Chromosome analysis of Endochironomus albipennis Meigen, 1830 and morphologically similar Endochironomus sp. (Diptera, Chironomidae) from water bodies of the Volga region, Russia

Abstract Based upon the detailed chromosome map of polytene chromosomes of the eurybiont species Endochironomus albipennis Meigen, 1830, the localization of the centromere regions using a C-banding technique is defined. Chromosomal polymorphism in populations from two water bodies in the Volga region has been studied, 17 sequences are described. Polytene chromosomes of Endochironomus sp. (2n=6), having larvae morphologically similar to those of Endochironomus albipennis Meigen, 1830 (2n=6) are described for the first time.


Introduction
Larvae of Endochironomus albipennis Meigen, 1830 inhabit water bodies of different types. They are typical epibiotic organisms inhabiting submerged objects in the littoral zone, sometimes occurring also inside strongly decomposed plant residues (Kalugina 1963, Belyanina 1981. In Russia, this species is widely spread in the South and Center of the European part, in Siberia and in Kamchatka (Kalugina 1963, Belyanina 1981, Petrova and Michailova 1989. The first data about the chromosome number of E. albipennis (2n=6) were reported by Konstantinov and Belyanina-Nesterova (1971). Later, a description of the karyotype and chromosomal polymorphism in a population from the Volga River was done by Belyanina (1981). This author indicated the chromosomes as: chromosome I (arms AB); chromosome II (arms CD); chromosome III (arms EF). Another description of chromosome arms including marking the chromosome regions was made by Michailova and Gercheva (1982) and Michailova (1987Michailova ( , 1989 for the Bulgarian and Swiss populations. Kiknadze et al. (1991) mapped E. albipennis chromosomes using the photomap of Michailova (1987). Nevertheless designation of arms in chromosome III in their article does not conform to this system, i.e. numeration of parts (from 1 to 12) begins from the arm defined as F, whereas the same arm in photomap of Michailova (1987) is defined as arm GE. Arm GE in the photomap of Kiknadze et al. (1991) conforms to arm F in photomap of Michailova (1987). Chromosomal polymorphism of E. albipennis is still poorly studied, but several types of inversions have been described by Belyanina (1981) and Petrova and Michailova (1989).
There is neither a unified system of chromosome mapping nor a catalogue of chromosome sequences for E. albipennis. The few available photomaps are partially incomparable with each other. Therefore it is impossible to establish the limits of chromosome rearrangements in the populations of this species.
The main objectives of the present work were to study the chromosome polymorphism in two populations of E. albipennis from the Volga region and to present the list of chromosome sequences of the species. In addition, our aim was to provide the first description of polytene chromosomes of Endochironomus sp., larvae of which are similar in morphology to those of E. albipennis.
The species were identifying using larval morphology (Pankratova 1983, Pinder andReiss 1983). The preparations of the polythene chromosomes were made from squashes of salivary glands cells stained with the ethanol-orcein method (Demin and Shobanov 1990). For detection of heterochromatin and centromere regions in chromosomes, a method of C-banding described by Belyanina and Sigareva (1978) was used.
Designation of the polythene chromosome arms was made according to Michailova (1987). In the chromosome map of E. albipennis (Figs 1a,2a,b,3a) we have saved the marking of large regions (marked by large numerals at the pictures) conforming to the mapping system developed by Michailova and Gercheva (1982) and Michailova (1987Michailova ( , 1989. We developed here a more detailed mapping including the separation of the small regions (marked by small numerals under the chromosome) of chromosomes ( Table 1, Figs 1-3).
Designation of the band patterns conforms to the order of their description: albA1, albA2 etc. Genotypic combinations of banding sequences in every arm were designated as A1.1, A1.2, A2.2, etc., respectively. For analyzing chromosomal polymorphism we calculated the frequencies for every combination of chromosome sequences in each chromosome arm and also the mean number of heterozygous inversions per individual. Chromosome arms (Belyanina 1981) Chromosome arms (Michailova 1987(Michailova , 1989 Banding sequences (Michailova 1987) Banding sequences (this study) Analysis of slides was performed under the microscope MBI-11У4.2. For photomicrography a digital photographic camera Panasonic LS80 LUMIX was used. In the description of the larval morphology the terminology by Saether (1980) was used.
Chromosome I (AD).The centromere region was detected using C-banding technique ( Fig. 4b) as a thin indistinct C-band on the boundary of sections 16 and 17.
Arm A (Fig. 1a) has the following band sequence: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 (Table 1, Fig. 1a). Balbiani Ring (BR 1 ) is located in section 15. Section 5 contains a weakly active puff; on the boundary of sections 10 and 11 there is a constriction. Sequence albA2 apparently was formed on sequence albA1 as a result of inversion of sections 4-14. Sequence albA2 was present both in homo-and heterozygous states (Fig. 1b). Homozygous inversion A2.2 was found for the first time; heterozygous inversion A1.2 was described previously as C/C1 by Belyanina (1981) and was observed with high frequency in larvae from the Volga River (Table 2). The chromosome sequence albA3 found here for the first time arose apparently as a result of inversion of sections 4-15 (Fig. 1c) and occurred only in a heterozygous state -A1.3 (Table 2).
Arm D (Fig. 1a) has the band sequence: 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33. In section 17, BR 2 is situated (Fig. 1a). The Nucleolus Organizer Region (NOR) is located in section 22 and shows a variable degree of activity. Sequence albD2 was apparently formed on sequence albD1 as a result of inversion of sections 24-31 and was present in the heterozygous state -D1.2 (Fig. 1b, c). The new sequence albD3 was apparently formed on the sequence albD1 as a result of inversion of sections 22-31. The homozygous inversion D2.2 and heterozygous inversion D1.3 were found for the first time, heterozygous inversion D1.2 was described previously as D/D1 by Belyanina (1981) and was also observed in larvae from Volga (Table 2).
Chromosome II (BC).The centromere region is detected using C-banding (Fig. 4  a) as an indistinct C-disc on the boundary between sections 18 and 19.
Arm B (Fig. 2a) has the band sequence: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18. The NOR is situated in section 5; no other active regions are present in this arm. For the first time we revealed the chromosome sequence albB3, that had apparently arisen on sequence albB1 as a result of inversion of sections 5-8 and was present both in the homozygous (Fig. 2b, c) and heterozygous states. The heterozygous inversion  Michailova (1987). The large regions of chromosome are presented according to Michailova (1987), small regions of chromosome done in this study were marked over the chromosome. The regions with inversions are marked by the brackets, Nucleolus Organizer (NOR), Balbiani ring (BR), puff (p), arrows indicates the centromere of the chromosome I (AD).    Belyanina (1981) and was not observed in our study (Table 2). Arm C (Fig. 2b) has the band sequence: 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35. The NOR is situated in section 31. Chromosome sequence albC3 was formed as a result of inversion in sections 24-34 and was present in both states, heterozygous -C1.3 (Fig. 2a) or homozygous -C3.3 (Fig. 2c). The inversions C1.3 and C3.3 were found for the first time; the heterozygous inversion C1.2 was previously described as B/B1 by Belyanina (1981) and was observed only in larvae from the Volga (Table 2).
Chromosome III (GEF). Previously it was suggested that this chromosome is the result of tandem fusion of the short chromosome IV with arm E of chromosome EF but the division into arms «GE» and F was made without using C-staining (Michailova 1987). C-banding in this chromosome has clearly detected a C-disc (Fig. 4) on the boundary of sections 6 and 7. This C-positive disc is possibly the active centromere suggesting thus the chromosome III is heterobrachial with short arm G and long arm EF.
Arm GE (Fig. 3a) has the band sequence: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16. The active regions in this arm were absent. We have discovered here three chromosome sequences, among them albGE1, accepted as a standard, and two sequences defined as albGE2 and albGE3 respectively. Sequence albGE2 was formed as a result of inversion of sections 3-10 and found in both states, heterozygous GE1.2 (Fig. 3c) and homozygous GE2.2 (Fig. 3b). Sequence albGE3 was found only in a heterozygous state (Fig. 3d); this is a complicated inversion formed through several inversion steps. The heterozygous inversions GE1.2 and GE1.3 were found in two reservoirs, whereas homozygous GE2.2 in the Sazanka Lake only (Table 2).
Arm F (Fig. 3a) has the band sequence: 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32. BR is situated in section 19. We have found one inversion sequence defined as albF2. Sequence albF2 was formed on sequence albF1 as a result of inversion of sections 21-25 and was present only in the heterozygous state. Frequency of this inversion was very low, 1.9%. It was only found in the population from the Sazanka Lake.
Analysis of chromosome polymorphism was performed in comparison with the data of Belyanina (1981) and Michailova (1987) (Table 2). A total of 17 chromosome sequences were recorded, which were found in the studied populations in homozygous and heterozygous states ( Table 2).The level of E. albipennis`s chromosomal polymorphism in populations from different water bodies was essentially lower (number of heterozygous inversions per individual was 1.6 in Sazanka Lake, and 1.3 in pond near Novo-Aleksandrovka village), than in the Volga River near Saratov -3.2 (Belyanina 1981).
Larva. Body is yellow, maximal length -10 mm. The head capsule is light yellow. Submentum of Endochironomus sp. (Fig. 5b) with a small pigment spot, as opposed to submentum of E. albipennis (Fig. 5a), which does not have a spot. Both species are similar in structure mental teeth (Fig. 5c, d), but differ significantly in structure of ventromental plates (VmP): VmP of E. albipennis (Fig. 5e) extend in width, the ratio of width to the length (VmPR) is 4.1-4.5 (4.2), VmP of Endochironomus sp. (Fig. 5f ) less elongated in width, VmPR is 2.3-3.6 (3.0). Anterior edge of the ventromental plate of E. albipennis with a row of small, not protruding teeth, anterior edge of the VmP of Endochironomus sp. with a well-visible row of teeth. Seta subdentalis (SSd) of E. albipennis is lanceolate and straight (Fig. 5g), but SSd of Endochironomus sp. (Fig.  5h) is lanceolate and slightly curved.
Karyotype. Centromeres are not distinct morphologically. Chromosome arms were designated in accordance with the photomap of E. albipennis: I (AD), II (BC), III (GEF), I<II=III.  Arm A (Fig. 6a) has the region including sections 15-16 which is homeologous with arm A of E. albipennis. There is an active BR 1 in section 15.
Arm B (Fig. 6b) has the small region including sections 5-6 which is homeologous with arm B of E. albipennis. In section 5, a NOR is situated.
Arm C (Fig. 6b) has the region including sections 29-35 which is homeologous with arm C of E. albipennis. In section 31, a NOR is situated.
Arm GE (Fig. 6c) has the only the region including sections 1-4 which is homeologous to arm GE of E. albipennis.
Arm F (Fig. 6c) has the sites including sections 17-19 and 30-32 which are homeologous with arm F of E. albipennis. In section 19, BR is situated.

Discussion
Among all Endochironomus species detailed cytophotomaps of polytene chromosomes have been earlier compiled only for E. tendens Fabricius, 1775 (Durnova 2009), so a comparative analysis of polytene chromosomes of E. tendens, E. albipennis and Endochironomus sp. is currently hampered. Karyotypes of E. tendens and E. albipennis differ strongly both in disc patterns and in distinctness of centromere regions: in E. tendens centromere regions appear as thick heterochromatin blocks, whereas in E. albipennis they are morphologically not distinct. With the differential staining of chromosomes of E. albipennis using C-technique described by Sigareva (1985), centromeric heterochromatin was only defined clearly and permanently as a thin C-disc in the chromosome III (Fig. 4). Centromere regions of the chromosomes I and II were stained indistinctly, which is apparently connected with the very low amount of paracentromeric heterochromatin in these chromosomes.
The evolution of E. tendens apparently proceeded as a narrow specialization because larvae of this species are the typical miners in the tissues of littoral macrophytes (Kalugina 1963, Durnova 2009). Larvae of E. albipennis are eurybiontic and inhabit different biotopes being epibiotic organisms of different submerged littoral substrata in the water bodies. Molecular data  have shown that by the nucleotide sequences of the mitochondrial gene COI E. tendens displays greater similarity to Synendotendipes kaluginae Durnova, 2010 than to E. albipennis, which indicates a high degree of divergence between E. tendens and E. albipennis not only at the chromosome level, but at the molecular level.
Larvae of Endochironomus sp. (2n=6) are morphologically similar to those of E. albipennis (Durnova et al. 2011). The degree of homeology in chromosome I (AD) between E. albipennis and Endochironomus sp. is relatively high; arms D are identical in banding patterns. These species differ in many sections of the chromosomes II (BC) and III (GEF), and only in few regions some common banding patterns can be seen. The number of discs in the central part of the chromosome II (BC) of Endochironomus sp., in which no homeology is observed, is much higher than in E. albipennis. Probably during a process of differentiation of these species, duplication of chromosome material took place. The degree of homeology between two species in chromosome I (GEF) is also low, length of arms F and GE of Endochironomus sp. exceeds considerably length of albF and albGE, which is probably related to the duplication of the chromosome material.
Thus, Endochironomus sp. distinctly differs from E. albipennis by the polytene chromosome band patterns, which undoubtedly argues for its separate species status. The chromosome differentiation of these two species was evidently accompanied not only by inversions, but also by duplications of chromosome material (in chromosome I and chromosome II), as indicated by larger number of discs in chromosomes of Endochironomus sp. as compared to E. albipennis.