Ecology

Maestre and colleagues collected data using a standardized field survey at 98 sites across 25 countries and 6 continents, fitted linear mixed models to data from all sites and grazing pressure levels, and then applied a multimodel inference procedure to select the set of best-fitting models. The authors found interactions between grazing and biodiversity in almost half of the best-fitting models, where increasing grazing pressure had positive effects on ecosystem services in colder sites with high plant species richness. However, increases in grazing pressure at warmer sites with high rainfall seasonality and low plant species richness interacted with soil properties to either increase or reduce the delivery of multiple ecosystem services. The authors’ findings highlight how increasing herbivore richness could enhance ecosystem service delivery across contrasting environmental and biodiversity conditions, enhancing soil carbon storage and reducing the negative impacts of increased grazing pressure. More

Sampling, bacterial isolation, and colony screeningSamples were collected from thermally active sediments as evidenced by temperature and/or emission of steam in November 2010 and 2012 from Harry’s Dream (HD2 and HD3), Warren Cave (WC1, WC2, WC3, WC4, WC7, and WC8), Haggis Hole, (HH), Mammoth Cave (MC), Hut Cave (Hut), and Heroine Cave (HC) (fumarolic ice caves on Mt. Erebus volcano [21]). The samples were divided into four types: Mainly weathered basaltic/phonolitic sand (HD2, HD3, WC3, WC4, WC8 and Hut); pebbles and rock fragments (WC7, HH, and MC); black porous glassy materials that appeared to be solidified lava (WC1, WC2, and HC); and ash sediment (WC10) with little to no organic material (Table S1). The sediments were collected aseptically using sterile 50 mm conical tubes and immediately sealed. Additional information and environmental parameters on the sampling locations are provided in Table S1 and in Tebo et al. [21].To isolate bacteria found in these oligotrophic environments, we employed a culture strategy of long-term incubation in a nutrient-poor medium and screening of slow-growing colonies by direct PCR identification. Reasoner’s 2 A gellan gum medium (10% R2AG) [23] and FS1VG medium [24] were used for bacterial isolation. The 10% R2AG is a 10-fold diluted R2A broth (Nihon Seiyaku, Tokyo, Japan), solidified with 15 g/L gellan gum (Kanto chemical, Tokyo, Japan) with 2 g/L CaCl2. The 10% R2AG and FS1VG media were adjusted to a pH of 4.5 or 6.0, with or without 30 mg/L sodium azide. Samples used for isolation were selected from Warren Cave and Harry’s Dream, where the presence of relatively large numbers of bacteria ( >107/g) was confirmed in a previous report [21], and a total of nine samples, WD1, 2, 3, 4, 7, 8, and 10 and HD2 and 3, were used without any other pretreatment such as drying or dilution. For the sandy and ash samples with fine particles (WC3, 4, 8, 10, and HD2, 3), approximately 50 mg of sample were spread directly on plates. Glassy materials (WC1 and WC2) were embedded directly in plates using 2-3 pieces (approx. 200 mg). Pebbles and rock fragments (WC7) were crushed and approximately 50 mg of debris were spread directly onto the plates. All plates were incubated at 15 °C, 30 °C or 37 °C under dark conditions. New colonies were marked as they appeared and selection of the isolates was performed by picking only colonies that appeared after four weeks of incubation. We selected colonies around the sediment or in the cracks created during spreading using a magnifying glass.For identification, colonies were picked with a sterile toothpick, re-streaked, stabbed on fresh medium, and subsequently suspended in 20 μL sterilized 0.05 M NaOH. Suspensions were heated at 100 °C for 15 min, and supernatants were used as template DNAs for PCR. Partial 16 S rRNA gene sequences were amplified by PCR using commonly used bacterial primer set 27 F (5′-AGATTTGATCCTGGCTCAG-3′) and 1492 R (5′-GGTTACCTTGTTACGACTT-3′) or 536 R (5′-GTA TTA CCG CGG CTG CTG-3′) with TaKaRa ExTaq DNA polymerase (Takara Bio, Shiga, Japan) as previously described [23]. Sequencing was performed at Eurofins Genomics (Louisville, KY, USA), using a 3730xl DNA analyzer (Applied Biosystems, CA, USA). Sequence similarities with closest species were calculated using EZbiocloud’s Identify Service (https://www.ezbiocloud.net/identify). Subsequently, cells in the stab identified as “Ca. Eremiobacterota” by the direct PCR identification were serially diluted and stabbed onto new plates until multiple pure cultures of Eremiobacterota were obtained. The isolate was designated as strain WC8-2.Whole genome sequencing and annotationGenomic DNA was extracted from WC8-2 cells grown in 10% R2A broth (pH6.0) with air/CO2 (90:10, v/v) at 30 °C for 30 days under a 12/12 h light/dark regime with incandescent light (250 μmol m–2s–1), using a Puregene Yeast/Bact. Kit B (Qiagen, Germantown, MD, USA) [25]. Sequencing was performed by Macrogen Japan Corp., on a NovaSeq 6000 (Illumina, Inc., San Diego, CA, USA) and PacBio RSII (Pacific Biosciences of California, Inc. Menlo Park, CA, USA). The gap-free complete genome was assembled de novo using the Unicycler version 0.4.8 hybrid assembly pipeline with default settings [26]. Completeness and contamination levels were estimated using CheckM [27]. The genome was annotated using the DDBJ Fast Annotation and Submission Tool (DFAST) [28] and the BlastKOALA web server version 2.2, and was visualized using CGView Server [29] (http://cgview.ca/).Phylogenetic analysis of “Ca. Eremiobacterota”Identification of strain WC8-2 was performed using the Genome Taxonomy Database Toolkit (GTDB-Tk) (ver. 2.1.0), which produces standardized taxonomic labels that are based on those used in the Genome Taxonomy Database [30]. Terrabacterial genomes including “Ca. Eremiobacterota” MAGs and related genomes were retrieved from the Genome Taxonomy Database (GTDB) (July 2022) and the NCBI RefSeq database (July 2022). Full-length 16 S rRNA gene sequences were retrieved from the WC8-2 genome (WPS_r00030) and the NCBI database (Table S2). Multiple sequences were aligned using SINA (version 1.2.11) [31]. IQ-TREE version 1.6.12 [32] was used to build the phylogeny. ModelFinder [33] was used to determine the optimal evolutionary model for phylogeny building, which selected the TNe+I + G4 model. Branch support was calculated using 1000 ultrafast bootstraps [34]. The pairwise 16 S rRNA gene sequence similarities were determined using SDT software. Phylogenomic analysis based on 400 marker proteins was carried out using PhyloPhlAn v3.0 [35]. Diamond v5.2.32 [36], MAFFT v7.453 [37], and TrimAI were utilized for orthologs searching, multiple sequence alignment within the superphylum Terrabacteria, and gap-trimming, respectively. Gappy sites and sequences with >50% gaps were deleted from the alignments. IQ-TREE version 1.6.12 [32] was used to build the phylogenomic tree. ModelFinder [33] was used to determine the optimal evolutionary model for phylogeny building, which selected the LG + F + R9 model. These analyses were conducted using the “AOBA-B” super- computer (NEC, Tokyo, Japan) with 2CPUs (EPYC7702, AMD, CA, US) and 256GB of RAM. The related similarity of genomes between strain WC8-2 and relatives was estimated using average nucleotide identity (ANI) values, which were calculated using OrthoANI calculator in the EzBio-Cloud web service [38]. The related similarity between strain WC8-2 and its sister phyla with one representative from each class was assessed by pairwise Average Amino acid Identity (AAI) values using the online tools at the Kostas Konstantinidis Lab website Environmental Microbial Genomics Laboratory (http://enve-omics.ce.gatech.edu/aai/). The MAGs used in the tree are listed in Table S3.Phylogeny of photosynthesis- and “atmospheric chemosynthesis”- associated genesWe retrieved phototrophy- and “atmospheric chemosynthesis”-related protein sequences from the WC8-2 genome, “Ca. Eremiobacterota” MAGs, and known phototrophs genomes (Table S3) using the local BLAST server (SequenceServer 1.0.14 [39]) with reference sequences (Table S3) or annotated sequences as queries. Sequences were aligned using MUSCLE and poorly aligned regions were removed using Gblocks version 0.91b [40] or by manual inspection. The alignment sequences were concatenated into a single sequence. The ML tree was constructed using IQ-TREE with the best-hit evolutionary rate model: LG + I + G4 for HhyL, CbbL, BchXYZ and CbbL, LG + F + I + G4 for BchLNB, and LG + F + G4 for PufML, BchI, and BchD. All trees were visualized using iTOL (version 5.0) [41]. All sequences used in the trees are listed in Table S3.Analysis of bacterial communities in the fumarolic ice cavesEnvironmental DNA was extracted from the samples (0.1 g) (WC7, WC8, HD3, MC, Hut, HH, HC) using a DNeasy PowerSoil Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The V4 region of the 16 S rRNA gene was PCR amplified using primers with adapter sequences (V3-V4f_MIX) [42]. The PCR cycling was carried out using the following parameters: 94 °C for 2 min followed by 25 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min with a final extension at 72 °C for 2 min. Library construction and sequencing were performed at the Bioengineering Lab (Kanagawa, Japan) using MiSeq (Illumina). Briefly, adaptor and primer regions were trimmed using the FASTX-Toolkit v0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit). Read sequences of ≤40 bp with ambiguous bases and low-quality sequences (quality score, ≤Q20), together with their paired-end reads, were filtered out using Sickle v1.33 (https://github.com/najoshi/sickle). High-quality paired-end reads were merged using PEAR v0.9.10 with default settings [43]. Merged sequences of ≤245 and ≥260 bp were discarded using SeqKit v0.8.0 [44]. Operational taxonomic units (OTUs) were classified using QIIME v1.9.1 and the SILVA database (release 132) with 97% identity. To study the phylogeny of the OTUs assigned to the “Ca. Eremiobacterota”, a neighbor-joining (NJ) tree of the OTUs was constructed [45] as described above.Microscopic observationElectron microscopic observations of the cells were performed at Tokai Electron Microscopy (Nagoya, Japan). Briefly, the cells grown in 10% R2A broth (pH6.0) with air/CO2 (90:10, v/v) at 30 °C for 15 days under a 12/12 h light/dark regime with incandescent light (250 μmol m–2s–1) were fixed with 4% paraformaldehyde (PFA) and 4% glutaraldehyde (GA) in 0.1 M phosphate buffer (PB) at pH7.4, and postfixed with 2% OsO4 in 0.1 M PB. Cells were then dehydrated using graded ethanol solutions. The dehydrated cells were polymerized with resin, ultrathin sectioned, stained with 2% uranyl acetate, then secondary-stained with Lead stain solution. A transmission electron microscope (TEM) (JEM-1400Pus; JEOL, Tokyo, Japan) was used to observe the ultrathin sectioned cells at 100 kV acceleration voltage. To observe the negative-stained cells, PFA- and GA-fixed cells were adsorbed on formvar film-coated copper grids and stained with 2% phosphotungstic acid solution (pH 7.0) and observed using a TEM at 100 kV. DAPI (4,6-diamidino2-phenylindole) and Nile-Red staining was performed by incubating 0.1 mL cell suspension with a 1 mL staining solution (1 mg/L DAPI and 1 mg/L Nile Red in PBS buffer) for 10 min. The stained cells were observed under a fluorescence microscope (Olympus AX80T; Olympus Optical; Tokyo, Japan).Growth assayUnless otherwise noted, all cultures were grown in 100 mL butyl stopper- and screw-cap-sealed glass vials containing 50 mL liquid medium (pH6.0) at 30 °C. Growth was monitored by optical density at 600 nm (OD600) using a spectrophotometer (BioSpectrometer Basic; Eppendorf; Tokyo, Japan). Initial cell density was adjusted to 0.005 (OD600). Specific culture conditions are described below. All anaerobic growth tests were conducted with 100% N2 gas in the headspaces and supplemented with a reducing agent (0.3 g/L cysteine-HCl) and a redox indicator (1 mg/L resazurin).Optimal culture conditionsCell growth in different media was examined using 1, 10, 20, 100% (a full strength) R2A broth and Basal_YE with air/CO2 (90:10, v/v) under a 12/12 h light/dark regime with incandescent light (250 μmol m–2s–1) for 25 days. Basal_YE contained (l−1) 0.44 g KH2PO4, 0.1 g (NH4)2SO4, 0.1 g MgSO4.7H2O, 0.3 g yeast extract, and 1 ml trace element SL-8 [refer to DSMZ745]. Cell growth at different temperatures (10, 13, 20, 25, 30, 33 and 37 °C; pH6.0), pH (3.7, 4.5, 6.0, 7.0, 8.0, and 9.0), and NaCl (0, 1, 10, 20, and 30 g [l−1]; pH6.0) was examined using Basal_YE with air/CO2 (90:10, v/v) for 25 days under a 12/12 h light/dark regime with incandescent light (250 μmol m−2s−1). The following pH buffer solutions were used: acetic acid/sodium acetate for pH 4–6, K2HPO4/KH2PO4 for pH 6–8, sodium bicarbonate/sodium carbonate for pH 9–10. To examine colony formation on solid media, WC8-2 was streaked or stabbed on 10% R2AG medium (pH 6.0) and incubated at 30 °C for 30 days under a 100% air atmosphere.Optimal oxygen and carbon dioxide conditionsTo determine the preferred O2 concentration, WC8-2 was grown in Basal_YE (pH 6.0, 30 °C, no NaCl) in butyl stopper-sealed glass bottles with the atmosphere in the headspace adjusted to different N2/O2/CO2 ratios (70:20:10%, 80:10:10%, 89:1:10%, 90:0:10% v/v) after removing oxygen with 100% N2 gas. Cultures were incubated for 25 days under a 12/12 h light/dark regime with incandescent light (250 μmol m–2s–1). To determine the CO2 preference, the gas phase was adjusted to different N2/O2/CO2 ratios (70:20:10%,75:20:5%, 80:20:0% v/v) in sealed bottles or 100% air (plugged with a BIO-SILICO N-38 sponge plug; Shin-Etsu Polymer Co., Ltd, Tokyo, Japan; breathable culture-plug) under the same conditions as the O2 preference test.Photoorganoheterotrophic (or photoorganoautotrophic) and chemoorganoheterotrophic (or chemoorganoautotrophic) growthThe utilization of organic compounds as carbon sources/organic electron donors was tested in Basal medium (Basal_YE without yeast extract, pH 6.0) supplemented with one of the following sources (l-1): 0.3 ml of glycerol, or 0.3 g of sucrose, d-glucose, d-ribose, maltose, l-leucine, l-isoleucine, l-valine, l-serine, l-lysine, taurine, yeast extract or gellan gum, 1 ml of vitamin B12 solution (2 mg/L). Utilization was assessed by measuring growth of the cultures during a 25-day incubation at 30 °C in continuous light (250 μmol m–2s–1) for photoorganoheterotrophy, or continuous dark for chemoorganoheterotrophy under aerobic (air/CO2 [90:10, v/v]) and anaerobic (N2/CO2 [90:10, v/v]) conditions.Photolithoautotrophic and chemolithoautotrophic growthCells were inoculated into amended PSB2 [46] as described below or Basal medium with 5 mM Na2S or Na2S2O3 or 1% H2 (v/v; in the gas phase) as electron donors, and cultivated in continuous light (250 μmol m–2s–1) for photolithoautotrophic growth, or continuous dark for chemolithoautotrophic growth under aerobic (air/CO2 [90:10, v/v]) and anaerobic (N2/CO2 [90:10, v/v]) conditions for 60 days at 30 °C. The amended PBS2 contained (L-1): 0.5 g NH4Cl, 1.0 g KH2PO4, 0.2 g NaCl, 0.4 g MgSO4.7H2O, 0.05 g CaCl2.2H2O, 4.2 g NaHCO3, 1 ml trace element SL-8 [refer to DSMZ745], and 1 ml vitamin B12 solution (2 mg/L), pH6.0.Fermentative or anaerobic growthAnaerobic growth was examined in continuous dark in 20% R2A broth (pH 6.0) with N2/CO2 (90:10, v/v) supplemented with 5 mM Na2SO4, NaNO3, or dimethyl sulfoxide (DMSO) as electron acceptors for 60 days at 30 °C.Pigment assaysCells grown in Basal_YE (pH6.0) with air/CO2 (90:10, v/v) at 30 °C for 14 days (exponential growth phase) under continuous light (250 μmol m–2 s–1) and continuous dark were used for the pigment assays. The absorption spectrum was determined in a cell suspension in 60% (w/v) sucrose and in a 100% methanol extract using a spectrophotometer (V-630; JASCO, Tokyo, Japan) at 350–1100 nm. The BChl a concentration was determined spectroscopically in 100% methanol [47]. Dry cell weight was measured after harvested cells were washed twice with Milli-Q water and dried at 80 °C for 3 days. The extract was also analyzed by HPLC (NEXERA X2; Shimadzu; Kyoto, Japan) equipped with a 4.6 × 250 mm COSMOSIL 5C18-AR (Nakarai Taque; Tokyo, Japan) with isocratic elution of 92.5% (v/v) methanol in water at a flow rate of 1.0 mL/min. BChl a was monitored at 766 nm using a diode-array spectrophotometer detector (SPD-M20A; Shimadzu; Kyoto, Japan).Observation of taxisTo study phototaxis in WC8-2, cells were grown in 20% R2A broth with air/CO2 (90:10, v/v) for 14 days under a 12/12 h light/dark regimen. Cultures were transferred to tissue culture flasks (175 cm2, canted neck, Iwaki, Shizuoka, Japan). Light sources were ultraviolet (UV) at 395 nm (Linkman, Fukui, Japan), blue at 470 nm (CREE, Durham, NC, USA), green at 502 nm (Linkman), red at 653 nm (LENOO, Shinpei, Taiwan), and near-infrared (NIR) at 880 nm (LENOO). The light-emitting device was constructed by assembling LEDs on a breadboard with a power supply. Cultivations were illuminated with each wavelength using a light-emitting device from the underside and incubated at 20 °C for 18 h. Cells aggregating toward a light source was taken to indicate phototaxis. As a control, culture vessels were wrapped in aluminum foil to block light. Images and time-lapse video were captured using an iPhone 6 S camera.Stable carbon isotope ratio mass spectrometry (IRMS)The WC8-2 cells were grown in Basal_YE (pH6.0) with air/13CO2 [90:10, v/v] and air/unlabeled CO2 [90:10, v/v] under continuous light (250 μmol m–2s–1) for photoheterotroph and continuous dark for chemoheterotroph at 30 °C for 14 days (exponential growth phase). Approximately 10 mg culture biomass was collected, washed in HCl overnight, rinsed three times with deionized water, and placed into tin capsules. Stable carbon isotope ratios (δ13C) were analyzed at Shoko Science (Saitama, Japan) using a Delta V Advantage (EA-IRMS; Thermo Fisher Scientific, Bremen, Germany). The standard for C isotope ratio analysis was Vienna PeeDee Belemnite (VPDB). The δ13C values of 13CO2-cultivated cells exceeded the optimum calibration range of the instrument, but were used in this study to provide conclusive evidence that inorganic carbon was incorporated into the biomass.Quantitative reverse transcription PCR (qRT-PCR)Total RNA extraction and cDNA synthesisTotal RNA was extracted from cells grown in Basal_YE (pH6.0) with air/CO2 [90:10, v/v] in continuous light (250 μmol m–2s–1) for photoheterotrophic conditions and in continuous dark for chemoheterotrophic conditions at 30 °C for 14 days (exponential growth phase) using the Total RNA Purification Kit (Norgen, Biotek Corp, Ontario, Canada). DNA was removed from the extracted nucleic acids using an RNase-Free DNase I Kit (Norgen, Biotek Corp) according to the manufacturer’s protocol. The absence of DNA in the RNA samples was confirmed by PCR without reverse transcriptase. cDNA was generated from 500 ng total RNA using a TaKaRa PrimeScript™ 1st strand cDNA Synthesis Kit (TaKaRa Bio) with random hexamers according to the manufacturer’s protocol.Primer design, specificity and efficiencyThe following three photosynthesis- and CO2-fixation-related genes in the WC8-2 genome were selected for qRT-PCR: bchM encoding an enzyme involved in BChl synthesis, pufL encoding the anoxygenic Type II photochemical reaction centers L-subunit, and cbbL encoding the large subunit of type IE RuBisCO. The RNA polymerase subunit beta (rpoB) was used as a housekeeping reference gene. Primers for qRT-PCR were designed with Primer3 (v. 0.4.0) [48] with the following criteria: product size ranging from 80 to 150 bp, optimum Tm of 60 °C and GC content about 50 to 55%. Standard RT-PCR confirmed that each primer set amplified only a single product with expected size (data not shown), and the product was also sequenced using Sanger sequencing at Macrogen Japan Corp to confirm the candidate products. Primer efficiency was calculated for qRT-PCR using the slope of the calibration curve based on a 20-, 40-, 80-, 160-fold dilution series of cDNA samples [49]. In addition, the specificity of the primers was determined by the confirmation of a single peak in the melting curve. All information about the primers is shown in Table S4.qRT-PCRqRT-PCR was performed using a MiniOpticon Real-Time PCR System (Bio-Rad, Marnes la Coquette, France). The reaction mixture contained 10 μL TB Green Premix Ex Taq II (Tli RNaseH Plus, Takara Bio), 0.8 μL 10 mM primer, 2 μL of a 20-, 40-, 80-, 160-fold dilution series of cDNA, and 6.4 μL water. qRT-PCR was performed using the following protocol: denaturation at 95 °C for 30 s; denaturation and amplification at 95 °C for 5 s and 60 °C for 30 s, respectively (40 cycles). Fluorescence was measured at the end of the amplification step, and amplified products were examined by melting curve analysis from 60 to 95 °C. Each reaction was performed in three independent cultivations. Relative gene expression fold change was calculated using the comparative Ct method (2−ΔΔCt) [49]. Normalized expressions were used for reaction in dark. The 2−ΔΔCt values ≤0.5 were defined as downregulated and values ≥2.0 as upregulated, with P  More

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Data sourcesThis study builds on two datasets; 666 livestock anthrax outbreaks collected over 60 years (1957–2017) by the Kenya Directorate of Veterinary Services (KDVS), and 13 reported anthrax outbreaks we investigated between 2017 and 201811,13. These datasets were combined with data from targeted active anthrax surveillance we conducted in 2019–2020 (see below) to define anthrax suitable areas in Kenya, including hotspots, and subsequently assessed effectiveness of livestock vaccination as a control strategy.Targeted active surveillance-collected anthrax data, 2019–2020Active anthrax surveillance was conducted for 12 months between 2019 and 2020 in randomly selected areas to ensure representation of all AEZs of the country. AEZs are land units defined based on the patterns of soil, landforms and climatic characteristics. Kenya has seven AEZs that include agro-alpine, high potential, medium potential, semi-arid, arid, very-arid and desert. In 2013, Kenya devolved governance into 47 semi-autonomous counties that are subdivided into 290 subcounties which are in turn divided into 1450 administrative wards, the smallest administrative units in the country. Using a geographic map that condensed Kenya into five AEZs; agro-alpine, high potential, medium potential, semi-arid, and arid/very arid zones, we randomly selected 4 administrative sub-counties from each AEZ (N = 20). To increase geographic spread of the study and enhance detection of anthrax outbreaks, we surveilled the larger administrative county (consisting of 20 to 45 administrative wards) where the randomly selected sub-counties were located. As shown in Fig. S1, we ultimately carried out the active anthrax surveillance in 18 counties, containing 523 administrative wards, the latter being used for measuring spatial association (see below).We conducted the surveillance between April 2019 and June 2020, through 523 animal health practitioners (AHPs), one in each ward, after intensive training to identify anthrax using a standard case definition, and to collect and electronically transmit the data weekly using telephone-based short messaging system (SMS) to a central server hosted by KDVS. Regarding case definition, any livestock death classified as anthrax through clinical or laboratory diagnosis was considered an anthrax event. Using standard guidelines issued by the KDVS, a clinical diagnosis was made by the AHPs across the country as an acute cattle, sheep or goat disease characterized by sudden death with or without bleeding from natural orifices, accompanied by absence of rigor mortis. Further, if the carcass was accidentally opened, failure of blood to clot and/or the presence of splenomegaly were included. In pigs, symptoms included swelling of the face and neck with oedema. A laboratory confirmed anthrax was diagnosed using Gram and methylene blue stains followed by identification of the capsule and typical rod-shaped B. anthracis in clinical specimens that the AHPs submitted to the central or regional veterinary investigation laboratories in Kenya. One case of anthrax in either species was considered an outbreak.During the surveillance, the programmed server sent prompting texts directly to the AHPs’ cell phones every Friday of each week for the 52 weeks. The AHPs interacted with the platform by responding to prompting questions sent via SMS to their telephones. Data were securely stored in an online encrypted platform which was subsequently downloaded into Ms Excel for analysis. This surveillance detected 119 anthrax outbreaks, whose partial data were used to model effects of climate change on future anthrax distribution in Kenya14. Here, we integrated these active surveillance data with other datasets to conduct detailed ENM and kernel-smoothed density mapping with a goal of refining suitable anthrax areas including crystalizing hotspots in the country.Anthrax outbreak incidence per livestock population by countyWe knew the total number of livestock per county and wards by species for the active surveillance period. The counties represented the level of disease management including vaccine distribution while the wards within counties represented the modeling unit for targeting control. Therefore, we estimated the outbreak incidence as the total number of outbreaks per livestock species per 100,000 head of that species.Ecological niche modeling and validationWe used boosted regression tree (BRT) algorithm as previously published13. In those studies, we estimated the geographic distribution of anthrax in southern Kenya using 69 spatially unique outbreak points (thinned from the 86 outbreaks in the records) and 18 environmental variables resampled to 250 m resolution. In this study, the final experiments were run with a learning rate (lr) = 0.001, bagging fraction (br) = 5, and maximum tree = 2500. We then mapped anthrax suitability as the mean output of the 100 experiments and the lower 2.5% and upper 97.5% mapped as confidence intervals. We determined variable contribution and derived partial dependence as previously described13. As BRTs are a random walk and each experiment randomly resamples training and test data, it was necessary to repeat those outputs along with the map predictions.Here, our goal was to evaluate the BRT models built with records data from 2011 to 2017 data and use the predict function to calculate model accuracy metrics using the 2017–2020 outbreaks as presence points and the sub-counties reporting zero outbreaks during the 2019–2020 active surveillance period as absence points. The model of southern Kenya was projected onto all of Kenya using climate variables clipped to the whole of Kenya. We tested the BRT models in two ways; first, evaluating 2011–2017 data models with holdout data using a random resampling and multi-modeling approach. Here, we report the area under curve (AUC) for each of the original training/testing split into the 69 historical points and the 2017–2020 data serving as independent data, the latter representing true model validation. Second, to determine the total percentage of surveillance data predicted and map areas of anthrax suitability to compare with kernel density estimates (see below), we produced a dichotomized map using the Youden index cutoff17 following Otieno et al.14.Outbreak concentrations from kernel density estimation (KDE)To describe the spatial concentration of reported outbreaks, we calculated descriptive spatial statistics, including the spatial mean, standard distance, and standard deviational ellipse of outbreak locations from the prospective surveillance dataset following Blackburn et al.18 These spatial statistics help to differentiate the geographic focus (spatial mean) and dispersion of outbreak reports from year to year and across the sampling period. We then conducted kernel density estimation (KDE) to visualize the concentration of anthrax outbreaks per square kilometer per year and across the study period18. We used the spatstat package for all KDE analyses using the quadratic kernel function19:$$fleft( x right) = frac{1}{{nh^{2} }} mathop sum limits_{i = 1}^{n} Kleft( {frac{{x – X_{i} }}{h}} right)$$where h is the bandwidth, x-Xi is the distance to each anthrax outbreak i. Finally, K is the quadratic kernel function, defined as:$$Kleft( x right) = frac{3}{4}left( {1 – x^{2} } right), left| x right| le 1$$$$Kleft( x right) = 0,x > 1$$This function was employed to estimate anthrax outbreak concentration across space using each outbreak weighted as one. We calculated the bandwidth (kernel) using hopt that uses the sample size (number of outbreaks) and the standard distance to estimate bandwidth. Finally, we estimated bandwidth for each year and then averaged them to apply the same fixed bandwidth for each year under study in Q-GIS version 3.1.8. The resulting outputs were map surfaces representing the spatial concentrations of outbreaks across the country per 1 km2 for each study year and all study years combined. For this study, we used the cutoff criteria of Nelson and Boots19 to identify outbreak hotspots as areas with density values in the upper 25%, 10%, and 5% of outbreak concentrations. The analyses identified these areas by year (2017–2020) and for all surveillance years combined.Local spatial clustering at the ward levelAnthrax outbreak incidence per livestock speciesThe ENM and KDE-derived maps provide a first estimate of potential risk and outbreak concentration, respectively. We were also interested in estimating anthrax outbreak intensity relative to livestock populations at a local level. For the active surveillance period, we knew the total number of outbreaks per ward (the smallest administrative spatial unit) by livestock species. For this two-year period, we estimated the ward-level outbreak incidence as the total number of outbreaks per livestock species per 10,000 head of that species. To estimate livestock population per ward, we extracted the values in the raster file of the areal weighted gridded livestock of the world data using the zonal statistic routine in Q-GIS version 3.1.8, into the polygon consisting of all pixels per ward as the total population19,20. We calculated outbreak incidence as the number of outbreaks per ward cattle population per 10,000 cattle for each administrative ward. We limited this analysis to those 18 counties participating in the active surveillance study (Fig. S1), as we could appropriately assume any ward with no reports was a ‘true zero’ for the estimation. Given that most reported outbreaks were in domestic cattle (see results below), we here report those results involving cattle alone. Given the overall high number of wards and the high number of wards without outbreaks, we performed the empirical Bayes smoothing and spatial Bayes smoothing routines in GeoDa version 1.12.1.161 to reduce the variance in anthrax incidence estimates20,21. To evaluate smoothing routine performance, we box plotted rates per ward and selected the method with the greatest reduction in outliers21. Smoothed rates were mapped as choropleth map in Q-GIS version 3.1.8 using the four equal area bins.Spatial cluster analysisWe used Local Moran’s I16 to test for spatial cluster of livestock anthrax in cattle using the smoothed outbreak incidence estimates. The Local Moran’s I statistic tests whether individual wards are part of spatial cluster, like incidence estimates surrounded by similar estimate (high-high or low-low) or spatial outliers where wards with significantly high or low estimates are surrounded by dissimilar values (high-low or low–high). The local Moran’s I is written as16:$$I_{i} = Z_{i} sum W_{ij} Z_{j}$$where Ii is the statistic for a ward i, Zi is the difference between the incidence at i and the mean anthrax incidence rate for all of wards in the study, Zj is the difference between anthrax risk at ward j and the mean for all wards. Wij is the weights matrix. In this study, the 1st order queen contiguity was employed. Here, Wij equals 1/n if a ward shared a boundary or vertex and 0 if not. For this study, Local Moran’s I was performed on the wards using 999 permutations and p = 0.05 using GeoDa version 1.12.1.161.Assessing effectiveness of cattle vaccination in burden hotspotsAs a first estimate of how we might scale up livestock anthrax vaccination efforts in Kenya, we slightly adjusted a simple published anthrax outbreak simulation model in a cattle population. For this study we applied an early mathematical approach of Funiss and Hahn22 to simulate anthrax at the ward level. While other recent models are available23,24, these are difficult to parameterize or require time series data we could not derive with the surveillance approach in this study. Like the more recent models, Funiss and Hahn22 assumed anthrax transmission was driven by cattle accessing spore-contaminated environments. Here the proportion of infected cattle each day depended on the population of susceptible animals in the population and probability of getting infected. This probability depends on environmental contamination (“a”), and a fraction of anthrax carcasses in the environment on a day (“f,”). Each day, the newly infected cattle are transferred to an incubation period vector, “d,” waiting to die following a probability “p”. In this model, all infected animals, “n,” die following the incubation periods given by the vector, “p”, in which pi is the probability of a cow dying i days after the infection. Following death, the cattle are transferred to a carcass state, providing a direct infection source to the susceptible cattle via environmental contamination. Environmental contamination “a,” is therefore defined as the number of spores ingested by an animal in a day. This environmental contamination depends on spores from carcasses and an assumed spore decay rate γ22.The complete set of difference equations with a daily time step is given by:$${text{S}}_{(t + 1)} = {text{S}}_{(t)} – {text{ S}}_{(t)} *left( {{1} – {text{e}}^{{ – left( {{text{a}}_{t} + gamma {text{f}}_{{{text{t}} + 1}} } right)}} } right)$$$${text{I}}_{(t + 1)} = {text{I}}_{(t)} + {text{ S}}_{(t)} *left( {{1} – {text{e}}^{{ – left( {{text{a}}_{{text{t}}} + gamma {text{f}}_{{{text{t}} + {1}}} } right)}} } right)$$where the expression (left( {{1} – {text{e}}^{{ – left( {{text{a}}_{t} + gamma {text{f}}_{{{text{t}} + 1}} } right)}} } right)) denotes the probability of an animal becoming infected and at + γft+1 is the mean number of spores ingested by a cow in a day. The equation for environmental contamination, a, is given by:$${text{a}}_{t + 1} {-}{text{a}}_{{text{t}}} = alpha {text{a}}_{{text{t}}} + beta {text{c}}_{{{text{t}} + {1}}}$$The newly infected animals die after a certain number of days. The distribution of incubation periods is given by the vector, p. On each day, the new cases are placed in a due-to-die vector, d, and when they die, they are subsequently moved down one step to fresh carcasses, ft. The fresh carcasses provide a direct source of infection to the susceptible cattle via the ‘fresh carcass term’, γ. These carcasses decay or are scavenged or disposed by man. The equation expressing the disseminating carcasses, c, is:$${text{C}}_{t + 1} – {text{c}}_{t} = {text{f}}_{t + 1} – delta {text{c}}_{t}$$The model parameters variables are provided in Table 1 and are similar to those used by Funiss and Hahn22 to generate a standard run. We ran the model for one year and extrapolated to cattle population in the identified hotspot wards.Table 1 Model parameters and variables.Full size table More

Site selection and sampling strategyField sites (Table 1) were selected to represent typical natural vegetation types showing little or no signs of disturbance. Although much of the natural vegetation of China has been altered by human activities, there are still extensive areas of natural vegetation. Access to these areas is facilitated by the existence of a number of ecological transects39,40, the ChinaFlux network (http://www.chinaflux.org) and the Chinese Ecosystem Research Network (http://www.cern.ac.cn/0index/index.asp).About half the sites in CPTDv1 used a stratified sampling approach and this approach was used at all of the new sites added in the CPTDv2. This sampling strategy involves sampling the dominant species within each vegetation stratum so as to be able to characterise trait values at community level18. Specifically, a total of 25 trees, 5 shrubs, 5 lianas or vines, and 5 understorey species (grasses, forbs) were sampled at each site. When there were less than 25 trees at a site, all of the tree species were sampled and additional examples from the other categories were included up to the maximum of 40 species. If there are more than the maximum sampling number in any one category, then the dominant (i.e. most common) representatives of each category were sampled. Sampled individuals of each species were mature, healthy plants. In principle, sun leaves (i.e. leaves in the canopy and fully exposed to sunlight) were sampled. For true shade-tolerant and understory species, the sampled individuals were those in well-lit environments and isolated to minimize interactions with other individuals.Nineteen sites from Xinjiang included in CPTDv1 used a simplified sampling strategy, where only canopy species were sampled. Sixteen sites from Xinjiang were particularly depauperate and thus only a limited number of species were sampled without consideration of abundance. These sites are retained in the database because they sample extremely arid location with α typically less than 0.25Species identification and taxonomic standardisationSampled plants were identified in the field by a taxonomist familiar with the local vegetation, most usually using a regional flora. Species names were subsequently standardised using the online version of the Flora of China (http://www.efloras.org/flora_page.aspx?flora_id=2). Where field-identified species were not accepted or included in the Flora of China, and thus could not be assigned unambiguously to an accepted taxonomic name, we cross-checked whether the species were listed in the Plant List (http://www.theplantlist.org/) (or alternative sources such as the Virtual Herbarium of China, Plants of the World Online or TROPICOS) in order to identify synonyms for these accepted names that were recognised by the Flora of China. In cases where we were unable to identify an accepted name consistent with the Flora of China, we retained the field-assigned name by default (Fig. 3). The decisions about taxonomy are described in the CPTDv2 table “Taxonomic Standardisation” (Table 2). The names assigned originally in the field and the accepted standardized names used in the database are given in the CPTDv2 table “Species Translations” (Table 3). When species were recognised in the Flora of China, we provide the Chinese translation of the species name. The written Chinese nomenclature system does not follow the Linnaean system, so this table of “Species Chinese Name” is designed to facilitate the use of the database by botanists in China (Table 4). There are no translations of names that are not recognized by the Flora of China and are used in the database by default.Fig. 3Flowchart showing the decision tree used to determine the names used in the China Plant Database (accepted names) and encapsulated in the Taxonomic Standardization table. ‘=1’ and ‘ >1’ indicate the number of Synonyms is equal or more than one.Full size imageDataset collection methodsPhotosynthetic pathwayInformation on photosynthetic pathway (Table 5) was obtained for each species from the literature. There are a large number of literature compilations on the photosynthetic pathway of Chinese plants (e.g.41,42,43,44,45,46. Where this information was not available from Chinese studies we used similar compilations from other regions of the world (e.g.47,48,49,50,51,52. Since C4 plants have much less carbon discrimination than C3 plants, the measurements on δ13C were also used as an indicator of the photosynthetic pathway53,54,55,56. δ13C value of –20‰ was applied as a threshold of C3 photosynthetic pathway distinction54. Information about photosynthetic pathway was not included for a species unless confirmed from the literature or δ13C measurements.Leaf physical and chemical traitsPhysical and chemical properties (Table 6) were measured on samples collected in the field following standard methods37. At least 10 g of leaves were collected for each species. Sunlit leaves of tree species were obtained with long-handled twig shears. The samples were subdivided for the measurement of specific leaf area, leaf dry matter content and the contents of carbon, nitrogen, phosphorus and potassium. Recorded values were the average of three replicates. Leaf area was determined by scanning five leaves (or more in the case of small leaves, to make up a total area ≥20 cm2 per species) with a laser scanner. Areas (Average LA) were measured using Photoshop on the scanned images. Leaf fresh weight was measured in the field. Dry weight was obtained after air drying for several days and then oven drying at 75 °C for 48 hours. Leaf dry matter content (LDMC) was expressed as leaf oven-dry weight divided by fresh weight. Specific leaf area (SLA) was then expressed as the ratio between leaf area and leaf dry mass. LMA is the inverse of SLA. Leaf carbon content (Cmass) was measured by the potassium dichromate volumetric method and leaf nitrogen content (Nmass) by the Micro-Kjeldahl method. Leaf phosphorus (Pmass) was analysed colorimetrically (Shimadzu UV-2550). Leaf potassium (Kmass) was measured by Flame Atomic Emission Spectrophotometry (PE 5100 PC). The area-based leaf chemical contents (Carea, Narea, Parea, Karea) were derived as a product of mass-based content and LMA. δ13C (d13C:12C) and δ15N (d15N:14N) were measured using the Isotope Ratio Mass Spectrometer (Thermo Fisher Scientific Inc., USA; Finnigan Corporation, San Jose, CA).Photosynthetic traitsSeveral different methods were used to characterise photosynthetic traits (Supplementary Table 1). Chlorophyll fluorescence measurements were made at the sites along Northeast China Transect. These measurements were recorded as the potential (Fv/Fm) and actual (QY) rates of photosynthetic electron transport. QY is correlated with photosynthetic rate, although it also includes the diversion of electrons to non-photosynthetic activities such as the elimination of reactive oxygen species57. Measurements of photosynthetic traits at most of the sites (about 68% of samples with photosynthetic measurements) were derived from leaf gas-exchange measurements in light-saturated conditions under either ambient or high CO2 levels, made with a portable infrared gas analyser (IRGA) system (LI-6400; Li-Cor Inc., Lincoln, NB, USA). Sunlit terminal branches from the upper canopy were collected and re-cut under water immediately prior to measurement. Measurements were made in the field with relative humidity and chamber block temperature close to that of the ambient air at the time of measurement, and a constant airflow rate (500 μmol s−1). The maximum capacity of carboxylation (Vcmax) and electron-transport (Jmax) were calculated from the light-saturated rate of net CO2 fixation at ambient and high CO2 level respectively using the one-point method for Vcmax58 and two-point method for Jmax59. Although it was indicated that applying one-point method could result in around 20% error in measuring photosynthetic capacity60, this time-saving method indeed allows much more samples to be measured in the field. For sites in CPTDv1, the Vcmax and Jmax values were made on a single specimen of each species at each site, due to the time-consuming nature of the measurement. For the newly collected sites in CPTDv2, for each species the Vcmax and Jmax were measured on three samples collected from three individual tress. The average values were recorded in the database. For Vcmax measurements, the CO2 level was set as the ambient atmospheric CO2 level, ranging from 380 ppm to 400 ppm. The leaves were exposed to a typical photosynthetic photon flux density (PPFD) of 1800 μmol m−2 s−1 with the light source. Pre-processing method was applied to determine the saturating PPFD for alpine plants, which goes up to 2000 μmol m−2 s−1 in the high elevation sites from Mountain Gonga. For Jmax measurements, the CO2 level was set as 1500 ppm or 2000 ppm to avoid any limitation on photosynthesis via carboxylation.There are a few cases (1 site from Cai, et al.61, and 8 sites from Zheng and Shangguan62, Zheng and Shangguan63), where field-measured ratio of leaf internal- to ambient-CO2 concentration (ci:ca) were not provided. In these cases, estimates of the ci:ca ratio were made from δ13C measurements using the method of64 to calculate isotopic discrimination (Δ) from δ13C (correcting for atmospheric δ13C, approximated as a function of time of collection and latitude), and the Ubierna and Farquhar65 method to calculate isotopic discrimination (Δ) from δ13C considering discrimination during stomatal diffusion and carboxylation. The R code for calculating Vcmax and Jcmax from original data was provided (seeing Code availability).Hydraulic traitsCPTDv2 contains information on four important hydraulic traits: specific sapwood conductivity, the sapwood to leaf area ratio (Huber value, vH), turgor loss point and wood density (Table 7). Hydraulic traits were measured on branches with a diameter wider than 7 mm, cut as close to the bifurcation point as possible to minimize any effect of measurement location on measured area. A section was taken from the part of the branch nearest to the bifurcation point, and the cross-sectional area of the xylem was measured at both ends of this section using digital calipers. Sapwood area was calculated as the average of these two measurements. All leaves attached to the branch were removed and dried at 70 °C for 72 hours before weighing. The total leaf area was obtained from dry mass and LMA. vH was calculated as the ratio of sapwood area and leaf area. The vH value recorded for each species at each site was the average of three measurements made on branches from different individuals.Five branches from at least three mature individuals of each species at each site were collected, wrapped in moist towels and sealed in black plastic bags, and then immediately transported to the laboratory. All the samples were re-cut under water, put into water and sealed in black plastic bags to rehydrate overnight. Sapwood-specific hydraulic conductivity, (KS) was measured using the method of Sperry, et al.66. Segments (10–15 cm length) were cut from the rehydrated branches and flushed using 20 mmol L−1 KCl solution for at least 30 minutes (to remove air from the vessels) until constant fluid dripped from the section. The segments were then placed under 0.005 MPa pressure to record the time (t) they took to transport a known water volume (W, m3). Length (L, m), sapwood area of both ends (S1 and S2, m2) and temperature (Tm, °C) were recorded. Sapwood-specific hydraulic conductivity at measurement temperature (KS,m, mol m−1 s−1 MPa−1) was calculated using Eq. (1). This was transformed to KS at mean maximum temperature during the growing season (KS,gt) and standard temperature (KS25) following Eqs. (2–3):$${K}_{S,m}={W,L{rho }_{w}/[0.005,t({S}_{1}+{S}_{2})/2]}(1000/,18)$$
(1)
$${K}_{S,t}={K}_{S,m}{eta }_{m}/{eta }_{t}$$
(2)
$$eta =1{0}^{-3}exp[A+B/,(C+T)]$$
(3)
where ηm and ηt (Pa s) are the water viscosity at measurement temperature and transformed temperature (i.e. mean maximum daytime temperature during the growing season and at a standard temperature of 25 °C), respectively, and ρw (kg m−3) is the density of water. The parameter values used in Eq. (3) were A = −3.719, B = 580 and C = −13867.A small part of each sapwood segment was used to measure wood density, the ratio of dry weight to volume of sapwood. After removal of bark and heartwood, the volume of sapwood was measured by displacement and the sapwood dry weight was obtained after drying at 70 °C for 72 hours to constant weight.The method described by Bartlett, et al.68 was used for the rapid determination of turgor loss point (Ψtlp). After rehydration overnight, discs were sampled using a 6-mm-diameter punch from mature, healthy leaves collected on each branch, avoiding major and minor veins. Leaf discs wrapped in foil were frozen in liquid nitrogen for at least 2 minutes and then punctured 20 times quickly with sharp-tipped tweezers. Five repeat experiments using leaves from multiple individuals were carried out for every species at each site. The osmotic potential (Ψosm) was measured with a VAPRO 5600 vapor pressure osmometer (Wescor, Logan, UT, USA) and Ψtlp (in MPa) was calculated as:$${Psi }_{tlp}=0.83{2Psi }_{osm}-0.631$$
(4)
Morphometric traitsThe morphometric trait data (Supplementary Table 2) were measured systematically by the same people (SPH and ICP) at all the sites. A standardized template for the field measurement of morphometric traits was used (Supplementary Table 5). This template provides a checklist of the traits and the categories used to describe them. The leaf traits assessed were texture, colour, size, thickness, orientation, display, shape, margin form, the presence of hairs, pubescence, pruinosity or rugosity, the presence of surface wax, hypostomatism, marginal curling (involute, revolute), smell (aromatic or fetid), the presence of a terminal notch or drip-tip, surface patterning, succulence, the presence and positioning of spines or thorns on the leaves. Illustrations of the various categories used in the classification of leaf margin and leaf shape are provided in supplementary materials, together with the template for leaf size categories (Supplementary Figs. 1–3). Although the distinction between spines and thorns is sometimes based on the source material (where thorns are derived from shoots and buds, and spines from any part of the leaf containing vascular material), here the differentiation is based on the shape of the protrusion (where thorns are triangular in shape and can be branched, and spines are unbranched and linear features). The checklist template also includes a limited amount of information on stem traits, such as form, colour, whether the stem is photosynthetic, the presence of stem hairs, pubescence, or pruinosity, and the presence of spines or thorns. For woody plants (trees, shrubs, climbers), the checklist also includes information on bark type (deciduous or not, with an indication of whether the bark is strip or chunk deciduous), the presence of furrowing, and also the presence of spines or thorns.Plant Functional TypesThe database includes information on life form, plant phenology, leaf form and leaf phenology (Table 8). Although these four pieces of information are used by many modellers in the definition of plant functional types (PFTs)69,70, they are not strictly species-specific traits. Thus, some species can occur as a tree, a small tree or a shrub (e.g. Cyclobalanopsis obovatifolia), or as a shrub or liana (e.g. Smilax discotis), depending on environmental conditions. Similarly, some species can behave as an evergreen or deciduous plant, depending on moisture availability (e.g. Ulmus parvifolia). Thus, this information is recorded for individual species at each site and no attempt was made to ensure that a given species was classified identically at all sites. In total 20 distinct life forms were recognized, including tree, small tree, low to high shrub, erect dwarf shrub, prostrate dwarf shrub, trailing shrub, liana, climber, forb, cushion forb, rosette forb, graminoid, bamboo, cycad, geophyte, stem succulent, succulent, pteridophyte, epiphyte, parasite. Plant phenology is recorded as perennial, biennial or annual. The primary distinction in leaf phenology is between deciduous and evergreen, but the classification used in the database also recognizes facultative deciduousness (semi-deciduous) and leaf-exchangers (i.e. plants that retain their leaves for nearly the whole year but drop and replace all of the leaves in a single short period, rather than replacing some leaves continuously through the year as evergreens do). The concept of leaf phenology is only relevant for woody plants (trees, shrubs, lianas) and so is not recorded for e.g. forbs or climbers.VegetationThe local vegetation was not recorded in the field at each site, and in any case such descriptions are hard to standardize. The CPTDv2 database contains information on vegetation type extracted from the digital vegetation map of China at the scale of 1:1 million71, which uses 55 plant communities (48 natural plant communities and seven cropping systems). CPTDv2 further provides information on vegetation clusters aggregated from those fundamental plant communities from the Vegetation Atlas of China based on their bioclimatic context72. CPTDv2 also contains information on potential natural vegetation (PNV), derived from an updated version of the73 global mapping of PNV. This PNV map was produced using pollen-based vegetation reconstructions as a target, a set of 160 spatially explicit co-variate data sets representing the climatic, topographic, geologic, and hydrological controls on plant growth and survival, and an ensemble machine-learning approach to account for the relationships between vegetation types and these covariates (Table 9). The original version of the map had a spatial resolution of 1 km; the updated version used here (https://github.com/Envirometrix/PNVmaps) has a resolution of 250 m.ClimateClimatological estimates of monthly temperature, precipitation and fraction of sunshine hours were derived from records from 1814 meteorological stations (740 stations have observations from 1971 to 2000, the rest from 1981 to 1990: China Meteorological Administration, unpublished data), interpolated to a 0.01 grid using a three-dimensional thin-plate spline (ANUSPLIN version 4.36;74. These monthly climatological data were used directly to calculate the mean temperature of the coldest month (MTCO), mean annual temperature (MAT), mean monthly precipitation (MMP) and mean annual precipitation (MAP). Bioclimatic variables at each site were calculated from the interpolated monthly temperature, precipitation and fraction of sunshine hours using the Simple Process-Led Algorithms for Simulating Habitats (SPLASH) model75. The bioclimatic variables include total annual photosynthetically active radiation during the growing season when mean daily temperatures are >0 °C (PAR0), the daily mean photosynthetically active radiation during the growing season (mPAR0), growing degree days above a baseline of 0 °C (GDD0), the daily mean temperature during the growing season (mGDD0), the ratio of actual to equilibrium evapotranspiration (α), and a moisture index (MI) defined as the ratio of mean annual precipitation to potential evapotranspiration. We also calculated the timing of peak rainfall and rainfall seasonality, using metrics described in Kelley, et al.76 (Supplementary Table 3).The topography in the Gongga region is complex, and the standard climate data set is inadequate to capture the elevation impacts of local climate at the sites there13. We therefore also provide alternative estimates of climatic variables for the Gongga elevation transects using 17 weather stations from the region with records from January 2017 to December 2019 (Supplementary Table 4). These 17 stations range in elevation from 422 m to 3951 m, in latitude from 28° to 31° N, and in longitude from 99.1° to 103.8° E. The climatological records for each station were downloaded from China Meteorological Data Service Centre, National Meteorological Information Centre (http://data.cma.cn/data/detail/dataCode/A.0012.0001.html). The monthly maximum and minimum temperature, precipitation, percentage of possible sunshine hours were extracted. The monthly mean temperature was calculated as the average of maximum and minimum temperature. The elevationally-sensitive ANUSPLIN interpolation scheme74 was used to provide estimates of meteorological variables at each site as described above. The bioclimatic variables were calculated following the same methodology as the 0.01 grid data described above.SoilSoil was not sampled in the field, but to facilitate analyses we provide soil information extracted from the Harmonized World Soil Database (HWSD) v1.277 (Table 10). The HWSD v1.2 is a high-resolution (0.05°) soil database with soil characteristics determined from real soil profiles. The soil properties were estimated in a harmonized way, where the actual soil profile data and the development of pedotransfer rules were undertaken in cooperation with ISRIC and ESBN drawing on the WISE soil profile database and some earlier works78,79. The HWSD v1.2 provides information for the uppermost soil layer (0–30 cm) and the deeper soil layer (30–100 cm). Although HWSD v1.2 contains information on a large number of soil properties, we only extracted information on soil texture (sand fraction, silt fraction and clay fraction), the content of organic carbon, soil pH in water, and cation exchange capacity. More

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