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Background The aim of this study was to identify hub genes, related transcription factors (TFs) and miRNAs from the miRNA–TF–gene interaction network in necrotizing enterocolitis (NEC). Methods Three expression data sets from GEO database that compared NEC with surgical negative controls were used to calculate differentially expressed miRNAs (DEMis) and genes (DEGs). A protein-protein interaction (PPI) network was constructed using DEGs and was used to determine hub genes. miRNAs related to hub genes were identified from the intersection between DEMis and predictions of hub gene-miRNA pairs using Starbase, TFs were predicted by hub genes, TF-miRNA pairs were predicted using miRNet. Finally, the miRNA–TF–hub gene interaction network was formed using these predicted pairs. Results A total of 14 DEMis and 123 DEGs were identified from the GEO datasets. One hundred and twenty DEGs were found in the PPI network. A pathogenic-associated interaction network was created by intersecting miRNAs, predicted TFs and hub genes. Article-published RNAs such as hsa-miR-7 or TLR4 were shown in this network, and novel RNAs and TFs (Hsa-miR-200a, GATA3, CXCL5) were shown in the network as important regulator. Conclusions This analysis displayed several important hub genes, TFs and miRNAs, some of which were not fully understood in previous studies of NEC. These results may play an important role in future studies on the etiology or treatment of NEC.

There is a debate about unmatched results between manipulative warming using constant warming rates every year (CW) and long-term observations warming affect temperature sensitivity of flowering phenology. This may be because long-term observations represent the actual yearly increase in temperature (i.e. a yearly stepwise warming rate per year, SW) which would differ from CW and their effects would be regulated by precipitation alteration. We conducted a warming experiment with CW (temperature increase by +1 °C and sustained this elevated temperature for the duration of the study) and SW (temperature increase by + 0.25 °C progressively each year) with precipitation addition in an alpine grassland for four years. Our results showed that neither warming rate affected community flowering phenology. However, precipitation addition advanced onsets of flowering for early-spring flowering (ESF) and mid-summer flowering (MSF) groups, and advanced the end date of flowering for ESF but delayed it for the MSF group. Therefore, flowering duration remained stable for the ESF group and prolonged for the MSF group, and further prolonging the flowering duration of the community. There were no interactions between warming rates and precipitation addition on the community’s flowering phenology. A severe drought in a year significantly decreased the maximal number of community flowers in the following year. Therefore, a change in precipitation has a greater effect than warming on the community flowering phenology in the semi-arid alpine grassland.

The impacts of human-driven environmental changes on the stability of natural grasslands have been assessed by comparing differences between manipulative warming and grazing plots and reference plots. However, little is known about whether or how ambient climate regulates the effects of manipulative treatments. A 36-year observational dataset shows that there is a nonlinear response of community stability to ambient climate. Manipulative warming and grazing decrease community stability with experiment duration through an increase in legume coverage and/or decrease in species asynchrony, due to exceeding the threshold of background annual mean air temperature with decreasing background annual mean air temperature through time during the 10-year experiment period. Moreover, the temperature sensitivity of community stability is more sensitive under the ambient treatment than under the manipulative treatments. Therefore, our study emphasizes the importance of the context dependency of the response of community stability to human-driven environmental changes.

Evolved gases from AdBlue decomposition with different catalysts (Ti, Zr, Pt, SiO2, and γ-Al2O3) were analyzed with a thermogravimetric analyzer coupled to a mass spectrometer (TG-MS). Kinetic parameters of AdBlue decomposition process were calculated by Kissinger–Akahira–Sunoe (KAS) and Flynn–Wall–Ozawa (FWO) methods. The results show that γ-Al2O3 is the best pyrolysis catalyst, and it promotes the decomposition of urea. The gaseous products (H2O, NH3, CHNO, CO2) were detected by MS at different reaction stages, resulting from the decomposition of urea and the formation of intermediate products. With the KAS method, the calculated activation energies with the same fractional conversion for three stages are 133.20 ± 3.71–151.14 ± 7.78 kJ/mol, 128.93 ± 3.13–186.09 ± 3.92 kJ/mol, and 326.00 ± 17.58–465.09 ± 17.62 kJ/mol, respectively.

Dissolved organic nitrogen (DON) has been increasingly recognized as a crucial component of the terrestrial nitrogen (N) cycle that regulates the ecosystem feedback to climate change. Yet, little information is available about the factors that control soil DON in the alpine ecosystems of the Tibetan Plateau, a region that is extremely sensitive to climate change. Here, we examined the relationship between DON and climate, plant, and soil attributes along a precipitation gradient in five alpine ecosystems (alpine wetland, alpine meadow, alpine shrub, alpine steppe, and alpine desert) across 20 sites that were up to 4000 km apart on the Tibetan Plateau. Results showed that soil DON concentration varied significantly across these alpine ecosystems, and had a positive relationship with mean annual precipitation. Belowground biomass, soil moisture, micro biomass carbon (MBC), and soil total phosphorus (TP) explained 84% of the variation in DON concentrations. The percentage of DON in soil total dissolved N (TDN) varied with vegetation type, with the lowest percentage observed in the alpine meadow (52%) and the highest in the alpine desert (77%), indicating the slow turnover of DON into inorganic N in systems with low litter input and microbial activity. Precipitation and plant biomass input determine the concentration and turnover of soil DON on the Tibetan Plateau. A warmer and wetter climate that has been predicted for the Tibetan grasslands may lead to a larger and more active DON pool.

Limited understanding of the effects of enhanced nitrogen (N) addition and grazing exclusion (E) on greenhouse gases fluxes (GHGs: CO2, CH4, and N2O) in grasslands constrains our ability to respond to the challenges of future climate change. In this study, we conducted a field experiment using a static closed opaque chamber to investigate the response of GHG fluxes to N addition (69 kg N ha−1 year−1 applied in 3 splits) and grazing exclusion in an alpine meadow on the Tibetan Plateau during the growing seasons from 2011 to 2013. Our results showed that winter grazing significantly raised soil temperature (ST), while grazing exclusion (E) had no effect on soil moisture (SM), and N fertilizer (F) had no effect on ST or SM. Aboveground biomass (AB) and root biomass (RB) were not significantly affected by E in 2011-2013 (p > 0.05), but F significantly affected AB and RB (p 2 emissions (by about 20.1%) during the experimental period. E did not significantly directly affect CH4 uptake, whereas N addition reduced seasonal mean CH4 uptake by about 6.5%, and N addition changed seasonal average absorption of N2O into an emission source. CO2 flux is the major contributor to CO2 equivalent emissions in this area. Our results indicate that exclosure from livestock grazing might be a promising measure to reduce CO2 emissions, while enhanced N addition might reduce CH4 uptake and increase N2O emission in the alpine meadow under future climate change.

Soil respiration (Rs) is expected to positively feedback to climate warming. The strength of this feedback is uncertain as numerous environmental factors, such as precipitation and soil moisture, can moderate the warming response of Rs. We combined seven-year Rs measurements in a warming experiment in the Tibetan alpine grassland with a meta-analysis on grassland warming experiments globally to investigate how precipitation and soil moisture influences the warming response of Rs. We further analyzed the warming responses of heterotrophic (Rh) and autotrophic (Ra) components of Rs. Warming enhanced growing-season Rs in the wet years but decreased it in the dry years in the field experiment at the Tibetan grassland. Precipitation modulated the warming responses of growing-season Rs via Rh, but not Ra. Consistent with the field experiment, a positive relationship between precipitation and the warming response of growing-season Rs was also observed in the global-scale meta-analysis on grassland warming experiments. Precipitation influences the warming effects on Rs and could result in variation in warming response of Rs across years and experimental systems. Empirical functions provided by this study could be used to reduce the uncertainty in predicting Rs in a warmer future.

Interactive effect of warming and precipitation addition on litter decomposition is still scarce. Moreover, nonlinear response of its temperature sensitivity to warming is not demonstrated due to lack of warming gradient experiment in situ. we performed a warming gradient experiment (0, ~0.25–0.5, ~1, ~2, and ~4 °C, respectively) using infrared heaters and a precipitation addition treatment in a fully-factorial design for 2-years on the Tibetan Plateau. The responses of mass loss of litter components (i.e. organic matter (OM), soluble cell content (SCC), lignin, cellulose (Ce), hemi-cellulose (Hce), total organic carbon (OC)) and nutrients (total nitrogen (TN) and total phosphorus (TP)) to warming and precipitation addition were determined over 2-years. We found that warming significantly increased annual mass losses of all litter components in both treatment years. Precipitation addition significantly increased annual mass losses of OM, SCC, OC, TN and TP only in the first year due to drought. There were no interactive effects between warming and precipitation addition on litter component decomposition in the semi-arid alpine region. Temperature sensitivities were lowest when soil temperature increased by a mean of about 2.3 °C. There was an inverse relationship between the temperature sensitivity of organic matter decomposition and quality of litter carbon compounds. Our results suggest that soil temperature effects may override soil moisture effects on litter decomposition in the alpine region, and the nonlinear temperature sensitivity of litter decomposition should be estimated using warming gradients. Lower quality of litter carbon compounds had higher temperature sensitivity of organic matter decomposition.

Leaf senescence is the final stage in the life cycle of leaves and is critical to plants' fitness as well as to ecosystem carbon and nutrient cycling. To date, most understanding about the responses of leaf senescence to environmental changes has derived from research in forests, but the topic has been relatively neglected, especially under grazing conditions, in natural grasslands. We conducted a 3-year manipulative asymmetric warming with moderate grazing experiment to explore the responses of leaf senescence of five main species in an alpine meadow on the Qinghai-Tibetan Plateau. We found that warming prolonged leaf longevity through earlier leaf-out and later leaf senescence, and grazing prolonged it through a greater advance in leaf-out than first leaf coloration for all plants. Warming did not affect leaf nitrogen (N) content or N resorption efficiency (NRE), but grazing increased N content in coloring leaves for P. anserine and P. nivea and decreased NRE for K. humilis, P. anserine under no-warming, and for P. nivea under warming. The interactive effects of warming and grazing on leaf phenology and leaf traits depended on species identity and year. There were positive relationships between leaf-out and leaf senescence mainly derived from grazing, and positive relationships between NRE from old leaves and leaf senescence for three out of five plant species. Therefore, our results indicated that earlier leaf-out could result in earlier leaf senescence only under grazing, but depending on plant species. Delayed leaf coloring increased NRE from old leaves for some plant species measured under warming and grazing. Our results suggested that alpine plants may develop strategies to adapt to warming and grazing to assimilate more carbon through prolonged leaf longevity rather than increased NRE through earlier leaf coloring in the alpine meadow.

Grazing intensity and global warming are expected to increase in the forthcoming decades, with uncertain consequences for their interaction on grassland ecosystems and their functions. We investigated the effects of warming, grazing and their interaction in a factorial warming (+1.2–1.7 °C) and grazing (moderate intensity with ca. 50% vegetation consumption) experiment in a Tibetan alpine meadow on microbial communities by studying functional genes involved in soil carbon and nitrogen cycles, using GeoChip technology. Our results showed that microbial functional gene structure and abundances were largely affected by the interactive effect of grazing and warming, rather than the main effect of warming or grazing. Compared to the control, grazing alone significantly increased the functional gene alpha diversity, changed the overall functional community structure, and increased the abundances of C fixation, C degradation, N mineralization and denitrification genes, likely due to the stimulating impact of urine and dung deposition. Warming alone did not change these microbial properties, possibly related to the unchanged soil nutrient status. Despite an increase in soil NO3− concentrations and the deposition of urine and dung, the combined treatment did not change functional gene alpha diversity, community structure, or C/N cycling gene abundances, possibly resulting from the limiting effect of water depletion in the combined treatment. Our study revealed antagonistic interactions between warming and grazing on microbial functional gene structure and abundances, which remained stable under the moderate intensity of grazing in future warming scenario in the Tibetan alpine meadow, raising potentially important implications for predicting future soil carbon and nitrogen processes in these systems.

Intact Tibetan meadows provide significant defense against soil-borne pathogen dispersal. However, dramatic meadow degradation has been observed due to climate change and pika damage, but their impacts on soil-borne pathogens are still unclear. With approximately 40% of the world’s population living in Tibetan Plateau and its downstream watersheds, this lack of knowledge should be of great concern. Here, we used Illumina amplicon sequencing to characterize the changes in potential human, domestic animal, plant, and zoonotic bacterial and fungal pathogens in nondegraded, desertified, and pika-burrowed meadows. The relative abundance of bacterial domestic animal pathogens and zoonotic pathogens were significantly increased by desertification. Pika burrowing significantly increased the relative abundance of bacterial human pathogens and zoonotic pathogens. The species richness and relative abundance of fungal pathogens was significantly increased by desertification and pika burrowing. Accordingly, fungal plant and animal pathogens categorized by FUNGuid significantly increased in desertified and pika-burrowed meadows. Soil chemical and plant properties explained 38% and 64% of the bacterial and fungal pathogen community variance, respectively. Therefore, our study indicates for the first time that both alpine meadow desertification and pika burrowing could potentially increase infectious disease risks in the alpine ecosystem, especially for fungal diseases.

Changes in ecological processes over time in ambient treatments are often larger than the responses to manipulative treatments in climate change experiments. However, the impacts of human-driven environmental changes on the stability of natural grasslands have been typically assessed by comparing differences between manipulative plots and reference plots. Little is known about whether or how ambient climate regulates the effects of manipulative treatments and their underlying mechanisms. We collected two datasets, one a 36-year long-term observational dataset from 1983 to 2018, and the other a 10-year manipulative asymmetric warming and grazing experiment using infrared heaters with moderate grazing from 2006 to 2015 in an alpine meadow on the Tibetan Plateau. The 36-year observational dataset shows that there was a nonlinear response of community stability to ambient temperature with a positive relationship between them due to an increase in ambient temperature in the first 25-years and then a decrease in ambient temperature thereafter. Warming and grazing decreased community stability with experiment duration through an increase in legume cover and a decrease in species asynchrony, which was due to the decreasing background temperature through time during the 10-year experiment period. Moreover, the temperature sensitivity of community stability was higher under the ambient treatment than under the manipulative treatments. Therefore, our results suggested that ambient climate may control the directional trend of community stability, while manipulative treatments may determine the temperature sensitivity of the response of community stability to climate relative to the ambient treatment. Our study emphasizes the importance of the context dependency of the response of community stability to human-driven environmental changes.

Phenology has a great effect on the carbon cycle. Significant relationships have been well demonstrated between phenology and photosynthesis. However, few studies have been undertaken to characterize relationships between phenology and ecosystem respiration (Re). We conducted a reciprocal transplant experiment among three elevations for two-years to measure Re over six phenological sequences throughout the growing seasons. Our results showed that changes in phenological duration were mainly determined by the onset of phenology, as one day advance of phenological onset could lengthen 0.13 days of phenological duration. Advances in early spring phenophases (i.e., first leaf-out, first bud/boot-set and first flowering) under warming strengthened the temperature sensitivity of Re. However, the late phenophases (i.e., first seeding-set, first post-fruiting vegetation and first leaf-coloring) had non-significant relationships with Re. In total, after pooling all the data, one day advance of phenophases would increase Re by 2.23% under warming. In particular, Re would increase by 29.12% with an advance of phenophases by 8.46 days of under a 1.5 °C warming scenario. Our analysis of the coupling between temperature/moisture–phenology–Re may further supplement evidence that warmer spring temperature increases carbon emission by advancing early phenophases. This points to a faster and easier way to investigate how aboveground functional traits (phenology) affect unseen functional traits (Re) on the Tibetan Plateau.

International audience; Warming in cold regions alters freezing and thawing (F–T) of soil in winter, exposing soil organic carbon to decomposition. Carbon-rich permafrost is expected to release more CO2 to the atmosphere through ecosystem respiration (Re) under future climate scenarios. However, the mechanisms of the responses of freeze–thaw periods to climate change and their coupling with Re in situ are poorly understood. Here, using 2 years of continuous data, we test how changes in F–T events relate to annual Re under four warming levels and precipitation addition in a semi-arid grassland with discontinuous alpine permafrost. Warming shortened the entire F–T period because the frozen period shortened more than the extended freezing period. It decreased total Re during the F–T period mainly due to decrease in mean Re rate. However, warming did not alter annual Re because of reduced soil water content and the small contribution of total Re during the F–T period to annual Re. Although there were no effects of precipitation addition alone or interactions with warming on F–T events, precipitation addition increased total Re during the F–T period and the whole year. This decoupling between changes in soil freeze–thaw events and annual Re could result from their different driving factors. Our results suggest that annual Re could be mainly determined by soil water content rather than by change in freeze–thaw periods induced by warming in semi-arid alpine permafrost.

There is ample experimental evidence for shifts in plant community composition under climate warming. To date, however, the underlying mechanisms driving these compositional shifts remain poorly understood. The amount and form of nitrogen (N) available to plants are among the primary factors limiting productivity and plant coexistence in terrestrial ecosystems. We conducted a short-term 15N tracer experiment in a ten-year warming and grazing experiment in an alpine grassland to investigate the effects of warming and grazing on plant uptake of NO3−-N, NH4+-N, and glycine-N. Four dominant plant species (Kobresia humilis, Potentilla anseria, Elymus nutans, Poa annua) were selected. Results We found that 10-years of warming decreased plant uptake of inorganic N by up to 80% in all species. In contrast, warming increased the uptake of organic N in K. humilis, P. anseria, and E. nutans but not in P. annua. Results showed that plant relative biomass increased hyperbolically with the ratio of the plant species total uptake of available N and plant community uptake of available N. And a significant positive correlation between plant species uptake of soil glycine-N and the uptake of total available N. The stable relative biomass of plant species is largely dependent on organic N uptake by plants. We conclude that plant organic N uptake maintains species dominance under long-term warming.

Lack of understanding of the effects of warming and winter grazing on soil fungal contribution to the nitrous oxide (N2O) production process has limited our ability to predict N2O fluxes under changes in climate and land use management, because soil fungi play an important role in driving terrestrial N cycling. A controlled warming and winter grazing experiment that included control (C), winter grazing (G), warming (W) and warming with winter grazing (WG) was conducted to investigate the effects of warming and winter grazing on soil N2O production potential in an alpine meadow on the Tibetan Plateau. Our results showed that soil bacteria and fungi contributed 46 ± 2 % and 54 ± 2 % to nitrification, and 37 ± 3 % and 63 ± 3 % to denitrification in the control treatment, respectively. We conclude that soil fungi could be the main source of N2O production potential for the Tibetan alpine grasslands. In our results, neither warming nor winter grazing affected the activity of enzymes responsible for overall nitrification and denitrification. However, warming significantly increased the enzyme activity of bacterial nitrification and potential of N2O production from denitrification to 53 ± 2 % and 55 ± 3 %, respectively, but decreased them to 47 ± 2 % and 45 ± 3 %, respectively. Winter grazing had no such effects. Warming and winter grazing may not affect the soil N2O production potential, but climate warming can alter biotic pathways responsible for N2O production process. These findings confirm the importance of soil fungi in the soil N2O production process and how they respond to environmental and land use changes in alpine meadow ecosystems. Therefore, our results provide some new insights into ecological controls on the N2O production process and contribute to the development of an ecosystem nitrogen cycle model.

Increased microbial respiration (Rm) usually decreases with warming time in incubation and field manipulative experiments, and substrate depletion and/or microbial thermal acclimation to warming is hypothesized to be responsible for this decrease. However, few studies have been conducted to determine the mechanism for this decrease, especially in alpine regions. First, soils taken from an alpine meadow on the Tibetan Plateau were incubated at three different temperatures (i.e., 5, 15, and 25 °C) with two soil moistures (i.e., 30 and 60% water holding capacity (WHC)) for 58 days, then another two experiments were conducted with incubation at high-low-high temperature (i.e., 25–15–25 °C) for 2 weeks and glucose-induced respiration (GIR) for 4 h. Rm increased with soil temperature increase at 60% WHC, but there were no significant differences between incubation at 5 and 15 °C with 30% WHC. Higher Rm was found at 60% WHC than at 30% WHC only when incubated at 15 and 25 °C. Both the high-low-high temperature incubation and GIR experiments indicated that decreased soil substrate availability weakened the responses of Rm to high temperature at 60% WHC, and there was no microbial thermal acclimation to temperature over the incubation period. Temperature sensitivity of Rm (Q10) was not affected by soil moisture, high-low-high temperature incubation, or glucose addition. Our findings suggest that the increase in Rm at higher temperature disappeared over the incubation period, and that this could be due to depletion of soil substrate availability.

Lack of understanding of how plant diversity of different flowering functional groups mediates response patterns of community phenophases to climate change limits our ability to predict future phenology. We used reciprocal transplant experiments across four elevations (i.e., 3200, 3400, 3600 and 3800 m) on the Tibetan Plateau for three years to investigate how temperature change (i.e., warming and cooling) affects the temperature responses of plant functional diversity and community phenophases and their relationships. Our results showed that a nonlinear regression model was the best fitting model for most temperature responses of SDI and community phenophases under warming and cooling. Meanwhile, decreased diversity of early-spring flowering (ESF) and mid-summer flowering (MSF) groups under warming, and increased diversity of ESF under cooling, reduced temperature sensitivities of nearly all community phenophases. These results illustrate that changes in plant diversity should be taken into account when predicting the response pattern of temperature sensitivities of community phenophases.

The distribution of annual plant species greatly expands with degradation and desertification of natural grass-lands caused by climate change and overgrazing. However, limited understanding of how their life history traits respond to climate change limits our ability to predict their adaptive strategies in the future. Here we explored effects of warming on phenological sequences and seed production of annual Koenigia islandica using an open top chamber on the Tibetan plateau from 2014 to 2016. Our results indicate that warming significantly delayed first budding-set and complete leaf coloring, and prolonged the durations of leaf coloring and the total activity period in the alpine region. Warming significantly reduced ratio of reproductive and vegetative phases relative to no warming in 2016. Warming significantly increased plant height at first flowering, seed number and mass, but had no significant effect on seed size in 2016. There was a positive correlation between seed germination time and seed number, and there were negative correlations between seed size and first fruiting-set and flowering duration. Soil temperatures and soil moisture in May and June had positive and negative correlations with seed number and mass per plant, respectively. Our results suggest that the annual plant primarily promotes its fitness through an increase in seed number and mass per plant due to an increase in plant height, rather than by altering phenophases or prolong duration of reproductive phase under future warming in the alpine meadow.

We evaluated the N2O emissions from four land use types (a native alpine meadow with winter grazing (NAM), an abandoned pasture (APL), a perennial Elymus nutans Griseb. pasture (PEN) and an annual Avena sativa L. pasture (AAS)) with and without three management practices (nitrogen (N) fertilizer, sheep manure and no tillage (NT)) in a Gelic Cambisol soil underlying an alpine meadow on the Qinghai-Tibetan Plateau in 2009 and 2010. Our results show that, compared with NAM, APL had significantly higher cumulative-average seasonal N2O emissions. Converting unmanaged APL to PEN or AAS significantly increased cumulative-average seasonal N2O emissions by 35% and 75%, respectively. Sheep manure and N fertilizer application significantly increased N2O emissions due to increased soil inorganic N concentration. The effect of sheep manure addition on N2O emissions was lower than that of N fertilizer. For AAS, tillage significantly decreased the effect of sheep manure application on N2O emissions. Compared with tillage, NT significantly decreased N2O emissions from AAS. Therefore, our results suggest that cultivating natural grassland would increase N2O emissions, and fertilizer application would amplify the magnitude of emissions, whereas NT could mitigate the fertilizer impact on N2O emission. Furthermore, the structural equation analysis revealed that land use change affected N2O emissions directly by influencing the number of plant species and soil characteristics. There were two different underlying mechanisms regulating N2O emissions in response to N fertilizer and sheep manure addition.

Few studies have investigated the effect of phosphate (P) fertilizer on net ecosystem CO2 exchange (NEE) and its components under mixed legume and grass pastures sown, which will limit our understanding about their effects on carbon (C) sequestration in soils. Our aim was to test the hypothesis that a combination of legumes with grass and P fertilizer increases NEE due to greater increases in gross primary productivity (GPP) compared to increases in ecosystem respiration (Re).We measured NEE, GPP and Re under 5 different seeding ratios of oat (Avena sativa L.) and common vetch (Vicia sativa L.) according to their thousand kernel weight and germination rate (i.e., seeding rate were: M1: 0:150, M2: 150:105, M3: 300:75, M4: 450:45 and M5: 600:0) with and P unfertilized on the Tibetan Plateau from 2011 to 2013. NEE was measured at 1-week intervals from June to September each year using a transparent chamber technique (Licor-6400). Our results showed that both P fertilizer and mixed ratio had significant effect on NEE, Re and GPP, but their interactive effects were not significant during the growing season. P fertilizer primarily increased NEE due to increased GPP for the monoculture and mixtures with a higher proportion of common vetch. NEE, Re and GPP were higher for the mixed pastures only compared with legume monoculture. From the perspective of forage yield and C sequestration, planting mixed legume-grass pastures with P fertilizer is a preferable way to balance the twin objectives of forage production and C sequestration in alpine regions.

Nitrogen (N) limitation leads to intense competition between plants and soil microorganisms for available N. However, it is unclear how grazing affects the acquisition of N by plants and microorganisms. We conducted short-term 15N tracer experiments during the growing season (June, early growing season; July, mid-growing season; and September, late growing season) in an alpine grassland on the Tibetan Plateau to investigate the effects of grazing on the acquisition of NO3 −-N, NH4 +-N, and glycine-N by plants and soil microorganisms. Three dominant plant species (one graminoid, Kobresia pygmaea, and two forbs, Potentilla bifurca and Potentilla multifida) were selected for the study. As these species represented >90% of the vegetation, the plant recovery of 15N reflected competition at the plant community. Grazing decreased the recovery of 15N by soil microorganisms and plants by 46 and 69%, respectively, indicating that grazing strongly reduced the uptake of 15N by plants and microorganisms and altered the partitioning of 15N between them. Significant interactions were found between grazing, season and the different forms of N. In the absence of grazing, plants acquired relatively more N than soil microorganisms for the three forms of N in July and September, whereas the microorganisms obtained relatively more 15N glycine in July and all three forms of N in September under grazing conditions. Under grazing, the plant root biomass played an important role in controlling plant–microbial N acquisition. Grazing alters the partitioning of inorganic and organic N between plants and soil microorganisms by reducing microbial 15N recovery to a lesser extent than plant 15N recovery. This indicates that heterotrophic microorganisms play an important part in N cycling in N-limited ecosystems.

Our ability to predict how temperature modifies phenology at the community scale is limited by our lack of understanding of responses by functional groups of flowering plants. These responses differ among species with different life histories. We performed a reciprocal transplant experiment along four elevation gradients (e.g., 3,200, 3,400, 3,600 and 3,800 m) to investigate the effects of warming (transferred downward) and cooling (transferred upward) on plant flowering functional groups (FFGs) and community phenological sequences (i.e., seven phenological events). Warming significantly decreased early-spring-flowering (ESF) plant coverage and increased mid-summer-flowering plant (MSF) coverage, while cooling had the opposite effect. All community phenological events were advanced by warming and delayed by cooling except for the date of complete leaf-coloring, which showed the opposite response. Warming and cooling could cause greater advance or delay in early-season phenological events of the community through increased coverage of MSF species, and warming could delay late-season phenological events of the community by increased coverage of ESF species. These results suggested that coverage change of FFGs in the community induced by temperature change could mediate the responses of the community phenological events to temperature change in the future. The response of phenological events to temperature change at the species level may not be sufficient to predict phenological responses at the community-level due to phenological compensation between species in the community.

There is still great uncertainty about the response of global carbon dynamics to land use change and nitrogen (N) fertilizer, especially how to balance between forage production and CO2 emission under land use change and N fertilizer times in alpine meadows. This objective of this study was to determine the effects of three land uses (i.e., abandoned cropland/pastureland (APL), perennial Elymus nutans (PEN), and annual Avena sativa L. pasture (AO)) and nitrogen fertilizer applied three times a year with the same N dose on aboveground net primary productivity (ANPP) and ecosystem respiration (Re) from 2011 to 2013. We found that AO had the highest average ANPP and lowest average Re compared with APL and PEN and that there were no significant differences for them between APL and PEN during the experimental period. Nitrogen fertilization did not significantly affect ANPP, root biomass, and seasonal mean Re. Soil temperature could explain about 21.1 and 13–15% of the variation in daily Re for APL and PEN, respectively, but 20.7 and 15.0% of the variation in daily Re for APL and PEN under both with and without N fertilization, respectively. ANPP explained about 49.4% of the variation in seasonal average Re for AO, and the ratio of root biomass and ANPP explained about 45% of the variation in seasonal average Re for AO-N treatment during 2011–2013. Land use significantly affected ANPP, root biomass, and seasonal mean Re. However, nitrogen addition with different times did not significantly affect seasonal average Re or ANPP and root biomass for all treatments during the experimental period. Therefore, our results suggested that N could not be a limiting factor for plant production in this alpine region.

Grazing exclusion is widely used globally to restore degraded grasslands. Plant diversity has important impacts on grassland ecosystem functions, including grassland productivity and carbon storage. In this study, we selected a Kobresia meadow on the Qinghai-Tibetan Plateau to investigate how grazing exclusion affects plant diversity. Inorganic nitrogen (NH4 (+) and NO3 (-)) was also measured because its availability impacts plant growth. We found that plant diversity in the meadow was significantly lower under grazing exclusion (fenced meadow) for 9 years compared with moderate grazing. Accumulated litter was significantly higher under grazing exclusion (386.41 g m(-2)) compared with grazing (58.77 g m(-2)). Soil inorganic nitrogen at 0-5 cm depth was significantly higher under grazing exclusion (13.60 x 10(-2) g kg(-1)) than under grazing (9.40 x 10(-2) g kg(-1)). The composition of the four functional groups (grasses, sedges, legumes, and forbs) might alter in response to significant changes in the amount of litter and soil available nitrogen content under grazing exclusion compared with grazing. However, the enhanced soil available nitrogen content showed weak feedbacks on plant diversity. In conclusion, light limitation induced by increased amounts of litter may be the main factor causing decreased plant diversity in grazing-excluded meadows compared with moderately grazed meadows.

The assumption of a linear relationship between temperature and phenophases may be misleading. Furthermore, a lack of understanding of the changes in temperature sensitivity of phenophases to changes in temperature strongly limits our ability to predict phenological change in response to climate change. We investigated the timing of seven phenophases of six alpine plant species to test the hypothesis of nonlinear responses in their temperature sensitivities to warming and cooling, using a reciprocal transplant experiment along a 3200–3800 m mountain slope gradient. Our results supported our hypothesis and showed that there were different thresholds in temperature sensitivity of phenophases to warming and cooling. Moreover, linear temperature sensitivity models significantly underestimated advances and delays of phenophases within the thresholds of temperature change. The nonlinear temperature sensitivity of phenophases is best explained by plastic and adaptive responses of phenophases to temperature change gradients. Therefore, our results suggest that the thresholds of temperature sensitivity for different species should be determined and that nonlinear models of temperature sensitivity may be essential to predict accurately phenological responses to climate change.

The production and fate of inorganic N as regulated by environmental change are relatively well understood, but we know comparatively little about how these factors influence dissolved organic nitrogen (DON). We measured total N, DON and dissolved inorganic nitrogen (DIN) concentrations in the soil solution and plant N uptake in a factorial warming x grazing experiment in a Tibetan alpine meadow. Results showed that warming significantly decreased DON concentration by up to 36%. The effect of warming on DON concentration in the soil solution varied with sampling date and soil depth. However, our results showed that moderate grazing set off the effect of warming on DON concentration in the soil solution. Grazing increased soil DON, opposite to the effect of warming. Previous studies have found warming to increase a range of factors which contribute to the supply of DON in soil. Our results show that the observed decrease of DON under warming was correlated with plant N uptake, suggesting that warming stimulated processes making DON available to plants. Our study highlights the complex interaction of land management regime and climate warming in the regulation of DON cycling in N limiting environments. (C) 2016 Elsevier B.V. All rights reserved.

Change in individual species phenology is often unsuitable for predicting change in community phenology because of different responses of different species to temperature change. However, few studies have observed community phenological sequences in the field. Here we explore the changes in timing and duration of the community phenological sequence (i.e. onset of leaf-out (OLO), first flower bud (FB), first flowering (FF), first fruiting-set (FFS), post-fruiting vegetation (OPFV), first leaf-coloring (FLC) and complete leaf-coloring (CLC)) along an elevation gradient from 3200 to 3800 m in an alpine meadow on the Tibetan plateau. Our results indicate that OLO and FFS significantly advanced and other timings of phenological events significantly delayed at 3200 m compared with higher elevations (3600 and 3800 m). The flowering duration of the community was shortest and other phenological durations (except budding stage and post-fruiting vegetation stage) were longest at 3200 m. The duration of the growing season decreased as elevation increased, and the ratio of the durations of the reproductive period and growing season was smallest at 3200 m. There were negative correlations between the proportion of early-spring flowering functional group plants and FB, and the durations of leafing and post-fruiting vegetation of the community. Positive correlations were found between the proportion of mid-summer flowering functional group plants in the community and these variables. There were significant negative correlations between flowering duration of the community and annual mean air temperature and soil moisture. Therefore, our results suggest that different community compositions might respond differently to climate change. (C) 2016 Elsevier B.V. All rights reserved.

Information about the potential effects of climate change, especially cooling, on ecosystem respiration (Re) in alpine meadows is scarce. We determined the effects of warming and cooling on Re on the Tibetan Plateau using a 2-year reciprocal translocation experiment with 4 different vegetation types (3 alpine meadows and 1 alpine shrub differentiated by plant community composition) along an elevation gradient from 3200 to 3800 m (with vegetation types E2, E4, E6 and E8 at 3200, 3600, 3800 and 3800 m, respectively) during the growing seasons in 2008 and 2009. Mean growing seasonal Re decreased by 13.6, 30.3 and 40.7% per 200 m rise in elevation (cooling) for vegetation types E2, E4 and E6, but increased by 1.3, 35.9 and 58.8% per 200 m decrease in elevation (warming) for vegetation types E4, E6 and E8, respectively. Soil temperature explained 49.3–64.0% of daily Re variation and aboveground biomass explained 21.5–61.6% of average Re variation of the growing season for all vegetation types, but the effect of soil moisture on Re was small over 2-year. The values of Re temperature sensitivity increased with an increase in elevation for both warming (3.3, 24.3 and 53.5% °C −1 for vegetation types E4, E6 and E8) and cooling (8.0, 19.1 and 24.4% °C −1 for vegetation types E2, E4 and E6), suggesting that alpine meadow at higher elevation was more sensitive to both warming and cooling. Based on the values of Re temperature sensitivity for all pooled vegetation types (25.4, 5.6 and 19.6% °C −1 for warming, cooling and pooled warming and cooling), it could be over-estimated by 23% for warming alone compared with pooled warming and cooling. Therefore, asymmetrical responses of Re to warming and cooling should be taken into account when we evaluate the effect of temperature change on Re using models in the future.

Containerless technique has many advantages for the density measurement of melts, such as high temperature and deep undercooling measurement. At present, many researchers focus on density measurement of melts with containerless technique and projection imaging method. In containerless experimental processes, the sphere melt droplet rotates and changes to spheroid, due to the asymmetrical of pressure of laser light and electrostatic force. In addition, there was no depth information is included in the image acquired from the droplet. Therefore, the accurate volume of droplet is difficult to obtain. Therefore, we proposed a new 3D reconstruction algorithm with two cameras that acquired the droplet images simultaneously at different angles. The deviation of the novel algorithm is less than 1.1%. With the proposed algorithm, the density of liquid Zr is measured from 1800 K to 2500 K, the combined relative standard uncertainty in density is 1.80%.