Comparative analysis of karyotypes of Chironomus solitus Linevich & Erbaeva, 1971 and Chironomus anthracinus Zetterstedt, 1860 (Diptera, Chironomidae) from East Siberia

Abstract A comparative chromosome banding analysis of Chironomus solitus Linevich & Erbaeva, 1971 and Chironomus anthracinus Zetterstedt, 1860 from East Siberia (Lakes Baikal, Gusinoe, Arakhley and Irkutsk Reservoir) showed close similarity of banding sequences. Chironomus solitus differs from Chironomus anthracinus in one species-specific sequence of arm B. Arms C (43%) and D (30%) had inversion banding sequences previously reported in Chironomus anthracinus The similarity of karyotypic features of Chironomus solitus and Chironomus anthracinus in combination with morphological features of larvae provide evidence in favour of including Chironomus solitus in the Chironomus anthracinus group of sibling species long with Chironomus reservatus Shobanov, 1997.

Ch. solitus and Ch. anthracinus live in the single type water environments (lakes, water reservoirs), and are characterized by similar larval morphology in the features used in the distinction of Chironomus species, which makes their differentiation complicated. Thus, accurate identification of these species requires analysis of their karyotypes, rather than only external larval morphology. Until recently, the Ch. solitus karyotype had only been examined in one population from the Irkutsk Reservoir. The first data were reported by Bukhteeva (1979); later, banding chromosome patterns and polymorphisms were described (Proviz 2009). Karyological analysis was made of Ch. anthracinus from many Palearctic and Nearctic regions (Belyanina 1983;Kiknadze et al. 1991Kiknadze et al. , 1996Shobanov 1996;Petrova and Rakisheva 2003;Kiknadze et al. 2005). In East Siberia, karyotypes of larvae from Lake Shchuchie (Buryatia) were briefly reported by Bukhteeva (1979Bukhteeva ( , 1980. Later, Kiknadze and co-authors (Kiknadze et al. 1991(Kiknadze et al. , 1996 described the karyotypic of Ch. anthracinus from the Vilyuy Reservoir (Yakutia).
The present work is aimed at comparative analysis of Ch. solitus and Ch. anthracinus karyotypes from the largest lakes of East Siberia, Baikal, Gusinoe, Arakhley and Irkutsk Reservoir, and determination of cytogenetic features for their identification.

Material and methods
Fourth instar larvae of Ch. solitus were collected in January 1992 in the Irkutsk Reservoir (depth 3 m, 52 larvae), and in June 2008 in Lake Baikal opposite the Bolshye Koty Settlement (depth 6 m, 12 larvae). Ch. anthracinus were collected in May 2013 in Lake Gusinoe (10-22 m, 65 larvae), and in March 2014 in Lake Arakhley (10-17 m, 78 larvae). Larvae were fixed in a 3:1 mixture of 96% ethanol and glacial acetic acid. Karyological preparations were made using the ethyl-orcein method (Demin and Shobanov 1990). In 1992 and 2008, chromosomes were photographed by a micro-camera unit MCU-1 with 90× zoom magnification; in 2013-2014, this was performed using an Axiostar plus (Zeiss) microscope (Centre for Microscopic Analysis LIN SB RAS) with AxioVision Rel. 4.7.1 software. Mapping of arms A, C, D, E, and F of Ch. anthracinus chromosomes was performed according to Kiknadze et al. (2005) based on piger-standard (Keyl 1962, Devai et al. 1989, while standard map of Ch. plumosus suggested by Shobanov (1994Shobanov ( , 1996 was used for mapping of arm B. Symbols designating banding sequences are as follow: distribution areas marked by p' for Palearctic, n' for Nearctic, and h' for Holarctic zoogeographical regions (Kiknadze et al. 2005) and followed by abbreviated species name (sol), arm designation (A) and banding sequence number-p'solx1 (in homozygote-p'solA1.1).

Larval morphology
Both species have a light yellow (from the dorsal part) cephalic capsule, including the frontal sclerite. Abdominal segment VIII bears two pairs of long ventral appendages; lateral appendages on segment VII are absent (bathophilus type after: Lenz 1926). Premandible with two uneven teeth. Fourth lateral cusp of mentum is smaller than fifth cusp. Third antennal segment is shorter than the fourth. The colour of the fourth lower mandibular tooth varies; that of Ch. solitus is dark yellow, while the remaining teeth are dark brown. The results of our examination of the population from Lake Arakhley showed that Ch. anthracinus tooth was either dark yellow or of the same colour as the rest of the teeth.

Karyotype characteristics
Karyotypes of Ch. solitus (Fig. 1) and Ch. anthracinus (Fig. 2) have common morphological features: 2n=8. A combination of chromosome arms is typical for species from "thummi" cytocomplex. Chromosomes AB and CD are metacentric, EF, submetacentric, and G, telocentric. The species differ in the size of cenromeric heterochromatin. The centromeric areas of Ch. solitus are well defined, and the centromeres of Ch. anthracinus look like thin disks. Arm G homologues are unconjugated and carry a Balbiani Ring (BR) and a nucleolus (N). In Ch. anthracinus there is a second nucleolus in the arm F.
Arms G of Ch. anthracinus and Ch. solitus (Figs 1, 2) have similar morphology: unconjugated homologues with a constriction, unclear banding pattern, similar location of Balbiani Ring and nucleolus. In general, homologues have ectopic contacts in active loci.

Discussion
As a result of comparative analysis of banding patterns of Ch. solitus and Ch. anthracinus from East Siberia, the similarity of these species in morphological features of larvae as well as karyotypes was revealed. Most of the chromosomal arms, A, D, E and F, have identical banding sequences, and a similar structure of arm G. The principal distinctive features of Ch. solitus karyotype are the species-specific p'solB1 sequence and the absence of a nucleolus in arm F. Previous investigators (Belyanina 1979, Kiknadze et al. 1991, 1996, Shobanov 1996, Petrova and Rakisheva 2003, Kiknadze et al. 2005 reported a low level of chromosome polymorphisms in Ch. anthracinus. Analysis of the populations with standard banding sequences from Lakes Gusinoe and Arakhley also confirmed these observations. The overall banding sequence pool of Ch. anthracinus from other regions includes h'antC2, h'antC1 and p'antD2 sequences, which are identical to p'solC1, h'solC2 and p'solD2 from East Siberia; this is suggestive of karyological similarity of Ch. solitus and Ch. anthracinus. There is one more species of the genus Chironomus -Ch. reservatus Shobanov, 1997, which has close similarity of karyotypic and morphological features at all developmental instars of Ch. anthracinus (Shobanov, 1997). Based on these results, the author included the two species in the C. anthracinus group. Banding sequence p'resB1, alongside h'antB2, localised close to p'solB1, and is regarded one of the species-specific markers: p'resB1 25s-24i 18c-16b 22b-18d 24h-23d 15m-16a 22c-23c 15l-12v C The morphology of Ch. anthracinus and Ch. solitus imagines from East Siberia is insufficiently studied (Linevich and Erbaeva 1971), therefore, it is possible to compare only several characteristics of these species. For instance, AR of Ch. solitus (3.8) is most closely related to Ch. anthracinus (4.14-4.43) from the European part (Shobanov 1996), and Ch. anthracinus from East Siberia (5.0) -to Ch. reservatus (4.8-5.6) (Shobanov 1997). Further research into metamorphosis of these species should be conducted to make reliable conclusions.
The results of our investigation, similarity of karyotypic features of Ch. solitus and Ch. anthracinus in combination with morphological features of larvae provide evidence in favour of their close similarity and enable us to include Ch. solitus as well as Ch. reservatus in the C. anthracinus group.