Limnology and cyanobacterial diversity of high altitude lakes of Lahaul-Spiti in Himachal Pradesh, India

1 1, 1 2 2 YSINGH , JIS KHATTAR *, DP SINGH , PRAHI and A GULATI 1Department of Botany, Punjabi University, Patiala 147 002, Punjab, India 2Plant Pathology and Microbiology Laboratory, CSIR – Institute of Himalayan Bioresource Technology, Palampur 176 061, Himachal Pradesh, India

*Corresponding author (Fax, +91-175-304-6265; Email, [email protected])

Limnological data of four high altitude lakes from the cold desert region of Himachal Pradesh, India, has been correlated with cyanobacterial diversity. Physico-chemical characteristics and nutrient contents of the studied lakes revealed that Sissu Lake is mesotrophic while Chandra Tal, Suraj Tal and Deepak Tal are ultra-oligotrophic. Based on morphology and 16S rRNA gene sequence, a total of 20 cyanobacterial species belonging to 11 genera were identified. Canonical correspondence analysis distinguished three groups of species with respect to their occurrence and nutrient/physical environment demand. The first group, which included Nostoc linckia, N. punctiforme, Nodularia sphaerocarpa, Geitlerinema acutissimum, Limnothrix redekii, Planktothrix agardhii and Plank. clathrata,was characteristic of water with high nutrient content and high temperature. The second group, including Gloeocapsopsis pleurocapsoides, Leptolyngbya antarctica, L. frigida, Pseudanabaena frigida and N. spongiaeforme, occurred in oligotrophic water with high pH and low temperature. The distribution of third group of Cyanobium parvum, Synechocystis pevalekii, L. benthonica, L. foveolarum, L. lurida, L. valderiana, Phormidium autumnale and P. chalybeum could not be associated with a particular environmental condition because of their presence in all sampling sites.

[Singh Y, Khattar JIS, Singh DP, Rahi P and Gulati A 2014 Limnology and cyanobacterial diversity of high altitude lakes of Lahaul-Spiti in Himachal Pradesh, India. J. Biosci. 39 643–657] DOI 10.1007/s12038-014-9458-4

1. Introduction dry and strong winds blow most of the year. This area is characterized by various types of micro-environments and Lahaul-Spiti, covering an area of ca. 13,883 km2 between unique ecosystems with high altitude mountain lakes being 31°44′57′′ and 32°59′57′′ N, and 76°29′46′′ and 78°41′34′′ one of extreme habitats. According to high altitudinal lake E, is a part of the Indian cold desert in the north-western inventory of Indian Space Research Organisation (2011), a Himalayas. Geographically it is the largest district in the total of 272 high altitude lakes covering an area of 617 ha are state of Himachal Pradesh and is divided into Lahaul present in Himachal Pradesh which account for less than 1% Valley and Spiti Valley. Climatic conditions here are typical of the total wetland area of the state. of dry temperate and alpine zones. Altitudinal range in this High altitude lakes characterized by low temperature, region is between 2,400 m and 6,400 m, rainfall is scanty, generally low buffering capacity and low level of nutrients and the area remains covered with snow for more than 6 act as reference systems for global climate change (Psenner months in a year. The temperature during winter falls to 2002; Catalan et al. 2006). Due to the extreme environmen- nearly −30°C, and even the temperature of summer nights tal conditions, the biodiversity of these lakes is scanty, with is sub-zero. Except for periods of rain or snowfall, the air is prokaryotes as the major component. Cyanobacteria, the

Keywords. Canonical correspondence analysis; cold desert; cyanobacterial diversity; high altitude lakes; limnology http://www.ias.ac.in/jbiosci J. Biosci. 39(4), September 2014, 643–657, * Indian Academy of Sciences 643

Published online: 8 July 2014 644 Y Singh et al. most diverse group of prokaryotes, are predominant 2. Materials and methods phototrophs of such ecosystems. These organisms even dominate as primary producers in hot water springs and 2.1 Study sites and sampling Antarctic permafrost aquatic systems (Jungblut et al. 2005; Waterbury 2006). Their ecological tolerance to a broad The four lakes Chandra Tal, Suraj Tal, Deepak Tal and Sissu range of temperatures, high salinity, and adaptations to Lake located in the Lahaul-Spiti cold desert region of north- high and low light conditions contributes to their compet- western Himalayas, Himachal Pradesh, were visited twice itive success as planktonic or benthic organisms in a between August 2009 and October 2011 for sampling. variety of environments (Sommaruga and Garcia-Pichel During each visit, three samples from two locations each of 1999; Badger et al. 2006; Cohen and Gurevitz 2006). A Chandra Tal, Suraj Tal and Deepak Tal and three locations diverse range of cyanobacteria have been found in polar, of the Sissu Lake were collected. Thus, a total of 54 samples, alpine habitats as benthic mats (Vincent et al. 1993; Elster 27 during each visit, were collected. Each sample compris- et al. 1997; McKnight et al. 1999; Fernández-Valiente ing soil, algal mats and water was taken with the help of et al. 2007; Zakhia et al. 2008), films in shallow trowel, spatula and net mesh in sterilized polyethylene bags thermokarst lakes (Vézina and Vincent 1997; Rautio and and brought to the laboratory for examination. The water Vincent 2006; Biondi et al. 2008), ice shelf ponds of the sample of each lake was also collected separately in 2 L pre- Arctic (Vincent et al. 2004a, b; Mueller et al. 2005) and sterilized plastic bottles. Antarctica (Howard-Williams et al. 1989; Sabbe et al. 2004; Jungblut et al. 2005), and in ice-covered lakes (Hawes and Schwarz 2001; Taton et al. 2006a, b; Vopel 2.2 Physico-chemical characteristics and Hawes 2006). The occurrence of cyanobacteria- dominated mats in cryoconite holes and pools on glaciers Water and soil samples collected from sampling sites were continue to be of great interest to microbial ecologists to physico-chemically examined. Water temperature was noted address questions concerning microbial distribution and in the field by a thermometer at the site of collection. The pH diversity, biogeochemical processes in microbial consortia of the water samples was noted by a digital pH meter (Orion, and microbial strategies for survival and success in the Thermo Fisher Scientific, Waltham, MA, USA) and conduc- cryosphere (Christner et al. 2003; Mueller and Pollard tivity was determined by a conductivity bridge (Systronics, 2004; Tranter et al. 2004; Porazinska et al. 2004; Stibal Norcross, GA, USA). Water samples were analysed for et al. 2006). High altitude lakes have received little atten- hardness, alkalinity, chloride, calcium, sodium, potassium, tion so far in terms of their limnology, biodiversity, con- magnesium, total phosphate, ammonium nitrogen and nitrate servation and water management, but they are becoming nitrogen by standard methods available in the literature increasingly important due to the possible consequences of (Mackereth et al. 1978; APHA 2005). the global climate change (Bhat et al. 2011). Life in the aquatic environment is largely governed by 2.3 Isolation and purification of cyanobacteria physico-chemical characteristics and their stability. Limnological studies of water bodies also provide informa- Samples were initially examined under a light microscope for tion about the trophic status which may help in management the presence of cyanobacteria, and a small portion of each and conservation (Marchetto et al. 1995). Limnological stud- sample was inoculated in 20 mL sterilized liquid nutrient ies on fresh water bodies from India have been undertaken Chu-10 medium (Safferman and Morris 1964) for culture (Rao and Mahmood 1995; Naganandini and Hosmani 1998; enrichment. The medium was supplemented with Pandey et al. 2000;PatelandSinha2000; Kumar et al. cyclohexamide (100 μgmL−1) to eliminate eukaryotic con- 2012), but little information is available on the limnological taminants. From these cultures, clonal and axenic cultures of studies on high altitude lakes of north-western Himalayas cyanobacterial strains were established by repeated sub- (Kumar et al. 2012; Nautiyal et al. 2012). Although few culturing on petriplates containing solidified Chu-10 medium reports on freshwater algal diversity of north-western (Stanier et al. 1971). The isolated and purified cyanobacterial Himalayas are available (Seth et al. 2006; Sidhu and strains are being maintained in liquid culture medium in a Ahluwalia 2011), no information is available on BOD incubator (NSW, India) at 15±2°C under 45 μmol pho- −2 −1 cyanobacterial diversity of high altitude lakes of the ton flux cm s light intensity with 14/10 h light/dark cycle. Lahaul-Spiti area. The present study was undertaken to study the limnology parameters and cyanobacterial diversity of 2.4 Identification of cyanobacteria four high altitude lakes of Lahaul-Spiti cold desert area of Himachal Pradesh, India, to determine a relationship be- 2.4.1 Morphological identification: All cyanobacterial isolates tween cyanobacterial diversity and limnology of these lakes. were observed under light microscope and identified following

J. Biosci. 39(4), September 2014 Limnology and cyanobacterial diversity of cold lakes 645 the taxonomic descriptions in monographs, floras and books 3. Results (Desikachary 1959; Anagnostidis and Komárek 1985; Komárek and Anagnostidis 1998, 2005; Komárek and Hauer 3.1 Characteristics of selected lakes 2013). Morphological taxonomic features included trichomes/ filaments shape; cell dimensions; presence/absence of sheath, The lakes of the present study are located at altitudes thickness; shape, size and position of akinetes/heterocyst, if ranging from 3010 m to 4883 m above sea level. The present, etc. Images were captured using a Nikon Eclipse 80i geographical location of these lakes is shown in the map Microscope fitted with a digital imaging system. (figure 1). Chandra Tal (32°28′30.65′′ N 77°37′1.42′′ E), situated at 4300 m, is the source of river Chandra. The lake has been suggested as a Ramsar site due to its 2.4.2 16S r RNA gene-sequence-based identification: Genomic TM biological, cultural and geological values. Suraj Tal DNA extraction was done by HiPurA plant genomic (32°45′02.09′′ N 77°24′52.79′′ E), located at the height DNA Miniprep Purification Spin Kit (HIMEDIA®, of 4883 m near Baralacha pass in Lahaul region, is the Mumbai, India). 16S rRNA gene was amplified using source of Bhaga river. Deepak Tal (32°45′11′′ N 77°15′ cyanobacteria specific primers CYA106F and CYA781R 20′′ E) is situated at 3752 m on way to Suraj Tal, while μ (Nübel et al. 1997). Total volume of 50.0 LPCR Sissu Lake (32°28′36.48′′ N 77°07′37.94′′ E) is a μ −1 reaction mixture was comprised of 200 mol L manmade lake situated at 3010 m in the village Sissu μ −1 dNTPs, 50 mol L each primer, 1X PCR buffer, 3 of Lahaul region. Chandra Tal, Suraj Tal and Deepak Tal U Taq polymerase, and 100 ng genomic DNA. The are clear water lakes devoid of any macrophyte even on thermo-cycling conditions involved an initial denatur- their littoral sides, while Sissu Lake supports growth of ationat94°Cfor4min,followedby35cyclesof1 some green algal forms like Spirogyra, Oedogonium and min at 94°C, 1 min at 52°C, 2 min at 72°C and final Scenedesmus, etc. These lakes remain totally frozen form extension at 72°C for 8 min. The gel-purified product November to February. was obtained using GeneJETTM Gel Extraction Kit (Fermentas, Vilnius, Lithuania). The gene sequencing was done using BigDye® Terminator v3.1 cycle se- quencing kit and ABI Prism 310 Genetic Analyzer (Applied 3.2 Physico-chemical characteristics of lakes Biosystems, CA, USA). The sequences were analyzed using the gapped BLASTn (http://www.ncbi.nlm.nih.gov/Blast) The physico-chemical characteristics of selected lakes are search algorithm and aligned to the nearest neighbours. presented in table 1. Water temperature of these lakes at the time of sampling ranged from 6°C to 18°C, with the lowest temperature (6±2°C) at Suraj Tal and the highest temperature (18±2°C) at Sissu Lake. The pH was alkaline, ranging from 2.5 Cyanobacterial diversity analysis 8.5±0.2 to 9.1±0.1. Total alkalinity, which is taken as tem- porary hardness of CaCO3, was in the range of 208±5.4 mg The cyanobacterial diversity in terms of relative abundance, L−1 (Deepak Tal) to 480±12.4 mg L−1 (Sissu Lake). species richness (Gleason 1922) and evenness (Pielou 1966), Conductivity ranged from 130±5.5 μscm−1 (Deepak Tal) was studied by applying the Shannon index (Shannon 1948) to 200±12.6 μscm−1 (Sissu Lake). Total hardness and TDS and Simpson index (Simpson 1949). The similarity analysis were in the range of 120±4.5 mg L−1 to 286±14.5 mg L−1 between the sites was determined by Jaccard similarity index and 98±5.0 mg L−1 to 245.5±12.6 mg L−1, respectively. (Jaccard 1912). Calcium content in these lakes was comparatively more than magnesium and the mean values of calcium and magnesium ranged from 30.46±4.6 mg L−1 to 53±5.0 2.6 Statistical analysis mg L−1 and 8.76±0.5 mg L−1 to 30±2.8 mg L−1, respec- tively. Sodium concentration in these lakes was compar- The relationship between cyanobacterial species distribution atively higher (2.60±0.5 mg L−1 to 10.50±1.7 mg L−1)as and physico-chemical variables of water was assessed by compared to potassium (0.29±0.05 mg L−1 to 2.70±0.5 canonical correspondence analysis, followed by Monte mg L−1). Mean concentration of chloride in the lakes was Carlo Test (1000 premutations, α=0.05) to estimate the sta- in the range of 3.55±0.5 mg L−1 to 56±2.5 mg L−1. tistical significance of these relationships. The analysis was Nitrate-nitrogen, ammonium-nitrogen and total phosphate carried out using Microsoft Excel Add-Ins Software in the lakes were recorded with mean concentrations between XLSTAT 2012. Water temperature, pH, total P, total N and 0.130±0.03 mg L−1 and 4.13±1.6 mg L−1, 0.02±0.005 mg L−1 conductivity were the environmental variables included in and 1.57±0.2 mg L−1, 0.004±0.001 mg L−1 and 14.5±5.5 mg the analysis. L−1, respectively.

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Figure 1. Map showing the geographical location of Chandra Tal, Suraj Tal, Deepak Tal and Sissu Lake.

Table 1. Physico-chemical characteristics of water at sampling locations from the high altitude lakes of Lahaul-Spiti

Locality

Parameter Chandra Tal Suraj Tal Deepak Tal Sissu Lake

Temperature (°C) 10±2 6±2 8±2 18±2 pH 8.6±0.2 9.1±0.1 8.8±0.2 8.5±0.2 Ammonium (mg L−1) 0.04±0.005 0.02±0.005 0.025±0.002 1.57±0.2 Calcium (mg L−1) 52.91±5.2 32.06±4.5 30.46±4.6 53.00±5.0 Chloride (mg L−1) 3.55±0.5 4.97±0.7 4.26±0.8 56.0±2.5 Conductivity (μscm−1) 180.0±10.4 140.0±9.5 130.0±5.5 200.0±12.6 Magnesium (mg L−1) 14.20±1.2 10.5±1.3 8.76±0.5 30.0±2.8 Nitrate (mg L−1) 0.148±0.05 0.130±0.03 0.131±0.02 4.13±1.6 Total P (mg L−1) 0.024±0.002 0.004±0.001 0.006±0.001 14.5±5.5 Potassium (mg L−1) 0.50±0.02 0.31±0.01 0.29±0.05 2.70±0.5 Sodium (mg L−1) 5.30±0.7 2.60±0.5 2.80±0.3 10.50±1.7 Sulphate (mg L−1) 6.25±0.9 22.0±2.2 20.0±2.0 86.0±4.0 −1 Total alkalinity as CaCO3 (mg L ) 286.0±8.4 224.0±10.4 208.0±5.4 480.0±12.4 −1 Total Hardness as HCO3 (mg L ) 150.0±8.6 130.0±5.5 120.0±4.5 286.0±14.5 TDS (mg L−1) 114.0±5.4 104.0±4.2 98.0±5.0 245.5±12.6

Data are mean±standard deviation.

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3.3 Morphology and 16S rRNA gene-sequence-based 3.5 Cyanobacterial diversity identification Cyanobacterial diversity of the lakes was studied by A total of 90 cyanobacterial isolates comprising 18 employing diversity indices. The maximum value for unicellular (20%), 11 heterocystous filamentous Shannon’s index (H) (2.654) was observed for Sissu Lake (12.22%) and 61 non-heterocystous filamentous (highest heterogeneity), while the minimum value (1.695) (67.77%) forms were isolated from the high altitude corresponded to Deepak Tal. Sissu Lake also exhibited max- lakes, represented by 20 species belonging to the 11 imum value (13.698) for Simpson’s index, while Deepak Tal genera Cyanobium, Gloeocapsopsis, Synechocystis, exhibited minimum value (5.0). Maximum species richness Geitlerinema, Leptolyngbya, Limnothrix, Phormidium, index (3.834) was also obtained for Sissu Lake, while min- Planktothrix, Pseudanabaena, Nostoc and Nodularia of imum (2.603) richness index was for Deepak Tal. The min- 6 families (Chroococcaceae, Synechococcaceae, imum evenness index value (0.945) was obtained for the Merismopediaceae, Pseudanabaenaceae, Phormidiaceae, Deepak Tal, while the maximum value (0.980) for this index Nostocaceae) and 3 Orders (Chroococcales, Oscillatoriales was obtained for the Sissu Lake. The Jaccard similarity and Nostocales) of the class Cyanophyaceae. The unicellular, index between Sissu Lake and other lakes was low as com- non-heterocystous filamentous and heterocystous filamentous pared to the similarity indices among other three lakes. The forms were represented by 3, 13 and 4 species, respectively Suraj Tal showed maximum similarity index (55.55) with (table 2;figure2 and 3). Partial 16S rRNA gene sequences Deepak Tal, while its similarity indices with Chandra Tal were obtained for all the morphologically identified species. and Sissu Lake were 50 and 27.77, respectively (table 4). These sequences were compared with the sequences available in the NCBI database using BLASTn (table 3). The 16S rRNA gene sequences deposited in NCBI GenBank have been 3.6 Relationship between cyanobacterial occurrence accorded the accession numbers KJ705090-KJ705107, and environmental variables KJ721218 and KJ721219. These cyanobacterial species are beingmaintainedinourculture collection under code Canonical correspondence analysis employed to study rela- PUPCCC. tionship between species composition and environmental variables showed that temperature, total nitrogen and total 3.4 Distribution and abundance of cyanobacteria phosphorus were strongly correlated with the first CCA axis (0.972, −3.607 and 4.057, respectively) while pH and con- Leptolyngbya foveolarum with 17.5% relative abundance ductivity were strongly correlated with the second CCA axis was the most abundant species occurring in all the sites. (−0.842 and 1.202, respectively) (figure 4). Axis 1 and axis 2 λ λ The distribution and richness of other taxa varied signif- of the CCA, with eigenvalues of 1=0.531 and 2=0.207, icantly from site to site. Some species were found to be explained 48.26% of cumulative percentage variance of site specific with Geitlerinema acutissimum, cyanobacterial species composition while all environmental Leptolyngbya benthonica, Limnothrix redekii, variables considered for analysis accounted for 70.96% of total Planktothrix agardhii, Plank. clathrata, Nostoc linckia, variation of cyanobacterial species composition (table 5). The N. punctiforme and Nodularia sphaerocarpa recorded two axes alone explained a large proportion of variance in only from Sissu Lake and Gloeocapsopsis cyanobacterial species composition–environment relationship pleurocapsoides and N. spongiaeforme only from (68.01%). Nine sampling sites are well separated on both axes Chandra Tal, and Cyanobium parvum was present only by physico-chemical features (figure 4). in Deepak Tal and Sissu Lake. Deepak Tal and Suraj Tal exhibited almost the same pattern of taxa formation having 6 and 8 taxa, respectively, whereas maximum 4. Discussion number (15 taxa) was recorded from Sissu Lake (table 3). Oscillatoriales was the dominant order in terms of num- The water temperature and ambient air temperature of the ber of species and abundance, as it contributed 13 spe- sampling sites were close to one another’s, mainly due to the cies (65%), followed by Nostocales represented by 4 standing nature of the water in the lakes. The water temper- species (20%), and Chroococcales represented by 3 spe- ature in streams generally shows variation with respect to air cies (15%). Genus Leptolyngbya (Oscillatoriales) with 6 temperature because of the running nature of water (Hynes species was dominant at species level (30%) followed by 1979; Williamson et al. 2008). Further, the temperature of Nostoc (Nostocales) with 3 species (15%), and water was dependent upon the altitude of the lake with the Phormidium and Planktothrix (Oscillatoriales) with 2 lowest temperature (6±2°C) being that of Suraj Tal, located species each (10%) (table 2). at a height of 4883 m above sea level, and the highest

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Table 2. Taxonomic assignment and morphological characteristics of cyanobacterial species isolated from the high altitude lakes of Lahaul-Spiti

Taxonomic PUPCCC Cell dimensions assignment code (μm) Filament/Trichome/Cell description Figure

Chroococcales Synechococcaceae Cyanobium parvum 007.1 B=2.8±0.8 Cells elongated, solitary, sometimes in chain up to 2-a L=2.4±0.8 4 cells. Cells mostly cylindrical, straight or slightly curved. Blue-green or pale colour Chrococcaceae Gloeocapsopsis 008.2 Ø =6.1±1.9 Cells rounded with irregular outline, blue green, 2-b pleurocapsoides finely granulated cell content. Cell envelope lamellated, firm, undiffluent, yellowish in colour. Colonies of irregular shape Merismopediaceae Synechocystis pevalekii 062.1 Ø =3.0±0.6 Spherical cells, solitary or forming pairs, bright 2-c blue-green colour Oscillatoriales Pseudanabaenaceae Geitlerinema acutissimum 110.4 B=2.0±0.4 Trichomes straight, blue-green, intensely motile, 3-k, 3-l L=2.4±0.8 unconstricted at cross wall, attenuated at the ends and bent, cell content granulated. Apical cell hooked, acuminate-rounded. Leptolyngbya antarctica 112.2 B=2.3±0.7 Filaments short, with indistinct sheath. Trichome 2-d L=3.2±1.0 densely constricted at cross wall, with rounded apical cell. Leptolyngbya benthonica 112.5 B=2.8±0.6 Long filaments formed by thin and hyaline sheath 3-a L=2.0±0.4 attached to the trichome. Flexuous trichomes composed of unconstricted or feebly constricted cells at the crosswalls, with rounded apical cells. Leptolyngbya foveolarum 112.8 B=2.0±0.4 Filaments long, parallel arranged, with thin, firm 2-h L=2.4±0.8 sheath. Trichome pale to bright blue-green in colour, constricted at cross wall, with rounded apical cell. Leptolyngbya frigida 112.1 B=2.0±0.4 Long and flexuous filaments formed of a thin sheath, 2-f L=2.4±0.8 hyaline, and homogeneous. Thick sheaths sometimes observed and separated from the filament. Cells strongly constricted at the cross-walls. Rounded apical cell. False branching observed Leptolyngbya lurida 112.6 B=2.0±0.4 Filaments nearly erect to variously curved, densely 2-e L=3.6±1.2 entangled, with thin sheath. Trichomes pale blue- green to yellow brown, constricted at cross wall, with cylindrical to rounded apical cell. Leptolyngbya valderiana 112.7 B=2.4±0.8 Straight, thin and long filaments and sometimes 2-g L=3.2±1.8 arranged in parallel. Sheaths very thin, mucilaginous and hyaline. Long cells, slightly constricted and elongated conical apical cell Limnothrix redekii 116.2 B=2.0±0.8 Trichomes solitary, slightly curved, pale blue-green, 2-i L=3.2±1.6 without sheath, unconstructed at cross wall, with two small polar aerotopes at the septa. Apical cell rounded. Pseudanabaena frigida 106.7 B=1.5±0.5 Filaments with cylindrical and constricted cells, with 3-m L=2.4±0.8 a very thin sheath, with truncated rounded end cells. Blue-green or pale green cells with polar aerotopes Phormidiaceae Phormidium autumnale 118.4 B=5.6±2.4 Filaments straight, entangled, with thin sheath. 3-b, 3-c L=4.6±1.4 Trichome bright blue-green, motile, attenuated towards ends, slightly constricted at cross walls. Apical cells somewhat elongated, rounded, with calyptras.

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Table 2 (continued)

Taxonomic PUPCCC Cell dimensions assignment code (μm) Filament/Trichome/Cell description Figure

Phormidium chalybeum 118.8 B=7.4±2.6 Filaments straight to curved, long, with thin sheath, 3-d, 3-e L=1.3±0.3 motile. Trichome dark blue-green, constricted at cross wall, slightly attenuated towards ends and hooked. Cell content finely granulated. Apical cell widely rounded, obtuse. Planktothrix agardhii 108.6 B=3.4±0.8 Trichomes solitary, long, straight, olive blue-green, 3-f to 3-h L=1.8±0.6 without sheath, not constricted at granulated cross walls, attenuated at the ends. Cell content with numerous aerotopes. Apical cell convex, bluntly conical to pointed. Planktothrix clathrata 108.8 B=5.6±2.4 Trichomes solitary, straight to slightly flexuous, 3-i, 3-j L=1.3±0.3 slightly constricted at cross walls, cylindrical, not attenuated towards ends, slightly motile. Cells blue-green, with aerotopes. Apical cells rounded. Nostocales Nostocaceae Nostoc linckia 405.2 VC: Filaments densely entangled, flexuous or highly 2-j B=4.0±0.8 coiled with diffluent and colourless sheath. L=2.0±0.4 Trichome pale blue-green, cells barrel shaped. Heterocyst: Spherical heterocysts and akinetes subspherical. Ø =4.0±0.8 Akinetes: B=3.6±0.4 L=4.6±1.4 Nostoc punctiforme 405.6 VC: Filaments densely entangled, flexuous with hyaline 2-k B=4.0±0.8 sheath. Trichome blue-green, cylindrical cells and L=4.8±1.8 very compact in thick sheaths with a heterocyst Heterocyst: outside the sheath. Spherical heterocysts and Ø =5.2±1.2 subspherical akinetes. Akinetes: B=5.6±0.8 L=2.0±0.4 Nostoc spongiaeforme 405.8 VC: Filaments loosely entangled, flexuous with diffluent 2-l B=3.6±0.8 sheath. Trichome blue-green, cells subcylindrical, L=3.6±1.2 Heterocysts and akinetes subspherical. Heterocyst: B=7.2±0.8 L=7.6±2.0 Akinetes: B=6.5±0.7 L=10.4±0.8 Nodularia sphaerocarpa 420.1 VC: Filaments solitary, straight, bent or spirally coiled, 3-n, 3-o B=4.0±0.8 with colourless thick sheath. Trichome blue-green, L=2.4±0.8 cells short, discoid. Heterocysts subspherical, broader Heterocyst: than vegetative cells and akinetes spherical, in series. B=4.0±0.8 L=2.8±0.4 Akinetes: Ø =6.4±1.6

VC: vegetative cells; Ø: cell diameter; B: breadth; L: length; PUPCCC: Punjabi University Patiala Cyanobacterial Culture Collection. Data are mean±standard deviation. temperature (18±2°C) being that of Sissu Lake, situated at macrophytic growth, it seems that geological features of the 3010 m above sea level. catchment and basin areas, and the evaporation pattern of The pH of all the lakes was alkaline, in the range 8.5±0.2 water from the lake surface, rather than photosynthesis, are to 9.1±0.1. Since all the lakes have clear water without any responsible for alkaline pH of water. Usually pH is on the

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Figure 2. Microphotographs of cyanobacterial species isolated from high altitude lakes of Lahaul-Spiti: (a) Cyanobium parvum, (b) Gloeocapsopsis pleurocapsoides, (c) Synechocystis pevalekii, (d) Leptolyngbya antarctica, (e) L. lurida, (f) L. frigida, (g) L. valderiana, (h) L. foveolarum, (i) Limnothrix redekii, (j) Nostoc linckia, (k) N. punctiforme and (l) N. spongiaeforme. Scale bar, 10 μm.

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Figure 3. Microphotographs of cyanobacterial species isolated from high altitude lakes of Lahaul-Spiti: (a) Leptolyngbya benthonica, (b– c) Phormidium autumnale, (d–e) P. chalybeum, (f–h) Planktothrix agardhii, (i–j) Plank. clathrata, (k–l) Geitlerinema acutissimum, (m) Pseudanabaena frigida and (n–o) Nodularia sphaerocarpa. Scale bar, 10 μm.

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Table 3. 16S rRNA gene-sequence-based identity of cyanobacterial species, distribution pattern and their relative abundance from the high altitude lakes of Lahaul-Spiti

Relative PUPCCC NCBI accession Similarity abundance code number Nearest neighbour (%) Distribution (%)

007.1 KJ705090 Cyanobium sp. UAM 406 (JQ070060) 98 III, IV 7.5 008.2 KJ705091 Gloeocapsopsis crepidinum LEGE 06123 (FJ589716) 98 I 5.0 062.1 KJ705092 Synechocystis sp. LMECYA 68 (EU078508) 99 I,II,III,IV 12.5 110.4 KJ705093 Geitlerinema sp. Sai004 (GU935348) 99 IV 5.0 112.2 KJ705094 Leptolyngbya antarctica ANT.BFI.1 (AY493590) 99 I,II,III 10.0 112.5 KJ721218 Leptolyngbya sp. LEGE 07296 (HM217082) 99 IV 10.0 112.8 KJ705095 Leptolyngbya foveolarum ( X84808) 99 I,II,III,IV 17.5 112.1 KJ705096 Leptolyngbya frigida ANT.L52.2 (AY493575) 98 I,II,III 10.0 112.6 KJ705097 Leptolyngbya sp. 690-AC125 (EU282439) 99 I,II,IV 15.0 112.7 KJ705098 Leptolyngbya sp. LEGE 06308 (HM217060) 99 I,II,III,IV 7.5 116.2 KJ705099 Limnothrix sp. CENA545 (KF246506) 99 IV 2.5 118.4 KJ705100 Phormidium autumnale SAG 35.90 (EF654081) 98 I,IV 5.0 118.8 KJ705101 Phormidium sp. 195-A12 (EU282429) 99 II,IV 5.0 108.6 KJ705102 Planktothrix agardhii CCAP 1460/13 (HF678485) 98 IV 7.5 108.8 KJ721219 Planktothrix sp. VUW25 (GQ451423) 97 IV 5.0 106.7 KJ705103 Pseudanabaena sp. ACCS 013 (GU250784) 99 I,II 7.5 405.2 KJ705104 Nostoc sp. KK-01 (AB187508) 97 IV 5.0 405.6 KJ705105 Nostoc sp. 'Peltigera sp. K40 cyanobiont' (KC514663) 99 IV 7.5 405.8 KJ705106 Nostoc sp. SKS5 (EU022707) 99 I 5.0 420.1 KJ705107 Nodularia sphaerocarpa UTEX B 2092 (AJ781151) 98 IV 5.0

I: Chandra Tal; II: Suraj Tal; III: Deepak Tal; IV: Sissu Lake.

− alkaline side when the CO3 system prevails in water bodies 1984). Sissu Lake is comparatively rich in these nutrients − and total alkalinity is higher than the HCO3 system (Toma compared to other lakes because of human activity. Vass 2011). The high pH is also not ascribable to the release of (1980) and Zutshi et al. (1980) have reported that the distri- carbohydrates from sediments and their conversion to bicar- bution of nitrate, ammonium and phosphate are governed by bonates by carbonic acid formed by decomposition of or- physical factors such as input source to the lake (catchment ganic matter as these lakes do not have high amount of characteristics), changes in mixing depth during stratification organic matter (Hannon et al. 1979). The alkaline pH of and complete mixing during isothermal conditions. The ni- the lakes in the present study is also supported by high trogenous compounds in the water bodies are derived to an alkalinity (208–480 mg L−1). Zutshi et al. (1980) reported appreciable degree from the atmosphere, whereas ammoni- decrease in water conductivity with the increase in altitude, um is the chief product of decomposition of plant and animal which holds true for the present study also. Conductivity and proteins (Sharma 2000). As the decomposition of organic alkalinity are strongly influenced by the nature of incoming water and its periodicity (Kiplagat et al. 1999). Table 4. Jaccard similarity index (J) of cyanobacterial species The source of water in high altitude Himalayan lakes is from the high altitude lakes of Lahaul-Spiti mostly snowmelt, usually low in ionic content, and scanty ++ ++ Deepak Sissu rains. The cation progression in the lakes was Ca >Mg > Chandra Tal Suraj Tal Tal Lake Na+ >K+. Zutshi et al. (1980) have reported that calcium is generally the dominant cation in Himalayan lakes of Chandra Tal 50 45.45 25.0 Kashmir region because of the predominance of lime-rich Suraj Tal 55.55 27.77 rocks in the catchment areas. Deepak Tal 23.52 Ammonium-nitrogen, nitrate-nitrogen and total phosphate Sissu Lake are good indicators of anthropogenic pressures (Zutshi et al.

J. Biosci. 39(4), September 2014 Limnology and cyanobacterial diversity of cold lakes 653

Figure 4. Canonical correspondence analysis biplot showing the relationships between sites, occurrence of cyanobacterial species and environmental variables in high altitude lakes of Lahaul-Spiti. CT1: Chandra Tal location 1; CT2: Chandra Tal location 2: ST1: Suraj Tal location 1; ST2: Suraj Tal location 2; DT1: Deepak Tal location 1; DT 2: Deepak Tal location 2; SS1: Sissu Lake location 1; SS2: Sissu Lake location 2; SS3: Sissu Lake location 3. CYPA: Cyanobium parvum; GEAC: Geitlerinema acutissimum; GLPL: Gloeocapsopsis pleurocapsoides; LEFO: Leptolyngbya foveolarum; LEAN: L. antarctica; LEBE; L. benthonica; LEFR: L. frigida; LELU: L. lurida; LEVA: L. valderiana; LIRE: Limnothrix redekii; NOLI: Nostoc linckia; NOPU: N. punctiforme; NOSP: N. spongiaeforme; NODS: Nodularia sphaerocarpa; PLAG: Planktothrix agardhii; PLCL: Plank. clathrata; PSFR: Pseudanabaena frigida; SYPA: Synechocystis pevalekii; PHAU: Phormidium autumnale; PHCH: P. chalybeum. Temp: Temperature; Cond: Conductivity; TN: Total N; TP: Total P. matter in these lakes except Sissu Lake is negligible, catch- closed nature of the lakes seem to be responsible for ob- ment characteristics (geological features), atmosphere and served level of nitrogen, ammonium and phosphate. On the

Table 5. Eigenvalues for CCA axes, and correlation coefficients between CCA ordination axes versus environment factors at high altitude lakes of Lahaul-Spiti

Axis F1 F2 F3 F4 F5 Total inertia

Eigenvalues 0.531 0.207 0.160 0.108 0.080 1.086 Cumulative percentage variance Of species data 34.731 48.262 58.706 65.753 70.961 Of species-environment relation 48.944 68.012 82.729 92.661 100.000 Sum of all eigenvalues 1.085 Sum of all canonical eigenvalues 0.444 Temperature 0.972 -1.858 0.404 6.445 -0.717 pH -0.239 -0.842 1.129 1.778 2.231 Conductivity -0.741 1.202 0.361 0.036 2.685 Total P 4.057 4.817 6.219 1.033 -11.317 Total N -3.607 -4.494 -6.243 -6.125 11.637

J. Biosci. 39(4), September 2014 654 Y Singh et al. basis of nutrient content, Sissu Lake is classified as meso- cyanobacterial species are well separated along both axes trophic while other three are ultra-oligotrophic. of CCA biplot, indicating different physico-chemical de- Information on biological diversity of any aquatic ecosys- mands of different species (figure 4). The CCA data were tem may provide a more sensitive time-integrated assess- differentially distributed, which allowed separation of the ment of a water body than physical or chemical variables. cyanobacterial species into three groups on the basis of water Of the total cyanobacterial diversity in these high altitude quality parameters at the sampling sites. Group 1 included lakes, 80% diversity was represented by non-heterocystous the heterocystous species N. linckia, N. punctiforme and cyanoprokaryotes (Chroococcales and Oscillatoriales), Nod. sphaerocarpa as well as non-heterocystous species G. whereas 20% diversity comprised hererocystous acutissimum, Limn. redekii, Plank. agardhii and Plank. cyanoprokaryotes (Nostocales). Members of Oscillatoriales clathrata, placed on the right side of the graph, with high were dominant, followed by members of Nostocales. It is temperature, high nutrient levels and low pH. In contrast, reported that Oscillatoriales and Nostocales are commonly Group 2 included Gloeo. pleurocapsoides, L. antarctica, L. present in polar benthic habitats (Vincent and James 1996; frigida, Pseudanabaena frigida and N. spongiaeforme plot- Broady and Weinstein 1998; Singh and Elster 2007). ted on the left side of the graph with high pH, low temper- Diversity indices provide important information about the ature and low nutrient levels. Group 3 comprised C. parvum, rarity and commonness of the particular biotype in a com- S. pevalekii, L. benthonica, L. foveolarum, L. lurida, L. munity. Sissu Lake, which is comparatively rich in nutrients valderiana, P. autumnale and P. chalybeum and is located with average temperature higher than other three lakes, ex- in the middle of the graph. Distribution of species of this hibited high values of Shannon’s index, Simpson’s index group could not be associated with a specific environmental and species richness index. The calculated ecological indices condition or sampling site. Conductivity and lake trophy of these lakes revealed that the cyanobacterial diversity of have been identified as the main factors regulating species composition of benthic communities (Vincent and James these lakes was influenced by the physico-chemical charac- 1996; Roberts et al. 2001; Sabbe et al. 2004), but in the teristics of water. In shallow, clear water polar and high present study pH, temperature and nutrient level of lakes altitude lakes, benthic phototrophs are major phototrophs seem to be major factors which influence distribution of (Ellis-Evans 1996;Tanget al. 1997). The cyanobacteria cyanobacterial species. have the capacity to grow at high altitudes because they show tolerance to bright, continuous radiation and posses defence mechanism against UV-damage (Tang et al. 1997; Acknowledgements Ehling-Schnlz and Scherer 1999;Mataloniet al. 2000; Leavitt et al. 2003). In cold water ecosystems, mat-forming The authors are thankful to Head, Department of Botany, cyanobacteria of orders Chroococcales, Oscillatoriales and Punjabi University, Patiala, and Coordinator, SAP-II of Uni- Nostocales are the conspicuous members (Howard-Williams versity Grant Commission, New Delhi, for laboratory facil- – et al. 1989; Taton et al. 2003, 2006a, b; Jungblut et al. ities. Laboratory facilities provided by the Director, CSIR 2005). During the present investigation it was observed that Institute of Himalayan Bioresource Technology, Palampur, taxa belonging to Oscillatoriales were the most abundant in are also acknowledged. YS thanks the Council of Scientific all the four lakes, followed by member of Nostocales and and Industrial Research, New Delhi, for providing financial Chroococcales. In particular, morphospecies of assistance in the form of Senior Research Fellowship. Leptolyngbya, Pseudanabaena, Phormidium, Oscillatoria and Nostoc are characteristic of polar mats (Vincent 2000). The diversity of Chroococcales observed during this study References was similar to freshwater ponds in the Larsemann and Vestfold Hills region, Antarctica (Wharton et al. 1983; Anagnostidis K and Komárek J 1985 Modern approach to the Taton et al. 2003; Jungblut et al. 2005). Interestingly, the classification system of cyanophytes.1. Introduction. Arch. cyanobacterial communities of lakes investigated in the pres- Hydrobiol. /Algol. 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MS received 28 January 2014; accepted 17 June 2014

Corresponding editor: S SHIVAJI

J. Biosci. 39(4), September 2014