Philosophical Transactions of the Royal Society B: Biological Sciences

Drumming--rhythmic, percussive sound production using body parts or external objects--is rare among non-human animals, with confirmed tool-assisted cases previously limited to primates and Palm Cockatoos. Here, we report the first documented instance of spontaneous, tool-assisted drumming in a Galah (Eolophus roseicapilla). A captive, male Galah produced rhythmic tapping by striking a coconut shell against a metal bowl. Across 14 recorded sessions, the bird displayed consistent temporal structure characterised by two stable tapping rates (approximately 0.8 s and 0.2 s inter-onset intervals) arranged into recurring phrases. This pattern indicates a simple hierarchical rhythmic organisation with a 4:1 ratio between metrical levels. The birds behaviour emerged without training, apparent reinforcement, or known exposure to conspecific or human drumming models, suggesting an intrinsic capacity for rhythmic tool use. Although the function of the behaviour remains unclear--play, nutrient extraction, or communicative signalling--these observations extend known rhythmic and tool-using abilities within cockatoos and raise new evolutionary questions. Our findings highlight the potential for rhythmically structured, instrumental behaviour to arise in a broader range of avian taxa than previously recognised, motivating further comparative and experimental work on the cognitive and biomechanical foundations of drumming in parrots.

A populations social structure, often represented as a social network, shapes fundamental biological processes including the spread of disease, information, and behaviour. The Friendship Paradox is a network phenomenon whereby the average individual has fewer friends than their friends do. This effect can be quantified as relationship disparity (the difference between an individuals connectedness and those they are connected to) which captures the local social environment. Previous work has shown that such relationship disparity can be exploited in effective outbreak monitoring, targeted health interventions and optimized contact tracing. Yet, how its magnitude varies across real-world social networks remains poorly understood. Here, we analyse relationship disparity across 391 empirical animal social networks to test how intrinsic network properties and biological attributes predict its extent. We find that smaller and sparser networks exhibit stronger relationship disparity, and that mammalian and avian social systems generally showed stronger relationship disparity than reptilian systems. After controlling for variation in individual sociability, mammalian and reptilian social networks displayed weaker relationship disparity than expected based on network structure alone. Together, these findings demonstrate that both network structure and biological attributes shape relationship disparity in natural social systems, providing a foundation for predicting how higher-order network architecture influences social processes such as contagion. Significance StatementIn natural populations, social connections are unevenly distributed, often resulting in a small subset of individuals that are highly connected while many are relatively peripheral. The Friendship Paradox is a measure of relationship disparity between individuals and their local social environment. Understanding how features of the social network and biological system are associated with relationship disparity can contribute to understanding what shapes social behaviour. Relationship disparity may not just be an emergent network property but could reflect a higher level of social structuring, and therefore shape processes that depend on social contacts. Our findings demonstrate the value of comparative network analysis for generating insights into fundamental principles structuring real-world societies.

Interpersonal neural synchrony (INS) in mother-child dyads is often interpreted as a neural marker of relational quality and sensitive caregiving, yet findings on its predictors remain heterogeneous. One possible source of this variability is the diversity of interactional paradigms used in hyperscanning research. This study examined how maternal personality, child temperament, and affective states relate to INS across interaction contexts varying in social interactivity. Thirty-three mother-child dyads (n = 20 female children) participated in a functional near-infrared spectroscopy hyperscanning experiment involving passive video co-exposure, a structured cooperative task, and free interaction. Fronto-temporal activity was recorded simultaneously, and INS was computed using wavelet transform coherence. Above-chance levels of INS emerged in inter-brain region combinations primarily involving the mothers left inferior frontal gyrus (IFG) and the childs right IFG (adjusted ps < 0.030, Cohens d range = 0.14-0.31). Maternal neuroticism was the only significant predictor of INS, with higher levels associated with increased synchrony during passive video co-exposure (adjusted p = 0.012) and free interaction (adjusted p = 0.021), but not during the structured game. These findings indicate that maternal dispositional traits shape INS in a context-dependent manner. Notably, the positive association between neuroticism and INS suggests that heightened neural synchrony may reflect over-attunement in more anxious caregivers, rather than optimal coordination. Excessive synchrony may therefore index tightly coupled, over-monitoring interaction dynamics, consistent with models of affiliative vigilance in anxious parenting. Overall, INS may follow a non-linear pattern in which moderate levels are most adaptive, highlighting its flexible, dynamic, and context-sensitive nature.

Pretend play is a hallmark behavior in childhood where children create nonliteral meanings. Empirical data supporting the role of social cognition and the decoupling from literality are still scarce during early development. We explored here how the comprehension of pretense affects the visual exploratory behavior of toddlers (n = 44) and adults (n = 65) when they were exposed to short video clips in which an actress performed either real actions (e.g., eating jelly) or pretend actions (e.g., pretending to eat with imaginary food), while varying the complexity of those actions. We analyzed participants exploration of the face in the videos as exploitation of social information. We showed that all observers paid more attention to the face in pretend scenarios than in real ones, measured as longer total looking time in adults and more fixations and revisits to the face in both age groups. We also found more gaze shifts (a measure of information sampling) between the face and the moving hand in the pretend videos in both age groups, mainly at the initial stages of the actions. Additionally, analyses of the scanpaths structure using gaze entropy showed less order in the exploration of pretend videos in both age groups, suggesting that pretense involved greater uncertainty and increased information seeking. The less structured trajectories were observed again mainly in complex pretend scenarios. Taken together, our gaze results indicate that from its developmental origins, the comprehension of pretense relies on social processes linked with information seeking and exploration. Significance StatementDevelopmental theories have long debated whether pretend games are born in conjunction with social capacities in the second year or become integrated later in life. Our study shows that, much like adults, toddlers visually explore pretend scenes gathering more social information and in a less structured manner compared to real-world scenarios, suggesting that the emerging capacity to play with the meaning of things is linked with that of thinking of other minds early in life.

Global populations are ageing at an unprecedented rate. For many diseases, age is a strong indicator of susceptibility, morbidity, or mortality. Principles of evolutionary biology can be leveraged to understand how pathogens may optimally exploit new populations, and the impact of this on the global burden of infectious-disease-induced mortality. We parameterised an age-specific R0 model with 2017 epidemiological data on Measles, Tuberculosis, Meningitis, and Ebola, and age-specific demographic estimates for 2017 and 2050, for the seven Global Burden of Disease super-regions. We explored the theoretical trade-offs between pathogen virulence & transmission, and virulence & host recovery, parameterising trade-off parameters using Latin Hypercube Sampling. Population ageing between 2017-2050 saw an increase in virulence induced mortality in four settings: 1) Ebola in sub-Saharan Africa, 2) Measles in central/eastern Europe & central Asia region, 3) Measles in North Africa & the Middle East and 4) Tuberculosis in the central/eastern Europe & central Asia region. The decrease in infection duration due to an increase of elderly people drives pathogen virulence down for diseases in the remaining settings. Understanding the mechanisms that shape pathogen dynamics and leveraging this to predict future challenges is key to endemic disease management in a rapidly changing world. Author SummaryKey aspects of disease transmission including susceptibility to infection, the severity of infection, and the probability of dying from that infection, are affected by host age. Global populations are rapidly ageing, so that the mean age of most populations is generally higher than it used to be and is set to continue on this trajectory. This suggests that the dynamics of infectious diseases are also likely to change, although infectious disease dynamics tend to be non-linear as these key parameters interact. We have developed a dynamic modelling framework to explore how changes in population age structure might impact the optimal level of pathogen virulence in a population. We have chosen four infectious diseases as case studies, that differentially impact certain age classes to illustrate these dynamics. We have parameterised this framework with open access data for each of the seven Global Burden of Disease super-regions and show that population ageing can increase virulence for several diseases in differing global regions, whilst increased background rates of mortality can drive virulence down in others.

Movement is costly, and animals are under strong selective pressure to move efficiently, yet, in patchy, dynamic landscapes, decision-making is inherently uncertain. We quantify the energetic savings achieved by using up-to-date information presented within social cues for reducing movement costs. We use an agent-based model, founded on realistic aeronautical rules and parametrised on the Andean condor (Vultur gryphus), to study movement in patchy landscapes. By explicitly considering altitude, flight results in a sequence of soaring and gliding in the 3D space. We investigate how the cost of movement to an overall goal varies when birds use social information from others that are either fixed in space or moving collectively to the common goal, and under different risk-taking speed strategies, from slow and cautious to fast and risky. The value of social information is operationalised as energetic savings in units of basal metabolic rate. Under low predictability, agents with intermediate risk and high social-information use exhibit lowest movement costs, with up to 41% energy savings over asocial movement. By extending classical aeronautical theory to social and variable environments we demonstrate the adaptive value of social information for efficient movement in patchy, unpredictable landscapes.

Orca, wolves, chimpanzees and humans share a similarly impressive capacity for group hunting, where individuals coordinate behaviour together to capture prey. Studying hunting behaviours has important implications for understanding how behaviour in group contexts may be indicative of cognitive decline. Despite growing interest in brain circuits for prey capture, the brain regions involved in tracking prey during a hunt and the behaviours in group hunt linked to success remain unclear. Here we combined functional near infrared spectroscopy (fNIRS) and a virtual minecraft world to examine behaviour, brain dynamics and brain synchrony involved in group hunting behaviour. We focused on the prefrontal cortex (PFC) due to its known role in planning and social coordination and recorded from pairs of individuals as they either cooperated to hunt another person (prey) or simply followed another person. Hunters were more successful if they managed to keep a smaller distance to the prey and moved at speeds that were more synchronised with their co-predator. At high-range frequencies for fNIRS (0.1-0.2Hz), we found greater brain-to-brain synchrony in lateral and medial (frontopolar) PFC regions during hunting compared with chance levels. Together, these findings provide insights into what behaviours and brain dynamics associated with successful group hunting.

Protogynous sex change, where individuals first function as females and later as males, is a key life-history strategy among polygynous reef fishes. In haremic systems, sex change is typically socially regulated, with dominants suppressing subordinates sex change through aggression. Females within a harem form a size-based hierarchy that can remain stable in most species through the threat of eviction. We studied a different situation in the cleaner wrasse Labroides dimidiatus, where larger females have incomplete control, as they spend most of their time alone at their own cleaning territory. We tracked over 400 individuals for 12 months, recording growth, behavior, social organization, and sex change. We confirmed earlier reports that both sexes direct aggression primarily at those ranked immediately below them. However, we observed 30 cases where smaller females outgrew larger ones, revealing hierarchy instability. Of 42 sex change events, 43% occurred in presence of the male, and half of these early sex changers were not the largest female, but individuals overlooked by the male. Fast growth relative to harem-mates and harem switching increased the likelihood of sex change. Local population densities also influenced growth and sex change, with individuals in high-density demes growing faster and changing sex at larger sizes. Our findings reveal flexible sex change dynamics in a system with incomplete social dominance. Such incomplete control and observations that becoming male confers both higher reproductive success and survival highlight the need to expand game-theoretical and life-history frameworks to encompass such strategic flexibility. Lay summaryDominant cleaner wrasse cannot fully control subordinates as individuals occupy distinct core areas. Tracking 400 fish for a year, we found that smaller females could outgrow initially larger ones, and early sex change despite a larger male. Fast growth and harem switching increased the chances of becoming male. Population density also shaped these strategies. Our findings reveal flexible sex change dynamics in a system where becoming male confers both higher reproductive success and survival.

O_LISeasonal patterns of species appearances constitute a major component of diversity variation. Theory attributes this phenological structuring of communities to the alignment of life cycles to suitable moments and to constraints of seasonality on development, yet the specific mechanisms operating across taxa remain largely unresolved. In insects, body size and colour are key functional traits that contribute to driving spatial community assembly through their link to thermoregulatory performance and development. C_LIO_LIHere we analyse variation in mean body size and colour lightness of 483 butterfly assemblages across Great Britain and throughout the season to test whether trait alignment with seasonal environment and developmental constraints may shape the phenological structuring of communities. C_LIO_LIBoth body size and body colour varied more along season than across space, emphasizing the importance of phenology on diversity variation. Body size was larger early and late in the season, i.e. under conditions of low temperature and solar radiation. This pattern contrasted with the spatial trends found and was driven by species overwintering as adults, which we interpret as being likely due to energetic constraints. Body colour, conversely, was darker early and late in the season, mirroring the spatial pattern found, and suggesting a thermoregulatory alignment with seasonal conditions. Furthermore, covariation between body size and colour suggests a thermoregulatory interaction between both traits. C_LIO_LIOur findings suggest that life-cycle constraints and seasonal thermoregulatory alignment contribute to shaping the phenological structure of insect communities. C_LI

Collective behavior in animal societies can buffer individual costs and confer resilience to environmental challenges. However, the mechanisms by which groups sustain function when members are compromised remain poorly understood. In the presented study, we investigate how social context shapes collective fanning, a thermoregulatory behavior critical for colony function, in Western honeybees (Apis mellifera). Using oxytetracycline (OTC), a known physiologically disruptive antibiotic to honeybees, to selectively impair certain group members, we tested our hypothesis that the presence of untreated bees would rescue the fanning response in mixed-composition groups. We show that groups containing untreated individuals fan at levels comparable to fully untreated groups, despite the presence of OTC-impaired bees. This preservation of collective thermoregulatory function was correlated with both treated and untreated individuals in mixed groups shifting their interaction dynamics and social network positions. These findings reveal a decentralized mechanism of collective resilience, whereby behavioral compensation by individuals sustains group-level thermoregulation under partial disruption. Our results provide a framework for understanding how social insect colonies maintain function in the face of individual-level perturbations, with broader implications for predicting the limits of collective resilience in animal societies experiencing increasing environmental pressures.

In the search for universals shaping acoustic communication across species, we increasingly look for patterns known from human languages and music in non-human animals. These parallels are often explored separately and with limited ecological context. Here, we take a deep dive into the temporal structure of a complex call used by the little auk (Alle alle), a pelagic seabird with elaborate vocal behaviour and socially complex colonial life. Based on syllable durations, intervals and silences, we examine its conformance to linguistic laws, rhythmic structure and information content. This reveals intricate problems of temporal organisation: while the calls conform not only to linguistic laws of brevity but also to the initial and final lengthening known from human prosody, these effects interact with the internal structure of the call and information carried within it. To our knowledge, this is the first time that conformance to multiple linguistic laws, exceeding simple vocal efficiency, has been described for a non-human, non-vocal learning animal. The calls rhythmic structure shows a progressive rallentando -- a systematic slowing driven by changes in syllable and silence durations and the intervals between syllable onsets. The exact patterns of this rallentando are indicative of the callers sex and individually specific. These results reveal how seabird communication is shaped not only by efficiency universals, but also the specific pressures of colonial life. Our work highlights the temporal structure as an important axis of communication evolution, but also serves as a reminder to consider the species ecological reality and the function, not only presence, of temporal organisation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/713940v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@13de3a8org.highwire.dtl.DTLVardef@2d64adorg.highwire.dtl.DTLVardef@2ca53aorg.highwire.dtl.DTLVardef@113c38d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cross-feeding interactions are pervasive in microbial communities and profoundly shape community structure, stability, and function. While previous studies have explored how cross-feeding affects evolvability, this work has predominantly focused on bidirectional mutualistic interactions in engineered auxotrophic systems where both partners reciprocally exchange essential metabolites. However, most metabolic interactions in natural microbial communities are unidirectional, with organisms feeding on the metabolic waste products of other species. Our study addresses this gap by examining how a unidirectional cross-feeding interaction affects the evolutionary dynamics of both the producer (Acinetobacter johnsonii) and consumer (Pseudomonas putida) over 800 generations of experimental evolution. We found that co-culture constrained adaptive evolution in both species. Co-cultures exhibited lower {pi}N/{pi}S ratios (0.75 for P. putida; 1.04 for A. johnsonii) than monocultures (1.44 and 2.02, respectively) indicating stronger purifying selection against nonsynonymous mutations in the community context. Lineage tracking through whole genome sequencing of populations and clones revealed greater lineage diversity and complexity in monocultures, with more mutations showing significant parallelism across replicate populations. Additionally, P. putida evolved increased dependence on its partner; co-culture-evolved P. putida grew significantly worse than its ancestor when A. johnsonii was removed. These findings demonstrate that ecological interactions fundamentally reshape fitness landscapes and constrain adaptive evolution even when fitness benefits are unidirectional, with implications for understanding microbial community stability and predicting evolutionary dynamics in complex communities.

O_LIUnderstanding functional community structure and the niche-based mechanisms that enable coexistence among sympatric species is essential for explaining how biodiversity is maintained in natural systems, and for anticipating how ecological communities will respond to ongoing environmental change. Stable isotope analysis provides a process-oriented perspective on resource use by integrating information across time and space, thereby allowing reconstruction of realised isotopic niches that reflect multiple dimensions of ecological differentiation. C_LIO_LIWe applied this framework to a community of ungulates in the Central-Eastern Italian Alps, including red deer (Cervus elaphus), roe deer (Capreolus capreolus), and Alpine chamois (Rupicapra rupicapra). Using stable isotope ratios in summer-grown hair segments ({delta}13C, {delta}15N, {delta}34S, {delta}18O, {delta}2H), we quantified species-specific n-dimensional niche hypervolumes within a Bayesian framework and estimated niche regions, overlap probabilities, univariate differentiation and multivariate structure. C_LIO_LIDespite broad dietary overlap typically observed among these ungulates, we found clear isotopic niche segregation, with mean pairwise overlap consistently remaining below 40%. Three dimensions emerged as primary drivers of differentiation: water sourcing ({delta}18O), diet quality ({delta}15N), and habitat openness ({delta}13C). Specifically, chamois appeared to derive more water from plants in their diet rather than from drinking, and to consume a higher-quality diet compared to Cervids. Red deer relied more heavily on forested habitats for resource use compared to roe deer and chamois, and additional isotopic differences between red deer and roe deer may stem from fine-scale abiotic conditions like microclimate and topography. We found no isotopic evidence for differential niche breadth among the three ungulate species. C_LIO_LITogether, these patterns highlight functional differentiation across multiple ecological axes, offering mechanistic insight into how these ungulates segregate realised niche space despite substantial potential for resource overlap. This multi-element isotope perspective underscores the value of integrative, process-based approaches for understanding current coexistence as well as improving predictions of how mammal communities may reorganise under accelerating environmental change. C_LI

Biological diversity driven by endosymbiosis arises from the intertwined evolution of microbes and their hosts. Each partner affects the fitness and therefore the evolution of the other. Here, we tested a further question: does the history of symbiosis affect evolution even after the partnership is dissolved? We analyzed phenotypic data from experimentally evolved strains of Dictyostelium discoideum hosts, each of which had had its symbiont removed, to study how their traits evolved. We found that host trait evolution was affected by the prior history of infection, specifically by which of three Paraburkolderia bacterial symbionts had been removed. Thus, symbionts affect not only current evolution but also generate path dependence that affects the subsequent evolutionary trajectories even after the symbionts are lost. Impact statementThe evolution of partner dependence in host-microbial symbioses has fundamentally shaped biological diversity and ecosystem function. To examine variation in symbiont dependence in the social amoeba, we compared how different strains of Dictyostelium discoideum respond evolutionarily after the loss of their bacterial symbionts. We analyzed phenotypic data from experimentally evolved strains and found that the absence of different symbiont species leads to distinct changes in the subsequent evolution of key traits like cell proliferation, slug migration, and spore production. This research expands our current understanding of microbial symbiosis by revealing that symbiont species may impact the evolution of their hosts even after the symbiont is gone. Data summaryWe used phenotypic traits data from our previous experimental-evolution dataset from the open-access repository Dryad (https://doi.org/10.5061/dryad.kkwh70s97). Scripts for the statistical analyses are available in a GitHub repository (https://github.com/jahanisrat/SymbiontLoss). The accompanying R project includes code to reproduce the graphs in the results section.

Population ageing increases the importance of cognitive capacity for making decisions about retirement and living independently beyond it. We tested whether post-war educational expansion and working-life social mobility eliminate the association between social class of origin and cognition in early old age using the 1958 National Child Development Study. Two outcomes were analysed at age 62: standard episodic memory (immediate + delayed word recall) and long-term episodic memory, capturing accurate half-century recall of childhood household facts (rooms and people at age 11 validated against mothers' responses). Social mobility trajectories derived in prior work were classified into predominantly manual versus non-manual class trajectories. Models were estimated separately for women and men across three specifications: (i) social origin and controls, (ii) adding social mobility, and (iii) adding weighting to address healthy survivor bias. Education was consistently associated with both outcomes. For long-term episodic memory, social origin gradients were clearer than for short-term episodic memory, with men from service/professional origins showing a 13 percentage-point higher probability of accurate half-century recall than men from manual origins. These findings indicate that education expansion and working-life social mobility failed to release the grip of social origin on long-term episodic memory.

The benefits of routines for daily functioning are widely acknowledged, yet, despite their apparent importance, methods for quantifying routine maintenance and the causes of their disruption remain lacking. Here, we propose a novel means of defining and quantifying routines (transition entropy). Using the transition entropy, we show that routines can be robustly elicited on tasks that require searching through a grid of squares for a hidden target. Over two experiments (N=100 each), we show that use of routines--as quantified by transition entropy--is robustly perturbed by frequent switches between search grids, as locations specific to the currently irrelevant grid become competitive for selection. Using a normative model that tracks task dynamics, we show that disruption to routines can be attributed to reduced sensitivity to the odds of success for completing a task. This suggests that routine maintenance may be disrupted by over-sensitivity to a lack of reward early in routine performance, or increased expectations regarding the utility of pursuing other tasks.

Brain function is organised in distributed circuits in which regional engagement unfolds over time, reflecting coordinated and temporally ordered patterns of neural computation and information flow. Neural activity also depends on a reliable and scalable supply of glucose. Yet, the temporal order and direction of metabolic signalling in brain circuits remain unknown. Here, we combine functional Positron Emission Tomography (fPET) with 18F-flurodeoxyglucose and Granger causality analysis to characterise directed metabolic connectivity in cognitive control, memory and affective regulatory circuits in 86 healthy adults. We observed widespread directed metabolic influences within the circuits, with the strength of connections a significant predictor of cognition and affect. The behavioural value of the connections was also governed by the efficiency with which baseline glucose metabolism was converted into adaptive functional connections. We conclude that the brain is organised into metabolic circuits that coordinate temporally ordered connectivity to enable information transfer and modulate cognition and psychosocial function. Directed connections vary in their efficiency of glucose use and functional benefit, suggesting that metabolic signalling does not follow a simple "more is better" rule but reflects context-dependent optimisation across cognitive systems.

Our bodies have evolved to maintain homeostasis through regulatory systems that continuously adapt to keep physiological processes within a normal range. From this perspective, complex chronic disease can be understood as a breakdown of compensatory mechanisms, resulting in loss of homeostasis. Here we propose that adaptive receptor expression dynamics may serve as one such compensatory mechanism, increasing receptor surface expression when external ligand is insufficient, and clearing it when signaling is excessive. To explore this, we adapt a previously published agent-based model and simulate it under a range of scenarios. We find that the system of adaptive receptor expression is robust to oscillatory perturbations but not to chronic stress. We propose that receptor turnover dynamics may be better understood as an adaptive, environmentally responsive process rather than a fixed biological property, and that in some cases, disease manifests only after compensatory mechanisms have been pushed past their limits. We conclude with a discussion of implications for understanding complex chronic diseases, for thinking about epigenetic and mutational change as escalating layers of adaptation, and for how we model receptor dynamics in the context of receptor-mediated drug activity.

Non-invasive brain stimulation (NiBS) studies frequently report exploratory correlations between individual-level changes in neurophysiological and behavioural measures. However, these analyses are typically underpowered and rely on ratio-based change scores with known statistical limitations. We addressed these limitations by pooling individual data from three independent studies (total N = 69), providing adequate power to detect small-to-medium effects. All studies applied 20 Hz transcranial alternating current stimulation (tACS) targeting the pre-supplementary motor area (preSMA) and right inferior frontal gyrus (rIFG), regions central to inhibitory control. Changes in preSMA-rIFG connectivity measured with EEG imaginary coherence (ImCoh) and response inhibition (stop-signal reaction time, SSRT) were quantified using reliable change indices (RCI), which were z-standardised within studies to enable pooled mixed-effects regression. No meaningful association was found between tACS-induced ImCoh change and SSRT change (r = .013, marginal R{superscript 2} = .004), with project-wise correlations that were small, non-significant, and inconsistent in direction. Sensitivity analysis using ratio-based change scores converged on the same null result (r = .014), though ratio scores showed severe distributional violations relative to the approximately normal RCI distributions, supporting the methodological case for RCI on statistical grounds. These results provide no support for a systematic individual-level brain-behaviour coupling between preSMA-rIFG connectivity and response inhibition following 20 Hz tACS, and suggest that any true effect is likely to be small. The present work offers a methodological benchmark for quantifying individual-level brain-behaviour coupling in NiBS research, and highlights the need for more sensitive neural markers and adequately powered design.

Plant leaves harbor diverse microbial communities influenced by environmental inputs and host traits, yet it remains unclear whether leaves act as passive substrates or active ecological filters that reorganize microbial functional capacity. Phylloplane pH regulation is one hostplant trait that has been traditionally underexplored. We used metatranscriptomics to examine microbial gene expression on the phylloplane and within whole leaves of five plant species spanning the extremes of baseline phylloplane pH, including hyperalkalinizing Gossypium species, weakly buffering Beta vulgaris, and hyperacidifying Nepenthes species. Young leaves were inoculated with a common soil-derived microbial community to quantify host-associated restructuring of taxonomic and functional profiles, and short-term pH perturbations were applied to test the effect of transient abiotic stress. Across both phylloplane and whole-leaf datasets, host species identity was the primary axis structuring microbial taxonomic composition and expressed functional repertoires. Leaf-associated communities diverged from the source inoculum, but retained a substantial shared functional backbone enriched for central biosynthetic and core metabolic pathways. Host-associated differentiation reflected selective retention and redistribution of reactions within this shared environmental pool rather than acquisition of novel metabolic capacity. Enriched pathway subsets were metabolically coherent and taxonomically distributed across multiple bacterial orders, consistent with functional redundancy and trait-based assembly. Among hosts, Gossypium exhibited the strongest restructuring relative to inoculum, suggesting comparatively stronger host-associated filtering. In contrast, short-term pH manipulation did not induce consistent community-wide functional reorganization. Microbial physiological responses to the phylloplane environment and external pH were observed at the organismal level. Together, these results position leaves as active ecological filters that reorganize microbial functional landscapes through host-specific trait regimes. This work begins to implicate some role of phylloplane pH regulation in microbial assembly and function.