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    Climate-induced range shifts drive adaptive response via spatio-temporal sieving of alleles

    Study populations and sequencing strategyDNA libraries were prepared for 1261 D. sylvestris individuals from 115 populations (5–20 individuals per population) under a modified protocol49 of the Illumina Nextera DNA library preparation kit (Supplementary Methods S1.1, Supplementary Data 1). Individuals were indexed with unique dual-indexes (IDT Illumina Nextera 10nt UDI – 384 set) from Integrated DNA Technologies Co, to avoid index-hopping50. Libraries were sequenced (150 bp paired-end sequencing) in four lanes of an Illumina NovaSeq 6000 machine at Novogene Co. This resulted in an average coverage of ca. 2x per individual. Sequenced individuals were trimmed for adapter sequences (Trimmomatic version 0.3551), mapped (BWA-MEM version 0.7.1752,53) against a reference assembly54 (ca. 440 Mb), had duplicates marked and removed (Picard Toolkit version 2.0.1; http://broadinstitute.github.io/picard), locally realigned around indels (GATK version 3.555), recalibrated for base quality scores (ATLAS version 0.956) and had overlapping read pairs clipped (bamUtil version 1.0.1457) (Supplementary Methods S1.1). Population genetic analyses were performed on the resultant BAM files via genotype likelihoods (ANGSD version 0.93358 and ATLAS versions 0.9–1.056), to accommodate the propagation of uncertainty from the raw sequence data to population genetic inference.Population genetic structure and biogeographic barriersTo investigate the genetic structure of our samples (Fig. 2A, Supplementary Fig. S2), we performed principal component analyses (PCA) on all 1261 samples (“full” dataset) via PCAngsd version 0.9859, following conversion of the mapped sequence data to ANGSD genotype likelihoods in Beagle format (Supplementary Methods S1.2). To visualise PCA results in space (Supplementary Fig. S4), individuals’ principal components were projected on a map, spatially interpolated (linear interpolation, akima R package version 0.6.260) and had the first two principal components represented as green and blue colour channels. Given that uneven sampling can bias the inference of structure in PCA, PCA was also performed on a balanced dataset comprising a common, down-sampled size of 125 individuals per geographic region (“balanced” dataset; Fig. 2B, Supplementary Fig. S3; Supplementary Methods S1.2; Supplementary Data 1). Individual admixture proportions and ancestral allele frequencies were estimated using PCAngsd (-admix model) for K = 2–6, using the balanced dataset to avoid potential biases related to imbalanced sampling22,23 and an automatic search for the optimal sparseness regularisation parameter (alpha) soft-capped to 10,000 (Supplementary Methods S1.2). To visualise ancestry proportions in space, population ancestry proportions were spatially interpolated (kriging) via code modified from Ref. 61 (Supplementary Fig. S5).To test if between-lineage admixture underlies admixture patterns inferred by PCAngsd or if the data is better explained by alternative scenarios such as recent bottlenecks, we used chromosome painting and patterns of allele sharing to construct painting palettes via the programmes MixPainter and badMIXTURE (unlinked model)28 and compared this to the PCAngsd-inferred palettes (Fig. 2B, C; Supplementary Methods S1.2). We referred to patterns of residuals between these palettes to inform of the most likely underlying demographic scenario. For assessing Alpine–Balkan palette residuals (and hence admixture), 65 individuals each from the French Alps (inferred as pure Alpine ancestry in PCAngsd), Monte Baldo (inferred with both Alpine and Balkan ancestries in PCAngsd) and Julian Alps (inferred as pure Balkan ancestry in PCAngsd) were analysed under K = 2 in PCAngsd and badMIXTURE (Fig. 2C). For assessing Apennine–Balkan admixture, 22 individuals each from the French pre-Alps (inferred as pure Apennine ancestry in PCAngsd), Tuscany (inferred with both Apennine and Balkan ancestries in PCAngsd) and Julian Alps (inferred as pure Balkan ancestry in PCAngsd) were analysed under K = 2 in PCAngsd and badMIXTURE.To construct a genetic distance tree (Supplementary Fig. S1), we first calculated pairwise genetic distances between 549 individuals (5 individuals per population for all populations) using ATLAS, employing a distance measure (weight) reflective of the number of alleles differing between the genotypes (Supplementary Methods S1.2; Supplementary Data 1). A tree was constructed from the resultant distance matrix via an initial topology defined by the BioNJ algorithm with subsequent topological moves performed via Subtree Pruning and Regrafting (SPR) in FastME version 2.1.6.162. This matrix of pairwise genetic distances was also used as input for analyses of effective migration and effective diversity surfaces in EEMS25. EEMS was run setting the number of modelled demes to 1000 (Fig. 2A, Supplementary Fig. S8). For each case, ten independent Markov chain Monte Carlo (MCMC) chains comprising 5 million iterations each were run, with a 1 million iteration burn-in, retaining every 10,000th iteration. Biogeographic barriers (Fig. 2A, Supplementary Fig. S7) were further identified via applying Monmonier’s algorithm24 on a valuated graph constructed via Delauney triangulation of population geographic coordinates, with edge values reflecting population pairwise FST; via the adegenet R package version 2.1.163. FST between all population pairs were calculated via ANGSD, employing a common sample size of 5 individuals per population (Supplementary Fig. S6; Supplementary Methods S1.2; Supplementary Data 1). 100 bootstrap runs were performed to generate a heatmap of genetic boundaries in space, from which a weighted mean line was drawn (Supplementary Fig. S7). All analyses in ANGSD were performed with the GATK (-GL 2) model, as we noticed irregularities in the site frequency spectra (SFS) with the SAMtools (-GL 1) model similar to that reported in Ref. 58 with particular BAM files. All analyses described above were performed on the full genome.Ancestral sequence reconstructionTo acquire ancestral states and polarise site-frequency spectra for use in the directionality index ψ and demographic inference, we reconstructed ancestral genome sequences at each node of the phylogenetic tree of 9 Dianthus species: D. carthusianorum, D. deltoides, D. glacialis, D. sylvestris (Apennine lineage), D. lusitanus, D. pungens, D. superbus alpestris, D. superbus superbus, and D. sylvestris (Alpine lineage). This tree topology was extracted from a detailed reconstruction of Dianthus phylogeny based on 30 taxa by Fior et al. (Fior, Luqman, Scharmann, Zemp, Zoller, Pålsson, Gargano, Wegmann & Widmer; paper in preparation) (Supplementary Methods S1.3). For ancestral sequence reconstruction, one individual per species was sequenced at medium coverage (ca. 10x), trimmed (Trimmomatic), mapped against the D. sylvestris reference assembly (BWA-MEM) and had overlapping read pairs clipped (bamUtil) (Supplementary Methods S1.3). For each species, we then generated a species-specific FASTA using GATK FastaAlternateReferenceMaker. This was achieved by replacing the reference bases at polymorphic sites with species-specific variants as identified by freebayes64 (version 1.3.1; default parameters), while masking (i.e., setting as “N”) sites (i) with zero depth and (ii) that didn’t pass the applied variant filtering criteria (i.e., that are not confidently called as polymorphic; Supplementary Methods S1.3). Species FASTA files were then combined into a multi-sample FASTA. Using this, we probabilistically reconstructed ancestral sequences at each node of the tree via PHAST (version 1.4) prequel65, using a tree model produced by PHAST phylofit under a REV substitution model and the specified tree topology (Supplementary Methods S1.3). Ancestral sequence FASTA files were then generated from the prequel results using a custom script.Expansion signalTo calculate the population pairwise directionality index ψ for the Alpine lineage, we utilised equation 1b from Peter and Slatkin (2013)31, which defines ψ in terms of the two-population site frequency spectrum (2D-SFS) (Supplementary Methods S1.4). 2D-SFS between all population pairs (10 individuals per population; Supplementary Data 1) were estimated via ANGSD and realSFS66 (Supplementary Methods S1.4), for unfolded spectra. Unfolding of spectra was achieved via polarisation with respect to the ancestral state of sites defined at the D. sylvestris (Apennine lineage) – D. sylvestris (Alpine lineage) ancestral node. Correlation of pairwise ψ and (great-circle) distance matrices was tested via a Mantel test (10,000 permutations). To infer the geographic origin of the expansion (Fig. 3), we employed a time difference of arrival (TDOA) algorithm following Peter and Slatkin (2013);31 performed via the rangeExpansion R package version 0.0.0.900031,67. We further estimated the strength of the founder of this expansion using the same package.Demographic inferenceTo evaluate the demographic history of D. sylvestris, a set of candidate demographic models was formulated. To constrain the topology of tested models, we first inferred the phylogenetic tree of the three identified evolutionary lineages of D. sylvestris (Alpine, Apennine and Balkan) as embedded within the larger phylogeny of the Eurasian Dianthus clade (note that the phylogeny from Fior et al. (Fior, Luqman, Scharmann, Zemp, Zoller, Pålsson, Gargano, Wegmann & Widmer; paper in preparation) excludes Balkan representatives of D. sylvestris). Trees were inferred based on low-coverage whole-genome sequence data of 1–2 representatives from each D. sylvestris lineage, together with whole-genome sequence data of 7 other Dianthus species, namely D. carthusianorum, D. deltoides, D. glacialis, D. lusitanus, D. pungens, D. superbus alpestris and D. superbus superbus, that were used to root the D. sylvestris clade (Supplementary Methods S1.5). We estimated distance-based phylogenies using ngsDist68 that accommodates genotype likelihoods in the estimation of genetic distances (Supplementary Methods S1.5). Genetic distances were calculated via two approaches: (i) genome-wide and (ii) along 10 kb windows. For the former, 110 bootstrap replicates were calculated by re-sampling over similar-sized genomic blocks. For the alternative strategy based on 10 kb windows, window trees were combined using ASTRAL-III version 5.6.369 to generate a genome-wide consensus tree accounting for potential gene tree discordance (Supplementary Methods S1.5). Trees were constructed from matrices of genetic distances from initial topologies defined by the BioNJ algorithm with subsequent topological moves performed via Subtree Pruning and Regrafting (SPR) in FastME version 2.1.6.162. We rooted all resultant phylogenetic trees with D. deltoides as the outgroup70. Both approaches recovered a topology with the Balkan lineage diverging prior to the Apennine and Alpine lineages (Supplementary Fig. S9). This taxon topology for D. sylvestris was supported by high ASTRAL-III posterior probabilities ( >99%), ASTRAL-III quartet scores ( >0.5) and bootstrap values ( >99%). Topologies deeper in the tree were less well-resolved (with quartet scores More

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    Adaptations of Pseudoxylaria towards a comb-associated lifestyle in fungus-farming termite colonies

    Genome reduction is associated with a termite comb-associated lifestyleFor our studies, we collected fungus comb samples originating from mounds of Macrotermes natalensis, Odontotermes spp., and Microtermes spp. termites and were able to obtain seven viable Pseudoxylaria cultures (X802 [Microtermes sp.], Mn132, Mn153, X187, X3-2 [Macrotermes natalensis], and X167, X170LB [Odontotermes spp.], Table S1-S3).To test if a fungus comb-associated lifestyle of Pseudoxylaria was reflected in differences at the genome level, we sequenced the genomes of all seven isolates using a combination of paired-end shotgun sequencing (BGISEQ-500, BGI) and long-read sequencing (PacBio sequel, BGI or Oxford Nanopore Technologies, Oxford, UK). In addition, we sequenced the transcriptomes (BGISEQ, BGI) of two isolates (X802, X170LB). Eleven publicly available genomes of free-living Xylaria (Fig. 2A, B) were used as reference genomes (Table S4). Hybrid draft genomes were comprised on average of 33–742 scaffolds with total haploid assembly lengths of 33.2–40.4 Mb, and a high BUSCO completeness of genomes ( > 95 %) with a total number of predicted proteins ranging from 8.8 to 12.1 × 103. The GC content was comparable to reference genomes with 49.7–51.6%. To verify the phylogenetic placement of the isolates, different genetic loci encoding conserved protein sequences (α-actin (ACT), second largest subunit of RNA polymerase (RPB2), β-tubulin (TUB) and the internal transcribed spacer (ITS) were used as genetic markers [7, 13].Fig. 2: Geographic and comparative phylogenomic analysis of termite-associated Pseudoxylaria isolates (strains 1-7) and free-living Xylaria (strains 8–18).A Geographic origins of genome-sequenced free-living Xylaria and termite-associated Pseudoxylaria isolates, B phylogenomic placement based on single-copy ortholog protein sequences, and C comparison of genome assembly length, and numbers of predicted proteins per genome.Full size imagePhylogenies were reconstructed from ITS sequences and three aligned sequence datasets (RPB2, TUB, ACT) using reference sequences of twelve different taxa (Table S4–S7). Consistent with previous findings, all isolates grouped within the monophyletic termite-associated Pseudoxylaria group [9,10,11,12,13], which diverged from the free-living members of the genus Xylaria (Fig. 2B, Figure S1–S4).As our seven isolates covered a larger portion of the previously reported phylogenetic diversity of the termite-associated subgenus, we elaborated on genomic characteristics of our isolates to uncover features of the termite-associated ecology of Pseudoxylaria. Indeed, comparative genome analysis of the South African Pseudoxylaria isolates with publicly available genomes of free-living Xylaria species of similar genome quality revealed significantly reduced genome assembly lengths in Pseudoxylaria with reduced numbers of predicted genes per genome (Table S4). Comparison of the annotated mitochondrial (mt) genomes (Figure S5, Table S8) also indicated that all seven mt genomes were shorter in length (assembly lengths: 18.5–63.8 kbp) compared to the, albeit few, publicly available mitochondrial genomes of free-living species (48.9–258.9 kbp). The reduction in mitochondrial genome size also corresponded to a significantly reduced mean number of annotated genes (7.6) and tRNAs (14.3) in Pseudoxylaria spp. compared to on average 30.0 (annotated genes) and 25.8 (tRNAs) found in free-living species.Analysis of the abundance and composition of transposable elements (TEs), which account for up to 30–35% of the genomes of (endo)parasitic fungi due to the expansion of certain gene families [20, 21], showed that the mean total numbers of TEs across Pseudoxylaria spp. genomes were comparable (1530), but the numbers were reduced compared to free-living Xylaria species (3690) (Table S9). We also identified high variation in the TE composition across genomes (1.5–9.9 %), comparable to what was observed in free-living Xylaria spp. (1.3–8.1 %), with reductions in long terminal repeat retrotransposons (LTRs: Copia and unknown LTRs) in two inverted tandem repeat DNA transposons (TIRs; CACTA, Mutator and hAT). As Pseudoxylaria spp. contained increased numbers of non-ITR transposons of the helitron class and LTRs of the Gypsy class compared to Xylaria strains, we concluded that Pseudoxylaria exhibits no typical features of an (endo)parasitic lifestyle, but that the overall composition and the reduced numbers of TEs could serve as a fingerprint to distinguish the genetically divergent Pseudoxylaria taxa.Repertoire of carbohydrate-active enzymes indicates specialized substrate useAs the fungus comb is mostly composed of partially-digested plant material interspersed with fungal mycelium of the termite mutualist [3], we anticipated that Pseudoxylaria should exhibit features of a substrate specialist similar to the fungal mutualist Termitomyces, which should be reflected in a Carbohydrate-Active enzyme (CAZyme) repertoire distinguishable from  free-living saprophytic Xylaria species [22,23,24]. In particular, numbers and composition of redox-active enzymes (e.g., benzoquinone reductase (EC 1.6.5.6/EC 1.6.5.7), catalase (EC 1.11.1.6), glutathione reductase (EC 1.11.1.9), hydroxy acid oxidase (EC 1.1.3.15), laccase (EC 1.10.3.2), manganese peroxidase (EC 1.11.1.13), peroxiredoxin (EC 1.11.1.15), superoxide dismutase (EC 1.15.1.1), dye-decolorization or unspecific peroxygenase (EC 1.11.2.1), Table S10), which catalyze the degradation of lignin-rich biomass, were expected to differ between free-living strains and substrate specialists [22].Identification of CAZymes using Peptide Pattern Recognition (PPR) revealed that Pseudoxylaria genomes encoded on average a reduced number of CAZymes (mean 264) compared to the free-living taxa in the family Xylaria (mean 367 CAZymes, pANOVA; F = 41.4, p = 3.5 × 10–8, pairwise p = 1.69 × 10–7) (Fig. 3A, B, Figure S6), but similar numbers to those identified in Termitomyces (mean 265, pairwise p = 0.949).Fig. 3: Comparison of carbohydrate-active enzymes (CAZymes) in Xylaria, Pseudoxylaria and the fungal mutualist Termitomyces.A Predicted CAZymes, B Principal Coordinates Analysis (PCoA) of predicted CAZyme families, and C heatmap of representatives CAZyme families in the predicted proteomes of free-living Xylaria, Termitomyces and Pseudoxylaria species.Full size imageOverall, significant differences in the composition of CAZymes were observed [8], most notably in the reduction of auxiliary activity enzymes (AA), carbohydrate esterases (CE), glycosyl hydrolases (GH), and polysaccharide lyases (PL). The most significant reduction was observed in the AA3 family (Fig. 3C), which typically displays a high multigenicity in wood-degrading fungi as many  enzymes of this family catalyze the oxidation of alcohols or carbohydrates with the concomitant formation of hydrogen peroxide or hydroquinones thereby supporting lignocellulose degradation by other AA-enzymes, such as peroxidases (AA2). Similarly, although to a lesser extent, reduced numbers within the related AA1 family were detected, which included oxidizing enzymes like laccases, ferroxidases, and laccase-like multicopper oxidases. Along these lines, glycosyl hydrolases of the GH3 and GH5 family, including enzymes responsible for degradation of cellulose-containing biomass and xylose, were less abundant. We also noted that all Pseudoxylaria lacked homologs of the unspecific peroxygenases (UPO; EC 1.11.2.1), while almost all free-living Xylaria spp. and the fungal symbiont Termitomyces harbored at least one or two copies of similar gene sequences.
    Pseudoxylaria shows reduced biosynthetic capacity for secondary metabolite productionA healthy termite colony is engulfed in several layers of social immunity [5, 6], which pose a constant selection pressure on associated and potentially antagonistic microbes. As Pseudoxylaria evolved measures to remain inconspicuously present within the comb environment, we hypothesized that one of the possible adaptations to evade hygiene measures of termites could be reflected in a reduced biosynthetic capability to produce antibiotic or volatile natural products, which often serve as infochemicals triggering defense mechanisms [25,26,27], or as alarm pheromones [4, 28].The biosynthesis of secondary metabolites is encoded in so called Biosynthetic Gene Cluster (BGC) regions. We explored the abundance and diversity of encoded BGCs using FungiSMASH 6.0.0 and manually cross-checked the obtained data set by BLAST to account for possible biases due to varying genome qualities across strains of both groups [29]. Overall, the herein investigated Xylaria genomes harbored on average 90 BGCs per genome, while Pseudoxylaria encoded on average 45 BGCs (Fig. 4, Figure S7). Fig. 4: Similarity network analysis of biosynthetic gene clusters.Comparative analysis of termite associated-associated Pseudoxylaria isolates (strains 1–7, red circles) and free-living Xylaria (strains 8–18, green circles) with BiG-SCAPE 1.0 annotations (blue hexagon) ACR ACR toxin, Alt alternariol, Bio biotin, Chr chromene, Cyt cytochalasins, Cur curvupalide, Dep depiudecin, Fus fusarin, Gri griseofulvin, Mon monascorubin, MSA 6-methylsalicylic acid, Pho phomasetin, Sol solanapyrone, Swa swasionine, Xen xenolozoyenone, Xsp xylasporins, Xyl xylacremolide. Singletons are not shown.Full size imageThe nature and relatedness of the BGCs were analyzed by creating a curated similarity network analysis using BiG-SCAPE 1.0 [30]. Overall, 28 orthologous BGCs were shared across all genomes, including the biosynthesis of polyketides like 6-methylsalicylic acid (MSA), chromenes (Chr) and polyketide-non-ribosomal peptide (PKS-NRPS) hybrids like the cytochalasins (Cyt) [31]. Furthermore, five BGC networks, which were shared by Pseudoxylaria and Xylaria, contained genes encoding natural product modifying dimethylallyltryptophan synthases (DMATS). In contrast, and despite the significant reduction in the biosynthetic capacity within Pseudoxylaria genomes [29], about 29 BGC networks were unique to Pseudoxylaria and thus could possibly relate to the comb-associated lifestyle (Figure S8 and S9). Notably, Pseudoxylaria genomes lacked genes encoding ribosomally synthesized and posttranslationally modified peptides (RiPPs) or halogenases. In comparision, free-living Xylaria spp. harbored at least one sequence encoding a RiPP, and up to two orthologous sequences encoding putative halogenases. In contrast, a reduced average number of terpene synthases (TPS) in Pseudoxylaria (9 TPS) compared to free-living Xylaria (18 TPS) was detected, which included three BGCs encoding TPSs that were unique to Pseudoxylaria.  In comparison, genomes of the fungal mutualist Termitomyces were reported to encode for about 20-25 terpene cyclases, but haboured only about two loci containing genes for a PKS and NRPS each [24].Manual BLAST searches were conducted to identify BGCs that could be putatively assigned to previously isolated metabolites from Pseudoxylaria (vide infra Fig. 7, Figure S8) [32, 33]. Using e.g., the known NRPS-PKS-hybrid cluster sequence ccs (Aspergillus clavatus) of cytochalasins as query, an orthologous BGC, here named cytA, was identified in the cytochalasin-producing strain X802 [34]. Although the putative PKS-NRPS hybrid and CcsA shared 60 % identical amino acids (aa), the sequences of the accessory enzymes were less related to CcsC-G (45–47% identical aa) and the BGC in X802 lacked a gene of a homologue to ccsB. Similarly, five free-living Xylaria species carried orthologous gene loci (Xylaria sp. BCC 1067, Xylaria sp. MSU_SB201401, X. flabelliformis G536, X. grammica EL000614, and X. multiplex DSM 110363) supporting previous isolation reports of cytochalasins with varying structural features. Furthermore, three Pseudoxylaria strains (X187, and closely related Mn153, and Mn132) were found to share a highly similar PKS-NRPS hybrid BGC (99–100 % identical aa, named xya), which likely encodes for the enzymatic production of previously identified xylacremolides [32]. Four Pseudoxylaria strains (X802, Mn132, Mn153, and X187) also shared a BGC (50–98 % amino acid identity) resembling the fog BGC (Aspergillus ruber) [35, 36], which putatively encodes the biosynthetic machinery to produce xylasporin/cytosporin-like metabolites. In this homology search, we also uncovered that fog-like BGC arrangements are likely more common than previously anticipated, as clusters with similar arrangements and identity were also found in genomes of Rosellinia necatrix, Pseudomasariella vexata, Stachybotrys chartarum, and Hyaloscypha bicolor (Fig. 4, Figure S8).A detailed analysis of the fog-like cluster arrangements within Pseudoxylaria genomes revealed – similar to homologs of the ccs cluster – variation in the abundance and arrangement of several accessory genes coding for a cupin protein (pxF), a short chain oxidoreductase (pxB; SDR), and an additional SnoaL-like polyketide cyclase (pxP), which could account for the production of strain-specific structural congeners (vide infra, Fig. 7).Change of nutrient sources causes dedicated transcriptomic changes in Pseudoxylaria
    To further solidify our in silico indications of substrate specialization with comb material as preferred substrate and fungus garden as environment, we analyzed Pseudoxylaria growth on different media (PDA, and reduced medium 1/3-PDA) including comb-like agar matrices (wood-rice medium (WRM), agar-agar or 1/3-PDA medium containing lyophilized (dead) Termitomyces sp. T112 biomass (T112, respectively T112-PDA), PDB covering glass-based surface-structuring elements (GB), Table S11–S14).Cultivation of Pseudoxylaria on agar-agar containing lyophilized biomass of Termitomyces (T112) as the sole nutrient source allowed Pseudoxylaria to sustain growth, although to a reduced extent compared to growth on nutrient-rich PDA medium (Table S3). Wood-rice medium (WRM) induced comparable growth rates as observed on PDA and also the appearance of phenotypic stromata.To investigate the influence of these growth conditions on the transcriptomic level, we harvested RNA from vegetative mycelium after growth on comb-like media (WRM, T112, T112-PDA, and GB), PDA, and reduced medium 1/3-PDA (Fig. 5A). The most significant transcript changes (normalized to data obtained from growth on PDA) were observed for genes coding for specific CAZymes including several redox active enzymes (Fig. 5B). The 30 most variable transcripts coded for specific glycoside hydrolases (GH), lytic polysaccharide monooxygenases (AA), ligninolytic enzymes, and a glycoside transferase (GT). Similarly, chitinases (CHT2; CHT4; CHI2; CHI4) were upregulated (up to 243-fold on T112) under almost all conditions compared to PDA, but some of these specific transcript changes were exclusive to growth on Termitomyces biomass or artificial comb material (WRM) suggesting the ability to regulate and increase chitin metabolism if necessary [37].Fig. 5: Transcriptomic analysis of Pseudoxylaria sp. X802 in dependence of growth conditions.A Representative pictures of Pseudoxylaria sp. X802 growing on PDA, PDB on glass beads (GB), wood-rice medium (WRM), and agar-agar medium containing lyophilized Termitomyces sp. T112 biomass (T112). B Heatmap of the most variable transcripts coding for CAZymes (red), redox enzymes (orange), secondary metabolite-related core genes (green), and more specifically on key genes within the boundaries of cytochalasin (turquoise) and xylasporin/cytosporin BGCs (blue). RNA was obtained from vegetative mycelium after growth on PDA, reduced medium (1/3-PDA), PDB on glass beads (GB), wood-rice medium (WRM), 1/3-PDA-medium enriched with Termitomyces sp. T112 biomass (T112-PDA) and agar-agar medium containing lyophilized Termitomyces biomass (T112). Transcript counts are shown as log10 transformed transcripts per million (top; TPM). Significance of the changes in transcript counts are compared to control (X802 grown on PDA) and depicted in log-10 transformed p values.Full size imageWhen X802 was grown on T112 (agar matrix containing lyophilized Termitomyces sp. T112 biomass), we observed a >400-fold increase in the expression of transcripts encoding glycoside hydrolases in the GH43 family, GH7 (~140-fold), GH3, and GH64 (5–12-fold). Similarly, transcripts for a putative mannosyl-oligosaccharide-α-1,2-mannosidase (MNS1B; 8.2-fold), chitinase CHT4 (2.9-fold), β-glucosidase BGL4 (5.7-fold), and copper-dependent lytic polysaccharide monooxygenase AA11 (1.6-fold) were significantly upregulated. Growth on WRM (wood-rice medium) or T112 (Termitomyces sp. T112 biomass) also caused a significant upregulation of genes coding for glycoside transferase GT2, glycoside hydrolases GH15, GH3, and aldehyde oxidase AOX1, which indicated the ability to expand the degradation portfolio if necessary. Along these lines, specific transcript levels were reduced when X802 was grown on T112, in particular class II lignin-modifying peroxidases (AA2), carbohydrate-binding module family 21 (CBM21), multicopper oxidases (AA1), secreted β-glucosidases (SUN4), and glycoside hydrolases GH16, and GH128.When the fungus was challenged with lignocellulose-rich WRM medium, higher transcript levels putatively assigned to glutathione peroxidase (GXP2), superoxide dismutase (SOD2), and laccases (LCC5) were observed, which indicated that despite the reduced wood-degrading capacity, Pseudoxylaria activates available enzymatic mechanisms to degrade the provided material and respond to the resulting oxidative stress. Cultivation on GB (glass-based surfaces covered in liquid PD broth) influenced the expression of certain genes coding for glycoside hydrolases (GH64, GH76, GH72, GH128, BGL4) and lytic polysaccharide monooxygenases (AA1, AA2, AA11), presumably enabling the fungus to utilize soluble carbohydrates.To test the hypothesis that the presence of Termitomyces biomass stimulates secondary metabolite production in Pseudoxylaria to eventually displace the mutualist, we also analyzed changes in the transcript levels of core BGC genes that encode the production of bioactive secondary metabolites. Overall, only slight transcript variations were detectable within the  most variable expressed genes. (Fig. 5B). Cultivation on GB, WRM, and T112 media caused lower transcript levels of genes coding for terpene synthase TC1, polyketide synthases (PKS7, PKS8), and the NRPS-like1, while an upregulation of NRPS-like2 on WRM (2.5-fold), and of PKS7 (1.7-fold) on reduced 1/3-PDA medium was observed.Transcript levels of core genes within BGCs assigned to cytochalasines (cyt) or xylasporins/cytosporins (px), e.g., remained nearly constant, while minor transcript level variations of neighboring genes and reduced transcript levels for pxI (flavin-dependent monooxygenase), pxH (ABBA-type prenyltransferase), pxF (cupin fold oxidoreductase), and pxJ (short-chain dehydrogenase) were detectable. Hence, it was concluded that the presence of Termitomyces biomass only weakly triggers secondary metabolite production in general, but varying transcript levels coding for decorating enzymes could cause substantial structural alterations within the produced natural product composition. It was also notable that transcript levels of the terpene synthase TC1 were downregulated, which could cause a reduced production level of specific volatiles.
    Pseudoxylaria antagonizes Termitomyces growth and metabolizes fungal biomassThe growth behavior of Pseudoxylaria isolates was also analyzed in co-culture assays with Termitomyces. As expected from prior studies, both fungi showed reduced growth when co-cultured on agar plates, often causing the formation of zones of inhibition (ZOI) between the fungal colonies (Fig. 6A–D, Table S11–S14) [7]. When fungus-fungus co-cultures were maintained for longer than two weeks on agar plates, Pseudoxylaria started to overcome the ZOI and overgrew Termitomyces via the extension of aerial mycelium. The observation was even more pronounced when co-cultures were performed on wood-rice medium (WRM), where Pseudoxylaria remained the only visible fungus after two weeks.Fig. 6: Co-cultivation of Pseudoxylaria sp. X170LB and Termitomyces sp. T112 and results of isotope fractionation experiments.Representative pictures of fungal growth and co-cultivation of A Termitomyces sp. T112, B Pseudoxylaria sp. X170LB, C co-culture of Pseudoxylaria sp. X802 and Termitomyces sp. T153 exhibiting a ZOI, in which X802 overgrowths T153 in proximity to the interaction zone (red arrow), and D Pseudoxylaria sp. X802 growing on the surface of a living Termitomyces sp. T153 culture. E, F Shown is the relative change in the carbon isotope pattern (δ13C values, ± standard deviation, with n = 3) of lipid and carbohydrate fractions isolated from fungal biomass of Termitomyces sp. T112, Pseudoxylaria sp. X170LB, and Pseudoxylaria sp. X170LB cultivated on vegetative Termitomyces sp. T112 biomass (T112ǂ), or on lyophilized Termitomyces sp. T112 biomass (T112). Fungal strains were grown on E medium with natural 13C abundance and F medium artificially enriched in 13C content.Full size imageTo verify whether Pseudoxylaria consumes Termitomyces or even partially degrades specific metabolites present within the fungal biomass, we pursued stable isotope fingerprinting commonly used to analyse trophic relations [38, 39]. This diagnostic method relies on measurable changes in the bulk stable isotope composition, because biosynthetic enzymes preferentially convert lighter metabolites enriched in 12C compared to their heavier 13C-enriched congeners. This intrinsic kinetic isotope effect results in an overall change in the 13C/12C ratio of the respective educts and products, in particular in biomarkers such as phospholipid fatty acids, carbohydrates, and amino acids. Using this isotope enrichment effect, we determined the natural trophic isotope fractionation of 13C in lipids and carbohydrates produced by Termitomyces sp. T112 and Pseudoxylaria sp. X170LB. For clearer differentiation, both fungi were cultivated on PDA medium containing naturally abundant 13C/12C, Fig. 6E) and on PDA medium enriched with 13C-glucose (Fig. 6F). Lipids and carbohydrates were isolated from mycelium harvested after 21 days (Fig. 6E, Table S15).Analysis of fungal carbohydrate and lipid-rich metabolite fractions by Elemental Analysis-Isotope Ratio Mass Spectrometry (EA-IRMS) [40, 41] uncovered that under normal growth conditions (full medium), Termitomyces sp. T112 and Pseudoxylaria sp. X170LB showed only a slight negative trophic fractionation of stable carbon isotopes (δ13C/12C ratio (expressed as δ13C values [‰]), Fig. 6F) within the carbohydrate fractions (T112: −1.2 ‰; for X170LB: −1.3 ‰), and expectedly a stronger depletion in the lipid fraction (T112: −6.7 ‰, and less pronounced for X170LB: −3.1 ‰). To determine if Pseudoxylaria metabolizes Termitomyces biomass, the isotope pattern of metabolites derived from Pseudoxylaria thriving on living biomass of Termitomyces (T112ǂ) was analysed next. Here, an overall positive carbon isotope (13C/12C) fractionation by approximately +0.6 ‰ relative to the control medium was detectable, while the δ13C values of lipids remained largely unchanged (Fig. 6F, Table S15). These results suggested that Pseudoxylaria might pursue a preferential uptake of Termitomyces-derived carbohydrates.In a last experiment, Pseudoxylaria was grown on lyophilized (dead) Termitomyces biomass (T112) as sole food source. In this experiment, the isotope fingerprint showed converging δ13C values of −1.9 ‰ (relative to the media) for both carbohydrate and lipid fractions, which indicated that Pseudoxylaria is able to simultaneously metabolize and cycle carbohydrates as well as lipids resulting in the equilibration of isotopic levels between carbohydrates and lipids. Thus, it was concluded that in nature, Pseudoxylaria likely harvests nutrients firstly from vegetative Termitomyces, and then—if possible—subsequently degrades dying or dead mycelium.
    Pseudoxylaria produces antimicrobial secondary metabolitesBased on the observation that Pseudoxylaria antagonizes growth of Termitomyces, we questioned if the formation of a ZOI might be caused by the secretion of Pseudoxylaria-derived antimicrobial metabolites [26, 42]. Thus, we performed an ESI(+)-HRMS/MS based metabolic survey using the web-based platform “Global Natural Product Social Molecular Networking” (GNPS) [43] to correlate the encoded biosynthetic repertoire of Pseudoxylaria with secreted metabolites.A partial similar metabolic repertoire across the six analyzed strains was detectable and allowed us to match some of the detectable chemical features and previously isolated metabolites to the predicted shared BGCs, such as antifungal and histone deacetylase inhibitory xylacremolides (Xyl; X187/Mn132) [32, 33], pseudoxylaramides (Psa; X187/Mn132) [32], antibacterial pseudoxylallemycins (Psm; X802/OD126) [18], xylasporin/cytosporins (Xsp; X802/OD126/X187/Mn132) [36], and cytotoxic cytochalasins (X802/OD126) (Fig. 7A and B) [18].Fig. 7: Comparative metabolomic analysis of six Pseudoxylaria strains (OD126 (red), Mn132 (orange), X170 (black), X187 (green), X3.2 (yellow) and X802 (blue)).A Overview of the GNPS network. Identified metabolite clusters xylacremolides (Xyl; X187/Mn132) [32, 33], pseudoxylaramides [32] (Psa; X187/Mn132), pseudoxylallemycins (Psm; X802/OD126) [18], xylasporin/cytosporins (Xsp; X802/OD126/X187/Mn132) and cytochalasins (X802/OD126) [18]. B xylasporin/cytosporin-related cluster formed by nodes from X802 (blue), OD126 (red), X187 (green) and Mn132 (orange). C Chemical structures of natural products isolated from Pseudoxylaria species and related compounds. Red box highlights proposed structures of isolated xylasporin G and I in this study.Full size imageA cluster that contained MS2 signals of molecular ions assigned to the cytosporin/xylasporin family, which was shared by at least four strains, caught our attention as a certain degree of structural diversity of xylasporin/cytosporin family was predicted from the comparison of their respective BGCs. The assigned nodes of this GNPS cluster split into two subclusters with only very little overlap between both regions. Analysis of the mass fragment shifts suggested that both subclusters belong to two different families of xylasporin/cytosporin congeners (Figure S9). To verify these deductions, we pursued an MS-guided purification of xylasporin/cytosporins from chemical extracts of Pseudoxylaria sp. X187, which yielded xylasporin G (3.23 mg, pale-yellow solid) and xylasporin I (1.75 mg, pale-yellow solid). The sum formulas of xylasporin G and xylasporin I were determined to be C17H22O5 (calcd. for [M + H]+ C17H23O5+ = 307.1540, found 307.15347, −1.726 ppm) and C17H24O5 (calcd. for [M + H]+ C17H25O5+ = 309.1697, found 309.1691, −1.68 ppm) by ESI-(+)-HRMS and were predicted to have six degrees of unsaturation (Fig. 7B, Figure S10, Table S16-S17). Planar structures were deduced by comparative 1D and 2D NMR analyses, which revealed the presence of an unsaturated polyketide chain that matched the unsaturation degree and the anticipated structural variation from cytosporins (Fig. 7C, Figure S11-S25).To evaluate if Pseudoxylaria-derived culture extracts and produced natural products (e.g., cytochalasins) are responsible for the observed antimicrobial activity, standardized antimicrobial activity assays were performed (Table S17, S18 and Figure S26). As neither culture extracts nor single compounds exhibited significant antimicrobial activity, they could not be held fully accountable for the antagonistic behavior in co-cultures. Thus, we hypothesized that the observed ZOI might be caused by yet unknown effects like nutrient depletion or bioactive enzymes.
    Pseudoxylaria has a negative impact on the fitness of insect larvaeDue to the production of structurally diverse and weakly antimicrobial secondary metabolites, we questioned if mycelium of Pseudoxylaria exhibits intrinsic insecticidal or other insect-detrimental activities, which could discourage or ward off grooming behavior of termite workers. Due to the technical challenges associated with behavioral studies of termites, we evaluated instead the effect of Pseudoxylaria biomass on Spodoptera littoralis, a well-established insect model system and a destructive agricultural lepidopterous pest [44, 45]. When S. littoralis larvae were fed with mycelium-covered agar plugs of Pseudoxylaria sp. X802, a clear decrease of the relative growth rate (RGR) and decline in survival was observed (Fig. 8: treatment D (green), Table S19, S20) compared to feeding with untreated agar plugs (treatment A (black)). In comparison, when larvae were fed with agar plugs covered with the fungal mutualist Termitomyces sp. T153 (treatment B (blue)) an increased growth rate of larvae was observed.Fig. 8: Effect of Termitomyces sp. T153 and Pseudoxylaria sp. X802 mycelia on the relative growth rate and survival of S. littoralis larvae.Insects were fed with either A PDA, B PDA agar plug covered with vegetative Termitomyces sp. T153, C PDA agar plug from which vegetative Termitomyces sp. T153 was removed prior to feeding, D PDA agar plug covered with vegetative Pseudoxylaria sp. X802 mycelium, and E PDA agar plug from which vegetative Pseudoxylaria sp. X802 mycelium was removed prior to feeding. All experiments were performed with 25 replicates per treatment, a duration of 10 days, and larval weights and survival rates were recorded every day. Statistical significances were determined using ANOVA on ranks (p  More

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    A density functional theory for ecology across scales

    Modular components of the DFTe energy functionalThe central ingredient of DFTe is an energy functional E, assembled according to Eq. (1). The methodology of DFTe can be understood by inspecting the dispersal and environmental energies in Eqs. (2) and (3) without interactions. In our first case study, illustrated in Fig. 2 and Supplementary Fig. 2, we demonstrate that equation (3), in conjunction with Eq. (2), can realistically describe the influence of the environment on species’ distributions. Mechanisms that alter the trade-off between dispersal and environment can be introduced as part of Eint. For instance, back reactions on the environment could be modelled with a bifunctional Ebr[Venv, n] that yields the equilibrated modified environment ({V}_{s}^{{{{{{{{rm{env}}}}}}}}}+delta {E}_{{{{{{{{rm{br}}}}}}}}}[{{{{{{{{bf{V}}}}}}}}}^{{{{{{{{rm{env}}}}}}}}},{{{{{{{bf{n}}}}}}}}]/delta {n}_{s}({{{{{{{bf{r}}}}}}}})), cf. Eq. (5).In the following we make explicit the interaction and resource energies that enter Eq. (1) and are used in our case studies of Figs. 2–7. We let Eint[n] include all possible bipartite interactions$${E}_{gamma }[{{{{{{{bf{n}}}}}}}}]=mathop{sum }limits_{{s,{s}^{{prime} }!=!1}atop {{s}^{{prime} }ne s}}^{S}{int}_{A}({{{{{{{rm{d}}}}}}}}{{{{{{{bf{r}}}}}}}})({{{{{{{rm{d}}}}}}}}{{{{{{{{bf{r}}}}}}}}}^{{prime} }){n}_{s}{({{{{{{{bf{r}}}}}}}})}^{{alpha }_{s}},{gamma }_{s{s}^{{prime} }}({{{{{{{bf{r}}}}}}}},, {{{{{{{{bf{r}}}}}}}}}^{{prime} }){n}_{{s}^{{prime} }}{({{{{{{{{bf{r}}}}}}}}}^{{prime} })}^{{beta }_{{s}^{{prime} }}},$$
    (6)
    which include amensalism, commensalism, mutualism, and so forth. Here, ({alpha }_{s},, {beta }_{{s}^{{prime} }}ge 0), and the interaction kernels ({gamma }_{s{s}^{{prime} }}) are assembled from fitness proxies of species s and ({s}^{{prime} }) (Supplementary Table 1). Higher-order interactions can be introduced, for example, through (i) terms like ({n}_{s},{gamma }_{s{s}^{{prime} }},{n}_{{s}^{{prime} }},{gamma }_{{s}^{{prime} }{s}^{{primeprime} }}^{{prime} },{n}_{{s}^{{primeprime} }}) that build on pairwise interactions or (ii) genuinely multipartite expressions like ({gamma }_{s{s}^{{prime} }{s}^{{primeprime} }}{n}_{s},,{n}_{{s}^{{prime} }},{n}_{{s}^{{primeprime} }}). Multi-partite interactions based on bipartite interactions do not seem to be an uncommon scenario48. However, there may be systems where nonzero coefficients ({gamma }_{s{s}^{{prime} }{s}^{{primeprime} }}) couple all species. This poses a challenge for mechanistic theories in general. Then, ‘simpler subsystems’ that have to be included in the DFTe workflow of Fig. 1a can only refer to situations where other energy components are absent, such as resource terms or complex environments. For example, the coefficients ({gamma }_{s{s}^{{prime} }{s}^{{primeprime} }}) could be extracted in an experiment with a controlled simple environment and then used to model the interacting species in a real-world setting. For (({alpha }_{s},, {beta }_{{s}^{{prime} }})=(1,1)) we identify the contact interaction in physics as ({gamma }_{s{s}^{{prime} }}propto delta ({{{{{{{bf{r}}}}}}}}-{{{{{{{{bf{r}}}}}}}}}^{{prime} })) with the two-dimensional delta function δ( ), while the Coulomb interaction amounts to setting ({gamma }_{s{s}^{{prime} }}propto 1/|{{{{{{{bf{r}}}}}}}}-{{{{{{{{bf{r}}}}}}}}}^{{prime} }|). The mechanistic effect of these interaction kernels on the density distributions is the same in ecology as it is in physics—a mathematical insight that inspired us to build ecological analogues to the phenomenology of quantum gases, which feature functionals of the kind in Eq. (6). Note that we do not introduce any quantum effects into ecology despite the fact that the mathematical structure of DFTe is borrowed in part from quantum physics. While the contact interaction is a suitable candidate for plants and especially microbes52, we expect long-range interactions (for example, repulsion of Coulomb type) to be more appropriate for species with long-range sensors, such as eyes. Both types of interactions feature in describing the ecosystems addressed in this work.In a natural setting the equilibrium abundances are ultimately constrained by the accessible resources. It is within these limits of resource availability that environment as well as intra- and inter-specific interactions can shape the density distributions. An energy term for penalising over- and underconsumption of resources is thus of central importance. Each species consumes resources from some of the K provided resources, indexed by k. A subset of species consumes the locally available resource density ρk(r) according to the resource requirements νks, which represent the absolute amount of resource k consumed by one individual (or aggregated constituent) of species s. The simple quadratic functional$${E}_{{{{{{{{rm{Res}}}}}}}}}[{{{{{{{bf{n}}}}}}}}]={int}_{A}({{{{{{{rm{d}}}}}}}}{{{{{{{bf{r}}}}}}}})mathop{sum }limits_{k=1}^{K}{{{{{{{{mathcal{L}}}}}}}}}_{k}left({{{{{{{bf{n}}}}}}}},, {rho }_{k}right)equiv zeta {int}_{A}({{{{{{{rm{d}}}}}}}}{{{{{{{bf{r}}}}}}}})mathop{sum }limits_{k=1}^{K}{w}_{k}({{{{{{{bf{r}}}}}}}}){left[mathop{sum }limits_{s=1}^{S}{nu }_{ks}{n}_{s}({{{{{{{bf{r}}}}}}}})-{rho }_{k}({{{{{{{bf{r}}}}}}}})right]}^{2}$$
    (7)
    proves appropriate. Here, νksns is the portion of resource density ρk that is consumed by species s. That is, νks  > 0 indicates that s requires resource k. If Eq. (7) is the total energy functional, then a single-species system with a single resource equilibrates with density n1(r) = ρ1(r)/ν11 at every position r, and additional DFTe energy components would modify this equilibrium. Predator–prey relationships are introduced by making species k a resource ({rho }_{k}=left]{n}_{k}right[), where (left]nright[) declares n a constant w.r.t. the functional differentiation of E, that is, the predator tends to align with the prey, not the prey with the predator. In view of the energy minimisation, the quadratic term in Eq. (7) entails that regions of low resource density ρk are less important than regions of high ρk. The different resources k have the same ability to limit the abundances, such that the limiting resource k = l at r has to come with the largest of weights wl(r), irrespective of the absolute amounts of resources at r. For example, the weights wk have to ensure that an essential but scarce mineral has (a priori) the same ability to limit the abundances as a resource like water, which might be abundant in absolute terms. To that end, we specify the weights$${w}_{k}({{{{{{{bf{r}}}}}}}})=frac{1}{{bar{rho }}_{k}^{2}}mathop{sum}limits_{s}eta ({nu }_{ks})exp left[sigma left(frac{{lambda }_{ks}}{{lambda }_{ls}}-1right)right],$$
    (8)
    which are inspired by the smooth minimum function, where σ  λls irrelevant at r. Using ({E}_{{{{{{{{rm{Res}}}}}}}}}), we show that an analytically solvable minimal example of two amensalistically interacting species already exhibits a plethora of resource-dependent equilibrium states (see Supplementary Notes and Supplementary Fig. 1).We specify the DFTe energy functional in Eq. (1) by summing Eqs. (2), (3), (6), and (7) and by (optionally) constraining the abundances to N via Lagrange multipliers μ:$$E[{{{{{{{bf{n}}}}}}}},, {{{{{{{boldsymbol{mu }}}}}}}}]({{{{{{{bf{N}}}}}}}}) equiv E[{{{{{{{bf{n}}}}}}}}]+{E}_{{{{{{{{boldsymbol{mu }}}}}}}}}[{{{{{{{bf{n}}}}}}}}]({{{{{{{bf{N}}}}}}}})\ equiv {E}_{{{{{{{{rm{dis}}}}}}}}}[{{{{{{{bf{n}}}}}}}}]+{E}_{{{{{{{{rm{env}}}}}}}}}[{{{{{{{bf{n}}}}}}}}]+{E}_{gamma }[{{{{{{{bf{n}}}}}}}}]+{E}_{{{{{{{{rm{Res}}}}}}}}}[{{{{{{{bf{n}}}}}}}}]+mathop{sum }limits_{s=1}^{S}{mu }_{s}left({N}_{s}-{int}_{A}({{{{{{{rm{d}}}}}}}}{{{{{{{bf{r}}}}}}}}),{n}_{s}right).$$
    (9)
    Uniform situations are characterised by spatially constant ingredients ns = Ns/A, ρk = Rk/A, coefficients τs, etc. for the DFTe energy, such that Eq. (9) reduces to a function E(N) with building blocks$${E}_{{{{{{{{rm{dis}}}}}}}}}longrightarrow frac{1}{2,A}mathop{sum }limits_{s=1}^{S}{tau }_{s},{N}_{s}^{2},$$
    (10)
    $${E}_{{{{{{{{rm{env}}}}}}}}}longrightarrow mathop{sum }limits_{s=1}^{S}{V}_{s}^{{{{{{{{rm{env}}}}}}}}},{N}_{s},$$
    (11)
    $${E}_{gamma }longrightarrow mathop{sum }limits_{{s,{s}^{{prime} }!=!1}atop {{s}^{{prime} }ne s}}^{S}frac{{N}_{s}^{{alpha }_{s}},{gamma }_{s{s}^{{prime} }},{N}_{{s}^{{prime} }}^{{beta }_{{s}^{{prime} }}}}{{A}^{{alpha }_{s}+{beta }_{{s}^{{prime} }}-1}},$$
    (12)
    $${E}_{{{{{{{{rm{Res}}}}}}}}}longrightarrow Amathop{sum }limits_{k=1}^{K}{{{{{{{{mathcal{L}}}}}}}}}_{k}left({{{{{{{bf{N}}}}}}}}/A,, {R}_{k}/Aright).$$
    (13)
    Ecosystem equilibria from the DFTe energy functionalThe general form of Eq. (9) gives rise to two types of minimisers (viz., equilibria): First, we term$${{{{{{{mathcal{H}}}}}}}}({{{{{{{bf{N}}}}}}}})equiv E[tilde{{{{{{{{bf{n}}}}}}}}}]equiv mathop{min }limits_{{{{{{{{bf{n}}}}}}}}}left{E[{{{{{{{bf{n}}}}}}}}],left|,{int}_{A}({{{{{{{rm{d}}}}}}}}{{{{{{{bf{r}}}}}}}}),{{{{{{{bf{n}}}}}}}}({{{{{{{bf{r}}}}}}}})={{{{{{{bf{N}}}}}}}},{{{{{{{rm{(fixed)}}}}}}}}right.right}$$
    (14)
    the ‘DFTe hypersurface’, with (tilde{{{{{{{{bf{n}}}}}}}}}) the energy-minimising spatial density profiles for given (fixed) N. Second, the ecosystem equilibrium is attained at the equilibrium abundances (hat{{{{{{{{bf{N}}}}}}}}}={int}_{A}({{{{{{{rm{d}}}}}}}}{{{{{{{bf{r}}}}}}}}),hat{{{{{{{{bf{n}}}}}}}}}({{{{{{{bf{r}}}}}}}})), which yield the global energy minimum$${{{{{{{mathcal{H}}}}}}}}(hat{{{{{{{{bf{N}}}}}}}}})=mathop{min }limits_{{{{{{{{bf{N}}}}}}}}},{{{{{{{mathcal{H}}}}}}}}({{{{{{{bf{N}}}}}}}}),$$
    (15)
    where the minimisation samples all admissible abundances, that is, ({{{{{{{bf{N}}}}}}}}in {left({{mathbb{R}}}_{0}^{+}right)}^{times S}) if no further constraints are imposed.The direct minimisation of E[n] is most practical for uniform systems, which only require us to minimise E(N) over an S-dimensional space of abundances. For the general nonuniform case, we adopt a two-step strategy that reflects Eqs. (14) and (15). First, we obtain the equilibrated density distributions on ({{{{{{{mathcal{H}}}}}}}}) for fixed N from the computational DPFT framework26,27,28,29,30,31. Second, a conjugate gradient descent searches ({{{{{{{mathcal{H}}}}}}}}({{{{{{{bf{N}}}}}}}})) for the global minimiser (hat{{{{{{{{bf{N}}}}}}}}}). Technically, we perform the computationally more efficient descent in μ-space. Local minima are frequently encountered, and we identify the best candidate for the global minimum from many individual runs that are initialised with random μ. Note that system realisations with energies close to the global minimum, especially local minima, are likely observable in reality, assuming that the system can equilibrate at all. There is always an equilibrium if the energy functional is bounded from below, together with the fact that the support (abundances/densities) of the energy functional is finite in any practical application. If some DFTe energy components are chosen (too) negative, the system can be unstable, in which case the energy functional has no minimum and is inappropriate for modelling the equilibrium in question. This means that another energy functional has to be considered, or, in the worst case, that DFTe is incapable of simulating this system. We also caution that no numerical optimisation algorithms for non-convex black-box functions can guarantee to find the global minimum, not even approximately. Without analytically available characteristics of the global minimum, all one may hope for are candidates of the minimiser, and those may not even be local minima—there is no way to be certain that an optimum proposed by a numerical optimisation algorithm is stable.Density-potential functional theory (DPFT) in Thomas–Fermi (TF) approximationDefining$${V}_{s}({{{{{{{bf{r}}}}}}}})={mu }_{s}-frac{delta {E}_{{{{{{{{rm{dis}}}}}}}}}[{{{{{{{bf{n}}}}}}}}]}{delta {n}_{s}({{{{{{{bf{r}}}}}}}})}$$
    (16)
    for all s, we obtain the reversible Legendre transform$${E}_{{{{{{{{rm{dis}}}}}}}}}^{{{{{{{{rm{L}}}}}}}}}[{{{{{{{bf{V}}}}}}}}-{{{{{{{boldsymbol{mu }}}}}}}}]={E}_{{{{{{{{rm{dis}}}}}}}}}[{{{{{{{bf{n}}}}}}}}]+mathop{sum }limits_{s=1}^{S}{int}_{A}({{{{{{{rm{d}}}}}}}}{{{{{{{bf{r}}}}}}}}),({V}_{s}-{mu }_{s}),{n}_{s}$$
    (17)
    of the dispersal energy and thereby supplement the total energy with the additional variables V:$$E[{{{{{{{bf{V}}}}}}}},, {{{{{{{bf{n}}}}}}}},, {{{{{{{boldsymbol{mu }}}}}}}}]({{{{{{{bf{N}}}}}}}})={E}_{{{{{{{{rm{dis}}}}}}}}}^{{{{{{{{rm{L}}}}}}}}}[{{{{{{{bf{V}}}}}}}}-{{{{{{{boldsymbol{mu }}}}}}}}]-{int}_{A}({{{{{{{rm{d}}}}}}}}{{{{{{{bf{r}}}}}}}}),{{{{{{{bf{n}}}}}}}}cdot ({{{{{{{bf{V}}}}}}}}-{{{{{{{{bf{V}}}}}}}}}^{{{{{{{{rm{env}}}}}}}}})+{E}_{{{{{{{{rm{int}}}}}}}}}[{{{{{{{bf{n}}}}}}}}]+{{{{{{{boldsymbol{mu }}}}}}}}cdot {{{{{{{bf{N}}}}}}}}.$$
    (18)
    This density-potential functional is equivalent to (but more flexible than) the density-only functional E[n,  μ](N). The minimisers of E[n] are thus among the stationary points of Eq. (18) and are obtained by solving$${n}_{s}[{V}_{s}-{mu }_{s}]({{{{{{{bf{r}}}}}}}})=frac{delta {E}_{{{{{{{{rm{dis}}}}}}}}}^{{{{{{{{rm{L}}}}}}}}}[{V}_{s}-{mu }_{s}]}{delta {V}_{s}({{{{{{{bf{r}}}}}}}})}$$
    (19)
    and$${V}_{s}[{{{{{{{bf{n}}}}}}}}]({{{{{{{bf{r}}}}}}}})={V}_{s}^{{{{{{{{rm{env}}}}}}}}}({{{{{{{bf{r}}}}}}}})+frac{delta {E}_{{{{{{{{rm{int}}}}}}}}}[{{{{{{{bf{n}}}}}}}}]}{delta {n}_{s}({{{{{{{bf{r}}}}}}}})}$$
    (20)
    self-consistently for all ns while enforcing ∫A(dr) ns(r) = Ns. Specifically, starting from V(0) = Venv, such that ({n}_{s}^{(0)}={n}_{s}[{V}_{s}^{(0)}-{mu }_{s}^{(0)}]), we iterate$${n}_{s}^{(i)}mathop{longrightarrow }limits^{{{{{{{{rm{equation}}}}}}}},(20)}{V}_{s}^{(i+1)}={V}_{s}[{{{{{{{{bf{n}}}}}}}}}^{(i)}]mathop{longrightarrow }limits^{{{{{{{{rm{equation}}}}}}}},(19)}{n}_{s}^{(i+1)}=(1-{theta }_{s}),{n}_{s}^{(i)}+{theta }_{s},{n}_{s}left[{V}_{s}^{(i+1)}-{mu }_{s}^{(i+1)}right]$$
    (21)
    until all ns are converged sufficiently. This self-consistent loop establishes a trade-off between dispersal energy and effective environment V by forcing an initial out-of-equilibrium density distribution to equilibrate at fixed N. We adjust ({mu }_{s}^{(i)}) in each iteration i such that ({n}_{s}^{(i)}) integrates to Ns. Small enough density admixtures, with 0  More

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    Horses discriminate human body odors between fear and joy contexts in a habituation-discrimination protocol

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    Extraction of total active proteomes from sediment samplesWe sampled 14 sediments along the coastlines of the Irish Sea, the Mediterranean Sea, and the Red Sea (from 16°N to 53°N), applying uniform sampling and storage procedures. Location details and sediment temperature fluctuations are summarized in Supplementary Table S1. We collected sediments (5 Kg) in triplicate and extracted the total proteins using a well-established microbial detachment procedure67, with some modifications. We mixed 100 g of sediment with 300 ml of sterilized saline solution (5 mM sodium pyrophosphate and 35 g L−1 of NaCl) containing 150 mg L−1 of Tween 80 (from Merck Life Science S.L.U., Madrid, Spain) in an ice water bath. After re-suspension, samples were kept in a water bath ultra-sonicator (Bandelin SONOREX, Berlin, Germany) on ice and sonicated (60 W output) for 120 min. We repeated this procedure twice, with an ice water bath incubation of 60 min between each cycle. We then centrifuged the samples at 500 g for 15 min at 4 °C to remove the sediments in a centrifuge 5810 R (Eppendorf AG, Hamburg, Germany). Supernatants were carefully transferred to a new tube, minimizing disruption of the sediments, and the resulting supernatants were centrifuged at 13,000 g for 15 min at 4 °C to produce microbial cell pellets. We used the resulting cell mix to extract the total protein by mixing the cells with 1.2 ml BugBuster® Protein Extraction Reagent (Novagen, Darmstadt, Germany) for 30 min with shaking (250 rpm). Subsequently, samples were disrupted by sonication using a pin Sonicator® 3000 (Misonix, New Highway Farmingdale, NY, USA) for a total time of 2 min (10 watts) on ice (4 cycles × 0.5 min with 1 min ice-cooling between each cycle). Extracts were centrifuged for 10 min at 12,000 g at 4 °C to separate cellular debris and intact cells. Supernatants were carefully aspirated (to avoid disturbing the pellet), transferred to new tubes, and stored at –80 °C until use. The protein solution was filtered at 15 °C for 7 h using Vivaspin filters (Sartorius, Goettingen, Germany) with a molecular weight (MW) cut-off of 3,000 Da to concentrate the proteins up to a final concentration of 10 mg ml−1, according to the Bradford Protein Assay (Bio-Rad Laboratories, S.A., Madrid, Spain)68. The average total amount of proteins extracted per each 100 g of sediment was 612 µg (interquartile range, 31 µg, see details in Supplementary Fig. S2). In all cases, extensive dialysis of protein solutions against 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer was performed using a Pur-A-LyzerTM Maxi 1200 dialysis kit (Merck Life Science S.L.U., Madrid, Spain)69, and active proteins stored at a concentration of 10 mg ml−1at –86 °C until use. As reported previously70, 2DE was performed using GE Healthcare reagents and equipment, 11 cm IPG strips in the pH range of 3–10 and molecular weight ranging from 10 to 250 kDa (Precision Plus Protein Dual Color Standards #1610374, Bio-Rad Laboratories, S.A., Madrid, Spain). The 2-DE was performed using a validated pooling strategy71, in which proteins extracted from three independent biological replicates (i.e., sediments) were mixed in equal amounts and a total of 150 µg of protein were further loaded per gel. Staining was performed with SYPRO Ruby Protein Gel Stain (Invitrogen, Waltham, MA, USA). The two-dimensional SDS-PAGE (12% acrylamide) gels of extracted proteins are reported in Supplementary Fig. S2 (original gels in Source Data). The same protocol was applied to extract and analyse by SDS-PAGE the total active proteins extracted from sediment samples with different temperature variability levels (HTV, ITV, and LTV) collected in the Red Sea (Supplementary Table S4). The total amount of protein extracted per each 100 g of sediment is given in Supplementary Table S8. Coomassie-stained one-dimension SDS-PAGE (1-DE) gels of extracted proteins are shown in Supplementary Fig. S9 (original gel in Source Data).Source, expression and purification of esterases and EXDOs from a wide geographical rangeWe recovered 83 enzymes (78 esterases and 5 EXDO) from microbial communities inhabiting marine sediments across ten distinct locations from the latitudinal transect described above: Ancona harbour (Anc), Priolo Gargallo (Pri), Gulf of Genoa, Messina harbour (Mes), Milazo harbour (Mil), Mar Chica lagoon (MCh), Bizerte lagoon (Biz), El-Max site (ElMax), Gulf of Aqaba (Aq), and Menai Strait (MS); further details are provided in Supplementary Data S3. Sources of the enzymes were the corresponding shotgun metagenomes (see Supplementary Table S3) and the metagenome clone libraries generated from the extracted DNA71. The sediment sample from the Gulf of Genoa was not used for activity tests and metaproteome analysis because no raw sample material was available; however, because of the possibility to access its shotgun metagenome (see Supplementary Table S3) and a metagenome clone library72, we used the sample for screening esterases to incorporate an additional latitude in our transect. In the case of Menai Strait (Irish Sea), five additional esterases were retrieved from a metagenome obtained from enriched cultures prepared with samples collected on 22nd June 2019 from Menai Strait (School of Ocean Sciences, Bangor University, St. George’s Pier, Menai Bridge, N53°13′31.3″; W4°09′33.3”). The water temperature was 14 °C and the salinity was 32 p.s.u. Two enrichment cultures were set up at 20 °C: (i) SW: seawater enrichment with 0.1% lignin; the enrichment was set up using 50 ml of the sample as inoculum with the addition of 0.1% lignin (Sigma-Aldrich, Gillingham, United Kingdom) (w/v); (ii) AW: algal surface wash-off in seawater, enriched with 0.1% lignin; the enrichment was set up using 50 ml of surface wash-off after mixing of ca. 10 g of Fucus (brown algae) in the seawater and removal of plant tissue, 0.1% lignin (w/v) was added. After 92 days of incubation, 5 ml of each enrichment cultures were transferred into the new flask containing 45 ml autoclaved and filtered seawater with 0.1% lignin. This procedure was repeated on days 185 and 260, and the incubation was stopped on day 365. The DNA was extracted using 12 months using MetaGnome extraction kit (EpiCentre, Biotechnologies, Madison, WI, USA), sequenced on Illumina MiSeq™ platform (Illumina Inc., San Diego, CA, USA) using paired-end 250 bp reads at the Centre for Environmental Biotechnology (Bangor, UK), and sequencing reads were processed and analysed as described previously73.The screening, cloning and activity of a subset of 35 identified esterases have been reported previously72. The remaining 48 enzymes are reported for the first time in this study and were identified using naive and in silico metagenomic approaches, as detailed below. The environmental site from which each enzyme originated and the method employed for its identification are detailed in Supplementary Data S3. For naive screens addressing the recovery of new sequences encoding esterases and EXDO, the large-insert pCCFOS1 fosmid libraries made using the corresponding DNA samples, the CopyControl Fosmid Library Kit (Epicentre Biotechnologies, Madison, WI, USA) and the Escherichia coli EPI300-T1R strain were used. The nucleic acid extraction, construction and the functional screens of such libraries have been previously described72. In brief, fosmid clones were plated onto large (22.5 × 22.5 cm) Petri plates with Luria Bertani (LB) agar containing chloramphenicol (12.5 µg ml−1) and induction solution (Epicentre Biotechnologies; WI, USA), at a quantity recommended by the supplier to induce a high fosmid copy number. Clones were scored by the ability to hydrolyze α-naphthyl acetate and tributyrin (for esterase activity), and catechol (for EXDO activity)72,74. Positive clones presumed to contain esterases and EXDOs were selected, and their DNA inserts were sequenced using a MiSeq Sequencing System (Illumina, San Diego, USA) with a 2 × 150-bp sequencing v2 kit at Lifesequencing S.L. (Valencia, Spain). After sequencing, the reads were quality-filtered and assembled to generate nonredundant meta-sequences, and genes were predicted and annotated via BLASTP and the PSI-BLAST tool72. For in silico screens, addressing the recovery of new sequences encoding esterases, the predicted protein-coding genes, obtained after the sequencing of DNA material from resident microbial communities in each of the samples, were used. The meta-sequences are available from the National Center for Biotechnology Information (NCBI) nonredundant public database (accession numbers reported in Supplementary Data S3). Protein-coding genes identified from the DNA inserts of positive clones (naive screen) or from the meta-sequences were screened for enzymes of interest using the Blastp algorithm via the DIAMOND v2.0.9 program with default parameters (percentage of identity ≥60%; alignment length ≥70; e-value ≤1e−5)29, against the Lipase Engineering sequence databases (to screen for esterases) and AromaDeg database (for EXDO)74. Since the collection of sediments across locations experiencing different MATs was limited by our sampling capacity, to expand our range of exploration at a global scale and to validate our dataset, we added our single enzyme analysis to the seawater metagenomes retrieved from the Tara Ocean Expedition database (accession number in Supplementary Data S4). Due to the volume of sequences generated, this database provides access to a large number of enzymes, including those studied here through homology search. Esterases were selected as target sequences, and the following pipeline was used. First, we selected a sequence encoding an esterase reported as one of the most substrate-ambiguous esterases out of 145 tested (EH1, Protein Data Bank acc. nr. 5JD4) and well-distributed in the marine environment72. Second, we performed a homology search of this sequence against the Tara Ocean metagenome21 to retrieve similar sequences, using the Blastp algorithm via the DIAMOND v2.0.9 program30 (e-value 98% using SDS-PAGE analysis in a Mini PROTEAN electrophoresis system (Bio-Rad Laboratories, S.A., Madrid, Spain). Purified protein was stored at –86 °C until use at a concentration of 10 mg ml−1 in 40 mM HEPES buffer (pH 7.0). A total of approximately 5–40 mg of total purified recombinant protein was obtained from 1 L of culture. Supplementary Fig. S1 illustrates a schematic representation of the pipeline implemented in this work to investigate enzyme activities in a large set of marine samples, starting from samples collected (sediments) and available metagenomes.Enzyme activity assessmentsAll substrates used for activity tests were of the highest purity and, if not indicated otherwise, were obtained from Merck Life Science S.L.U. (Madrid, Spain): 4-nitrophenyl-propionate (ref. MFCD00024664), 4-nitrophenyl phosphate (ref. 487663), 4-nitrophenyl β-D-galactose (ref. N1252), bis(p-nitrophenyl) phosphate (ref. 123943), benzaldehyde (ref. B1334), 2-(4-nitrophenyl)ethan-1-amine (ref. 184802-5G), pyridoxal phosphate (ref. P9255), acetophenone (ref. A10701), NADPH (ref. N5130) and catechol (ref. PHL82372). We directly tested total protein extracts for esterase, phosphatase, beta-galactosidase, and nuclease activity using 4-nitrophenyl-propionate, 4-nitrophenyl phosphate, 4-nitrophenyl β-D-galactose, and bis(p-nitrophenyl) phosphate, respectively, by following the production of 4-nitrophenol at 348 nm (extinction coefficient [ε], 4147 M−1 cm−1), as previously described69. For determination: [total protein]: 5 μg ml−1; [substrate]: 0.8 mM; reaction volume: 200 μl; T: 4–85 °C; and pH: 8.0 (50 mM Tris-HCl buffer). The hydrolysis of 4-nitrophenyl-propionate was used to determine, under these standard conditions, the effects of temperature on the purified esterase. Transaminase activity was determined using benzaldehyde as amine acceptor, 2-(4-nitrophenyl)ethan-1-amine as amine donor, and pyridoxal phosphate as a cofactor, by following the production of a colour amine at 600 nm (extinction coefficient, 537 M−1 cm−1), as previously described75. For determination, [total protein]: 5 μg ml−1; [substrates]: 25 mM; [pyridoxal phosphate]: 1 mM; reaction volume: 200 μL; T: 4-85 °C; and pH: 8.0 (50 mM Tris-HCl buffer). Aldo-keto reductase activity was determined using acetophenone as a substrate and NADPH as a cofactor, by following the consumption of NADPH at 340 nm (extinction coefficient, 6220 M−1 cm−1), as described76. For determination, [total protein]: 5 μg ml−1; [substrate]: 1 mM; [cofactor]: 1 mM; reaction volume: 200 μL; T: 4–85 °C; and pH: 8.0 (50 mM Tris-HCl buffer). We determined EXDO activity using catechol as substrate, by following the increase of absorbance at 375 nm of the ring fission products (extinction coefficient, 36000 M−1 cm−1), as previously described74. For determination, [protein]: 5 μg ml−1; [catechol]: 0.5 mM; reaction volume: 200 μL; T: 4–85 °C; and pH: 8.0 (50 mM Tris-HCl buffer). The hydrolysis of catechol was used to determine, under these standard conditions, the effects of temperature on the purified EXDOs. All measurements were performed in 96-well plates (ref. 655801, Greiner Bio-One GmbH, Kremsmünster, Austria), in biological triplicates over 180 min in a Synergy HT Multi-Mode Microplate Reader (Biotek Instruments, Winooski, VT, USA) in continuous mode (measurements every 30 s) and determining the absorbance per minute from the slopes generated and applying the formula (1). All values were corrected for nonenzymatic transformation.$${Rate}left(frac{mu {mol}}{{{min }}{mg},{protein}}right)= frac{frac{triangle {{{{{rm{Abs}}}}}}}{{{min }}}}{{{{{{rm{varepsilon }}}}}},{{{{{rm{M}}}}}}-1{{{{{rm{cm}}}}}}-1}*frac{1}{0.4,{cm}}*frac{{10}^{6},mu M}{1{{{{{rm{M}}}}}}}\ *0.0002,L*frac{1}{{mg},{protein}}$$
    (1)
    Shotgun proteomicsProteomics was performed by using total active proteins (extracted as above), which were then subjected to protein precipitation, protein digestion and Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS/MS) analysis, as previously described77. High-quality reference metagenomes corresponding to each sample (BioProject number in Supplementary Table S3) were used for protein calling, with a threshold of only one identified peptide per protein identification because False Discovery Rates (FDR) controlled experiments counter-intuitively suffer from the two-peptide rule. The confidence interval for protein identification was set to ≥95% (p  50 °C for which the second phase transition was chosen to focus on the decomposition of the core. It is important to note that applying CNA to MD simulations at room temperature may lead to an evening out of Tp values for esterases that transition around this temperature, i.e., systems with a Tp at or below room temperature might all be influenced similarly by loosening their bonding network. By contrast, systems with a transition temperature at or above room temperature would still be discriminated against. The data generated in this study for analyzing Tp values have been deposited at researchdata.hhu.de under accession code DOI: 10.25838/d5p-42101 [https://doi.org/10.25838/d5p-42].Relationship of temperature-induced changes in enzymeRelationship between MAT and enzyme response to temperature (i.e., Topt, Td and Tp) were evaluated by performing linear regression in R. In the case of enzymes retrieved from the Tara ocean dataset we calculated first the break point (flexus) using the package segmented in R102 and then we computed separately the linear model describing the two linear regressions before and after the breakpoint. To evaluate the possible relation between enzyme thermal response and other environmental parameters, salinity and pH data were retrieved from Bio-ORACLE52 using GPS coordinates of each location.Environmental characterization and sediment collection from different temperature variability levels in the Red SeaWe recorded the temperatures of surface sediments from March 2015 to September 2016 along the coast of the Red Sea using HOBO data loggers (Onset, USA) in nine stations located at 3, 25, and 50 m depth. Details on the location, depth and temperature fluctuations of the studied sediments are reported in Supplementary Table S4 and Source Data. We first assess the differences in the homogeneity of the temperature variance in the three types of sediments to evaluate the magnitude of thermal variation and then we test the difference among their MATs using a non-parametric ANOVA (Dunnett’s multiple comparisons tests). We identified three different levels of temperature variability (Fig. 3a–c; Supplementary Table S5): high, intermediate, and low thermal variability (HTV, ITV, and LTV, respectively), where sediments experienced temperature variations of 12.8 °C, 8.8 °C, and 6.7 °C, respectively. From each station, we sampled 200 g of surface sediment (0–5 cm depth) in triplicate in August and December 2015 with a Van der Venn grab (1 dm3) equipped with a MicroCat 250 Seabird CTD (Conductivity, Temperature, Depth), which was assembled on board the research vessel R/V Explorer (KAUST). During sampling, we measured the temperature of the sediments and the water layer covering the sediments using a digital thermometer and the CTD, respectively. We conducted all sampling in compliance with the guidelines specified by KAUST and Saudi Arabian authorities.Sediment processing for analysis of bacterial communitiesFrom each sample (in triplicate), we immediately removed subsamples of sediment (n = 54, ~10 g) and stored them at –20 °C for molecular analysis. Separately, sediment 25 ± 1 g was transferred to 50 ml tubes and added 30 ml of filtered (0.2 µm) water from the Red Sea. The tubes were shaken at 500 rpm for one hour and then centrifuged them at 300 g for 15 min to detach the microbial cells in the sediments without affecting their vitality103,104. The supernatant containing the extracted cells was collected in sterile tubes and was immediately used to measure microbial growth rates.Evaluation of bacterial growth in sediments at different temperaturesWe evaluated the microbial growth rate of the heterotrophic community extracted from the sediments under HTV, ITV, and LTV at 10 °C, 20 °C, 30 °C, 40 °C and 50 °C, using Marine Broth as the cultivation medium (Zobell Marine Broth 2216) supplemented with 0.1 g/L cycloheximide; a rich-medium was selected to avoid the nutrient limitation effect that can affect bacterial physiology63,105. We inoculated 96-well plates with 200 µl of cultivation medium and 25 µl of the cell suspension extracted from the sediments. We inoculated the three biological replicates from each station and each level of temperature variability in eight wells, giving a total of 72 wells for each plate, with 24 wells used as a negative control inoculated with water. We assembled a total of three plates for each incubation temperature from August and December. Plates were spectrophotometrically measured at 3 h intervals using an optical density of 600 nm (Spectramax® M5) for 72 h. Wells with optical density 90%) for further analysis (Supplementary Tables S9 and S10). We calculated the compositional similarity matrix (Bray-Curtis of the log-transformed OTU table) with Primer 6109. Using the same software, canonical analysis of principal coordinates (CAP)110 was used to compare the temperature variability samples (temperature variability levels: HTV, ITV, and LTV; season levels: August and December) based on the compositional similarity matrix. We applied permutational multivariate analyses of variance to the matrix (PERMANOVA; main and multiple comparison tests). We tested the occurrence of thermal-decay patterns in sediments with different temperature variability levels using linear regression (Prism 9.2 software, La Jolla California USA, www.graphpad.com) between the bacterial community similarities (Bray-Curtis) and the temperature differences among sediments (∆T°C) at the time of sampling. We calculated alphadiversity indices (richness and evenness) using the paleontological statistics (PAST) software, and their correlation with temperature was modelled using linear regression in Prism 9.2. Spearman correlation among temperature and relative abundance of OTUs within each sediment sample was evaluated; OTUs were classified based on their positive (enriched) and negative (depleted) correlation with sediment temperature.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article. More

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    Environmental changes associated with drying climate are expected to affect functional groups of pro- and microeukaryotes differently in temporary saline waters

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