tance between Polish and other Palearctic popu-lations by applying Nei’s method (Nei 1972). Adendogram was made using UPGMA clusteranalysis (Sneath and Sokal 1973) withPHYLIP (Felsenstein 1993). Heterozygote fre-quencies of C.plumosus were tested for confirm-ing to expectation under Hardy − Weinbergequilibrium using X2 test.

RESULTS

On the basis of external larvae morphology thesamples were divided into three forms (A, B, C).Form A has a long tubule located ventrally andwas therefore called the “plumosus” type; form Cwith a ventral short tubule − “semireductus” type,while form B exhibits an intermediate state be-tween the two forms. Ollik and Zbikowski

(2002) also differentiated the C form on the basisof length of the body of the larva as well as on thesize of the head capsule.

Cytogenetically, the studied material was con-sidered as three species: C. plumosus, C. muraten-sis and C.annularius. Every species has specificchromosome markers by which the species weredifferentiated. They belong to cytocomplex“thummi” with chromosome arm combinations:AB CD EF G.

Chironomus plumosus L. - It has 2n = 8. Chromo-somes AB and CD are metacentric, chromosomeEF is submetacentric and chromosome G is telo-centric.Chromosome G has a nucleolar organizer(NOR) and two Balbiani rings (BR2, BR3). OneBalbiani ring (BR1) is located in arm B. (Figs. 1a,b, 2a, b, 3a, b, c).

Chromosome arms A, B, C, D are very poly-morphic. It is interesting to note that on thesearms the standard band sequences were convertedinto another homozygous band sequences through

346 paraskeva, warchałowska-sliwa, krastanov and kownacki

heterozygotization (Table 1 and table 2) (Fig.1a,b). In arms A and B standard and inverted bandsequences occurred almost in the same frequency(Table 2). In arms C, D the standard sequences(C1, D1) were predominated (Table 2) (Fig. 2b).The heterozygous (C1.2) and homozygous inver-sions (C2.2, D2.2) appeared very rarely (Table 1)(Fig.2a). The arms E, F and G have the standardband sequences (Table 1 and table 2) (Fig.3a, b, c).In a mosaic state an asysnapsis was found in chro-mosome arms E and F (Fig. 3a, b).

BR1 in arm B occurred in a very active state inall studied individuals, although not in all cells.

The studied individuals differed with respectof their centromeric heterochromatin In somespecimens (about 50%) centromere regions of thepolytene chromosomes were represented by welldefined large heterochromatin blocks (Figs. 2b,3b). In five individuals, in few cells the centromereheterochromatin in chromosomes AB, CD and EFappeared in a heterozygous state (Figs. 1b, 3b).

Four individuals have a “B” chromosome,which appeared in a compact as well as in a net-work state (Fig. 4 a, b). Every type is observed in

different individuals and within the salivary glandcells of an individual. It doesn’t have a clear band-ing structure and it is often oriented towardschromosome G.

Fig. 1 — Polytene chromosomes of Chironomus plumosus L. a. Chromosome AB (Chromosome arm A with invertedhomozygous sequences - A2.2; Chromosome arm B with standard sequences - B1.1); The centromere regions ex-pressed by thin band. b. Chromosome AB (Chromosome arm A with inverted heterozygous sequences - A1.2; Chro-mosome arm B with inverted homozygous sequences - B2.2); The centromere region in heterozygous state; Q cen-tromere region; BR - Balbiani ring; Bar = 10 µm.

Table 1 — Types and frequency of chromosome inver-sions of C. plumosus.

Inversionsequences Frequency �2

A1.1 0.348A1.2 0.304 3.52A2.2 0.348B1.1 0.435B1.2 0.217 7.25*B2.2 0.348C1.1 0.826C1.2 0.130 0.41C2.2 0.044D1.1 0.522D1.2 0.304 1.23D2.2 0.174E1.1 1.000 0F1.1 1.000 0G1.1 1.000 0

* P < 0.01

cytogenetic variability in species of genus chironomus 347

The studied population of C.plumosus had ahigh level of chromosome polymorphism − thelarvae with heterozygous inversions were 87%and the average number of heterozygous inver-sions per larva was 0.87.

Chironomus muratensis Ryser, Scholl &

Wulker. - Its chromosome complement is 2n = 8.

Chromosomes AB, CD are metacentric, chromo-some EF is submetacentric and chromosome G istelocentric. There are two nucleolar organizers(NOR) in arms C and G, and three Balbiani ringsin arms B(BR1) and G (BR2, BR3) (Fig. 5 a, b, c,d). Arms A, C, E, F, G appeared in standard bandsequences A1, C1, E1, F1 and G1 respectively.Arm B had an inverted band sequence B2, whilearm D was in heterozygous and standard ho-mozygous state (D1.2 and D1.1). The hetero-zygous state of this arm has been documented inRussia (Petrova and Michailova 1986;Kiknadze et al. 1991a), Finland (Michailova

2001), and Poland (Michailova et al. 2002).

Chironomus annularius Meigen - This species has2n = 8. Chromosomes AB and CD are metacen-tric, chromosome EF is submentacentric, andchromosome G is telocentric. One Balbiani ring(BR) is located in arm G and a nucleolar organizer(NOR) appears in arms A, C, E and G (Fig. 6a, b,c, d, e, f, g).

Fig. 2 — Polytene chromosomes of Chironomus plumosus L. a. Chromosome CD (Chromosome arm C, with in-verted heterozygous sequence - C1.2; Chromosome arm D, with standard sequences - D1.1); The centromere regionsexpressed by thin band. b. Chromosome CD (Both arms are with standard sequences - C1.1 and D1.1); The centro-mere regions expressed by large heterochromatin block. Q centromere region; Bar = 10 µm.

Table 2 — Frequency of band sequences in polytenechromosomes of C. plumosus.

Band sequences Frequency

A1 0.500A2 0.500B1 0.543B2 0.457C1 0.891C2 0.109D1 0.674D2 0.326E1 1.000F1 1.000G1 1.000


Arm A has two band sequences (A1 and A2)(Table 4). They appeared in three states, standardA1.1 (Fig. 6 a) inverted homozygous state A2.2(Fig. 6 c), and heterozygous state A1.2 (Fig.6 b).The nucleolar organizer (NOR) in this arm as wellas in arm E was slightly expressed and in 10% ofthe individuals the NOR in arm A was in a hetero-zygous state.

In arm B has three band sequences: B1, B2 anda new one B4 (Table 4). From the standard, twosteps of inversions have formed the new band se-quence of arm B:

22 21 20 19 18a 18b 17 16 15 14 1322 21 20 19 18a 13 14 15 16 17 18b

22 21 20 19 18a 13 14 15 18b 17 16 (the newhomozygous band sequences) (the bold numberswere included in the inversions) (Fig. 6d). Thestandard band sequences of this arm were pre-dominated (Table 3 and table 4).

With exception of arm D, the arms C, E, G ap-peared in standard homozygous state only (C1.1,E1.1, G1.1) (Fig.6e, f, g) (Table 3 and table 4). Inarm F, standard band sequences F1 was predomi-nated (Fig.6f). Only 3 individuals had a hetero-zygous inversion (F1.2). It is important to under-line that in all studied individuals in the mosaicstate, the homologues of chromosome G were un-paired due to different functions of both homo-

Fig. 3 — Polytene chromosomes of Chironomus plumosus L. a. Chromosome EF (Both arms have standard se-quences - E1.1 and F1.1, with unpaired sections in arm F); The centromere regions expressed by thin band. c. Chro-mosome EF (Both arms are with standard sequences − E1.1 and F1.1, with unpaired arm E); The centromere regionsexpressed by large heterochromatin block. d. Chromosome G with standard sequences − G1.1, the both homologuesare unpaired; The centromere regions expressed by large heterochromatin block. Q centromere region; BR − Balbi-ani ring; NOR − Nucleolar organizer; Bar = 10 µm.


logues. In C. annularius 61% of studied individu-als were with heterozygous inversions. The aver-age number of heterozygous inversions per larvawas 0.71.

DISCUSSION

A total of 24 and 15 band sequences werefound in 30 Palearctic and several Nearctic popu-

lations of C. plumosus, respectively (Butler et al.1999; Gunderina et al. 1999). Eight were com-mon for both Palearctic and Nearctic regions. De-tailed cytogenetic and statistical analysis showedclear cytogenetic differentiation between Palearc-tic and Nearctic populations (Butler et al. 1999).On the other hand, Gunderina et al. (1999) docu-mented considerable cytogenetic diversity in Eu-ropean and Asian (Siberian) populations of C. plu-

Fig. 4 — Chromosome “B” in Chironomus plumosus L. a. Compact chromosome “B”; b. Decondensed chromosome“B”; Bar = 10 µm.


Fig. 5 — Polytene chromosomes of Chironomus muratensis Ryser, Scholl & Wulker. a. Chromosome AB (Chro-mosome arm A with standard sequences - A1.1; Chromosome arm B with inverted homozygous sequences - B2.2); b.Chromosome CD (Both arms are with standard sequences - C1.1 and D1.1); c. Chromosome EF (Both arms are withstandard sequences - E1.1 and F1.1); d. Chromosome G (with standard sequences - G1.1); Q centromere region; BR- Balbiani ring; NOR - Nucleolar organizer. Bar = 10 µm.


mosus, which form the Palearctic parts of its Hol-arctic range. These populations are distinguishedby both the type and frequency of sequences. It isworth noting that an eastern population in Siberia(Yakutia, Beloe Lake) was the most cytogeneti-cally divergent from other Palearctic populations(Butler et al. 1999). Also, Gunderina et al.(1999) underlined that in central Palearctic popu-lations cytogenetic differences were not as sub-stantial, although they increased toward the pe-riphery of the range of C. plumosus. SequencesA1, A2, B1, B2, C1, D1, D2, E1, F1 and G1 foundin the Polish population were represented in allEuropean populations cytogenetically studied byGunderina et al. (1999) and Butler et al. (1999).Some of these (A2, B1, B2, D2) have been re-corded in the Nearctic. However, in the Nearcticpopulations the sequence D2 occurred as the mostfrequent sequence in this region (Butler et al.1999). Seven of the sequences found in the Polishpopulation (A1, B1, C1, D1, E1, F1 and G1) aredistributed in all Palearctic populations and areconsidered as the main common Palearctic se-

quences (MCP) (Gunderina et al.1999). There-fore, concerning the band sequences, the Polishpopulation of C. plumosus was not different fromother Palearctic and some Nearctic populations.However, the sequence frequencies showed con-siderable inter population variation. The standardsequences of arms C (C1), D (D1), E (E1), F (F1)and G (G1) predominated in Poland. However,the inverted homozygous state in arm A (A2.2)and arm B (B2.2) respectively, occurred in highfrequencies. Even more, statistically significantdeviations were observed in arm B. It is quite pos-sible that the heterozygous state of this arm havesome advantages in studied are, characterized byalkaline reaction and oxygen demand. These dataconfirm the idea done by Michailova andPetrova (1991) that inversions have varying se-lective priorities in different ecological condi-tions.

The dendrogram (Fig. 7) showed that the Pal-earctic populations of C. plumosus were clusteredinto several groups. C. plumosus from geographi-cally close regions was separated into differentclusters. For instance, samples from Altai lakeswere put into different clusters. On the otherhand, populations from geographically isolatedsites were closely grouped together. The studiedPolish population formed an independent group,which was most closely related to the Novosibirskpopulation (NSK-K), with the smallest cytoge-netic distance 0, 0528 (Table 5). The highest cy-togenetic distance exists between Poland and Vol-gograd — Vu populations — 0, 2165 (Table 5).The results confirmed Gunderina’s et al. (1999)idea that there isn’t any geographical gradient ofthe observed cytogenetic variability of the species.It is quite possible that in all these populationsthere are variations in the frequency of aberra-tions depending on the specific conditions of thehabitat.

In the arms A, B, C, D of C. plumosus and inarm A of C. annularius the polymorphic system,described by Michailova (1989) was evident −one homozygous inversion converted into anotherhomozygous state through heterozygotization.This polymorphic system has been found in C.plumosus from Hungary and Switzerland(Michailova and Fisher 1986), although it wasfirst detected in C. annularius. It is quite possiblethat each chromosome band sequence has someadvantage in a specific ecological niche availableto a population. It is worth underlining that thestandard sequences of arms C, D, E, F of C. plu-mosus predominated, while in C.plumosus and inC. annularius inverted homozygous (A2.2) and

Table 3 — Types and frequency of chromosome inver-sions of C. annularius.

Inversion sequences Frequency

A1.1 0.238A1.2 0.381A2.2 0.381B1.1 0.619B4.4 0.095B1.2 0.286C1.1 1.000D1.1 0.952D2.2 0.048E1.1 1.000F1.1 0.857F1.2 0.143G1.1 1.000

Table 4 — Frequency of band sequences in polytenechromosomes of C. annularius.

Band sequences Frequency

A1 0.429A2 0.571B1 0.762B2 0.143B4 0.095C1 1.000D1 0.952D2 0.048E1 1.000F1 0.929F2 0.071G1 1.000


Fig. 6 — Polytene chromosomes of Chironomus annularius Meigen a. Chromosome AB (Both arms are with stand-ard sequences - A1.1 and B1.1); b. Chromosome AB (Chromosome arm A with inverted heterozygous sequences -A1.2; Chromosome arm B with standard sequence - B1.1); c. Chromosome AB (Chromosome arm A with invertedhomozygous sequences - A2.2; Chromosome arm B with standard sequences - B1.1); d. Chromosome AB (Chromo-some arm A with inverted heterozygous sequences - A1.2; chromosome arm B with new band sequences - B4.4); e.Chromosome CD (Both arms are with standard sequences - C1.1 and D1.1); f. Chromosome EF (Both arms are withstandard sequences - E1.1 and F1.1); g. Chromosome G (with standard sequences - G1.1). Q centromere region; BR- Balbiani ring; NOR - Nucleolar organizer. Bar - 10 µm.


Fig. 7 — The dendrogram of C.plumosus showing the cytogenetic distance (Dcg) between Palearctic populations,studied by Golygina and Kiknadze (2001) and C.plumosus of Polish population (this study). The symbols indicatedthe region (the first symbol) and lake (the second symbol): DK − Denmark; RAI − Republic Altai; ALT − Altai; LEN− Leningrad; CHE − Chelyabinsk; PAV − Pavlovsk; NSK − Novosibirsk; VKA − East Kasachstan; OMS − Omsk;VLA − Vladimirovsk; NNG − Nijegorodsk; SAR − Saratov; SVE − Sverdlovsk; KUR − Kurgan; VOL - Volgograd;POL − Ryb. − Poland; YAK - Yakutia. The scale below the tree demonstrates the length of the tree branches in cy-togenetic distances.

VOL-VU

NSK-KR

DK-ES

RAI-TE

ALT-TR

LEN-SP

ALT-BA

ALT-BY

ALT-RA

CHE-KA

CHE-KU

PAV-ZH

PAV-DA

NSK-RE

NSK-3R

NSK-2R

VKA-VO

OMS-OM

OMS-PO

ALT-TG

KUR-YO

SVE-SH

SAR-VO

ALT-KO

ALT-BE

NNG-YU

VLA-ST

NSK-K

POL-RUB

YAK-BE

KUR-IS

0.000.020.040.060.08 Dcg


Tab

le5

—C

ytog

enet

icdi

stan

ces

betw

een

natu

ralp

opul

atio

nsof

C.p

lum

osus

Polan

dV

OL-

VU

0.21

65

VLA

-ST

0.05

670.

1575

NN

G-Y

U0.

1395

0.22

400.

0216

SVE-

SH0.

1076

0.16

680.

0161

0.00

96

NSK

-K0.

0528

0.23

130.

0189

0.04

380.

0521

NSK

-RE

0.07

780.

0593

0.03

740.

0797

0.04

030.

0908

ALT

-BY

0.11

600.

0703

0.04

630.

0664

0.02

900.

1075

0.00

72

ALT

-BA

0.15

240.

0847

0.08

120.

0971

0.04

790.

1594

0.02

040.

0061

YA

K-B

E0.

1812

0.18

410.

1482

0.20

710.

2185

0.10

630.

1998

0.23

200.

3138

DK

-ES

0.14

560.

1066

0.08

040.

0974

0.04

780.

1633

0.02

280.

0109

0.00

320.

3578

LEN

-SP

0.13

860.

1092

0.07

820.

0944

0.04

680.

1526

0.02

320.

0117

0.00

520.

3469

0.00

26

SAR-

VO

0.07

420.

1511

0.01

670.

0284

0.00

620.

0557

0.02

370.

0193

0.03

340.

2332

0.03

030.

0290

CH

E-K

A0.

0738

0.09

820.

0320

0.06

070.

0236

0.08

190.

0068

0.00

620.

0147

0.23

120.

0143

0.01

420.

0084

CH

E-K

U0.

0625

0.14

610.

0323

0.05

790.

0222

0.07

590.

0203

0.01

920.

0265

0.26

390.

0219

0.02

030.

0051

0.00

48

KU

R-IS

0.13

480.

0809

0.06

300.

1028

0.10

080.

0705

0.07

850.

0967

0.15

020.

0359

0.17

470.

1711

0.11

140.

1054

0.13

67

KU

R-Y

O0.

1008

0.14

880.

0154

0.01

490.

0010

0.05

610.

0297

0.02

080.

0378

0.22

150.

0370

0.03

650.

0036

0.01

610.

0165

0.09

80

OM

S-O

M0.

1087

0.06

500.

0725

0.11

240.

0573

0.14

260.

0091

0.00

840.

0075

0.27

470.

0094

0.01

030.

0347

0.01

040.

0225

0.12

950.

0452

OM

S-PO

0.10

130.

0611

0.06

710.

1070

0.05

410.

1294

0.00

870.

0085

0.01

030.

2458

0.01

470.

0146

0.03

330.

0100

0.02

230.

1153

0.04

330.

0012

PAV

-ZH

0.06

090.

0931

0.07

850.

1469

0.08

390.

1327

0.01

840.

0318

0.03

440.

2708

0.03

250.

0313

0.04

660.

0193

0.02

440.

1480

0.06

920.

0131

0.01

41

PAV

-DA

0.07

440.

1067

0.07

520.

1274

0.06

730.

1343

0.01

870.

0234

0.02

140.

2999

0.01

860.

0178

0.03

450.

0126

0.01

480.

1626

0.05

460.

0087

0.01

050.

0031

VK

A-V

O0.

1253

0.05

420.

0786

0.11

550.

0610

0.15

170.

0101

0.00

680.

0057

0.26

730.

0103

0.01

150.

0404

0.01

370.

0297

0.12

250.

0483

0.00

160.

0032

0.01

900.

0143

NSK

-2R

0.06

590.

0649

0.05

070.

1066

0.05

760.

1044

0.00

310.

0150

0.02

500.

2199

0.02

560.

0255

0.03

210.

0098

0.02

100.

0989

0.04

480.

0078

0.00

800.

0071

0.00

990.

0107

NSK

-3R

0.08

810.

0443

0.04

520.

0912

0.05

040.

1009

0.00

120.

0096

0.02

380.

1883

0.02

860.

0293

0.03

400.

0128

0.03

090.

0699

0.03

870.

0112

0.01

070.

0224

0.02

490.

0104

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48

NSK

-KR

0.11

460.

0162

0.10

300.

1845

0.12

880.

1560

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890.

0506

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930.

1587

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280.

0828

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210.

0589

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080.

0680

0.11

120.

0409

0.03

780.

0424

0.05

850.

0365

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660.

0197

ALT

-TG

0.15

910.

0480

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090.

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420.

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210.

0106

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950.

2397

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190.

0214

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150.

0248

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540.

1103

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380.

0121

0.01

080.

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280.

0065

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750.

0204

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36

ALT

-TR

0.13

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120.

0986

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940.

1514

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230.

0071

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3164

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270.

0051

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090.

0117

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320.

1450

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730.

0048

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0251

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630.

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490.

0155

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0.13

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400.

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700.

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0887

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0895

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ALT

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0.11

160.

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0079

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760.

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440.

2027

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740.

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690.

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640.

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540.

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730.

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98


heterozygous (A1.2) states appeared almost inequal frequency. These results support Peder-

sen's (1978) idea that “polymorphism can beused as a biological system that very quicklyrecords change in the environments”. In both spe-cies the polymorphous system could be treated asan “adaptive population strategy” determined bythe fluctuation of homo-and heterokaryotypes.

C. annularius also possesses substantial chro-mosome variability in its range. The standardbanding patterns of arm A have been describedonly from Germany (Keyl and Keyl 1959), whileheterozygous and inverted homozygous stateshave been observed in most populations in Ger-many (Keyl and Keyl 1959; Keyl 1962), Russia,Bulgaria, Hungary (Beljanina 1981; Kiknadze etal. 1996; Petrova and Michailova 1986) andPoland (Michailova et al. 2002). Also, in arm Fheterozygotes have been found in these popula-tions. Beljanina (1981) and Petrova andMichailova (1986) noted that microdifferentia-tion was occurring in Russian populations due toseveral fixed homozygous inversions in the karyo-type of the species. In the studied area C. annular-ius was found in a water body characterized by anincreased level of organic contaminants. In thisarea we observed a new homozygous inversion inarm B in a low frequency.

Interestingly, differences in the amount of cen-tromeric heterochromatin of chromosomes wereobserved in C. plumosus. Differences in the quan-tity of heterochromatin show how this part of thegenome evolves. Quantitative variation in hetero-chromatin among species is probably due to sev-eral mechanisms such as multiple replication, un-equal exchange, amplification, accumulation anddeletion (John 1988). Such differences have alsobeen observed in other geographically isolatedPalearctic populations of C.plumosus: as in Russia(Kiknadze et al. 1991b; Petrova 1991; Golygina

and Kiknadze 2001), Hungry, Switzerland(Michailova and Fisher 1986), and Finland(Michailova 2001). Variation may arise from am-plification events in centomeric heterochromatinand illustrates the process of cytogenetic differen-tiation. Molecular methods employed in the fu-ture will provide additional information on theevolution of this species. However, this cytoge-netic mechanism is not yet involved in postmatingisolation factors, which was proven by a hybridi-zation test (Michailova and Fisher 1986).

C. annularius was found in a water body char-acterized by an increased level of organic contami-nants (Michailova et al. 2001). Changes were ob-served in the functional activity of BRs in chromo-

some G and the nucleolar organizer (NOR) inchromosome arms A and E. The nucleolar organ-izer in chromosome AB is active in Russian(Kiknadze et al. 1991a, 1996) and other Polishpopulations (Michailova et al. 2002). Also,Kiknadze et al. (1996) discerned several well-ex-pressed Balbiani rings in chromosome G, whichwere not seen in our material. We observed onlyone slightly active BR in the telomeric region ofchromosome G. Both structures (BRs and NOR)are very sensitive to different stress conditions andare used as a model to study the response of thegenome to different environmental factors (Diez

et al. 1990; Michailova et al. 1998). BRs are sitesof intense gene transcription for silk proteins (Wi-

eslander 1994), important for the constructionof larval tubes. Obviously the BR system seems tobe very sensitive to high levels of organic contami-nants detected in this area by Michailova et al.(2001). Most BRs observed in Russian popula-tions (Kiknadze et al. 1996) were not seen in thestudied area. Only one BR, located in the telomereregion of chromosome G, appeared slightly activeor in heterozygous state. Also, the nucleolar or-ganizers (NOR) in arms A and E were suppressedor appeared in a heterozygous state,which mightbe due to the organic contaminants, established inthe studied area. The NOR contains genes re-sponsible for the synthesis of rRNA. Suppressionis probably due to inhibition of RNA polymerase I(Horgen and Greffin 1971) or to the synthesis ofhighly conservative proteins (HSPs) which have aprotective role in the cells (Multohoff et al.1998). However, it is still unclear which mecha-nism is involved in the suppression of NOR activ-ity. The same reaction was observed in naturalpopulations of C. riparius in different anthropo-genic sites (Michailova et al. 1996; 2000).

Different hypotheses on the evolutionary ori-gin of “B” chromosomes have been proposed.The most widely accepted view is that they origi-nated from A chromosomes because of homologybetween the two types (Camacho et al. 2000).The molecular cytogenetic characteristics of Chi-ronomid “B” chromosome was studied by (Siirin

et al. 2003). Applying in situ hybridization, theseauthors found that “B” chromosome are com-posed of repetitive DNA sequences homologousto sequences of centromere and telomere DNA ofA chromosomes and also these of mobile elementsNLRCth1. No ribosomal DNA repeats were iden-tified in “B” chromosome. They postulated that“B” chromosome and chromosome G have simi-lar telomeric and centromeric organization whichinfluence on their very repeated association. In


the studied species (C. plumosus) “B” chromo-some frequently pair with chromosome G.

The polytene “B” chromosome has been es-tablished in different populations of C. plumosus(Keyl and Hagele 1971; Ilynskaja and Petrova

1985; Michailova and Mettinen 2000; Goly-

gina and Kiknadze 2001). Ilinskaja andPetrova (1985); Michailova and Mettinen

(2000) considered that “B” chromosome in-creased the chances of survival for individuals un-der extreme, polluted conditions and in this wayproposed their selective value. “B” chromosomewas found by us in both groups of C. plumosus inthe studied area, which is characterized by high al-kaline reaction and oxygen demand.

Acknowledgements — This research was sup-ported by a grant from an exchange program betweenthe Bulgarian Academy of Sciences and the PolishAcademy of Sciences. Thanks are due to Dr. L. Gunde-rina and Dr. V. Golygina (Novosibirsk), Institute forCytology and Genetics for their useful suggesting thecluster analysis. The authors thank Dr. Ollik and Dr.Zbikowski for collecting some of the studied material.

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Received II.14.2005; accepted VII.07.2005


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Cytogenetic variability in species of genus Chironomus (Dip-tera, Chironomidae) from Poland