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    Allometry reveals trade-offs between Bergmann’s and Allen’s rules, and different avian adaptive strategies for thermoregulation

    Bergmann’s ruleVariation in avian body size has arisen through millions of years of evolution43, and our data reflects this by showing that log body mass is strongly predicted by phylogeny (Supplementary Table 1). Yet, avian body size also shows large geographical variation (Fig. 1a), and our analysis provides strong support for Bergmann’s rule across the global community of birds. Phylogenetic linear mixed models indicated that the temperature variables explain from 9.0% to 11.8% of the variance in log-transformed body size (estimated with r-squared; Fig. 1b). These models are substantially better supported than the null model and the model with latitude alone (Fig. 1b), suggesting that the observed geographical pattern is linked to thermoregulation. All of these temperature models indicate that temperature negatively correlates with body size (Fig. 1c and Supplementary Fig. 1), as predicted by Bergmann’s rule.Fig. 1: Global test of Bergmann’s rule across 9962 (99.7%) avian species.Distribution of log-transformed body mass across species geographic ranges, shown as their geometric centroids (a). Model selection procedure for predicting log body mass (b), with six temperature measures assessed within species geographic ranges, as sole fixed effects; AIC—Akaike Information Criterion, r2—coefficient of determination. An exemplar Bergmann’s model (c), showing decreasing body size with max temperature of all months; see Supplementary Fig. 1 for surrogate models based on the other temperature measures (evaluated in b). The shaded area around the trend line is simple shading to facilitate reading. The p values refer to the significance of temperature effect and whether it differs from zero as derived from a two-tailed test. The results were obtained with phylogenetic linear regression by phylolm models on a single maximum clade credibility phylogenetic tree.Full size imageAllometry of appendagesAllen’s hypothesis3 implies that the length of animal’s appendages varies with temperature in relative (not absolute) terms, thus when asking how the appendage length vary across temperature gradient, we always need to control for body size. Phylogenetic log-log regression models revealed that body mass explains 72.7% and 72.5% of variance in beak and tarsus length (estimated with r-squared of models shown in Fig. 2a and Supplementary Fig. 3a), respectively, confirming that the evolution of absolute avian appendage size is substantially constrained by body size. These null allometric models predict that log-transformed beak length (Fig. 2a) and tarsus length (Fig. 3a) scale with log-transformed body mass in a linear manner:$${{{{rm{log }}}}}_{e}left({{{{rm{Beak}}}}},{{{{rm{Length}}}}}right)=1.4345+0.3362,{{{{rm{log }}}}}_{e}{{{{rm{Body}}}}},{{{{rm{Mass}}}}}$$
    (1)
    $${{{{rm{log }}}}}_{e}left({{{{rm{Tarsus}}}}},{{{{rm{Length}}}}}right)=2.1141+0.2883,{{{{rm{log }}}}}_{e}{Body},{{{{rm{Mass}}}}}$$
    (2)
    Fig. 2: Global test of Allen’s rule on avian beak length across 9962 (99.7%) bird species.The null allometric model (a) used to scale the absolute (log-transformed) beak length with log body size, the residuals from which were used as the relative beak length. Distribution of relative beak length across species geographic ranges (b). Model selection procedure for predicting log beak length (c), involving models with log body mass and either of six temperature measures within species geographic ranges included as fixed and interaction terms; AIC—Akaike Information Criterion, r2—coefficient of determination. An exemplar Allen’s model (d) showing increasing beak length with max temperature of all months, while controlling for body size as fixed term. An exemplar model with interaction of body size and max temperature of all months (e) illustrating how Allen’s rule operates across steeping quantiles of body size (left) and how allometry varies across quantiles of temperature (right). See Supplementary Fig. 2 for surrogate models based on the other temperature measures (evaluated in c). The p values refer to the significance of model’s fixed (d) or interaction terms (e) derived from two-tailed tests. The shaded area around the trend line is simple shading to facilitate reading. The results were obtained with phylogenetic linear regression by phylolm models on a single maximum clade credibility phylogenetic tree.Full size imageFig. 3: Global test of Allen’s rule on avian tarsus length across 9962 (99.7%) bird species.The null allometric model (a) used to scale the absolute (log-transformed) tarsus length with log body size, the residuals from which were used as the relative tarsus length. Distribution of relative tarsus length across species geographic ranges (b). Model selection procedure for predicting log tarsus length (c), involving models with log body mass and either of six temperature measures within species geographic ranges included as fixed and interaction terms; AIC—Akaike Information Criterion, r2—coefficient of determination. An exemplar Allen’s model (d) showing decreasing tarsus length with max temperature of all months, while controlling for body size as fixed term. An exemplar model with interaction of body size and max temperature of all months (e) illustrates how Allen’s rule operates across steeping quantiles of body size (left) and how allometry varies across steeping quantiles of temperature (right). See Supplementary Fig. 3 for surrogate models based on the other temperature measures (evaluated in c). The p values refer to the significance of model’s fixed (d) or interaction terms (e) derived from two-tailed tests. The shaded area around the trend line is simple shading to facilitate reading. The results were obtained with phylogenetic linear regression by phylolm models on a single maximum clade credibility phylogenetic tree.Full size imagethe normalized formulas of which give us the logarithmic equations:$${{{{rm{Beak}}}}},{{{{rm{Length}}}}}={4.1975,{{{{rm{Body}}}}},{{{{rm{Mass}}}}}}^{0.3362}$$
    (3)
    $${{{{rm{Tarsus}}}}},{{{{rm{Length}}}}}={8.2821,{{{{rm{Body}}}}},{{{{rm{Mass}}}}}}^{0.2883}$$
    (4)
    Because these allometric plots (Figs. 2a and 3a) relate the length of the appendage (one dimensional linear measure) to the body mass (three-dimensional volumetric measure) it means that the size of appendages would scale isometrically (proportionally) with the body size if the allometric coefficient was 0.3333. Thus, beak length equals to body mass to a power of 0.3362 means that the beak elongates almost exactly proportionally with body size. However, tarsus length equals to body mass to a power of 0.2883 means that the extent to which tarsus elongates with body size is slightly more pronounced in smaller species and weaker in larger species.These allometric relationships have implications for how we interpret subsequent patterns. For example, consider a species that experience a temporal increase in temperature, or invades a warmer climate. Then, if only Bergmann’s rule is operating (and in the absence of other confounds), a gradual decrease in body size should result in a proportional decrease in absolute beak length, and a gradually larger decrease in absolute tarsus length. Conversely, if species follow only Allen’s rule (and not follow Bergmann’s rule), then the increase in beak length should be similar between larger and smaller species, while the increase in tarsus length should be weaker in larger species and stronger in smaller species. Thus, Allen’s assumption that the increase in the ratio of body width to body length is steeper in larger species3, should not be a direct effect of the allometric rules, as appendages tend to increase proportionally with body size (beak) or increase milder at larger body sizes (tarsus).Allen’s ruleAfter excluding the effect of allometry, relative beak length is still tightly associated with phylogeny (Supplementary Table 1), while showing an impressive geographic variation (Fig. 2b). Our phylogenetic analysis concurs with an array of existing studies12,16,17,44,45 that found that the length of avian beak follows Allen’s rule, and is a general pattern across birds as a whole. Among our models predicting beak length, those with temperature variables among fixed terms are more informative than the null allometric model, where log body mass (allometry) is put as sole predictor (Fig. 2c). Most of the temperature variables also predict beak length better than latitude (Fig. 2c), again confirming the thermoregulatory basis of the observed pattern. Each of the temperature variables are positively associated with longer beaks (Fig. 2d and Supplementary Fig. 2a), which remains in agreement with Allen’s rule.Some studies have reported the ambiguous46 or very weak16 Allen’s pattern for avian legs. While relative tarsus length is also well conserved in avian phylogeny (Supplementary Table 1) and shows a high geographic variation (Fig. 3b), surprisingly, our global phylogenetic analysis indicates that avian tarsus length follows the inverse of Allen’s rule. Among models explaining tarsus length, those with temperature variables are better than the null allometric model (Fig. 3c). However, these models indicate a negative correlation—thereby shorter tarsi are associated with warmer temperatures (Fig. 3d).Allen’s vs Bergmann’s rule in allometryOur analyses support the hypothesis that the way in which avian appendages size varies across temperature regimes, depends on body size and vice versa. First, among models of beak length, those with an interaction of body size and the temperature consistently perform better than models without that interaction (Fig. 2c). The interaction of temperature and body size loads positively on beak length, indicating that larger-bodied species show stronger increases in beak size with temperature (Fig. 2e, left plot). Notably, beak length does not co-vary with temperature in the smallest birds (Fig. 2e, left plot), which is in agreement with Allen’s speculations3 that being smaller reduces the need to develop elongated appendages in hot climates, as effective heat exchange is already enabled through small body size (according to Bergmann’s rule). The positive interaction between body size and temperature also indicates that the higher the temperature, the steeper the allometric relationship between beak size and body size (Fig. 2e, right plot), meaning that in warmer climates beak size increases more strongly with body size than in colder climates, exactly as Allen hypothesized.An interaction between body size and temperature is also consistently supported in models of tarsus length (Fig. 3c). This interaction has strong positive effect on tarsus length, thereby reversing the trend by which tarsus shortens with temperature (Fig. 3e, left plot). This means that despite the overall decrease of tarsus size with temperature in smaller birds (the inverse of Allen’s rule), the opposite is true for larger birds that show increasing tarsus size with temperature (Fig. 3e, left plot). The interaction holds regardless of the temperature measure examined (Supplementary Fig. 3b, upper row), even if those previously did not co-vary with tarsus length when included as simple independent term with body size (Supplementary Fig. 3a). The case of larger birds thus fits Allen’s rule, and agrees with Allen’s further speculations3 that appendages are more likely to increase in larger- than in smaller-bodied animals. However, Allen did not predict the possibility of shortening appendages toward hot temperatures as seen in small birds. Given the extent of our sampling, the effect of shortening tarsi toward the equator in small-bodied species is presumably not an artefact, but relies on yet unknown mechanisms (possibly unrelated to thermoregulation). Nevertheless, if there is an evolutionary pressure to develop a smaller tarsus in hot climates, the increased thermoregulatory needs of larger-bodied species possibly overwhelm this selective process. This may be because large species acquire higher heat loads when the ambient temperature is hot, hence necessitating the development of longer legs as cooling organs. As with beak size, the interaction also indicates substantial changes in allometry, with much more millimeters of tarsus per each gram of body in warm conditions compared to cold conditions (Fig. 3e, right plot).Our analyses also support the mirror scenario, that the extent to which body size decreases with temperature (Bergmann’s rule) depends on the length of appendage. In models predicting body size, the temperature does not interact with relative beak length (Supplementary Fig. 4a), but interacts with tarsus length (Supplementary Fig. 4b). This interaction indicates that the strongest shrinkage in body size with temperature occurs in shorter-legged birds, while in longer-legged birds body size increases with temperature (inverse Bergmann’s rule). This again supports Allen’s speculations that variation in body shape allows birds to evolve body sizes less restricted (or even unrestricted) to environmental temperature. Thus, the results support the theory that Bergmann’s and Allen’s rules are two distinct, albeit analogous strategies to deal with thermoregulation.Allen’s vs Bergmann’s rule in climatic adaptationsOur analysis shows that the interactions of body size (Bergmann’s rule), beak length and tarsus length (Allen’s rule) predict the thermal environment across birds (e.g. the max temperatures of all months across species ranges, Fig. 4a). As with body size and shape, the temperatures experienced by species within their geographic ranges are finely conserved in the avian phylogeny (Supplementary Table 1), suggesting that thermal preferences of avian species have been established through evolutionary history. Evolution of these preferences then occurred when temperature changes affected their native environments (thus causing extinctions or adaptations), or when birds invaded novel environments (thus adapting to newly-encountered climates). Log-transformed body mass, relative beak length and tarsus length clearly predict the species ambient temperature (Fig. 4b), suggesting that the phenotype changes as animals adapt to suit different climates. However, of particular note is that the addition of an interaction between body size and relative beak length substantially improves model performance (Fig. 4b). This interaction shows that for longer-beaked birds, temperature associations are unrelated to body size, but the shorter the beak, the more pronounced is the shrinking in body size in warmer temperatures (Fig. 4c, left plot). In the case of smaller-bodied birds, the adaptation to different temperatures is independent of beak length, but with larger birds, the adaptation to warmer temperatures is more likely associated with elongated beaks (Fig. 4c, right plot). These results indicate that living in warmer temperatures tends to be associated either with smaller body size (Bergmann’s rule) or longer beak (Allen’s rule), rather than both rules simultaneously, thus again supporting the hypothesis of an evolutionary compromise between shifts in body size and shape as alternative adaptations to thermal environment.Fig. 4: Global test for avian adaptation to maximum temperature across all months by shifts in body size (Bergmann’s rule) and appendage size (Allen’s rule) across 9962 (99.7%) avian species.Distribution of environmental temperature across species geographic ranges (a). Model selection procedure for predicting max temperature all months (b), involving models with different combinations of log body mass, relative beak and tarsus length as fixed and interaction terms; AIC—Akaike Information Criterion, r2—coefficient of determination. Exemplar models with two-way interaction of body size and relative beak length (c) or tarsus length (d) illustrate how Bergmann’s rule operate across steeping quantiles of relative appendage length (left plots) and how Allen’s rule operate across steeping quantiles of body size (right plots). An exemplar model with two-way interaction of relative beak length and tarsus length (e) illustrates how Allen’s rule based on the relative length of one appendage operates across steeping quantiles of the relative length of second appendage. An exemplar model with three-way interaction of log body mass, relative beak and tarsus length (f) illustrates how shifts in body size and two measures of body shape depend on each other when animals adapt to novel climates; the trend lines indicate relationships between y and x1 (axes) across combinations of min and max values of x2 and x3 (colors); see also Supplementary Fig. 5 for more detailed visualization of the model f. The p values refer to significance of two-way (c–e) and three-way (f) interaction terms derived from two-tailed tests. The shaded area around the trend line is simple shading to facilitate reading. Obtained with phylogenetic linear regression by phylolm models on a single maximum clade credibility phylogenetic tree.Full size imageThe interaction of body size and relative tarsus length also substantially improves the model predicting ambient temperature of the species (Fig. 4b). This interaction indicates that living in warmer climates is associated with smaller body size (Bergmann’s rule) only in shorter-legged birds, while in longer-legged birds the environmental temperature increases with body size (inverse Bergmann’s rule; Fig. 4d, left plot). Simultaneously, the avian environmental temperature increases with tarsus length (Allen’s rule) only in larger species, while the opposite is true for smaller species (Fig. 4d, right plot). This suggests that larger-legged avian lineages may be resistant to Bergmann’s rule and become larger when habituating to warm climates, while shorter- and average-legged birds become smaller with temperature, as predicted by Bergmann’s rule. These findings again converge with Allen’s speculations on trade-off in the evolution of body size and appendage length in relation to temperature.We found that the length of the two different appendages—beak and tarsus—show independent evolutionary patterns (Fig. 4e). The environmental temperature of a species increases with beak length independently from tarsus length, and decreases with tarsus length independently from beak length (Fig. 4e). These outcomes reject the possibility of an evolutionary compromise in climatic adaptation of two types of appendages, at least when we do not control for body size (Bergmann’s rule) as additional type of climatic adaptation.Finally, the model with a three-way interaction between body size, relative beak and tarsus length predicting temperature performs the best among all considered candidate models (Fig. 4b) and this interaction is statistically significant (Fig. 4f), suggesting that evolutionary adaptation to novel climates depends on various configurations of body size, beak, and tarsus length. This model indicates various Bergmann’s rule slopes across different settings of body shape (Fig. 4f, top-left). Namely, the steepest decrease in environmental temperature with body size (i.e. strongest Bergmann’s rule) is observed in smaller-billed and smaller-legged birds (Fig. 4f, top-left, brown trend line), whereas in longer-billed and shorter-legged birds (Fig. 4f, top-left, green trend line) body size is not associated with environmental temperature. This model also indicates that in shorter-billed, longer-legged birds (Fig. 4f, top-left, purple trend line) body size increases across temperature gradient (inverse Bergmann’s pattern). This thus strengthens the support for Allen’s theory that having bodies with elongated appendages may enable species to circumvent or even reverse Bergmann’s pattern; whereas compact bodies are more prone to decrease in size with temperature in order to deal with overheating in warm climates. Counteracting this argument, however, is that longer-billed and longer-legged birds show (moderate) typical Bergmann’s pattern (Fig. 4f, leftmost plot, bluish trend line).The three-way interaction model also shows other mixtures of expected and unexpected results. For example, the strongest increase in environmental temperature with beak length occurs in larger-bodied and shorter-legged birds (Fig. 4f, top-right plot, orange trend line), which clearly suggests a trade-off in evolution of body size and beak length and a similar trade-off in the evolution of the two types of appendages, presumably reflecting different adaptive responses for thermoregulation. However, a similar increase in beak length also occurs in tiny-bodied and longer-legged birds (Fig. 4f, top-right plot, blue trend line), which stands in contrast to this trade-off hypothesis. Likewise, the steepest increase in environmental temperature with tarsus length (Allen’s rule) occurs in larger-bodied and shorter-billed birds (Fig. 4f, bottom plot, pink trend line), again suggesting a compromise scenario, with elongated tarsus evolving as thermoregulatory organ to compensate for insufficient heat exchange due to large body and small beak. It also suggests that, in large birds, having a short beak in hot climates requires longer tarsi (Fig. 4f, top-right, pinkish and orange trend lines) and vice versa (Fig. 4f, bottom plot, rose and yellowish trend lines), indicating that in large species, the summarized length of two types of appendages is important for thermoregulation. However, by contrast, it seems that in small bodied species, beak and tarsi length evolved in a correlated way (Fig. 4f, top-right and bottom plots, green and blue trend lines) across environmental temperature (occurrences in warmer temperatures are associated with simultaneously both longer beaks and tarsi, or else simultaneously shorter beaks and tarsi). This may indicate a general tendency to correlated evolution of relative beak and tarsus lengths, perhaps for functional reasons, e.g. longer beaks may allow long-legged birds to explore substrate more efficiently, as longer necks also do47.Allen’s vs Bergmann’s rules in causal modelsOur hypothesis consequently holds within phylogenetic path analysis, where the best causal models integrate Bergmann’s and Allen’s rules to explain both the size of avian appendages (Fig. 5a) and the avian thermal environment (here, maximum temperature across all months) (Fig. 5b). The best model predicting beak and tarsus length includes the causal effect of temperature on body size (Bergmann’s rule) and then body size on beak and tarsus length (allometry), as well as the direct effect of temperature on the size of appendages (Allen’s rule) (Fig. 5a). This joint Bergmann’s and Allen’s model is substantially better than the model assuming that temperature does not affect body size before scaling for the length of appendages (Fig. 5a, Allen’s rule only). The combined Bergmann’s and Allen’s model is also better than one assuming no direct effect of temperature on appendages (Fig. 5a, Bergmann’s rule only). This again indicates that how the length of avian appendages co-varies with the ambient temperature partially depends on how avian body size co-varies with temperature, yielding results aligned with the trade-off hypothesis. This notably argues against the possibility that the increase in the length of appendages (relative to body size) with temperature is an artefact of decreased body sizes at hot temperatures (see26). However, interestingly, the model including only Allen’s rule (and allometry) explains the length of appendages with similar accuracy to the model with only Bergmann’s rule (Fig. 5a).Fig. 5: Phylogenetic path analysis with responses of the length of avian appendages (beak and tarsus) (a) and the maximum temperature of all months within species range (b) across 9962 species (99.7% of global community).In both cases model candidates include different combinations of allometry (relationship between the length of appendages and body size), Bergmann’s rule (the relationship between body size and the temperature) and Allen’s rule (relationship between the length of appendages and temperature). ∆CIC—delta C statistic Information Criterion. The results were obtained with phylogenetic path analysis by using phylopath models on a single maximum clade credibility phylogenetic tree and scaled covariates (mean = 0 ± 1 SD) to compare their effect sizes (see numbers on path diagrams).Full size imageThe best model predicting the temperature associations includes the indirect effect of body size on the length of appendages (allometry), and then the length of appendages on temperature (Allen’s rule), as well as the direct effect of body size on temperature (Bergmann’s rule) (Fig. 5b). These results again demonstrate that how the temperature varies across species ranges depends on both the size of body and appendages, suggesting that Bergmann’s and Allen’s rules describe two distinct evolutionary ways to cope with thermoregulation. Moreover, the similar performance of Allen’s model compared to Bergmann’s model (Fig. 5b) again suggests that shifts in the animal’s body size and shape represent roughly equally influential in the evolution of adaptations to novel climates.Excluding possible confounding factorsTo ensure the reliability of our findings, first we show that when explaining the phenotype (Supplementary Figs. 2b and 3b), or the temperature within species geographic ranges (Supplementary Fig. 6), the main results remain consistent whichever of the five temperature measures is included. Second, despite the fact that the relationships with relative length of appendages and the experienced temperatures are strongest in resident birds, followed by partial- and full- migrants, our results still hold when accounting for these three categories of avian migratory habits (Supplementary Fig. 7); and the compromise scenario remains similar in each of these groups independently (Supplementary Figs. 8–10). It aligns with previous studies22,46, which found that ecogeographical rules are valid regardless of variation in avian migratory habits. However, it is worth to notice that the most prominent trade-offs are found in resident species (in case of explaining environmental temperature, see Supplementary Fig. 10) or in partial migrants (in case of explaining beak length, see Supplementary Fig. 8). Third, the trade-offs in thermoregulatory strategies also hold after controlling for geographic range size (Supplementary Fig. 11) and remains quantitatively (Supplementary Figs. 12–13) or qualitatively (Supplementary Fig. 14) stable across the gradient of endemic-cosmopolitan species. Thus, even if ecogeographical rules operate within widespread species (across distanced populations, as well documented9,10,11,12,13), this does not appear to influences the results of our cross-species analysis. Fourth, the predictions of temperature within species geographic ranges are also not specific to the way by which we account for the allometry of appendages (by using residual appendage length). Parallel analyses with ratios of appendage length to body mass (Supplementary Fig. 15) or principal components of all phenotypic traits (Supplementary Fig. 16) give qualitatively similar outcomes. Fifth, we also show that results of both phylogenetic regression (Supplementary Fig. 17) and phylogenetic path models (Supplementary Fig. 18) remain consistently valid across 100 randomly chosen phylogenetic trees32, mitigating concerns regarding phylogenetic uncertainty influencing our results.Notably, there is a wider list of important ecological factors constraining or favoring variation in body size and shape, e.g. tropic levels or foraging techniques21,23,47,48, although they are also themselves constrained by phylogeny to some extent, which we control for. Nevertheless, we believe that it is likely that these constraints influenced (or were influenced by) the Bergmann-Allen trade-off. Understanding of this issue would benefit from a deeper dive into the relationship between climatic, phenotypic and ecological variation across animals.Our findings in the context of eco-evolutionary processes driven by climateTo the best of our knowledge, this study is the largest (taxonomically and geographically) simultaneous test of ecogeographical rules and it provides a first empirical evidence for a trade-off in the evolution of body size (Bergmann’s rule2) and the size of appendages (Allen’s rule3) across global temperature gradients. Our results confirm what Allen3 speculated—the larger the body, the stronger the increase in appendage size with temperature; and the larger the appendages, the milder the decrease in body size with temperature. Thus, the evolution of body size under temperature regimes likely depends on the size of appendages and, on the other hand, the extent to which temperature drives the size of appendages depends on body size. This means that these two thermoregulatory adaptations are not independent of each other, but the phenotype has at least two ways to adapt to novel climates, i.e. by the shifts in body size or the shifts in the size of appendages (or both to a lesser extent).The evolution of appendages (e.g. avian beaks49) was a dynamic process believed to overtake the changes in body size across evolutionary time50. Our analyses do not indicate, however, that shifts in body size have been more frequent than shifts in appendage size (or vice versa), at least not because of thermoregulation. Rather, they indicate that shifts in body size and shape are intertwined through avian evolutionary history, agreeing with the theory that animals select the most convenient strategy of thermoregulation to maintain functional traits of its phenotype. For functional reasons animal lineages tend to increase in body size over evolutionary time (Cope’s rule43), thus it is not surprising that strategies allowing species to maintain/develop larger bodies (i.e. over-increase in appendage size) are to be expected evolutionarily. On the other hand, some lineages may be constrained in appendage size (e.g. to forage21,47 or communicate23 effectively), hence those may favor the shifts in body size to reconcile optimal thermoregulation with a desired functionality.We found that the compromise in thermoregulatory strategies may also involve two distinct types of appendages, here beak versus tarsus. However, this is true only for larger-bodied species (see Fig. 4f top-right and bottom plots, trends for large bodies), that are more likely to acquire higher heat loads in warmer environments, thus the summarized size of many appendages may be for them crucial to disperse heat loads. Both beak and legs have been confirmed to act as key regions of heat transfer on the avian body37,38,51,52, thus both may be sensitive to thermal conditions when body size is too large to deal alone with too hot temperatures. Yet, in small-bodied species both appendages seem to evolve in concerted way across temperature gradients, and this may be in a way that conforms with Allen’s rule or not (see Fig. 4f, top-right and bottom plots, trends for small bodies), indicating that the small body ensures good temperature exchange in hot climates, thus the evolution of appendages in these species may be correlated, but independent of thermoregulatory selection pressures.It is worthwhile emphasizing that apart from shifts in body size and shape, many other elements combine to help birds meet their thermoregulatory requirements53, e.g. through variation in insulation (feathers)54, coloration55,56 metabolism57, blood circulation58 or behavior59,60,61. Extrapolating our results, these thermoregulatory strategies might also co-evolve under a trade-off to ensure optimal thermoregulation along with desired functionality. This is presumably a reason for the relatively low performance of our models; e.g. physical phenotype explains up to only 20% of the variance in ambient temperature (Fig. 4b, upper model), therefore unexplained variance must be attributed to other thermoregulatory strategies.In this study, we demonstrate that Allen’s rule may be attributed to the varying allometric functions across temperature gradients. Although logical and argued elsewhere26, it has never been addressed by any empirical research. Our findings clearly indicate the importance of considering body mass as both a fixed and interaction term in studies of Allen’s rule, but also might suggest that ambient temperature should be included in other allometric studies of animals’ morphology. That said, temperature explains very little of the variance in the size of appendages compared to body size (Figs. 2c and 3c), thus thermal conditions are unlikely to be a very crucial confounding factor for allometry in comparative analyses.In this study, we empirically confirm for the first time an evolutionary compromise theory that was first proposed almost 150 years ago3– the evolution of body size and appendages are two distinct and interacting ways to cope with thermoregulation. This may explain why many studies fail to detect Allen’s or Bergmann’s rules independently which has led to questioning of the generality of these ecogeographical patterns13,24,25. Here, our findings suggest that Bergmann’s and Allen’s rules should not necessarily be considered in isolation. We believe that these thermoregulatory strategies might intertwine through the evolutionary history of animals, as the evolution of phenotype possibly interacts to confound ecogeographical rules to evolve functional traits. This explanation also highlights the diverse mechanisms that animals may employ to expand across the world’s multiple environments. It also raises the speculation that with observed and future anticipated warming of Earth’s climate, we should expect mainly large animals to elongate in appendages, while mainly compact-bodied animals to shrink in size. More

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    Better incentives are needed to reward academic software development

    Department of Ecology and Evolutionary Biology and Eversource Energy Center, University of Connecticut, Storrs, CT, USACory MerowDepartment of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USABrad Boyle & Brian J. EnquistDepartment of Geography, Florida State University, Tallahassee, FL, USAXiao FengBiodiversity and Biocomplexity Unit, Okinawa Institute of Science and Technology Graduate University, Onna, JapanJamie M. KassDepartment of Geography, University at Buffalo, Buffalo, NY, USABrian S. Maitner & Adam M. WilsonSchool of Biology and Ecology, University of Maine, Orono, ME, USABrian McGillMitchell Center for Sustainability Solutions, University of Maine, Orono, ME, USABrian McGillCenter for Macroecology, Evolution and Climate, Globe Institute, University of Copenhagen, Copenhagen, DenmarkHannah OwensFlorida Museum of Natural History, University of Florida, Gainesville, FL, USAHannah OwensDepartment of Biological Sciences, Purdue University, West Lafayette, IN, USADaniel S. ParkPurdue Center for Plant Biology, Purdue University, West Lafayette, IN, USADaniel S. ParkDepartment of Environmental Systems Science, Institute of Integrative Biology, ETH Zürich, Zurich, SwitzerlandAndrea PazDepartment of Biology, City College of the City University of New York, New York, NY, USAGonzalo E. Pinilla-BuitragoPhD Program in Biology, Graduate Center of the City University of New York, New York, NY, USAGonzalo E. Pinilla-BuitragoDepartment of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT, USAMark C. UrbanCenter of Biological Risk, University of Connecticut, Storrs, CT, USAMark C. UrbanDepartamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, Vigo, SpainSara Varela More

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    Spatio-temporal visualization and forecasting of $${text {PM}}_{10}$$ PM 10 in the Brazilian state of Minas Gerais

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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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    Differential global distribution of marine picocyanobacteria gene clusters reveals distinct niche-related adaptive strategies

    Different picocyanobacterial communities exhibit distinct gene repertoiresTo analyze the distribution of Prochlorococcus and Synechococcus reads along the Tara Oceans transect, metagenomic reads corresponding to the bacterial size fraction were recruited against 256 picocyanobacterial reference genomes, including SAGs and MAGs representative of uncultured lineages (e.g., Prochlorococcus HLIII-IV, Synechococcus EnvA or EnvB). This yielded a total of 1.07 billion recruited reads, of which 87.7% mapped onto Prochlorococcus genomes and 12.3% onto Synechococcus genomes, which were then functionally assigned by mapping them onto the manually curated Cyanorak v2.1 CLOG database [19]. In order to identify picocyanobacterial genes potentially involved in niche adaptation, we analyzed the distribution across the oceans of flexible (i.e. non-core) genes. Clustering of Tara Oceans stations according to the relative abundance of flexible genes resulted in three well-defined clusters for Prochlorococcus (Fig. 1A), which matched those obtained when stations were clustered according to the relative abundance of Prochlorococcus ESTUs, as assessed using the high-resolution marker gene petB, encoding cytochrome b6 (Fig. 1A; [24]). Only a few discrepancies can be observed between the two trees, including stations TARA-070 that displayed one of the most disparate ESTU compositions and TARA-094, dominated by the rare HLID ESTU (Fig. 1A). Similarly, for Synechococcus, most of the eight assemblages of stations discriminated based on the relative abundance of ESTUs (Fig. 1B) were also retrieved in the clustering based on flexible gene abundance, except for a few intra-assemblage switches between stations, notably those dominated by ESTU IIA (Fig. 1B). Despite these few variations, four major clusters can be clearly delineated in both Synechococcus trees, corresponding to four broadly defined ecological niches, namely (i) cold, nutrient-rich, pelagic or coastal environments (blue and light red in Fig. 1B), (ii) Fe-limited environments (purple and grey), (iii) temperate, P-depleted, Fe-replete areas (yellow) and (iv) warm, N-depleted, Fe-replete regions (dark red). This correspondence between taxonomic and functional information was also confirmed by the high congruence between distance matrices based on ESTU relative abundance and on CLOG relative abundance (p-value  0.01) are marked by a cross. Φsat: index of iron limitation derived from satellite data. PAR30: satellite-derived photosynthetically available radiation at the surface, averaged on 30 days. DCM: depth of the deep chlorophyll maximum.Full size imageIdentification of individual genes potentially involved in niche partitioningTo identify genes relevant for adaptation to a specific set of environmental conditions and enriched in specific ESTU assemblages, we selected the most representative genes from each module (Dataset 5; Figs. 3, S2). Most genes retrieved this way encode proteins of unknown or hypothetical function (85.7% of 7,485 genes). However, among the genes with a functional annotation (Dataset 6), a large fraction seems to have a function related to their realized environmental niche (Figs. 3, S2). For instance, many genes involved in the transport and assimilation of nitrite and nitrate (nirA, nirX, moaA-C, moaE, mobA, moeA, narB, M, nrtP; [6]) as well as cyanate, an organic form of nitrogen (cynA, B, D, S), are enriched in the Prochlorococcus blue module, which is correlated with the HLIIA-D ESTU and to low inorganic N, P, and silica levels and anti-correlated with Fe availability (Fig. 2A–C). This is consistent with previous studies showing that while only a few Prochlorococcus strains in culture possess the nirA gene and even less the narB gene, natural Prochlorococcus populations inhabiting N-poor areas do possess one or both of these genes [40,41,42]. Similarly, numerous genes amongst the most representative of Prochlorococcus brown, red and turquoise modules are related to adaptation of HLIIIA/IVA, HLIA and LLIA ESTUs to Fe-limited, cold P-limited, and cold, mixed waters, respectively (Fig. 3). Comparable results were obtained for Synechococcus, although the niche delineation was less clear than for Prochlorococcus since genes within each module exhibited lower correlations with the module eigenvalue (Fig. S2). These results therefore constitute a proof of concept that this network analysis was able to retrieve niche-related genes from metagenomics data.Fig. 3: Violin plots highlighting the most representative genes of each Prochlorococcus module.For each module, each gene is represented as a dot positioned according to its correlation with the eigengene for each module, the most representative genes being localized on top of each violin plot. Genes mentioned in the text and/or in Dataset 6 have been colored according to the color of the corresponding module, indicated by a colored bar above each module. The text above violin plots indicates the most significant environmental parameter(s) and/or ESTU(s) for each module, as derived from Fig. 2.Full size imageIdentification of eCAGs potentially involved in niche partitioningIn order to better understand the function of niche-related genes, notably of the numerous unknowns, we then integrated global distribution data with gene synteny in reference genomes using a network approach (Datasets 7, 8). This led us to identify clusters of adjacent genes in reference genomes, and thus potentially involved in the same metabolic pathway (Figs. 4, S3, S4; Dataset 6). These clusters were defined within each module and thus encompass genes with similar distribution and abundance in situ. Hereafter, these environmental clusters of adjacent genes will be called “eCAGs”.Fig. 4: Delineation of Prochlorococcus eCAGs, defined as a set of genes that are both adjacent in reference genomes and share a similar in situ distribution.Nodes correspond to individual genes with their gene name (or significant numbers of the CK number, e.g. 1234 for CK_00001234) and are colored according to their WGCNA module. A link between two nodes indicates that these two genes are less than five genes apart in at least one genome. The bottom insert shows the most significant environmental parameter(s) and/or ESTU(s) for each module, as derived from Fig. 2.Full size imageeCAGs related to nitrogen metabolismThe well-known nitrate/nitrite gene cluster involved in uptake and assimilation of inorganic forms of N (see above), which is present in most Synechococcus genomes (Dataset 6), was expectedly not restricted to a particular niche in natural Synechococcus populations, as shown by its quasi-absence from WGCNA modules. In Prochlorococcus, this cluster is separated into two eCAGs enriched in low-N areas (Fig. S5A, B), most genes being included in Pro-eCAG_002, present in only 13 out of 118 Prochlorococcus genomes, while nirA and nirX form an independent eCAG (Pro-eCAG_001) due to their presence in many more genomes. The quasi-core ureA-G/urtB-E genomic region was also found to form a Prochlorococcus eCAG (Pro-eCAG_003) that was impoverished in low-Fe compared to other regions (Fig. S5C, D), in agreement with its presence in only two out of six HLIII/IV genomes. We also uncovered several other Prochlorococcus and Synechococcus eCAGs that seem to be involved in the transport and/or assimilation of more unusual and/or complex forms of nitrogen, which might either be degraded into elementary N molecules or possibly directly used by cells for e.g. the biosynthesis of proteins or DNA. Indeed, we detected in both genera an eCAG (Pro-eCAG_004 and Syn-eCAG_001; Fig. S6A, B; Dataset 6) that encompasses speB2, an ortholog of Synechocystis PCC 6803 sll1077, previously annotated as encoding an agmatinase [29, 43] and which was recently characterized as a guanidinase that degrades guanidine rather than agmatine to urea and ammonium [44]. E. coli produces guanidine under nutrient-poor conditions, suggesting that guanidine metabolism is biologically significant and potentially prevalent in natural environments [44, 45]. Furthermore, the ykkC riboswitch candidate, which was shown to specifically sense guanidine and to control the expression of a variety of genes involved in either guanidine metabolism or nitrate, sulfate, or bicarbonate transport, is located immediately upstream of this eCAG in Synechococcus reference genomes, all genes of this cluster being predicted by RegPrecise 3.0 to be regulated by this riboswitch (Fig. S6C; [45, 46]). The presence of hypA and B homologs within this eCAG furthermore suggests that, in the presence of guanidine, these homologs could be involved in the insertion of Ni2+, or another metal cofactor, in the active site of guanidinase. The next three genes of this eCAG, which encode an ABC transporter similar to the TauABC taurine transporter in E. coli (Fig. S6C), could be involved in guanidine transport in low-N areas. Of note, the presence in most Synechococcus/Cyanobium genomes possessing this eCAG of a gene encoding a putative Rieske Fe-sulfur protein (CK_00002251) downstream of this gene cluster, seems to constitute a specificity compared to the homologous gene cluster in Synechocystis sp. PCC 6803. The presence of this Fe-S protein suggests that Fe is used as a cofactor in this system and might explain why this gene cluster is absent from picocyanobacteria thriving in low-Fe areas, while it is present in a large proportion of the population in most other oceanic areas (Fig. S6A, B).Another example of the use of organic N forms concerns compounds containing a cyano radical (C ≡ N). The cyanate transporter genes (cynABD) were indeed found in a Prochlorococcus eCAG (Pro-eCAG_005, also including the conserved hypothetical gene CK_00055128; Fig. S7A, B). While only a small proportion of the Prochlorococcus community possesses this eCAG in warm, Fe-replete waters, it is absent from other oceanic areas in accordance with its low frequency in Prochlorococcus genomes (present in only two HLI and five HLII genomes). In Synechococcus these genes were not included in a module, and thus are not in an eCAG (Dataset 6; Fig. S7C), but seem widely distributed despite their presence in only a few Synechococcus genomes (mostly in clade III strains; [6, 47, 48]). Interestingly, we also uncovered a 7-gene eCAG (Pro-eCAG_006 and Syn-eCAG_002), encompassing a putative nitrilase gene (nitC), which also suggests that most Synechococcus cells and a more variable fraction of the Prochlorococcus population could use nitriles or cyanides in warm, Fe-replete waters and more particularly in low-N areas such as the Indian Ocean (Fig. 5A, B). The whole operon (nitHBCDEFG; Fig. 5C), called Nit1C, was shown to be upregulated in the presence of cyanide and to trigger an increase in the rate of ammonia accumulation in the heterotrophic bacterium Pseudomonas fluorescens [49], suggesting that like cyanate, cyanide could constitute an alternative nitrogen source in marine picocyanobacteria as well. However, given the potential toxicity of these C ≡ N-containing compounds [50], we cannot exclude that these eCAGs could also be devoted to cell detoxification [45, 47]. Such an example of detoxification has been described for arsenate and chromate that, as analogs of phosphate and sulfate respectively, are toxic to marine phytoplankton and must be actively exported out of the cells [51, 52].Fig. 5: Global distribution map of the eCAG involved in nitrile or cyanide transport and assimilation.A Prochlorococcus Pro-eCAG_006. B Synechococcus Syn-eCAG_002. C The genomic region in Prochlorococcus marinus MIT9301. The size of the circle is proportional to relative abundance of each genus as estimated based on the single-copy core gene petB and this gene was also used to estimate the relative abundance of other genes in the population. Black dots represent Tara Oceans stations for which Prochlorococcus or Synechococcus read abundance was too low to reach the threshold limit.Full size imageWe detected the presence of an eCAG encompassing asnB, pyrB2, and pydC (Pro-eCAG_007, Syn-eCAG_003, Fig. S8), which could contribute to an alternative pyrimidine biosynthesis pathway and thus provide another way for cells to recycle complex nitrogen forms. While this eCAG is found in only one fifth of HLII genomes and in quite specific locations for Prochlorococcus, notably in the Red Sea, it is found in most Synechococcus cells in warm, Fe-replete, N and P-depleted niches, consistent with its phyletic pattern showing its absence only from most clade I, IV, CRD1, and EnvB genomes (Fig. S8; Dataset 6). More generally, most N-uptake and assimilation genes in both genera were specifically absent from Fe-depleted areas, including the nirA/narB eCAG for Prochlorococcus, as mentioned by Kent et al. [36] as well as guanidinase and nitrilase eCAGs. In contrast, picocyanobacterial populations present in low-Fe areas possess, in addition to the core ammonium transporter amt1, a second transporter amt2, also present in cold areas for Synechococcus (Fig. S9). Additionally, Prochlorococcus populations thriving in HNLC areas also possess two amino acid-related eCAGs that are present in most Synechococcus genomes, the first one involved in polar amino acid N-II transport (Pro-eCAG_008; natF-G-H-bgtA; [53]; Fig. S10A, B) and the second one (leuDH-soxA-CK_00001744, Pro-eCAG_009, Fig. S10C, D) that notably encompasses a leucine dehydrogenase, able to produce ammonium from branched-chain amino acids. This highlights the profound difference in N acquisition mechanisms between HNLC regions and Fe-replete, N-deprived areas: the primary nitrogen sources for picocyanobacterial populations dwelling in HNLC areas seem to be ammonium and amino acids, while N acquisition mechanisms are more diverse in N-limited, Fe-replete regions.eCAGs related to phosphorus metabolismAdaptation to P depletion has been well documented in marine picocyanobacteria showing that while in P-replete waters Prochlorococcus and Synechococcus essentially rely on inorganic phosphate acquired by core transporters (PstSABC), strains isolated from low-P regions and natural populations thriving in these areas additionally contain a number of accessory genes related to P metabolism, located in specific genomic islands [6, 14, 30,31,32, 54]. Here, we indeed found in Prochlorococcus an eCAG containing the phoBR operon (Pro-eCAG_010) that encodes a two-component system response regulator, as well as an eCAG including the alkaline phosphatase phoA (Pro-eCAG_011), both present in virtually the whole Prochlorococcus population from the Mediterranean Sea, the Gulf of Mexico and the Western North Atlantic Ocean, which are known to be P-limited [30, 55] (Fig. S11A, B). By comparison, in Synechococcus, we only identified the phoBR eCAG (Syn-eCAG_005, Fig. S11C) that is systematically present in warm waters whatever the limiting nutrient, in agreement with its phyletic pattern in reference genomes showing its specific absence from cold thermotypes (clades I and IV, Dataset 6). Furthermore, although our analysis did not retrieve them within eCAGs due to the variability of gene content and synteny in this genomic region, even within each genus, several other P-related genes were enriched in low-P areas but partially differed between Prochlorococcus and Synechococcus (Figs. 3, S2, S11; Dataset 6). While the genes putatively encoding a chromate transporter (ChrA) and an arsenate efflux pump ArsB were present in both genera in different proportions, a putative transcriptional phosphate regulator related to PtrA (CK_00056804; [56]) was specific to Prochlorococcus. Synechococcus in contrast harbors a large variety of alkaline phosphatases (PhoX, CK_00005263 and CK_00040198) as well as the phosphate transporter SphX (Fig. S11).Phosphonates, i.e. reduced organophosphorus compounds containing C–P bonds that represent up to 25% of the high-molecular-weight dissolved organic P pool in the open ocean, constitute an alternative P form for marine picocyanobacteria [57]. We indeed identified, in addition to the core phosphonate ABC transporter (phnD1-C1-E1), a second previously unreported putative phosphonate transporter phnC2-D2-E2-E3 (Pro-eCAG_012; Fig. 6A). Most of the Prochlorococcus population in strongly P-limited areas of the ocean harbored these genes, while they were absent from other areas, consistent with their presence in only a few Prochlorococcus and no Synechococcus genomes. Furthermore, as previously described [58,59,60], we found a Prochlorococcus eCAG encompassing the phnYZ operon involved in C-P bond cleavage, the putative phosphite dehydrogenase ptxD, and the phosphite and methylphosphonate transporter ptxABC (Pro-eCAG_0013, Dataset 6; Fig. 6B, [60,61,62]). Compared to these previous studies that mainly reported the presence of these genes in Prochlorococcus cells from the North Atlantic Ocean, here we show that they actually occur in a much larger geographic area, including the Mediterranean Sea, the Gulf of Mexico, and the ALOHA station (TARA_132) in the North Pacific, even though they were present in a fairly low fraction of Prochlorococcus cells. These genes occurred in an even larger proportion of the Synechococcus population, although not found in an eCAG for this genus (Fig. S12; Dataset 6). Synechococcus cells from the Mediterranean Sea, a P-limited area dominated by clade III [24], seem to lack phnYZ, in agreement with the phyletic pattern of these genes in reference genomes, showing the absence of this two-gene operon in the sole clade III strain that possesses the ptxABDC gene cluster. In contrast, the presence of the complete gene set (ptxABDC-phnYZ) in the North Atlantic, at the entrance of the Mediterranean Sea, and in several clade II reference genomes rather suggests that it is primarily attributable to this clade. Altogether, our data indicate that part of the natural populations of both Prochlorococcus and Synechococcus would be able to assimilate phosphonate and phosphite as alternative P-sources in low-P areas using the ptxABDC-phnYZ operon. Yet, the fact that no picocyanobacterial genome except P. marinus RS01 (Fig. 6C) possesses both phnC2-D2-E2-E3 and phnYZ, suggests that the phosphonate taken up by the phnC2-D2-E2-E3 transporter could be incorporated into cell surface phosphonoglycoproteins that may act to mitigate cell mortality by grazing and viral lysis, as recently suggested [63].Fig. 6: Global distribution map of eCAGs putatively involved in phosphonate and phosphite transport and assimilation.A Prochlorococcus Pro-eCAG_012 putatively involved in phosphonate transport. B Prochlorococcus Pro-eCAG_013, involved in phosphonate/phosphite uptake and assimilation and phosphonate C-P bond cleavage. C The genomic region encompassing both phnC2-D2-E2-E3 and ptxABDC-phnYZ specific to P. marinus RS01. The size of the circle is proportional to relative abundance of Prochlorococcus as estimated based on the single-copy core gene petB and this gene was also used to estimate the relative abundance of other genes in the population. Black dots represent Tara Oceans stations for which Prochlorococcus read abundance was too low to reach the threshold limit.Full size imageeCAGs related to iron metabolismAs for macronutrients, it has been hypothesized that the survival of marine picocyanobacteria in low-Fe regions was made possible through several strategies, including the loss of genes encoding proteins that contain Fe as a cofactor, the replacement of Fe by another metal cofactor, and the acquisition of genes involved in Fe uptake and storage [14, 15, 36, 39, 64]. Accordingly, several eCAGs encompassing genes encoding proteins interacting with Fe were found in modules anti-correlated to HNLC regions in both genera. These include three subunits of the (photo)respiratory complex succinate dehydrogenase (SdhABC, Pro-eCAG_014, Syn-eCAG_006, Fig. S13; [65]) and Fe-containing proteins encoded in most abovementioned eCAGs involved in N or P metabolism, such as the guanidinase (Fig. S6), the NitC1 (Fig. 5), the pyrB2 (Fig. S8), the phosphonate (Fig. 6, S12), and the urea and inorganic nitrogen eCAGs (Fig. S5). Most Synechococcus cells thriving in Fe-replete areas also possess the sodT/sodX eCAG (Syn-eCAG_007, Fig. S14A, B) involved in nickel transport and maturation of the Ni-superoxide dismutase (SodN), these three genes being in contrast core in Prochlorococcus. Additionally, Synechococcus from Fe-replete areas, notably from the Mediterranean Sea and the Indian Ocean, specifically possess two eCAGs (Syn-eCAG_008 and 009; Fig. S14C, D), involved in the biosynthesis of a polysaccharide capsule that appear to be most similar to the E. coli groups 2 and 3 kps loci [66]. These extracellular structures, known to provide protection against biotic or abiotic stress, were recently shown in Klebsiella to provide a clear fitness advantage in nutrient-poor conditions since they were associated with increased growth rates and population yields [67]. However, while these authors suggested that capsules may play a role in Fe uptake, the significant reduction in the relative abundance of kps genes in low-Fe compared to Fe-replete areas (t-test p-value  More