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Karyotype characteristics and polymorphism peculiarities of Chironomus bernensis Wülker & Klötzli, 1973 (Diptera, Chironomidae) from the Central Caucasus and Ciscaucasia

Abstract Data about the karyotype characteristics, features of chromosomal polymorphism and larval morphology of populations of Chironomus bernensis Wülker & Klötzli, 1973 (Diptera, Chironomidae) from the Central Caucasus (the northern macroslope) and Ciscaucasia are presented. The characteristics of the pericentromeric regions of the long chromosomes of this species from Caucasian populations were very similar to the ones from some European populations (from Poland and Italy), but differed from Swiss and Siberian populations. In the North Caucasian populations 10 banding sequences were found: two in arms A, C, and E, and one in arms B, D, F, and G. Nine of them were already known for this species, and one, berC2, is described for the first time. Cytogenetic distances between all the studied populations of Chironomus bernensis show that close geographical location of all studied populations from the Central Caucasus and Ciscaucasia is reflected in their similar cytogenetic structure, but on the other hand, that they are more closely related to populations from Europe than to populations from Western Siberia. At the same time, all studied larvae from Caucasian populations have a four-bladed premandible, instead of a two-bladed one, as in the description of Chironomus bernensis from Switzerland (Wülker and Klötzli 1973, Polukonova 2005c). These peculiarities may indicate the relative isolation of the Caucasus from the viewpoint of microevolution. Further research on karyological and morphological characteristics of Chironomus bernensis from geographically distant regions is necessary as there is a possibility that the presently known species is actually polytypic and consists of several sibling species.

The karyotype of Ch. bernensis was studied early-on from Switzerland (Wülker and Klötzli 1973), Bulgaria, Poland, Northern Italy (Michailova 1989, Petrova and Michailova 2002, Michailova et al. 2009) and Spain (Real et al. 2000). In Russia this species was known only from Western Siberia and the chromosomal polymorphism of those populations was described by Istomina and Kiknadze Kiknadze 2004, Kiknadze et al. 2007).
The aim of this work is to present the description of karyotype, chromosomal polymorphism and larval morphology of Ch. bernensis from the Central Caucasus (the northern macroslope) and Ciscaucasia -Republic of Kabardino-Balkaria (RKB), Republic of North Ossetia-Alania (RNO-Alania), Karachai-Cherkess Republic (KCR) and Stavropol Krai. It was also important to compare characteristics of chromosomal polymorphism of Ch. bernensis from Caucasus, Western Europe and Western Siberia.

Methods
Fourth instar larvae were used in the karyological study. The larvae were collected from 12 sites of the Central Caucasus and Ciscaucasia: seven sites from Republic of Kabardino-Balkaria (RKB), one site from Republic of North Ossetia-Alania (RNO-Alania), one site from Karachai-Cherkess Republic (KCR), and four sites from Stavropol Krai (Table 1). In the aspect of the vertical zonation the site in KCR belongs to the Kuban variant, all sites in Stavropol Krai belong to the steppe zone and all sites in RKB and RNO-Alania belong to the Terek variant (typification of the zone variants are given according to Sokolov and Tembotov 1989).
The identification of chromosome banding sequences for arms A, E and F was performed with use of photomaps of Wülker and Klotzli (1973) in the system of Keyl (Keyl 1962) and chromosome mapping for arms C and D was performed according to Istomina and Kiknadze (2004) in the system of Dévai et al. (Dévai et al. 1989). Micro-scope Carl Zeiss Axio Imager.A2 was used to study chromosome slides. Software packages PAST 2.17 and STATISTICA 10 were used for statistical analysis (cluster analysis).
The following parameters were used for comparison of characteristics of chromosomal polymorphism: the number of zygotic combinations, percentage of heterozygous larvae, number of heterozygous inversions per specimen, number of inversions per arm, number of banding sequences in a population.
Cytogenetic distances between populations were calculated according to Nei (Nei 1972).

Results
The larvae of the genus Chironomus Meigen, 1803 in all studied sites of the Central Caucasus and Ciscaucasia were attributed to Ch. bernensis by chromosomal and morphological characteristics. Morphological characteristics are presented on Fig. 1a-g. In general, the larval characters of Ch. bernensis from Caucasian sites are similar to those described previously for this species by Wülker and Klötzli (1973), however, some noticeable distinctions were found. Thus, it was stated by Wülker and Klötzli (1973) that larva of Ch. bernensis was not different from that of Ch. commutatus. Indeed, both species have the same type of larva ("bathophilus"), degree of gular sclerite pigmentation and structure of mentum and antenna. However, the fourth tooth of mandible of Ch. bernensis from Caucasian populations was dark brown or dark (Fig. 1e), while it is pale brown in Ch. commutatus according to Shobanov (2000). It is possible that Wülker and Klötzli (1973) did not notice this distinction. Another morphological peculiarity that was revealed was the presence of four-bladed premandibles in all studied larvae (Fig. 1f ) instead of the two-bladed ones of Ch. commutatus (Laville 1971, Polukonova 2005c. The exterior tooth of the premandible in Ch. bernensis larvae of the North Caucasian populations was 2-2.5 times narrower than the inner one, longer and awl-shaped at the edge, the inner tooth was split into two small additional teeth near its basis (Fig. 1f ).

Karyotype of Ch. bernensis from the Central Caucasus and Ciscaucasia
The diploid number of chromosomes in Ch. bernensis karyotype is 2n=8, chromosome arm combination is AD, BC, EF, G -"lacunarius" cytocomplex ( Fig. 2). Chromosomes AD and BC are metacentric, EF is submetacentric and G is telocentric. Two well developed nucleoli (N) are located on arms A and E. There are two Balbiani rings (BR) in the karyotype: one is situated in arm B and the other -in arm G, but in populations that we have studied the activity of both both BR was greatly reduced (Fig. 2). The centromeric bands of long polytene chromosomes of Ch. bernensis from the studied populations are large and belong to n-type (according to the classification by Shobanov (2002)). One of the peculiarities of the karyotype of Ch. bernensis, as indicate before by Istomina and Kiknadze (2004), is comparatively large telomeres of all chromosomes that often results in a presence of ectopic pairing between different chromosomes. We also observed such ectopic pairing with very low frequency and without any clear pattern between arms B and D in some specimens from different collection sites of Caucasus.

Banding sequences and chromosomal polymorphism of Ch. bernensis from the Central Caucasus and Ciscaucasia
Up until now, 16 banding sequences have been described in the banding sequences pool of Ch. bernensis (Table 2). In populations studied in this paper only 9 of those banding sequences were present, and one banding sequence has been found for the first time, so in total 10 banding sequences were found in Caucasian populations (Table 3).
Arm B was monomorphic. Banding sequence berB1 remain unmapped due to the complex rearrangements that differ the banding pattern in the arm B of Ch. bernensis from the standard one of Ch. piger. Table 2. Catalog of banding sequences in the banding sequences pool of Ch. bernensis.
In all three North Caucasian populations the number of banding sequences was identical and equal to 10 ( Table 3). The number of zygotic combinations found in studied populations varied from 11 to 12 (Table 4). From 59 to 82% of larvae were heterozygous (Table 4).
In total, 12 genotypic combinations have been found (

Comparison of chromosomal polymorphism of Ch. bernensis from the Central Caucasus and Ciscaucasia and other parts of the range
As stated above, in all the long polytene chromosomes of Ch. bernensis from the studied North Caucasian populations the centromere bands are large and belong to n-type   according to the classification by Shobanov (2002) (Fig. 6). In Siberian populations Kiknadze 2004, Kiknadze et al. 2007) and in the photo of chromosomes in the first description of Ch. bernensis from Swiss populations (Wülker and Klötzli 1973), the centromere bands are thin and belong to s-type. The large centromeric bands of this species were found in the populations of Bulgaria, Poland, Northern Italy (Michailova 1989, Petrova and Michailova 2002. Data for Polish and Italian populations are presented on the basis of publications of Michailova (1989); Michailova and coauthors , Petrova and Michailova (2002), data for Siberian populations are presented on the basis of publications of Istomina and Kiknadze (2004), Kiknadze and coauthors (Kiknadze et al. 2007).
Unfortunately, because of the low number of specimens of Ch. bernensis found in most populations of Central Caucasus and Ciscaucasia water bodies studied, only three populations with a significant number of larvae -the former riverbed in the mouth of the Cherek River near Oktyabrskaya village, the long-term water body near Zhemtala village, the backwater in the main riverbed of Malyi Zelenchuk River near Adyl-Khalk villagewere used for comparison with populations from other geographic regions (Table 3, 4).
Arm A. The populations from the North Caucasus, as well as populations from Europe-Switzerland (Wülker and Klötzli 1973) and Italy (Petrova and Michailova 2002) -are characterized by the presence of two banding sequences in this arm, berA1 and berA2 (Table 3, 4), whereas only berA1 was present in populations of Western Siberia (Istomina and Kiknadze 2004). At the same time it should be noted that populations from the North Caucasus and Europe differ significantly by the frequencies of banding sequence berA1 and berA2: while the former was dominant in Western Europe, the latter dominated in North Caucasian populations, occurring there in both the heterozygote and homozygote state.
Arm B and D of Ch. bernensis were monomorphic in all studied populations. Arm C of Ch. bernensis were monomorphic in populations from Europe and Siberia but showed high level of inversion polymorphism in studied Caucasian populations due to the presence of a new banding sequence berC2 that might be endemic for this region. However, for Ch. bernensis from Spain unmapped chromosomal rearrangement in the arm C was early indicated (Real et al. 2000). The high frequencies of heterozygotes berC1.2 and homozygotes berC2.2 in Caucasian populations (Table 3, 4) clearly distinguishes them from all other populations.
In the arm E all studied populations of Ch. bernensis share the same dominant banding sequence berE1. At the same time populations from all regions differ from each other by sets of additional banding sequences found in heterozygote state. Thus, in Switzerland this arm was completely monomorphic (Wülker and Klötzli 1973), in Italy two banding sequences -berE2 and berE3 (Petrova and Michailova 2002) -were found with low frequencies in heterozygotes with berE1, while only heterozygotes berE1.3 were found in Caucasian populations and berE1.4 -in populations from Western Siberia (Istomina and Kiknadze 2004). The comparison of the inversion banding sequences of the arm E from different populations shows the most similarity between Caucasian and Italian populations.
Arm F of Ch. bernensis in Caucasian populations was monomorphic and presented only by the standard banding sequence berF1 unlike the populations from other regions. In the population of Switzerland (Wülker and Klötzli 1973) the approximately equal number of homo-(ber F1.1) and heterozygotes (ber F1.2) was observed. In the Siberian population banding sequence berF2 was strictly dominant with the only other banding sequence being berF4 that was present with a low frequency in a heterozygote state (berF2.4) (Istomina and Kiknadze 2004), which clearly distinguishes the Siberian population of Ch. bernensis.
Arm G of Ch. bernensis was monomorphic in both European and Caucasian populations and was presented by the standard banding sequence berG1. At the same time in the Siberian population three banding sequences were found in different zygotic combination (Istomina and Kiknadze 2004) with berG1.2 being the dominant one, which clearly distinguishes this population from the other ones.
Thus, summarizing all data it can be concluded that a significant degree of divergence can be seen between populations of Europe, Caucasus and Western Siberia.
The inversion polymorphism of populations of Ch. bernensis from the North Caucasus has much higher level of heterozygous inversions per specimen in comparison with the early studied populations, i.e. 0,65 to 1,11 (Tables 3-5). In the number of genotypic combinations (11), number of banding sequences per population (10) and number of inversions per arm (0,43), the Caucasian populations of this species are intermediate between European (respectively: 10, 9 and 0,29) and Siberian (respectively: 13, 11 and 0,71) populations.
Cytogenetic distances (Table 6), was measured by Nei criteria (1972) on basis of the original data and data of other authors on inversion polymorphism of the species in Europe and Siberia (Fig. 7). These distances indicate the significant distance of the Siberian populations of Ch. bernensis and of intermediate position of the Caucasian populations between the populations of Western Europe and Western Siberia.
The dendrogramm was constructed on the basis of Nei criteria (1972) using NJmethod.  In establishing of cytogenetic distances for populations of Siberia, Switzerland and Italy data of other authors were used (Wülker and Klötzli 1973, Petrova and Michailova 2002, Istomina and Kiknadze 2004).

Discussion
In the Central Caucasus (the northern macroslope) and Ciscaucasia Ch. bernensis has been found for the first time. At present, 17 banding sequences including berC2 are known in the banding sequences pool of Ch. bernensis. The comparative analysis of chromosomal polymorphism between the Caucasian populations and populations of other regions has revealed specific peculiarities: the presence of sequence berA2 in homozygous state, which was not registered in the populations studied earlier, and the presence of banding sequence berC2, which is probably endemic for the region.
The morphological characteristics such as the number of premandible teeth are diagnostic for Chironomus species. Thus, among the species of this genus more than two teeth of the premandible can be found in larvae dwelling in the brackish water bodies, i.e. Ch. behningi Goetgh. with five teeth (Pankratova 1983, Polukonova andBeljianina 2002); Chironomus albidus Konst. (Konstantinov 1956) and Ch. sp. (sibling species of Chironomus albidus, apparently belonging to Ch. paraalbidus ) with three teeth (Polukonova et al. 2004, Beljianina et al. 2005b, Polukonova 2007. It can be suggested that this morphological peculiarity emerged due to such special feature of the chemical composition in water bodies of the Caucasus as increased mineralization. However, such an assertion needs additional research on the water mineralization level in the collection sites of Ch. bernensis in the Central Caucasus (the northern macroslope) and Ciscaucasia.
The other significant diagnostic characteristic that allows differentiating the species of genus Chironomus is the centromere type (Shobanov 2000(Shobanov , 2002. Thus, several pairs of sibling species with identical banding sequences in the polytene chromosomes (homosequential species), such as Ch. piger and Ch. riparius (Keyl and Strenzke 1965, Polukonova et al. 1996, Karmokov et al. 2011 or Ch. nuditarsis and Ch. curabilis (Polukonova et al. 2003, 2005, Polukonova 2005a, were found to be different in the size of the pericentromeric heterochromatin. Although it is necessary to note that intra-and interpopulation chromosomal polymorphism can be observed for this characteristic (Iliynskaya 1984, Kiknadze et al. 1991b, which can complicate its use as a species-specific criteria especially in the cases when the difference in centromere size of different species is not very significant.
The dominance of different genotypic combinations at various sites of the Caucasus probably can be explained by the fact that in some areas some combinations can be more adaptive than the others. Perhaps this is happening due to a different level of mineralization, temperature and degree of eutrophication in the different collection sites.
Caucasian populations on the dendrogram occupy an intermediate position between Italian and Swiss populations, on the one hand, and Western Siberian popula-tion, on the other. Such arrangement agrees rather well with the geographic location of the studied regions and may reflect the true course of settlement of the species (either from west to east or from east to west). For more specific allegations more researches are needed.
In the context of the data mentioned above, further researches on Ch. bernensis from geographically distant regions are necessary, as there is possibility that the presently known species is actually polytypic and consists of several sibling species.