ISSN 19954255, Contemporary Problems of Ecology, 2012, Vol. 5, No. 4, pp. 434–442. © Pleiades Publishing, Ltd., 2012. Original Russian Text © V.V. Zykov, D.Yu. Rogozin, I.A. Kalugin, A.V. Dar’in, A.G. Degermendzhi, 2012, published in Sibirskii Ekologicheskii Zhurnal, 2012, No. 4, pp. 585–595.

Carotenoids in Bottom Sediments of Lake Shira as a Paleoindicator for Reconstruction of Lake States in Khakassiya, Russia V. V. Zykova, D. Yu. Rogozina, b, I. A. Kaluginc, A. V. Dar’inc, and A. G. Degermendzhia a

Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Akademgorodok 5, build. 50, Krasnoyarsk, 660036 Russia b Siberian Federal University, Svobodnyi pr. 79, Krasnoyarsk, 660041 Russia c Sobolev Institute of Geology and Mineralogy, Siberian Branch, Russian Academy of Sciences, Acedemika Koptyuga pr. 3, Novosibirsk, 630090 Russia email: [email protected] Abstract—The concentrations of carotenoids buried in the bottom sediments of Lake Shira (Siberia, Kha kassiya) have analyzed for the period of the last 2300 years. The bottom sediments were found to contain car otenoids, which are molecular markers of the corresponding groups of Phototrophic organisms. The bottom sediments of Lake Shira were shown to be a promising object for climate reconstructions of the Late Holocene in southern Siberia. Keywords: okenone, meromixis, holomixis, anaerobiosis, bottom sediments DOI: 10.1134/S199542551204018X

Lake ecosystems are responsive to different envi ronmental changes, which is reflected in variations in the biological, physical, and chemical characteristics of bottom sediments. It has been shown in many works that undisturbed layers of bottom sediments serve as good archives of the state of a water body. Therefore, they can be used for the chronological reconstruction of climate scenarios that caused changes in the ecosys tem of the lake [1–4]. Reconstructions of nonanthro pogenic climate changes of the past allow one to deter mine the basic and background range of climate varia tions in the area under study; they also yield valuable data for predicting possible changes. The most valuable archives are bottom sediments with pronounced seasonal layering, which is formed in horizontal annual zones (varves) and allows making reconstructions for a oneyear period. This type of lay ering is common for sediments in freezing water bod ies located in a temperate zone, where the summer and winter differ dramatically in the production of the allochthonous organic matter, deposition conditions of mineral components, etc. [4]. A layered structure of bottom sediments is best retained in meromictic lakes because their nearbottom water layers are isolated from turbulent and convection processes at the sur face. In addition, constant anaerobiosis and high sul phide concentrations in nearbottom waters hinder the activity of bottom invertebrates, i.e., there is no bioturbation, and the original series of bottom sedi ments remains [1]. In the case when there are no anthropogenic flow offs or tributaries, the water level in closed lakes

located in semiarid climatic zones changes dramati cally depending on the precipitation balance and evaporation in the area under consideration. In the semiarid zone of the northern part of the Minusinsk hollow (Khakassiya), the water layer of lakes changed significantly over the last century. In particular, in 1910–1930s, Lake Shira dried up to the minimum level, then increased up to the current level. The min imum level detected was 7 m lower than the current one (1926) [5]. The mineralization of the lake changed inversely to the volume of water. It was about 27 g L–1 [5, 6] at a minimum level. Similar synchronous changes were common for all lakes of this region [7]. Sharp changes in the water level of lakes observed in the beginning of the last century correlate with changes in the annual amount of precipitation for this region [5]. Therefore, the chronology of changes in the water level of lakes reflects the chronology of dry and wet periods in the area under consideration; with certain marker characteristics, it can be reconstructed using bottom sediments. Lake Shira, which is salt, was found to have a yearly layered structure of bottom sed iments [8, 9]. Changes in the water level of the lake can signifi cantly influence its ecosystem. Thus, mineralized water bodies can change their seasonal circulation regime depending on the water level; a lake becomes holomictic at a relatively low water level, i.e., it is sub jected to a complete overturning from the surface to the bottom once or twice a year (in spring and/or autumn). A lake becomes meromictic when the water level increases, i.e., spring and autumn overturnings

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only proceed in the surface layers (mixolimnion), while the nearbottom layers (monimolimnion) stay in the depths. The monimolimnion favors the develop ment of anaerobiosis and, as a rule, the accumulation of hydrogen sulphide. These transitions from a holo mictic to meromictic state and back are described for a number of lakes located in different parts of the world [10, 11]. In addition, a decrease in the water level and volume of the lake favors an increase in the salinity and vice versa, which is reflected in the com position of biota and sedimentary material. A special feature of meromictic lakes is the pres ence of dense accumulations of anoxygenic pho totrophic bacteria (APB) in a chemocline zone; i.e., it is one the limit of aerobic and anaerobic conditions [12]. This group of bacteria can only exist when hydro gen sulphide and light are present simultaneously. These conditions are formed in the chemocline of meromictic water bodies, including Lake Shira. The molecular remains of APB (such as carotenoids and bacteriochlorophylls and their derivatives (phaeopig ments)) and DNA can remain in the bottom sedi ments of lakes for many years and serve as indicators of anaerobic conditions in the photic zone of a lake at some stage of its existence [1]. Representatives of APB develop in the chemocline of Lake Shira. They are purple sulfur bacteria (PSB) Chromatiaceae, the major carotenoid of which is okenone. In 2011, for the first time, we analyzed the composition of carotenoids in the bottom sediments of Lake Shira. It was found that, in the bottom sediments of the last 450 years, along with more common caro tenoids, such as lutein and zeaxanthin, there is also okenone, which indicates anaerobiosis in the photic zone of the lake [13]. The goal of this work is to evaluate the vertical dis tribution of carotenoids with high accuracy in the upper bottom sediments of Lake Shira and compare it to available data on the dynamics in the water level of the lake, as well as to evaluate the composition of car otenoids in deeper sediment layers formed over the last 2300 years. MATERIAL AND METHODS Lake Shira (lat. 54°30′ N, long. 90°11′ E) is located in the northern part of the Republic of Khakassiya, 15 km away from Shira Village, which is a salty water body. The mineral composition is chloride–sodium– magnesium. The lake has an elliptic form of 9.35 × 5.3 km [14], a watersurface area of 35.9 km2, an aver age depth of 11.2 m, and a maximum depth of 24 m (2007–2009) [6]. The lake is closed and its main trib utaries are the Son river (12.9 million m3 per year, 40% of the inflow), groundwater (2.4 million m3 per year), and anthropogenic inflows [7]. The lake freezes at the end of November and breaks up in the beginning of May. At present, the lake is meromictic, its average CONTEMPORARY PROBLEMS OF ECOLOGY

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mixolimnion salinity during the study period was 15 g L–1, and the monimolimnion salinity was about 19 g L–1 [6]. Nevertheless, the depth of the mixolim nion is unstable and changed from year to year and from season to season in the range of 11–16 m, which also depends on the weather conditions [6]. The lake is a popular place for resorts due to its balneological properties. Lake Shira, a popular sanatorium, has operated shores for more than a century. The bottom sediments were sampled in the deepest part of the lake near the place of hydrobiological mon itoring at 54°30′350 N, 90°11′350 E. The depth of the sampling area was 24 m. In order to avoid disturbing the bottom areas during early field works, we chose a location at a distance of 100 m from this site in a dif ferent direction. Two samples of the bottom sediments were used to analyze carotenoids, i.e., (1) a core with a length of 1550 mm (K1550) (Fig. 1) obtained using a gravita tion core extractor with a movable plastic working tube 60 mm in internal diameter in July 2009; (2) a core with a length of 400 mm (V400) obtained using the box bottom grab taking a bottom area of 160 × 160 mm. The core was picked out of the box bottom grab by inserting plastic tubes with an internal diame ter of 45 mm. K1550 core and V400 container tubes were sealed and stored vertically at a temperature of +4°C. Each core was sliced lengthwise and divided into halves by two thin plates made out of stainless steel inserted into the section. When the core was divided, the plates were removed by a transverse shift, which enabled the sur face of the cut to remain undisturbed with clear lay ered horizontal heterogeneities (Fig. 1). The core halves were kept in air under faint light for 24 h (to make color differences more visible). A color photo of each core was taken with a millimeter ruler. The V400 core was cut into transverse samples in increments of 5–10 mm and they were used to analyze carotenoids (see below). One half of the K1550 core was cut into transverse samples in increments of 5–10 mm. The samples were used to analyze the moisture content and content of organic matter (see below). The second half of the K1550 core was used to selectively take 11 sam ples with widths of 20–100 mm at 200mm intervals. The samples were taken according to color differences (dark and light layers were cut out) (Figs. 1 and 4). Only carotenoids were analyzed in these samples. All of the samples were stored at a temperature of –20°C in the dark and compressed plastic bags. All proce dures were performed under faint, diffuse light. The surface layers in K1550 and V400 cores were par tially eroded or lost during the sampling process. For this reason, the upper limit of the first white layer at a depth of 130 mm from the surface of a division between water and bottom sediments fixed according to the undisturbed Core Sample Box2010 taken in 2010 [9] was considered as a defined point to reduce both cores to a common scale. The layers in the Core No. 4

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Sample Box2010 were counted visually and the upper limit of the first white layer is the same as that in 1945 (Fig. 1). In July 2010, water samples were taken at the same point (see above) to analyze carotenoids in the water layer using a hose, one end of which was at a certain depth, while the other was connected to a vacuum hand pump. The water samples with a volume of 400– 700 mL were filtered through GF/F fiberglass filters (Whatman), dried at room temperature in the dark, placed in plastic bags, and stored at –20°C until they were processed. The rate of accumulation of bottom sediment was measured based on K1550 cores and the Core Sample Box2010 [9]. The age of the intervals under study was evaluated by counting the individual layers, the annual character of which is proved by their position at the section with a peak of a radioactive isotope, such as

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The layer limits were determined visually and according to the peaks in the concentrations of Sr, Br, etc. obtained over a scanning elementary analysis of the in situ core performed using the Xray fluorescent method based on synchrotron radiation (XFA) at the Budker Institute of Nuclear Physics, Siberian Branch, Russian Academy of Sciences, Novosibirsk [9]. The age and rate of sedimentation were also controlled according to the radiocarbon dates obtained in the Poznan Radiocarbon Laboratory (Poland) for three samples from the K1550 core. As a result, the average rate of sedimentation was measured to be 1.9–2 mm year–1 for the upper intervals of the core and gradually decreased to 0.5–0.65 mm year–1 [9, 13]. A visual evaluation and counting of the layers showed that different cores obtained from the same area of Lake Shira (central part) have a similar pattern with the same characteristic light and dark layers (Fig. 1), which allows one to compare data from dif ferent cores on a common time scale. Extraction of carotenoids. The samples of bottom sediments were thawed at a room temperature. Then, we removed 1.5 g of the substance from the internal part of the sample (surfaces in contact with air were not studied). The substance was homogenized by a palette knife, divided into three parts, and weighed. One part was used to determine the airdry weigh and organics (see below), the other two parts were used for two replication analyses of carotenoids. The following extraction of carotenoids from the bottom sediments and water suspension was per formed according to Overmann [1] using a methodol ogy modified by us. The bottom sediments or GF/F fiberglass filters used to filter the water samples were placed in 10 mL of 6% KOH ethanol solution, tritu rated by a glass rod, and kept in a water bath at 60°C for 20 min. The mixture was centrifugalized under 10000 g and supernatant was poured off. 2.5 mL of acetone were added to the remained pellet. Next, it was resuspended and settled at a room temperature for 10 min, then centrifugalized at 10000 g; supernatant was added to the mixture obtained earlier. The proce dure with the pellet was replicated. Then, 10 mL of distilled water and 3 mL of diethyl ether/hexane mix ture at a ratio of 1 : 10 were added using a separating funnel to the mixture of alcohol and acetone superna tants. After intensive mixing and sedimentation were carried out, the upper (hydrophobic) fraction was removed. Carotenoids were isolated from the bottom (hydrophilic) part using the same funnel by adding 3 mL of the hexane/ether mixture. All hexane/ether extracts were mixed and purified from remains of the water fraction by making them run through a water absorber (Na2SO4). The extracts of carotenoids were evaporated with a rotavap and embedded in acetoni

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trile (Panreac). All operations were carried out under faint light to prevent carotenoids from degradation. Chromatographic analysis of carotenoids. The anal ysis was carried out using a highperformance liquid chromatography (HPLC) on the Agilent 1200 Installa tion (Agilent Technologies, United States) with mass spectrometric (MS) and diodearray (DAD) detectors, as well as on the Eclipse XDBC18 Column with an average diameter of particles of 5 μm and a size of 4.6– 150 mm. Eluent and samples were first passed through the Eclipse XDVC18 Precolumn, the average particle diameter was 5 μm, the size was 4.6 × 12.5 mm, and division was performed at a temperature of 40°C. Two phases were used, i.e., phase A with deionized water and phase B with acetonitrile. The eluent was originally half A and half B at a flow rate of 0.5 mL min–1; at 0–3 min, a linear increase occurred in the length of the Bphase to 100% under the same flow; at 3–10 min, a linear increase occurred in the low rate to 1.2 mL min–1 at the same ratio of eluents; at 10–23 min, a steady state was observed; and, at 23–25 min, a linear increase was observed in eluent A up to 50% and decrease in the flow rate up to 0.5 mL min–1. In K1550 samples, the signal was registered by both DAD and MS detectors. For an analysis of the V400 core and water samples, only a DAD detector was used because the MS detector was faulty. The set tings of the diode array were as follows: the length of the absorption wave was 466 nm and the length of the comparison wave was 360 nm. Identification of carotenoids and evaluation of con centrations. The okenone standard for identifying car otenoids in samples and evaluating their concentra tions was obtained from the biomass of the PSB Thio capsa sp. Shira 1 strain grown on a liquid medium [15]. The obtained extract was purified using the method of thinlayer chromatography on glass plates covered with silica gel. The acetone/hexane mixture at a ratio of 1 : 10 was taken as a mobile phase. The concentra tion of the okenone standard was measured in spectro photometrically taking into consideration the molar extinction coefficient for okenone in alcoholic solu tion equal to 134000 L mole–1 [16]. The concentration obtained was used to calibrate the DAD detector and MS detector of the chromatograph. Other carotenoids were identified using the database according to the optical characteristics and molecular weights measured by the MS detector. The settings of the MS detector were as follows: SIM regime (Selected Ion Method) (detec tion of the ions selected) at 6 min, it was 564 (alloxan tin); at 9 min, it was 584 (loroxanthin); at 9 min 30 s, it was 569 (lutein and zeaxanthin); at 10 min, it was 529 (isoreniranthine); and, at 11 min 30 s, it was 579 (transokenone and cisokenone) [13]. The concen trations of all carotenoids were evaluated based on the calibration for okenone because the optical character istics of all xanthophylls are almost the same [16].

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Fig. 2. Vertical distribution of temperature, dissolved oxy gen, and carotenoids in Lake Shira in July 2011.

All samples of the bottom sediments were analyzed in two replications. The concentration rates were mostly similar. For this reason, average rates are given on the profiles (Figs. 3 and 4). The concentrations of carotenoids in bottom sediments were calculated per unit of dry matter. Weight loss after drying at 105°C over 24 h weight loss after ignition at 550°C (Loss On Ignition, LOI550) over 1 h were determined in all samples of K1550 core [17]. RESULTS AND DISCUSSION Carotenoids in the water layer of Lake Shira. Only trace amounts of lutein and zeaxanthine were found in the aerobic zone. The concentration of lutein only exceeded the detection threshold at a depth of 1 m (Fig. 2). The volume of the samples was insufficient to perform a quantitative evaluation of carotenoids in the aerobic zone. The following carotenoids were found in the aerobic zone at a depth of more than 13 m: okenone (cis and transisomers), alloxantin, lutein, zeaxanthin, and loroxanthin (Fig. 2). No. 4

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Fig. 3. Carotenoids in bottom sediments of Lake Shira, V400 core. Right diagram shows the dynamics of the water level (maxi mum depth) in the lake. Shaded area shows carbonate (white) layer.

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Fig. 4. Carotenoids and LOI550 in bottom sediments of Lake Shira. The absence of okenone in the lower sample is shown by an arrow (see explanation in text).

Okenone is a pigment of purple sulfur bacteria. Okenoncontaining PSB are common in Lake Shira, which is described in a number of works [18, 19, etc.]. Alloxantin is a pigment of cryptophyte algae. Their mass accumulation near the chemocline zone is com mon for many stratified water bodies, including the

meromictic Lake Shunet, which is located near Lake Shira [20]. There are no data on cryptophytes in Lake Shira in the literature, mostly likely because they are insufficiently studied. In 2011, this group of microal gae was first found in Lake Shira (Khromechek, Barkhatov, oral report).

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Lutein is a pigment of green algae and higher plants. Zeaxanthin is common to green algae and cyanobacteria and, in the latter, it dominates over lutein [1, 21]. Both green algae and cyanobacteria are permanent inhabitants of the lake under study [22]. Loroxanthin is a carotenoid of green algae, in particu lar Botryococcus braunii [23], which is also a common representative of phytoplankton in Lake Shira [22]. Okenone was mostly common in the lake. The content was maximum in the chemocline zone and gradually decreased towards the bottom (Fig. 2), which completely agrees with the ecophysiology of purple sulfur bacteria, which show the highest activity in the chemocline. The second most common caro tenoid was alloxantin, the carriers of which also favor the chemocline zone (see above). Lutein, zeaxanthin, and loroxanthin were less common in the anaerobic zone. Their profile gradually increases to the bottom (Fig. 2). Because these pigments are produced in the aerobic zone, their elevated concentrations in the monimolimnion are caused by accumulation during the sedimentation process and slow destruction under the monimolimnion conditions. These conditions are oxygen and light poverty, and low temperature [21]. The low concentrations of carotenoids observed in the aerobic zone are in turn explained by a rapid destruc tion, which depends on the content of oxygen, light, and higher temperature. Analysis of pigments in the bottom sediments. LOI550 varied significantly over the whole interval of bottom sediments under study. At the same time, the minimum LOI550 is clearly seen in white layers (Fig. 4). As a rule, this value is indicative of the con tent of organic matter in the sedimentary material [17]. Therefore, white layers are characterized by a low content of organic matter. The qualitative composition of carotenoids buried in the bottom sediments of the lake corresponded completely to the composition of carotenoids in the water layer. Okenone was common in all sediment lay ers of the V400 core (Fig. 3). It was also found in all samples of the K1400 core except one, a deeper sam ple located at a depth of 1430 m (about 2350 years old) (Fig. 4). Other carotenoids were found in all layers without exception. The following five zones can be singled out on the profiles of the V400 core for convenience (Fig. 3): Zone I is 90–130 mm; i.e., it goes from the upper limit to the white carbonate layer. It is characterized by a clear maximum of okenone and local maximums of other carotenoids near the upper limit of the carbon ate layer. The maximum concentration of okenone was much higher than the concentrations of all other carotenoids. Zone II is a white carbonate layer in which the con centrations of all carotenoids decreased. Small caro tenoid peaks were observed in the upper limit of the carbonate layer. CONTEMPORARY PROBLEMS OF ECOLOGY

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Zone III is made up of layers below the white layer to a depth of 240 mm. These layers were characterized by trace amounts of okenone and a relatively even dis tribution of other carotenoids. A slight trend towards an increase in the concentrations towards the bottom was observed. Zone IV contains increased concentrations of all pigments, except okenone, in the interval of 240– 290 mm. The highest increase was in zeaxanthin. A small local maximum of okenone in the thin dark layer at a depth of about 290 mm (Fig. 3, photo of the core). Zone V demonstrates a slight trend of the content of all carotenoids to a decrease is observed below 290 mm. The data fit from V400 and K1400 cores is stud ied more roughly (with a resolution of 50–100 mm), which indicates the high concordance of the concen trations (Fig. 4). It should be noted that the profiles of carotenoids were not associated definitely with the sediment color. Thus, the number of carotenoids was minimum in the first white layer (Fig. 3), while there was a peak of all carotenoids, except zeaxanthin, which is found in the white layer at a depth of 1000 mm (Fig. 4). This layer shows the first significant concen tration of okenone after the maximum it demonstrated in the upper layers. The clear maximum of all pig ments is observed in the dark layers at a depth of 350 mm. Unfortunately, the rough interval of sam pling does not allow one to describe the profiles of car otenoids in more detail. Nevertheless, it should be noted that okenone was clearly absent in the sample taken from the lowest level at a depth of 1430 mm (Fig. 4). As for the reconstruction of climate, the most sig nificant for the reconstruction of the lake history is the period for which there are data on the dynamics of the water layer in the lake, i.e., that extend from 1890 to the present. The period in which the white layer was formed (Fig. 1 and 3) corresponds to the lake depres sion, i.e., the decrease in the water level and its mini mization, then a switch in the water level. The switch in the water level of the lake coincides in time with the formation of the white layer, the upper limit of which is at a depth of 130 mm from the sediment surface (about 1945 or so). There are five more white layers with a width of 45–120 mm in the core K1550; they are 200–250 mm apart (Fig. 1). They are also most likely to indicate the periods when the water layer of the lake decreased. Nevertheless, an analysis of some other indicators (chemical composition and diatom frustules) is needed to prove that this assumption is right. This analysis is currently being performed. The content of organic matter is lower in the white layers than in the dark layers (Fig. 4), which is indicative of accelerated destruction, i.e., it shows the oxygen con tent in the nearbottom waters (holomixis). The pres ence of okenone allows one to insist that the anaerobic zone existed in the lake the period from the fourth century BCE to the present. Nevertheless, the absence No. 4

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of okenone in the earlier sample can be evidence that there was no anaerobiosis in the nearbottom layers during the earlier period. It should be pointed out that the character of the sedimentary material changes dra matically at a depth of 1100 mm below the bright white layer. If the silts before this level were almost dark, then their color changes to lighter gray (not shown in the figures). This transition was also previously discussed by other researchers [24]. This limit most likely marks the lake’s transition from the aerobic hypolimnion to the anaerobic hydrogen–sulphidous hypolimnion or stable monimolimnion. Nevertheless, an analysis of the deeper core layers is needed to prove it. As was shown for other lakes, okenone is found in bottom sediments during either meromixis or holo mixis if anaerobiosis develops in summer hypolimnion [2, 25]. As a rule, meromixis favors the high produc tion of PSB and the better preservation of carotenoids [1, 2, 21]. Therefore, a peak in the concentration of okenone in the layers near 110–130 mm (1945–1970) indicates the pronounced meromictic properties of the lake during this period. Reasons for the current meromixis of Lake Shira are unknown, but the influence of an ectogenic factor is the most probable, which is in agreement with Hutchinson’s classification [26]. Due to the inflow of large amounts of fresh water with the surface runoff into the saltier lake, which it was in the period when the water level (1920s–1930s) was minimum, there could be a difference in salinity that favored a stable density gradient and permanent meromixis [6]. Another factor that significantly influences the salinity gradient (and so meromixis) is a thick (more than 1 m) ice cover [6], the width of which determines the salin ity profile in the next summer period. Obviously, all major factors that determine the overturning regimes of the lake should be considered, which is only possi ble by applying mathematical models [6, 27]. The low content of okenone in the layers until the 1920s–1930s indicates the meromixis at a high water level, which is almost the same as the current content, could be weak or completely absent. Unfortunately, the lack or absence of the substance prevented on from carrying out a more detailed analysis of carotenoids near all white and upper sediment (0–90 mm) layers, for which there are data on the PSB biomass [19]. Therefore, it was shown that the concentration of okenone in the core layers does not correlate directly with the water level of the lake, but the concentration profile can change dramatically near the level fluctua tions (Fig. 3). The low concentration of okenone in the layers that correspond to the minimum water level of the lake is indicative of the weakness or absence of the meromixis during that time. One of the mecha nisms for reducing the stratification is as follows: upon the evaporative reduction of the lake’s volume, the salinity in the mixolimnion becomes similar to the salinity in monimolimnion, which makes the density gradient disappear and, thus, provokes the holomixis.

The similar mechanism of the transition into a holo mictic regime was described for the salty Mono lake (United States) [11]. An increase in the concentration of okenone could be caused by the growing production of PSB in a more favorable environment. As a rule, a major factor that restricts the growth of PSB in deep water bodies is a lack of light [12]. Intensified brightness in the chemocline zone can result from a decrease in the water depth and/or increase in the transparence of mixolimnion. The increase in transparence is condi tioned by the lower biomass of phytoplankton, which occurs due to a lack of biogenic elements, in particular phosphorus. The latter is absorbed by the anaerobic zone and falls out of the cycle, as was shown for other lakes [11]. In the period when the concentration of okenone is maximum, the water level of the lake was about 2 m lower than the current one. Thus, it is quite possible that the depth of the mixolimnion (i.e., the area of the chemocline) was lower than it is now, which could in its turn favor higher brightness. At present, the low brightness in the chemocline of the lake (about 2 μE m–2 s–1) explains the limitation of PSB by light [19]. The increased concentration of pigment in a given layer can indicate not only its increased production, but also more favorable condition for its preservation [21]. The increased concentration of all carotenoids in the layers of about 110–130 mm (1945–1970) can be caused by conditions favorable for preservation, which includes oxygen poverty [21]. This indicates stable meromixis. Therefore, purple sulfur bacteria lived in the lake over the last 2300 years. This means that the photic zone contained hydrogen sulphide. Nevertheless, okenone was definitely absent in the most ancient sample from 340 BCE (Fig. 3). Anaerobiosis was likely to fail in the deep layers of the lake at the time, but fur ther study is required to prove or deny this assumption. CONCLUSIONS (1) A definite peak in the concentration of okenone indicates conditions favorable for the development of purple sulfur bacteria in 1945–1970, which is most likely caused by stable meromixis. (2) The presence of okenone in other layers indi cates that there were anaerobic conditions in the hypolimnion of the lake from about the fourth century BCE until now. (3) In order to reconstruct the lake states, further study with due consideration of data on chemical and biological paleoindicators, a more detailed analysis of the layers, and the use of mathematical models of water circulation and the production of phototrophic organisms should be performed.

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ACKNOWLEDGMENTS This work was supported by the Russian Founda tion for Basic Research, project no. 110500552a; the Fundamental Research Program of the Presidium of the Russian Academy of Sciences, no. 30 (Micro bial Communities in Stratified Lakes of Southern Siberia: Monitoring and Ecological Forecast); and the Joint Project of the Siberian Branch of the Russian Academy of Sciences and the Taiwan Academy of Sci ences, project no. 149. REFERENCES 1. Overmann, J., Sandmann, G., Hall, K.G., and North cote, T., Fossil Carotenoids and Paleolimnology of Meromictic Mahoney Lake, British Columbia, Can ada, Aquat. Sci., 1993, vol. 55, no. 1, pp. 1015–1621. 2. Schmidt, R., Psenner, R., Muller, J., Indinger, P., and Kamenik, C., Impact of Late Glacial Climate Varia tions on Stratification and Trophic State of the Mero mictic Lake Landsee (Austria): Validation of a Concep tual Model by Multi Proxy Studies, J. Limnol., 2002, vol. 61, no. 1, pp. 49–60. 3. Mackay, A., The Paleoclimatology of Lake Baikal: a Diatom Synthesis and Prospectus, EarthSci. Rev., 2007, vol. 82, pp. 181–215. 4. Tylmann, W., Szpakovwska, K., Ohlendorf, C., Woszc zyk, M., and Zolitschka, B., Conditions for Deposition of Annually Laminated Sedimanets in Small Meromic tic Lakes: a Case Study of Lake Suminko (Northern Poland), J. Paleolimnol., 2012, vol. 47, pp. 55–70. 5. Kuskovskii, V.S. and Krivosheev, A.S., Mineral’nye ozera Sibiri (Mineral Lakes of Siberia), Novosibirsk: Nauka, 1989. 6. Rogozin, D.Y., Genova, S.V., Gulati, R.D., and Deger mendzhy, A.G., Some Generalizations on Stratifica tion and Vertical Mixing in Meromictic Lake Shira, Russia, in the Period 20022009, Aquat. Ecol., 2010, vol. 44, no. 3, pp. 485–496. 7. Prirodnye vody Shirinskogo raiona Respubliki Khakasiya (Natural Waters of the Republic of Khakassia), Par nachev, V.P., Ed., Tomsk: Izd. Tomskogo Univ., 2003. 8. Vologina, E.G., Tolomeev, A.P., and Fedorin, M.A., Sedimentation Rate and Annual Layering of Bottom Sediments of Lake Shira as an Instrument for Paleo Proc. Conference ecological Reconstructions, “IV Vereshchaginskaya baikal’skaya konferentsiya” (IV Vereshchagin Baikal Conference), Irkutsk: Izd. Sochava Inst. Geogr. SO RAN, 2005, pp. 35–36. 9. Kalugin, I.A., Dar’in, A.V., Babich, V.V., Smolyani nova, L.G, Vologina, E.G., Ptitsyn, A.B., Ovchin nikov, D.V., and Rogozin, D.Yu., Reconstructions of the Annual Climatic Changes and Water Level of Mountain Lakes in Siberia Basing on Geochemistry of Their Bottom Sediments, Proc. Conference “III Vseros. Konf. S Mezhdunarodnym Uchastiem: Fundamental’nye Problemy Vody I Vodnykh Resursov. 2428 Avgusta 2010” (III AllRussia Conference with International Par ticipation: Fundamental Problems of Water and Water Resources. August 24–28, 2010), Barnaul: Izd. ART, 2010, pp. 130–133. CONTEMPORARY PROBLEMS OF ECOLOGY

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