Ecology

Combined culture-dependent and culture-independent approach identifies the prevalent and viable bacterial community members of the human lung post-transplantTo characterize the bacterial community composition of the lung microbiota post-transplant, we performed 16S rRNA gene amplicon sequencing of 234 longitudinal BALF samples from 64 lung transplant recipients collected over a 49-month period (Fig. 1a, Supplementary Table 1). A total of 7164 operational taxonomic units (OTUs) were identified, excluding OTUs contributing to reads in 11 negative control samples32 (see “Methods”, Supplementary Fig. 1a, Supplementary Data 1 and 2). In accordance with previous studies on BALF samples from healthy non-transplant individuals4,5,6,26, we found that Bacteroidetes and Firmicutes followed by Proteobacteria and Actinobacteria are the most abundant phyla in the post-transplant lung (Fig. 1b). Prevalence analysis across all BALF samples showed that the community composition is highly variable with only 22 OTUs shared by ≥50% of the samples (Supplementary Fig. 1b, Supplementary Data 3). However, these 22 OTUs constituted 42% of the total number of rarefied reads, indicating that they are predominant members of the post-transplant lung microbiota (Fig. 1c, Supplementary Fig. 1c, Supplementary Table 2, Supplementary Data 3). They belonged to the genera Prevotella 7, Streptococcus, Veillonella, Neisseria, Alloprevotella, Pseudomonas, Gemella, Granulicatella, Campylobacter, Porphyromonas and Rothia, the majority of which are also prevailing community members in the healthy human lung3,5,7,26, suggesting a considerable overlap in the overall composition of the lung microbiota between the healthy and the transplanted lung.Fig. 1: Combining BALF amplicon sequencing and bacterial culturing to deduce the microbial ecology of deep lung microbiota.a Schematic of the sampling of Bronchoalveolar lavage fluid (BALF) from lung transplant recipients over time (months post-transplant). b Relative abundances (%) of most abundant phyla across BALF samples. Box plots show median (middle line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) as well as outliers (single points). c Prevalence (% samples) vs contribution to total reads across samples for most abundant phyla. Dot color shows different genera and size show total rarefied reads. Gray dashed horizontal line shows prevalence ≥50%. d Scatter plot shows correlation between number of observed OTUs and bacterial counts per BALF sample obtained by quantifying 16S rRNA gene copies with qPCR. Linear regression is shown by the blue line with gray shaded area showing 95% confidence interval (n = 234, two-sided, F(1, 232) = 91.04, P = 2.2 × 10−16), Coefficient of correlation; R2 = 0.28. e Bar chart shows lung taxa (genera; OTU IDs) that contributed ≥75% of total bacterial biomass across samples (n = 234). Venn diagram inset shows overlap (yellow) between the most prevalent (≥50% incidence, light blue) and the most abundant (≥75% total count, red) taxa in the transplanted lung. Bar colors also show the same.Full size imageDifferences in bacterial loads between samples can skew community analyses when based on relative abundance profiling alone. Therefore, we used qPCR to determine the total copies of the 16S rRNA gene as an estimate for bacterial counts, and normalized the abundances of each OTU across the 234 samples (absolute abundance). We found that the bacterial counts vastly differed between samples, ranging between 101 and 106 gene copies per ml of BALF (Supplementary Fig. 1d). The number of observed OTUs increased with decreasing counts (Fig. 1d) suggesting that a large fraction of the OTUs were detected in samples of low bacterial biomass and hence represent either transient or extremely low-abundant community members, or sequencing artefacts and contaminations. In turn, 19 of the 7164 OTUs constituted >75% of the total bacterial biomass detected across the 234 BALF samples (Fig. 1e). This included 11 of the 22 most prevalent OTUs (see above) plus eight OTUs that were detected in only a few samples but at very high abundance (Staphylococcus; OTU_2, Corynebacterium 1; OTU_16 and OTU_24, Anaerococcus; OTU_49 and OTU_234, Haemophilus; OTU_78, Streptococcus; OTU_6768, Peptoniphilus; OTU_63, Supplementary Table 2). It is important to differentiate these opportunistic colonizers from other community members with low incidence, as they reached very high bacterial counts in some samples with potential implications for lung health.To demonstrate the viability of prevalent lung microbiota members and to establish a reference catalogue of bacterial isolates from the human lung for experimental studies, we complemented the amplicon sequencing with a bacterial culturing approach (Supplementary Fig. 2). We cultivated 21 random BALF samples from 18 individuals, on 15 different semi-solid media (both general and selective) in combination with 3 oxygen concentrations; aerobic, 5% CO2, and anaerobic (See “Methods” and Supplementary Table 3), representing 26 different conditions. We cultured fresh BALF immediately upon extraction (within 2 h), as we observed loss in bacterial diversity upon cultivating frozen samples. This resulted in a total of 300 bacterial isolates, representing 5 phyla, 7 classes, 13 orders, and 17 families from which we built an open-access biobank called the Lung Microbiota culture Collection (LuMiCol, Supplementary Data 4, https://github.com/sudu87/Microbial-ecology-of-the-transplanted-human-lung).To examine the extent of overlap between bacteria in LuMiCol and the diversity obtained by amplicon sequencing, we included 16S rRNA gene sequences from 215 isolates that passed our quality filter into the community analysis, which allowed for the retrieval of OTU-isolate matching pairs32 (Methods). We found that 213 isolates matched to 47 OTUs (Fig. 2a, c, Supplementary Data 5), including 17 of the most prevalent and abundant bacteria (Fig. 1e, Supplementary Table 2). As expected, bacteria with high abundance in the amplicon sequencing-based community analysis were isolated more frequently, with Firmicutes revealing the highest isolate diversity (Fig. 2a–c, Supplementary Data 4, 5) and being recovered under the most diverse culturing conditions.Fig. 2: A lung microbiota culture collection (LuMiCol) reveals extended diversity and phenotypic characteristics of the lower airway bacterial community.a Phylogenetic tree of the 47 OTU-isolate matching pairs inferred with FastTree. Branch bootstrap support values (size of dark gray circles) ≥80% are displayed. b Growth characteristics of each OTU-isolate matching pair in three different oxygen conditions (Anaerobic – light brown, 5% CO2-yellow, aerobic-light blue, n = 3). Column with pie charts shows growth on semi-solid agar. Heatmap shows median change in Optical Density (OD) at 600 nm growth in three different liquid media (THY, RPMI, RPMI without glucose) over 3 days. c Cumulative counts of each OTU-isolate matching pair across all BALF samples (gray). d Number of isolates in Lumicol (black) per OTU-isolate matching pair. Taxa are labeled as genus; OTU ID, with an indication of whether they are prevalent (gray rectangle) or opportunistic (magenta rectangle) in the lower airway community. The names of the closest hit in databases: eHOMD and SILVA are used as species descriptor.Full size imageIn summary, our results from the combined culture-dependent and culture-independent approach show that the lung microbiota post-transplant is highly variable in terms of both bacterial load and community composition with many transient and low-abundant bacterial taxa. However, a few community members display relatively high prevalence and/or abundance suggesting that they represent important colonizers of the human lung.LuMiCol informs on the diversity and metabolic preferences of culturable human lung bacteriaWe characterized the culturable community members of the lower respiratory tract contained in LuMiCol by testing a wide range of growth conditions and phenotypic properties (see “Methods”). The majority of the cultured isolates could taxonomically be assigned at the species level based on genotyping of the 16S rRNA gene V1-V5 region. However, the limited taxonomic resolution offered by this method does not allow to discriminate between closely related strains, which can include both pathogenic and non-pathogenic bacteria. Hence for Streptococcus, we additionally tested for type of hemolysis (alpha, beta, or gamma) and resistance to optochin, which differentiates the pathogenic pneumococcus and the non-pathogenic viridans groups (Fig. 2a, Supplementary Fig. 2b, c). This demonstrated that the 16 Streptococcus OTU-isolate pairs belong to the viridans group of streptococci (VS)33. Interestingly, these isolates exhibited the highest genotypic and phenotypic diversity throughout our collection and belonged to five OTUs among the 22 most prevalent community members, with Streptococcus mitis (OTU_11) present in 93.6% of all samples.BALF from healthy individuals contains amino acids, citrate, urate, fatty acids, and antioxidants such as glutathione but no detectable glucose34, which is associated with increased bacterial load and infection35,36,37. To get insights into basic bacterial metabolism, we assessed the growth of all 47 isolates matching an OTU under different oxygen concentrations. We used undefined rich media (Todd-Hewitt Yeast extract) and defined low-complexity liquid media (RPMI 1640), including a glucose-free version to mimic the deep lung environment (see “Methods”). Despite the presence of oxygen in the human lung, the majority of the isolates were either obligate or facultative anaerobes (Fig. 2a), including some of the most prevalent members (Prevotella melaninogenica (OTU_3), Streptococcus mitis (OTU_11), Veillonella atypica (OTU_6) and Granulicatella adiacens (OTU_17). A similar trend was also observed in liquid media under anaerobic conditions, with the exception of the genera Prevotella, Veillonella and Granulicatella. Most streptococci from the human lung grew best in complex liquid media containing glucose under anaerobic conditions, including the most prevalent species in our cohort, S. mitis (OTU_11) (Fig. 2b). However, noticeable exceptions were S. vestibularis (OTU_34), S. oralis (OTU_3427 and OTU_1567), and S. gordonii (OTU_10031), which grew equally well in the presence of oxygen and in low-complexity liquid medium (Fig. 2b). Most Actinobacteria grew best on rich medium in the presence of 5% CO2, with an exception of Actinomyces odontolyticus (OTU_39), which required anaerobic conditions. Some Actinobacteria grew equally well in anaerobic conditions as in the presence of 5% CO2, i.e., Corynebacterium durum (OTU_501), Actinobacteria sp. oral taxon (OTU_328 and OTU_228).The two most predominant opportunistic pathogens in our lung cohort, P. aeruginosa (OTU_1) and S. aureus (OTU_2), grew best in rich liquid medium in the presence of oxygen (Fig. 2c), although these also grew to lower degree under anaerobic conditions. These results indicate that changes in the physicochemical conditions in the lung may favor the growth of these two opportunistic pathogens. In summary, our observations from the bacterial culture collection provide first insights into the phenotypic properties of human lung bacteria and will serve as a basis for future experimental work.Identification of four compositionally distinct pneumotypes post-transplant using machine learning based on ecological metricsTo detect and characterize differences in bacterial community composition between BALF samples from transplant patients, we clustered the samples using an unsupervised machine learning algorithm based on pairwise Bray–Curtis dissimilarity32 (beta diversity, See “Methods”, Supplementary Data 6). This segregated the samples into four partitions around medoids (PAMs) at both phylum and OTU level (Fig. 3a, b, Supplementary Fig. 3a, b). We refer to these clusters as “pneumotypes” PAM1, PAM2, PAM3, and PAM4 (Supplementary Table 4). PAM1 formed the largest cluster consisting of the majority of samples (n = 115) followed by PAM3 (n = 76), PAM2 (n = 19), and PAM4 (n = 24) (Supplementary Data 7). Examination of various diversity measures (Species occurrence, OTU diversity, OTU richness, Fig. 3c–e), distribution of the dominant community members (Fig. 3f), and bacterial counts (16S rRNA gene copies, Fig. 3g) revealed distinctive characteristics between the four pneumotypes.Fig. 3: Bacterial communities of the lung post-transplant fall into four ‘pneumotypes’ with distinct community characteristics.a, b Principal component analysis shows Partition around medoids (PAMs) at phylum and OTU level respectively generated by k-medoid-based unsupervised machine learning using Bray–Curtis dissimilarity (occurrence and abundance). Pneumotypes are color coded: Balanced (red, n = 115), Staphylococcus (green, n = 19), Microbiota-depleted (MD, blue, n = 76), and Pseudomonas (orange, n = 24). c–g Violin plots show distributions of pairwise species occurrence (Sorenson’s index, PERMANOVA, two-sided, F(3, 229) = 8.49, P = 9.9 × 10−5), OTU diversity (Kruskal–Wallis test, χ2 = 89.2, df = 3, two-sided, P = 2.2 × 10−16), OTU richness (ANOVA, F(3, 229) = 43.9, two-sided, P = 2.2 × 10−16), proportion of most dominant OTUs (Kruskal–Wallis test, χ2 = 94.45, df = 3, two-sided, P = 2.2 × 10−16), and total bacterial counts (ANOVA, F(3, 229) = 43.9, two-sided, P = 2.2 × 10−16), respectively, across the four pneumotypes. h, i Enrichment analysis of prevalence (green dotted line ≥50%) and absolute abundance across all samples of the 30 most dominant taxa (i.e., OTUs) in PneumotypeBalanced and PneumotypeMD respectively, when each was compared to the other three combined pneumotypes (gray boxes). Differential abundances after enrichment analysis was calculated between each PAM and the other three PAMs combined, using ART-ANOVA. j Heatmap shows relative percentage of taxa (right colored panel) cultured from paired samples of Bronchial aspiration (BA) and Bronchoalveolar lavage fluid (BALF) from each pneumotype (left colored panel). Oropharyngeal flora mainly corresponds to PneumotypeBalanced (i.e., Streptococcus, Prevotella, Veillonella). All box plots including insets show median (middle line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) as well as outliers (single points). Multiple comparison of beta diversity indices was done by pairwise PERMANOVA (adonis) with False Discovery rate (FDR). Post hoc analyses (95% Confidence Interval) were done by using Tukey’s test (ANOVA) or Dunn’s test (Kruskal test) with False Discovery Rate (FDR) or least-squares means (ART-ANOVA) with False Discovery Rate (FDR). * P  More

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The experiment underlying the study provides a diversity gradient of 1–60 plant species, established in assemblages randomly chosen from a pool of species typical of Arrhenatheretum grasslands. Recently sown on fertile arable soil and maintained by weeding, this experiment is a highly artificial system that fails to meet the definition of semi-natural grasslands7. Four years after establishment, a management intensity gradient of one to four annual cuts and three fertilization levels was established in subplots randomly assigned to the 1–60-species plots. Data presented in this study were collected in the following year.Intensive management was thus imposed on plant species typical of Arrhenaterethum meadows, a plant community characterized by two annual cuts8. The potential effect size of increased management intensity is thus underestimated by applying the management to a plant community not adapted to it. More importantly, it is unlikely that the species-richness of high-diversity plots could be maintained under increased management intensity over longer periods. In fact, 22% of these subplots managed at very high intensity had to be excluded for missing or insufficient yield after only two years, indicating that their species did not persist under high defoliation frequency and fertilizer levels, even when competitors were excluded by weeding.While the discussion hardly addresses this crucial trade-off between management intensity and plant diversity, Schaub et al.6 do indicate that repeated resowing is likely to be necessary to maintain high diversity under increased management intensities. In contrast to permanent grasslands, whose species composition is shaped by site conditions and management, species selection in (re-)sown grasslands is a conscious choice. To be advantageous, mixtures have to show larger yields than the most productive monoculture, so-called transgressive overyielding. Transgressive overyielding is one of the reasons why mixtures, especially grass-clover mixtures, are frequently used in sown grasslands. A European-scale experiment demonstrated that four-species mixtures showed transgressive overyielding at a wide range of sites under intensive agricultural management9,10. Although Schaub et al.6 generally quantify the diversity effects in comparison to monocultures, they argue that grasslands with the high-diversity characteristic of semi-natural grasslands have benefits not only over monocultures but over low-diversity grasslands, such as the 1–8 species standard mixtures shown in Fig. 6 of their paper. However, their results fail to demonstrate that their high-diversity plots show any transgressive overyielding even over monocultures, not to speak of low-diversity mixtures. As species assemblages of the experiment are randomly drawn from the species pool, monocultures and low-diversity mixtures cannot be expected to include the most productive species or species combinations and thus cannot be used to assess transgressive overyielding. When transgressive overyielding was quantified for one- to eight-species plots of the same experiment under extensive management in 2003, it decreased with species number. While two-species mixtures showed a mean transgressive overyielding of 5%, eight-species mixtures were only 70% as productive as the corresponding best monoculture, on average11.Accordingly, the experimental design fails to capture the real trade-offs faced by grassland managers, either in permanent or in sown grassland. It cannot answer if high levels of diversity and the associated biodiversity benefits can be maintained under intensive management for a longer period than just a few years. Neither can it show a productivity benefit of high-diversity grassland assemblages compared to species-poor mixtures, or even monocultures, when in practice the sown species are deliberately chosen rather than randomly drawn from a species pool. While the underlying biodiversity experiment has made valuable contributions to our fundamental understanding of plant diversity effects on ecosystem functioning, it thus cannot be used to derive direct management recommendations for managed grassland. More

Study areaThis study was conducted using tree census data collected from 119 forest inventory plots (73 tropical, 46 subtropical) situated across a latitudinal range of 7.1°N (Colombia) to 27.8°S (Argentina), a longitudinal range of 79.5° to −63.8° W, and an elevation range of 500–3511 m asl (Fig. 1). The mean annual temperature (MAT) of plots ranged from 7.3 to 23.8 °C (mean = 16.7 ± 4.1 °C; mean ± SD) and mean annual precipitation (MAP) of the plots ranged from 608 to 4313 mm y−1 (mean = 1405.0 ± 623.9 mm y−1) (External Databases 1). The number of plots sampled in each country was: Argentina = 46, Bolivia = 26, Peru = 16, Ecuador = 21, and Colombia = 10 (Fig. 1). The 119 forest plots ranged in size from 0.32 to 1.28 ha and represent a cumulative sample area of 104.4 ha (horizontal areas corrected for slope) that containe more than 63,000 trees with a diameter at breast height (DBH, 1.3 m) ≥10 cm (External Database 1). Ninety-four of the plots (79.0%) were ≥1 ha in size. Neither secondary forests nor plantations were included. However, only seven of the plots (five in Argentina and two in Bolivia) were located in forests >100 km2 in extent41, which suggests that at least the edges and borders of some plots could have experienced some degree of disturbance or degradation. All plots were censused at least twice between 1991 and 2017 (census intervals ranged between 2 and 9 years).In each plot, we tagged, mapped, measured, and collected vouchers of all trees and palms (DBH ≥ 10 cm). DBH was measured 50 cm above buttresses or aerial roots when present (where the stem was cylindrical). During the second or subsequent set of censuses, DBH growth, recruitment, and mortality were recorded. In cases where the recorded DBH growth of the second census was less than −0.1 cm y−1 or greater than 7.5 cm y−1, the DBH of the second census was augmented/reduced in order to match these minimum/maximum values42. To homogenize and validate species names of palms and trees recorded in each country and plot, we submitted the combined list from all plots to the Taxonomic Name Resolution Service (TNRS; http://tnrs.iplantcollaborative.org/) version 3.0. Any species with an unassigned TNRS accepted name or with a taxonomic status of ‘no opinion’, ‘illegitimate’, or ‘invalid’ was manually reviewed. Families and genera were changed in accordance with the new species names. If a full species name was not provided or could not be found, the genus and/or family name from the original file was retained.Aboveground carbon stocksThe aboveground biomass (AGB) of each tree was estimated using the allometric equation proposed by Chave et al43., defined as: AGB = 0.0673 × (WD × DBH2 × H)0.976 where AGB (kg) is the estimated aboveground biomass, DBH (cm) is the diameter of the tree at breast height, H (m) is the estimated total height, and WD (g cm−3) is the stem wood density. To estimate WD, we assigned the WD values available in the literature44 to each species found in each plot. In cases where we could not assign a WD value at the species level, we used the average value at the genus- or family level. For unidentified individuals, we used the average WD value of all other species in the plot. Tree height (H) was estimated (see below) based on the heights measured on a subset of the individual stems in each plot using digital hypsometers or clinometers. The estimated AGB of each tree was then converted to units of aboveground carbon (AGC) by applying a conversion factor of 1 kg AGB = 0.456 kg C45. The AGC per ha was then determined by converting kg to Mg, summing the values for all trees in a plot, and extrapolating or interpolating to a sample area of 1 ha.Estimates of AGB and AGC are highly dependent on tree height. Unfortunately, tree height was difficult or impossible to measure on all stems due to physical and logistical constraints. Therefore, we estimated the height of each stem based on allometric relationships between DBH and tree height that we developed for each plot based on height and DBH measurements taken on a subset of individuals. Although the AGB/AGC estimates are only for trees with DBH ≥ 10, we used trees with DBH ≥ 5 cm to construct the H:DBH models when possible in order to be as comparable as possible with the existing pantropical H:DBH models46. In total, 44,442 trees had their heights measured in the field and were employed to construct the H:DBH models. The percentage of trees with direct field measurements of H (DBH ≥ 5 cm) in each country was: Argentina = 19%, Bolivia = 98%, Peru = 96%, Ecuador = 97%, and Colombia = 46%. In Argentina, 32 of 46 plots did not have any field measurements of H, while all plots in all other countries had field measurements of H for at least a subset of trees.We tested and compared the expected effects of using H:DBH models constructed using the local (plot), country, or pantropical (regional) level data. To select the best model to estimate H from DBH at the plot and country level, we used the function modelHD available in the BIOMASS package for R47. We chose the best allometric model from four candidate models (two log-log polynomial models, the three-parameter Weibull model, and a two-parameter Michaelis-Menten model (Supplementary Table 7)) by selecting the model with the lowest RSE and bias (Supplementary Table 8). At the regional level, we used a pantropical model46. The use of country and pantropical H:DBH allometries underestimates tree heights in the lowlands and overestimates tree heights in highlands, thereby homogenizing AGB estimates along elevational gradients10,48 (Supplementary Figs. 11, 12, 13). Using plot level allometries eliminates this problem. However, in the 32 plots in Argentina where we had no information about tree height, we used the country-level H:DBH model developed with the data available in the remaining 14 plots to estimate the height of each tree, which could have homogenized the AGC estimates along the Argentinian elevational gradient (Supplementary Figs. 11, 12, 13).Aboveground carbon dynamicsThe AGC dynamics of each plot was estimated from the annualized values of AGC mortality, AGC productivity (AGC change due to recruitment + growth), and AGC net change3. The calculations of the separate AGC dynamic components was performed as follows: (i) AGC mortality (Mg ha−1 y−1) = the sum of the AGC of all individuals that died between censuses divided by the time between measurements. (ii) AGC recruitment (Mg C ha−1 y−1) = the sum of the AGC of individuals that recruited into DBH ≥ 10 cm between censuses divided by the time between measurements. However, for each tree recruited (DBH ≥ 10 cm), we subtracted the corresponding AGC associated with a tree of 9.99 cm (i.e. just below the detection limit) in order to avoid overestimations of the overall increase in AGC due to recruitment49. (iii) AGC growth (Mg ha−1 y−1) = the sum of the increase in AGC of all individuals with DBH ≥ 10 cm that survived between censuses divided by the time between censuses. (iv) AGC net change (Mg ha−1 y−1) = the difference between AGC stock in the last census (AGCfinal) and AGC stock in the first census (AGC1) divided by the elapsed time (t; in years) between measurements [(AGC net change = AGCfinal − AGC1)/t]. We recognize that these methods exclude C stored in soils or in belowground tissues9,48; however, quantifying just aboveground C stocks and fluxes provides valuable information about the overall status of these forests as net C sinks or sources.ClimateClimate variables at each plot location were extracted from the CHELSA28 bioclimatic rasters at a resolution of 30-arcsec (~1 km2 at the equator). The climate variables extracted were: Mean Annual Temperature (MAT), Mean Diurnal Range (MDR), Isothermality (Isoth), Temperature Seasonality (TS), Maximum Temperature of Warmest Month (MaxTWarmM), Minimum Temperature of Coldest Month (MinTCM), Temperature Annual Range (TAR), Mean Temperature of Wettest Quarter (MeanTWarmQ), Mean Temperature of Driest Quarter (MeanTDQ), Mean Temperature of Warmest Quarter (MeanTWetQ), Mean Temperature of Coldest Quarter (MeanTCQ), Mean Annual Precipitation (MAP), Precipitation of Wettest Month (PWetM), Precipitation of Driest Month (PDM), Precipitation Seasonality (PS), Precipitation of Wettest Quarter (PWetQ), Precipitation of Driest Quarter (PDQ), Precipitation of Warmest Quarter (PWarmQ), Precipitation of Coldest Quarter (PCQ). We separated all variables associated with temperature (°C) from those associated with precipitation (mm y−1) and applied a Principal Component Analysis (PCA) to the 11 variables associated with temperature (PCAtemp) and a separate PCA to the eight variables associated with precipitation (PCAprec). The first two principal components of both PCAtemp and PCAprec (four PCA axes in total) were selected for use in subsequent analyses. Plot elevations were estimated based on their coordinates and the SRTM 1 ArcSec Global V3 (https://lta.cr.usgs.gov) 30 m resolution digital elevation model (DEM).PCAtemp1 (Supplementary Fig. 1a) explained 53.0% of the total variance of the temperature variables and had high loading from Isothermality and Maximum Temperature of Warmest Month, which was primarily associated with changes in elevation (r = −0.97, p  More

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Species threat abatement and restoration (STAR) metricWe developed and analysed a STAR metric that evaluates the potential benefit for threatened species of actions to reduce threats and restore habitat. Like the Red List Index7,8, STAR is derived from existing data in the IUCN Red List and is intended to help address an urgent need. STAR is spatially explicit, enabling identification of specific threat abatement and habitat restoration opportunities in particular places, which, if implemented, could reduce species extinction risk to levels that would exist without ongoing human impact. Abatement of threats to species encompasses reduction in threat intensity and/or action to mitigate the impacts of threats. Positive population and/or distribution changes, along with the resulting reduction of species extinction risk, have been documented in response to threat abatement13. STAR assumes that, for the great majority of species (see Supplementary Discussion), complete alleviation of threats would reduce extinction risk through halting the decline and/or permitting sufficient recovery in population and distribution, such that the species could be downlisted to the IUCN Red List category of Least Concern. We recognize that complete threat reduction is difficult, incremental conservation gains will need to be tracked at the species level14 and species recovery will vary across a species’ range14.For each species, a global STAR threat abatement (START) score is defined. This varies from zero for species of Least Concern to 100 for Near Threatened, 200 for Vulnerable, 300 for Endangered and 400 for Critically Endangered species (using established weighting ratios7,8). The sum of START values across all species represents the global threat abatement effort needed for all species to become Least Concern. START scores can be disaggregated spatially, based on the area of habitat (AOH) currently available for each species in a particular location (as a proxy for population proportion). This shows the potential contribution of conservation actions in that location to reducing the extinction risk for all species globally. The local START score can be further disaggregated by threat, based on the known contribution of each threat to the species’ risk of extinction (see Methods). This quantifies how actions that abate a specific threat at a particular location contribute to the global abatement of extinction risk for all species.The STAR metric also includes a complementary habitat restoration component to reflect the potential benefits to species of restoring lost habitat. During the United Nations Decade on Ecosystem Restoration (2021–2030), restoration efforts are likely to expand. The STAR restoration component applies a similar logic to the STAR threat abatement component, but for habitat that has been lost and is potentially restorable (that is, restorable AOH). The STAR restoration component does not make assumptions about the extent of habitat restoration required for individual species, but instead quantifies the potential contribution that habitat restoration activities could make to reducing species’ extinction risk. For a particular species at a particular location, the STAR restoration (STARR) score reflects the proportion that restorable habitat at the location represents of the global area of remaining habitat for that species. Importantly, a multiplier is applied to STARR scores to reflect the slower and lower success rate in delivering benefits to species from restored habitat compared with conserved existing habitat15. Again, STARR scores can be disaggregated by threat and summed across species within the location.STAR is intended to provide a metric to underpin the establishment of science-based targets as explicit contributions from individual actors towards the post-2020 biodiversity framework, by allowing assessment of actions and locations according to their potential ability to deliver towards international conservation targets. Individual spatially based START and STARR scores, for all species present in a particular location or country, represent a proportion of the global opportunity to reduce species’ extinction risk through threat abatement and restoration, respectively. For each species, the total START score could be achieved by the complete abatement of all threats in remaining habitat, or an equivalent value of the STAR metric can be achieved by a combination of threat abatement in the remaining habitat and restoration of lost habitat (with concomitant threat abatement therein). The metric can support establishment of science-based targets by a range of actors across spatial scales. By enabling governments and non-state actors to quantify their potential contributions, STAR, along with other tools, could facilitate achievement of global policy goals, notably the species component of the Sustainable Development Goals and the expected post-2020 Global Biodiversity Framework.STAR uses existing publicly available datasets: species’ extinction risk categories and threats available from the IUCN Red List6 (or, for country endemics not yet assessed globally, from national red lists); and species’ AOH estimated using species’ ranges, habitat associations, and elevation limits, along with digital elevation models and current and historical land cover maps (here, we used backcast land cover maps of the distribution of habitat pre-human impact, as in ref. 16). To demonstrate the utility of STAR, we calculated global STAR scores for the groups of terrestrial vertebrate species that are comprehensively assessed on the IUCN Red List (that is, threatened and Near Threatened species of amphibians, birds and mammals globally; n = 5,359).Potential to reduce species extinction riskGlobally, the greatest contribution that could be made to reduce the extinction risk of these groups is tackling threats from annual and perennial non-timber crop production, which account for 24.5% of the global START score (Fig. 1). A further 16.4% is contributed by logging and wood harvesting. There are likely to be specific targets for reducing agriculture and forestry threats in the post-2020 framework3, and applying STAR quantifies the large potential contribution that mitigating these threats could make to the goal for species conservation. Appropriate activities to deliver on such targets range along a continuum from land sharing through to land sparing17.Fig. 1: Contribution to the global START score of different threats and the potential contribution of habitat restoration.The total global START score represents the global threat abatement effort needed for all Near Threatened and threatened (Vulnerable, Endangered and Critically Endangered, according to the IUCN Red List) amphibian, bird and mammal species to be reclassified as Least Concern. This score can be disaggregated by threat type, based on the known contribution of each threat to species’ risk of extinction. The STARR score quantifies the potential contribution that habitat restoration activities could make to reducing overall species’ extinction risk. The total START score could thus be achieved by the complete abatement of all threats in existing natural habitat, or through a combination of threat abatement in existing habitat and restoration of lost habitat (with concomitant threat abatement therein).Full size imageSTAR can be used in combination with existing policy and planning tools to quantify the potential contribution of action targets towards species conservation outcomes. The proposed post-2020 framework includes an action target for the protection of sites of particular importance to biodiversity3. Key Biodiversity Areas11, which include Important Bird and Biodiversity Areas18 and Alliance for Zero Extinction sites19, correspond to such sites. Key Biodiversity Areas so far cover 8.8% of the terrestrial surface (www.keybiodiversityareas.org; identification is ongoing), but already capture 47% of the global START score for the vertebrate groups analysed. They represent large proportions of some national START scores: >70% in Mexico and Venezuela and >50% in Madagascar, Ecuador, the Philippines and Tanzania.START scores can also support target setting at national and sub-national scales to help meet international policy goals. The control and eradication of invasive species forms one of the CBD’s proposed post-2020 action targets3. New Zealand has already set a Predator Free 2050 goal that aims to eradicate three invasive mammal species by 2050. New Zealand contributes 0.8% to the global START score for the three vertebrate groups included in this study. Achieving the Predator Free 2050 goal would contribute 30% of the total START score for New Zealand, amounting to 0.2% of the global START score.All countries contribute towards the global START score, but scores are highly skewed, with a few countries having high START scores and most having low scores for the vertebrate groups analysed (Fig. 2a and Extended Data Fig. 1). The highest-scoring countries are located in biodiverse regions with many threatened endemic species20: Indonesia contributes 7.1% of the global START score, Colombia 7.0%, Mexico 6.1%, Madagascar 6.0% and Brazil 5.2%. These top five countries contribute 31.3% of the global START score. In contrast, the lowest-scoring 88 countries together contribute only 1% of the global START score. This does not imply that these low-scoring countries have negligible species conservation responsibilities; the global decline in even common species indicates that all countries must act to reverse the degradation of nature and restore the diversity and abundance of species and integrity of ecosystems21, as well as preventing extinctions at a national scale. Moreover, most countries have a Red List Index22, or an equivalent, quantifying their progress or failure in reducing the global extinction risk of assessed species relative to their national responsibility for global species conservation. STAR provides a means to guide the reduction of extinction risk and so assist all countries in meeting national species conservation targets.Fig. 2: Global distribution of START and STARR scores.a,b, Global STAR scores for amphibians, birds and mammals at a 50-km grid cell resolution for START scores (a) and STARR scores (b). Each species has a global START score, weighted relative to their extinction risk. This global START score can be disaggregated spatially, based on the AOH currently available for each species in a particular location. The total START score per grid cell (a) is thus the sum of the individual species’ START scores per grid cell across all Near Threatened and threatened species of amphibians, birds and mammals included in this study. The global STARR score per species reflects the potential contribution that habitat restoration activities could make to reducing species’ extinction risk, and is spatially disaggregated based on the availability of restorable habitat. Thus, the total STARR score per grid cell (b) is the sum of the individual species’ STARR scores per grid cell across all species included in this study. For the legends in a and b, each range excludes the lower bound and includes the upper bound.Full size imageAt the global level, we estimated that an equivalent to 55.9% of the global START score for vertebrates could, theoretically, be achieved by restoring lost habitat within the current range (Fig. 1). Ecosystem restoration objectives have been identified in many national biodiversity strategies for the CBD, as well as in many countries’ commitments under the Bonn Challenge, and as part of Nationally Determined Contributions under the United Nations Framework Convention on Climate Change. The STAR metric has the potential to support restoration initiatives alongside species conservation targets by quantifying the potential benefit to particular species of restoring habitat in specific places23 (Fig. 2b). Restoration may be particularly important for some species, including those assessed under Red List sub-criteria D/D1 (with a very small population) or Bac (with a small range with severe fragmentation, plus extreme fluctuations). For species uniquely assessed under these criteria (2.8% of those included in this study), threat abatement alone is unlikely to eliminate extinction risk, so this might need to be complemented by restoration in order to achieve Least Concern status (see Supplementary Discussion). Moreover, depending on habitat loss and threat type, restoration of habitat may be beneficial for a larger proportion of threatened species.Application of STAR at the landscape scaleWe tested the landscape-scale application of the STAR metric in the southern part of Bukit Tigapuluh landscape, in central Sumatra, Indonesia (Fig. 3a). The Bukit Tigapuluh Sustainable Landscape and Livelihoods Project is a sustainable commercial rubber initiative. The study area (approximately 88,000 ha) includes a 5-km buffer (which is set aside to support local livelihoods, wildlife conservation areas and forest protection and restoration) and two ecosystem restoration areas (which form a conservation management zone that protects the Bukit Tigapuluh National Park from encroachment).Fig. 3: STAR results for the Bukit Tigapuluh Sustainable Landscape and Livelihoods Project.The Bukit Tigapuluh Sustainable Landscape and Livelihoods Project is a sustainable commercial rubber initiative. The study area (approximately 88,000 ha) includes a 5-km buffer, which is set aside to support local livelihoods, wildlife conservation areas and forest protection and restoration, and two ecosystem restoration areas, which form a conservation management zone that protects the Bukit Tigapuluh National Park from encroachment. a, Mapped START scores in areas with remaining forest (green) and STARR scores in areas where forest has been lost (purple) at the 30-m grid cell resolution. b, START scores per threat for the top five highest-scoring threats across the study area (the concession, 5-km buffer and ecosystem restoration areas combined).Full size imageThe total START score for the study area represents 0.2% of the START score for Sumatra, 0.04% of the START score for Indonesia and 0.003% of the global START. The major threats are from annual and perennial non-timber crops, logging and wood harvesting, and the collection of terrestrial animals (Fig. 3b). The proximate causes of these pressures in the project area are rubber cultivation, oil palm cultivation, industrial logging, subsistence wood cutting and hunting. STAR analysis shows that areas with the greatest potential to contribute to species conservation through threat mitigation are in remaining natural habitat close to the national park, with a small area of high potential also to the west, where the relatively small distribution of the orbiculus leaf-nosed bat (Hipposideros orbiculus) overlaps the site (Fig. 3a). Additionally, due to recent forest loss, 47% of the START score for the study area could be achieved through habitat restoration (that is, STARR). Investment in these management actions has the potential to deliver these quantified contributions to national and global biodiversity targets.Operationalization and future developmentThe STAR metric makes use of the best available data, producing results that are relevant to policy and practice. However, there is scope for future refinement as the underlying data improve. Here, the STAR metric covers amphibians, birds and mammals globally, constituting a well-studied but small proportion of taxonomic diversity (see Extended Data Figs. 2 and 3 for variation among taxa). STAR can be expanded to other taxonomic groups, including freshwater and marine species, as data become available (reptiles, cacti, cycads, conifers, freshwater fish and reef-building corals are among the groups imminently available for incorporation). Global application of STAR will require comprehensive assessment of taxonomic groups, testing of the transferability of the STAR metric assumptions among taxa as Red List coverage expands, and further development of methods to calculate AOH. AOH calculation does not currently capture spatial variation in species’ population density, which will be important for many species14; such data have not been gathered on a global scale yet and could be incorporated as available.The completeness of threat data in the IUCN Red List is uneven but is continually improving. The STAR metric does not currently reflect spatial variation in threat magnitude within species’ ranges; more broadly, there is a lack of information on the spatial distribution of threats24. Most species included in this study have relatively small ranges; the total current AOH is More

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