18S rRNA amplicon sequencing of the eukaryotic communities of Pangong, Tsomoriri and Tsokar lake showed V4 region as a good marker for successfully capturing the eukaryotic diversity using eDNA (Jo et al. 2019; Ruppert et al. 2019). OTU analyses showed the presence of several eukaryotes which included phytoplanktons, zooplanktons and invertebrates. Previous studies have suggested that the food web in saline lakes are simple involving microbes, phytoplanktons, zooplanktons, macroinvertebrates and birds as compared to the fresh water bodies (Wurtsbaugh 1992; Pedrós-Alió et al. 2000; Vidal et al. 2021); birds being the top most consumers (Figure graphical abstract) (Vareschi. 1987; Wollheim and Lovvorn 1995). The details of the major contributors of food web and biogeochemical cycles in these lakes are discussed and represented in Table S1. However, the variation in physiochemical parameters could also attribute to the difference among three studied lakes which in turn will affect the community composition.
4.1 Physiochemical and elemental analysis of water
The findings from the three hypersaline lakes concluded that the water samples were found to be free crystal clear in IPGL and ITRL. The high concentration of Iron (Fe) in ITRL and IPGL could be one of the main reasons behind the clear water as high concentration of Fe results in oxidative stress, disruption of the cell membrane and damage to DNA ultimately causing death (Sinha et al. 2013). Whereas, in ITKL comparatively low concentration of Fe favours and increases the primary producers hence resulting in the milky colouration of water due to the growth of organism (Søndergaard et al. 2007). Further, the temperature of the lake(s) does not show much variation with the atmospheric temperature due to the strong radiation and standing feature of water bodies. All the three lakes are devoid of macrophytes and therefore the rate of photosynthesis could not affect the pH of lakes. Rather the rate of evaporation seems to be responsible for the high pH range in all the studied lakes. These lakes provide suitable ecological conditions for the several indicator species of Phylum Ciliophora living in freshwaters with slightly eutrophic conditions, at pH values between 9 and 8.8 (Kulaš et al. 2021). DO act as a vital factor and is the source of oxygen not only for life sustenance but also in regulating their metabolic activities as well as subsequent trophodynamics (Hull et al., 2008; Banerjee et al. 2019). The critical value of 4.5mg/L− 1 is an appropriate concentration for survival of eukaryotes whereas, below this value will causes hypoxia which in turn cause the significant reduction in fish survival, algal bloom and loss of diversity (Howarth et al. 2000). However, few zooplanktons can survive below critical concentration of DO by regulating their mechanism. Such members include genus Tokophrya which frequently occur in alpha-mesosaprobic (highly polluted) to beta-mesosaprobic (moderately polluted) waters with sufficient DO content suggesting that the lake water to be moderately polluted (Kulaš et al. 2021). Therefore, the presence of genus Tokophrya in ITKL and IPGL suggests that these two lakes are mesosaprobic, polluted with sufficient DO content. The TDS value of IPGL and ITRL samples was above 500ppm and this value was beyond the guidelines of Bureau of Indian Standards (BIS) (2012) implying that the water is hard in these two lakes. Literature as well as physical survey of the lakes has clearly showed the absence of major aquatic vertebrates such as fishes, amphibians, reptiles, etc., which could be due to the high salinity (greater than 4%) or salinity intolerance or winterkill from low DO in the lakes under study (Hammer 1986; Peterka 1989). These findings are in concordance with the actual collected specimen from the study sites (Figure S2). The predominance of alkaline earth metals (Mg and Ca) and alkali metals (Na and K) in these water samples highly contributes to the TDS increment (Witherow and Lyons, 2011). The source of these metal elements was the deposition due to weathering of Cenozoic igneous rocks (Guillot et al. 2019) or lime rock in the catchment area (Wünnemann et al. 2010). Hence, the TDS value was low in ITKL indicating that the water is more corrosive and can leach the metals containers (Tam and Elefsiniotis 2009). As observed by Jellison et al. (2004), saline alkaline lakes are exclusively abundant in bicarbonate (HCO3−) ions and carbonate (CO32−) ions, often characterized by “low species diversity but high populations of aquatic organisms”. So, due to the loss of carbonates from the bottom sediments and subsequent convergence to bicarbonates, these lakes have high total alkalinity (Bhat et al. 2011; Verspagen et al. 2014; Cao et al. 2020). ITKL encompass the highest diversity implying that all the organisms inhibiting the lakes are salt tolerant and have acclimatised their body according to their surroundings. Salinity has a major impact on the occurrence of species because of its relationship with osmoregulatory physiology (Hart et al. 1991; Thompson and Withers, 1992). Also, high salinity has been reported to hamper the bird’s egg survival (Lee and Gerking 1980) by affecting its osmoregulatory (Verschuren et al 2000; Barnes and Wurtsbaugh 2015). The high concentration of Li > Cu > Mn > Cd > Co content in IPGL lake indicated that it is the most anthropogenically affected lake as the tourist footfall is greater in IPGL followed by ITKL and ITRL. These elements could be deposited from automobiles exhaust, washing of vehicles, dumping of plastics, water bottles, usage of Li based batteries as well as microplastics deposition (Tsering et al. 2019; 2022). Hg, Al, Cr, Ni, Mo, Sb, etc. have been enlisted as the high potential ecological risk elements and their concentrations are mostly affected by human activities. Hence, the abiotic factor also plays a very crucial role in maintaining the overall diversity and production in an aquatic ecosystem.
4.2 Community structure and their role in food web:
a. Microeukaryotes:
The microeukaryotic community in the lakes showed SAR supergroup/clad to be dominant. It comprises various phytoplanktons and zooplanktons such as Stramenopiles (diatoms, golden/ brown algae, Oomycetes), Alveolates (dinoflagellates, apicomplexa and ciliates) and Rhizaria (cercozoa, foraminifera and radiolaria). In the studied samples phytoplanktons such as Phylum Bacillariophyta (diatoms) and Chlorophyta (green algae) are found to be present in all the lakes. They form a crucial component as a primary producer in the aquatic ecosystem. They make up the cell membranes of rotifers and ciliates that form the primary consumers (Barnes and Mann 2009). Members of Cryptomonads (algae with plastids), order Fragilariales, Naviculales and genus Dunaliella were detected in all the three lakes. All the above members have been documented to be very well adapted to grow in alkaline or marine environment or polar region (Melkonian and Preisig 1984; Katano et al 2009; Luddington et al. 2012; Oren 2014). The class Cryptophyta is a dominant bacterivore in freshwater ecosystems (Grujcic et al. 2018). The presence of this class reveals that among all the studied lakes ITRL has the highest percentage of bacteria preferred by cryptophytes followed by IPGL. They grow well in the saline habitat-serving as a perfect food in the saline water food chain. They also contribute to CO2 fixation as photosynthetic organisms and as non-toxic planktonic flagellates and act as significant prey in the food chain (Bellinger and Sigee 2015).
Zooplankton acts as an important trophic intermediate that connects phytoplankton with higher trophic levels (Li et al., 2010; Striebel et al., 2012). Presence of Ciliophora as the most abundant phylum suggested that ciliated protozoa such as Philasterida constitute one of the common components of the aquatic food web ( Pérez et al. 2000; Elloumi et al. 2006). They act as significant energy transfer mediators from pico and nanoplanktonic production to higher trophic levels (Sime-Ngando 1995; Elloumi et al. 2006; Lepere et al. 2010). Members of phylum Perkinsozoa are known to feed on dinoflagellates and are also responsible increased mortality of molluscs. (Mangot et al. 2011). So, one of the reasons for the absence of molluscs in these hypersaline lakes could be the presence of phylum Perkinsozoa. Members of phylum Cercozoa are well known bacterial grazers (Ekelund et al. 2004). The copious presence of phylum Cercozoa in all the samples confirms the rich occurrence of bacterial and archaeal diversity in all the studied lakes (paper communicated). Similarly other members of this phylum such as Bodomorpha, Cercomonas, Heteromita and Thaumatomonas are heterotrophic flagellates that survive by consuming bacteria, yeast, fungi, and algae. They also contribute towards the decomposition of soil organic matter and maintenance of overall productivity of the lake ecosystem.
b. Macroinvertebrate
The presence of Phylum Bryozoa and Rotifers (wheel animalcules) indicated the presence of high concentration of dead and decomposing organic matter in these lakes (Franch-Gras et al. 2019; Carballeira et al. 2020). Genus Plumatella found in ITKL sample has already been documented in alkaline shallow marshes with high concentration of organic matter (Massard and Geimer 2008; Carballeira et al. 2020). High pH (8.61) and low DO (2.11 mg/ml) in ITKL seem to support the growth of genus Plumatella. The order Ploimida of phylum Rotifera present in the ITRL are filter feeders, primarily omnivorous. They become prey to the secondary carnivorous consumers, including shrimp and crabs in this case Gammarus. The Order Monhysterida was detected only in IPGL sample. And they are generally present in mass and supplemented with dissolved and particulate organic matter existing on macrophytes (Trotter and Webster 1983; Mokievsky et al. 2005) but can also live on the mouth parts or inside the intestine of some decapods (Tchesunov et al. 2015). This order is marine in origin and adapted to cold and harsh environmental conditions (Udalov et al. 2021; Zhang et al. 2021). Class Turbellaria (flatworms) was present prominently in ITKL sample and appear to affect the zooplankton population dynamics as they get their nourishment by consuming protozoans, rotifers, and algae, hence, they help to keep the above populations in check. The order Rhabdocoela constitutes a few genera that are mostly preferred by the birds of high altitude i.e., the Black-necked crane (Grus nigricollis), Ruddy Shelduck (Tadorna ferruginea) and Great-crested Grebe (Podiceps cristatus) etc. The phylum Arthropoda (Ostracoda, Gammarus, Daphnia, Water fleas, etc.) and Platyhelminthes constitutes the most abundant phylum in ITKL followed by IPGL. They contribute as important food source for other invertebrates as well as the resident, summer visiting or migratory birds providing them necessary protein essential for their growth and reproduction (Swanson and Meyer 1977; Kramer et al. 2014; Chandra et al. 2021). In the saline environment, the phylum Arthropoda inhabits all trophic levels from the herbivore to the detritivore and in some cases, it also inhabits up to the top aquatic predator (Labandeira and Beall 1990; Balsamo et al. 2020). Chandra et al. (2017; 2021) confirmed that in these studied lakes the diversity of Arthropoda was maximum but their relative percentage and taxonomical classification were unknown. Therefore, this study is the first attempt to identify the total diversity of eukaryotes present in these three hypersaline lakes. However, the morphological characterization of these organisms is usually challenging as it is labour intensive, time consuming and requires technical expertise in the field (Thorp and Covich, 2009; Grattepanche et al. 2018; Weisse and Montagnes, 2021). Therefore, the findings clearly showed that eDNA metabarcoding is a robust and efficient method for elucidating the micro and macro eukaryotic assemblage (Zhang et al. 2020).
4.3 Community richness & diversity: Alpha and Beta diversity
The Shannon Weiner index of ITKL represents more species richness and equitability in the distribution as compared to the other two lakes which may be due to the high pH, salinity and low TDS in ITKL. The Inverse Simpson's Diversity Index (1/D) analysis showed that the total number of eukaryotic diversities was highest in ITKL followed by ITKL and IPGL. Chao1 and observed OTUs analysis supported that enough sample was sequenced to cover the overall diversity of the lakes. Results from various alpha diversity indices suggested that the diversity increases with the increase in salinity (Saccò et al. 2021). In UPGMA tree the lower the difference score more closely the two lakes i.e., IPGL and ITRL indicating more divergence in terms of diversity in these two samples. Weighted and Unweighted UPGMA cladogram showed the close clustering of the IPGL and ITRL samples in one clade while ITKL forms a different clade suggesting its community structure to be different from the other two lakes. Thereby, indicating that the structural composition of these three lakes were different. This also suggests that lake IPGL and ITRL are more closely related than IPGL and ITKL. Also, lake IPGL and ITRL were similar in composition and are equally different in terms of diversity with that of ITKL, these inferences are in line with the results of clustered heatmap and PCoA. This result implies that the diversity of all the lakes is distinctive and that could be due to the variation in their physiochemical and elemental composition.
4.4 Future Prospects:
Phylum Ciliophora was highest in IPGL and ITRL and its members have been already established as good environmental indicators (Abo-Taleb et al. 2016; Xu et al. 2018). For example, genus Vorticella of the phylum Ciliophora is heterotrophic and feeds on a variety of organisms ranging from bacteria to smaller protozoan, thereby helping in regulating the population of these organisms in the environment. Also, this genus is known as a biological indicator of pollution (Buhse and Clamp 2001; El-Serehy et al. 2014). Also the occurrence of Vorticella in ITRL and ITKL sample was in measurable percentages indicating that there is some level of pollution in these lakes. Similarly, the presence of order Cyclopida in IPGL and ITKL samples shows that they can endure high pH with low TDS (Lanzén et al. 2016; Noor et al. 2018). Also, order Cyclopida has been shown to be resilient to hydrocarbons (Quiñones 2005; Sun et al. 2019). Therefore, it can be used as a biomonitoring indicator for pollution due to oil spills. The genus Daphnia is a well-established bioindicator, whose survival is affected by the presence of trace elements causing variation in its survival rate (Issa 2021). These eukaryotic invertebrates are also used as tractable models to study the extremophile physiology, since their diversity is affected by environmental responses such as desiccation, freezing, extreme heat, and extreme pH (Reardon et al. 2010; Sapir et al. 2014). Hence, these organisms can be used as an ecological biomonitoring tool in these hypersaline lakes. Members of class Chrysophyceae have medicinal value as they contain fucoxanthin, a carotenoid that has been proven to have many medicinal as well as nutritional values such as antiobesity, antidiabetic, antioxidant, anti-inflammatory and hepatoprotective activities, as well as cardiovascular and cerebrovascular protective effects (Zhang et al. 2015). In the present study, orangish red colour was observed in Tsokar site during sampling. Few species of the genus Dunaliella contain relatively large amounts of β-carotenoids and glycerol to survive in very harsh conditions (involving high intensity UV light, increased salt concentrations and inadequate oxygen and nitrogen levels) (Oren, 2005). Due to the presence of higher β-carotenoids composition these sites appear to be red to orange in colour (can be seen with our naked eyes). Therefore, in near future, these sites may prove to be useful for future biotechnological and medicinal purposes.