Philippa Kaur

arising from Y. Wang et al. Nature https://doi.org/10.1038/s41586-021-04016-x (2021)A unique challenge for environmental DNA (eDNA)-based palaeoecological reconstructions and extinction estimates is that organisms can contribute DNA to sediments long after their death. Recently, Wang et al.1 discovered mammoth eDNA in sediments that are between approximately 4.6 and 7 thousand years (kyr) younger than the most recent mammoth fossils in North America and Eurasia, which they interpreted as mammoths surviving on both continents into the Middle Holocene epoch. Here we present an alternative explanation for these offsets: the slow decomposition of mammoth tissues on cold Arctic landscapes is responsible for the release of DNA into sediments for thousands of years after mammoths went extinct. eDNA records are important palaeobiological archives, but the mixing of undatable DNA from long-dead organisms into younger sediments complicates the interpretation of eDNA, particularly from cold and high-latitude systems.All animal tissues, including faeces, contribute DNA to eDNA records2, but the durations across which tissues can contribute genetic information must vary depending on tissue type and local rates of destruction and decomposition. On high-latitude landscapes, soft tissues and skeletal remains of large mammals may persist, unburied, for millennia3,4,5. For example, unburied antlers of caribou (Rangifer tarandus) from Svalbard (Norway) and Ellesmere Island (Canada) have been dated3,4 to between 1 and 2 cal kyr bp (calibrated kyr before present). Elephant seal (Mirounga leonina) remains near the Antarctic coastline5,6 can persist for more than 5,000 years. This is in contrast to bones in warmer settings, which persist for only centuries or decades7,8. Because bones are particularly resistant to decay, quantifying how their persistence changes across environments enables us to constrain the durations that dead individuals generally contribute to eDNA archives. To do this, we consolidated data on the oldest radiocarbon-dated surface-collected bones from different ecosystems. We included bones that we are reasonably confident persisted without being completely buried (‘never buried’), and bones for which exhumation cannot be confidently excluded (‘potentially never buried’). Pairing bone persistence with mean annual temperatures (MAT) from their sample localities, we find a strong link between the local temperature and the logged duration of bone persistence (Fig. 1, never buried bones: R2 = 0.94, P  More

Rearing O. biroi pupae in social isolation and collecting pupal fluidIn O. biroi colonies, larvae and pupae develop in discrete and synchronized cohorts26. Ten days after the first larvae had entered pupation in a large stock colony, the entire colony was anaesthetized using a CO2 pad, and white pupae were separated using a paintbrush. Pupae were individually placed in 0.2 ml PCR tubes with open lid. These tubes were then placed inside 1.5 ml Eppendorf tubes with 5 µl sterile water at the bottom to provide 100% relative humidity. The outer tubes were closed and kept in a climate room at 25 °C. The inner tube in this design prevents the pupa from drowning in the water reservoir. The outer tubes were kept closed throughout the experiment, except for once a day when the tubes were opened to remove pupal social fluid. Pulled glass capillaries were prepared as described elsewhere29, and used to remove and/or collect secretion droplets. We were careful to leave no remains of the secretion behind on the pupae or the inside of the tubes. To ensure that all secretion had been removed, pupae were taken out of the tube after fluid collection and briefly placed on a tissue paper to absorb any excess liquid. The inner tubes were replaced if needed—for example, if fluid traces were visible on the old tube after collection. Each pupa was checked daily for secretion (absent or present), onset of melanization and eclosion, and whether the pupa was alive (responding to touch). Control groups of 30 pupae and 30 adult ants from the same stock colony and cohort as the isolated pupae were placed in Petri dishes with a plaster of Paris floor, and the same parameters as for the isolated pupae were scored daily. Experiments ended when all pupae had either eclosed or died. Newly eclosed (callow) workers moved freely inside the tube and showed no abnormalities when put in a colony. A pupa was declared dead if it did not shed its pupal skin and did not respond to touch three days after all pupae in the control group had eclosed.To calculate the average secretion volume per secreting pupa (Fig. 1d), the total volume collected daily from a group of isolated pupae (142–166 pupae) was divided by the number of pupae from which fluid had been collected that day. The total volume was determined by multiplying the height of the fluid’s meniscus in the capillary by πr², where r is the inner radius of the capillary (0.29 mm). While pupae were secreting, pupal whole-body wash samples were collected daily. The pupae were removed from colonies with adults and washed promptly with 1500 µl LC–MS grade water. Whole-body wash samples were lyophilized and reconstituted in 15 µl LC–MS grade water.Collecting additional ant species and honeybees, rearing pupae in social isolation, and collecting pupal fluidsColonies of the ants N. flavipes, T. sessile, P. pennsylvanica and Lasius neoniger were collected in NY state, USA (Central Park, Manhattan; Pelham Bay Park, Bronx; Prospect Park, Brooklyn; and Woodstock). Solenopsis invicta colonies were collected in Athens, GA, USA. M. mexicanus colonies were collected in Piñon Hills, CA, USA. Colonies comprised of queens, workers and brood were maintained in the laboratory in airtight acrylic boxes with plaster of Paris floors. Colonies were fed a diet of insects (flies, crickets and mealworms). White pupae were socially isolated, cocoons were removed in the case of P. pennsylvanica, and secretion droplets were collected from melanized pupae as described for O. biroi. A. mellifera pupae of unknown age were socially isolated from hive fragments (A&Z Apiaries, USA) and reared as described for O biroi, except that the rearing temperature was set to 32 °C. Relative humidity was set to either 100% to replicate conditions used for the different ant species, or to 75% as recommended in the literature30.Injecting dye and tracking pupal fluidInjection needles were prepared as in previous studies31. Injections were performed using an Eppendorf Femtojet with a Narishige micromanipulator. The Femtojet was set to Pi 1000 hPa and Pc 60 hPa. Needles were broken by gently touching the capillary tip to the side of a glass slide. To inject, melanized pupae were placed on ‘Sticky note’ tape (Post-it), with the abdomen tip forward and the ventral side upward. Pupae were injected with blue food colouring (McCormick) into the exuvium for 1–2 s by gently piercing the pupal case at the abdominal tip with the needle. During successful injections, no fluid was discharged from the pupa when the needle was removed, and the moulting fluid inside the exuvium was immediately stained. Pupae were washed in water three times to remove any excess dye. Following injections, 10 pupae were reared in social isolation to confirm the secretion of dyed droplets. For experiments, injected pupae were transferred to colonies with adult ants (Figs. 1f and  4c) or to colonies with adult ants and larvae (Figs. 3b and  4c) to track the distribution of the pupal social fluid.After spending 24 h with dye-injected pupae, adults were taken out of the colony, briefly immersed in 95% ethanol, and transferred to PBS. Digestive systems were dissected in cold PBS and mounted in DAKO mounting medium. Crop and stomach images (Fig. 1f, inset and Fig. 4c, inset) were acquired with a Revolve microscope (Echo). Larvae are translucent, and the presence of dye in the digestive system can be assayed without dissection. Whole-body images of larvae were acquired with a Leica Z16 APO microscope equipped with a Leica DFC450 camera and Leica Application Suite version 4.12.0 (Leica Microsystems). In the experiment on larval growth (Fig. 3c), larval length was measured from images using ImageJ32.Occluding pupaeTen pupae were placed on double-sided tape on a glass coverslip with the ventral side up. The area between the pupae was covered with laser-cut filter paper to prevent adults from sticking to the tape. The pupae were then placed in a 5 cm diameter Petri dish with a moist plaster of Paris floor. To block pupal secretion, the tip of the gaster was occluded with a drop of oil-paint (Uni Paint Markers PX-20), which has no discernible toxic effect7. Secreting pupae received a drop of the same paint on their head to control for putative differences resulting from the paint. Pupae were left in isolation for one day before adults were added to the assay chamber.Behavioural tracking of adult preference assayVideos were recorded using BFS-U3-50S5C-C: 5.0 MP, 35 FPS, Sony IMX264, Colour cameras (FLIR) and the Motif Video Recording System (Loopbio). To assess adult preference (Fig. 1g), physical contact of adults with pupae was manually annotated for the first 10 min after the first adult had encountered (physically contacted) a pupa.Protein profilingWe extracted 30 µl of pupal social fluid and whole-body wash samples with 75:25:0.2 acetonitrile: methanol: formic acid. Extracts were vortexed for 10 min, centrifuged at 16,000g and 4 °C for 10 min, dried in a SpeedVac, and stored at −80 °C until they were analysed by LC–MS/MS.Protein pellets were dissolved in 8 M urea, 50 mM ammonium bicarbonate, and 10 mM dithiothreitol, and disulfide bonds were reduced for 1 h at room temperature. Alkylation was performed by adding iodoacetamide to a final concentration of 20 mM and incubating for 1 h at room temperature in the dark. Samples were diluted using 50 mM ammonium bicarbonate until the concentration of urea had reached 3.5 M, and proteins were digested with endopeptidase LysC overnight at room temperature. Samples were further diluted to bring the urea concentration to 1.5 M before sequencing-grade modified trypsin was added. Digestion proceeded for 6 h at room temperature before being halted by acidification with TFA and samples were purified using in-house constructed C18 micropurification tips.LC–MS/MS analysis was performed using a Dionex3000 nanoflow HPLC and a Q-Exactive HF mass spectrometer (both Thermo Scientific). Solvent A was 0.1% formic acid in water and solvent B was 80% acetonitrile, 0.1% formic acid in water. Peptides were separated on a 90-minute linear gradient at 300 nl min−1 across a 75 µm × 100 mm fused-silica column packed with 3 µm Reprosil C18 material (Dr. Maisch). The mass spectrometer operated in positive ion Top20 DDA mode at resolution 60 k/30 k (MS1/MS2) and AGC targets were 3 × 106/2 × 105 (MS1/MS2).Raw files were searched through Proteome Discoverer v.1.4 (Thermo Scientific) and spectra were queried against the O. biroi proteome using MASCOT with a 1% FDR applied. Oxidation of M and acetylation of protein N termini were applied as a variable modification and carbamidomethylation of C was applied as a static modification. The average area of the three most abundant peptides for a matched protein33 was used to gauge protein amounts within and between samples.Functional annotation and gene ontology enrichmentTo supplement the current functional annotation of the O. biroi genome34, the full proteome for canonical transcripts was retrieved from UniProtKB (UniProt release 2020_04) in FASTA format. We then applied the EggNog-Mapper tool35,36 (http://eggnog-mapper.embl.de, emapper version 1.0.3-35-g63c274b, EggNogDB version 2) using standard parameters (m diamond -d none –tax_scope auto –go_evidence non-electronic –target_orthologs all –seed_ortholog_evalue 0.001 –seed_ortholog_score 60 –query-cover 20 –subject-cover 0) to produce an expanded annotation for all GO trees (Molecular Function, Biological Process, Cellular Components). The list of proteins identified in the pupal fluid was evaluated for functional enrichment in these GO terms, P-values were adjusted with an FDR cut-off of 0.05, and the network plots were visualized using the clusterProfiler package37.Metabolite profilingFor bulk polar metabolite profiling, we used 10 µl aliquots of pupal social fluid and whole-body wash (pooled samples). For the time-series metabolite profiling, 1 µl of pupal social fluid and whole-body wash was used. Samples were extracted in 180 µl cold LC–MS grade methanol containing 1 μM of uniformly labelled 15N- and 13C-amino acid internal standards (MSK-A2-1.2, Cambridge Isotope Laboratories) and consecutive addition of 390 µl LC–MS grade chloroform followed by 120 µl of LC–MS grade water.The samples were vortexed vigorously for 10 min followed by centrifugation (10 min at 16,000g and 4 °C). The upper polar metabolite-containing layer was collected, flash frozen and SpeedVac-dried. Dried extracts were stored at −80 °C until LC–MS analysis.LC–MS was conducted on a Q-Exactive benchtop Orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe, which was coupled to a Vanquish UPLC system (Thermo Fisher Scientific). External mass calibration was performed using the standard calibration mixture every three days.Dried polar samples were resuspended in 60 µl 50% acetonitrile, and 5 µl were injected into a ZIC-pHILIC 150 × 2.1 mm (5 µm particle size) column (EMD Millipore). Chromatographic separation was achieved using the following conditions: buffer A was 20 mM ammonium carbonate, 0.1% (v/v) ammonium hydroxide (adjusted to pH 9.3); buffer B was acetonitrile. The column oven and autosampler tray were held at 40 °C and 4 °C, respectively. The chromatographic gradient was run at a flow rate of 0.150 ml min−1 as follows: 0–22 min: linear gradient from 90% to 40% B; 22–24 min: held at 40% B; 24–24.1 min: returned to 90% B; 24.1 −30 min: held at 90% B. The mass spectrometer was operated in full-scan, polarity switching mode with the spray voltage set to 3.0 kV, the heated capillary held at 275 °C, and the HESI probe held at 250 °C. The sheath gas flow was set to 40 units, the auxiliary gas flow was set to 15 units. The MS data acquisition was performed in a range of 55–825 m/z, with the resolution set at 70,000, the AGC target at 10 × 106, and the maximum injection time at 80 ms. Relative quantification of metabolite abundances was performed using Skyline Daily v 20.1 (MacCoss Lab) with a 2 ppm mass tolerance and a pooled library of metabolite standards to confirm metabolite identity (via data-dependent acquisition). Metabolite levels were normalized by the mean signal of 8 heavy 13C,15N-labelled amino acid internal standards (technical normalization).The raw data were searched for a targeted list of ~230 polar metabolites and the corresponding peaks were integrated manually using Skyline Daily software. We were able to assign peaks to 107 compounds based on high mass accuracy ( More

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As humans warm the planet, biodiversity is plummeting. These two global crises are connected in multiple ways. But the details of the intricate feedback loops between biodiversity decline and climate change are astonishingly under-studied.It is well known that climate extremes such as droughts and heatwaves can have devastating impacts on ecosystems and, in turn, that degraded ecosystems have a reduced capacity to protect humanity against the social and physical impacts of such events. Yet only a few such relationships have been probed in detail. Even less well known is whether biodiversity-depleted ecosystems will also have a negative effect on climate, provoking or exacerbating weather extremes.For us, a group of researchers living and working mainly in Central Europe, the wake-up call was the sequence of heatwaves of 2018, 2019 and 2022. It felt unreal to watch a floodplain forest suffer drought stress in Leipzig, Germany. Across Germany, more than 380,000 hectares of trees have now been damaged (see go.nature.com/3etrrnp; in German), and the forestry sector is struggling with how to plan restoration activities over the coming decades1. What could have protected these ecosystems against such extremes? And how will the resultant damage further impact our climate?
Nature-based solutions can help cool the planet — if we act now
In June 2021, the Intergovernmental Panel on Climate Change (IPCC) and the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) published their first joint report2, acknowledging the need for more collaborative work between these two domains. And some good policy moves are afoot: the new EU Forest Strategy for 2030, released in July 2021, and other high-level policy initiatives by the European Commission, formally recognize the multifunctional value of forests, including their role in regulating atmospheric processes and climate. But much more remains to be done.To thoroughly quantify the risk that lies ahead, ecologists, climate scientists, remote-sensing experts, modellers and data scientists need to work together. The upcoming meeting of the United Nations Convention on Biological Diversity in Montreal, Canada, in December is a good opportunity to catalyse such collaboration.Buffers and responsesWhen lamenting the decline in biodiversity, most people think first about the tragedy of species driven to extinction. There are more subtle changes under way, too.For instance, a study across Germany showed that over the past century, most plant species have declined in cover, with only a few increasing in abundance3. Also affected is species functionality4 — genetic diversity, and the diversity of form and structure that can make communities more or less efficient at taking up nutrients, resisting heat or surviving pathogen attacks.When entire ecosystems are transformed, their functionality is often degraded. They are left with less capacity to absorb pollution, store carbon dioxide, soak up water, regulate temperature and support vital functions for other organisms, including humans5. Conversely, higher levels of functional biodiversity increase the odds of an ecosystem coping with unexpected events, including climate extremes. This is known as the insurance effect6.The effect is well documented in field experiments and modelling studies. And there is mounting evidence of it in ecosystem responses to natural events. A global synthesis of various drought conditions showed, for instance, that forests were more resilient when trees with a greater diversity of strategies for using and transporting water lived together7.

Dead trees near Iserlohn, Germany, in April 2020 (left) and after felling in June 2021 (right).Credit: Ina Fassbender/AFP via Getty

However, biodiversity cannot protect all ecosystems against all kinds of impacts. In a study this year across plots in the United States and Canada, for example, mortality was shown to be higher in diverse forest ecosystems8. The proposed explanation for this unexpected result was that greater biodiversity could also foster more competition for resources. When extreme events induce stress, resources can become scarce in areas with high biomass and competition can suddenly drive mortality, overwhelming the benefits of cohabitation. Whether or not higher biodiversity protects an ecosystem from an extreme is highly site-specific.Some plants respond to drought by reducing photosynthesis and transpiration immediately; others can maintain business as usual for much longer, stabilizing the response of the ecosystem as a whole. So the exact response of ecosystems to extremes depends on interactions between the type of event, plant strategies, vegetation composition and structure.Which plant strategies will prevail is hard to predict and highly dependent on the duration and severity of the climatic extreme, and on previous extremes9. Researchers cannot fully explain why some forests, tree species or individual plants survive in certain regions hit by extreme climate conditions, whereas entire stands disappear elsewhere10. One study of beech trees in Germany showed that survival chances had a genomic basis11, yet it is not clear whether the genetic variability present in forests will be sufficient to cope with future conditions.And it can take years for ecosystem impacts to play out. The effects of the two consecutive hot drought years, 2018 and 2019, were an eye-opener for many of us. In Leipzig, tree growth declined, pathogens proliferated and ash and maple trees died. The double blow, interrupted by a mild winter, on top of the long-term loss of soil moisture, led to trees dying at 4–20 times the usual rate throughout Germany, depending on the species (see go.nature.com/3etrrnp; in German). The devastation peaked in 2020.Ecosystem changes can also affect atmospheric conditions and climate. Notably, land-use change can alter the brightness (albedo) of the planet’s surface and its capacity for heat exchange. But there are more-complex mechanisms of influence.Vegetation can be a source or sink for atmospheric substances. A study published in 2020 showed that vegetation under stress is less capable of removing ozone than are unstressed plants, leading to higher levels of air pollution12. Pollen and other biogenic particles emitted from certain plants can induce the freezing of supercooled cloud droplets, allowing ice in clouds to form at much warmer temperatures13, with consequences for rainfall14. Changes to species composition and stress can alter the dynamics of these particle emissions. Plant stress also modifies the emission of biogenic volatile organic gases, which can form secondary particles. Wildfires — enhanced by drought and monocultures — affect clouds, weather and climate through the emission of greenhouse gases and smoke particles. Satellite data show that afforestation can boost the formation of low-level, cooling cloud cover15 by enhancing the supply of water to the atmosphere.Research prioritiesAn important question is whether there is a feedback loop: will more intense, and more frequent, extremes accelerate the degradation and homogenization of ecosystems, which then, in turn, promote further climate extremes? So far, we don’t know.One reason for this lack of knowledge is that research has so far been selective: most studies have focused on the impacts of droughts and heatwaves on ecosystems. Relatively little is known about the impacts of other kinds of extremes, such as a ‘false spring’ caused by an early-season bout of warm weather, a late spring frost, heavy rainfall events, ozone maxima, or exposure to high levels of solar radiation during dry, cloudless weather.Researchers have no overview, much less a global catalogue, of how each dimension of biodiversity interacts with the full breadth of climate extremes in different combinations and at multiple scales. In an ideal world, scientists would know, for example, how the variation in canopy density, vegetation age, and species diversity protects against storm damage; and whether and how the diversity of canopy structures controls atmospheric processes such as cloud formation in the wake of extremes. Researchers need to link spatiotemporal patterns of biodiversity with the responses of ecosystem processes to climate extremes.
Biodiversity needs every tool in the box: use OECMs
Creating such a catalogue is a huge challenge, particularly given the more frequent occurrence of extremes with little or no precedent16. Scientists will also need to account for the increasing likelihood of pile-ups of climate stressors. The ways in which ecosystems respond to compound events17 could be quite different. Researchers will have to study which facets of biodiversity (genetic, physiological, structural) are required to stabilize ecosystems and their functions against these onslaughts.There is at least one piece of good news: tools for data collection and analysis are improving fast, with huge advances over the past decade in satellite-based observations for both climate and biodiversity monitoring. The European Copernicus Earth-observation programme, for example — which includes the Sentinel 1 and 2 satellite fleet, and other recently launched missions that cover the most important wavelengths of the electromagnetic spectrum — offer metre-scale resolution observations of the biochemical status of plants and canopy structure. Atmospheric states are recorded in unprecedented detail, vertically and in time.Scientists must now make these data interoperable and integrate them with in situ observations. The latter is challenging. On the ground, a new generation of data are being collected by researchers and by citizen scientists18. For example, unique insights into plant responses to stress are coming from time-lapse photography of leaf orientation; accelerometer measures of movement patterns of stems have been shown to provide proxies for the drought stress of trees19.High-quality models are needed to turn these data into predictions. The development of functional ‘digital twins’ of the climate system is now in reach. These models replicate hydrometeorological processes at the metre scale, and are fast enough to allow for rapid scenario development and testing20. The analogous models for ecosystems are still in a more conceptual phase. Artificial-intelligence methods will be key here, to study links between climate extremes and biodiversity.Researchers can no longer afford to track global transformations of the Earth system in disciplinary silos. Instead, ecologists and climate scientists need to establish a joint agenda, so that humanity is properly forewarned: of the risks of removing biodiversity buffers against climate extremes, and of the risk of thereby amplifying these extremes. More

Differences in environmental factorsEnvironmental factors showed value differences between forest types, while the significance of differences differed among variables, which were both found with corrected values and original measurements (Fig. 1).Figure 1The differences in environmental factors between naturally regenerating forests (NF) and planted forests (PF) in China. The environmental factors include three annual climatic factors (a–c), three seasonal temperature factors (d–f), three seasonal precipitation factors (g–i), three biotic factors (j–l), and two soil factors (m,n). Three annual climatic factors include mean annual air temperature (MAT, a), mean annual precipitation (MAP, b), and aridity index (AI, c) defined as the ratio of MAP to annual potential evapotranspiration. Three seasonal temperature factors include the temperature of the warmest month (Tw, d), the temperature of the coldest month (Tc, e), temperature annual range (TR, f). Three seasonal precipitation factors include precipitation of the wettest month (Pw, g), precipitation of the driest month (Pd, h), and precipitation seasonality (Ps, i) defined as the standard deviation of monthly precipitation during the measuring year. Three biological factors include the mean annual leaf area index (LAI, j), the maximum leaf area index (MLAI, k), and stand age (SA, l). Two soil factors include soil organic carbon content (SOC, m) and soil total nitrogen content (STN, n). The differences are tested for each variable with one-way analysis of variance (ANOVA), where * and ** indicate significant differences between forest types at significance levels of α = 0.05 and α = 0.01, respectively. The corrected values are mean values during 2003–2019 after correcting the original measurements with the interannual trend (See methods), which are listed in each panel, while original measurements are mean values during the measuring period of each ecosystem, which are not shown in each panel.Full size imageFor annual climatic factors, the significant difference between NF and PF only appeared in MAT (Fig. 1a). The mean MAT of NF was 10.50 ± 7.81 °C, which was significantly lower than that of PF (15.65 ± 6.23 °C) (p  0.05) (Fig. 2c). Even considering the significant effects of MAT on ER, ANCOVA results obtained by fixing MAT as a covariant also suggested that ER values did not significantly differ between forest types (F = 0.01, p  > 0.05). Fixing other variables as a covariant also drew a similar result.Therefore, NF showed a lower NEP resulting from the lower GPP than PF, while their differences were not statistically significant (Fig. 2).Differences in NEP latitudinal patternsCarbon fluxes showed divergent latitudinal patterns between NF and PF, while their latitudinal patterns varied among carbon fluxes, which were both found with corrected values and original measurements (Fig. 3).Figure 3The latitudinal patterns of carbon fluxes over Chinese naturally regenerating forests (NF) and planted forests (PF). The carbon fluxes include net ecosystem productivity (NEP, a,b), gross primary productivity (GPP, c,d), and ecosystem respiration (ER, e,f). Each panel is drawn with the corrected values (blue points) and original measurements (grey points), respectively. The blue and black lines represent the regression lines calculated from the corrected values and original measurements, respectively, with their regression statistics listed in blue and black letters. Only the regression slope (Sl) and R2 of each regression are listed. The grey lines represent the regressions between carbon fluxes added by random errors and latitude. Only significant (p  0.05).The ER of NF showed a significant decreasing latitudinal pattern (Fig. 3e), while that of PF exhibited no significant latitudinal pattern (Fig. 3f). The increasing latitude caused the ER of NF to significantly decrease. Each unit increase in latitude led to a 28.71 gC m−2 year−1 decrease in ER, with an R2 of 0.31. However, the increasing latitude contributed little to the ER spatial variation of PF (p  > 0.05).In addition, the latitudinal patterns of carbon fluxes and their differences between forest types were also obtained with the original measurements (Fig. 3, grey points). The latitudinal patterns of random error adding carbon fluxes were comparable to those of our corrected carbon fluxes (Fig. 3), which confirmed that the latitudinal patterns of carbon fluxes and their differences between forest types would not be affected by the uncertainties in generating the corrected carbon fluxes.Therefore, among NFs, the similar decreasing latitudinal patterns of GPP and ER meant that NEP showed no significant latitudinal pattern, while the significant decreasing latitudinal pattern of GPP and no significant latitudinal pattern of ER caused NEP to show a decreasing latitudinal pattern among PFs.Differences in the environmental effects on NEP spatial variationsEnvironmental factors, including the annual climatic factors, seasonal temperature factors, seasonal precipitation factors, biological factors, and soil factors, exerted divergent effects on the spatial variations of NEP and its components, which also differed between forest types (Table 1). No factor was found to affect that the spatial variation of NEP among NFs, while most annual and seasonal climatic factors were found to affect that among PFs. The spatial variations of GPP and ER among NFs were both affected by most annual and seasonal climatic factors and LAI, while those among PFs were primarily shaped by most annual and seasonal climatic factors. Though LAI showed no significant effect on GPP and ER spatial variations among PFs, SA exerted a significant negative effect. In addition, the spatial variations of soil variables contributed little to the spatial variations of carbon fluxes. Therefore, among NFs, most annual and seasonal climatic factors and LAI were found to affect GPP and ER spatial variations, while no factor was found to significantly influent the NEP spatial variation. However, among PFs, most annual and seasonal climatic factors were found to affect the spatial variations of NEP and its components, while LAI showed no significant effect. Using the original measurements also generated the similar correlation coefficients (Supplementary Table S1).Table 1 Correlation coefficients between carbon fluxes and environmental factors in naturally regenerating forests (NF) and planted forests (PF).Full size tableGiven the high correlations among annual climatic factors and seasonal climatic factors (Supplementary Table S2), the partial correlation analysis was applied to determine which factors should be employed to reveal the mechanisms underlying the spatial variations of NEP. Partial correlation analysis showed that MAT and MAP exerted the most important roles in spatial variations of NEP and its components (Table 2). After controlling MAT (or MAP), other factors seldom showed significant correlation with carbon fluxes, especially fixing MAT (Table 2). In addition, MAT and MAP exerted similar effects on the spatial variations of NEP and its components (Table 1). Using the original measurements also generated the similar partial correlation coefficients (Supplementary Table S3). Therefore, we only presented the effects of MAT on carbon flux spatial variations and their differences between forest types in detail.Table 2 Partial correlation coefficients between carbon fluxes and environmental factors in naturally regenerating forests (NF) and planted forests (PF) with fixing mean annual air temperature (MAT) or mean annual precipitation (MAP).Full size tableThe increasing MAT increased carbon fluxes, while the increasing rates differed between forest types (Fig. 4). The increasing MAT contributed little to the NEP spatial variation of NF but raised the NEP of PF (Fig. 4a,b). Each unit increase in MAT caused the NEP of PF to increase at a rate of 27.77 gC m−2 year−1, with an R2 of 0.31 (Fig. 4b). The increasing MAT significantly raised GPP in NF and PF (Fig. 4c,d). For NF, each unit increase in MAT increased GPP at a rate of 43.76 gC m−2 year−1, with an R2 of 0.49 (Fig. 4c), while each unit increase in MAT increased the GPP of PF at a rate of 69.18 gC m−2 year−1, with an R2 of 0.57 (Fig. 4d). The GPP increasing rates did not significantly differ between NF and PF (F = 1.52, p  > 0.05). The increasing MAT also raised ER in both NF and PF (Fig. 4e,f), whose increasing rates were 38.97 gC m−2 year−1 (Fig. 4e) and 36.79 gC m−2 year−1 (Fig. 4f), respectively, while their differences were not statistically significant (F = 0.01, p  > 0.05). In addition, using the original measurements also generated the similar spatial variations and their differences between forest types (Fig. 4). Furthermore, the random error adding carbon fluxes responded similarly to those of our correcting carbon fluxes (Fig. 4), indicating that the effects of MAT on carbon fluxes would not be affected by the uncertainties in our correcting carbon fluxes. Therefore, the similar responses of GPP and ER to MAT made MAT contribute little to NEP spatial variations among NFs, while GPP and ER showed divergent response rates to MAT, which made NEP increase with MAT among PFs.Figure 4The effects of mean annual air temperature (MAT) on the spatial variations of carbon fluxes over Chinese naturally regenerating forests (NF) and planted forests (PF). The carbon fluxes include net ecosystem productivity (NEP, a,b), gross primary productivity (GPP, c,d), and ecosystem respiration (ER, e,f). Each panel is drawn with the corrected values (blue points) and original measurements (grey points), respectively. The blue and black lines represent the regression lines calculated from the corrected values and original measurements, respectively, with their regression statistics listed in blue and black letters. Only the regression slope (Sl) and R2 of each regression are listed. The grey lines represent the regressions between carbon fluxes added by random errors and latitude. Only significant (p  More