Ecology

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The Santa Barbara Coastal Long Term Ecological Research program has monitored benthic communities in five kelp forests seasonally since 2008 using fixed transect diver surveys, and moored sensors at each reef have recorded bottom temperatures every 15 min. Blob-associated positive bottom temperature anomalies began in winter 2014 and persisted through autumn 2016 (Fig. 1a)18. Peak temperature anomalies occurred during the summer and autumn of 2014 and 2015 (Fig. 1a), and the average temperature anomaly in autumn 2015 was +3.1 °C, equivalent to an average daily temperature of 19.6 °C. In 2014 and 2015, 91 and 69% of autumn days, respectively, were classified as heatwave days as defined by Hobday et al.20. Seasonal chlorophyll-a concentration, a proxy for phytoplankton abundance, was obtained from satellite imagery at each of the five reefs over the 14-year period. The average chlorophyll-a concentration was anomalously low throughout the warming period, and exceptionally low during the springs of 2014 and 2015 (Fig. 1a), when upwelling-driven nutrient enrichment typically supports dense phytoplankton blooms.Fig. 1: Average seasonal bottom temperature anomaly, chlorophyll-a concentration anomaly, and percent cover and species richness of sessile invertebrates across five sites.The Blob, an anomalous warming period from spring of 2014 to winter of 2016, is highlighted in gray, coincident with (a) positive temperature anomalies (°C; solid line), negative chlorophyll-a anomalies (mg/m3; dashed line), and declines in (b) invertebrate cover (solid line) and species richness (number of unique species/taxa/80 contact points; dashed line). Seasons are denoted by Sp (Spring), Su (Summer), A (Autumn) and W (Winter).Full size imageMean sessile invertebrate cover averaged across all sites declined 71% during the Blob, reaching a 14-year minimum of 7% in autumn of 2015 (Fig. 1b and Supplementary Fig. 1). Species richness declined 69% during the same period (Fig. 1b and Supplementary Fig. 1). The responses of invertebrates to warming were not consistent across time even though the duration and intensity of warming was similar in 2014 and 2015, suggesting that extended periods of elevated seawater temperature were not solely responsible for the most severe loss of invertebrates. For ectotherms, increases in ambient seawater temperature should be met with increases in metabolic rate and food requirements to sustain metabolism21. Because of their sedentary lifestyle, sessile invertebrates cannot actively forage for food or seek spatial refuge from thermal extremes, and limitations in their planktonic food supply can result in metabolic stress over extended periods22,23. Anomalously low chlorophyll-a concentrations during the Blob (Fig. 1a), particularly in the spring of 2015, indicated that food limitation was a likely driver of invertebrate decline. Results from piecewise structural equation modeling (Fig. 2) that incorporated biological interactions with space competitors (understory macroalgae), predators (sea urchins), and foundation species (giant kelp) showed that the severity of warming had both a direct and indirect effect on the sessile invertebrate community. The proportion of heatwave days was a direct negative predictor of sessile invertebrate cover (−0.11) and species richness (−0.21). The proportion of heatwave days was an even stronger negative predictor of chlorophyll-a concentration (−0.26), yielding negative indirect effects on invertebrate cover (−0.07) and species richness (−0.05) due to the positive influence of chlorophyll-a concentration on sessile invertebrate cover (+0.26) and richness (+0.20).Fig. 2: Piecewise structural equation modeling (SEM) for sessile invertebrate cover and species richness.Arrows indicate directionality of effects on (a) invertebrate cover and (b) species richness. Red arrows show negative relationships; black arrows show positive relationships. R2 values are conditional R2. Arrow widths are proportional to effect sizes as measured by standardized regression coefficients (shown next to arrows). ***p  More

Conception of the workflow to demonstrate the microbial associations from co-occurrence networks with microbial cultivationMicrobial co-occurrence networks are composed of nodes and edges, which usually represent microbes and statistically significant associations between microbes, respectively. We hypothesized that the microbial associations could be validated if the topological properties of networks are simplified, and if the microbes representing the nodes can be cultivated. To test this hypothesis, we designed a workflow as shown in Fig. 1. A total of 12,096 wells from 126 96-well plates were inoculated with droplets of series diluted environmental samples, wells from each 96-well plate represented the same combination of given culture condition, sample type (plants, roots, and sediments) and dilution rate (from 10–1 to 10–7). After being cultivated at 30 °C for 10 days, 69 effective (Supplementary Table S3) plates with  > 30% wells showing microbial growth were retained for downstream microbial community analysis. Microbial DNA in each well was extracted, bar-coded, and sequenced for the inference of co-occurrence networks. The wells of plates showing high abundances of target Zotus were targeted for microbial isolations. Lastly, the cultivated microbial isolates were matched to Zotus in the network and used for demonstration of microbial interactions.Figure 1Overview of experimental demonstration of microbial interactions in co-occurrence networks. For detailed description, please refer to the method section.Full size imagePrevalent Zotu pairs in the co-occurrence networksDepending on the microbial density in samples, the 96-well plates harbored different numbers of wells with microbial growth. We obtained 65 96-well plates (6,091 wells) that were effective with microbial growth and data analysis for co-occurrence network reconstruction. After quality control and denoise, we obtained 130 Gbp sequence data. A total of 14,377 Zotus were annotated (Supplementary Table S4). There were 217 ± 94 (average ± standard deviation) prevalent Zotus, i.e., these Zotus appeared at frequencies ≥ 30% of wells in a given 96-well plate.Next, we analyzed Zotus compositions and abundances in each well of the 65 plates. Accordingly, we reconstructed 65 independent microbial co-occurrence networks and further retrieved the robust (Spearman’s |ρ| > 0.6 and P  More

“This new study is inspired by some our earlier theoretical work applied to killer whales that suggests that age-related changes in relatedness are important for the evolution of menopause,” says Samuel Ellis, the first author of the study. “Reproduction can be thought of as a form of generalized harm as the birth of an offspring increases within-group competition for resources. Kinship dynamics — the ways in which local relatedness changes over an individual’s lifetime — are one way that menopause could be favored, because older females are more inclined to cease reproduction to not harm their group mates than younger females. Here we wanted to generalize this concept to both sexes, and to other species without menopause.” More

After massive parallel sequencing of the nucleic acids obtained from fur seal scats, a wide variety of invertebrate and vertebrate viral hosts assignations with low nucleotidic and amino-acidic identities were obtained, most of them corresponding to animal species not described before in Antarctica. These results make us reconsider the use of closed RefSeq databases for viral discovery, especially because the studied area was a remote geographical area where a high number of new viral species is expected to occur22.After repeating the analysis of the contigs obtained using BLASTn, a high number of miss-assignments was observed, corresponding almost entirely to contigs newly assigned as unclassified Eukaryotic Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viral sequences. CRESS viruses have been detected ubiquitously in many different animals without any recognised role in the development of any disease23,24,25,26.These results are in accordance with the recent reporting of CRESS sequences also being ubiquitous in a wide variety of environments and at high proportions, including Antarctica, where they have been described to represent more than 50% of sequences obtained from glacier waters27.Viral-host distributionVirome studies in other Arctocephalus species from subantarctic and South American regions revealed a 5% of viral sequences with predominance of bacteriophages followed by viruses from the Parvoviridae family28. The methodology here applied provided an increase of 12–25% viral reads when probe-based Target Enrichment Sequencing (TES) was applied, that in comparison with Untargeted Viral Metagenomics (UVM) approaches conducted in these type of samples28 could be considered an optimal result.Most of the viral species detected in feces corresponded to unknown viruses, 83.59% from the total of sequences, followed by viruses that infect invertebrates, 8.75%, bacteriophages, 4.46%, and vertebrate viruses, 3.11% (Fig. 1).Figure 1Host distribution of viral assignations sequenced from fecal (A) and serum (B) samples collected from male A. gazella.Full size imageAs expected, when applying both targeted and untargeted sequencing methodologies, TES approach resulted in a recovery of many vertebrate viral assignations (Table 1) whereas untargeted sequencing enabled a better detection of viruses known to infect invertebrates (Table 2). To describe the complete A. gazella fecal virome, sequences obtained by both sequencing methodologies were considered all together, representing a total of 2.62 million reads.Table 1 Vertebrate viral assignations obtained from fecal samples sequencing from male A. gazella. Ranges of Genome coverage, nucleotide identity and aminoacidic identity are expressed in percentages.Full size tableTable 2 Invertebrate viral assignations obtained from fecal samples sequencing from male A. gazella. Colours represent the presence of each assignation in the processed pools. Ranges of Coverage, NT ID and AA ID are represented in percentages.Full size table
A. gazella virusesFur seal picorna-like virusFur seal picorna-like virus was firstly described in a fecal sample obtained from A. gazella in King George Island in the South Shetland Islands, Antarctica by Krumbholz and co-workers16.In this study, we report a total of 19 contigs resulting after assembling 2671 reads obtained from 4/4 fecal pools analysed being the most prevalent virus described in this study. One of the contigs covered 96.91% of the fur seal picorna-like virus genome and presented a nucleotide homology of 99.38% with the reference strain described in 2017. The other contigs coverage ranged from 19.75 to 21.22% with a 45.92 to 90.5% nucleotide identity with reference strain NC_035110. Four contigs matching the ORF2 polyprotein are represented in Fig. 2 where differences among them and with the reference strain are showed.Figure 2Nucleotide alignment of ORF2 sequences from the A. gazella picorna-like contigs compared to the ORF2 from RefSeq NC_0351110. In consensus strain, position 1 represents position 6523 from RefSeqs genome.Full size imagePicornaviruses are known to cause a wide variety of diseases in vertebrate hosts, especially mammals29, but the role of Fur seal picorna-like virus in pathogenesis development is still unknown30. Many picornaviruses are transmitted horizontally via fecal–oral or airborne routes29. The fact that these sequences were detected in all the fecal pools obtained from animals with no evidence of disease may that suggest the virus may have a stable endemic relationship within that seal population.Torque teno pinniped virusLambdatorquevirus is a genus within the Anelloviridae family. The genus comprises 8 species named Torque teno pinniped virus 2 to 9 isolated from different pinniped species: A. gazella (Torque teno pinniped virus 6 and 7)17, Phoca vitulina (Torque teno pinniped virus 2, 3, 4)31, Zalophus californianus (Torque teno pinniped virus 5)32 and Leptonychotes weddellii (Torque teno pinniped 8 and 9)33.One contig with a nuleotide similarity of 95.12% against Torque teno pinniped virus 7 was obtained from one of the fecal pools. This virus had been described in these animals inhabiting Livingston Island in 2016, using rolling circle amplification and subsequent Sanger sequencing from buccal swabs17. However, sequences obtained in this study belong to partial ORF2 which is not the optimal genome region for typing purposes or phylogenetic analysis.These members of the Anelloviridae represent the more abundant viruses found in human, animals and environmental samples although their etiological role in any disease has not been clearly identified being considered a persistent virus ubiquitous to several different tissues34,35No Torque teno virus sequences were detected in serum samples which agree with what was observed for Zalophus californianus anellovirus prevalently detected in different tissues, like lung and liver, but not in blood samples. Interestingly, other known anelloviruses are typically found in blood or plasma samples32.MamastrovirusTwo of the fecal pools analyzed presented Mamastrovirus sequences. The presence of these viruses in humans and other mammals is widely known, as well as their involvement in gastroenteritis development36. The four contigs obtained (comprising 1008 sequences) showed homologies against reference genomes, ranging from 45.70% to 59.37% when compared at nucleotide level and 36.69% to 46.69% when compared at aminoacidic level. Phylogenetic analysis of partial OFR2 regions of these contigs indicate its closer similarity with sequences from California Sea Lion astroviruses, a virus that was determined as to be the most prevalent in fecal samples from these animals (Z. californianus)37. This finding suggests that these sequences may belong to a yet unknown virus like Z. californianus astrovirus and may indicate that such virus is prevalent in the sampled area (detected in 2/4 fecal pools studied) and the second more abundant virus (1008 reads) in the studied fecal samples (Fig. 3).Figure 3Phylogenetic consensus tree based on partial ORF2 sequences from the Mamastrovirus contigs sequenced from A. gazella scats (in bold). Bootstrap resampling with 1000 replicates.Full size imageAdeno associated virus 2Two of the studied fecal pools presented 138 sequences, forming 3 contigs with nucleotide identities ranging from 46.91 to 48.04% (Table 1), that matched adeno associated viruses previously described in Z. californianus, humans and other mammals with and unknow etiologic role (Fig. 4). The detected sequences probably correspond to fur seal adeno associated viruses never described before. The detection of these viruses is quite common in other mammals suggesting they could cause persistent infections in their hosts, but no etiological role has been attributed to them38.Figure 4Phylogenetic consensus tree of the Adeno-associated virus contigs sequenced from A. gazella scats (in bold). Bootstrap resampling with 1000 replicates.Full size imageNorovirusA norovirus contig was obtained in one of the four pools analyzed. Noroviruses are the most relevant non-bacterial gastroenteritis etiological agents in humans39, with its presence widely described in other mammals40. The contig detected in the fecal samples, represented the 4.43% of the viral genome, was in the VP1 region and comprised 56 reads with an identity  > 99% to California sea lion norovirus described by Teng and collaborators in 201841 (Fig. 5). Results obtained suggest these sequences belong to a putative new norovirus specie.Figure 5Phylogenetic consensus tree of the Norovirus contig sequenced from A. gazella scats (in bold). Bootstrap resampling with 1000 replicates.Full size imageViruses in serum samplesAll the viral sequences obtained from serum samples (970 reads) matched to CRESS-DNA viral sequences from unknown hosts.The fact that no other viruses were identified in serum samples suggests the animals tested were not under active viremia at the time of sample collection or it was not detectable by the applied methodology.Diet related virusesSeveral virus sequences similar to viruses known to have invertebrate animals as hosts were detected in fecal pools, mainly by UVM although some also by TES. These viruses are probably present in fur seal feces because of dietary habits although, since scats were collected from the ground nearby the animals, environmental cross-contamination should not be ruled out.Sequences with high coverage or similarities to any described virus are showed in Table 2.The high prevalence of virus sequences from crustaceans in the feces analyzed is hardly surprising because A. gazella inhabiting the Antarctic peninsula and the Atlantic sector of the Southern Ocean feed mostly on Antarctic krill Euphasia superba during the summer months42,43,44,45,46,47,48. Sequences from cephalopod viruses were also detected, although were much scarcer than those from crustaceans. This also agrees with current knowledge about the diet of A. gazella in the Atlantic sector of the Southern Ocean, where octopuses and squids are regularly consumed, although in low numbers44,45,46. It is worth noting than not cephalopod beak was recovered from the scats analyzed here48. Among all invertebrate viruses identified, some sequences present low identities with genomes from available databases, probably because Antarctica wildlife has been scarcely explored, forcing bioinformatic analysis to match them with the most similar viruses from these databases.No fish viruses were found in this study. Hard skeletal remains of fishes are often recovered from the scats of A. gazella from the Atlantic sector of the Southern Ocean42,43,44,45,46,47 and occurred indeed in the samples analysed here48, but stable isotope analysis of blood and whiskers revealed a negligible contribution of fish to the assimilate diet of juvenile and subadult male A. gazella49, which likely explain the absence of fish viruses in the samples analized here. Additionaly, no data on the virome present in the fish species regularly consumed by A. gazella has been published to our knowledge, with information limited to the bacteriome32, so even in case fish viruses were sequenced, it might not be correctly assigned to a fish host. Nevertheless, the methodology applied in this study had been successfully applied to the identification of the virome of Atlantic fishes50. Furthermore, Li and coworkers.37 and Wille and coworkers.22 also observed viral sequences probably corresponding to fish when analyzing the fecal virome of the California sea lions and Antarctic penguins.On the other hand, sequences highly similar to Coelho and Khabarov viral polymerases (greater than 98% of aminoacid identity), previously described in chinstrap penguins (Pygoscelis antarcticus) by Wille and coworkers22, were found in this study. The consumption of penguins by A. gazella during the summer months has been reported widely51,52,53,54,55, penguins feathers were reported from the scats analyzed in this study48 and stable isotope analysis of blood and whiskers revealed penguins as the second most relevant prey from juvenile and subadult male A. gazella in the population studied here49. This evidence is consistent with the presence of virus from chinstrap penguins in the samples analysed here. All in all, the study of fecal virome constitutes a very promising tool to explore the consumers’ diet. More

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OverviewWe compiled systematically sampled empirical taxa occurrence across the landscape, and inferentially assembled respective blue and green local food webs by combining these data with a metaweb approach. We quantified key properties of the inferred food webs, then analysed with GIS-derived environmental information how focal food-web metrics change along elevation and among different land-use types in blue versus green systems. Details are given below.Assemble food webs using a metaweb approachWe applied a metaweb method to obtain the composition and structure of multiple local food webs across a landscape spatial scale10. A metaweb is an accumulation of all interactions (here, trophic relationships) among the focal taxa. In this study, we built our metaweb based on known trophic interactions derived from literature and published datasets, which themselves were all based on primary empirical natural history observations. We further complemented or refined the trophic interactions in the metaweb based on expert knowledge of primary observations that are not yet published or only accessible in grey literature. The expert knowledge covers authors and collaborators who have specific natural history knowledge on Central European plants, herbivorous insects, birds, fish, and aquatic invertebrates. Importantly, these observations were all based on empirical observations and/or unpublished data accumulated over considerable field research experience. The respective literature we referred, as well as the metaweb itself with information source of each trophic link (online repository), are provided in Supplementary Methods. By assuming that any interaction in the metaweb will realise if the interacting taxa co-occur, the metaweb approach allows an inference of local food webs if taxa occurrence is known. Such an assumption of fixed diets may lead to an over-estimation of the locally realised trophic links32, as it essentially ignores the possible intraspecific diet variation caused by resource availability61,62, predation risk63, temperature64, ontogenetic shift65, or other genetic and environmental sources66. Therefore, the food webs we inferred systematically using this method capture trophic relationships driven by community composition (species presence versus absence) but not the above-mentioned processes. Nonetheless, since the trophic interactions were based on empirical observations, the fixed diets can be seen as collapsing all intraspecific variations of diet-determining traits (or trait-matching) at species level, within which we know realisable interactions surely exist. This, together with co-occurrence as a pre-requisite, gives realistic boundaries for the potential interaction realisation, which is plausible and non-biased when applying to localised sites. With this approach, we were addressing a systematic comparison among potential local food webs between the blue and green systems and across the selected gradients. For sensitivity analyses considering the potential inaccuracy of the metaweb approach mentioned here, please see further below Food-web metrics and analyses and Supplementary Discussion.We compiled taxa occurrence of four terrestrial and two aquatic broad taxonomic groups (“focal groups”) to assemble local green and blue communities, respectively and independently, based on the well-resolved data available. Each focal group referred to a distinct taxonomic group, and the within- and among-group trophic relationships captured most of the realised interactions. These focal groups were vascular plants, butterflies, grasshoppers, and birds in the green biome, and stream invertebrates and fishes in the blue biome. Notably, with “butterflies” we refer to their larval stage and accordingly their mostly-herbivorous trophic interactions throughout this study. Larval interactions were also the predominant interaction assessed for stream invertebrates (i.e., all interactions of stream invertebrates focussed on their aquatic stage, which is predominant larval). The occurrence data of these focal groups were compiled from highly standardised multiple-year empirical surveys of various authorities, all conducted by trained biologists with fixed protocols (Supplementary Methods). The information across sites should thus be representative and can be up-scaled to the landscape. The occurrences of plants, butterflies, birds, and stream invertebrates were from the Biodiversity Monitoring Switzerland programme (BDM Coordination Office67) managed by the Swiss Federal Office for the Environment (BAFU/FOEN). The occurrences of grasshoppers and fishes were from the Swiss database on faunistic records, info fauna (CSCF), where we further complemented fish occurrence from the data of Progetto Fiumi Project (Eawag). In terms of biological resolution, taxa were resolved to species level in most cases, while the plant and butterfly groups included some multi-species complexes. Insects of the order Ephemeroptera, Plecoptera, and Trichoptera were resolved to species, while all other stream invertebrates were resolved to family level. These were each treated as a node later in our food-web assembly, and referred to as “species”, as the species within such complexes and families mostly share the same trophic role. Spatially, the occurrence datasets adopted coordinates resolved to 1 × 1 km2. The species that were recorded in the same 1 × 1 km2 grid were considered to co-occurred. We took the co-occurring four/two focal groups to form local green/blue local communities, respectively. To obtain better co-occurrence across group-specific data from different sources (e.g., BDM and info fauna), we intentionally coarsened the grasshopper and fish occurrence to 5 × 5 km2 coordinates. This is arguably a biologically acceptable approximation considering the high mobility of these two groups. Also, we only included known stream-borne fishes and dropped pure lake-borne ones to match our stream-only invertebrate occurrence data. Across all 462 green and 465 blue communities we assembled, we covered 2016 plant, 191 butterfly, 109 grasshopper, 155 bird, 248 stream invertebrate, and 78 stream fish species. Unlike the knowledge of plant occurrence in green communities, we did not have detailed occurrence information of the basal components (e.g., primary producers) in blue ones. Therefore, we assumed three mega nodes—namely plant (including all alive or dead plant materials), plankton (including zooplankton, phytoplankton, and other algae), and detritus—as the basal nodes occurring in all blue communities, without further discrimination of identities or biology within. These adding to our focal groups thus cover major taxonomic groups as well as trophic roles from producers to top consumers in both blue and green systems.Taking the above-assembled local communities then drawing trophic links among species (nodes) according to the metaweb yielded the local food webs (illustrated in Fig. 1), representatively covering the whole Swiss area. Notably, although our understanding of trophic interactions indeed encompassed some links across the blue and green taxa (e.g., between piscivorous birds and fishes), our occurrence datasets did not present sufficient spatial grids where these taxa co-occur. We, therefore, did not include such links, nor assembled blue-green interconnected food webs, but the blue and green food webs separately instead (but see Supplementary Discussion). Also, we dropped isolated nodes, i.e., basal nodes without any co-occurring consumer and consumer nodes without any co-occurring resource, from the inferred food webs. These could possibly be passing-by species that were recorded but had no trophic interaction locally, or those that interact with non-focal taxa whose occurrence information was unknown to us. We thus had to exclude them to focus on evidence-supported occurrences and trophic interactions. Nonetheless, across all cases, isolated nodes were rather rare (averaged less than 3% of species occurred in either blue or green communities).Environmental dataWe acquired environmental data across all of Switzerland (42,000 km2) on a 1 × 1 km2 grid basis (i.e., values are averaged over the grid) from GIS databases, with which we mapped environmental conditions to the grids where we assembled food webs. These included: topographical information from DHM25 (Swisstopo, FOT), land-cover information from CLC (EEA), and climate information (averaged over the decade of 2005–2015) from CHELSA. Among environmental variables, elevation and temperature are essentially highly correlated. In this study, we took elevation as the focal environmental gradient throughout, as after accounting for the main effects of elevation on temperature, the residual temperature was not a good predictor of the food-web metrics we looked at (see next section, and Supplementary Table 4). In other words, by analysing along the elevation gradient, we already captured most of the temperature influences on food webs. Based on the labels provided by the GIS databases, we categorised the originally detailed land cover into the five major land-use types that we used in this study, namely forest, scrubland, open space, farmland, and urban area. Forest includes broad-leaved, coniferous, and mixed forests. Scrub includes bushy and herbaceous vegetation, heathlands, and natural grasslands. Open space encompasses sparsely vegetated areas, such as dunes, bare rocks, glaciers and perpetual snow. Farmland include any form of arable, pastures, and agro-forestry areas. Finally, urban area is where artificial constructions and infrastructure prevail. As each grid could contain multiple land-use types, we then defined the dominant land-use type of the grid as any of the five above that occupied more than 50% of the grid’s area. Analyses separated by land-use types with subsetted food webs (land-use-specific analyses) were based on the grids’ dominant land-use type. There were a few grids where the dominant land-use type did not belong to the focal major five, e.g., wetlands or water bodies, and a few where no single land-use type covered more than 50% of the area. Food webs of these grids were still included in the overall analyses but excluded from any land-use-specific analyses (as revealed in the difference in sample sizes between all versus land-use type subsetted food webs in Fig. 2; analyses details below).Food-web metrics and analysesWe quantified five metrics as the measures of the food webs’ structural and ecological properties. For the fundamental structure of the food webs, the number of nodes (“No. Nodes”) reflects the size of the web, meanwhile represents local species richness (though the few isolated nodes were excluded as above-mentioned). Connectance is the proportion of realised links among all potential ones (thus bounded 0–1), reflecting how connected the web is. We also derived holistic topological measures, namely nestedness and modularity. Nestedness of a food web, on the one hand, describes the tendency that some nodes’ narrower diets being subsets of other’s broader diets. We adopted a recently developed UNODF index68 (bounded 0–1) that is especially suitable for quantifying such a feature in our unipartite food webs. On the other hand, modularity (bounded 0–1 with our index) reflects the tendency of a food web to form modules, where nodes are highly connected within but only loosely connected between. Nestedness and modularity are two commonly investigated structures in ecological networks and have been considered relevant to species feeding ecology24 and the stability of the system69. Finally, we measured the level of consumers’ diet niche overlap of the food webs (Horn’s index70, bounded 0–1), which essentially depends on the arrangement of trophic relationships (thus the structure of the webs), and could have strong ecological implications as niche partitioning has been recognised to be a key mechanism that drives species coexistence71,72. We selected these fundamental and holistic properties as they are potentially more relevant to the processes that may have shaped food webs across a landscape scale (e.g., community assembly), in comparison to some node- or link-centric properties. Also, addressing similar metrics as in the literature13,69 would facilitate potential cross-study comparison or validation.To first gain a glimpse of the structure of the blue and green food webs, we performed a principal component analysis (PCA; Fig. 3a) on the inferred food webs (n = 462 and 465 in green and blue, respectively) taking the four structural metrics (number of nodes, connectance, nestedness, and modularity) as the explaining variables of blue versus green system types. We then confirmed that system type, elevation, and land-use type were all important predictors of food-web metrics (whereas the residual temperature after accounting elevation effects was not) by conducting general linear model analyses, taking the former as interactive predictors while the latter response variables (Supplementary Tables 3, 4). To check how elevation influences food-web properties in blue and green systems separately, and how food-web properties depend on each other, we ran a series of piecewise structural equation modelling (SEM)73 analyses on inferred food webs (Fig. 3b, c) whose dominant land use can be defined (n = 421 and 430 in green and blue, respectively). This was also conducted on subsetted webs of each of the five major land-use types (Supplementary Figs. 1 and 2). The SEM relationships were derived from linear mixed model analyses with dominant land-use type as a random effect (assumption tests see Supplementary Figs. 12–17). The SEM structure of direct effects was set according to the literature13,69 and is illustrated in Fig. 3b. In short, this structure tests the dependencies from elevation (an environmental predictor) to food-web metrics (ecological responses). The further dependencies among food-web metrics themselves were assigned with the principle of pointing from relative lower-level properties to higher-level ones. That is, from number of nodes (purely determined by nodes) to connectance (determined by numbers of nodes and links), further to nestedness and modularity (holistic topologies, determined further by the arrangement of links), then to diet niche overlap (ecological functional outcome).Finally, to check and visualise the exact changing patterns of food webs, we applied generalised additive models (GAMs) to reveal the relationships between food-web metrics and the whole-ranged elevation (Figs. 4 and 5), as well as a particular comparison between food webs in forests and farmlands below 1500 m a.s.l. (Supplementary Fig. 5), as this elevation segment covered most of the sites belonged to these two land-use types. We also performed a series of linear models (LMs) and least-squared slope comparisons based on land-use-specific subsets of food webs (Figs. 4 and 5; Supplementary Figs. 3 and 4), to investigate whether food-web elevational patterns are different among land-use types (assumption tests see Supplementary Tables 5 and 6). In the GAMs analyses, specifically, we simulated two sets of randomised webs, i.e., “keep-group” and “fully”, as the null models to compare with the inferred ones74. Both randomisations generated ten independently simulated webs from each input inferred local food web, keeping the same number of nodes and connectance as of the latter. On the one hand, the keep-group randomisation shuffled trophic links from an input local web but only allowed them to realised fulfilling some pre-set within- and among-group relationships. That is, in green communities, birds can feed on all groups, grasshoppers on any groups but birds, while butterflies only on plants; in blue communities, fishes can feed on all groups, while invertebrates on themselves and the basal resources. These pre-set group-wide relationships captured the majority of realistic trophic interactions compiled in our metaweb. On the other hand, the fully randomised webs shuffled trophic links disregarding the biological identity of nodes. The GAMs of nestedness, modularity, and niche overlap illustrated the patterns of these randomised webs (Fig. 5). Comparing among the three types of webs, the patterns exhibited already by fully randomised webs should be those contributed by variations in web size and connectance, while the difference between keep-group and fully randomised webs by the focal-group composition of local communities, and the difference between inferred and keep-group randomised webs further by the realistic species-specific diets. In addition, we also applied the same GAMs and LMs approach to analyse node richness, as well as both realised and potential diet generality (vulnerability for plants) of each focal group (Supplementary Figs. 6–11). These analyses provided hints about the changes in community composition and species diet breadths along elevation and among land-use types, which helped explain the detected food-web responses in mechanistic ways.In addition, to check if our findings were shaped or strongly influenced by the potential inaccuracy of using the metaweb, we repeated the above PCA, SEM, and GAM analyses as a series of sensitivity analyses. We generated food webs based on our locally inferred ones (i.e., the observations) but with random 10% link removal. This procedure mimics the effect of potential intraspecific diet variation (mentioned earlier) so that some trophic interactions in the metaweb do not realise locally. Overall, these analyses with link removal showed that our conclusions are qualitatively and quantitatively highly robust, and only very minorly affected by the such potential inaccuracy of metawebs, which is also in accordance to other food-web studies (see e.g., Pearse & Altermatt 201575). All details and outcomes of these additional analyses are given in Supplementary discussion.All metric quantification and analyses were performed under R version 4.0.3 (R Core Team76). All applied packages and functions were described in Supplementary Methods, while the R scripts performing these tasks can be accessed at the online repository provided.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. More