Effects Of Nitrogen Enrichment And Warming On Plant Endophytic Fungal Community In An Alpine Meadow Ecosystem

Global nitrogen deposition has increased approximately tenfold in the last 150 years.In addition,anthropogenic greenhouse gas emissions are causing significant climate change.Nitrogen deposition and warming alter community structure in biome and reduce terrestrial biodiversity and are hence considered to be important threats to ecosystem functions and services.The ecological environment of the Qinghai-Tibet Plateau,where the productivity has greatly limited by nitrogen and temperature,is extremely sensitive to global climate change and human activities.At present,human activities have affected the stability of ecosystems in the Tibetan Plateau region,accordingly,there is an urgent need to explore the response of ecosystems in the region to global change.Leaves are among the largest terrestrial habitats in the world,and thus an important place for the interaction between plants and their symbiotic microbial communities.Endophytic fungi in leaves are crucial symbiotic microorganisms in plants.The interaction between host plants and endophytic fungi depends on factors such as genotype,environmental conditions,and host health,but the mechanisms of these complex interactive networks still exist in the black box.Whether leaf endophytic fungi are host-specific has long been debated,and most such studies have been conducted in the tropics.The size,structure,and age of host plant may strongly constrain the growth,reproduce,and transmission of endophytes within their hosts.In addition,leaves shape the biotic and abiotic environments in which fungi live,thus the promotion of plant growth stem from changes environmental variables may further lead to variations in the structure of plant-associated fungal communities.It is still unclear to us which of the two,host identity and environmental variables,is the more dominant factor driving changes in endophytic fungal communities in plant leaves.In this study,long-term nitrogen enrichment and warming sites were artificially set up in the alpine meadow ecosystem of the Qinghai-Tibet Plateau.4-year nitrogen(N)fertilization and warming experiments were nested,with plant foliar endophytic fungi(FEFs)as the target in representative plants in different sites(in N-addition site:Anemone rivularis(Ranunculaceae),Elymus nutans(Poaceae)and Thermopsis lanceolate(Fabaceae); in warming site: E.nutans,Saussurea pulchra(Asteraceae),Gentiana straminea(Gentianaceae)and Oxytropis ochrocephala(Fabaceae)).The functional characteristics and community structure of endophytic fungi in common plant leaves were analyzed by high-throughput sequencing in an attempt to test the following three hypotheses:1.Host specificity hypothesis,i.e.,the diversity and community structure of endophytic fungi in leaves of different plant species differed significantly;2.Resource supply hypothesis,i.e.,the community structure of endophytic fungi changed significantly along the settled environmental gradients;3.Core microbiome hypothesis,i.e.,some endophytic fungi appear in all hosts and/or environmental gradients,which are,the ”shared” microorganisms.We hypothesized that in the harsh and N-deficient area in the Qinghai-Tibet Plateau,diversity and community composition of FEFs varied substantially among plots with experimentally elevated temperature and levels of macronutrients,and thus,N availability and/or temperature,instead of host species identity,would have a greater influence in structuring fungal communities at different scales.We also expected an important subset of taxa shared among numerous host species and N gradients to form a communitywide core microbiome.We found that plant FEFs in alpine meadows are highly diverse.In the nitrogen addition experiment,a total of 927 operational taxonomic units(OTUs)of endophytic fungi were collected from leaves,among which T.lanceolate had the least number of OTUs,at 280,and A.rivularis had the most OTUs at 497,and the OTUs of endophytes in all three plants decreased after N input; in the warming experiment,a total of 1463 OTUs of FEFs were collected in the four focal plants,and warming increased the OTUs of endophytic fungi in the leaves.Further analysis revealed that host plant identity was a powerful factor driving the endophytic fungal community in leaves(in nitrogen addition plots: p < 0.001; in warming plots: p < 0.05),even in habitats where productivity was strongly limited by N and temperature.We also found that within the same host,both N and temperature were decisive driving forces for the composition of endophytic fungal community(p < 0.05),though with varied influences on varied hosts,and thus the effects of N input and warming on the plant symbiotic microbiomes may depend on host identity and tissue type.In addition,leaf carbon content was the most important functional trait that limited the diversity of endophytic fungi(p < 0.001).Also,we documented a distinct core microbiome shared across focal species and N gradients.These “core microbes”were also found to occur in a variety of other habitats upon inquiry,suggesting that core microbiota tend to have a high degree of habitat adaptation and a broader ecological niche.We also observed that some soil factors played an important role in the community structure of endophytic fungi in plant leaves(e.g.,soil organic carbon,total nitrogen,total phosphorus,etc.),and the important factors differed between hosts and temperatures.The increase of spatial distance significantly changed the community structure of endophytic fungi.The results reveal that the effects of nitrogen addition and warming on the plant symbiotic microbiome are multifarious in alpine meadow ecosystems of the QinghaiTibetan Plateau,and that fungal communities may have adapted to different types of hosts on an evolutionary time scale,i.e.,host plant identity replaces nitrogen availability and temperature as an important driver of endophytic community assemblage patterns.This study also highlights the general relationship between carbon source availability and plant symbiotic microbes,and the importance of the core microbiome for conducting the study of plant stress tolerance.While understanding the diversity of endophytic fungi symbiotic with dominant plant species in alpine meadows on the Tibetan Plateau,studying the effects of environmental change,especially the increasing prominence of warming and nitrogen deposition,on the community structure of endophytic fungi unravel the mystery of the plant-endophytic fungal interaction network and increase our insights of the response of plant symbiotic microbes to global change.This research implements the theoretical framework and research methodology of community ecology and provides a new perspective on the dynamics of endophytic fungal communities of grasses and forbs driven by environmental and host functional traits.