The role of chromosomal polymorphism in microevolution of chironomid midges (Diptera, Chironomidae) has been repeatedly considered in members of the genera Chironomus and Camptochironomus with benthic larvae. In particular, the effect of intercontinental isolation on cytogenetic divergence was demonstrated for the natural populations of these species (Gunderina et al., 1996; Kiknadze et al., 1996, 2006), and a correlation was revealed between chromosomal variability and certain living conditions of their larvae (Shobanov, 1994a, 1994b; Petrova et al., 2000). Studying chromosomal polymorphism in phytophilous chironomids, in particular the members of Endochironomus, Glyptotendipes, Phaenopsectra, and some other genera is important since the zone of periaquatic vegetation provides a unique diversity of ecological niches; therefore, colonization of different macrophytes by chironomid larvae even within the same water body may be related to changes in the spectrum and incidence of inversion sequences.

Endochironomus tendens (Fabricius, 1794) is a phytophilous chironomid species which may serve as a good model for studying interpopulation chromosomal polymorphism. First, as shown earlier, in Saratov Province the larvae of this species are the most common miners of macrophytes, well adapted to development in both living and decomposing parts of various plants (Durnova et al., 2011, 2012). Second, the reference photomap of its polytene chromosomes has been previously created, and data on the inversion polymorphism in its populations have been obtained. The karyotype of E. tendens was originally described from Bulgaria and Hungary (Michailova and Gercheva, 1982); two cytotypes (I, II) were distinguished in its populations, and a high level of chromosomal polymorphism due to homo- and heterozygous inversions was observed (Michailova and Gercheva, 1982; Michailova, 1989). Later, the available data on the karyotype variation of E. tendens in different geographic zones were summarized (Belyanina, 1978, 1983; Michailova and Gercheva, 1982; Michailova, 1987, 1989, 1992), a more detailed photomap of its polytene chromosomes was proposed, and 22 banding sequences were described in the larval karyotypes of the species from the territory of Russia (Durnova, 2009a).

The populations of E. tendens from Bulgaria and Hungary (Michailova, 1992) revealed a certain correlation between some homozygous inversions and the occurrence of larvae in different plant species: the larvae of cytotype I preferred mining the leaves and stems of Typha latifolia L., while those of cytotype II more often mined the tissues of Trapa natans L. and very rarely developed in T. latifolia. A detailed description of the banding sequences in the karyotype pool of E. tendens, corresponding to the reference photomap, is needed for studying the composition of inversions in different populations and for analyzing the chromosomal polymorphism of the species in relation to the occurrence of its larvae in different macrophytes.

The objective of this work was to study the karyotype and to describe the chromosome banding sequences and their combinations in E. tendens larvae collected in different water bodies of Saratov and Volgograd provinces.

MATERIALS AND METHODS

Our material comprised larvae from 22 samples collected in the summer seasons of 2012–2015 in several water bodies of Saratov and Volgograd provinces of Russia (Table 1). Karyotypes were studied in the IV instar larvae collected from partly decomposed leaves of Butomus umbellatus L., Sparganum erectum L., Typha angustifolia L., Sagittaria sagittifolia L., and Stratiotes aloides L. Altogether, 673 individuals were collected and studied.

Table 1. Localities and dates of collection of Endochironomus tendens F. larvae
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The larvae were identified by morphological characters (Makarchenko and Makarchenko, 2006). Preparations of polytene chromosomes from salivary gland cells were made using the ethyl-orcein method (Demin and Shobanov, 1990) and examined using an Axioskop2 Plus microscope, an AxioCamHRc CCD camera, and the AxioVision 4 software (Zeiss, Germany), at 10 × 20; 10 × 40, and 10 × 100 magnifications. The images were processed in Adobe Photoshop CS6 and Corel Draw 13.

The banding sequences were described and named according to the reference photomap for this species (Durnova, 2009a).

RESULTS

The karyotype of E. tendens (2n = 6) is characterized by the following chromosome arm combination: I (EF), II (CDG), III (AB) (Michailova and Gercheva, 1982; Michailova, 1987, 1989). The centromeric zones are morphologically well defined and represented by large heterochromatin blocks.

Chromosome I (EF). Five banding sequences were previously described in arm E: tendE1, tendE2, tendE3, tendE4, and tendE5. Sequence tendE5 was earlier found only in the heterozygous state tendE1.5 and therefore remained unmapped (Durnova, 2009a). We found this inversion in the larvae mining B. umbellatus in the Tereshka River (Table 1), both in the homogygous state tendE5.5 (Fig. 1, a) and in the heterozygous one tendE4.5 (Fig. 1, b). Sequence tendE5 probably originated from tendE4 and differs from the latter in a simple inversion in the segment comprising regions 14–17a–d (since the break occurred within region 17, this region was additionally divided into subregions a–e):

tendE4: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17abcd e

tendE5: 1 2 3 4 5 6 7 8 9 10 11 12 13 17dcba 16 15 14 17e

The combination tendE1.5 of two sequences, E1 and E5, was described for the first time in the studied populations. The conjugated homologs formed a complex double loop (Fig. 1, c).

Of the four known banding sequences of arm F (Durnova, 2009a), we found three previously described sequences tendF1, tendF3, and tendF4 and their combinations: tendF1.3, tendF1.4, tendF3.3, and tendF4.4.

Fig. 1.
figure 1

Homo- and heterozygous inversions in chromosome I (EF) of E. tendens: (a) sequence combination E5.5F1.1; (b) E4.5F1.11; (c) E1.5F1.1; N, nucleolus; BR, Balbiani ring; p, puff; arrows mark the centromeres.

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Chromosome II (CDG). Arm C in most of the studied larvae contained the sequence combination tendC1.1, while tendC1.2 and tendC3.3 were rare combinations; they were described by us for the first time. The nucleolar organizer region was located in region 10 of arm C (Fig. 2; Fig. 3). A characteristic feature of arm DG was the varying level of activity of BR1, BR2, and p regions, depending on the inversions affecting different parts of this arm. All the four sequences that have been described previously for arm DG, namely tend(DG)1, tend(DG)2, tend(DG)3, and tend(DG)4 (Durnova, 2009a), were recorded in our material in different combinations. Banding sequence tend(DG)4 was recorded for the first time in the homozygous state in the population developing in B. umbellatus (Fig. 2, b); the previous mapping of this sequence should be regarded as preliminary, because this sequence was earlier found only in the heterozygous states tend(DG)1.4 and tend(DG)2.4 (Durnova, 2009a). Sequence tend(DG)4 probably originated from tend(DG)2 as the result of a simple inversion of region 16–20 (since the break occurred within region 16, this region was additionally divided into subregions a–e):

tend(DG)2: 11 12 13 14 15 16abced 17 18 26 25 24 23 22 21 20 19 27

tend(DG)4: 11 12 13 14 15 16abc 20 21 22 23 24 25 26 18 17 16de 19 27

This sequence was found by us not only in the homozygous state tend(DG)4.4 (Fig. 2, b), but also in vari-ous heterozygous combinations: tend(DG)1.4 (Fig. 3, a), tend(DG)2.4 (Fig. 2, c), tend(DG)4.5 (Fig. 3, b), and tend(DG)4.6 (Fig. 3, c).

Fig. 2.
figure 2

Homo- and heterozygous inversions in chromosome II (CDG) of E. tendens: (a) sequence combination C1.1(DG)2.2; (b) C1.1(DG)4.4; (c) C1.1(DG)2.4. Designations as in Fig. 1.

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Sequence tend(DG)5 was found by us for the first time. It probably originated from tend(DG)4 as the result of a simple inversion of region 23-16ed:

tend(DG)4: 11 12 13 14 15 16abc 20 21 22 23 24 25 26 18 17 16ed 19 27

tend(DG)5: 11 12 13 14 15 16abc 20 21 22 16de 17 18 26 25 24 23 19 27

Sequence tend(DG)6 was found for the first time and only in the heterozygous state tend(DG)4.6 (Fig. 3, c). It probably originated from tend(DG)4 as the result of an inversion of region 12b-25a:

tend(DG)4: 11 12ab 13 14 15 16abc 20 21 22 23 24 25abc 26 18 17 16ed 19 27

tend(BG)6: 11 12a 25a 24 23 22 21 20 16cba 15 14 13 12b 25bc 26 18 17 16ed 19 27

Fig. 3.
figure 3

Homo- and heterozygous inversions in chromosome II (CDG) of E. tendens: (a) sequence combination C1.1(DG)1.4; (b) C1.1(DG)4.5; (c) C1.1(DG)4.6. Designations as in Fig. 1.

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Combination tendDG3.4 of two inversion sequences was described for the first time from the studied populations (Fig. 4, a–c). These sequences originated by inversions in different parts of sequence tend(DG)2. When the two inversions co-occurred in the chromosome, the homologs formed a complex heterozygous inversion; the conjugated segments are marked with brackets in Fig. 4, a, b.

Fig. 4.
figure 4

Homo- and heterozygous inversions in chromosome II (CDG) of E. tendens: (a) sequence combination C1.1(DG)3.3; (b) C1.1(DG)4.4; (c) C1.1(DG)3.4. Designations as in Fig. 1.

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Chromosome III (AB). Four sequences were previously described in arm A: tendA1, tendA2, tendA3, and tendA4 (Durnova, 2009a). The populations studied by us did not reveal any new sequences; tendA2 and tendA3 were found both in homogygous states and in heterozygous ones: tendA2.2, tendA2.3, and tendA3.3. A new combination tendA1.3 was recorded in the population inhabiting B. umbellatus in the Tereshka River (Fig. 5).

Fig. 5.
figure 5

A heterozygous inversion in arm A of chromosome III (AB) of E. tendens: A1.3.

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Arm B in the studied populations included only two previously described sequences tendB1 and tendB2 (Durnova, 2009a) and the following combinations: tendB1.1, tendB1.2, and tendB2.2.

DISCUSSION

The level of chromosomal polymorphism varies greatly between chironomid species and depends on different factors. For instance, chromosomal transformations are almost completely absent in some narrowly specialized species, such as Xenochironomus xenolabis (Kieffer, 1916) and Demeijerea rufipes (Linnaeus, 1761) developing as miners of sponges, and also Stenochironomus gibbus (Fabricius, 1794) inhabiting the bast layer of submerged tree trunks and branches; this fact may be related to the relatively stable living conditions of these species (Durnova, 2009b, 2010a, 2010b).

Comparative analysis of karyotype pools in the species with a high level of chromosomal polymorphism (members of the genera Chironomus, Camptochironomus, and Glyptotendipes) is difficult due to the nonuniform level of knowledge of these species, in particular, the number of the populations examined. To date, the highest level of chromosomal polymorphism (estimated only by the number of chromosome banding sequences) has been observed in the benthic species Ch. balatonicus (Devai et al., 1983) and Ch. plumosus (Linnaeus, 1758), with 60 and 54 sequences recorded in the Palaearctic, respectively (Kiknadze, 2008). The larvae of Ch. plumosus from the Rybinsk Reservoir revealed 15 sequences forming 64 genomic combinations, with 1.3–2.2 heterozygous inversions per individual on average. The appearance of various genomic combinations was probably related to the microenvironmental conditions (Bolshakov and Shobanov, 2017).

The phytophilous chironomids developing in various plant substrates were also shown to have a varying level of chromosomal polymorphism. For example, the eurybiotic species Glyptotendipes glaucus (Meigen, 1818), inhabiting various submerged substrates, revealed 20 chromosome banding sequences forming 30 genomic combinations in Saratov and Kaliningrad provinces; the mean number of heterozygous inversions per individual was 0.55–0.93 (Belyanina and Durnova, 1998; Durnova et al., 2012, 2014; Vinokurova et al., 2016). In the highly specialized G. mancunianus (Edwards, 1929), inhabiting only living plant tissues, 17 sequences were described, the mean number of heterozygous inversions per individual being 0.11–0.53 (Durnova and Oglezneva, 2015). In the genus Endochironomus, the karyotype pool of E. albipennis (Meigen, 1830) was found to comprise 17 chromosome banding sequences and 20 zygotic combinations; that of Endochironomus sp. comprised 6 sequences (Durnova et al., 2015).

Altogether, 24 chromosome banding sequences were described for E. tendens larvae collected from the water bodies of Saratov and Volgograd provinces (Table 2); the results of analysis of their chromosomal polymorphism will be reported in the next communication. Two sequences, tend(DG)5 and tend(DG)6, were found and described for the first time; they occurred in both homo- and heterozygous states. The new data have allowed us to clarify the banding pattern of sequence tend(DG)4 and to map sequence tendE5; both sequences were previously found only in the heterozygous state.

Table 2. Frequency of occurrence of chromosome banding sequences in E. tendens F. from water bodies of Saratov and Volgograd provinces
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Our karyological analysis of E. tendens has shown that this species is the most polymorphic of all the presently studied phytophilous chironomids, in terms of the number of banding sequences and their combinations.