Protists are a major component of freshwater lakes communities, forming the base of the food web on which all other aquatic organisms depend . Here, we present a detailed analysis of changes in protistan communities from the ice-covered period to ice-free period in a freshwater mountain lake with seasonal freezing and thawing. From ice cover to ice melting, the lake water environment undergoes dramatic seasonal fluctuations (Table 1). Changes in composition and abundance are often regarded as a response of protistan communities to climate fluctuations and changes in nutrient availability [4, 10], especially in mountainous lakes .
Effects of seasonal freezing-thawing on protistan communities
We found that the abundances of the protistan communities during the two periods showed clear differences from the phylum to genus levels, driven by multiple environmental factors, especially T, pH, DO, and nutrients (NO3− and TOC). Temperature change was the main factor directly influencing the freeze-thaw process. In general, water temperature clearly rises after ice melting, and this was seen in our study (Table 1). The influence of temperature on microorganisms includes many aspects. First, temperature changes directly affect the metabolic activity and abundance of microorganisms [2, 16, 17]; Second, temperature also affects microorganisms by mediating the release of nutrients in sediments . Moreover, temperature is closely related to other environmental factors , and our results also confirm that temperature is collinear with other environmental factors (Table S1). The relative abundances of Ochrophyta, Choanoflagellida, and Stramenopiles X were higher in the ice-covered period, while the relative abundance of Chlorophyta increased significantly during the ice-free period (Fig. 3A). This was because the abundances of the genera Stephanodiscus and unclassified_F_Polar-centric-Mesophyceae (belonging to the Ochrophyta phylum) and genus unclassified_C_Choanoflagellatea (belonging to the Choanoflagellida phylum) were higher during the ice-covered winter. The physical stability of the lake under the ice was identified as the key factor behind the initiation of these particular groups, which are well adapted to winter temperature and low light conditions . Stephanodiscus readily adapts to low temperature, with its membrane lipid composition changing in cold environments , to ensure that the activity of its cells can be sustained. The abundances of the genera unclassified_f__Cryptomonadales, Pedinellales X, Cryptomonas, and Chlamydomonas increased significantly after ice melting. With the melting of the ice cover, the abundances of these groups with higher demands for temperature and light increased in spring . Meanwhile, other physical and chemical parameters of the water were changing. The increase in nutrients and DO promoted the increase of aerobic and heterotrophic protozoa (Ciliophora, Cercozoa), which further indicates that the environmental changes caused by freezing and thawing had an important impact on protistan community composition.
The alpha diversity of the protistan communities increased significantly in the ice-free period compared with that in the ice-covered period (Fig. 5). Our results are consistent with previous studies, and microbial diversity in the water was lower during the ice-covered period [5, 21]. Ice cover acts as a shield over the lake surface, which reduces light intensity and the input of terrestrial nutrients and organic matter, and hinders gas exchange with the atmosphere [5, 10]. During the ice-free period, material exchange can occur between water and the surrounding environment. The increase in nutrients (mainly from rain erosion) in water promoted the photosynthesis of phytoplankton, thus changing the balance of free carbon dioxide and carbonate in the water, and finally increased the pH of the water. Precipitation in the ice-covered period was 9.4 mm, whereas precipitation during the ice-free period was three times that amount (Supplementary Table S3), which indicates that rainfall is also an important factor causing physico-chemical changes in the aquatic environment. With Gonghai Lake being a basin lake, nutrients from the surrounding land enter the lake with rainfall, which increases the concentration of nutrients in the lake water. Thus, the diversity of microbial communities increased in an environment with abundant nutrients.
Seasonal freezing and thawing resulted in significant changes in the distribution pattern of protistan communities (P < 0.01, Fig. 6A), and their distribution was affected by multiple factors, especially TOC and NO3− (Table 3). Within lakes, some organic carbon originates from terrestrially derived dissolved organic matter from surrounding soils. Many studies have confirmed that microbial community composition, structure, and metabolic strategies are driven by soluble organic matter [22–24]. In this study, the concentration of organic carbon increased significantly during the ice-free period. This suggests that terrestrial carbon subsidies make an important contribution to seasonal trends in microbial communities in the mountain lakes. TOC can be utilized by aquatic heterotrophic microorganisms [25, 26]. Because organic carbon is an important energy material for heterogeneous microorganisms, changes in organic carbon lead to changes in protistan community structure. Eukaryotic algae are the main group of protists in water, and changes in nutrient concentrations have the greatest effect on their communities . It is generally accepted that nitrate will accumulate in water bodies in the winter [2, 10]. In this study, however, the increase in terrestrial nitrate nitrogen may have led to the change in the bacterial community structure involved in the nitrogen cycle. Bacteria and protists are closely linked through the food web, and changes in the bacterial community will inevitably lead to changes in the protistan community .
Effects of water depth on protistan communities
Water depth greatly influences the composition and diversity of biological communities . Solar radiation penetrates the ice layer, and the surface photosynthetic autotrophs increase, leading to changes in nutrient concentrations at different depths. Coupled with the release of heat from lake-bottom sediments, a vertical temperature gradient is formed, resulting in the downward mixing of water bodies . In these ways, convective mixing can make microorganisms circulate under the ice and improve their access to nutrients . Therefore, it is necessary to study different depth gradients to further understand the changes in microbial communities caused by freezing and thawing. Our results also confirm that water depth affected the composition and diversity of the protistan communities. Stratification of the lake will hinder the vertical material exchange at different water depths, and then affect various biochemical processes of the lake water, biological metabolism, and material decomposition . The relative abundances of the dominant groups from phylum to genus varied with sampling depth, and the variation trend of the same group differed by season. The heterogeneity of the physico-chemical environment at different depths directly caused this change (Fig. 4). Light availability, along with water mass characteristics, strongly impacts microbial communities in the vertical gradients . There will also be stable stratification of water below the ice, with the main driving factors being the heat flux of sediments and the solar radiation infiltrating the ice . Winter climatic fluctuations proved to be a key element in a linked chain of causal factors including the cooling of hypolimnetic waters, deep vertical mixing, and epilimnetic nutrient replenishment.
In contrast to the ice-covered period, the OTUs of protists were highest in the surface water; however, the Shannon index did not differ significantly at different depths during the ice-free period (Fig. 5). During the ice-free period, the nutrient concentrations in the surface layer and the vertical mixing intensity of the water body increased significantly with the aggravation of external disturbances (such as rainfall and gales), which resulted in the vertical difference in alpha diversity for the protistan communities. The OTU number did not differ significantly at different depths during the ice-covered period. This was mainly due to the following reasons. First, different protist groups have different requirements for light intensity and nutrient concentration . Second, in winter, external disturbances are much smaller, and the water body is relatively stable.
The results of PERMANOVA analysis showed that temperature was the main reason for the differences in protistan communities at different depths during the ice-covered period (Table 3). The temperature gradient is the main factor leading to water stratification at different depths, and it forms nutrient gradients. Althoug Bertilsson  showed that temperature usually does not limit the growth of phytoplankton in winter in shallow lakes, we found that the vertical difference in temperature and sampling depth together affect the spatial distribution of protistan communities. Under the ice, photoautotrophic protist activities are often limited by the availability of photosynthetically active radiation [2, 5, 30]. Limited light penetration reduces the photodegradation of organic matter under ice and changes the quality and biodegradability of organic carbon available to microorganisms. Some phototrophic phytoplankton can perform aerobic photosynthesis in the upper waters, yet the light in the deeper waters is more limited, and the distribution of photoautotrophs at the bottom is lower. Owing to the reduced availability of light and the reduction of bacterial biomass during the ice-covered period, the life history strategies of protists became more diverse. During the ice-free period, the bottom layer (8 m) and other depths (0–6 m) differed significantly (Fig. 6). The water at the depth of 8 m is directly connected with the sediment, which is rich in organic and inorganic substances. The release of nutrients in sediments can promote the growth and diversity of microorganisms in water. For example, the release of nitrate and organic carbon in sediments is an important source of microbial nutrients in deep water . Because the concentration of heat and mixed energy near the sediment–water interface is conducive to the activity of microorganisms in the bottom boundary layer, the characteristics of the bottom-water environment led to the obvious division of its community from those of other layers.