Karyotype characteristics, larval morphology and chromosomal polymorphism peculiarities of Glyptotendipes salinus Michailova, 1983 (Diptera, Chironomidae) from Tambukan Lake, Central Caucasus

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 characters of Glyptotendipes 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 Glyptotendipes barbipes than to ones provided for European larvae of Glyptotendipes salinus by Contreras-Lichtenberg (1999). Obtained morphological data make possible to conclude that Caucasian population of Glyptotendipes 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 Glyptotendipes salinus were measured using Nei criteria (1972). The population of the central Caucasus occupies a distinct position on the dendrogram 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.


Introduction
Glyptotendipes salinus was first described by Michailova (1987) from Bulgaria. According 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 , 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 Glyptotendipes (Michailova 1987a(Michailova , 1989. The species G. salinus differs from G. barbipes by chromosomal rearrangements in the chromosome 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 chromosomes 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 I 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. salinus 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 important to compare chromosomal polymorphism characteristics of G. salinus from the Caucasus with earlier studies.

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 a.s.l. Tambukan Lake is a lake with bitter salt water (salinity varies from 30 to 100 grams per liter) located 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 km 2 , 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 Saether (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 Ilyinskaya 1988, Dyomin andShobanov 1990). The banding sequences were designated 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, Wülker 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 chromosomal polymorphism: the number of banding sequences in a population, the percentage 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. 1a-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),  and Contreras-Lichtenberg (1999), but there are some differences. The head 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. 1e), reaching the top of 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 µm according to Contreras-Lichtenberg (1999), but varies from 235 to 266 µm in Caucasian population, which is closer to G. barbipes measurements (256 µm). The ratio of the width of the mentum to the width of the middle 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 µm (307.5 µm according to Contreras-Lichtenberg 1999), and its value is much higher than 288 µm of G. barbipes. The inter-plate distance (IPD) of G. salinus from Caucasian population is similar to data of Contreras-Lichtenberg (1999) -36.9 µm -and varies from 34.5 to 37.5 µm, IPD of G. barbipes is much higher -68 µm. 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 metacentric, 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 studied populations are large and belong to v-type according to the classification by Shobanov (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 D is monomorphic with banding sequence salD1 (Fig. 5, Table 2). Arm E had two banding sequences, salE1 and salEX ( Table 2). The banding sequence 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  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 Caucasus and other regions
Data for Russian (Altai Krai) and Kazakhstan populations are presented on the basis of publication of Andreeva et al. (1998). Arm A. The populations from Altai and Kazakhstan  were characterized by the presence of single banding sequence in the arm, salA1 (Table 2). Same banding sequence is dominating in population from North Caucasus but one new for the species sequence salA2 was also found with very low frequency (0.008). The new banding sequence might be endemic for this region.
Arm B was monomorphic in populations of Altai and Kazakhstan and presented only by the banding sequence salB1 (Table 2). In the Caucasian population another banding sequence new for the species -salB2, was predominant. This new banding sequence is probably endemic for this region.
Arm C of G. salinus in all the studied populations was polymorphic. However in Altai populations the predominant banding sequence was salC1, whereas in Kazakhstan population dominated salC2. The population of the North Caucasus is closer to populations of Kazakhstan with salC2 dominating with even higher frequency (Table 2).
Arm D of G. salinus was monomorphic in all the studied populations. Arm E was monomorphic in populations of Altai and Kazakhstan and low polymorphic in Caucasian population with the same dominant banding sequence salE1. A new banding sequence salEX was found in the Caucasian population with very low frequency (0.008) and might be endemic for the region.  Arm F of G. salinus was monomorphic in all the studied populations and presented only by the sequence salF1.
Arm G of G. salinus was monomorphic in populations of Altai and low polymorphic in populations of Kazakhstan and the Caucasus, although in all populations the dominant banding sequence was salG1. At the same time Kazakhstan and Caucasian populations differ by the set of rare inversions: salG2 was found in Kazakhstan while salG3 occurred in Caucasian population.
The inversion polymorphism of populations of G. salinus from the North Caucasus has a high level of heterozygous inversions per specimen and is similar to those of the Altai populations (Table 2). By the number of banding sequences per population (12), the Caucasian population is closer to Kazakhstan populations, but by the percentage of heterozygous larvae (51%) the studied population is more close to that of the Altai populations (48-62%). Genetic distances (Table 3) measured by Nei criteria (1972) on the basis of original and previous data ) on inversion polymorphism of the species in Altai region and Kazakhstan (Fig. 8) indicate the significant distance and distinct position of the Caucasian population of G. salinus in comparison with populations of Altai and Kazakhstan. The dendrogram was constructed on the basis of Nei criteria (1972) using NJ-method.

Discussion
In the northern Caucasus (central part of the northern macroslope) as well as in European Russia, G. salinus has been found for the first time.
As mentioned above, the diagnostic larval characters of G. salinus from the Caucasus in general are similar to those described by Michailova (1987), Andreeva et al. (1998) andContreras-Lichtenberg (1999), but there are some significant differences as by some morphological characteristics Caucasian larvae of G. salinus are actually closer to G. barbipes. The data on larval morphology from the Caucasus are close to data provided by Contreras-Lichtenberg (1999) for G. salinus by PSR, width of ventramental plate and inter-plate distance (IPD). However, by width of mentum, MR and total length of larva body the Caucasian material is closer to G. barbipes. Also the length of the body of larva of G. salinus from Caucasian site is strikingly different from the data provided by Michailova (1987) and Contreras-Lichtenberg (1999) where it is said to be 25-27 mm, while Caucasian larvae are twice shorter (12-14 mm) and are very similar to G. barbipes (12 mm). Considering the data of Contreras-Lichtenberg (1999) the length of a larva body is the most different character of G. salinus in comparison to other species from the subgenus Phytotendipes Goethjebuer, 1934. The length of a larva body of G. salinus is the biggest (25-27 mm) among all other species: G. pallens (Meigen, 1804) -10 mm, G. glacus (Meigen, 1818) -12 mm, G. gropekoveni (Kieffer, 1913) -13 (11-18) mm, G. ospeli Contreras-Lichtenberg, 1999 -11 mm, G. barbipes (Staeger, 1839) -12 m and G. paripes (Edwards, 1929) -11-13 mm. At the same time the significant difference between larval length in previously described and Caucasian population of G. salinus suggest that further study of these species is necessary to determine the true characteristics of its larvae.
On the basis of morphological data one can conclude that the Caucasian population of G. salinus can be a markedly diverged population of the species, probably even subspecies. This conclusion is also supported by comparative analysis of inversion polymorphism between the Caucasian population and populations of other regions.
At present, 15 banding sequences including four new ones -salA2, salB2, salEX, and salG3 -are known in the banding sequences pool of G. salinus.
By frequencies of the banding sequences Caucasian population are closer to the Kazakhstan populations than to populations from Altai, but it clearly differ from populations from both other regions by the presence of four new banding sequences. The inversion polymorphism in population of G. salinus from the North Caucasus has a high level of heterozygous inversions per specimen and is similar to those of the Altai populations (Table 2). By the number of banding sequences per population, the Caucasian population is close to Kazakhstan populations, but in the percentage of heterozygous larvae, the studied population is more close to that of the Altai populations.
The population of the central Caucasus on the dendrogram of genetic distances (Fig.  8) occupies a distinct position while populations of Altai and Kazakhstan form their own clusters. All the obtained morphological and cytogenetic data may indicate the plausible relative isolation and complexity of the Caucasus from the viewpoint of microevolution. Such arrangement agrees rather well with the geographic location of the studied region. One can say that the north Caucasus is a relatively isolated territory, a special place, or a kind of "island" situated in the "sea" of steppes. Considering the presence in the Ciscaucasia and Greater Caucasus of a large number of saltwater lakes and rivers (Efremov et al. 2010), one can expect a large number of new records of this species in southern Russia. More researches are required in other parts of Caucasus, i.e. Western and Eastern Caucasus and other geographically distant regions for more specific allegations.