SCIENCE ADVANCES | RESEARCH ARTICLE ECOLOGY Copyright © 2020 The Authors, some Ozone affects plant, insect, and soil microbial rights reserved; exclusive licensee communities: A threat to terrestrial ecosystems American Association for the Advancement and biodiversity of Science. No claim to original U.S. Government Works. Distributed Evgenios Agathokleous1*, Zhaozhong Feng1*, Elina Oksanen2, Pierre Sicard3, Qi Wang1, under a Creative Costas J. Saitanis4, Valda Araminiene5, James D. Blande6, Felicity Hayes7, Vicent Calatayud8, Commons Attribution Marisa Domingos9, Stavros D. Veresoglou10, Josep Peñuelas11,12, David A. Wardle13, NonCommercial Alessandra De Marco14, Zhengzhen Li15, Harry Harmens7, Xiangyang Yuan15, License 4.0 (CC BY-NC). Marcello Vitale16, Elena Paoletti17 Elevated tropospheric ozone concentrations induce adverse effects in plants. We reviewed how ozone affects (i) the composition and diversity of plant communities by affecting key physiological traits; (ii) foliar chemistry and the emission of volatiles, thereby affecting plant-plant competition, plant-insect interactions, and the composition of insect communities; and (iii) plant-soil-microbe interactions and the composition of soil communities Downloaded from http://advances.sciencemag.org/ on August 13, 2020 by disrupting plant litterfall and altering root exudation, soil enzymatic activities, decomposition, and nutrient cycling. The community composition of soil microbes is consequently changed, and alpha diversity is often reduced. The effects depend on the environment and vary across space and time. We suggest that Atlantic islands in the Northern Hemisphere, the Mediterranean Basin, equatorial Africa, Ethiopia, the Indian coastline, the Himalayan region, southern Asia, and Japan have high endemic richness at high ozone risk by 2100. INTRODUCTION changes in the environment may affect biodiversity and the func- Above- and belowground trophic interactions play pivotal roles in tioning of terrestrial ecosystems (2, 4, 5). maintaining plant diversity. Plants respond to herbivores by various Ground-level concentrations of ozone (O3) increased consider- physiological mechanisms, affecting plant performance and plant-­ ably in the second half of the 20th century due to increased levels of microbe interaction and potentially regulating ecosystem processes NOx, volatile organic compounds (VOCs), and radical precursors and community dynamics (1–3). Plant-soil feedbacks (PSFs) like- responsible for its production (6–8). For example, current O3 levels wise involve interactions among plants, soil microbiota, and abiotic in rural areas of the temperate and polar zones of the Northern factors, affecting structural and functional features at different scales Hemisphere (NH) have increased by 30 to 70% compared to 1896–1975 of biological organization. These effects allow plants to readily re- (6, 7). The O3 concentrations have remained elevated (see the Sup- spond to environmental changes and mediate ecosystem processes plementary Materials) but are also projected to remain high through- (4). Trophic interactions depend on environmental conditions, so out the 21st century (9), raising concerns about O3 phytotoxicity, despite policies for reducing precursor emissions. Many programs 1 Key Laboratory of Agrometeorology of Jiangsu Province, Institute of Ecology, have documented the incidence of visible injury from O3 in numer- School of Applied Meteorology, Nanjing University of Information Science and ous field-grown species of forbs, shrubs, and trees worldwide, but Technology, Nanjing 210044, China. 2Department of Environmental and Biological O3 has often not been included in global assessments of threats to Sciences, University of Eastern Finland, POB 111, 80101 Joensuu, Finland. 3ARGANS, 260 route du Pin Montard, 06410 Biot, France. 4Lab of Ecology and Environmental biodiversity (5, 10). Preliminary analyses suggest that O3 will continue Science, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece. to pose risks to terrestrial biodiversity at various trophic levels and 5 Institute of Forestry, Lithuanian Research Centre for Agriculture and Forestry, Girionys ecosystem processes and feedbacks in the future (10). 53101 Kaunas District, Lithuania. 6Department of Environmental and Biological Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland. Despite the progress in our understanding of numerous physio- 7 UK Centre for Ecology and Hydrology, Environment Centre Wales, Deiniol Road, logical mechanisms specific to plants for responding to O3 (11, 12), Bangor, Gwynedd LL57 2UW, UK. 8Fundación CEAM, c/Charles R. Darwin 14, Parque we still know little about the responses of communities and eco- Tecnológico, Paterna, Valencia 46980, Spain. 9Instituto de Botânica, Núcleo de systems to O3. Identifying plant mechanisms that may drive the Pesquisa em Ecologia, PO Box 68041, 04045-972 São Paulo, Brazil. 10Freie Universität Berlin-Institut für Biologie, Dahlem Center of Plant Sciences, Plant Ecology, Berlin, structure and function of plant communities and interactions with Germany. 11Consejo Superior de Investigaciones Científicas, Global Ecology Unit insect and soil communities in O3-polluted atmospheres is challeng- CREAF-CSIC-UAB, Bellaterra, Catalonia E-08193, Spain. 12CREAF, Cerdanyola del ing, and the effects on the functioning and biodiversity of terrestrial Vallès, Catalonia E-08193, Spain. 13Asian School of the Environment, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore. 14Italian ecosystems are underexplored (5, 10, 13). National Agency for New Technologies, Energy and the Environment (ENEA), This paper addresses how O3 affects plant communities, plant-­ C.R. Casaccia, S. Maria di Galeria, Rome I-00123, Italy. 15State Key Laboratory of Urban insect interactions, and PSFs and thus plant, insect, and microbial and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road 18, Haidian District, Beijing 100085, China. diversity (Figs. 1 and 2). We have four aims. (i) Review the relation- 16 Department of Environmental Biology, Sapienza University of Rome, Piazzale Aldo ships between plant ecological traits and susceptibility to O3. We thus Moro 5, Rome I-00185, Italy. 17Institute of Research on Terrestrial Ecosystems (IRET), collated empirical evidence to determine whether O3 affects the National Research Council of Italy (CNR), Via Madonna del Piano 10, I-50019 Sesto structure and diversity of plant communities depending on func- Fiorentino, Italy. *Corresponding author. Email: evgenios@nuist.edu.cn (E.A.); zhaozhong.feng@ tional groups. (ii) Discuss how O3 affects plant-insect interactions nuist.edu.cn (Z.F.) and whether ecosystem functioning and the structure and diversity Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 1 of 17 SCIENCE ADVANCES | RESEARCH ARTICLE Downloaded from http://advances.sciencemag.org/ on August 13, 2020 Fig. 1. Effects of elevated ozone (O3) on aboveground ecosystem processes. Ecological processes occurring at the ecosystem and foliar levels in a natural (not polluted) ecosystem (A) versus an ecosystem disturbed by increased levels of O3 (B). Gray icons represent the loss of insect or plant diversity but not for particular species. O3 reduces the growth rate and biomass of plants (including forest trees) (I). Deciduous broadleaf species are usually more susceptible than evergreen broadleaf and needle-leaf species (I). O3 can also reduce plant species richness and alter community composition (II). O3 reduces the abundance of insect species but not species rich- ness in forest ecosystems (III). O3 and OH degrade biogenic VOC (BVOCs), thereby impeding plant-pollinator communication (IV). O3-plant-insect interactions may be quite complex and species specific. O3 inhibits isoprene emissions, increases monoterpene emissions in tolerant and evergreen species, reduces foliar size, induces foli- age prematurity (V and I), and increases plant susceptibility to insects and pathogens (I and VI). In other cases, O3 induces the accumulation of phenolic compounds in leaves, discouraging herbivory by insects (thus reducing insect abundance), increases insect mortality, and inhibits the growth of insect body mass (VII). O3 also alters foliar phytochemistry, thereby impeding insect oviposition (VIII). of insect communities are threatened by O3-caused changes in VOCs Plant-plant interactions: Are plant-community composition and foliar chemical composition. (iii) Assess how O3 affects plant- and diversity at risk from O3? soil interactions and thus soil ecosystem functioning, with special Indicators of plant susceptibility to O3 reference to plant litter, decomposition, nutrient cycling, and The main indicators of the susceptibility of plants to O3 are stomatal microbial biomass. The hypothesis that O3 can affect microbial conductance, specific leaf area, and genetically controlled defensive communities and diversity was verified by a series of studies demon- capacity (14, 15). Classification of plant species into tolerant and strating that PSF responses to O3 can lead to changes in the community susceptible, however, is difficult because of their high intraspecific structure and diversity of soil microbes. (iv) Analyze global endemic variation in tolerance (5, 16, 17). High intraspecific genetic varia- richness of vascular plants versus predicted O3 exposure by 2100 for tion has been detected in grasses and herbaceous and woody plants assessing whether foci of the endemic richness of various biomes [e.g., Phleum alpinum, Trifolium repens, and Plantago major (16), as overlap with areas of high O3 risk, with adverse effects on plant pro- well as Betula pendula and Populus spp. (18)] and has been studied ductivity. We use these discussions to seek to identify important gaps in detail in, for example, willows and silver birches (18). The genetic in our knowledge and to draw some conclusions about how O3 can variation of O3 tolerance in these species has been attributed to a affect plant interactions with insects and microbiota and, thus, alter range of factors, including foliar phenolic composition (19), effective PSFs and community composition and diversity of plants, insects, pathways of ascorbate-glutathione detoxification, remobilization of and microbiota. amino acids (20), genetic plasticity, chemical composition of foliar Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 2 of 17 SCIENCE ADVANCES | RESEARCH ARTICLE Downloaded from http://advances.sciencemag.org/ on August 13, 2020 Fig. 2. PSFs under elevated ozone (O3). A healthy holobiont in a clean atmosphere (with natural background O3 levels), where mutually beneficial PSFs occur (A), versus a suppressed holobiont and disturbed PSFs due to O3 (B). Gray icons represent the loss of microbial biomass but not for particular species. O3 decreases root biomass, reduces the quantity, and affects the quality of foliar and root litter, potentially affecting litter-feeding soil macrofauna, decomposition, and cycling of nutrients. O3 may influence the chemical composition of roots and soluble root exudates, including reduced exudation of some extracellular enzymes (e.g., -glucosidase). The rate of de- composition can be increased or decreased species-specifically. Soil microbial biomass also decreases. O3 alters the composition and structure of soil microbial commu- nities, with fungi being likely more susceptible to O3 than bacteria. Some N-fixing bacteria are promoted by O3, but N fixation is reduced by O3 in other studies. Some denitrifying bacteria are likewise promoted by O3, and the abundance of some nitrifying bacteria can be either reduced or increased by O3. The decrease in microbial biomass disturbs the rates of N and C cycling as feedback, potentially reducing N2O and storing less C in the rhizosphere. The changes in C and N cycling in PSFs may occur in tandem with changes in the cycling of other nutrients due to poor leaf and root litter as well as affected decomposition processes. surface waxes (21, 22), stomatal closure, and foliar structural traits, that O3 tolerance at the foliar level was associated with LMA, and a such as apoplastic volume, thickness, and leaf dry mass per unit area reduction in whole-tree biomass was linked to high stomatal flux (LMA) (18, 23). Model species such as Arabidopsis thaliana clearly per unit foliar mass, indicating high O3 uptake per unit mass (29). indicate that O3 tolerance is due to a unique set of alterations of High LMA and sclerophyllia can provide O3 tolerance by (i) cross-­ various defensive mechanisms, e.g., signaling pathways, regulatory protection (e.g., resistance to several stress factors such as drought), genes, plant hormones, antioxidants, and physiological processes (ii) the so-called dilution effect (lower O3 load per unit foliar mass), (function and regulation of stomata) (24, 25). These indicators of and (iii) a large apoplastic compartment, which is often associated tolerance can be useful for individual plants or plant mixtures but with high antioxidative capacity (5, 15, 30, 31). Trichomes are foliar-­ are challenging in studies at evolutionary and population levels. The surface structures that may provide protection against high levels of selection for O3 tolerance in the field is further complicated by the O3 by acting as physical barriers or by physiological detoxification multiple environmental factors and stressors that usually affect plants, (32). LMA, sclerophyllia, and trichomes are relatively easy to measure such as drought, salt, heavy metals, light, nutrient availability, and and can, therefore, be widely used for assessing the threat of O3 in changeable meteorological conditions (16, 26, 27). Agrobiodiversity the field. and the compositions of seminatural plant communities, among Relationships between O3 susceptibility and plant diversity other factors, are often affected by management practices such as Studying how elevated O3 affects plant diversity requires focusing on grazing, cutting, and fertilization, all of which further obscure the functional traits associated with ecological fitness, such as repro- influence of O3. ductive fitness (seed output), plant health, and competitiveness. Plant leaves are most exposed to elevated O3 and have therefore The responses of plant communities to O3, however, are ultimately been extensively examined for structural modifications and adapta- driven by changes in growth, physiology, biochemistry, or genetics tions to elevated O3 and associated plant susceptibility (5, 15, 16). at the level of individuals or species (10). Many indirect and well- The O3 susceptibility in silver birch and trembling aspen (Populus known effects of O3 on plants may lead to changes in community tremuloides) has been associated with foliar thickness, i.e., the most composition and diversity, including impacts on growth, plant vi- susceptible genotypes had thinner leaves (28). An extensive survey tality (assessed as crown defoliation), photosynthesis, stomatal con- of subtropical, temperate, and Mediterranean tree species found ductance, water balance, the trade-off between biomass production Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 3 of 17 SCIENCE ADVANCES | RESEARCH ARTICLE and defensive processes, flowering, competition, and susceptibility to pests and pathogens (5, 10, 33, 34). These impacts can ultimately Table 1. Evidence that the susceptibility of plants to O3 is conserved reduce the ability of species of a plant community to compete (inter- at relatively high taxonomic levels. The reader may refer to the specific competition) and the ability of communities to compete. references for further details. Specific indicators of biodiversity, however, may not represent the Susceptibility Reference direct effects of O3 such as reduced biodiversity within a plant com- Angiosperms > gymnosperms (204) munity but rather variations in the competition for resources Pioneer species > climax species (205) among plant species. Temperate species > subtropical Visible foliar injury induced by O3 is an important attribute for (12) species epidemiological assessments because it manifests susceptibility of plants Therophytes > other Raunkiaer life to O3 and is the only indicator of adverse effects of O3 that can be forms (chamaephytes and (14, 43) used for routine field surveys (10, 35). The visible foliar injury hemicryptophytes) appears as chlorosis, necrosis, flecks, stipples, bronzing, and/or red- Deciduous trees > evergreen dening (see the Supplementary Materials for more information). (5) conifers While it is difficult to distinguish whether chlorosis and necrosis are Deciduous broadleaf species > induced by O3 or normal senescence in the field, a characteristic of evergreen broadleaf and (12) O3-induced symptoms is that they usually occur on interveinal needle-leaf species areas of the upper leaf surface only and older leaves show the most Fabaceae (legumes), Poaceae, and Downloaded from http://advances.sciencemag.org/ on August 13, 2020 symptoms. Species-specific visible injuries have been widely found (5, 43) Asteraceae > Brassicaeae in nature (10, 35–37), although evidence for the relationship be- Fabaceae > Asteraceae, tween O3-induced visible injuries and damage is contradictory and (43) Caryophyllaceae, and Poaceae uncertain especially for forest trees (38). Visible injuries may be Myrtaceae and Salicaceae > associated with negative impacts on fitness traits [e.g., the reduction (5) Boraginaceae and Brassicaceae of carbon (C) assimilation/allocation limits growth and seed pro- Legumes > other forbs and grasses (14) duction, making the species uncompetitive] (38). Little is known, Species grown under favorable however, about the relationship between short-term phenomena growth conditions or productive such as visible foliar injury and longer-term processes affecting (14) habitats > species grown in less plant-community structure and ecosystem biodiversity (5, 10). productive habitats Ozone susceptibility varies among plant functional groups Light-loving plants > Plants that On the basis of ecological strategies of competition and survival, (43) normally occur in the shade some susceptible plants may be affected more than nonsusceptible Plants grown at dry sites > plants plants by O3-induced stress and may thus be competitively penalized (43) grown in moister soils (17, 39–42). The degree of susceptibility differs widely among species Plants tolerant to moderate salinity but also functional groups (Table 1), although current rankings of (43) > plants of no saline habitats O3 susceptibility are based on the susceptibility of individual species (5, 17, 43) and the modeling of different indicators (44). For example, elevated O3 may decrease the aboveground biomass of therophytes (annuals) more than non-annual plants (e.g., chamaephytes) (43), for reduced plant species richness (total number of species), diver- suggesting that variations in response of annual and perennial species sity (Shannon-Weaver index), and evenness of an early successional in a community under O3 are also important for long-term bio- community due to exposure to ambient and elevated O3 levels over diversity effects. High oxidative stress induced by O3 can also ad- two growing seasons (49). These elevated O3 concentrations occur versely affect the fitness of O3-susceptible genotypes when combined in highly polluted areas of NH nowadays. Solardome experiments with harsh inter- and intraspecific competition within communities, have also suggested potential changes in the structure of grassland ultimately altering the timing of flowering and seed development communities (33, 45). An O3 treatment accounted for ≈40% of the and reducing the number and biomass of flowers in some species in variation in species composition (41). Reduced species richness (total a community (33, 45, 46). The community composition of terrestrial number of species), diversity (Shannon-Weaver index), and even- ecosystems may thus exhibit long-term changes, and plant diversity ness due to exposure to O3 were particularly important over two may be at risk (Fig. 1), especially in areas where O3 occurs at potentially growing seasons in an early successional community (49). The phytotoxic levels (Fig. 3). species richness of communities with different histories of O3 expo- Empirical evidence for O3 threats to plant-community sure was similar, but the relative abundance of the dominant species composition and diversity differed among the communities (50). Changes in community Empirical evidence from several experiments conducted in different structure due to past exposure to O3 suggest that O3 might have regions of the world indicates that O3 can affect the structure and already contributed to changes in natural communities. diversity of plant communities (table S1). Several experiments with The use of free-air O3 concentration enrichment (FACE) exper- open-top chambers (OTCs) in pasture communities exposed to O3 iments can generate more realistic estimates of the impacts of O3 on (commonly up to twofold above the ambient O3 concentrations) for vegetation because the plants fully interact with the surrounding two to four growing seasons have demonstrated changes in species natural environment. A few FACE studies have assessed the impacts composition (table S1), with some species being lost over time, and of O3 (commonly up to 1.6-fold higher than the ambient O3 con- in community structure (42, 47–51). For example, there is evidence centrations) in subalpine and other types of grasslands after two to Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 4 of 17 SCIENCE ADVANCES | RESEARCH ARTICLE Downloaded from http://advances.sciencemag.org/ on August 13, 2020 Fig. 3. Ozone exposure levels and global plant endemic richness. Surface mean AOT40 [parts per million (ppm)·hour] for 2000–2003 (A) and for RCP2.6 (B), RCP4.5 (C), and RCP8.5 (D) by 2100, overlapping the global patterns of the endemic richness of vascular plants (number of species of vascular plants per 10,000 km2) across bio- geographic regions worldwide (except Antarctica). RCP represents a representative concentration pathway, and AOT40 represents accumulated ozone exposure above a threshold of 40 parts per billion (ppb). Data sources: (9) and (197). The ozone maps are from (9). seven growing seasons (table S1). Some experiments found O3-­ in the legume fraction, but not the grass fraction, of harvested bio- induced shifts in the fractions of plant functional groups, changes in mass as a result of elevated O3 exposure occurred in intact and species composition depending on the functional component, and managed pasture (54, 55), T. repens–Lolium perenne mixtures increased abundance of some species such as Nardus stricta (52). established under field conditions from seed (56), and T. repens–­ Empirical evidence from long-term FACE experiments, therefore, L. perenne established in mesocosms (57). Similarly, reductions in supports the suggestions from OTC experiments that O3 induces the clover/grass ratio due to elevated O3 appeared in T. repens and alterations to the structure and species composition of grassland L. perenne (58) and for T. repens and Festuca arundinacea (59). communities. Comparable reductions can also occur if the aboveground biomass Forbs are generally more susceptible to O3 than grasses, but the of the grass component increases due to reduced competition from relative abundance of a grass species may decrease, depending on the legume. Altered pasture quality from samples analyzed from potential interacting environmental factors (table S1). Note that seven component studies also occurred (60). This was the result of some species may increase in abundance and others may decrease species composition alterations and the nutritional quality change but without necessarily a large loss of species richness or a shift of individual species within the community. in their distributional evenness (53). These changes in community The hypothesis of impacts on community composition associated composition may also lead to changes in the nutritional value of with O3 was also supported by an assessment of the impacts on vegetation used for animal consumption. For instance, reductions community composition at 64 field sites selected from the U.K. National Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 5 of 17 SCIENCE ADVANCES | RESEARCH ARTICLE Vegetation Classification of U4 “Festuca ovinae–Agrostis capillaris–­ enemies of insect pests via herbivore-induced plant volatiles and Galium saxatile grasslands,” which represent calcifugous grasslands, extra floral nectar (67, 79, 80). For example, the mortality of gypsy widely distributed in the British Isles (table S1) (53). The use of moths increased when fed with leaves treated with O3 (81). A recent genetic markers and DNA fingerprinting in wild populations also study also found that the increase in body mass in Samia ricini larvae demonstrated that genetic diversity is declining in areas with high was inhibited when the larvae were fed with cauliflower (Brassica levels of O3 [reviewed in (5)]. These studies suggest potentially oleracea) leaves treated with O3 (65), similarly to Pieris brassicae changing diversity in the real world. Recent technological develop- reared on Brassica nigra (82). This inhibition was due to the effect ments are providing powerful tools for monitoring the loss of bio- of O3 on foliar quality and could, thus, affect herbivory levels by diversity in vegetation with various plant functional groups, e.g., overcompensating for the poor nutritional quality of the tissues remote sensing and hyperspectral imaging techniques combined with (65, 83). These effects suggest potential shifts between generalist drones (61); drone techniques are particularly useful in wetland eco- species, which can adapt easier to plant defensive compounds and systems. These techniques can potentially more robustly assess the thrive in a wide variety of environmental conditions, and specialist global threat of O3 to biodiversity in the future. species, which have a limited diet and can thrive only within a Some studies have reported negligible or no significant effects narrow range of environmental conditions (84). on species composition, community structure, or diversity. Several Shifts in secondary metabolites under elevated O3 may lead to factors, which can affect the estimated impacts of O3, such as OTC changes in relative insect performances, including feeding and ovi- environments, can affect the species composition of pastures (55). positional preferences, longevity, and the ability to reproduce, Within-family competition can also be a major driver of the general potentially influencing the population density and community Downloaded from http://advances.sciencemag.org/ on August 13, 2020 response of a community (42), and competition between species composition of the insects (73, 81, 85). Recent studies of a community can be affected by both O3 and the mixing ratio of the individuals of trees grown in a FACE system for 5 years, however, found that within a community (62). The compositions of species with low populations of different species of insects, with different host plants, abundances may also not greatly change, even if the species are decreased significantly in elevated O3 (74, 86). These studies suggest strongly affected by O3 (52, 63). The outcome of inter- or intraspecific that these decreases were not due to foliar palatability, also confirmed competition also depends on other environmental factors such as by laboratory assays (74, 86, 87), or to direct negative effects of O3 soil-water content (62), soil nitrogen (N) content (42, 52), and spatial on insects (88), leading to the suggestion that VOCs played an heterogeneity (64). A mixture of contaminants may also occur in important role in plant-herbivore interactions under O3 (89). These the environment, and multiple co-occurring environmental con- observations agree with those from other studies of different plant-­ taminants may have larger effects than the effects of single contam- insect systems (85). inants (53). A new generation of studies accounting for various Impacts of O3-induced changes in VOC emissions on co-occurring factors is therefore needed to provide a basis for as- insect dynamics sessing real-world risks. Biogenic VOCs (BVOCs), i.e., VOCs emitted by plants, play important roles in a range of conspecific and heterospecific interactions and Plant-insect interactions: Is insect community composition plant survival (90–92). They are emitted by most plant components, and diversity at risk from O3? both above- and belowground (roots), and provide pivotal ecological Foliar quality drives plant-insect interactions cues detectable over a range of distances (93–96). Typical BVOCs Ozone can affect both the foliar content of N, a major nutrient driving include isoprene, terpenes, green leaf volatiles, carbonyls, organic insect dynamics (65), and secondary metabolites. For example, acids, halides, sulfurous compounds, and benzenoids (97). Ozone several studies show that elevated O3 enhanced the concentration of can induce, reduce, or have no effect on BVOC emissions, depend- lignin, a key secondary metabolite determining the palatability of ing on the species and conditions of exposure (98–100). Isoprene biomass to insects (66). Secondary metabolites play important roles emissions are typically inhibited under elevated O3, but monoterpene in the defense of plants against herbivores by deterring feeding and emissions of O3-tolerant and evergreen species are stimulated (101). reducing digestibility by being toxic at high concentrations (2, 67–71) Interactions involving O3, BVOCs, and insects are highly complex. but may also attract herbivores at lower concentrations (70, 72). Ozone can react with a multitude of VOCs in the atmosphere, Elevated O3 doses that exceed the toxicological threshold inhibit breaking them down into mostly unknown reaction products, which photosynthesis and thus the biosynthesis of secondary metabolites, may impair communication between plants and insects mediated whereas low O3 doses that are below the toxicological threshold by volatiles and may compromise pollination (82, 93, 102–105). For stimulate defensive signaling pathways and induce the biosynthesis example, a laboratory study assessing the effects of O3 on the orien- of secondary metabolites (21, 73). Insect herbivores are expected to tation of a beetle (Acalymma vittatum) to flowers reported that O3 damage plants more if O3 decreases the rate of C assimilation so that levels <80 parts per billion (ppb) did not affect orientation, where- less C is available for C-based defensive chemicals (74). Numerous as levels >80 ppb disrupted orientation toward floral volatiles studies have demonstrated that O3 affects phenolics and terpenes, (106). Similar results were found for the attraction of other beetles especially in angiosperms (75), although different groups of phenolics (Agelastica coerulea) to leaves (89). Both elevated O3 (107) and diesel and terpenes may respond differently to O3 (19, 76, 77). Despite the exhaust (108), which comprises some precursors of O3, have recently recent advancements in the understanding of O3 effects on foliar been reported to rapidly degrade floral volatiles. The distance over quality, O3 effects on latex and other constituents of saps exuded which floral scents can be detected by pollinators thus decreased, from damaged tissues remain completely unknown, although plant negatively affecting the orientation of the pollinators toward floral latex plays an important role in defense against herbivores (78). food sources (107). Plant defense modulates insect growth and development directly via Behavioral tests run at elevated O3 concentrations indicated that toxic secondary metabolites or indirectly by recruiting the natural elevated O3 could also considerably alter the composition of volatile Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 6 of 17 SCIENCE ADVANCES | RESEARCH ARTICLE blends induced by herbivory and consequently alter tritrophic inter- to acquire water and nutrients, defend against pathogenic microbes, actions by influencing the behavior of the natural enemies of the tolerate abiotic stress (including drought and O3 and other air herbivores (109–111). The results from these studies, however, were pollutants), adapt to environmental changes, establish mycorrhizae, inconsistent. For example, O3 affected plant volatile bouquets in- and regulate plant growth (117–121). Microbes can regulate plant duced by herbivory, but the changes did not affect the orientation of growth directly by releasing phytohormones or organic growth parasitoids, indicating a minimal role of oxidation products in signal promoters, contributing to ecosystem processes, such as N fixation, perception for this system (109). A combination of elevated O3 and ammonia oxidation, and phosphate solubilization, or indirectly by P. brassicae larval feeding on B. nigra, however, induced substantially producing biocontrol compounds, such as antibiotics, siderophores, larger volatile emissions than either stress alone, which decreased and enzymes, or signaling compounds (121–123). Microbes, in the attractiveness of the plants to the parasitoid Cotesia glomerata return, benefit from a stable niche, and the supply of primary and relative to plants exposed to herbivores alone (111). Exposure to secondary metabolites and C from root exudates (123, 124). Micro- elevated O3 may also typically reduce insect oviposition on host organisms are evolutionarily older than land plants, and our current plants (86, 112), which may be influenced by multiple mechanisms understanding is that plants are coevolving species assemblages— such as the degradation by O3 of specific VOCs that stimulate ovi- holobionts (Fig. 2), consisting of plants and their microbiota (125). position (105, 113). The long coevolution of plants and their associated microbiota has Understanding the knock-on effects on insect diversity and the likely led to phylogenetically and functionally divergent microbiomes impact of O3-induced VOC emissions on insect diversity is needed, in different environments and climatic zones. Our current knowl- despite the growing number of studies addressing the effects of O3 edge of the impacts of O3 on plant microbiota and biodiversity is Downloaded from http://advances.sciencemag.org/ on August 13, 2020 on the composition of BVOC emissions and specific ecological mainly restricted to rhizospheric processes. interactions. The impact of O3-induced VOC emissions on insect Plant-soil feedbacks diversity should also be studied further. An extensive body of literature addresses the interactions between Empirical evidence for O3 risks of insect community plants and biotic and abiotic soil properties, known as PSFs (4). The composition and diversity effect of increasing atmospheric concentrations of O3 on these in- Numerous studies have addressed how O3 alters insect performance teractions, however, has rarely been studied (10, 13). A few studies by modifying secondary metabolism and VOC emissions, but the have investigated the effects of manipulated O3 levels on community impacts of O3 on insect diversity remain relatively understudied. structure and composition and the function of soil microbial com- The fecundity of individual insects does not predict the responses of munities, but the results have been inconclusive (126–131), perhaps populations or communities to O3 (114), but three studies of the due to varying durations of the experiments or other experimental effects of O3 on insect diversity suggest that O3 can alter species factors such as the facilities used for exposing plants to O3, ecosystem abundance and community structure, thereby threatening diversity type, and type of management (132). Many of these new studies, (50, 115, 116). For example, an examination of >47,000 insects from however, suggest potentially important impacts of elevated O3 on four orders and 83 families sampled in a FACE system at regular PSFs, including soil microbial diversity and decoupled PSF interac- intervals over four growing seasons found that O3 tended to in- tions (Fig. 2). crease the abundance of phloem-feeding herbivores and decreased Soil ecosystem functioning the abundance of chewing herbivores and parasitoids in aspen Elevated O3 can modify resource allocation between above- and (115). This study also found that elevated O3 reduced the total belowground parts of the plant, an important response in all plants abundance of insects (17%) compared to ambient O3, with prominent under O3 stress (13, 133). A meta-analysis showed that O3 generally effects on parasitoids such as Braconidae (−33%), Chalcidoidea inhibits the allocation to roots, relatively to shoots, albeit a variable (−26%), Figitidae (−59%), and Ichneumonidae (−41%) (115). phenomenon (133). A more recent analysis of 239 data entries of Arthropod communities also changed in two experiments when seeds dry root mass of woody plants revealed that 40% of the entries re- from a community of natural plants previously exposed to filtered flected a statistically significant decrease and only 3% of the entries air, 90-ppb O3 or 120-ppb O3, with episodic patterns of varying daily reflected a statistically significant increase (13). Likewise, an analysis peak concentrations over four growing seasons, were used to re- of 104 data entries of root/shoot biomass ratio showed that 27% of establish the plant community in a new environment with low-level the entries reflected a statistically significant decrease and only 5% O3 pollution (50). The carnivore/herbivore ratio particularly increased of the entries reflected a statistically significant increase (13). Hence, (increased abundance of carnivores) with increasing historical levels it appears that elevated O3 generally reduces the allocation of re- of O3 in the 2 years following historical exposures (50). The effects sources to roots more than to shoots, suggesting potential feedbacks on insect abundance and species richness are specific to plant species to the soil ecosystem. and vary temporally (116). More studies are needed, but these Rhizospheric microbial communities are highly diverse and have results suggest that O3 may influence biotic communities and pose a fundamental role in nutrient acquisition, water economy, growth, a threat to biodiversity even years after exposure. and disease tolerance (118). Elevated O3 reduces the allocation of C derived from the soil, which reduces the amount of resources for Interactions between plant and soil microbiota: heterotrophic microbes and thereby affects belowground processes Are microbial community composition and diversity driven by microbes (132, 134, 135). O3 can thus modify decomposi- at risk from O3? tion rates, activities of soil enzymes, root turnover, rhizodeposition Plant-microbe coevolutionary relationship (all material lost from plant roots and deposited into the soil) and Diverse communities of bacteria and fungi live near and on the sur- belowground cycling of C and N, although the magnitude and faces of plant roots (rhizosphere) and leaves (phyllosphere) and in direction of the influence are plant specific and depend on various internal plant tissues (endosphere). Beneficial microbes help plants environmental conditions other than O3 (136–139). The role of Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 7 of 17 SCIENCE ADVANCES | RESEARCH ARTICLE rhizodeposition is particularly important in northern biomes, where soybean fields (159, 160) and rice and wheat fields (161–163), productivity is often strongly limited by the uptake and cycling of N suggesting that reduced denitrification due to decreases in plant-­ (140). Exudation of other molecules such as chelators or organic acids derived C inputs induced by O3 may play a dominant role. The is also crucial for the acquisition of phosphorus or microelements, direction of the effects of O3 on N2O emissions is also driven by the but they lack experimentation regarding their role in O3 effects. system of cultivation (163). Recent advances suggest that O3 can Many studies have reported that O3 accelerates foliar senescence, reduce the expression of microbial genes involved in N fixation, thereby changing the timing of litter deposition, and reduces the denitrification, and N mineralization associated with legumes (129), amount of leaf litter due to diminished foliar area (13, 141, 142). O3 including decreased abundances of nirK, nirS, and nosZ (which are can also affect litter quality (12, 13, 143). A series of experiments on widely used to describe denitrifier communities) and amoA (an in- the effects of O3 on nutrient translocation from senescing to younger dicator of nitrifier communities) (160). Reduced availability of soil leaves identified species-, soil-, and study-specific changes in the levels N and/or labile C for nitrifiers or denitrifiers may account for these of micro- and macronutrients in the foliar litter, which appeared observations in the abundances of genes associated with N cycling. early during stress, altering ecological stoichiometry (144, 145). More The effects of O3 on the cycling of soil N, however, clearly vary studies are needed to draw general conclusions about these highly among plant genotypes or cultivars depending on their susceptibility complex phenomena. These changes in the amount and quality of to O3 (139, 149, 163). These findings demonstrate the decoupling of litter could affect soil macrofauna that feeds on litter, including PSF interactions by O3. detritivores [e.g., (143)]. Several studies suggest that O3 usually decreases soil microbial Ozone reduces rhizodeposition because ecosystems exposed to biomass (127, 150, 152, 164–166). The negative response of micro- Downloaded from http://advances.sciencemag.org/ on August 13, 2020 elevated O3 support a lower net primary productivity, similar to bial biomass to O3 is most likely due to a reduction in root biomass leaves. The rate of turnover of fine roots may also be higher, which and substrate availability (13). O3 can reduce both fungal biomass may also affect decomposition (13). Many experiments have found and the ratio of fungi to bacteria, suggesting that fungi may be more that O3 affects the chemical composition of fine roots, such as susceptible than bacteria to O3 (149, 165, 166). Elevated O3 can sig- decreasing or increasing the contents of monosaccharides, total sol- nificantly decrease microbial biomass in microaggregates (inhabited uble carbohydrates, and total sugars, depending on the severity of predominately by bacteria) relative to macroaggregates (132), sug- stress (13). The levels of fatty acids, starches, and nutrients were also gesting that bacteria in microaggregates may be more susceptible to affected but not consistently in direction or magnitude, which are O3. Ozone, however, increased microbial biomass in a community expected to vary with time and stress level (13)). Ozone influences dominated by sedges (167), perhaps due to an increase in substrate the chemical composition of both roots and soluble root exudates availability, because O3 slightly increased the total number of sedge (146, 147). All these alterations can lead to species-specific changes leaves toward the end of the experiment, and/or to a faster turnover in the rates of decomposition (138, 148). of fine roots caused by O 3 (168). A decrease in soil microbial Recent advances show that elevated O3 alters the expression of biomass would also contribute to a suppressed holobiont and dis- microbial genes involved in C cycling, which, in turn, likely affects turbed PSFs (Fig. 2). C cycling regulated by microbes (149). For instance, in two Chinese Empirical evidence for O3 threats to microbial community wheat cultivars exposed to elevated O3 in a FACE system, the abun- composition and diversity dance of C cycling genes was generally decreased in both cultivars Elevated O3 can alter the composition and structure of soil microbial (including fhs genes involved in the reductive acetogenesis pathway), communities (127, 129, 130, 165, 166, 169–171). For example, 11 years although the abundance of a few genes increased in both cultivars of exposure to elevated O3 doubled the ratio of Basidiomycota to (mcrA and mannanase and xylanase genes) or one of the cultivars Ascomycota in soil microbial communities (128). The relative abun- (amyX, nplT, and lip genes in one cultivar; pcc, aceA/B, bacterial dances of bacteria at the order level in the phylum Actinobacteria ara, and carbon monoxide dehydrogenase and phenol oxidase (which may promote the degradation of recalcitrant substances) genes in the other cultivar) (149). Ozone can also reduce the ability increased under O3 (130). O3 also reduced the relative abundance of of microbes to use C sources, especially in the rhizosphere, as indi- bacterial groups belonging to the family Rhodospirillaceae and the cated by the reduced signal intensity of some C degradation genes order Clostridiales in rice systems (169). Further studies have recently (e.g., related to hemicellulose, aromatics, and chitin) detected in a reported the effects of O3 on bacteria that facilitate the oxidation of soil depth of 0 to 5 cm (129). The excretion of some extracellular ammonia, the rate-limiting step of nitrification. Elevated O3 decreased enzymes involved in C metabolism (e.g., -glucosidase) can be similarly the relative abundances of some nitrifiers (e.g., Proteobacteria and suppressed by O3 (150, 151), reducing the availability of C sources, Nitrospira) but increased those of some denitrifiers (e.g., Acremonium which can also decrease the methanogenic activity of microbes in and Bacillus) in soils growing Machilus ichangensis and Taxus chinensis paddy fields (152). Ozone generally decreases C cycling by decreasing (127). O3 likewise increased the relative abundances of some nitrify- C-based exudation caused by a reduced translocation of photo- ing bacteria (e.g., Nitrososphaeraceae, Nitrospiraceae, Nocardioidaceae, synthates to the roots (13). and 0319-6A21) and N-fixing bacteria (e.g., Sphingomonadaceae, Ozone can also impair N cycling in soil driven by microbial Rhizobiaceae, Termomonosporaceae, Micromonosporaceae, Strepto- activity (139, 150, 153–157). For example, N fixation by legumes can mycetaceae, and Bradyrhizobiaceae) in the soil microbial community decrease in response to increasing O3 concentrations (158). Several of a maize field (131). The abundance of microbes is an important studies show that N-fixing plants (e.g., legumes) grown within mul- indicator of N mineralization (172), and these results suggest tispecies plant communities can be more sensitive to O3 than their that the effects of O3 on microbial structural diversity can affect co-occurring species (table S1), although it remains elusive how O3 N mineralization. affects their N-fixing capacity in these multispecies communities. The structure of microbial communities may be affected by the Ozone can also negatively affect N2O emissions from meadows and soil environment and plant functions. Decreases in root exudation Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 8 of 17 SCIENCE ADVANCES | RESEARCH ARTICLE caused by elevated O3 represent a plausible mechanism by which plants Atmosphérique à Grande Echelle) among the 16 ACCMIP models could modulate their interaction with microbes. Root exudates have to assess the worldwide threats of O3 to plant endemic richness. an important role in plant-microbe interactions and help to deter- MOCAGE is a global 3D chemistry transport model with a high mine the composition of the rhizospheric microbiome (173–175). grid resolution (0.5° by 0.5°), which provides numerical simulations Plants generally exude up to 20% of the fixed C and 15% of the N, of the interactions between dynamical, physical, and chemical pro- which includes an array of simple molecules such as sugars, organic cesses in the troposphere and lower stratosphere (190). It uses a acids, and secondary metabolites and complex polymers such as semi-Lagrangian advection scheme to transport the chemical spe- mucilage (174, 176). The amount and composition of root exudates cies (190). MOCAGE reproduces well the spatial pattern of O3 vary among plant genotypes. Root exudation is modulated by various exposure at the global scale, e.g., in high-elevation areas or areas abiotic stresses (177, 178). For example, microbial composition dif- downwind of O3 precursor sources (9). The MOCAGE historical fered between maize genotypes, a phenomenon linked to differences runs cover the period 2000–2003, while the time slice of RCPs is in the amount of root exudation induced by O3 (131). Few studies, centered around 2100. MOCAGE can simulate 110 gas species however, have focused on the effect of O3 on the relationships between including VOC species as well as soil and lightning NOx, with a root exudates and soil bacterial communities, so our understanding horizontal resolution of 2° by 2°, and includes 47 vertical pressure of the response of soil microorganisms to O3 is incomplete. levels from the surface up to 5 hPa. MOCAGE simulates biogenic The alpha diversity of soil fungi (especially ectomycorrhizae), emissions of hydrocarbons from vegetation (isoprene, monoterpenes, bacteria, and archaea is often reduced by O3 as an indirect con- and other VOCs) and also parameterizes dry deposition of hundreds sequence of changes in plant (and rhizospheric) structure and func- of compounds including O3 (190). A shortfall of the MOCAGE Downloaded from http://advances.sciencemag.org/ on August 13, 2020 tion caused by elevated O3 (130, 179–182). Ozone in other cases, model is that it overestimates O3, especially near sea surfaces, essen- however, has increased microbial alpha diversity (130, 183, 184), tially in NH (190, 191). This phenomenon was due to limitations of highlighting the degrees to which the literature remains inconclusive the thermodynamic equilibrium hypothesis in a marine atmosphere and additional studies are needed for a comprehensive mechanistic and an overestimation of NOx emissions in the lowest part of the understanding. troposphere, especially in the winter months (190, 191). It may re- sult from a positive bias in OH and CO emissions at the south of the Ozone risks by 2100 and global biodiversity: Equator, which are mainly from a biomass burning origin, and are Model predictions too strong (191). Rationale The O3 exposure–based index AOT40 [parts per million (ppm)·hour] The previous sections documented that the effects of O3 on insects is a metric used to assess the potential O3 risk to vegetation from and microbiota were mediated by plants and that the effects of O3 local to global scales and adopted by European regulatory agencies on plants could affect the composition and diversity of plant, insect, (192–194). AOT40 is less biologically relevant than flux-based metrics and microbial communities (Figs. 1 and 2). The impacts of O3 on because, among other reasons, it does not incorporate species-specific plant diversity is not clear, but the evidence of indirect impacts on O3 influx (11). However, O3 flux information can be obtained for only the diversity of plant microbiota is much clearer, suggesting that few ecotypes of (semi)natural plants, thus not permitting reliable O3 microbial diversity may be at a higher O3 risk than plant diversity. flux estimates across large regions of the globe. Global flux data for Plant biomass, a trait critical to fitness that also indicates permanent (semi)natural vegetation, which is the focus of this study, are not adverse effects of O3 (17), can be used as an indicator of the threat available. Furthermore, mapping global-scale O3 flux at high spatial of O3 to the health of the plant, insect, and microbial communities. resolution cannot be practically performed because of physical scarcity The productivity of plant communities, such as in predominant global of data, such as hourly meteorological data, hourly soil moisture, forests, is also positively correlated with species richness (185). We dominant tree species per pixel, and stomatal conductance per identified areas with overlapping O3 risk and high terrestrial en- dominant species. Moreover, world-leading agencies base regulatory demic richness for vascular plants under scenarios of representative ecological risk assessment upon estimated environmental concen- concentration pathways (RCPs). trations (exposures) and not upon dose intake by organisms (influx), Methodology including the Deterministic Approach and the Probabilistic Approach Projected changes in O3 vary considerably among models (186) and [e.g., U.S. Environmental Protection Agency, Scientific Advisory Panel scenarios of emission of O3 precursors (9). The latest emission of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), scenarios, the RCPs, were developed for the Fifth Assessment Ecological Committee on FIFRA Risk Assessment Methods; www.epa. Report of the Intergovernmental Panel on Climate Change (187). gov/]. AOT40 is computed as the sum of the hourly exceedances above The RCP scenarios include various assumptions on climate, policies 40 ppb, for daylight hours (radiation, >50 W m−2) over vegetation- or of energy access, and changes in land cover and use (188). Different species-specific growing seasons (195), namely, April to September for RCP scenarios lead to different concentrations and emissions, e.g., temperate climates (e.g., Europe) and all year round for Mediterranean, CH4, O3 (even stratospheric O3 inputs), and emissions of NOx from subtropical, and tropical-type climates (196). On the basis of (193), we lightning, associated with climate change, all of which affect O3 levels. calculated AOT40 for a model grid (8:00 to 20:00, local time) for all The RCPs have been described elsewhere (9, 186, 189). days of the year. A critical level of 5 ppm·hour calculated over the Sixteen global or regional chemical models within the Atmo- growing season has been recommended for the protection of trees spheric Chemistry and Climate Model Intercomparison Project (5% reduction, total biomass) and (semi)natural vegetation dominated (ACCMIP) were validated and used to evaluate projected changes by perennials (10% reduction, above- or belowground biomass and/or in air quality under various scenarios of emission and climate cover of individual species) (195). We selected this level as a stringent (186, 189). On the basis of (9), we selected the global three-dimensional reference point for both annual and perennial (semi)natural vegeta- (3D) chemistry transport model MOCAGE (Modèle de Chimie tion; a level of 3 ppm·hour (195) would produce exceedances of critical Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 9 of 17 SCIENCE ADVANCES | RESEARCH ARTICLE level throughout NH. Details of the O3 data used and the procedure where a high increase was observed (Fig. 3C). The surface O3 levels followed are provided by (9). and AOT40 were higher for RCP8.5 than historical runs and in- Kier et al. (197) analyzed the “endemism richness” of vascular creased the most in northwestern United States, Greenland, the plants (i.e., ferns, gymnosperms, and angiosperms) in large datasets Mediterranean Basin, the Near East, and eastern Asia (Fig. 3D). and maps across 90 biogeographic regions worldwide (excluding AOT40 greatly exceeded 25 ppm·hour worldwide, except in Central Antarctica) using a standard area of 10,000 km2. This index (endemic and South America and Australia (Fig. 3D). richness) combines both endemic and species richness and is con- AOT40 for the three RCPs did not change significantly in SH. sidered superior to species richness or species endemism, and it can MOCAGE indicated that the critical levels for the protection of indicate the specific contribution of an area to global biodiversity perennial (semi)natural vegetation for RCP8.5 based on AOT40 (197). The dataset contains the full spectrum of abiotic conditions (5 ppm·hour) would be exceeded over many areas of NH by as and includes all major biomes. We used the dataset provided by much as 10-fold, despite improvements for RCP2.6 and RCP4.5. Kier et al. (197) to map the endemism richness of <5, 5 to 10, 10 to AOT40 is expected to be high (>5 ppm·hour) by 2100 for 23 to 51% 20, 20 to 50, and >50 species of terrestrial vascular plants per of the land areas with an endemic richness of 20 to 50 or >50 species 10,000 km2. Mean richness for mainland areas was 18.2 species per per 10,000 km2, depending on the RCP (Table 2). Only 4% of the land 10,000 km2, so we created these arbitrary categories and considered an areas with an endemic richness of >200 species per 10,000 km2, how- endemic richness of ≥20 species per 10,000 km2 to be high, i.e., exceed- ever, is projected to be exposed to high AOT40 values (>5 ppm·hour) ing the average endemic richness (see the Supplementary Materials). and only for the most pessimistic scenario, RCP8.5 (Table 2). All NH areas with very high endemic richness (>50 species Downloaded from http://advances.sciencemag.org/ on August 13, 2020 per 10,000 km2) in the 2000s overlapped with high O3 exposures RESULTS AND DISCUSSION (>5 ppm·hour), except Central America, northern South America, AOT40 averaged 31.8 ppm·hour in NH and 3.5 ppm·hour in the and the Philippines. Plant endemic richness was high in regions Southern Hemisphere (SH) in the 2000s (Fig. 3A). In the early 2000s, with a Mediterranean climate, and regions in NH where endemic AOT40 below the critical level of 5 ppm·hour occurred mainly in the richness was high (e.g., California, the Mediterranean Basin, and SH: South America, Pacific islands, the Cape region of South Africa, Ethiopia) had high O3 levels in summer (198, 199). Madagascar, Polynesia-Micronesia, Melanesia, Indonesia, the A comparison of the global distributions of the endemic richness Philippines, Australia, Tasmania, New Caledonia, and New Zealand of vascular plants (>20 plant species per 10,000 km2) where AOT40 (Fig. 3A). Higher O3 burdens (AOT40, >25 ppm·hour) have been was >5 ppm·hour for the three RCPs indicated that both endemic simulated for areas at high elevations, e.g., the Rocky and Appalachian richness and O3 risk were high for NH Atlantic islands in the latitude Mountains and the Tibetan Plateau, and for Greenland, northeastern band 15° to 45°N (e.g., the Canary Islands, Azores, and the Caribbean), Siberia, the Mediterranean Basin, and areas downwind of precursor the Mediterranean Basin, equatorial Africa, Ethiopia, the Indian coast- sources, e.g., southern Asia (Fig. 3A). line, the Himalayan region, southern Asia, and Japan. Changes in AOT40 compared to the early 2000s were −60% (NH) and +69% (SH) for RCP2.6 (most optimistic), −28% (NH) and Unresolved questions +203% (SH) for RCP4.5, and +67% (NH) and +449% (SH) for A gap of knowledge exists in O3 effects on natural and seminatural RCP8.5 (most pessimistic). Surface AOT40 for RCP2.6 would de- communities in tropical and subtropical environments; thus, these crease worldwide, except in equatorial Africa, where AOT40 was studies are encouraged. The long-term impacts of O3 on biodiversity higher (Fig. 3B). Mean surface O3 concentrations decreased most remain completely unknown, especially in terms of global biodiversity. where historical O3 concentrations were high. The O3 foci (AOT40, A new generation of long-term “real-world” experiments designed >25 ppm·hour) for RCP2.6 were in Greenland, India, the Near East, to study the effects of O3 on biodiversity are greatly needed. The and equatorial Africa (Fig. 3B), but the vegetation in Greenland was complex structure, physiognomy, and high biodiversity of southern very limited. AOT40 for RCP4.5 was slightly lower than historical biomes are obstacles to conducting field or laboratory studies of runs, except over Canada and eastern Asia (particularly Siberia) this subject. Table 2. Land areas for three ranges of the endemic richness of vascular plants exposed to high O3 exposures (AOT40, >5 ppm·hour) by 2100 estimated using three RCPs. Land area exposed to AOT40 > 5 ppm·hour (%) Historical RCP2.6 RCP4.5 RCP8.5 Area with 20–50 species per 10,000 km2 (15.1% of total 41.7 23.9 29.9 41.7 land area) Area with >50 species per 10.000 km2 (11.4% of total 37.7 23.2 23.2 51.3 land area) Area with >200 species per 10,000 km2 (1.8% of total 0.0 0.0 0.0 4.4 land area) Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 10 of 17 SCIENCE ADVANCES | RESEARCH ARTICLE Can elevated O3 alter the ecological plasticity of wild and semi- community composition but if and to what extent O3 may contribute wild plant species? Potential selection pressure due to plant-plant to changes in biodiversity in terrestrial ecosystems remain unclear. competition under O3 is likely weak and will be difficult to demon- Climatic scenarios for 2100 suggest that regions with high endemic strate. Previous studies have reported controversial results (16, 200) richness, e.g., NH Atlantic islands in the latitude band 15° to 45°N, due to the high intraspecific variation of many species (5). Little the Mediterranean Basin, equatorial Africa, Ethiopia, the Indian evidence has been found for the impact of temporal changes in O3 coastline, the Himalayan region, southern Asia, and Japan, are most concentrations on diversity. Understanding and improving the threatened by high levels of O3. tolerance to O3 are more advanced for crop plants than wild and Ozone affects the foliar chemical composition and the composi- semi-wild species, but the tolerance of genetically improved plants tion of BVOC emissions, altering plant-insect interactions and thus to O3 has rarely been demonstrated in the field (201). The use of threatening key ecosystem functions (e.g., plant-insect communica- genetic markers and DNA fingerprinting in wild populations has tion). O3 can indirectly affect species abundance and the structure demonstrated that genetic diversity is decreasing in areas with high of insect communities. O3 levels [reviewed in (5)], which may lead to reduced ecological Ozone also alters plant properties and soil processes that define plasticity in changing environments. Population differentiation plant-soil-microbe interactions and PSFs, such as the input of plant studies at high-risk areas under future scenarios may provide litter, plant exudation, root turnover, nutrient cycling, activities of further insights and are thus encouraged. Limited information is soil enzymes, and decomposition, threatening the functioning of the yet available for the impacts of O3 on mosses, ferns, lichens, algae, soil ecosystem. Soil microbial communities can be indirectly affected, and fungi, although current data suggest high tolerance to O3 for including decreasing microbial biomass, altering the composition Downloaded from http://advances.sciencemag.org/ on August 13, 2020 many of these organisms (5). More research is needed to understand and structure of communities and usually reducing alpha diversity. the role of belowground processes and phyllospheric microbiota for The responses of plant-insect interactions and PSFs to O3 are assessing biodiversity. species specific and affected by several factors, such as the spatial Can O3 affect insect diversity? The effects of elevated O3 on variation of O3, temperature, relative humidity, degree of urbaniza- insects could be indirect and would depend on the magnitude of tion, and the quality of control services in urban, rural, and forested change in the quality of the host plant (bottom-up factors) and/or areas. Ozone has, nonetheless, been demonstrated to decouple the impact on natural enemies (top-down factors) (83). Elevated O3 plant-insect interactions and PSFs, which should be considered may affect populations of natural enemies by shifts in the diversity, when predicting the impacts of climate change. abundance, and quality of prey or changes in behavior that may affect Potential threats of elevated O3 to biodiversity and ecosystem finding hosts (85, 115). Information about the temporary and long- services should be considered when adopting the post-2020 global term impacts of elevated O3 on insect diversity remains elusive. biodiversity initiative “Roadmap for EU Biodiversity Strategy to 2030” Can elevated O3 alter the diversity of phyllospheric microbiota? at the UN Biodiversity Conference in Kunming, China (October The differences in the composition of microbiota from different plant 2020), for curtailing biodiversity loss and preserving and restoring tissues represent an adaptation to different plant niches and the its ecosystems. specific function of the tissue, such as C assimilation in leaves and the uptake of water and nutrients by roots. The atmospheric envi- SUPPLEMENTARY MATERIALS ronment has direct contact with the phyllosphere, which forms the Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/6/33/eabc1176/DC1 largest area of bioactive surfaces on Earth, at the interface between the plant and atmosphere, mediating plant responses to a changing environment (202). The phyllosphere is a habitat for a large variety of REFERENCES AND NOTES 1. A. Biere, A. E. Bennett, Three-way interactions between plants, microbes and insects. microbes (phyllobiome), with microbial densities of up to 107 cells cm−2 Funct. Ecol. 27, 567–573 (2013). (203), but the impacts of elevated O3 or other air pollutants on the 2. J. K. Holopainen, J. D. Blande, Where do herbivore-induced plant volatiles go? Front. Plant phyllobiome are poorly known (169). Rhizospheric and phyllo- Sci. 4, 185 (2013). spheric microbiotas are interconnected and overlap with endophytic 3. M. M. Howard, J. 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Ashmore, The effects of ambient air on a semi-natural grassland E.O. and E.A. each drafted a version of the plant section, with inputs from F.H., and generated community. Agric. Ecosyst. Environ. 38, 91–97 (1992). the final section. Z.F., E.P., M.D., C.J.S., V.C., M.V., and H.H. reviewed the plant section. P.S. and 249. K. Rämö, T. Kanerva, S. Nikula, K. Ojanperä, S. Manninen, Influences of elevated ozone A.D.M. conducted the analyses of global O3 exposure and plant diversity and wrote the results and carbon dioxide in growth responses of lowland hay meadow mesocosms. with inputs from E.A. V.A., J.D.B., and E.A. drafted the section on insects, with inputs from Z.L. Environ. Pollut. 144, 101–111 (2006). and X.Y. V.C., E.P., and C.J.S. reviewed the section on insects. Q.W. drafted the section on 250. H. Pleijel, G. Pihl Karlsson, E. Sild, H. Danielsson, L. Skärby, G. Selldén, Exposure microbes, with inputs from E.A. S.D.V., J.P., and J.D.B. reviewed the section on the microbiota. of a grass-clover mixture to ozone in open-top chambers - Effects on yield, quality E.O., Z.F., and E.A. drafted the “Unresolved questions” section. E.A. created Table 1 and table S1 and botanical composition. Agric. Ecosyst. Environ. 59, 55–62 (1996). and drafted Abstract and Conclusions. P.S. created Fig. 3 and Table 2. C.J.S. created Figs. 1 and 251. M. Volk, P. Bungener, F. Contat, M. Montani, J. Fuhrer, Grassland yield declined by 2 with inputs from E.A. E.A. and Z.F. edited all reviewed sections and produced an integrated a quarter in 5 years of free-air ozone fumigation. Glob. Chang. Biol. 12, 74–83 manuscript. All co-authors reviewed the integrated manuscript, substantially contributed (2006). intellectual input, and approved the final version for publication. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All Acknowledgments: We are grateful to H. Kreft, Professor at the University of Bonn, Germany, data needed to evaluate the conclusions in the paper are present in the paper and/or the for providing the dataset of endemic richness and for helpful suggestions. This paper was Supplementary Materials. Additional data related to this paper may be requested from the prepared within the International Union of Forest Research Organizations (IUFRO) Research authors. Group 8.04.00 “Air Pollution and Climate Change” and under the Working Party 8.04.05 “Ground-level ozone.” Funding: Z.F., E.A., X.Y., and Z.L. acknowledge funding from the Submitted 7 April 2020 National Natural Science Foundation of China (nos. 41771034, 31950410547, 41907383, and Accepted 29 June 2020 Downloaded from http://advances.sciencemag.org/ on August 13, 2020 31901159). E.A. and Z.F. acknowledge multiyear support from The Startup Foundation for Published 12 August 2020 Introducing Talent of Nanjing University of Information Science and Technology (NUIST), 10.1126/sciadv.abc1176 Nanjing, China (no. 003080 to E.A. and no. 002992 to Z.F.). E.O. acknowledges funding from the Academy of Finland (BETUMICS project 284931). J.D.B. acknowledges funding from Citation: E. Agathokleous, Z. Feng, E. Oksanen, P. Sicard, Q. Wang, C. J. Saitanis, V. Araminiene, Academy of Finland decision number 309425. V.C. thanks the project ELEMENTAL (CGL J. D. Blande, F. Hayes, V. Calatayud, M. Domingos, S. D. Veresoglou, J. Peñuelas, D. A. Wardle, 2017-83538-C3-3-R, MINECO-FEDER). J.P. acknowledges the European Research Council A. De Marco, Z. Li, H. Harmens, X. Yuan, M. Vitale, E. Paoletti, Ozone affects plant, insect, and soil Synergy grant ERC-SyG-2013-610028 IMBALANCE-P. The sponsors were not involved in the microbial communities: A threat to terrestrial ecosystems and biodiversity. Sci. Adv. 6, eabc1176 study design; the collection, analysis, or interpretation of the data; the preparation of the (2020). Agathokleous et al., Sci. Adv. 2020; 6 : eabc1176 12 August 2020 17 of 17 Ozone affects plant, insect, and soil microbial communities: A threat to terrestrial ecosystems and biodiversity Evgenios Agathokleous, Zhaozhong Feng, Elina Oksanen, Pierre Sicard, Qi Wang, Costas J. Saitanis, Valda Araminiene, James D. Blande, Felicity Hayes, Vicent Calatayud, Marisa Domingos, Stavros D. Veresoglou, Josep Peñuelas, David A. Wardle, Alessandra De Marco, Zhengzhen Li, Harry Harmens, Xiangyang Yuan, Marcello Vitale and Elena Paoletti Sci Adv 6 (33), eabc1176. DOI: 10.1126/sciadv.abc1176 Downloaded from http://advances.sciencemag.org/ on August 13, 2020 ARTICLE TOOLS http://advances.sciencemag.org/content/6/33/eabc1176 SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2020/08/11/6.33.eabc1176.DC1 MATERIALS REFERENCES This article cites 235 articles, 6 of which you can access for free http://advances.sciencemag.org/content/6/33/eabc1176#BIBL PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions Use of this article is subject to the Terms of Service Science Advances (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. The title Science Advances is a registered trademark of AAAS. Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).