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Field-data collectionFieldwork was conducted in DRC between January 2018 and March 2020. Ten transects (4–11 km long) were installed, identical to the approach in ref. 9, in locations that were highly likely to be peatland. These were selected to help test hypotheses about the role of vegetation, surface wetness, nutrient status and topography in peat accumulation (Fig. 1a and Supplementary Table 1). A further eight transects (0.5–3 km long) were installed to assess our peat mapping capabilities (Fig. 1a and Supplementary Table 1).Every 250 m along each transect, land cover was classified as one of six classes: water, savannah, terra firme forest, non-peat-forming seasonally inundated forest, hardwood-dominated peat swamp forest or palm-dominated peat swamp forest. Peat swamp forest was classified as palm dominated when >50% of the canopy, estimated by eye, was palms (commonly Raphia laurentii or Raphia sese). In addition, several ground-truth points were collected at locations in the vicinity of each transect from the clearly identifiable land-cover classes water, savannah and terra firme forest.Peat presence/absence was recorded every 250 m along all transects, and peat thickness (if present) was measured by inserting metal poles into the ground until the poles were prevented from going any further by the underlying mineral layer, identical to the pole method of ref. 9. In addition, a core of the full peat profile was extracted every kilometre along the ten hypothesis-testing transects, if peat was present, with a Russian-type corer (52 mm stainless steel Eijkelkamp model); these 63 cores were sealed in plastic for laboratory analysis.Peat-thickness laboratory measurementsPeat was defined as having an organic matter (OM) content of ≥65% and a thickness of ≥0.3 m (sensu ref. 9). Therefore, down-core OM content of all 63 cores was analysed to measure peat thickness. The organic matter content of each 0.1-m-thick peat sample was estimated via loss on ignition (LOI), whereby samples were heated at 550 °C for 4 h. The mass fraction lost after heating was used as an estimate of total OM content (% of mass). Peat thickness was defined as the deepest 0.1 m with OM ≥ 65%, after which there is a transition to mineral soil. Samples below this depth were excluded from further analysis. Rare mineral intrusions into the peat layer above this depth, where OM 4× the mean Cook’s distance were excluded as influential outliers. Mean pole-method offset was significantly higher along the DRC transects (0.94 m) than along those in ROC (0.48 m; P  More

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Our long-term surveillance of SARS-CoV-2 in Austria demonstrated that WBE alone yields a time-resolved map of the genetic dynamics during a pandemic. Yet one task of pathogenomic surveillance is to link genetic pathogen information with clinical manifestation and the immunological status of patients. WBE is limited in that regard since the available data are anonymized to start with. Nonetheless, WBE provides invaluable population-level guidance on epidemiological developments, which complements case-based surveillance and provides information for optimal resource allocation. This notion can also be transferred to a global perspective. WBE provides a tool to shed light on blind spots of pathogen surveillance in places and communities with poor healthcare accessibility. If carefully set up and used in respectful and coequal terms, WBE of infectious diseases could make an important contribution to global safety.To this end, several challenges must be overcome. Current WBE methods need to be expanded to other pathogens beyond SARS-CoV-2 and validated with case-based epidemiological data. Furthermore, current methods must be adapted and optimized to be applicable in locations without a centralized sewer infrastructure5. Finally, international sharing of wastewater-based pathogen sequencing data will be needed to unleash the full potential of WBE for global pathogen surveillance.We are confident that our study will support initiatives already working in these directions, as well as encouraging intensified efforts to exploit such population-level surveillance approaches in the global fight against infectious diseases.
Fabian Amman
1
& Andreas Bergthaler
2

1
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

2
Medical University Vienna, Vienna, Austria More

Predictors of microbial carbon stocksWe used a machine learning modeling approach to predict soil microbial carbon from a set of environmental covariates. To account for stochastic variability, we ran a set of models to assess the importance of environmental factors, which showed that the contribution of each variable to the model fit differed between runs, with some overlap between a number of them (Fig. 2b). Mean annual temperature was always the most important variable, with soil organic carbon and soil pH following. Clay content, precipitation, land-cover type, nitrogen content, and sand content contributed roughly equally to explaining variations in microbial carbon. Finally, NDVI and elevation had the lowest variable importance. Coniferous forests had the highest and most variable predicted values of microbial carbon (Supplementary Figs. 1, 2), which can be explained by high soil organic matter and a thick litter layer26. Tropical forests also had fairly high values of microbial carbon, while shrublands and croplands had the lowest values26. We used partial prediction response curves to evaluate the direction and range of effect of the predictor variables (Supplementary Figs. 1, 2). In agreement with the variable importance measure, variables that scored high often showed strong effects on the predicted microbial carbon values, while variables with a low variable importance score (e.g., elevation, NDVI, and sand content) only showed smaller responses. The only exception was for precipitation, which had a relatively high variable importance, although the response curves only showed a weak effect of precipitation for forests and grasslands, with limited effect on other land-cover types (Supplementary Fig. 2). The importance of precipitation might also indicate that this relationship involves interactions with other variables7,28. Overall, the differences in microbial carbon between land-cover types showed mostly similar patterns across the range of variables. Soil organic carbon and nitrogen content had a positive and mostly linear effect on microbial carbon (Supplementary Fig. 1). In contrast, clay content, soil pH, and mean temperature had non-linear relationships, with high microbial carbon in the low range of these variables and a rapid decrease that reached an asymptote at low microbial carbon values for the higher portion of the range. Soil pH patterns showed a decrease in microbial carbon for values between 4.1 and 5.8, and a constant pattern between 5.8 and 8.6. Contrary to our expectations, we did not find a parabolic effect of soil pH on microbial carbon26. Instead, our model predicted higher values in very acidic soils with a pH below 5.2, which are rare globally and almost only found in central Amazonia. Similarly, locations with a clay content lower than 16.9% had higher values in microbial carbon, and then stabilized until 51.0%.Fig. 2: Microbial carbon stock spatial predictions and temporal trends.a Microbial carbon stock predictions for 2013. b Variable importance from 100 random forest model runs, calculated by the mean decrease in accuracy after variable permutation. Variables were ordered by the median variable importance. SOC soil organic carbon, NDVI normalized difference vegetation index. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. c Relative microbial carbon stocks rate of change in percentage per year.Full size imageMean temperature showed an interesting shift with much higher microbial carbon values with a mean annual temperature below zero, but had otherwise a limited effect on microbial carbon values in the rest of the range above zero up to 28.9 °C. Based on partial predictions (Supplementary Figs. 1–2), microbial carbon decreased monotonically with an increase in temperature (with all other variables fixed to their median), with the relationship being mostly stable for parts of the range. We observed an especially sharp decrease at around 0°C, which is in agreement with the patterns observed in the data. The reason for sites with a mean annual temperature below the freezing point to have higher microbial carbon stocks is not fully understood. This could be due to a regime shift in which microbial communities are in a semi-dormant state for a major part of the year35. Moreover, it could also be in part explained by the soil organic carbon content that follows a similar trend and accumulates in higher latitude soils9, thus promoting higher microbial carbon stocks. Within these cold, high organic carbon soils, large microbial populations can be maintained, due to the low temperature that reduces metabolic requirements35. In contrast, at higher temperatures, metabolic activity increases and requires more resources and nutrients to maintain microorganisms alive. Experimental evidence is divided about the effects of warming on microbial carbon18,36, highlighting the strong context-dependency of this relationship, although global observations show a clear pattern, where low-temperature sites have higher soil microbial carbon stocks. Despite this uncertainty, there is a strong indication that a warming soil would tend to lose organic carbon17,37, and subsequent patterns in microbial carbon can also be expected, because of the dependency on organic substrate9,26,38. These dynamics were observed in Melillo et al.39, where the warming of sites in a mid-latitude forest ecosystem led to a decrease in soil carbon, followed by a decrease in microbial carbon12.Even with predictions being made for each grid location separately, microbial carbon values showed distinctive patterns and transitions over the globe (Fig. 2a). While temporal changes took place, broad spatial patterns were relatively constant over the range of years studied (Supplementary Movie 1). The highest microbial carbon stock values ranging from 1.50 to 7.00 t ha−1 were found at high latitudes in the Northern Hemisphere in areas of coniferous forest. Tropical humid regions also showed high microbial carbon values between 0.50 and 1.50 t ha−1 in the Amazon Rainforest and Central Africa. The main regions with low microbial carbon below 0.30 t ha−1 were in Eastern South America, areas directly south of the Sahara Desert, East Africa, and most of Australia, all of which mostly correspond to shrublands. Cropland areas as seen in India were also predicted with low microbial carbon values ranging from 0.06 to 0.38 t ha−1. A strong latitudinal gradient was visible for North America and Eurasia, with the highest microbial carbon stocks at high latitude, medium values in temperate ecosystems, and decreasing values towards the Equator. Positive coastal effects can also be observed, mostly on the Eastern South American and Australian coasts. In total, we estimated that there is 4.34 Gt of microbial carbon in the 5 to 15 cm layer for the predicted areas. Using the coefficient of variation calculated from the variability assessment set of models, we found that predictions made for the Amazon Basin, Northern Canada, and South-East Russia were more variable than for other regions (Supplementary Fig. 3a). Especially Western Europe, Central North America, and South-East Asia, however, showed high stability in the predictions between model runs.Drivers of changeThe analysis of the rate of change of microbial carbon stocks over time revealed that large regions of the globe experienced important changes in soil microbial carbon stocks between 1992 and 2013, with contrasting patterns across areas, and overall larger regions showed a decrease rather than an increase in microbial carbon stocks (Fig. 2c and Supplementary Fig. 3b). To account for spatial differences in microbial carbon stocks, we calculated the relative rate of change in percentage for each location (Fig. 2c). When considering all predictable regions together, microbial carbon stocks in the 5–15 cm layer showed a decrease of 7.09 Mt per year, summing to 148.80 Mt between 1992 and 2013, or 3.4% of the global microbial carbon pool predicted (Supplementary Fig. 4a; p = 0.038). The main regions with a microbial carbon loss higher than 0.7 kg ha−1 y−1 were in Northern Canada and a large continuous region in North-Eastern Europe. These northern regions accounted for an important part of the global loss in microbial carbon stocks, with large areas that had both a high soil microbial carbon stock and a fast decrease (Figs. 3 and 4). Other areas of high loss were in the Amazon basin, Western Argentina, the USA East Coast, Southern South Africa, and South-East Russia. The main continuous region of microbial carbon increase above 0.7 kg ha−1 y−1 was in central Russia, with smaller regions present in India, Europe, Central North America, and parts of Africa. Besides these general patterns, predictions vary at the local scale, and they consider the effects of parameters including soil properties, elevation, and land-cover type, which change between neighbor locations and affect the observed patterns. This is especially visible in the Americas, where both increases and decreases happen side-by-side.Fig. 3: Status of microbial carbon stocks between 1992 and 2013.Bivariate plot comparing the relative microbial carbon stock rate of change (% per year) with the amount of microbial carbon stock. The status groups were allocated using quantile distributions.Full size imageFig. 4: Distribution and classification of point values from the locations in Fig. 3.The assignment of points into the 9 groups was performed using quantile distributions. Areas in dark red are especially vulnerable to climate and land-cover change.Full size imagePatterns in the relative rate of change have a lot in common with that of absolute change, with a few notable differences (Fig. 2c and Supplementary Fig. 3b). Both positive and negative stock changes in tropical and subtropical regions are more prominent in relative terms, as these regions typically have low microbial carbon stocks. Similarly, regions in Central Russia with high microbial carbon stocks show less decrease in relative terms. To assess how stable these trends are over time, we show the p values of the rate of change for the 22 years (Supplementary Fig. 3c). The largest region with low p values is associated with more significant trends in Western Russia, and corresponds to an area with a fast loss of microbial carbon. India and Central Russia show high p values, and are informative of high variability compared to the strength of the signal. Considering that only up to 22 data points are available for each grid location and that especially climatic conditions vary considerably from year to year, p values are only provided as a complementary assessment. We can summarize the global situation by combining the two maps of microbial carbon stocks and relative rate of change to categorize and define vulnerable locations that experienced a high loss of microbial carbon (Figs. 3 and 4), and where the provision of soil functions is potentially at risk.It is informative to look at regional trends, by grouping grid locations using the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) sub-regions, and assessing regional-scale changes in microbial carbon stocks (Fig. 5, Supplementary Table 1). The main regions that contributed to microbial carbon loss were North America with a decrease of 62.49 Mt of microbial carbon and Eastern Europe with 60.88 Mt over the studied period, although both trends had high yearly variability and were non-significant. The region with the highest increase was North-East Asia with a gain of 4.49 Mt, but this change was also non-significant. The Caribbean was the only region to show a significant increase in soil microbial carbon stocks over time (+2.1% over 22 y, p = 0.017), while significant decreases in stocks were found in North Africa (−4.1%, p  More

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