Karyotype and chromosomal polymorphism of Chi- ronomus luridus Strenzke, 1959 (Diptera: Chironomidae) in European and Asian populations

The karyotype structure and chromosomal polymorphism were studied in several European (European part of Russia and the Netherlands) and Asian (Siberia and Kazakhstan) populations of Chironomus luridus Strenzke, 1959. Inversion polymorphism was detected in six of the seven chromosome arms: three banding sequences detected in arm A, six sequences in arm B, two sequences in arm C, three sequences in arm E, fi ve sequences in arm F, and two sequences in arm G. Only arm D was monomorphic in all studied populations. In total, 22 banding sequences were recorded in Ch. luridus; they form the banding sequence pool of this species. Thus, Ch. luridus can be regarded as a very polymorphic species. However, the European and Asian populations differed considerably in the levels of polymorphism: the Asian populations were less polymorphic, containing only 8 to 10 sequences, whereas the European populations had 11 to 16 sequences. The new banding sequences lurA3, lurB5, lurE3 were found in Asian populations, whereas the sequences lurB2, lurB3, lurB4, lurB6, lurE2, lurF1, lurF2a, lurF3, lurF4, and lurG2 аre lacked. The total level of inversion heterozygosity in the Asian populations was 12-25% versus 78-80% in the European populations.


INTRODUCTION
Inversions play a key role in the evolution of animal karyotypes.They change the normal order of genes within a chromosome, which has important consequences in the evolution of a species.Molecular analysis of genomes and proteomes has confi rmed that the genomes of distant species, such as man, domestic mouse, Drosophila Fallén, 1823, and Anopheles Meigen, 1818 differ mainly by the order of genes in chromosomes (linkage group) rather than the number and the set of genes (Zdobnov et al., 2002;Ayala, Coluzzi, 2005).The contributions of other chromosome rearrangements, in particular, reciprocal whole-arm translocations, are more limited (White, 1973;King, 1993).However, for the genus Chironomus Meigen, 1803, reciprocal whole-arm translocations proved to be the main rearrangements that have led to the formation of the so-called cytocomplexes of species, differing in the combinations of seven chromosome arms (Keyl, 1962;Martin, 1979Martin, , 2007;;Wülker, 1980Wülker, , 2007)).In particular, the karyotypes of the species belonging to the "thummi" cytocomplex have the chromosome arm combination AB CD Comp. Cytogenet., 2010 4(1) Comparative Cytogenetics EF G in the four chromosomes of the haploid set; "pseudothummi" cytocomplex -AE CD BF G; "camptochironomus" cytocomplex -AB CF ED G; "parathummi" cytocomplex -AC ED BF G; "maturus" cytocomplex -AF CD EB G; and so on.The "thummi" and "pseudothummi" cytocomplexes contain the most species.The karyotype structure and chromosomal polymorphism have been so far comprehensively studied only in the "thummi" cytocomplex.The species of the "pseudothummi" cytocomplex still require further study.In addition, the effect of reciprocal whole-arm translocations on the structure of the centromeric regions in AE and BF translocated chromosomes has been recently demonstrated in Ch. dorsalis Meigen, 1818, a member of the "pseudothummi" cytocomplex.The translocated chromosomes became dicentric (Kiknadze et al., 2008b).A loss of one of the centromeres and appearance of a neocentromere has been suggested in the translocated chromosome AE in another species of this cytocomplex, Ch. saxatilis Wülker, Ryser et Scholl, 1981(Shobanov, Petrova, 1995).It is still unclear whether such phenomena are characteristic of only some members of the "pseudothummi" cytocomplex or they are common to all the species of this cytocomplex.In addition, it has been demonstrated that the changes in chromosome arm combinations (linkage groups combinations in Diptera) result in emergence of new inversion breakpoints stimulating divergence of cytocomplexes during evolution (Kiknadze et al., 2003).
In this work, we have studied in detail the karyotype of Ch. luridus, a member of the "pseudothummi" cytocomplex.Earlier the karyotype of Ch. luridus was described by Keyl and Keyl (1959) with mapping of arms A, E, and F (Keyl, 1962).Additional information about the Ch.luridus karyotype has been reported by Belyanina (1983), Kiknadze et al. (1988Kiknadze et al. ( , 1991) ) and Michailova (1989).The chromosome polymorphism in arms A, E, and F in German populations was briefl y described by Keyl (1962).A high level of chromosome polymorphism in Western European Ch. luridus populations was noted by Acton (1957), although this author erroneously identifi ed this species as Ch.dorsalis.We have evaluated quantitatively the chromosome polymorphism in several European and Asian populations.We were the fi rst to map chromosome arms C and D and discover inversion polymorphisms in six of the seven chromosome arms (arms A, B, C, E, F, and G).Considerable differences between the levels of chromosome polymorphism of the European and Asian populations studied were found.Structural changes in the centromeric regions on translocated chromosomes were studied.

Karyotype
The karyotype of Ch. luridus (Fig. 1) has a haploid number n=4 with the chromosome arm combinations AE CD BF G (the "pseudothummi" cytocomplex).Chromosomes CD and BF are metacentrics; AE, submetacentric; and G, telocentric.The nucleolus is single and is localized on arm G near the centromeric-telomeric end.The karyotype contains four Balbiani rings: three in arm G and one in arm B (Fig. 1).The karyotype of Ch. luridus from different populations studied was identical with standard, described by Keyl, Keyl (1959) and Keyl (1962).

Banding sequences East European (Yaroslavl) population
Arm A is polymorphic and occurs in two banding sequences, lurA1 and lurA2, differing by a simple paracentric inversion (Tables 2-3; Figs 2, a; 3, a).The sequence lurA1 is identical to dorA1 in Ch. dorsalis and differs from the standard sequence pigA1 by only three overlapping inversions: The sequence lurA1 is predominant in the populations studied, whereas the sequence lurA2 is rare and observed only as heterozygotes (Tables 2-3).The additional sequence lurA3 formed by short pericentric inversion was found in Novosibirsk populations.
Arm E is polymorphic and has two banding

MATERIAL AND METHODS
Ch. luridus larvae of the last (fourth) instar were used in the work.The collection sites and sample sizes are listed in Table 1.The larvae were fi xed in a mixture of 96% ethanol and glacial acetic acid (3:1) and stored in a refrigerator.Squash preparations of the salivary gland polytene chromosomes were made conventionally, using aceto-orcein staining (Kiknadze et al., 1991).Polytene chromosome arms A, E, and F were mapped according to Keyl (1962) and arms C and D, according to Dévai et al. (1989), using the banding sequences of Chironomus piger Strenzke, 1959 polytene chromosomes as a standard.Arms B and G were not mapped due to complex chromosome rearrangements, as compared with Ch. piger.Inversion banding sequences of polytene chromosomes were designated using the abbreviated species name, arm designation, and banding sequence number (lurA1, lurA2, lurA3, lurD1, lurB2 etc.).Genotype combinations of banding sequences were designated as lurA1.1,lurB1.1,lurC1.1 etc., for homozygotes and as lurA1.2,lurB1.2, and lurB1.3,for heterozygotes.
The following cytogenetic characteristics of chromosomal polymorphisms in populations were used: the set, number and frequency of banding sequences and their genotypic combinations, the percent of the larvae with heterozygous inversions, and the mean number of heterozygous inversions per individual.
The studied cytological slides and fi xed larval body after dissection of salivary glands are preserved in the collection of the Institute of Cytology and Genetics of Russian Academy of Sciences, Novosibirsk, Russia.
Equipment of the Center of Microscopy Analysis of Biological Objects of SB RAS in the Institute of Cytology and Genetics (Novosibirsk) was used in this work: microscope Comp.Cytogenet., 2010 4(1) Comparative Cytogenetics  The sequence lurE1 is predominant, whereas lurE2 is very rare and has been detected only as heterozygotes.The former sequence is close to pigE1 and differs from it only by a simple inversion: The sequence lurE1 is identical with many Chironomus species (basic sequence).Characteristic of lurE1 is a loosened state of region 5 (dark puff), which suggests a transcriptional activity of this region.The additional sequence lurE3 formed by short pericentric inversion was found in Novosibirsk populations.
Arm C is polymorphic and occurs in two banding sequences, lurC1 and lurC2, differing by one large simple inversion (Tables 2-3; Figs 2, c-d; 3, d).The sequence lurC1 differs from the standard pigC1 by three included inversions in the distal part of the arm.The proximal part (regions 9a-22) is completely identical to pigC1: The sequence lurC1 is dominant, but lurC2 is still rather frequent (Table 2), mainly as heterozygotes (Table 3).
The banding sequences in arm B have not been mapped.The localization of heterozygous inversions is shown in Figs 2, f; 3, e-i.The inversion in lurB2 sequence covers the central part of the arm (Figs 2, f; 3, e); the inversion in lurB3 sequence is located at the end of the arm (Figs 2, f; 3, f); and the inversion in lurB4 is small and located at the proximal part of the arm (Figs 2, f; 3, g).
Arm G is weakly polymorphic with a predominance of lurG1 (Tables 2-3; Figs 2, i-k).Only one larva displayed the heterozygote lurG1.2.Arm G in Ch. luridus is very similar in the localization and molecular characteristics of transcriptionally active locinucleolus and Balbiani rings -with arm G in the standard pigG1 (Kiknadze et al., 1989).
Correspondingly, the designations of Balbiani rings (BRa, BRb, and BRc) (Figs 2, i-j) in these two species coincide.The inversion in lurG2 sequences covers the entire central part of the arm, including the nucleolus and two Balbiani rings, BRb and BRc (Fig. 2, k).However, the overall banding sequence in Ch. luridus arm G is considerably changed due to complex rearrangements in comparison with standard Ch.piger.
The Balbiani rings BRb and BRc function in all the cells of salivary glands, whereas the

Comp. Cytogenet., 2010 4(1)
Comparative Cytogenetics form 19 genotypic combinations.The level of chromosome polymorphism was also rather high, as up to 80% of individuals in the population were inversion heterozygotes (Table 4).Arms B and F, carrying four and three banding sequences, respectively, appeared to be the most polymorphic.
The specifi c feature of the Yaroslavl population, as compared with the earlier ring BRa develops only in four cells of the salivary gland special lobe (Fig. 2, j), where an additional secretory protein is synthesized (Kiknadze et al., 1989).
The overall pool of banding sequences of the Yaroslavl population appears rather large owing to the chromosomal polymorphism in arms A, B, C, E, F, and G.In total, 16 banding sequences were discovered, which studied German populations (Keyl, 1962), is the absence of the sequence lurF1.In addition, we discovered new banding sequences -lurA2, lurB2, lurB3, lurB4, lurC2, lurF4, and lurG2 -in this population.

Western European populations
Only nine Ch. luridus individuals from several water bodies of the Netherlands, Germany, and Belgium were available (Table 1).The main banding sequences in them were identical to those in the population studied in East Europe (Yaroslavl) (Tables 2-3).The western European populations were also similar to the Yaroslavl population in having a high level of chromosome polymorphism (Table 4) and polymorphic arms C, B, F, and G. Their main distinction was the discovery of heterozygotes with the sequence lurF1, undetected in the Yaroslavl population (Tables 2-3; Fig. 3, j).Among the individuals from the Netherlands, lurC2.2 and lurF3.3homozygotes (Figs 2,d,h,respectively) were detected as well as lurB1.6 heterozygote (Fig. 3, i), which has not been found in other populations.The sequence lurF3 was formed by long simple inversion from lurF2:

Novosibirsk populations
In general, the main banding sequences discovered in Novosibirsk populations are identical to the sequences found in the European population (Table 2).However, the Novosibirsk populations are considerably less polymorphic (Tables 2-4).In total, only 11 banding sequences were detected, and the level of heterozygosity in these populations was sevenfold lower.Four arms (A, B, C, and E) were heterozygous versus six arms in the Yaroslavl population.All the heterozygotes detected for these arms were rather rare.The characteristic features of the Novosibirsk populations studied are a complete domination of lurF2.2homozygotes and the absence of the wide range of heterozygotes in this arm, which is typical of European populations (Table 3).One larva displayed a unique pericentric inversion covering the centromeric region in chromosome AE (Fig. 3, c), which gave rise to the sequences lurA3 and lurE3.Inverted regions in both sequences, including centromeric band, are distinguished by square brackets In addition, the sequence lurB5 (Fig. 3, h) has been detected only in this population.It was not mapped.
Thus, the Novosibirsk populations are considerably less polymorphic compared with the European populations.
Kazakhstan populations These populations were very similar to the Novosibirsk populations in the range and frequencies of inversion banding sequences and in a low level of chromosomal polymorphism (Tables 2-4).Overall, eight banding sequences were detected in these populations.

Comp. Cytogenet., 2010 4(1)
Comparative Cytogenetics "pseudothummi" cytocomplexes (Kiknadze et al., 2008b).The translocated chromosomes AE and BF of Chironomus dorsalis Meigen, 1818, a member of the "pseudothummi" cytocomplex, appeared dicentric as compared with the untranslocated chromosomes AB and EF in species of "thummi" cytocomplex due to appearance of the centromeric characteristics of bands 19ef in chromosome AE and the bands 28de in chromosome BF.The neocentromeric activity of these bands was suggested.
We have shown that the karyotype of Ch. luridus has a high level of chromosomal polymorphism; six of seven chromosome arms (A, B, C, E, F, and G On the contrary, the Siberian and Kazakhstan populations display considerably smaller sets and lower frequencies of inversion sequences, as well as a lower total level of inversion heterozygosity.In these populations mainly arm C was polymorphic, but very rare heterozygotes in arm B were also recorded.Among the most pronounced distinctions between the European, Siberian, and Kazakhstan populations was a drastic decrease in the sets and frequencies of inversion polymorphism in arm F. The studied Western  The sequence lurF1, described by Keyl (1962), was not detected in any of the studied populations except for the population from the Netherlands.Keyl noted that the four German populations that he studied displayed differences connected with the presence of lurF1 and lurF2; unfortunately, he did not mentioned whether these differences were connected with the occurrence of homozygotes at lurF1.1 and lurF2.2 or only with lurF1.2heterozygotes.Strangely, Keyl did not give any photograph of either homozygote lurF1.1 or homozygote lurF2.2,which considerably hinders the comparison of the polymorphism in Western European populations with Eastern European, Siberian, and Kazakhstan populations.However, the fact that the difference connected with the inversion polymorphism of Ch. luridus populations is essentially associated with the polymorphism in arm F is undoubted.
In spite of some differences in banding sequences between populations we view these differences as falling within the range of intraspecies polymorphism as well in many other Chironomus species (Gunderina, Kiknadze, 1999;Kiknadze, 2008).
According to Dobzhansky (1970), central populations of a species are the most polymorphic.So, it is possible to suggest that European populations of Ch. luridus can be considered as central while Asian populations as peripheral.
The karyotypes of the species belonging to the "pseudothummi" cytocomplex differ from the karyotypes of the species belonging to the "thummi" cytocomplex by the presence of reciprocal whole-arm translocations in two chromosomes: AE and BF in the "pseudothummi" cytocomplex have become AB and EF in the "thummi" cytocomplex.
The specifi c feature of the Ch.luridus

Fig. 2
Fig. 2, a, b.Banding sequences in the arms A and E of Chironomus luridus.a -lurA1.1.b -lurE1.1.The designations are the same as in Fig. 1.

Fig. 2
Fig. 2, i, k.Arm G in Chironomus luridus.i -lurG1.1 from the cells of salivary gland main lobe.j -lurG1.1 from the cells of salivary gland special lobe.k -heterozygote lurG1.2.BRa, BRb, and BRc are Balbiani rings a, b, and c.The rest designations are the same as in Fig. 1.
, b).There are no two heterochromatinized bands (centromere and neocentromere) in the translocated chromosomes AE and BF in Ch. luridus.The centromeric region of chromosome AE has a very weak centromeric band (Fig. 4, a), bands 19ef are identical with standard.The centromeric region of chromosome BF has also very thin bands 28de (Fig. 4, b), which Comp.Cytogenet., 2010 4(1) Comparative Cytogenetics
) are polymorphic except arm D. In total, 22 banding sequences form the banding sequences pool of this species.Comparative study of the chromosomal polymorphism in different Ch.luridus populations has demonstrated signifi cant differences among the populations in the sets and frequencies of inversion banding Comp.Cytogenet., 2010 4(1) Comparative Cytogenetics sequences.The most polymorphic of the populations studied was the Yaroslavl population, which contained 16 banding sequences and six polymorphic chromosome arms.The overall level of polymorphism in this population was also high, as almost 80% of individuals in the population were inversion heterozygotes.The populations from Western Europe were similar to the Yaroslavl population in having a high level of polymorphism.

Fig. 4
Fig. 4, a, b.Mapping of the centromeric regions in chromosomes AE (a) and BF (b) of Chironomus luridus, Ch. piger, and Ch.muratensis Ryser, Scholl, Wülker, 1983.Solid arrows show the centromeric bands, and dashed arrows indicate bands 19ef on arm A (a) and 28de on arm B (b), which demonstrate neocentromeric characters in Ch. dorsalis

Table 1 .
Collection sites and number of Chironomus luridus larvae analyzed.

Table 2 .
Frequencies of banding sequences in natural populations of Chironomus luridus.*Keyl'sdata(1962), frequencies of banding sequences were not determined; presence of banding sequence is marked by +.N -the number of individuals.

Table 3 .
Frequencies of genotypic combinations of banding sequences in natural populations of Chironomus luridus.*Keyl'sdata(1962), frequencies of genotypic combinations of banding sequences were not determined; presence of a combination is marked by +.N -the number of individuals.

Table 4 .
Inversion polymorphism in Chironomus luridus populations.N -the number of individuals.