P limitation for bacteria in above-treeline lakes and light limitation for algae in below-treeline lakes
Compared to below-treeline alpine lakes, lowland lakes have much higher 1/K:TP ratios. For example, in low-elevation, eutrophic Lake Taihu (30°55'40"-31°32'58"N, 119°52'32"-120°36'10"E; 3.3 m above sea level), the 1/K:TP ratio was found to be 173.61. In oligotrophic Lake Lugu (27°36′55′′-27°47′2′′N, 100°43′36′′-100°54′20′′E; 2,685 m above sea level), the 1/K:TP ratio was 383.14. The studied alpine lakes located below treeline were encircled by forests and meadows, and the terrestrial organic matter in these regions mainly consisted of humic substances; thus, the lake water appeared brown. In contrast, the lakes located above treeline were mainly surrounded by exposed heaps of rocks with extremely low concentrations of humic substances and cDOM. As a result, the underwater attenuation coefficients were low in these lakes, and the water was highly transparent.
Both of these alpine lake types are oligotrophic because the amounts of nutrients input from terrestrial sources are much lower than those in catchments where human activities have great impacts. Zhang et al. investigated 38 plateau lakes and found no eutrophic lakes at elevations higher than ~ 4000 m ; in their study, the concentrations of TN and TP in oligotrophic lakes were 0.23 ± 0.11 mg/L and 0.012 ± 0.006 mg/L, respectively. The N:P ratio is extremely high in most oligotrophic lakes. In this study, the mean N:P value measured in the above-treeline lakes was 66.6, and this value was 31.7 in the below-treeline lakes, both of which were higher than the Redfield value (~ 22) of the algal N:P ratio, particularly in the above-treeline lakes. Therefore, the P limitation appears to be more remarkable in these lakes. P limitation for bacteria under high light:TP conditions can be explained by high light energy and low nutrient utilization promoting carbon exudation rather than biomass production by phytoplankton. This explanation seems to be based on the fact that bacteria mainly utilize autochthonous carbon such as algae. Although the carbon pools of the above-treeline lakes were mainly replenished by allochthonous carbon from glacial meltwater, the log(bacterial abundance) decreased with increasing Chl a concentrations (Fig. 2b, R2 = 0.62). The relatively high phytoplankton biomasses (Chl a concentrations) and relatively low bacterial abundances measured in the above-treeline lakes suggested that phytoplankton compete with bacteria for nutrients and obtain more P than bacteria do , thereby aggravating bacterial P limitation. Overall, the bacterial nutrient limitation in the above-treeline lakes with high light:nutrient (high 1/K:TP) conditions fit the light-to-nutrient hypothesis.
Sterner et al. proposed that under low light:TP conditions, planktonic bacteria are limited by C because phytoplankton have a high P utilization efficiency, promoting the production of phytoplankton biomass rather than the excretion of C . Again, this explanation implies that bacteria mainly utilize algal carbon. However, in this work, in below-treeline lakes with low light:nutrient (low 1/K:TP) conditions, the log(bacterial abundance) had little correlation with the Chl a concentrations (Fig. 2b, R2 = 0.0005). Notably, although the P limitation of bacteria was weakened under low light:TP conditions, bacteria did not appear to be subject to C limitation, but phytoplankton were subject to light limitation. In humic or turbid lakes, the influence of high cDOM concentrations or turbidity causes light to become a limiting factor for algae. The studied lakes located below treeline were rich in humic substances since they were surrounded by thick pinewood and evergreen broad-leaf forests, thereby presenting dark-brown colours. A high cDOM concentration results in rapid light attenuation in water. Consequently, weak light conditions limit the growth of algae, leading to low Chl a concentrations (Fig. 1c), and the growth of planktonic bacteria is strongly supported by allochthonous carbon. In the absence of C limitation and without significant P limitation, bacteria are likely to compete with algae for nutrients. Overall, the ecosystem processes in below-treeline lakes cannot be predicted using the light-to-nutrient hypothesis.
The traditional view holds that heterotrophic bacterioplankton greatly depend on the organic matter produced by phytoplankton, and the growth of phytoplankton is limited by light and nutrients such as N and P. The relationship between bacterial abundance and phytoplankton abundance in waters with low humic substance contents conforms to the classic interpretation described above. However, in some oligotrophic lakes, the C:P ratio of bacteria is 10-fold lower than that of phytoplankton, and when the organic carbon supply is sufficient, heterotrophic bacterioplankton are able to compete with algae for limited inorganic nutrients . This is most common in humic lakes located below treeline with high C:N (Fig. 2d) or C:P ratios. For example, the correlation between the bacterial abundance and chlorophyll concentration (a surrogate of algal abundance) was found to be weaker than that between bacteria and TP, reflecting the competition for P between bacteria and algae . When organic carbon is abundant and/or the P concentration is low, heterotrophic bacteria are likely to have an advantage over algae when competing for P . In general, we believe that this is the first study to verify the applicability of the light-nutrient hypothesis in alpine lake ecosystems, and the nutrient limitations of bacterioplankton showed different scenarios in above- and below-treeline lakes.
Bacterial community composition was sensitive to allochthonous and autochthonous carbon
The effect of the light:P ratio on a bacterial community is mainly reflected by the amounts and compositions of organic carbon and phosphorus. In addition to potential P limitations in lakes located above treeline, light limitation affecting algae growth and organic carbon photodegradation in below-treeline lakes impact the organic carbon composition, thereby affecting the bacterial community distribution. The intact communities demonstrated clear separation between lakes located below and above treeline. Furthermore, strong evidence was obtained that cryptic diversity in Polynucleobacter species is crucial when interpreting diversity studies on freshwater bacterioplankton conducted based on ribosomal sequences.
Polynucleobacter is a globally abundant freshwater bacteria, and its PnecC cluster is a particularly interesting taxon used to study diversification in freshwater  due to its cosmopolitan distribution  and high global abundance . Its ecological success has been shown to be the result of diversification rather than a generalist adaptation, as lineages within the PnecC cluster reveal distinct ecological characteristics [41, 43, 44]. Hahn et al. discovered that the PnecC cluster represents a broad pH spectrum, further emphasizing the differences among habitat-specific adaptations . Until now, no field evidence was available for the carbon source spectrum of this diversified cluster. In this study, we found clear habitat-specific adaptations in lakes located below and above treeline and in the substrate preference within the same OTU. A small cluster including 12 oligotypes was directed towards the phytoplankton biomass index (Chl a concentration); this cluster probably relied on the autochthonous carbon produced by phytoplankton as its major organic carbon source. Alga-released organic matter is composed of a variety of low- to high-molecular-weight compounds predominantly comprising carbohydrates, nitrogenous substrates, lipids and organic acids . The addition of extracellular and biomass-derived organic compounds initiated the growth of the Polynucleobacter PnecB strain . On the other hand, more diverse oligotypes were identified in the lakes located below treeline; these oligotypes might have adapted to the abundant structures of terrestrial carbon. Experiments have indicated that these bacteria live as chemoorganotrophs by mainly utilizing low-molecular-weight substrates derived from the photooxidation of humic substances .
Implications for bacterial nutrient limitation in high-elevation and polar lakes
With climate warming, the global area of glaciers has decreased by ~ 17% in the last 30 years . The proportion of glacier retreat on the Tibetan Plateau reached 95% (n = 116) from 1990 to 2005 . Future changes in glacial runoff will impact the bioavailability of OC in downstream ecosystems. It has been reported that the bioavailability of DOC in glacial meltwater is 2–5 times higher than that in forested and wetland streams . Therefore, it can be expected that the input of cryosphere-derived OC will increase in above-treeline lakes, subsidizing the carbon source of bacteria in these lakes.
In addition, the rapid development of industry and agriculture resulted in the nitrogen and phosphorus deposition rates doubling in Asia during the past 40 years . Climate warming together with enhanced nutrient loading may have two consequences. One involves the rapid growth of algae in lakes and bacteria that do not lack nutrients. At present, a few alpine lakes have shown moderate eutrophication . The other possible consequence is the rapid growth of terrestrial plants and the upward shift of treelines; under these circumstances, a catchment environment above treeline may gradually become more like the environment below treeline. A meta-analysis indicated that global treelines shifted upward during the last century at 52% . As a possible result, the presence of terrestrial humic substances will decrease the underwater light intensity and cause light limitation for algae. If the light:TP ratio decreases and the P limitation for bacteria further weakens in alpine lakes, bacteria will compete with algae. The conditions seem to be moving towards this scenario in below-treeline lakes.
However, lake ecosystems are complex and are affected by many factors other than climatic and environmental changes. We should also consider that different lakes have different topographical and hydrological characteristics and that predation has an extremely important effect on bacterial and algal community structures and their competitive relationships. Consequently, predicting how climatic and environmental changes in cryosphere ecosystems influence bacterial nutrient limitations and community structures remains a challenge. In the future, further studies of bacterial nutrient limitation changes and the driving forces that influence these changes will be of great significance for the stability and biodiversity of high-elevation and high-latitude aquatic ecosystems.