The total relative abundance of Bacteroidetes was negatively affected by warming (Figure 2C and Supplementary Figure 3a). The dominance index was significantly higher in C plots, because of the high relative abundance of two genera, Hymenobacter (T3) and Pedobacter (T8) under control conditions. The low relative abundance of those taxa in T plots led to a significantly higher diversity of Bacteroidetes on G. album leaves grown under elevated temperature (Supplementary Figure 3b). Hymenobacter and Pedobacter, contributed mostly to the differences between the G. album leaf microbiota from C and T plots (Supplementary Figure 3c). The boxplots of the ranked distance analysis demonstrated a high variability within the community composition in C and T plots (Supplementary Figure 11).

The total relative abundance of Alphaproteobacteria was also negatively affected by warming (Figure 2C and Supplementary Figure 4a). Significantly more single dominant phylogenetic groups, Sphingomonas (T1), Aureimonas (T7), and Rhizobium (T10), were detected in control compared to warmed plots (all p < 0.05; Supplementary Figures 4b,c). Due to the lower relative abundance of those genera, the diversity increased significantly under warmed conditions (Supplementary Figure 4b). Mesorhizobium (T11) occurred in high abundance on leaves from T plots (especially high in one replicate: T-1G) and contributed also to the differences between G. album leaf microbiota from C and T plots (Supplementary Figure 4c). The Alphaproteobacteria community compositions showed significant differences with respect to the similarities within C and T plot replicates. The Alphaproteobacteria communities were markedly more similar within control plots compared to warmed plots as shown by ranked distance analysis (Supplementary Figure 11).

In contrast to the Alphaproteobacteria, the total relative abundance of Gammaproteobacteria was positively affected by warming (Figure 2C and Supplementary Figure 5a). In three of four warmed plots, Gammaproteobacteria were more equally distributed with less single dominant phylogenetic groups. Differences in the diversity indices were not significant here (Supplementary Figure 5b). Within this subclass the genera Pseudomonas (T2), Erwinia (T4), Buchnera (T5), and Enterobacter (T9) contributed most to the differences between the G. album leaf microbiota from control and warmed plots and each of them were abundant on leaves from T plots (Supplementary Figure 5c). Ranked distance analysis showed more similarity of the Gammaproteobacteria community within C plots and a significant higher variability of the community in T plots (Supplementary Figure 11).

Warming had no significant effect on the total relative abundance of Betaproteobacteria communities (Figure 2C and Supplementary Figure 6a). Uncultured Comamonadaceae (T6) and Massilia (T13) were detected in high abundance in control plots. The dominance index was significantly higher under C compared to T (Supplementary Figure 6b). In consequence their absence led to a significantly more diverse community in the leaf phyllosphere of warmed plots (Supplementary Figure 6b). The phylogenetic groups assigned to SC-I-84 (T26) and Duganella (T37) were abundant in T plots and had together with T6 the highest contribution to the differences between leaf microbiota from C and T plots (Supplementary Figure 6c). The ranked distance analysis showed a high variability of Betaproteobacteria within C and T plots (Supplementary Figure 11).

The total relative abundance of Deltaproteobacteria was not influenced by warming (Figure 2C and Supplementary Figure 7a). In three of the four warmed plots, less single dominant phylogenetic groups, but a higher diverse community of Deltaproteobacteria was detected (Supplementary Figure 7b). The diversity indices were not significantly different. Within this subclass GR-WP33-30 (T29), Haliangium (T75), P3OB-42 (T138), uncultured Sandaracinaceae (T185) and group BIrii41 (T113) contributed mainly to the differences between the G. album leaf microbiota from C and T plots and were all abundant on leaves from T plots (Supplementary Figure 7c). The variability of the community was smaller within T plots compared to C plots (Supplementary Figure 11).

Actinobacteria and Firmicutes were the two main phyla, which occurred in significantly higher relative abundance in warmed plots (Figure 2C). Single dominant phylogenetic groups dominated Actinobacteria and Firmicutes communities of leaves from C plots, but not from T plots, which led to a higher diversity of Actinobacteria and Firmicutes under warmed conditions (Supplementary Figures 8a–j, 9a, and 10a, respectively).

Within the Actinobacteria, the genus Agreia (T45) was dominant under control conditions (Supplementary Figure 9b). Uncultured members of the Gaiellales (T12), Nocardioides (T15), Kineococcus (T17), uncultured group 480-2 (T18), Gaiella (T19), uncultured group MB-A2-108 (T20), Mycobacterium (T23) and Brevibacterium (T30) were abundant on leaves from T plots (Supplementary Figure 9b). All nine phylogenetic groups contributed to the differences between the leaf microbiota from C and T plots. Ranked distance analysis showed, that the community patterns were more similar among C plots and had a high variability among T plots (Supplementary Figure 11).

Four main phylogenetic groups of Firmicutes had the strongest impact on the differences between control and warmed plots (Supplementary Figure 10b). Streptococcus spp. (T41) were abundant under C. Bacillus (T22), Clostridium sensu stricto 7 (T25) and Paenibacillus (T71) were detected with a high relative abundance in T plots. Ranked distance analysis showed a high variability of Firmicutes within control and warmed plots with a higher variability under elevated temperature (Supplementary Figure 11).

Differences in the relative abundance of Sphingomonas (tax ID T1, Figures 5, 6) made the highest contribution (26.4%; Figure 6) to the differences between the phyllosphere microbiota of G. album leaves from C and T plots. Sphingomonas occurred in a high relative abundance on leaves grown in C plots (49.2 ± 10.3%) and in a lower relative abundance on leaves grown in T plots (6.0 ± 6.3%; Supplementary Table 4). Sphingomonas are well-known as phyllosphere inhabiting bacteria of different plants and for their plant protective potential against pathogens (Delmotte et al., 2009; Innerebner et al., 2011; Vorholt, 2012). Ren et al. (2015) found by a 16S rRNA gene based 454 pyrosequencing approach, that Sphingomonadaceae, which includes Sphingomonas as type genus, showed a slight reduction under elevated temperature in the rice phyllosphere. Their lower relative abundance on plants grown under elevated temperature may cause a negative effect on plant protection and in consequence enhance the growth of plant pathogens. In a laboratory study of Innerebner et al. (2011) it was shown, that the population size of the plant pathogens Pseudomonas syringae pv. tomato DC3000 and Xanthomonas campestris pv. campestris LMG 568 was reduced on Arabidopsis leaves after leaf inoculation with a Sphingomonas sp. strain, that was isolated previously from the phyllosphere of grasses.

The lower relative abundance of Sphingomonas in the leaf phyllosphere of G. album grown in T plots may be explained by changes in the substrate availability on G. album plants grown under elevated temperature. Metaproteome studies of the phyllosphere microbiota showed a high abundance of sugar uptake transporters of Sphingomonas which indicated, that plant-released carbohydrates are the main carbon source of phyllosphere inhabiting Sphingomonas spp. (Delmotte et al., 2009). Jansen-Willems et al. (2016) showed, that the plant biomass increased in T plots, which indicated changes of the plant physiology and morphology. A first insight into metabolome profiles of G. album leaves of C and T plots indicated significant differences among leave metabolomes (unpublished data). The impact of changed substrate profiles of the plants in general will be an important aspect for the shift of the phyllosphere microbiota. Bai et al. (2015) showed in a comparative genomic study of phyllosphere and root derived bacterial isolates, that phyllosphere bacteria contain a significantly higher fraction of annotated proteins linked to the carbohydrate and amino acid metabolism, indicating the importance of changes in substrate availability for phyllosphere inhabiting microbiota. If the changed substrate pattern available under elevated temperature did not match the optimal substrate utilization capacity of Sphingomonas, those may have been outcompeted by other phylogenetic groups, which could more efficiently use these substrates and occupy that novel ecological niche characterized by specific substrate profiles.

The second most abundant phylogenetic group, the genus Hymenobacter was significantly less abundant in the phyllosphere of G. album in warmed plots (tax ID T3, Figures 5, 6). It occurred with a high relative abundance in the G. album phyllosphere of C plots (C: 16.9 ± 11.7%) and with only 1.4 ± 1.5% relative abundance in the phyllosphere of G. album grown in T plots (Supplementary Table 4). The genus Hymenobacter contributed with 9.6% to the differences between the G. album leaf microbiota from C and T plots. Several studies showed that members of the genus Hymenobacter are common inhabitants of the phyllosphere (Ottesen et al., 2009; Leveau and Tech, 2011), but little is known about their function. Ding and Melchner (2016) described Hymenobacter as dominant endospheric bacteria in leaf samples from five plant species (Ambrosia psilostachya DC., Asclepias viridis Walt., Panicum virgatum L., Sorghastrum nutans (L.) Nash, and Ruellia humilis Nutt.).

Related to the microbial “hub” concept discussed by Agler et al. (2016), genera which occurred in a high relative abundance in the phyllosphere under control conditions, as Sphingomonas and Hymenobacter, may represent “hub” taxa within the phyllosphere microbiota of G. album plants. Their low relative abundance in the phyllosphere of G. album leaves grown under elevated temperature may be one reason for the high variability or high instability of the T microbiota, which was especially indicated by increased abundance of individual members of the Gammaproteobacteria including potential pathogenic genera, Pseudomonas or Erwinia. Copeland et al. (2015) described members of the genus Sphingomonas as leaf adapted taxa and Pseudomonas and Erwinia as taxa with moderate prevalence and variable abundance on leaves of different crop plants. Based on the microbial “hub” concept, the loss of central hub taxa may disturb the stability of the phyllosphere microbiota, if those strongly interacting taxa are functionally not replaced. Other taxa, which include potentially pathogenic bacteria may be able to grow in ecological niches which were previously occupied by the “hub” taxa without replacing their functionality, i.e., the high abundance of different Gammaproteobacteria in warmed plots. The localization of Hymenobacter in or at the surface of leaves of G. album is so far not known, but may be endospheric as indicated by Ding and Melchner (2016). Thereby Hymenobacter may be a “hub” taxon, which occupies another ecological niche than the mainly surface attached Sphingomonas (Delmotte et al., 2009; Vorholt, 2012).

Amplicon sequences assigned to the genus Aureimonas were also detected in significantly lower abundance on G. album leaves grown under elevated temperature with a relative abundance between 4.4 (±1.7)% in C and 0.08 (±0.03)% in T plots (Figures 5B, 6 and Supplementary Table 4). The contribution of this genus to the differences between the phyllosphere microbiota from C and T plots was 2.6% (Figure 6). Little is known and reported about the genus Aureimonas and its occurrence in the phyllosphere. The genus Aureimonas belongs to the Aurantimonadaceae, which are very closely related to the genus Rhizobium, which contains many plant growth-promoting bacteria. Among the currently described ten species with validated names, four were isolated from the phyllosphere. A. galii and A. pseudogalii (Aydogan et al., 2016) were both isolated from G. album leaves, the type stain of A. galii even from leaves of the current experiment from plants grown under C. Strains of the same species were not cultured from leaves of plants grown under T, which supported the finding of the reduced abundance of this genus in warmed plots (data not shown). A. phyllosphaerae and A. jatrophae (Madhaiyan et al., 2013) were isolated from surface-sterilized leaf tissues of Jatropha curcas L. cultivars, which indicated an endophytic life style.

The second phylogenetic group of Bacteroidetes, which was among the ten phylogenetic groups that showed main contribution to the differences among the bacterial community composition under warmed conditions and occurred with a slightly lower but not significant relative abundance in warmed plots, was the genus Pedobacter (Figures 5B, 6). Pedobacter spp. was detected also in other phyllosphere studies (Nissinen et al., 2012; Vorholt, 2012; Humphrey et al., 2014; Dees et al., 2015). Pedobacter infection was negatively correlated with herbivory and may affect plant–microbe–herbivore interactions in a similar manner as specific Pseudomonas spp. (P. fluorescence) (Humphrey et al., 2014).

Sequences assigned to the genus Rhizobium were also detected in significant lower relative abundance on G. album leaves grown in T (0.2 ± 0.2%) compared to C plots (2.5 ± 1.7%) (Figure 5B and Supplementary Table 4). They contributed with 1.4% to the differences between the leaf microbiota from C and T plots (Figure 6). Members of the genus Rhizobium were often reported as phyllosphere inhabiting bacteria (Wellner et al., 2011; Knief et al., 2012; Horton et al., 2014) with beneficial effects on the plant phyllosphere (Nandi et al., 1982). Rhizobium sp. are well known for their plant growth promotion and induction of systemic resistances, which positively affects plant fitness (Glaeser et al., 2016).

The genus Pseudomonas (tax ID T2, Figures 5, 6) had the second strongest contribution (13.4%) to the differences among the microbial community patterns of leaves after warming, due to an increased relative abundance of Pseudomonas on leaves derived from T plots (28.2 ± 31.9%) compared to C plots (6.6 ± 2.7%, Supplementary Table 4). The high abundance of sequences assigned to the genus were however, only detected on the leaf phyllosphere from one (T-2G: 83.5%) of four T plots with significantly higher abundance compared to C plots. The high abundance of Pseudomonas in one warmed plot sample was the main reason for the high dominance index value obtained for this T sample (Figure 4C), and had also a strong contribution to the high variability among the total bacterial community patterns of T plots (Figure 3B). Differences of the relative abundance of Pseudomonas were therefore not significant between the leaf microbiota from C and T plots. The genus Pseudomonas was often reported as a dominating phyllosphere inhabitant (Lopez-Velasco et al., 2011; Rastogi et al., 2012; Bodenhausen et al., 2013). Within the genus, some species are known as plant or human pathogens (Vorholt, 2012) while other species are well known for their plant protective function (Cronin et al., 1997; Mendes et al., 2011). Mendes et al. (2011) showed, that the presence of plant pathogenic fungi (Rhizoctonia solani) enhanced the abundance of the antagonistically acting Pseudomonadaceae haplotypes, and illustrated herewith the huge functional diversity even among strains of Pseudomonas. An increase in the abundance of distinct Pseudomonas spp. (especially P. syringae) was also positively correlated, whereas P. fluorescens infection was negatively correlated with herbivory (Humphrey et al., 2014). Positive or negative impacts of Pseudomonas in the phyllosphere were however, always strain dependent and could strongly vary among different Pseudomonas spp. isolated from plants (Mendes et al., 2011; Humphrey et al., 2014). The function of Pseudomonas present in the phyllosphere of G. album under control and warmed conditions needs to be further analyzed through an investigation of Pseudomonas spp. isolates obtained from the respective samples in parallel to the cultivation independent study (data not shown).

The phyllosphere microbiota of leaves derived from T plots contained a significantly higher abundance of the genus Enterobacter (Figure 5B). Enterobacter spp. contributed with 1.8% (Figure 6) to the differences between C (0.03 ± 0.02%) and T (3.0 ± 2.2%) derived phyllosphere bacterial communities, which was also shown with the respective biplot (tax ID T9, Enterobacter, Figure 5A). The genus Erwinia (biplot T4; Figure 5A) also had a strong contribution (9.6%) to the differences between leaf microbiota from C and T plots. Erwinia spp. occurred with a high relative abundance on G. album leaves obtained from one T plot (T-3G: 57.6%, Supplementary Table 4) of four, which led again to the strong variability of the bacterial community composition among leaf samples from elevated temperature plots (Figure 3B) and explained also the high dominance index value for the community composition on leaves from this plot (Figure 4C). Several species within the genus Erwinia are known to be plant pathogens (Starr and Chatterjee, 1972). Other low abundant members of the Enterobacteriaceae (Pantoea, Proteus, Escherichia-Shigella, Serratia) occurred in slightly higher abundances on G. album leaves from T plots, and other genera like Buttiauxella, Citrobacter, Cronobacter, Klebsiella, Kluyvera, Pectobacterium, Providencia, Raoultella, Tatumella, and uncultured Enterobacteriaceae were detected only on leaves grown in T plots (Supplementary Table 4) although the results were not statistically significant because of the variability within the bacterial community on leaves obtained from T plots. Enterobacteriaceae in general constituted 2.6% of all detected phylogenetic groups. In summary, the data suggests that an increase of the surface temperature by +2°C had a long-term effect on the microbial community of the G. album leaf phyllosphere with an increase of the relative abundance of Enterobacteriaceae. Rastogi et al. (2012) detected more Enterobacteriaceae and culturable coliforms in summer compared to winter samples on field grown lettuce leaves, and Ren et al. (2015) also detected slightly higher Enterobacteriaceae in the upper rice leaf phyllosphere with elevated temperature.

A significantly higher relative abundance of potential plant and/or human pathogens, as classified by the “Technische Regel für Biologische Arbeitsstoffe” (TRBA 466) for Germany as risk group 2 microbes, like members of Curtobacterium, Clostridium, Peptoclostridium, Aeromonas, Rhodococcus, Acinetobacter, Burkholderia, Bacillus, Staphylococcus, Cellulomonas, Corynebacterium and a slightly but not significantly higher relative abundance of the potential plant pathogen Xanthomonas were detected on leaves of G. album from T plots (Supplementary Tables 4, 5). In former studies it is already reported, that plants represent reservoirs for potential plant or human pathogens like Pseudomonas, Staphylococcus, Enterococcus, Burkholderia, and Enterobacteriaceae (Berg et al., 2014; Schikora and Schikora, 2014). Sydnor and Perl (2011) described genera like Acinetobacter, Enterobacter, Klebsiella (K. pneumonia, K. oxytoca), Proteus, Pseudomonas, and Serratia as antibiotic-resistant microorganisms, which play an important role in hospital-acquired infections. Acinetobacter baumannii is regarded as an emerging pathogen and one of most important multidrug-resistant bacteria in hospitals worldwide (Peleg et al., 2008; Antunes et al., 2014). These findings indicate a potential risk for an increase in potential plant and/or human pathogens on G. album leaves with elevated temperature due to global warming.

Galium album leaf phyllosphere from elevated temperature plots harbored a significantly higher abundance of sequences assigned to Buchnera (C: 0.002 ± 0.002%; T: 8.3 ± 5.0%; Figure 5 and Supplementary Table 4) with a contribution of 5.1% to the differences between the leaf microbiota from C and T plots (Figure 6). The strong impact of the high relative abundance of Buchnera on the microbial community differences among both treatments was also indicated by the respective biplot (Figure 5A; Buchnera, T5). Buchnera spp. are well known endosymbionts of aphids (Moran et al., 2008). The high relative abundance of Buchnera in the warmed plots therefore indicated an enhanced occurrence of aphids on leaves of G. album in T plots, as shown in the Supplementary Figure 12 for a G. album plant from the same field side. Either aphids or Buchnera only were coextracted with plant material. Aphids constitute a globally distributed agricultural pest as sap-sucking herbivores, using large amounts of phloem as a nutritional source (Gray and Fraenkel, 1954). Our findings were in congruence with the results obtained by Barton and Ives (2014). They detected an increased aphid population growth rate on sweet corn grown under elevated temperature (elevated daily temperature of 4.87°C ± 0.14°C). Dong et al. (2013) also described, that experimental warming led to an increase in pest aphid abundance on wheat fields.

The increase of the relative abundance of insects or at least the enhanced relative abundance of insect symbionts was demonstrated for the genus Wolbachia. However, Wolbachia spp. occurred generally only in a low abundance. A significantly (p ≤ 0.01, Student’s t-test) higher relative abundance of Wolbachia was detected in the samples analyzed from T (0.2 ± 0.1%) compared to C (0.001 ± 0.001%) plots (Supplementary Tables 4, 5). Wolbachia spp. are intracellular bacteria found in ∼40% arthropods and filarial nematodes (Ferri et al., 2011; Zug and Hammerstein, 2012). Most of the filarial nematodes can cause dangerous filariasis (river blindness and elephantiasis) in humans, and arthropods can lead to dengue fever, yellow fever, malaria and Chikungunya virus (as reviewed by Slatko et al., 2014). Both of them, arthropods and nematodes, can be found in the phyllosphere (Erb et al., 2008; as reviewed by Lindow and Brandl, 2003; Müller et al., 2016).

As reviewed by Robinet and Roques (2010) there is an impact of global warming on several insects, which respond with earlier flight periods, enhanced winter survival and acceleration of development rates. In contrast, a long-term monitoring study of insects feeding on mountain birch (Betula pubescens ssp. czerepanovii) demonstrated, that climate change influenced insect herbivory in heavily stressed compared to pristine forests differently, and that herbivorous insects showed diverse responses to climate variations (Kozlov et al., 2017). Kozlov et al. (2017) highlighted the problems of predicting effects of global change on forest damaging insects, which requires long-term observations.

The higher relative abundance of Buchnera and Wolbachia on leaves of G. album were an indicator of increased numbers of arthropods and filarial nematodes with elevated temperature, which can represent a vector for potential human diseases (Tobias, 2016).

Highly abundant and plant beneficial bacterial members of the phyllosphere microbiota are members of the genus Methylobacterium (Delmotte et al., 2009; Wellner et al., 2011; Knief et al., 2012; Vorholt, 2012). Methylobacterium spp. normally represent the most abundant methylotrophic bacteria. Sequences assigned to the genus Methylobacterium were only detected in low abundance (<0.1%, Supplementary Table 4) in the phyllosphere of G. album leaves. A non-significantly lower relative abundance of Methylobacterium sequences was observed with elevated temperature. One important reason for the low detection rate of Methylobacterium sequences may be due to the difficulties in the DNA extraction from this genus. Reisberg et al. (2012) mentioned the same problem by analyzing DNA extracts of the Arabidopsis phyllosphere. However, pure culture test experiments showed that DNA from Methylobacterium spp. could be efficiently extracted with the DNA extraction kit applied here for the leaf samples. Whether or not the relative abundance of Methylobacterium sp. in the G. album phyllosphere is common, needs to be investigated further.