Ecology

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Sets of interaction-associated mutants change across interactive conditionsTo investigate how microbial interactions are reorganized in a microbial community with increasing complexity, we reconstructed in vitro a modified bloomy rind cheese-associated microbiome on Cheese Curd Agar plates (CCA plates) as described in our previous work14 Growth as a biofilm on agar plates models the surface-associated growth of these communities, and allows inclusion of the filamentous fungus, P. camemberti, which grows poorly in shaken liquid culture. The original community is composed of the gamma-proteobacterium H. alvei, the yeast G. candidum and the mold P. camemberti. Using a barcoded transposon library of the model bacterium E. coli as a probe to identify interactions, we investigated microbial interactions in 2-species cultures (E. coli + 1 community member), in 3-species cultures (E. coli + 2 community members) and in 4-species cultures (or whole community: E. coli + 3 community members) (Fig. 1a).Figure 1Changes of E. coli’s genes associated with interaction-associated mutants in 2-species, 3-species and 4-species cultures. (a) Experimental design for the identification of interaction-associated mutants in 7 interactive conditions from the Brie community. The E. coli RB-TnSeq Keio_ML9 (Wetmore et al. 2015) is either grown alone or in 2, 3 or 4 species cultures to calculate E. coli gene fitness in each condition (in triplicate). Interaction fitness effect (IFE) is calculated for each gene in each interactive culture as the difference of the gene fitness in the interactive condition and in growth alone. IFE that are significantly different from 0 (two-sided t-test, Benjamini–Hochberg correction for multiple comparisons) highlight interaction-associated mutants in an interactive condition. (b) Volcanoplots of IFEs calculated for each interactive condition. Adjusted p-values lower than 0.1 highlight significant IFEs. Negative IFEs (blue) identify negative interactions and positive IFE (red) identify positive interactions. Numbers on each plot indicate the number of negative (blue) or positive (red) IFEs. (c) Functional analysis of the interaction-associated genes (significant IFEs). Genes of interaction-associated mutants have been separated into two groups: negative IFE and positive IFE. For each group, we represent the STRING network of the genes associated with interaction-associated mutants (Nodes). Edges connecting the genes represent both functional and physical protein association and the thickness of the edges indicates the strength of data support (minimum required interaction score: 0.4—medium confidence). Nodes are colored based on their COG annotation and the size of each node is proportional to the number of interactive conditions in which that given gene has been found associated with a significant IFE. Higher resolution of the networks with apparent gene names are found in Supplementary Figs. 2, 3.Full size imageQuantification of species’ final CFUs after 3 days of growth highlighted consistent growth for H. alvei and G. candidum independent of the culture condition and slightly reduced growth for E. coli in interactive conditions compared to growth alone (Dunnett’s test against growth alone; adjusted-p value ≤ 5%) except for the 2-species growth with P. camemberti (Supplementary Fig. 1). Although we were unable to quantify spores of P. camemberti after three days, growth of P. camemberti was visually evident in all of the expected samples. Quantitative analysis of E. coli’s library final growth using an epistatic model highlighted that the growth of E. coli in the 3-species and 4-species condition can be predicted from the corresponding 2-species growths (Supplementary Fig. 1).Previously, we developed an assay and a pipeline to identify microbial genes associated with interactions by adapting the original RB-TnSeq approach19 to allow for consistent implementation of biological replicates as well as for direct quantitative comparison of fitness values between different culture conditions15. More specifically, the original RB-TnSeq assay relies on the use of a dense pooled library of randomly barcoded transposon mutants of a given microorganism (RB-TnSeq library)19 containing multiple insertion mutants for each gene as well as intergenic insertion mutants. Measuring the variation of the abundance of each transposon mutant before and after growth, the pipeline allows the calculation of a fitness value for each insertion-mutant as well as a fitness value for each gene corresponding to the average of the insertion-mutants’ fitness of the associated genes across biological replicates. A negative fitness indicates that disruption of this gene decreases growth of the mutant relative to a wild type strain, whereas a positive fitness value indicates increased growth in the studied condition. Then, we infer the interactions based on the effects of insertion-mutants between interactive growth and growth alone. In other words, we measure and compare gene fitness across the different studied conditions. Any significant change in fitness values identifies an interaction-associated mutant. The subsequent analysis of interactions, including the inference of the interaction mechanisms and the comparison of interactions across the different interactive conditions, is mainly based on the nature of the disrupted genes by the transposon and their characterized function. Also, by measuring interactions as the difference of fitness value of a given gene between growth with other species and growth alone, we consider that interactions between insertion-mutants of the RB-TnSeq library are controlled and included in our calculation. Then, any interaction-associated mutant predominantly identifies inter-species interactions.In this work, we used the E. coli RB-TnSeq Keio_ML9 library19 and grew it for 3 days alone or in the seven different interactive conditions studied here (Fig. 1a). This library contains 152,018 pooled insertion mutants with an average of 16 individual insertion mutants per gene and many intergenic insertion mutants. For each interactive condition, we calculated the Interaction Fitness Effect (IFE) associated with 3699 E. coli genes as the difference between the gene fitness in the studied interactive condition and the gene fitness in growth alone (Supplementary Data 1). Negative IFE occurs when gene fitness decreases in the interactive condition, and positive IFE occurs when gene fitness improves in the interactive condition. We then tested for all the IFEs that are significantly different from 0 (adjusted p-value ≤ 0.1; two-sided t-test and Benjamini–Hochberg correction for multiple comparison20) to screen for interactions and to identify, in each condition, the insertion-mutants that are associated with inter-species interactions. Here, we identified between 6 (with P. camemberti) and 71 (with H. alvei + P. camemberti) significant IFEs per condition (Fig. 1b). Both negative IFEs and positive IFEs were found in each interactive condition except for the 2-species culture with P. camemberti, where only negative interactions were identified. A total of 330 significant IFEs associated with 218 unique genes were identified (as the same gene can be associated with a significant IFE in multiple conditions) including 125 genes associated with negative IFE and 120 genes associated with positive IFE (Supplementary Figs. 2, 3). Altogether, we didn’t notice any strong correlation between the number and type of IFE identified by condition and the overall growth impact measured on E. coli.
To gain insight into the interaction mechanisms among microbes, we next analyzed the functions of the genes of the interaction-associated mutants (i.e., genes associated with a significant IFE). Here, the vast majority of the genes associated with interaction-associated mutants are part of an interaction network (Fig. 1c). These STRING networks connect genes that code for proteins that have been shown or are predicted to contribute to a shared function, with or without having to form a complex21. A significant fraction of the interaction-associated mutants associated with a negative IFE are part of amino acid biosynthesis and transport (17%—Fig. 1c and Supplementary Figs. 2, 4), and more specifically with histidine, tryptophan and arginine biosynthesis. This points to competition for these nutrients between E. coli and the other species. Another large set of interaction-associated mutants is related to nucleotide metabolism and transport (14%—Fig. 1c and Supplementary Figs. 2, 5), highlighting competitive interactions for nucleotides and/or their precursors. The majority of the associated genes relate to purine nucleotides and more specifically to the initial steps of their de novo biosynthesis associated with the biosynthesis of 5-aminoimidazole monophosphate (IMP) ribonucleotide. Of the genes associated with interaction-mutants with a positive IFE, 15% are related to amino acid biosynthesis and transport (Fig. 1c and Supplementary Figs. 3, 4), suggesting cross feeding of amino acids between E. coli and the other species. More specifically, this includes phosphoserine, serine, homoserine, threonine, proline and arginine. The presence of amino acid biosynthetic genes among both negative and positive IFEs indicate that trophic interactions (competition versus cross-feeding) depend on the type of amino-acid and/or the species interacting with E. coli. For both negative and positive IFEs, numerous genes of the associated interaction-mutants were annotated as transcriptional regulators (Fig. 1c and Supplementary Figs. 2, 3) emphasizing the importance of transcriptional reprogramming in response to interactions. These transcriptional regulators include metabolism regulators as well as regulators of growth, cell cycle and response to stress. Finally, these interaction-associated mutants and the infered interaction mechanisms are consistent with previous findings in this microbiome14 as well as in a study of bacterial-fungal interactions involving E. coli and cheese rind isolated fungal species15. While this approach allows us to infer the interaction mechanisms that are happening between the transposon library and the other species, further experimental validation would be needed to confirm that these interactions more generally happen between a WT strain and the other species.Introduction of a third interacting species deeply reshapes microbial interactionsThe differences in the number and sign of significant IFEs observed among the different interactive conditions, with different numbers of interaction species, suggest that the number and type of interacting partners influence interaction mechanisms. To characterize how the interactions are reorganized with community complexity, we then investigated if and how the genetic basis of interactions changes when the number of interacting partners increases by comparing the genes associated with interaction-associated mutants with significant IFE in 2-species cultures, in 3-species cultures and then in 4-species cultures.First, we have identified 104 IFEs associated with 98 genes in 2-species cultures as well as 168 IFEs associated with 136 unique genes in 3-species conditions (Supplementary Fig. 6 and Supplementary Data 2). Comparing these gene sets, we can identify how the interaction-associated mutants change when a third-species is added to a 2-species culture. We identified 45 genes associated with 2-species interaction-associated mutants maintained in at least one 3-species condition (maintained interaction-mutants), 55 genes associated with 2-species interaction-associated mutants no longer associated with interaction in any 3-species condition (dropped interaction-mutants) and 100 genes associated with 3-species interaction-associated mutants that aren’t related to any 2-species interaction-associated mutants (emergent interaction-mutants) (Fig. 2a, Supplementary Fig. 6 and Supplementary Data 3). Both dropped and emerging interaction-associated mutants represent 3-species HOIs; the third species either removes an existing interaction or brings about a new one.Figure 2Comparison of the genetic basis of interaction for 2-species and 3-species conditions. (a) Venn Diagram of 2-species and 3-species sets of genes related to interaction-associated mutants. This Venn Diagram identifies 2-species interaction-mutants that are dropped when a third species is introduced (Left side; Dropped interaction-mutants = any 2-species gene that is not found in any 3-species condition), 2-species interaction-mutants that are maintained in at least one associated 3-species condition (Intersection; Maintained interaction-mutants) and interaction-mutants that are specific to 3-species condition (Right side; Emerging interaction-mutants). (b) Functional analysis of the genes associated with dropped, maintained and emerging interaction-mutants from 2-species to 3-species. Each dot represents the fraction of genes of the studied gene set associated with a given COG category (Number of genes found in the category / Total number of genes in the gene set). The color of the dots indicates the general COG group of the COG category: Teal: Metabolism; Blue: Information storage and processing; Orange: Cellular Processes and Signaling; Grey: Unknown or no COG category. (c) Species-level analysis of 3-species HOIs: for each 2-species condition, we measure the fraction of interaction-mutants that are dropped in associated 3-species cultures (Dropped in 3-species) or maintained in at least one of the 3-species cultures (Maintained in 3-species); for each 3-species condition, we measure the fraction of interaction-mutants that have been conserved from at least one associated 2-species condition (Maintained from 2-species) or that are emerging with 3-species (Emerging in 3-species).Full size imageWe further carried out functional analysis of the genes related to maintained, dropped and emerging interaction-mutants to elucidate whether maintained and HOIs interaction-mutants would be associated with specific functions and thus interaction mechanisms (Fig. 2b). For each set of genes, we calculated the fraction of genes of that set associated with a given COG ontology category. Metabolism and transport is the most observed COG group (Fig. 2b—teal dots). For genes related to maintained interaction-mutants, this indicates that some trophic interactions can be maintained from 2-species to 3-species conditions. For instance, serine biosynthetic genes serA, serB and serC as well as threonine biosynthetic genes thrA, thrB and thrC are associated with positive IFEs in the 2-species condition with G. candidum as well as in the 3-species conditions involving G. candidum (Supplementary Fig. 4). This suggests that, (i) G. candidum facilitates serine and threonine cross feeding and (ii) this cross-feeding is still observed when another species is introduced. However, metabolism-related genes identified among the dropped and emerging interaction-mutants indicate that many trophic interactions are also rearranged through HOIs. Genes associated with lactate catabolism (lldP and lldD) and lactate metabolism regulation (lldR) have a negative IFE in the 2-species culture with H. alvei, suggesting competition for lactate between E. coli and H. alvei. Yet, mutants of these genes are no longer associated with a significant IFE when at least another partner is introduced (Supplementary Fig. 7). Histidine biosynthesis genes hisA, hisB, hisD, hisH and hisI are associated with interaction-mutants with negative IFE in the 2-species culture with H. alvei and sometimes in the 3 species culture with H. alvei + P. camemberti. However, the negative IFE is alleviated whenever G. candidum is present, suggesting that potential competition for histidine between E. coli and H. alvei is alleviated by this fungal species (Supplementary Fig. 4). Also, genes related to the COG section “Information storage and processing” are mostly found among genes of HOIs-mutants suggesting a fine-tuning of specific cellular activity depending on the interacting condition. For instance, we identified many transcriptional regulators of central metabolism among the dropped interaction-mutants genes (rbsR and lldR) and the emerging interaction-mutants genes (purR, puuR, gcvR and mngR), highlighting again the reorganization of trophic interactions associated with HOIs. Also, many transcriptional regulators broadly associated with growth control, cell cycle and response to stress were found among the emerging interaction-mutants genes with 3-species (hyfR, chpS, sdiA, slyA and rssB), underlining a noticeable modification of E. coli’s growth environment with 3-species compare to with 2-species.Finally, we further aimed to understand whether HOIs are associated with the introduction of any specific species (Fig. 2c and Supplementary Fig. 8). We observe that interaction-associated mutants with H. alvei are more likely to be dropped, as 65% of them are alleviated by the introduction of a fungal species (Fig. 2c). This can be seen, for instance, with the reorganization of E. coli and H. alvei trophic interactions following the introduction of G. candidum (alleviation of lactate and histidine competition for instance). Also, we observe that 76% of the interactions in the 3-species cultures with H. alvei + P. camemberti and 65% in the 3-species culture with H. alvei + G. candidum are emerging interaction-mutants (compared to 38% of emerging interaction-associated mutants in the 3-species condition with G. candidum + P. camemberti) (Fig. 2c). For the interaction-associated mutans found in the 3-species with H. alvei + P. camemberti, they include for instance the genes associated with purine de novo biosynthesis (purR, purF, purN, purE, purC) and the genes associated with pyrimidine de novo biosynthesis (pyrD, pyrF, pyrC, carA and ulaD), suggesting important trophic HOIs. For the 3-species condition with H. alvei + G. candidum, emerging interaction-mutants include for example the transcriptional regulator genes chpS, sdiA and slyA, indicating the presence of a stress inducing environment. Together, these observations suggest that the introduction of a fungal partner may introduce multiple 3-species HOIs by both canceling existing interactions and introducing new ones.HOIs are prevalent in a 4-species communityTo further decipher whether microbial interactions continue to change with increasing community complexity, we investigated the changes in the genetic basis of interactions going from 3-species to 4-species experiments. We identified 58 interaction-associated mutants in the 4-species condition (E. coli with H. alvei + G. candidum + P. camemberti), compared with 145 interaction-associated mutants in any 3-species condition. Comparing the two sets of interaction-associated mutants and corresponding genes we identify: 26 3-species interaction-mutants that are maintained in the 4-species condition (including 16 directly from 2-species interactions), 115 3-species interaction-mutans that are no longer associated with interactions in the 4-species condition (dropped interaction-mutants) and 32 interaction-mutants that are observed solely in the 4-species condition (emerging interaction-mutants) (Fig. 3a, Supplementary Fig. 6 and Supplementary Data 3). Both dropped and emerging interaction-mutants represent 4-species HOIs. Here, HOIs are remarkably abundant when introducing a single new species and moving up from 3-species interactions to 4-species interactions. Functional analysis of the genes of maintained-mutants and HOI-mutants reveals the presence of many metabolism related genes in every gene set (Fig. 3), suggesting that some trophic interactions can be maintained from 3-species to 4-species interactions while some other trophic interactions are rearranged with HOIs. For instance, most of the genes of the initial steps of de novo purine biosynthesis have been found to be associated with a negative IFE in the 3 species condition with H. alvei + P. camemberti (purC, purE, purF, purL and purN) as well as in the pairwise condition with H. alvei for purH and purK (Supplementary Fig. 5), suggesting competition for purine initial precursor IMP in these conditions. Yet, the introduction of the yeast G. candidum as a fourth species cancels the negative IFE value, suggesting that the competition is no longer happening in its presence. Altogether, the observation of noticeable trophic HOIs moving up from 2 to 3 species and then from 3 to 4-species interaction highlights a consistent reorganization of trophic interactions along with community complexity. Also, genes related to Cell wall/membrane/envelope biogenesis are found abundantly among the 4-species emerging-mutants (Fig. 3b) and they represent the largest functional fraction of this gene set. These genes are associated with a negative IFE and are related to Enterobacterial Common Antigen (ECA) biosynthetic processes (wecG, wecB and wecA) (Supplementary Fig. 9). While the roles of ECA can be multiple but are not well defined22, they have been shown to be important for response to different toxic stress, suggesting the development of a specific stress in the presence of the four species.Figure 3Organization of the interactions in the 4-species community. (a) Venn Diagram of 3-species and 4-species sets of genes related to interaction-associated mutants. This Venn Diagram identifies 3-species interaction-mutants that are dropped when a fourth species is introduced (Left side; Dropped interaction-mutants = any 3-species interaction-associated mutant that is not found in the 4-species condition), 3-species interaction-mutants that are maintained in the 4-species condition (Intersection; Maintained interaction-mutants) and interaction-mutants that are specific to 4-species condition (Right side; Emerging interaction-mutants). (b) Functional analysis of the genes associated with dropped, maintained and emerging interaction-mutants from 3-species to 4-species. Each dot represents the fraction of genes of the studied gene set associated with a given COG category (Number of genes found in the category/Total number of genes in the gene set). The color of the dots indicates the general COG group of the COG category: Teal: Metabolism; Blue: Information storage and processing; Orange: Cellular Processes and Signaling; Grey: Unknown or no COG category. (c) Species-level analysis of 4-species HOIs: for each 3-species cultures we measure the fraction of interaction-genes that is conserved in the 4-species culture (Maintained in 4-species) and the fraction of interaction-genes that has been dropped (Dropped in 4-species). (d) Alluvial plots of the interaction genes across community complexity levels. (e) STRING network of the 4-species interaction genes (Nodes). Edges connecting the genes represent both functional and physical protein association and the thickness of the edges indicates the strength of data support (minimum required interaction score: 0.4—medium confidence). Nodes are colored based on the level of community complexity the genes are conserved from.Full size imageAs for the 2 to 3 species comparison, we investigated whether the introduction of a specific fourth species would be most likely associated with HOIs. The 3-species culture that appears to be the least affected by the introduction of a fourth member is with G. candidum + P. camemberti where 34% of the observed interactions are still conserved in the 4-species condition after the introduction of H. alvei (versus 22% for with H. alvei + G. candidum when P. camemberti is added and 21% for with H. alvei + P. camemberti when G. candidum is added) (Fig. 3c and Supplementary Fig. 10). Together, these observations suggest that, again, the introduction of a fungal partner may introduce multiple 4-species HOIs.Finally, by increasing the number of interacting species in our system and investigating interaction-mutants maintenance and modification with every increment of community complexity, we are able to build our understanding of the architecture of interactions in a microbial community. Altogether, we have observed a total of 218 individual interaction-associated mutants in any experiment. Only 16 of them (7%) were conserved across all levels of community complexity (Fig. 3d). Starting from 2-species interaction-mutants, 48% of them were maintained with 3-species and only 15% (16 out of 104) were still maintained with 4-species. Thus, we demonstrate here a progressive loss and replacement of 2-species interactions as community complexity increases and the prevalent apparition of HOIs. Tracking back the origins of the genetic basis of interactions in the 4-species experiment that represents the full community of our model, we identify that 28% of the full community interactions can be traced back to 2-species interactions, 18% are from 3-species interaction and 54% are specific to the 4-species interaction (Fig. 3d,e). Most of the maintained interaction-mutants from 2-species as well as from 3-species are associated with metabolism (Fig. 3d and Supplementary Fig. 11) while Signal transduction and cell membrane biosynthesis genes are most abundant among the 4-species interaction-mutants as previously mentioned. To conclude, this shows that the genetic basis of interactions and thus the sets of microbial interaction are deeply reprogrammed at every level of community complexity and illustrates the prevalence of higher order interactions (HOIs) even in simple communities.The majority of maintained 2-species interaction-mutants in the 4-species culture follows an additive conservation behaviorWhile HOIs are abundant in the 4-species condition, our data yet suggest that up to 28% of the interactions are maintained from 2-species interactions. However, we don’t know whether and how 2-species interactions are quantitatively affected by the introduction of other species and whether they would follow specific quantitative models of conservation. For instance, we can wonder how the strength of a given 2-species interaction is modified by the introduction of one or two other species, or how two 2-species interactions associated with the same gene will combine when all the species are present. In other words, can we treat species interactions as additive when we add multiple species? Such information would generate a deeper mechanistic understanding of the architecture of microbial interactions while allowing us to potentially predict some whole community interactions from 2-species interactions. Here, two main hypothetical scenarios can be anticipated. First, the conservation of 2-species interactions follows a linear or additive behavior, where the introduction of other species either doesn’t affect the strength of the conserved 2-species interaction or two similar 2-species interactions combine additively. The second scenario identifies non-linear or non-additive conservation of 2-species interactions, where the strength of the conserved 2-species interaction is modified by the introduction of other species or two similar 2-species interactions are not additive. The second scenario would encompass for instance synergistic effects or inhibitory effects following the introduction of more species. We next use an epistasis and quantitative genomics approach to understand whether interactions that are conserved follow a linear, or additive, pattern. For the 16 interaction-associated mutants that are associated with interaction in 2-species cultures, in associated 3-species cultures and in the 4-species condition, we use epistasis analysis to test the linear behavior of their IFE when the number of interacting species increases, as IFEs are quantitative traits related to the interaction strength. In multi-dimensional systems, an epistasis analysis quantifies the additive (or linear) behavior of conserved quantitative traits. In quantitative genetics, for instance, epistasis measures the quantitative difference in the effects of mutations introduced individually versus together18,23,24. Using a similar rationale, we can use IFEs as a quantitative proxy for interaction strength and test whether the IFEs of the maintained interaction genes in 3-species and in 4-species conditions result from the linear combination of associated 2-species IFEs (Fig. 4a). Nonlinear combination, or non-additivity of 2-species IFEs in higher community level also highlights higher-order interactions.Figure 4Quantitative analysis of IFE conservation for the interaction-associated mutants conserved from 2-species to 4-species conditions. (a) Schematized quantitative epistasis/non-linearity measured in 3-species conditions (with partner i and j). Epistasis (εij) is the difference between the individual IFE of partner i and partner j (red and orange bars) versus placing them together (green). Mathematically, we need three terms (IFEi, IFEj, and εij) to reproduce the observed IFE for the 3-species condition. (b) This analysis can be extended to higher levels of community complexity: 4-species (E. coli with 3-partners i, j, and k). The model first accounts for epistasis between i/j, i/k, and j/k. In this example, i and j exhibit epistasis; i/k and j/k are additive (dark blue and purple). The predicted IFE for the 4-species community is the sum of the individual 2-species effects (red, orange, light blue) and the 3-species epistatic terms (green). The 4-species epistatic coefficient is the difference between this low-order prediction and the observed IFE for the i,j,k community (pink). (c) Conservation profiles of the 16 2-species interaction-associated mutants conserved up to 4-species. 2-species conditions: a colored square indicates the 2-species condition(s) in which the interaction-associated mutant was identified; a grey square indicates non-significant 2-species IFEs. 3-species conditions: a teal square indicates that the associated IFE is associated with additive behavior from associated 2-species IFE (no εij epistatic coefficient), a red square indicates that the associated IFE displays non-additivity from 2-species IFE and thus epistasis, a grey square corresponds to a 3-species condition that is not associated with significant 2-species IFE (no epistasis analysis performed); 4-species condition: a teal square indicates that the associated IFE is associated with additive behavior (no εijk epistatic coefficient) , a red square indicates that the associated IFE is associated with non-additivity from lower-order IFE. (d) Comparison of the observed and predicted IFE for the genes and condition associated with 3-species and 4-species non-additive IFE.Full size imageWe adapted the pipeline Epistasis17, originally designed for quantitative genetics investigation. We implemented the linear model with the gene fitness values of the interaction-associated mutants for growth alone, for each of the 2-species conditions, for each of the 3-species cultures and for the 4-species condition. For each gene, the software finds the simplest mathematical model that reproduces the observed IFEs across all levels of community complexity. In the simplest case, the model will have a term describing the effects for adding each species individually to the E. coli alone culture; that term corresponds to the 2-species IFE. Then, if the IFE for two E. coli’s partners combined (3-species IFE) differs from the sum of their individual effects (corresponding 2-species IFE), the software adds a term capturing this epistasis (Fig. 4a). Here, we call that term 3-species epistatic coefficient or εi,j. Finally, if the IFE for the combined community (E. coli plus all three species; 4-species condition) differs from the prediction based on the 2-species and 3-species terms, the software will add a high-order interaction term to the model (Fig. 4b). Here, we name that term 4-species epistatic coefficient or εijk.We performed this analysis on the 16 interaction-associated mutants that are associated with interactions at every level of community complexity. To identify real additive behavior of IFE from non-additivity, we screen for 3-species epistatic coefficients and 4-species epistatic coefficients that are significantly different from 0 (adjusted p-value ≤ 0.01, Benjamini–Hochberg correction for multiple testing). We found that 13 interaction-associated mutants behaved additively from 2-species to 4-species culture, with no epistatic contributions in the 3-species conditions nor in the 4-species condition (Fig. 4c, (i)). One interaction-associated mutant (gene (gadW)) exhibited nonlinear conservation of IFE only in the 4-species condition, but additive IFE conservation from 2-species to 3-species (Fig. 4c, (ii)). Another interaction-associated mutant (gene (lsrG)) showed epistasis in one 3-species condition but no epistasis in the 4-species condition (Fig. 4c, (iii)) Finally, one interaction-associated mutant (gene (gltB)) displayed both non-additivity in 3-species and 4-species conditions (Fig. 4c, (iv)). If we look more closely at the genes related to interaction-associated mutant with an additive behavior, we find genes (betA, betT, purD and purH) that are associated with the conservation of negative IFEs (Supplementary Fig. 12). While betA and betT are associated with choline transport (betT) and glycine betaine biosynthesis from choline (betA)25, purD and purH are associated with de novo purine biosynthesis26. This suggests that requirements for glycine betaine biosynthesis from choline and for purine biosynthesis caused by microbial interactions, possibly due to competition for the nutrients used as precursors, are additively conserved from individual 2-species interactions requirements. Also, 5 genes associated with amino acid biosynthesis (serA, thrC, cysG, argG and proA) are associated with the additive conservation of positive IFE (Supplementary Fig. 12), suggesting that cross feeding can be additive when the community complexity increases. Altogether, this highlights the existence of 2-species interactions, including trophic ones, conserved in an additive fashion in the highest-level of complexity.This leaves 3 interaction-associated mutants (18%) of the maintained 2-species interaction-mutants, that are associated with non-additive behavior, and thus HOIs, at at least one higher level of community complexity (Fig. 4c—(ii), (iii) and (iv)). The interaction-associated mutant for the gene gadW is associated with non-additivity at the 4-species level, suggesting that while IFEs are additive in 3-species cultures, the introduction of a fourth species introduces HOI. Moreover, the observed 4-species IFE is greater than the IFE predicted by a linear model (Fig. 4d), highlighting a potential synergistic effect when the 4 species are together. The interaction-assoacited mutant for the gene lsrg is associated with non-additivity only at the 3-species culture w G.c + P.c. More specifically, this indicates that HOI arise when these 2 fungal species are interacting together with E. coli, but that no more HOI emerge when H. alvei is introduced (i.e., the 4-species IFE can be predicted by the linear combination of the lower levels IFEs). As the observed IFE for the 3-species condition w G.c + P.c is greater than the predicted IFE (Fig. 4c), this suggests a synergistic effect between the 2 fungal species. Finally, the interaction-associated mutants for the gene gltB is associated with non-additivity at both the 3-species and 4-species levels. For this interaction-associated mutant, the conservation of IFE is never associated with an additive model. Here, the observed 4-species IFE is not as negative as it would be as the result of the linear combination of the associated lower IFE (Fig. 4d), suggesting the existence of a possible IFE threshold, or plateau effect. Altogether, this indicates that maintained 2-species-interactions can follow nonlinear behaviors that could involve synergistic effects, inhibitory effects or constraints. More

Study areaThe study was carried out at the University of Dodoma (UDOM) and specifically at College of Natural and Mathematical Sciences (CNMS) and College of Education (COED) (Fig. 1). These two sites were considered because they have both afforested and non-afforested areas. Furthermore, unlike other places where afforestation is uncoordinated, the selected study area has proper management and records for the afforestation program that is taking place. The study area is located at latitude of 6° 57´ and 3° 82´ and longitudinal of 36° 26´ and 35° 26´. Its elevation is estimated to be 1120 m above the sea level. The site is semi-arid area dominated by sandy loam soil classified as Oxisol. The average annual rainfall of the areas is 447 mm. Temperatures vary depending on the season, with average minimum and maximum of (18^circ{rm C}) and 32 (^circ{rm C}) respectively.Figure 1Map showing the study area within the University of Dodoma (Created using QGIS 3.28.0 Firenze version, 2022). Note: CNMS-College of natural and mathematical science, CHS-College of humanities and social science, CIVE- College of informatic and virtual education, COED-College of Education.Full size imageThe bush is leafless and dry during the dry season, but comes to life during the rainy season, when the entire countryside turns a vibrant green19,20 The remaining land is covered in woodlands, with the highest concentrations in hills (URT 2014). The vegetation consists of dry savanna shrub-thicket areas with scattered trees and grassland patches interrupted by trees and shrubs.Study design on abundance and diversity of lizardsData on lizard abundance and diversity were collected at two sites, namely the CNMS and another site located at COED. These areas were purposely selected because the afforestation program is taking place. In the selected areas, trees have been planted for the past three years, which are 2019–2021. More effort is being made to plant more trees. Also, the areas have natural vegetation characterized by thickets, shrubs, and nature trees with species as described above in the study area. This makes the areas ideal for making comparisons between the afforested and un-afforested areas. In each site, two blocks were established, in which one block consisted of an afforested area while the other block was a non-afforested area.Data collectionDocumentation of planted tree speciesThe plants observed in the study areas were recorded. In addition to that, we worked with the restoration team, which provided the list of tree species that are grown in those study areas. Secondary data was collected from the restoration team regarding the tree species and how much has been planted in the last 5 years in the study areas.Sampling of lizard for abundance and diversity determinationPitfall trapsEach block had a size of 60 m by 60 m (2600 m2). In each block, two transects were established, each with a length of 60 m and a spacing of 20 m. In each transect, 4 points were identified, whereby 10 pitfall traps of 5 L each were set at an interval of 12 m. This makes 40 pitfall traps and eight walking transects. Emptying was performed every morning for 10 consecutive days in each pitfall. Thus, a total of 800 samples were collected from pitfall traps, with 400 samples being collected at each site.Direct searchingGeneral direct searching involving time-constrained observation was also used to collect data on the lizards found in the study area. Time constrained searches were conducted as an opportunistic means of finding animals hiding under cover and flushing them as the observer approached. Searching was conducted in an area of 20 m × 20 m at each sampling point where pitfalls were set. Searching was performed by an individual who is an ecologist and is an expert in reptiles for 10 min, 3 times a day for 10 days (n = 240). To ensure consistency, the same individual was employed in searching for each sampling point.At each site, the observed lizards were identified by their numbers and habitats. Photographs of captured or observed animals were taken to aid in identification. In addition, human activities such as cultivation, roads, tree cutting, building, and distance from roads and buildings, were recorded. Furthermore, more physical structures like rocks and distances from rocks were recorded. Identification of species of lizards was performed using a guide book for east African reptiles21.Sampling and interview for the assessment of awareness of the importance of afforestationA cross-sectional survey using a semi-structured questionnaire was used to collect data from undergraduate students in four colleges, which are CNMS, COED, CHSS, and CIVE. The respondents were selected randomly from each college. These students were selected based on their familiarity with the areas that are anticipated to see what is taking place within the University of Dodoma. It was anticipated that awareness would vary by college because the programs offered differed. For example, it was predicted that students from CNMS would be more aware than others because they have programs and courses that teach conservation, restoration, and afforestation knowledge. Both genders were included in the survey. A total of 394 interviewees were recruited; 100 participants were from CHSS, 103 from CIVE, 101 from CNMS, and 90 from COED. The questionnaire consisted of both closed and open-ended questions. The questions consisted of information on the demographic structure of students and their awareness of the afforestation program. Concerning awareness, the questions focused on their understanding of afforestation, their participation, and other stakeholders involved in the program.Some questions had to be ranked from 1 to 5, with the answers classified as very high, high, moderate, low, and very low if they scored 5, 4, 3, 2, and 1, respectively. The questions were designed to elicit responses from respondents regarding their knowledge of the ongoing afforestation program. In addition, information on the program’s participants and their level of involvement was requested.Human ethical guideline statementAll methods were carried out in accordance with relevant guidelines and regulations.Ethical approval and consent to participateThe ethics committee of University of Dodoma granted ethical approval for this study, with reference number MA.84/261/02.Informed consentInformed consent was obtained from all participants included in the study. More

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