CompCytogen 10(4):571—585 (2016) COMPARATIVE Arreeesiorenssssiourt doi: 10.3897/CompCytogen.v | 0i4.9400 Kas Cyto genetics http://compcytogen.pensoft.net International journal of Plant & Animal Cytogenetics, i Karyosystematics, and Molecular Systematics Karyotype characteristics, larval morphology and chromosomal polymorphism peculiarities of Glyptotendipes salinus Michailova, 1983 (Diptera, Chironomidae) from Tambukan Lake, Central Caucasus Mukhamed Kh. Karmokov', Azamat Y. Akkizov? | Zembotov Institute of Ecology of Mountain territories RAS, I. Armand str. 37a, Nalchik 360051, Russia 2 Institute of Biomedical Problems RAS, Center of Medico-Ecological Research, Shogentsukova str., 40, Nalchik 360051, Russia Corresponding author: Mukhamed Kh. Karmokov (lacedemon@rambler.ru) Academic editor: V. Golygina | Received 1 June 2016 | Accepted 20 September 2016 | Published 4 November 2016 http://zoobank. ore! FD456195-2F77-4280-846F-7799A86B67F2 Citation: Karmokov MKh, Akkizov AY (2016) Karyotype characteristics, larval morphology and chromosomal polymorphism peculiarities of Glyptotendipes salinus Michailova, 1983 (Diptera, Chironomidae) from Tambukan Lake, Central Caucasus. Comparative Cytogenetics 10(4): 571-585. doi: 10.3897/CompCytogen.v10i4.9400 Abstract Data on the karyotype characteristics, larval morphology and features of chromosomal polymorphism of a population of Glyptotendipes salinus Michailova, 1983 (Diptera, Chironomidae) from Tambukan Lake (on the northern macroslope of the central Caucasus) are presented. It was found that diagnostic larval char- acters of G. salinus from Caucasus in general are similar to those described in previous studies, but with some significant differences. By some morphological characteristics Caucasian larvae appeared to be closer to G. barbipes than to ones provided for European larvae of G. salinus by Contreras-Lichtenberg (1999). Obtained morphological data make possible to conclude that Caucasian population of G. salinus can be a markedly diverged population of the species, probably even subspecies. In the Caucasian population 12 banding sequences were found: two in arms A, B, C, E, and G, and one in arms D and F Eight of these are already known for this species, and four, salA2, salB2, salEX, and salG3, are described for the first time. Genetic distances between all the previously studied populations of G. salinus were measured using Nei criteria (1972). The population of the central Caucasus occupies a distinct position on the dendrogram Copyright M. Kh. Karmokov,A.Y. Akkizov. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 5/2 M.Kh. Karmokov & A.Y. Akkizov / Comparative Cytogenetics 10(4): 571-585 (2016) compared with populations from Altai and Kazakhstan. All the obtained morphological and cytogenetic data can indicate the plausible relative isolation and complexity of the Caucasus from the viewpoint of microevolution. More researches are required in other parts of Caucasus and other geographically distant regions for more specific allegations. Keywords Diptera, Chironomidae, Glyptotendipes salinus, larval morphology, polytene chromosomes, chromosomal polymorphism, genetic distances, Tambukan Lake, central Caucasus (northern macroslope) Introduction Glyptotendipes salinus was first described by Michailova (1987) from Bulgaria. Accord- ing to Fauna Europaea web source (http://www.faunaeur.org) the species is known in Europe from Austria, the British Isles and Bulgaria. The karyotype of G. salinus has been studied from Bulgaria (Michailova 1987), Russia and Kazakhstan (Andreeva et al. 1998, Aimanova et al. 2000). In Russia this species is known from several regions (Altai Krai, Omsk Oblast, and Chelyabinsk Oblast). In Kazakhstan the two studied populations of the species are situated in the area of the Semipalatinsk Test Site (STS). The species is a sibling species of G. barbipes that allows study of the earlier phases of divergence of the species in genus G/yptotendipes (Michailova 1987a, 1989). The species G. salinus differs from G. barbipes by chromosomal rearrangements in the chro- mosome arms A, C, D and E. Significant difference was also found in the amount and quality of the centromeric C- heterochromatin. Thus, some bands that were in the euchromatic state in G. salinus were in the heterochromatic state in G. barbipes (Michailova 1987b). The amount of centromeric DNA in the Ist, IInd and IIIrd chro- mosomes differs greatly between the two species (Michailova and Nikolov 1992). It was also found that C-heterochromatin of G. barbipes consists of two different types of C-bands: the dark ones at the periphery of the centromeres, which correspond to satellite II DNA, and pale C-bands corresponding to the satellite | DNA in the middle of the centromeric regions. Such heterochromatin differentiation was not expressed as prominently in the centromeric regions of G. salinus (Michailova 2014). Michailova et al. (2002) also provide research on the effects of lead on the polytene chromosomes of G. salinus. They found that exposure to lead results in a decrease in the activity of the nucleoli (NOR) and Balbiani Rings (BRs). Earlier it was shown that G. sal/inus occurs in brackish water, while sibling species G. barbipes prefers fresh water (Michailova 2014). The aim of the work was to present the description of karyotype characteristics, larval morphology peculiarities and chromosomal polymorphism of G. salinus from Tambukan Lake (northern macroslope of the central Caucasus). Also it was very im- portant to compare chromosomal polymorphism characteristics of G. salinus from the Caucasus with earlier studies. Karyotype characteristics, larval morphology and chromosomal polymorphism peculiarities... 573 Methods The fourth instar larvae of Glyptotendipes were used in the karyological study. The larvae were collected from one site of the central Caucasus: 17.05.13, 43°27.30'N; 43°09.75'E, southern shore of Tambukan Lake, altitude ca. 550 m az.s.l. Tambukan Lake is a lake with bitter salt water (salinity varies from 30 to 100 grams per liter) lo- cated in the northern macroslope of the central Caucasus, near the border of Stavropol Krai and the Republic of Kabardino-Balkaria of Russia. The lake’s surface area is 1.87 km7, and its depth ranges between 1.5—3.1 m. With regard to vertical zonation, the collection site belongs to the steppe zone (typification of the zone variants was given according to Sokolov and Tembotov 1989). The morphological terminology follows Sether (1980). Head capsule and body of 20 larvae were slide mounted in Fora-Berlese solution. The specimens have been deposited in Tembotov Institute of Ecology of Mountain territories RAS, Nalchik, Russia. The karyotype and chromosomal polymorphism has been studied in 63 larvae from the Tambukan Lake. Larvae for karyotype analysis were fixed in ethanol-glacial acetic acid (3:1). Slides of the chromosomes were prepared using the ethanol-orcein technique (see Dyomin and Ilyinskaya 1988, Dyomin and Shobanov 1990). The banding sequences were des- ignated according to the accepted rule specifying the abbreviated name of the species, symbol of chromosome arm, and sequence number, for example salC1, salC2, etc. (Keyl 1962, Wilker 1973). The identification of chromosome banding sequences was performed with the use of the photomaps of Michailova (1983) and Andreeva et al. (1998); chromosome mapping was performed according to Martin and Porter (1973) and Kiknadze et al. (1998), with corrections of Andreeva et al. (1998). Microscope Carl Zeiss Axio Imager.A2 was used to study the chromosome slides. The software package STATISTICA 10 was used for statistical analysis (cluster analysis). The following parameters were used for comparison of characteristics of chromo- somal polymorphism: the number of banding sequences in a population, the per- centage of heterozygous larvae, and number of heterozygous inversions per specimen. Genetic distances between populations were calculated according to Nei criteria (Nei 1972) on basis of the original data and data of Andreeva et al. (1998) on inversion polymorphism of the species in Russia and Kazakhstan. Results The larvae of Glyptotendipes in the studied site were attributed to G. salinus by both morphological and chromosomal characteristics. Morphological characteristics of larva are presented in Fig. la—g. The diagnostic larval characters of G. salinus from the Caucasian site in general are similar to those described previously for this species by Michailova (1987), Andreeva et al. (1998) and Contreras-Lichtenberg (1999), but there are some differences. The head 574 M.Kh. Karmokov & A.Y. Akkizov / Comparative Cytogenetics 10(4): 571-585 (2016) & Figure |. The larva of G. salinus from Tambukan Lake. a gular sclerite b mentum € antenna d premandible e mandible f ventromental plate g ventral tubuli on segment VIII. capsule is lightly colored as in specimens found in other localities. Ventral tubuli are shorter than in G. barbipes and do not exceed the length of IX segment of larva body (Fig. 1g). The seta subdentalis of mandible is leaf-shaped (Fig. le), reaching the top of Karyotype characteristics, larval morphology and chromosomal polymorphism peculiarities... 575 the last tooth. The ratio of the width of the ventramental plate to inter-plate distance (PSR) in G. salinus is more than 8 (8.34 according to Contreras-Lichtenberg (1999), from 8.02 to 9.04 in Caucasian population), while PSR of G. barbipes is 4.2. The width of the mentum of G. salinus was 219 um according to Contreras-Lichtenberg (1999), but varies from 235 to 266 um in Caucasian population, which is closer to G. barbipes measurements (256 um). The ratio of the width of the mentum to the width of the mid- dle tooth of mentum (MR) of G. salinus described by Contreras-Lichtenberg (1999) is 5.33, while in Caucasian population it varies from 6.8 to 7.17 and slightly exceeds MR=6.5 of G. barbipes. The width of the ventramental plate of G. salinus in Caucasian population varies from 297 to 340 pm (307.5 um according to Contreras-Lichtenberg 1999), and its value is much higher than 288 um of G. barbipes. The inter-plate distance (IPD) of G. salinus from Caucasian population is similar to data of Contreras-Lichten- berg (1999) — 36.9 um — and varies from 34.5 to 37.5 um, IPD of G. barbipes is much higher - 68 um. The most significant difference was observed for the length of the larva’s body: in first description of Michailova (1987) and in paper of Contreras-Lichtenberg (1999) it is said to be 25-27 mm, while Caucasian larvae are twice shorter — 12-14 mm — which is very similar to an average larva’s length of G. barbipes (12 mm). Karyotype of G. salinus from the Central Caucasus The diploid number of chromosomes in G. salinus karyotype is 2n = 8, chromosome arm combination is AB, CD, EF, and G (Fig. 2). Chromosomes AB and CD are meta- centric, EF is submetacentric, and G is telocentric. Three well-developed nucleoli (N) are located on arms B, C, and E. ‘There are two Balbiani rings (BR) in the karyotype: both are situated in arm G (Fig. 2). The centromeric bands of long polytene chromosomes of G. salinus from the stud- ied populations are large and belong to v-type according to the classification by Sho- banov (2002). Banding sequences and chromosomal polymorphism of G. salinus from the Tambukan Lake Until now, eleven banding sequences have been described in the banding sequences pool of G. salinus (Table 1). In the studied population only eight of those banding sequences were present, and four banding sequences have been found for the first time, providing a total of 12 banding sequences in the Caucasian population (Table 2). Arm A has two banding sequences, salA1 and salA2 (Figs 3—4, Table 2). The band- ing sequence salAl was predominant in the studied population (Table 2). The band- ing sequence salA2 was found only in the heterozygous state with very low frequency (salA1.1 — 0.968, salA1.2 — 0.032). 576 salAl.1 Wy M.Kh. Karmokov & A. Y. Akkizov / Comparative Cytogenetics 10(4): 571-585 (2016) salE1.1 salB2.2 salF 1.1 salD1.1 v salG1.1 BR2 | BRI Figure 2. Karyotype of G. salinus from the Tambukan Lake; salA1.1, salD 1.1 etc. — zygotic combinations of banding sequences; BR — Balbiani rings, N — nucleoli. Arrows indicate centromeric regions. Table |. Catalog of banding sequences in the banding sequences pool of G. salinus. Arm | Sequence Order of bands Authors of mapping lal la-b 5n-t 6a-n 2d-h 3ba 2u-i 3c-t 4a-v Pridhet ayer alrioes “i 5a-m 2cba It-n 1c-m 60-t 7a-s ealiXo lab 5n-t 6a-d 5h-a 4v-a 3t-c 2i-u 3ab 2h-d 6h-e 5i-m 2c-a 1t-n Oniginalidaea 1c-m Go-t 7a-s B salB1 13z-a 12t-a 1lo-a 10v-a 9n-a 8p-a 7c-a Andreeva et al.1983 salB2 13-z-j 9d-h 10a-v 1la-o 12a-t 13a-i 9cba 8p-a 7s-a Original data salCl la-o 4v-k 1p-r 2a-n 3a-p 4a-m 5a-z 6a-n 7a-i Andreeva et al.1983 salC2 la-o 5f-a 4m-a 3p-a 2n-a Ir-p 4n-v 5l-z 6a-n 7a-i Andreeva et al.1983 Cc salC3 la-o 5f-a 4m-a 3p-a 2n-a 4u-n lp-r 4v A Cidepen M1GR3 51-z 6a-n 7a-i salC4 la-d 4e-m 5a-k lo-e 4d-a 3p-a 2n-a Ir-p 4n-v 5l-z 6a-n 7a-i__| Andreeva et al.1983 D salD1 Brey tae bare ead Andreeva et al.1983 Ja-x 7i-a salE1 la-u 3a-q 4a-w 5a-t 6a-r 2l-a lv 6s-w 7a-l Andreeva et al.1983 E salEX Not mapped F salF1 11w-a 10s-a 9t-a 8m-a 7l-a Andreeva et al.1983 salG1 5t-a 4i-a 3q-a 2r-a lg-a Andreeva et al.1983 G salG2 5t-a 4i 2b-r 3a-g 4a-h 2a lg-a Andreeva et al.1983 salG3 5t-j 2h-r 3a-g 4a-i 5a-i 2g-a lg-a Original data Karyotype characteristics, larval morphology and chromosomal polymorphism peculiarities... 577 Table 2. Frequency of banding sequences in different populations of G. salinus. Populations Kazakhstan Altai Krai eel Banding oS ig os Altai, Bolshoe | Caucasus STS, Atomnoe | Shagan Lake | Bulatovo Lake | Gorkoe Lake roe ; sequence Utichie Lake | Tambukan Lake (Andreeva et | (Andreeva | (Andreevaet | (Andreeva (npaseeva ee alsledkeoteinal al. 1998) 50 larvae | et al. 1998) | al. 1998) 50 | et al. 1998) é Sedan intte astapaae 1998) 50 larvae | data) 63 larvae salAl 1 1 1 1 1 0.992 salA2 0 0 0 0 0 0.008 salB1 1 1 1 1 1 0.317 salB2 0 0 0 0 0 0.683 salCl 0.220 0.164 0.622 0.530 0.016 salC2 0.780 0.817 0.378 0.470 0.984 salC3 0 0.009 | o | 0 0 0 salC4 0 0.009 0 0 0 0 salD1 1 1 1 1 1 1 salE1 1 1 1 1 1 0.992 salEX 0 0 0 0 0 0.008 salF 1 1 1 1 1 1 1 salG1 0.950 0.991 1 1 0.968 salG2 0.050 0.009 | o | 0 0 0 salG3 0 o- | 6 | % 0 0.032 Number of banding 9 11 8 8 9 12 sequences 1n population Percentage of heterozygous 40 29 48 57 62 51 larvae Number of EserPayB OU 0.34 0.30 0.60 0.61 0.60 0.60 inversions per specimen It is new for the species and described for the first time here (Fig. 4, Table 2). It differs from salA1 by one simple inversion that involves region 6e-n 2d-h 3ba 2u-i 3c-t 4a-v 5a-h: salA2 lab 5n-t Ga-d 5Sh-a 4v-a 3t-c 2i-u 3ab 2h-d Gh-e 5i-m 2c-a I1t-n 1c-m Go-t 7a-s Arm B has two banding sequences, salB1 and salB2 (Figs 3-4). The banding se- quence salB2 was predominant in the studied population (Table 2). The sequence salB2 is new for the species and described for the first time (Figs 3—4, Table 2). It differs from salB1 by one simple inversion that involves region 13i-a 12t-a 11lo-a 10 v-a 9h-d: salB2 13-z-j 9d-h 10a-v 1la-o 12a-t 13a-i 9cba 8p-a 7s-a The banding sequence salB2 was found with high frequency in both homozygous (salB1.1 — 0.095, salB2.2 — 0.445) and heterozygous states (salB1.2 — 0.460). 578 M.Kh. Karmokov & A.Y. Akkizov / Comparative Cytogenetics 10(4): 571-585 (2016) salAl.1 salB2.2 salB1.1 Figure 3. Banding sequences in arms A and B of G. salinus; a homozygotes salA1.1 b homozygotes salB2.2 € homozygotes salB1.1, Designations as in Fig. 2. Arm C has two banding sequences, salC1 and salC2. The banding sequence salC2 was dominant in this population (Fig. 5, Table 2). The other banding sequence salC1 was found only in the heterozygous state (salC1.2 — 0.032, sal C2.2 — 0.968) (Fig. 4). Arm D is monomorphic with banding sequence salD1 (Fig. 5, Table 2). Arm E had two banding sequences, salE1 and salEX (Table 2). The banding se- quence salE1 was dominant in the population (Fig. 6, Table 2). The banding sequence salEX was found only in the heterozygous state (salE1.1 — 0.968, salE1.2 — 0.032). This banding sequence is new for the species and described here for the first time Karyotype characteristics, larval morphology and chromosomal polymorphism peculiarities... 579 A2 salA1.2 salB1.2 Bl N Al salE1.X salC 1.2 Figure 4. Chromosome inversions in different arms of G. salinus from Tambukan Lake. Heterozygous zygotic combination key: a salA1.2 b salB1.2 ¢€ salC1.2 d salE1.X. Designations as in Fig. 2. (Fig. 4, Table 2). Because banding structure of salEX was unclear it was impossible to map it and so no numerical designation was assigned to it. Arms F is monomorphic with banding sequence salF1 (Fig. 6, Table 2). Arm G had two banding sequences, salG1 and salG3. The banding sequence salG1 was dominant in the population (Fig. 7, Table 2). The banding sequence salG3 was found only in the heterozygous state (salG1.1 — 0.937, salG1.3 — 0.063). This banding sequence is new for the species and described for the first time (Fig. 4). It differs from salG1 by one simple inversion that involves region 5i-a 4i-a 3g-a 2r-h: salG3 5t-j 2h-r 3a-g 4a-i 5a-i 2g-a 1g-a Comparison of chromosomal polymorphism of G. salinus from the Central Cau- casus and other regions Data for Russian (Altai Krai) and Kazakhstan populations are presented on the basis of publication of Andreeva et al. (1998). 580 M.Kh. Karmokov & A.Y. Akkizov / Comparative Cytogenetics 10(4): 571-585 (2016) salC2.2 salD1.1