Comparative transcriptomic analysis delineates adaptation strategies of Rana kukunoris toward cold stress on the Qinghai-Tibet Plateau.

BACKGROUND: Cold hardiness is fundamental for amphibians to survive during the extremely cold winter on the Qinghai-Tibet plateau. Exploring the gene regulation mechanism of freezing-tolerant Rana kukunoris could help us to understand how the frogs survive in winter. RESULTS: Transcriptome of liver and muscle of R. kukunoris collected in hibernation and spring were assisted by single molecule real-time (SMRT) sequencing technology. A total of 10,062 unigenes of R. kukunoris were obtained, and 9,924 coding sequences (CDS) were successfully annotated. Our examination of the mRNA response to whole body freezing and recover in the frogs revealed key genes concerning underlying antifreeze proteins and cryoprotectants (glucose and urea). Functional pathway analyses revealed differential regulated pathways of ribosome, energy supply, and protein metabolism which displayed a freeze-induced response and damage recover. Genes related to energy supply in the muscle of winter frogs were up-regulated compared with the muscle of spring frogs. The liver of hibernating frogs maintained modest levels of protein synthesis in the winter. In contrast, the liver underwent intensive high levels of protein synthesis and lipid catabolism to produce substantial quantity of fresh proteins and energy in spring. Differences between hibernation and spring were smaller than that between tissues, yet the physiological traits of hibernation were nevertheless passed down to active state in spring. CONCLUSIONS: Based on our comparative transcriptomic analyses, we revealed the likely adaptive mechanisms of R. kukunoris. Ultimately, our study expands genetic resources for the freezing-tolerant frogs.

Microbial phosphorus-cycling genes in soil under global change.

The ongoing climate change on the Tibetan Plateau, leading to warming and precipitation anomalies, modifies phosphorus (P) cycling in alpine meadow soils. However, the interactions and cascading effects of warming and precipitation changes on the key "extracellular" and "intracellular" P cycling genes (PCGs) of bacteria are largely unknown for these P-limited ecosystems. We used metagenomics to analyze the individual and combined effects of warming and altered precipitation on soil PCGs and P transformation in a manipulation experiment. Warming and increased precipitation raised Olsen-P (bioavailable P, AP) by 13% and 20%, respectively, mainly caused by augmented hydrolysis of organic P compounds (NaOH-Po). The decreased precipitation reduced soil AP by 5.3%. The richness and abundance of the PCGs' community in soils on the cold Tibetan plateau were more sensitive to warming than altered precipitation. The abundance of PCGs and P cycling processes decreased under the influence of individual climate change factors (i.e., warming and altered precipitation alone), except for the warming combined with increased precipitation. Pyruvate metabolism, phosphotransferase system, oxidative phosphorylation, and purine metabolism (all "intracellular" PCG) were closely correlated with P pools under climate change conditions. Specifically, warming recruited bacteria with the phoD and phoX genes, which encode enzymes responsible for phosphoester hydrolysis (extracellular P cycling), strongly accelerated organic P mineralization and so, directly impacted P bioavailability in alpine soil. The interactions between warming and altered precipitation profoundly influenced the PCGs' community and facilitated microbial adaptation to these environmental changes. Warming combined with increased precipitation compensated for the detrimental impacts of the individual climate change factors on PCGs. In conclusion, warming combined with rising precipitation has boosting effect on most P-related functions, leading to the acceleration of P cycling within microbial cells and extracellularly, including mineralization and more available P release for microorganisms and plants in alpine soils.

Host plant height explains the effect of nitrogen enrichment on arbuscular mycorrhizal fungal communities.

Nitrogen (N) enrichment is widely known to affect the root-associated arbuscular mycorrhizal fungal (AMF) community in different ways, for example, via altering soil properties and/or shifting host plant functional structure. However, empirical knowledge of their relative importance is still lacking. Using a long-term N addition experiment, we measured the AMF community taxonomic and phylogenetic diversity at the single plant species (roots of 15 plant species) and plant community (mixed roots) levels. We also measured four functional traits of 35 common plant species along the N addition gradient. We found divergent responses of AMF diversity to N addition for host plants with different innate heights (i.e. plant natural height under unfertilized treatment). Furthermore, our data showed that species-specific responses of AMF diversity to N addition were negatively related to the change in maximum plant height. When scaling up to the community level, N addition affected AMF diversity mainly through increasing the maximum plant height, rather than altering soil properties. Our results highlight the importance of plant height in driving AMF community dynamics under N enrichment at both species and community levels, thus providing important implications for understanding the response of AMF diversity to anthropogenic N deposition.

Potential Effects of High Temperature and Heat Wave on Nanorana pleskei Based on Transcriptomic Analysis.

In the context of climate change, understanding how indigenous amphibians of the Qinghai-Tibet plateau react to stresses and their coping mechanisms could be crucial for predicting their fate and successful conservation. A liver transcriptome for Nanorana pleskei was constructed using high-throughput RNA sequencing, and its gene expression was compared with frogs acclimated under either room temperature or high temperature and also heat wave exposed ones. A total of 126,465 unigenes were produced, with 66,924 (52.92%) of them being annotated. Up to 694 genes were found to be differently regulated as a result of abnormal temperature acclimatization. Notably, genes belonging to the heat shock protein (HSP) family were down-regulated in both treated groups. Long-term exposure to high-temperature stress may impair the metabolic rate of the frog and trigger the body to maintain a hypometabolic state in an effort to survive challenging times. During heat waves, unlike the high-temperature group, mitochondrial function was not impaired, and the energy supply was largely normal to support the highly energy-consuming metabolic processes. Genes were more transcriptionally suppressed when treated with high temperatures than heat waves, and the body stayed in low-energy states for combating these long-term adverse environments to survive. It might be strategic to preserve initiation to executive protein activity under heat wave stress. Under both stress conditions, compromising the protection of HSP and sluggish steroid activity occurred in frogs. Frogs were more affected by high temperatures than by heat waves.

Warming reduced flowering synchrony and extended community flowering season in an alpine meadow on the Tibetan Plateau.

The timing of phenological events is highly sensitive to climate change, and may influence ecosystem structure and function. Although changes in flowering phenology among species under climate change have been reported widely, how species-specific shifts will affect phenological synchrony and community-level phenology patterns remains unclear. We conducted a manipulative experiment of warming and precipitation addition and reduction to explore how climate change affected flowering phenology at the species and community levels in an alpine meadow on the eastern Tibetan Plateau. We found that warming advanced the first and last flowering times differently and with no consistent shifts in flowering duration among species, resulting in the entire flowering period of species emerging earlier in the growing season. Early-flowering species were more sensitive to warming than mid- and late-flowering species, thereby reducing flowering synchrony among species and extending the community-level flowering season. However, precipitation and its interactions with warming had no significant effects on flowering phenology. Our results suggest that temperature regulates flowering phenology from the species to community levels in this alpine meadow community, yet how species shifted their flowering timing and duration in response to warming varied. This species-level divergence may reshape flowering phenology in this alpine plant community. Decreasing flowering synchrony among species and the extension of community-level flowering seasons under warming may alter future trophic interactions, with cascading consequences to community and ecosystem function.

Light controls alpine meadow community assembly during succession by affecting species recruitment from the seed bank.

Some research indicates that soil seed banks can promote species coexistence through storage effects. However, the seed bank mechanism that maintains plant assembly and its role in degraded grassland restoration are still not clear. We collected seed bank samples from early, mid and late secondary successional stages of an abandoned subalpine meadow on the Tibetan Plateau, and samples from each stage were exposed to full (i.e., natural), mid, and low light treatments in the field to represent light availability at the bottom/understory (soil surface) of a plant community in the early, mid and late stages of succession, respectively. Species richness, seed density, species composition, and community weighted mean values (CWMs) of seed mass of the species whose seeds germinated in soil samples were evaluated. In response to the light treatments, species richness increased significantly with increased light only for the late successional stage, seed density increased significantly with increased light only in the early and mid successional stages, and seed mass decreased significantly with increased light only in the mid and late successional stages. Species composition differed significantly among the light treatments only in the late successional stage. For the successional series, species richness and seed mass of the species that germinated increased significantly with succession only under mid and full light treatments. Seed density decreased significantly with succession in each light treatment. Species composition differed significantly between the early- and late stage and between the mid and late stage in each light treatment. Both the abiotic (light) and biotic (seed mass) factors influence seed bank recruitment to the plant community. Regeneration of small-seeded species in the seed bank was inhibited under low light in the late successional stage. The balance of stochastic and deterministic processes along a successional gradient was determined by regeneration from the seed bank depending on light intensity change. Differences in seed response to light intensity change largely determined plant community assembly. Our findings should help in the development of effective conservation and restoration strategies.

Does a Moderately Warming Climate Compensate for the Negative Effects of UV-B Radiation on Amphibians at High Altitudes? A Test of Rana kukunoris Living on the Qinghai-Tibetan Plateau.

Both the warming climate and ultraviolet-B radiation (UVBR) are considered to be notable environmental factors affecting amphibian population decline, with particular effects on tadpole development. However, the phenotypes of tadpoles living at high altitudes may be improved by moderately warming temperatures, reducing or eliminating the negative effects of oxidative damage caused by cool temperatures or strong UVBR at high altitudes. To verify this hypothesis, Rana kukunoris tadpoles, which live at high altitudes, were used to test the effect of the interaction of temperature and UVBR on their development and antioxidant systems in a fully factorial design. The tadpoles were exposed to UVBR and UVBR-free environments at 14 (cool temperature) and 22 °C (warm temperature), respectively. UVBR and a warm temperature had opposite influences on several traits of the tadpoles, including their survival, developmental rate, individual size, preferred body temperature, thermal tolerance temperature, oxidative damage, and enzymatic and nonenzymatic antioxidant systems. The moderate temperature could compensate for or override the negative effects of UVBR by increasing the tadpoles' preferred body temperature and critical tolerance temperature, thus enhancing the locomotion ability and thermal sensitivity of their antioxidant systems. Furthermore, the dark skin coloration and aggregation behavior of R. kukunoris tadpoles may also be effective strategies for allowing them to resist UVBR and helping them to better adapt to a warming environment with stronger UVBR. Thus, it is possible that a moderate degree of warming may increase the capacity of living organisms to adapt to environmental changes and thus have positive effects on the development of tadpoles living at high altitudes.

Direct and indirect effects of temperature and precipitation on alpine seed banks in the Tibetan Plateau.

Plant community responses to global environmental change focus primarily on aboveground vegetation; however, the important role of the seed bank is frequently neglected. Specifically, the direct and indirect effects of changes in temperature and precipitation on seed banks remain poorly understood, yet seed banks provide a vital source of ecosystem resilience to global environmental change. We used a structural equation model to explore the direct and indirect effects of temperature, precipitation, and other biotic and abiotic factors on soil seed bank community composition using 1,026 soil seed bank samples from 57 sites along an elevation gradient that served as a space-for-time substitution for changing climate in the Tibetan Plateau. Seed bank richness was negatively correlated with both precipitation and temperature, but neither climate factor affected seed bank density. Temperature was also negatively correlated with vegetation species richness, which was positively correlated with seed bank richness and density. Both precipitation and temperature were positively correlated with soil total N, and total N was negatively correlated with vegetation richness. Both precipitation and temperature were negatively correlated with soil pH, and soil pH was negatively correlated with vegetation richness, but positively correlated with seed bank richness and density. Increasing precipitation and temperature would decrease seed bank diversity through direct effects as well as indirectly by decreasing vegetation diversity. Soil pH and total N emerged as the most important soil abiotic factors for seed bank diversity. Increasing precipitation and temperature under climate change may increase the extinction risk of some species in the seed bank by altering bet-hedging and risk-spreading strategies, which will degrade natural restoration ability and ultimately ecosystem resilience. This research is important because it identifies the potential underlying mechanistic basis of climate change impacts on seed banks through effects of aboveground vegetation and belowground biotic and abiotic factors.

Precipitation controls seed bank size and its role in alpine meadow community regeneration with increasing altitude.

The Tibetan Plateau has undergone significant climate warming in recent decades, and precipitation has also become increasingly variable. Much research has explored the effects of climate change on vegetation on this plateau. As potential vegetation buried in the soil, the soil seed bank is an important resource for ecosystem restoration and resilience. However, almost no studies have explored the effects of climate change on seed banks and the mechanisms of these effects. We used an altitudinal gradient to represent a decrease in temperature and collected soil seed bank samples from 27 alpine meadows (3,158-4,002 m) along this gradient. A structural equation model was used to explore the direct effects of mean annual precipitation (MAP) and mean annual temperature (MAT) on the soil seed bank and their indirect effects through aboveground vegetation and soil environmental factors. The species richness and abundance of the aboveground vegetation varied little along the altitudinal gradient, while the species richness and density of the seed bank decreased. The similarity between the seed bank and aboveground vegetation decreased with altitude; specifically, it decreased with MAP but was not related to MAT. The increase in MAP with increasing altitude directly decreased the species richness and density of the seed bank, while the increase in MAP and decrease in MAT with increasing altitude indirectly increased and decreased the species richness of the seed bank, respectively, by directly increasing and decreasing the species richness of the plant community. The size of the soil seed bank declined with increasing altitude. Increases in precipitation directly decreased the species richness and density and indirectly decreased the species richness of the seed bank with increasing elevation. The role of the seed bank in aboveground plant community regeneration decreases with increasing altitude, and this process is controlled by precipitation but not temperature.

Seed banks trigger ecological resilience in subalpine meadows abandoned after arable farming on the Tibetan Plateau.

Although long-term agricultural activity frequently decreases biodiversity, it remains unclear whether such biodiversity losses are readily reversible. There is no doubt that the important ecological function of seed bank is ecological memory, but few researchers have explored the role of seed banks in grassland ecosystem resilience and threshold theory. We used a space-for-time subrogation method, i.e., a natural meadow (never farmed but used for moderate gazing) and meadows farmed for 30 yr and then abandoned for 1, 10, and 20 yr, to determine if the biodiversity/ecosystem of subalpine meadows could be reversed to the natural vegetation state and to investigate the role of soil seed banks in grassland ecosystem restoration and resilience. After 20 yr of natural regeneration, aboveground vegetation composition and properties had recovered to the natural meadow state, suggesting that critical thresholds were not crossed. Seed bank composition and structure exhibited almost no change after agricultural disturbance for decades. The persistent seed bank had the highest contribution to vegetation regeneration in the 1-yr abandoned field, which had the highest seed density. Similarity between the seed bank and aboveground vegetation and seed density decreased with years since abandonment. Since the seed bank still reflected the desired state, the system had inherent resilience and had not have crossed the transition threshold. Thus, high-diversity persistent seed banks are an important indicator of high resilience of this ecosystem. High similarity between the seed bank and vegetation in early-abandoned fields may indicate that ecological resilience is triggered and be a warning signal that interventions are needed to avoid a state transition. In applying alternative stable state theory to ecological restoration, much attention should be given to the soil seed bank.

Grazing disturbance increases transient but decreases persistent soil seed bank.

Very few studies have examined whether the impacts of grazing disturbance on soil seed banks occur directly or indirectly through aboveground vegetation and soil properties. The potential role of the seed bank in alpine wetland restoration is also unknown. We used SEM (structural equation modeling) to explore the direct effect of grazing disturbance on the seed bank and the indirect effect through aboveground vegetation and soil properties. We also studied the role of the seed bank on the restoration potential in wetlands with various grazing intensities: low (fenced, winter grazed only), medium (seasonally grazed), and high (whole-year grazed). For the seed bank, species richness and density per plot showed no difference among grazing intensities for each depth (0-5, 5-10, 10-15 cm) and for the whole depth (0-15 cm) in spring and summer. There was no direct effect of grazing disturbance on seed bank richness and density both in spring and summer, and also no indirect effect on the seed bank through its direct effect on vegetation richness and abundance. Grazing disturbance indirectly increased spring seed bank density but decreased summer seed bank density through its direct effect (negative correlation) on soil moisture and total nitrogen and its indirect effect on vegetation abundance. Species composition of the vegetation changed with grazing regime, but that of the seed bank did not. An increased trend of similarity between the seed bank and aboveground vegetation with increased grazing disturbance was found in the shallow depth and in the whole depth only in spring. Although there was almost no change in seed bank size with grazing intensities, grazing disturbance increased the quantity of transient seeds but decreased persistent seeds. Persistent seeds stored in the soil could play a crucial role in vegetation regeneration and in restoration of degraded wetland ecosystems. The seed bank should be an integral part of alpine wetland restoration programs.

Effects of water level on three wetlands soil seed banks on the Tibetan Plateau.

BACKGROUND: Although the effect of water level on germination in soil seed banks has been documented in many ecosystems, the mechanism is not fully understood, and to date no empirical studies on this subject exist. Further, no work has been done on the effect of water level on seed banks of drying and saline-alkaline wetlands in alpine areas on the Tibetan Plateau. METHODOLOGY: We examined the effects of water level (0 cm, 5 cm and 10 cm) on seed germination and seedling establishment from soil seed banks at 0-5 cm and 5-10 cm depths in typical, drying, and saline-alkaline wetlands. We also explore the potential role of soil seed bank in restoration of drying and saline-alkaline wetlands. PRINCIPAL FINDINGS: Species richness decreased with increase in water level, but there almost no change in seed density. A huge difference exists in species composition of the seed bank among different water levels in all three wetlands, especially between 0 cm and 5 cm and 0 cm and 10 cm. Similarity of species composition between seed bank and plant community was higher in 0 cm water level in drying wetland than in the other two wetlands. The similarity was much higher in 0 cm water level than in 5 cm and 10 cm water levels in all three wetlands. Species composition of the alpine wetland plant community changed significantly after drying and salinization, however, species composition of the seed bank was unchanged regardless of the environment change. CONCLUSIONS/SIGNIFICANCE: Water level greatly affects seed bank recruitment and plant community establishment. Further, different water levels in restored habitats are likely to determine its species composition of the plant community. The seed bank is important in restoration of degraded wetlands. Successful restoration of drying and salinization wetlands could depend on the seed bank.

Seasonal dynamics of the plant community and soil seed bank along a successional gradient in a subalpine meadow on the Tibetan Plateau.

BACKGROUND: Knowledge about how change the importance of soil seed bank and relationship between seed mass and abundance during vegetation succession is crucial for understanding vegetation dynamics. Many studies have been conducted, but their ecological mechanisms of community assembly are not fully understood. METHODOLOGY: We examined the seasonal dynamics of the vegetation and soil seed bank as well as seed size distribution along a successional gradient. We also explored the potential role of the soil seed bank in plant community regeneration, the relationship between seed mass and species abundance, and the relative importance of deterministic and stochastic processes along a successional gradient. PRINCIPAL FINDINGS: Species richness of seed bank increased (shallow layer and the total) and seed density decreased (each layer and the total) significantly with succession. Species richness and seed density differed significantly between different seasons and among soil depths. Seed mass showed a significant negative relationship with relative abundance in the earliest successional stage, but the relationships were not significant in later stages. Seed mass showed no relationship with relative abundance in the whole successional series in seed bank. Results were similar for both July 2005 and April 2006. CONCLUSIONS: The seed mass and abundance relationship was determined by a complex interaction between small and larger seeded species and environmental factors. Both stochastic processes and deterministic processes were important determinants of the structure of the earliest stage. The importance of seed bank decreased with succession. The restoration of abandoned farmed and grazed meadows to the species-rich subalpine meadow in Tibetan Plateau can be successfully achieved from the soil seed bank. However, at least 20 years are required to fully restore an abandoned agricultural meadow to a natural mature subalpine meadow.

Effect of diurnal fluctuating versus constant temperatures on germination of 445 species from the eastern Tibet Plateau.

Germination response to fluctuating temperatures is a mechanism by which seeds detect gaps in vegetation canopies and depth of burial in soil, and it is very important for plants. Thus, studies on the effect of fluctuating temperature on germination at the community level are valuable for understanding community structure and biodiversity maintenance. We determined the effects of two alternating temperatures (5/25°C and 10/20°C) and one constant temperature (15°C) on seed germination of 445 species in a grassland community on the eastern Tibet Plateau. Seed mass was determined for each species, and data on habitat, type of life cycle, altitudinal distribution and functional group (graminoids or forbs) were obtained from the literature. Taking all species into account, alternating temperatures increased germination percentages regardless of amplitude. Overall, species growing in disturbed ground showed a significant germination response to temperature fluctuation, but those living in Alpine/subalpine meadow, forest margin /scrub, marshland and dry sunny slope habitats did not. Species distributed only at high elevations (>2000m) did not show a significant germination response to temperature fluctuation, whereas those occurring at both high and low elevations had a significant positive response. Germination of annuals/biennials was significantly promoted by 5/25°C, but not by 10/20°C, whereas germination of perennials was significantly promoted by both 5/25°C and 10/20°C. Small-seeded species were more likely than large-seeded species to respond positively to fluctuating temperatures. Germination of forbs had a positive response to temperature fluctuation, but germination of graminoids did not. Regeneration ability by seeds for about 36% of the species studied in the grassland can be increased by temperature fluctuation. The differential response among species to alternating vs. constant temperatures helps maintain community structure and biodiversity. A positive germination response to temperature fluctuation can partly explain why there are more forbs in degraded meadows.