Deciphering the Molecular Mechanism Underpinning Phage Arbitrium Communication Systems.

Bacillus phages use a communication system, termed "arbitrium," to coordinate lysis-lysogeny decisions. Arbitrium communication is mediated by the production and secretion of a hexapeptide (AimP) during lytic cycle. Once internalized, AimP reduces the expression of the negative regulator of lysogeny, AimX, by binding to the transcription factor, AimR, promoting lysogeny. We have elucidated the crystal structures of AimR from the Bacillus subtilis SPbeta phage in its apo form, bound to its DNA operator and in complex with AimP. AimR presents intrinsic plasticity, sharing structural features with the RRNPP quorum-sensing family. Remarkably, AimR binds to an unusual operator with a long spacer that interacts nonspecifically with the receptor TPR domain, while the HTH domain canonically recognizes two inverted repeats. AimP stabilizes a compact conformation of AimR that approximates the DNA-recognition helices, preventing AimR binding to the aimX promoter region. Our results establish the molecular basis of the arbitrium communication system.

A genome-guided strategy for climate resilience in American chestnut restoration populations.

American chestnut (Castanea dentata) is a deciduous tree species of eastern North America that was decimated by the introduction of the chestnut blight fungus (Cryphonectria parasitica) in the early 20th century. Although millions of American chestnuts survive as root collar sprouts, these trees rarely reproduce. Thus, the species is considered functionally extinct. American chestnuts with improved blight resistance have been developed through interspecific hybridization followed by conspecific backcrossing, and by genetic engineering. Incorporating adaptive genomic diversity into these backcross families and transgenic lines is important for restoring the species across broad climatic gradients. To develop sampling recommendations for ex situ conservation of wild adaptive genetic variation, we coupled whole-genome resequencing of 384 stump sprouts with genotype-environment association analyses and found that the species range can be subdivided into three seed zones characterized by relatively homogeneous adaptive allele frequencies. We estimated that 21 to 29 trees per seed zone will need to be conserved to capture most extant adaptive diversity. We also resequenced the genomes of 269 backcross trees to understand the extent to which the breeding program has already captured wild adaptive diversity, and to estimate optimal reintroduction sites for specific families on the basis of their adaptive portfolio and future climate projections. Taken together, these results inform the development of an ex situ germplasm conservation and breeding plan to target blight-resistant breeding populations to specific environments and provides a blueprint for developing restoration plans for other imperiled tree species.

How to introduce a new bacteriophage on the block: a short guide to phage classification.

Bacteriophage (phage) studies established the field of molecular biology and continue to propel life science research forward due to their diversity, abundance, and potential applications. In this Gem article, we orient newcomers to four common ways phages are currently classified: infection cycle, morphology, taxonomy, and supergroup. By using these classifications, researchers can determine where any novel phage fits into the scheme of the known "phage-verse".

PhaTYP: predicting the lifestyle for bacteriophages using BERT.

Bacteriophages (or phages), which infect bacteria, have two distinct lifestyles: virulent and temperate. Predicting the lifestyle of phages helps decipher their interactions with their bacterial hosts, aiding phages' applications in fields such as phage therapy. Because experimental methods for annotating the lifestyle of phages cannot keep pace with the fast accumulation of sequenced phages, computational method for predicting phages' lifestyles has become an attractive alternative. Despite some promising results, computational lifestyle prediction remains difficult because of the limited known annotations and the sheer amount of sequenced phage contigs assembled from metagenomic data. In particular, most of the existing tools cannot precisely predict phages' lifestyles for short contigs. In this work, we develop PhaTYP (Phage TYPe prediction tool) to improve the accuracy of lifestyle prediction on short contigs. We design two different training tasks, self-supervised and fine-tuning tasks, to overcome lifestyle prediction difficulties. We rigorously tested and compared PhaTYP with four state-of-the-art methods: DeePhage, PHACTS, PhagePred and BACPHLIP. The experimental results show that PhaTYP outperforms all these methods and achieves more stable performance on short contigs. In addition, we demonstrated the utility of PhaTYP for analyzing the phage lifestyle on human neonates' gut data. This application shows that PhaTYP is a useful means for studying phages in metagenomic data and helps extend our understanding of microbial communities.

Transcription factor VRT2 reinitiates vernalization when interrupted by warm temperatures in a temperate grass model.

Vernalisation-responsive plants use cold as a cue to monitor the passing of winter. Winter cereals can remember how much cold they have experienced, even when winter is punctuated by warm days. However, in a seemingly unnatural process called 'devernalisation', hot temperatures can erase winter memory. Previous studies in bread wheat (Triticum aestivum) have implicated the MADS-box transcription factor VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2) in vernalisation based on transcriptional behaviour and ectopic expression. Here, we characterised three BdVRT2 loss-of-function alleles in the temperate model grass Brachypodium distachyon. In addition to extended vernalisation requirements, mutants showed delayed flowering relative to wild-type plants when exposed only briefly to warm temperatures after partial vernalisation, with flowering being unaffected when vernalisation was saturating. Together, these data suggest a role for BdVRT2 in both vernalisation and in its re-initiation when interrupted by warm temperatures. In controlled constant conditions, BdVRT2 transcription was not strongly affected by vernalisation or devernalisation. Yet, by monitoring BdVRT2 expression in seasonally varying and fluctuating conditions in an unheated greenhouse, we observed strong upregulation, suggesting that its transcription is regulated by fluctuating vernalising-devernalising conditions. Our data suggest that devernalisation by hot temperatures is not a peculiarity of domesticated cereal crops but is the extreme of the reversibility of vernalisation by warm temperatures and has broader biological relevance across temperate grasses.

Ecotype formation and prophage domestication during gut bacterial evolution.

How much bacterial evolution occurs in our intestines and which factors control it are currently burning questions. The formation of new ecotypes, some of which capable of coexisting for long periods of time, is highly likely in our guts. Horizontal gene transfer driven by temperate phages that can perform lysogeny is also widespread in mammalian intestines. Yet, the roles of mutation and especially lysogeny as key drivers of gut bacterial adaptation remain poorly understood. The mammalian gut contains hundreds of bacterial species, each with many strains and ecotypes, whose abundance varies along the lifetime of a host. A continuous high input of mutations and horizontal gene transfer events mediated by temperate phages drives that diversity. Future experiments to study the interaction between mutations that cause adaptation in microbiomes and lysogenic events with different costs and benefits will be key to understand the dynamic microbiomes of mammals. Also see the video abstract here: https://youtu.be/Zjqsiyb5Pk0.

Genomics of Secondarily Temperate Adaptation in the Only Non-Antarctic Icefish.

White-blooded Antarctic icefishes, a family within the adaptive radiation of Antarctic notothenioid fishes, are an example of extreme biological specialization to both the chronic cold of the Southern Ocean and life without hemoglobin. As a result, icefishes display derived physiology that limits them to the cold and highly oxygenated Antarctic waters. Against these constraints, remarkably one species, the pike icefish Champsocephalus esox, successfully colonized temperate South American waters. To study the genetic mechanisms underlying secondarily temperate adaptation in icefishes, we generated chromosome-level genome assemblies of both C. esox and its Antarctic sister species, Champsocephalus gunnari. The C. esox genome is similar in structure and organization to that of its Antarctic congener; however, we observe evidence of chromosomal rearrangements coinciding with regions of elevated genetic divergence in pike icefish populations. We also find several key biological pathways under selection, including genes related to mitochondria and vision, highlighting candidates behind temperate adaptation in C. esox. Substantial antifreeze glycoprotein (AFGP) pseudogenization has occurred in the pike icefish, likely due to relaxed selection following ancestral escape from Antarctica. The canonical AFGP locus organization is conserved in C. esox and C. gunnari, but both show a translocation of two AFGP copies to a separate locus, previously unobserved in cryonotothenioids. Altogether, the study of this secondarily temperate species provides an insight into the mechanisms underlying adaptation to ecologically disparate environments in this otherwise highly specialized group.

Temperature and water availability drive insect seasonality across a temperate and a tropical region.

The more insects there are, the more food there is for insectivores and the higher the likelihood for insect-associated ecosystem services. Yet, we lack insights into the drivers of insect biomass over space and seasons, for both tropical and temperate zones. We used 245 Malaise traps, managed by 191 volunteers and park guards, to characterize year-round flying insect biomass in a temperate (Sweden) and a tropical (Madagascar) country. Surprisingly, we found that local insect biomass was similar across zones. In Sweden, local insect biomass increased with accumulated heat and varied across habitats, while biomass in Madagascar was unrelated to the environmental predictors measured. Drivers behind seasonality partly converged: In both countries, the seasonality of insect biomass differed between warmer and colder sites, and wetter and drier sites. In Sweden, short-term deviations from expected season-specific biomass were explained by week-to-week fluctuations in accumulated heat, rainfall and soil moisture, whereas in Madagascar, weeks with higher soil moisture had higher insect biomass. Overall, our study identifies key drivers of the seasonal distribution of flying insect biomass in a temperate and a tropical climate. This knowledge is key to understanding the spatial and seasonal availability of insects-as well as predicting future scenarios of insect biomass change.

Genetic architecture of disease resistance and tolerance in Douglas-fir trees.

Understanding the genetic basis of how plants defend against pathogens is important to monitor and maintain resilient tree populations. Swiss needle cast (SNC) and Rhabdocline needle cast (RNC) epidemics are responsible for major damage of forest ecosystems in North America. Here we investigate the genetic architecture of tolerance and resistance to needle cast diseases in Douglas-fir (Pseudotsuga menziesii) caused by two fungal pathogens: SNC caused by Nothophaeocryptopus gaeumannii, and RNC caused by Rhabdocline pseudotsugae. We performed case-control genome-wide association analyses and found disease resistance and tolerance in Douglas-fir to be polygenic and under strong selection. We show that stomatal regulation as well as ethylene and jasmonic acid pathways are important for resisting SNC infection, and secondary metabolite pathways play a role in tolerating SNC once the plant is infected. We identify a major transcriptional regulator of plant defense, ERF1, as the top candidate for RNC resistance. Our findings shed light on the highly polygenic architectures underlying fungal disease resistance and tolerance and have important implications for forestry and conservation as the climate changes.

Diversity and functional differentiation of renewal buds in temperate herbaceous plants.

Spring regrowth in temperate perennials relies on renewal buds, which form a key component in the shoot growth cycle. Still, we possess almost no information on these renewal buds, which is becoming more pressing with the current climate change. Most existing studies concentrated on easy-to-study aboveground buds of woody plants, whose morphology has largely been linked to frost protection. It is not clear to what extent these findings apply also to herbaceous species. We therefore examined protective traits and preformation of winter renewal buds in 379 species of temperate herbs, and tested how these traits are distributed across the phylogeny and related to other bud bank and whole-plant traits. We identified a major gradient from few, large, highly preformed, scale-covered buds associated with larger belowground storage organs deep in the soil, to small, numerous, less preformed, and naked buds near the soil surface. Belowground renewal buds of temperate herbs show several distinct strategies for winter survival and spring regrowth that might affect their response to changing winter and early spring conditions. Renewal bud traits are driven not only by frost protection but also by protection of the apical meristem from mechanical disturbance in the soil.

Legacy effects of premature defoliation in response to an extreme drought event modulate phytochemical profiles with subtle consequences for leaf herbivory in European beech.

Extreme droughts can have long-lasting effects on forest community dynamics and species interactions. Yet, our understanding of how drought legacy modulates ecological relationships is just unfolding. We tested the hypothesis that leaf chemistry and herbivory show long-term responses to premature defoliation caused by an extreme drought event in European beech (Fagus sylvatica L.). For two consecutive years after the extreme European summer drought in 2018, we collected leaves from the upper and lower canopy of adjacently growing drought-stressed and unstressed trees. Leaf chemistry was analyzed and leaf damage by different herbivore-feeding guilds was quantified. We found that drought had lasting impacts on leaf nutrients and on specialized metabolomic profiles. However, drought did not affect the primary metabolome. Drought-related phytochemical changes affected damage of leaf-chewing herbivores whereas damage caused by other herbivore-feeding guilds was largely unaffected. Drought legacy effects on phytochemistry and herbivory were often weaker than between-year or between-canopy strata variability. Our findings suggest that a single extreme drought event bears the potential to long-lastingly affect tree-herbivore interactions. Drought legacy effects likely become more important in modulating tree-herbivore interactions since drought frequency and severity are projected to globally increase in the coming decades.

Weak local adaptation to drought in seedlings of a widespread conifer.

Tree seedlings from populations native to drier regions are often assumed to be more drought tolerant than those from wetter provenances. However, intraspecific variation in drought tolerance has not been well-characterized despite being critical for developing climate change mitigation and adaptation strategies, and for predicting the effects of drought on forests. We used a large-scale common garden drought-to-death experiment to assess range-wide variation in drought tolerance, measured by decline of photosynthetic efficiency, growth, and plastic responses to extreme summer drought in seedlings of 73 natural populations of the two main varieties of Douglas-fir (Pseudotsuga menziesii var. menziesii and var. glauca). Local adaptation to drought was weak in var. glauca and nearly absent in menziesii. Var. glauca showed higher tolerance to drought but slower growth than var. menziesii. Clinal variation in drought tolerance and growth species-wide was mainly associated with temperature rather than precipitation. A higher degree of plasticity for growth was observed in var. menziesii in response to extreme drought. Genetic variation for drought tolerance in seedlings within varieties is maintained primarily within populations. Selective breeding within populations may facilitate adaptation to drought more than assisted gene flow.

Forest plant indicator values for moisture reflect atmospheric vapour pressure deficit rather than soil water content.

Soil moisture shapes ecological patterns and processes, but it is difficult to continuously measure soil moisture variability across the landscape. To overcome these limitations, soil moisture is often bioindicated using community-weighted means of the Ellenberg indicator values of vascular plant species. However, the ecology and distribution of plant species reflect soil water supply as well as atmospheric water demand. Therefore, we hypothesized that Ellenberg moisture values can also reflect atmospheric water demand expressed as a vapour pressure deficit (VPD). To test this hypothesis, we disentangled the relationships among soil water content, atmospheric vapour pressure deficit, and Ellenberg moisture values in the understory plant communities of temperate broadleaved forests in central Europe. Ellenberg moisture values reflected atmospheric VPD rather than soil water content consistently across local, landscape, and regional spatial scales, regardless of vegetation plot size, depth as well as method of soil moisture measurement. Using in situ microclimate measurements, we discovered that forest plant indicator values for moisture reflect an atmospheric VPD rather than soil water content. Many ecological patterns and processes correlated with Ellenberg moisture values and previously attributed to soil water supply are thus more likely driven by atmospheric water demand.

Temporal trends in respiratory pathogens following the COVID-19 pandemic and climate variables: A unicentric retrospective evaluation of 24 pathogens in a temperate subtropical region.

Temperature and humidity are studied in the context of seasonal infections in temperate and tropical zones, but the relationship between viral trends and climate variables in temperate subtropical zones remains underexplored. Our retrospective study analyzes respiratory pathogen incidence and its correlation with climate data in a subtropical zone. Retrospective observational study at Moinhos de Vento Hospital, South Brazil, aiming to assess seasonal trends in respiratory pathogens, correlating them with climate data. The study included patients of all ages from various healthcare settings, with data collected between April 2022 and July 2023. Biological samples were analyzed for 24 pathogens using polymerase chain reaction and hybridization techniques; demographic variables were also collected. The data was analyzed descriptively and graphically. Spearman tests and Poisson regression were used as correlation tests. Tests were clustered according to all pathogens, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza viruses, rhinovirus, and respiratory syncytial virus (RSV). Between April 2022 and July 2023, 3329 tests showed a 71.6% positivity rate. Rhinovirus and RSV predominated, exhibiting seasonal patterns. Temperature was inversely correlated with the viruses, notably rhinovirus, but SARS-CoV-2 was positively correlated. Air humidity was positively correlated with all pathogens, RSV, rhinovirus, and atmospheric pressure with all pathogens and rhinovirus. Our results showed statistically significant correlations, with modest effect sizes. Our study did not evaluate causation effects. Despite the correlation between climate and respiratory pathogens, our work suggests additional factors influencing transmission dynamics. Our findings underscore the complex interplay between climate and respiratory infections in subtropical climates.

Rapid migration of Mongolian oak into the southern Asian boreal forest.

The migration of trees induced by climatic warming has been observed at many alpine treelines and boreal-tundra ecotones, but the migration of temperate trees into southern boreal forest remains less well documented. We conducted a field investigation across an ecotone of temperate and boreal forests in northern Greater Khingan Mountains of northeast China. Our analysis demonstrates that Mongolian oak (Quercus mongolica), an important temperate tree species, has migrated rapidly into southern boreal forest in synchrony with significant climatic warming over the past century. The average rate of migration is estimated to be 12.0 ± 1.0 km decade-1 , being slightly slower than the movement of isotherms (14.7 ± 6.4 km decade-1 ). The migration rate of Mongolian oak is the highest observed among migratory temperate trees (average rate 4.0 ± 1.0 km decade-1 ) and significantly higher than the rates of tree migration at boreal-tundra ecotones (0.9 ± 0.4 km decade-1 ) and alpine treelines (0.004 ± 0.003 km decade-1 ). Compared with the coexisting dominant boreal tree species, Dahurian larch (Larix gmelinii), temperate Mongolian oak is observed to have significantly lower capacity for light acquisition, comparable water-use efficiency but stronger capacity to utilize nutrients especially the most limiting nutrient, nitrogen. In the context of climatic warming, and in addition to a high seed dispersal capacity and potential thermal niche differences, the advantage of nutrient utilization, reflected by foliar elementomes and stable nitrogen isotope ratios, is also likely a key mechanism for Mongolian oak to coexist with Dahurian larch and facilitate its migration toward boreal forest. These findings highlight a rapid deborealization of southern Asian boreal forest in response to climatic warming.

Species interactions amplify functional group responses to elevated CO2 and N enrichment in a 24-year grassland experiment.

Plant functional groups (FGs) differ in their response to global changes, although species within those groups also vary in such responses. Both species and FG responses to global change are likely influenced by species interactions such as inter-specific competition and facilitation, which are prevalent in species mixtures but not monocultures. As most studies focus on responses of plants growing in either monocultures or mixtures, but rarely both, it remains unclear how interspecific interactions in diverse ecological communities, especially among species in different FGs, modify FG responses to global changes. To address these issues, we leveraged data from a 16-species, 24-year perennial grassland experiment to examine plant FG biomass responses to atmospheric CO2, and N inputs at different planted diversity. FGs differed in their responses to N and CO2 treatments in monocultures. Such differences were amplified in mixtures, where N enrichment strongly increased C3 grass success at ambient CO2 and C4 grass success at elevated CO2. Legumes declined with N enrichment in mixtures at both CO2 levels and increased with elevated CO2 in the initial years of the experiment. Our results suggest that previous studies that considered responses to global changes in monocultures may underestimate biomass changes in diverse communities where interspecific interactions can amplify responses. Such effects of interspecific interactions on responses of FGs to global change may impact community composition over time and consequently influence ecosystem functions.

The interacting effect of climate change and herbivory can trigger large-scale transformations of European temperate forests.

In many regions of Europe, large wild herbivores alter forest community composition through their foraging preferences, hinder the forest's natural adaptive responses to climate change, and reduce ecosystem resilience. We investigated a widespread European forest type, a mixed forest dominated by Picea abies, which has recently experienced an unprecedented level of disturbance across the continent. Using the forest landscape model iLand, we investigated the combined effect of climate change and herbivory on forest structure, composition, and carbon and identified conditions leading to ecosystem transitions on a 300-year timescale. Eight climate change scenarios, driven by Representative Concentration Pathways 4.5 and 8.5, combined with three levels of regeneration browsing, were tested. We found that the persistence of the current level of browsing pressure impedes adaptive changes in community composition and sustains the presence of the vulnerable yet less palatable P. abies. These development trajectories were tortuous, characterized by a high disturbance intensity. On the contrary, reduced herbivory initiated a transformation towards the naturally dominant broadleaved species that was associated with an increased forest carbon and a considerably reduced disturbance. The conditions of RCP4.5 combined with high and moderate browsing levels preserved the forest within its reference range of variability, defining the actual boundaries of resilience. The remaining combinations of browsing and climate change led to ecosystem transitions. Under RCP4.5 with browsing effects excluded, the new equilibrium conditions were achieved within 120 years, whereas the stabilization was delayed by 50-100 years under RCP8.5 with higher browsing intensities. We conclude that forests dominated by P. abies are prone to transitions driven by climate change. However, reducing herbivory can set the forest on a stable and predictable trajectory, whereas sustaining the current browsing levels can lead to heightened disturbance activity, extended transition times, and high variability in the target conditions.

Long-term soil warming decreases soil microbial necromass carbon by adversely affecting its production and decomposition.

Microbial necromass carbon (MNC) accounts for a large fraction of soil organic carbon (SOC) in terrestrial ecosystems. Yet our understanding of the fate of this large carbon pool under long-term warming is uncertain. Here, we show that 14 years of soil warming (+4°C) in a temperate forest resulted in a reduction in MNC by 11% (0-10 cm) and 33% (10-20 cm). Warming caused a decrease in the content of MNC due to a decline in microbial biomass carbon and reduced microbial carbon use efficiency. This reduction was primarily caused by warming-induced limitations in available soil phosphorus, which, in turn, constrained the production of microbial biomass. Conversely, warming increased the activity of soil extracellular enzymes, specifically N-acetylglucosaminidase and leucine aminopeptidase, which accelerated the decomposition of MNC. These findings collectively demonstrate that decoupling of MNC formation and decomposition underlie the observed MNC loss under climate warming, which could affect SOC content in temperate forest ecosystems more widespread.

Major tree species of Central European forests differ in their proportion of positive, negative, and nonstationary growth trends.

Temperate forests are undergoing significant transformations due to the influence of climate change, including varying responses of different tree species to increasing temperature and drought severity. To comprehensively understand the full range of growth responses, representative datasets spanning extensive site and climatic gradients are essential. This study utilizes tree-ring data from 550 sites from the temperate forests of Czechia to assess growth trends of six dominant Central European tree species (European beech, Norway spruce, Scots pine, silver fir, sessile and pedunculate oak) over 1990-2014. By modeling mean growth series for each species and site, and employing principal component analysis, we identified the predominant growth trends. Over the study period, linear growth trends were evident across most sites (56% increasing, 32% decreasing, and 10% neutral). The proportion of sites with stationary positive trends increased from low toward high elevations, whereas the opposite was true for the stationary negative trends. Notably, within the middle range of their distribution (between 500 and 700 m a.s.l.), Norway spruce and European beech exhibited a mix of positive and negative growth trends. While Scots pine growth trends showed no clear elevation-based pattern, silver fir and oaks displayed consistent positive growth trends regardless of site elevation, indicating resilience to the ongoing warming. We demonstrate divergent growth trajectories across space and among species. These findings are particularly important as recent warming has triggered a gradual shift in the elevation range of optimal growth conditions for most tree species and has also led to a decoupling of growth trends between lowlands and mountain areas. As a result, further future shifts in the elevation range and changes in species diversity of European temperate forests can be expected.

Even cooler insights: On the power of forests to (water the Earth and) cool the planet.

Scientific innovation is overturning conventional paradigms of forest, water, and energy cycle interactions. This has implications for our understanding of the principal causal pathways by which tree, forest, and vegetation cover (TFVC) influence local and global warming/cooling. Many identify surface albedo and carbon sequestration as the principal causal pathways by which TFVC affects global warming/cooling. Moving toward the outer latitudes, in particular, where snow cover is more important, surface albedo effects are perceived to overpower carbon sequestration. By raising surface albedo, deforestation is thus predicted to lead to surface cooling, while increasing forest cover is assumed to result in warming. Observational data, however, generally support the opposite conclusion, suggesting surface albedo is poorly understood. Most accept that surface temperatures are influenced by the interplay of surface albedo, incoming shortwave (SW) radiation, and the partitioning of the remaining, post-albedo, SW radiation into latent and sensible heat. However, the extent to which the avoidance of sensible heat formation is first and foremost mediated by the presence (absence) of water and TFVC is not well understood. TFVC both mediates the availability of water on the land surface and drives the potential for latent heat production (evapotranspiration, ET). While latent heat is more directly linked to local than global cooling/warming, it is driven by photosynthesis and carbon sequestration and powers additional cloud formation and top-of-cloud reflectivity, both of which drive global cooling. TFVC loss reduces water storage, precipitation recycling, and downwind rainfall potential, thus driving the reduction of both ET (latent heat) and cloud formation. By reducing latent heat, cloud formation, and precipitation, deforestation thus powers warming (sensible heat formation), which further diminishes TFVC growth (carbon sequestration). Large-scale tree and forest restoration could, therefore, contribute significantly to both global and surface temperature cooling through the principal causal pathways of carbon sequestration and cloud formation.