Evolutionary Biology

Statistical associations between two variables can arise via various causal mechanisms. For example, one variable may influence the other, or they may share common causes. Surprisingly little attention has been devoted to quantifying how such causal pathways contribute to the overall association between variables. Here, I present two methods for partitioning a covariance between variables X and Z into a causal component arising from the effect of X and Z and a spurious component arising via other pathways. Both methods rely on averaging causal decompositions over the distributions of the variables, but the decomposition and averaging are performed in different ways. The 'gradient' definition decomposes causal effects in the neighbourhood of naturally occurring values, making it relatively straightforward to estimate. It assumes that the cause variable X is normally distributed. In contrast, the 'two step' definition decomposes large ('step-wise') changes in values and makes no distributional assumptions. I describe applications of these methods to analysing natural selection and illustrate them using the covariance between body size and the number of eggs carried by pregnant male dusky pipefish, Syngnathus floridae. I partition this total covariance into a causal component due to the effect of body size on egg number and a spurious component arising from variation in body size and egg number among collection sites.

Three-dimensional imaging is rapidly increasing the scale of biological morphometric datasets, but establishing automated anatomical correspondence remains a major bottleneck. Conventional point-cloud registration methods transfer landmarks efficiently, yet poor initialization or high morphological variation can produce geometrically plausible but biologically implausible deformations. Here, we developed a statistical-shape-model (SSM)-regularized coherent point drift (CPD) estimator for automated morphometric landmark transfer. It replaces the unconstrained non-rigid CPD update with optimization in SSM coefficient space and regularizes coefficients by inverse population covariance, so deformations along low-variance biological modes are penalized more strongly. We compared pose-matched conventional CPD, SSM-CPD, and SSM-CPD with refinement in leave-one-out atlas experiments on laboratory mouse skulls (N=249), primate molars (N=115), and squamate vertebrae (N=254) under aligned, mildly perturbed, and severely perturbed initialization. We evaluated these formulations in terms of landmark accuracy, failure-tail behavior, runtime, and preservation of morphometric structure. Under severe perturbation, we also evaluated an integrated recovery procedure that optimizes the starting template, recovers global pose, and then applies SSM-CPD with refinement. Severe misalignment produced widespread failures in pose-matched conventional CPD, whereas SSM regularization reduced median error from 13 - 21% to approximately 1 - 3% of the target bounding-box diagonal. This improvement was highly consistent for skulls and molars. In the morphologically complex vertebral dataset, however, low median error concealed a substantial minority of catastrophic failures (28% of vertebrae above a 6% error threshold), which integrated recovery reduced to 4%. Across datasets, automated landmarks preserved the major axes and pairwise relationships of morphometric shape space, and local CPD refinement improved covariance preservation. Embedding learned biological variation directly into registration broadens the range of starting conditions and anatomies for which automated landmark transfer remains reliable. Our results also show that failure tails and downstream morphometric fidelity must be assessed alongside median accuracy when automated landmarking methods are evaluated.

Multifunctional anatomical structures must accommodate potentially competing performance demands, leading to functional tradeoffs. The mammal rostrum (snout) is involved in multiple functions, including feeding, respiratory conditioning, olfaction, and, in most bats, echolocation. Therefore, it serves as an ideal system to investigate whether and how functional tradeoffs promote or constrain morphological diversification. While the external morphology of the rostrum has been linked with mechanical demands of feeding in bats, the factors and processes influencing its internal morphology, including the turbinals in the nasal cavity, remain comparatively unexplored. Here we investigate how internal rostral morphology reflects specialization and tradeoffs among respiratory air conditioning, echolocation, and olfaction by quantifying turbinal morphological diversity in a phylogenetically and ecologically diverse sample of bat species. We find that echolocation mode predominantly influences both the ethmoturbinal complex (ETc) and maxilloturbinal (MT) systems, with nasal-emitting bats having reduced and less complex turbinals compared to oral and non-laryngeally echolocating bat species. MT complexity and surface area are not associated with bioclimatic variables representing thermal variability and aridity. However, the ETc exhibits higher complexity in plantivorous bats than faunivorous bats, suggesting higher olfactory reliance. While MT morphological disparity was predominantly explained by echolocation mode, ETc morphological disparity was associated with both echolocation mode and diet, suggesting potentially opposing demands favoring turbinal reduction for sound transmission and elaboration for olfaction. Among nasal emitters, which must accommodate both demands within the nasal cavity, ETc complexity exhibited higher relative disparity compared to MT complexity. These patterns suggest that competing functional demands may promote or maintain morphological diversity, possibly through ecological specialization and/or many-to-one mapping between morphology and performance.

Reliable morphological criteria for sexing birds without the need for invasive procedures are scientifically and ethically valuable. Smith et al (2005) reported that iris colour assigns the genetic sex of Starlings Sturnus vulgaris with 98% accuracy, and bill base colour with 100% accuracy when birds are in breeding condition, but these figures have not been tested independently. We applied their criteria to 31 captive Starlings in breeding condition, using three observers blind to genetic sex, two of them new to Starling sexing. Bill base colour correctly predicted genetic sex for all 29 birds scored, with complete observer agreement. Mean iris score was correct for 27 of 31 (87.1%), fewer than the 94 of 96 reported by Smith et al (2005) for birds in breeding condition (Fisher exact test, P = 0.031), although with so few errors in either study this comparison is imprecise. Observer agreement was high, so these errors reflect iris colouration conflicting with genetic sex rather than inconsistent scoring; bill base colour was correct for all four misassigned birds. Body mass and tarsus length classified 77% and 58% of birds correctly, their published thresholds missing in opposite directions. We conclude that bill base colour should be the primary criterion for sexing Starlings in breeding condition; the accuracy of iris colour should not be assumed to be 98%.

Sperm competition can occur when sperm from two or more males compete for fertilization of a batch of eggs. While it has been extensively studied in internal fertilizers, it remains poorly under-stood in external fertilizers. To investigate how sperm competition affects fertilization under sperm limitation, we conducted controlled experiments on two sea urchin species: Paracentrotus lividus and Dendraster excentricus. Eggs from a single female were exposed to sperm from three individ-ual males separately (non-competitive treatment) or to a pooled sample from three males (competi-tive treatment). We measured fertilization success as proportion of eggs fertilized after two hours and assessed sperm motility as percentage of motile sperm and curvilinear velocity. For P. lividus , we estimated the sperm concentrations where fertilization success was 50% of maximum observed fertilization success (F50), and the fertilization efficiency of the sperm (Fe), i.e. the likelihood that a sperm - egg encounter results in a fertilizing contact, using the fertilization kinetics model of Styan (1998). For D. excentricus, fertilization success was measured at a sperm concentration expected to produce 50% fertilization success. Sperm competition reduced fertilization success and increased F50 compared to non-competitive treatments, suggesting antagonistic effects that may compromise fertilization under sperm-limited conditions in external fertilizers.

Lineages that have undergone little apparent evolutionary change over long geological intervals are often described as "living fossils", with iconic examples including the coelacanth, tuatara, and Ginkgo. However, studies of morphological and molecular data have produced mixed evidence regarding whether living fossils have evolved more slowly than their relatives. We tested for reduced evolutionary rates using a sister-pairs approach, analysing a comprehensive set of morphological and molecular data for 45 living-fossil lineages and their close relatives across the Tree of Life. Living-fossil lineages showed substantially reduced rates of morphological evolution, whereas evidence for reduced molecular rates was weaker. To test whether this pattern could be explained by species poverty, we also analysed other depauperate lineages (species-poor clades contrasted with species-rich sister clades) and found a weak tendency towards reduced molecular rates but without a corresponding slowdown in morphological evolution. Our study reveals that the living-fossil concept captures a quantifiable pattern of prolonged morphological conservatism, but also suggests that living fossils lack other shared biological or life-history characteristics that might unite them as a group.

Social aggregation is widespread in vertebrate larvae, yet how genetic relatedness shapes group cohesion remains poorly understood. In particular, the association between genetic similarity and social aggregation may depend on the scale at which genetic relatedness is evaluated. Here, we quantitatively examined aggregation behaviour in tadpoles from three lineages of Japanese toads, Bufo japonicus, B. formosus, and B. gargarizans miyakonis . We experimentally combined tadpole cohorts differing in relatedness and lineage and quantified aggregation using spatial dispersion analyses. We then examined relationships between aggregation behaviour and genetic variation using mitochondrial DNA (mtDNA), major histocompatibility complex (MHC) class II variation, and microsatellite diversity. Overall degree of aggregation did not differ significantly between sibling and non-sibling cohorts, and Morisitas index provided no evidence that tadpoles formed separate clumps according to relatedness. However, the correlation between genetic variation and aggregation differed across scales of genetic relatedness: among non-sibling cohorts, greater mtDNA pairwise distance was associated with weaker aggregation, whereas among sibling cohorts, greater MHC amino-acid difference was associated with weaker aggregation. These results indicate that the degree of aggregation is related to genetic similarity across different scales of relatedness, rather than being explained by a simple sibling/non-sibling distinction. Our findings suggest that tadpole group cohesion is related to genetic similarity in a scale-dependent manner, highlighting the importance of considering the scale of genetic relatedness when investigating social discrimination in vertebrates, while the sensory and molecular mechanisms underlying these relationships remain to be determined.

Vertebrates cope with rapid temperature fluctuations by plastically adjusting their behaviour and physiology. This phenotypic plasticity is partly mediated by plastic changes in glucocorticoid levels; however, whether individual differences in endocrine plasticity are associated with fitness remains unknown. Here, we examined the relationship between individual variation in glucocorticoid plasticity in response to air temperature changes and reproductive success in a wild great tit population (Parus major). Over nine years, we collected repeated measurements of baseline and stress-induced corticosterone during nestling rearing. We found consistent reaction norm patterns in hormone response to temperature variation -- as shown in a previous study conducted over a shorter period. This provides robust evidence of individual differences in average hormone levels (intercepts) and the strength of the plastic response (slopes), indicating potential for evolution. However, neither intercepts nor slopes were associated with reproductive success (fledgling number or brood mass), suggesting that individual variation in endocrine plasticity was not under natural selection, at least within the environmental conditions experienced. This study provides a first empirical test of the evolutionary implications of endocrine plasticity in the wild and highlights the need to assess its fitness relevance under more extreme conditions, such as those expected under future climate scenarios.

Trade-offs are commonly assumed when the statistical parameters used to define biological traits are negatively correlated. However, parameter correlations can emerge from the geometry of the model, rather than underlying biological signal. We characterize this phenomenon generally, and develop a framework to parse biological signal from statistical artifact in parameter correlations. Using the widely reported trade-off between thermal sensitivity and static upper critical thermal limit as a case study, we show that an extreme correlation between these parameters is expected even in the absence of a real trade-off. However, our method reveals that there is, in fact, a biological correlation underneath this spurious artifact. We confirm this finding, as independent genome-wide association analysis reveals genetic variation among trade-off phenotypes, but none corresponding to net thermal tolerance, suggesting that adaptive capacity to a warming climate may be severely limited. This particular trade-off is therefore biologically supported, illustrating how biological signal can be obscured by estimation artifact, thus evading correlation-based detection. We broaden our analysis, showing that artifact is present across biological systems and model types, although its manifestation may vary. Analytical results and simulations reveal how sampling design, model choice and parameterization govern the artifact, providing practical guidance for experimental planning. Our findings identify both a problem and a solution: correlations among model parameters cannot be interpreted biologically without first accounting for the structure imposed by the model and data, but genuine biological relationships can still be recovered when that structure is explicitly modeled.

Quantitative analysis of biological shape is central to understanding how form encodes function. Despite rapid advances in volumetric imaging and segmentation, analysis and of three-dimensional morphology remains challenging due to complex geometries and topologies found in biology. Existing approaches either rely on handcrafted metrics that lack generalizability or employ data-driven neural models that sacrifice interpretability and robustness. Here, we introduce Minkowski Tensors (MT), a family of integral-geometric shape descriptors that extend classical scalar measures of volume, surface area, and curvature into tensor-valued quantities encoding direction and anisotropy. MT form a provably complete yet compact and interpretable feature set that captures volumetric and curvature-based information in a transformation- and scale-covariant, topology-aware manner. We benchmark MT on diverse segmented 3D biomedical images spanning vascular and adrenal structures from magnetic resonance angiography (MRA) and computed tomography (CT), and dividing cell nuclei imaged by confocal microscopy, demonstrating competitive or superior classification performance relative to classical geometric descriptors and neural baselines while retaining direct biological interpretability. Our results establish Minkowski tensors as a concise yet powerful framework for discriminative analysis of complex 3D biological morphology.

Schooling is a widespread behaviour among pelagic fish that reduces locomotion costs of migration and predation risk. It is characterised by coordinated movements, speed and orientation of group members. Although schooling can vary both between populations and among individuals of the same population, these levels of variation are seldom considered together. Here, we investigated whether schooling tendencies in three-spined sticklebacks (Gasterosteus aculeatus) vary between populations and among individuals within-populations depending on their phenotype. We first compared the schooling behaviour of anadromous (migrants) and recently landlocked (residents) populations and tested whether individual sociability explained within-population variation in schooling. We then studied whether schooling behaviour is modulated when migrants and residents are in mixed groups. Migrant groups displayed better schooling performance than residents with higher alignment. Within migrant groups, less social individuals displayed more leadership, were better aligned, swam closer to others and fatigued less than more social fish. These patterns were absent in residents. When the two populations were mixed, overall schooling performance declined, particularly in migrants, with schools exhibiting reduced compaction and alignment, resembling resident groups. Thus, individuals did not modulate their schooling behaviour to the group social composition and phenotype-dependent schooling patterns were disrupted in migrants when mixed. These findings suggest that migration-related behaviours can disappear quickly post-isolation. They also highlight the importance of individual sociability in self-organisation and shaping collective behaviour, especially in populations that retain migratory behaviour. Considering behavioural variation across multiple hierarchical levels is thus key to understanding complex social systems.

The Yampolsky-Stoltzfus (YS) model provided the first formal demonstration that a bias in the introduction of new mutations can shape the direction of adaptive evolution, even when mutation rates are small relative to selection coefficients. Despite its foundational role, the model has been characterized almost entirely by simulation and closed-form solutions for its central quantity, the probability PA that a mutationally favored but less-fit genotype fixes ahead of a rarer, fitter alternative, have been available only in the limiting regimes of vanishing mutation supply and infinite population size. Here we derive closed-form approximations for PA across the intermediate regime of finite population size and appreciable mutation supply that lies between these two limits. Under strong selection between the competing mutants, we combine an establishment-probability argument with clonal interference theory to obtain an explicit expression for PA that accounts for both the ongoing input of the mutationally favored mutation and the frequency-dependent establishment probability of its competitor. Under weak selection between competing mutants, we derive a complementary expression based on the fixation of a mixed population under Kimura's classical formula. Both expressions agree closely with direct simulation of the underlying Wright-Fisher process, recover the known limiting cases, and remain consistent with one another over a substantial intermediate range of parameters. These results are expected to provide a basis for assessing how the strength of mutation bias affects adaptation in empirical data.

Whether sexual selection facilitates or hinders adaptation to novel environments is a question of fundamental importance due to current rapid environmental change. Using phylogenetic comparative analysis in 42 Drosophila species, we found that sperm length - a sexually selected ornament - positively associates with the ability of males to survive high temperatures. However, the ability to remain fertile does not increase with sperm length to the same extent, meaning that longer sperm species experience a larger gap between temperatures that kill and temperatures that sterilize males. The findings are consistent with the idea that strong sexual selection on condition-dependent exaggerated traits may facilitate somatic resilience to environmental stressors, but that susceptibility to heat-induced infertility may be a cost of extreme sperm ornamentation. Overall, the data suggests multifaceted links between postcopulatory sexual selection and climate change.

Fluctuating asymmetry (FA) is widely used as a proxy for developmental instability, although reduced rather than increased FA, has occasionally been reported under particularly challenging conditions, possibly because environmental filtering removes less developmentally stable individuals. Here, we investigated temporal variation in wing shape asymmetry in males of Drosophila sturtevanti collected in 2022, 2024, and 2026 at the same sampling site, an urban Atlantic Forest fragment. Sampling in 2022 and 2026 were completed during single field expeditions in early April, whereas in 2024 repeated collections were required until mid-May because of markedly low capture success. We therefore examined whether this difference was associated with unusual environmental conditions and whether FA was higher in 2024, as expected under greater developmental perturbation. Daily temperature and relative humidity were examined from early March onward, encompassing approximately one month before adult emergence. The 2024 sampling period was warmer and drier than the corresponding periods in 2022 and 2026 and coincided with the final phase of the strong 2023-2024 El Ni&ntildeo event. Contrary to our prediction, FA was significantly lower in 2024. The magnitude of directional asymmetry remained similar among years, although the spatial pattern of the DA vector differed between 2024 and 2026. The coincidence of reduced capture success and reduced FA is consistent with the hypothesis that adverse conditions may impose demographic filtering, such that individuals expressing greater developmental instability are less likely to contribute to the sampled adult population.

Haldane's principle relates equilibrium mutation load to deleterious mutational input rather than to selection strength. We study this balance in a homeostatically regulated, damage-structured population where mutation occurs during reproduction, repair restores individuals toward lower-damage states, and nonlinear demographic feedback maintains constant population size. An exact demographic identity shows that load at every time equals excess reproductive effort weighted by mean relative fecundity. Without repair, the equilibrium branch with positive pristine-class frequency is exactly solvable, and along it the load is exactly the mutation probability times the realized per-capita reproductive turnover rate; when the terminal cap is removed, this is precisely the realized deleterious input, for every mutation probability below one. For rare mutation it reduces to the mutation probability times the pristine turnover rate, with the local fitness contrast canceling at leading order. With a finite terminal class the branch instead reaches a closure-dependent boundary collision. Repair modifies the rare-mutation balance by partitioning first-order removal from the first damaged class between selection and restoration, multiplying the leading load by the selective share of that removal. An exact binary solution extends these results to finite mutation pressure: the unrepaired interior equilibrium collides nonhyperbolically with the fully damaged boundary and converges algebraically at the threshold, whereas any positive repair rate restores a unique interior equilibrium for positive mutation; at the no-repair threshold, its displacement from the boundary scales as the square root of the repair rate as repair vanishes. These results separate the familiar competing-transition structure of mutation--selection balance from the specifically homeostatic effects of turnover-dependent mutation input and nonlinear demographic feedback.

The relative roles of genetic drift, inbreeding, and natural selection in island evolution have been fundamental themes in evolutionary biology for nearly a century. However, empirical studies have yet to reach a consensus, and research integrating both genotypes and phenotypes in primates remains limited. The present study aims to estimate the demographic history of an island primate population, the Yakushima macaque (Macaca fuscata yakui), and evaluate the genomic and morphological consequences of its isolation. To this end, we conducted population genetic analyses based on whole-genome sequencing and geometric morphometric analyses of cranial shape. Our results indicate that M. f. yakui has been isolated with an effective population size of several thousand individuals for over 100,000 years. Although we detected signatures of purifying selection, deleterious alleles have not been sufficiently purged. The degree of fluctuating asymmetry in males was slightly, yet significantly, higher in M. f. yakui than in mainland populations, whereas this trend was not observed in females. Marginal evidence of directional selection was found for cranial size, but not for shape. Overall, our findings suggest that due to long-term isolation, genetic drift and inbreeding have exerted a substantial influence on shaping genotypic and phenotypic variation in this island population.

The toxicity of chemical defences is well studied, but the potential contribution of compound structure to predator deterrence remains largely unexplored. Whether predation acts more strongly on toxicity or unpalatability remains largely untested, partly because few systems allow toxin structure to vary independently of quantity. Heliconius sara larvae provide such a system: those reared on Passiflora auriculata sequester cyclopentenyl cyanogenic glucosides (CGs), while those reared on P. biflora biosynthesise comparable quantities of aliphatic CGs. Using two invertebrate predators, Camponotus floridanus ants and Hierodula membranacea mantids, we tested whether this structural difference affects palatability independent of toxicity. Mantids rejected larvae with cyclopentenyl CGs more often than larvae with aliphatic CGs, despite no detectable difference in total CG content. This pattern was mirrored in extract-based assays with ants, independently of cyanide release: extracts with cyclopentenyl CGs remained deterrent, while extracts with aliphatic CGs did not differ in deterrence from water. Live larvae, by contrast, elicited similar responses from ants regardless of CG structure. These results show that variation in toxin structure can strongly affect palatability, with some compounds conferring greater protection than others. This demonstrates the importance of chemical structural diversity in the evolution of chemical defences.

Regular environmental cycles such as day and night or the lunar cycle are ubiquitous and drive many biological processes. Many organisms have evolved endogenous time-keeping mechanisms to anticipate these cycles to accurately time their behavior or synchronize physiological processes. The evolution of such synchronization to environmental cycles and their variability is not very well understood. As an example, we use the marine insect Clunio (Diptera, Chironomidae), which synchronize adult emergence and reproduction with the lunar cycle, and show different emergence patterns across populations. We use a model constructed with replicator-mutator dynamics to provide a novel outlook at the evolution of these emergence patterns. In particular, we investigate the effect of two traits of individual phenotypic variability of emergence, the spread and shape, on population-wide emergence patterns. We find that the semilunar and lunar emergence patterns are alternatively stable and occur when the phenotypic distribution has a narrow and is roughly flat. Arrhythmic emergence occurs when the phenotypic distribution is wide and nearly flat. The semi-lunar pattern can only be observed when the parameter of rhythmicity is changed. With our model, we introduce the concepts of frequency-dependent selection and individual phenotypic variability into the theory of chronobiology, which gives us insights into the evolution of rhythmicity in these organisms.

Artificial light at night (ALAN) disrupts the natural light-dark cycle, which is the most important cue for synchronizing circadian clocks. If populations most exposed to ALAN adapt to get less disturbed by it, we expect forest and city populations to differ genetically. We tested this prediction in a common garden experiment using a songbird, the great tit (Parus major), originating from forest and city populations. We simulated ALAN exposure during the breeding season by keeping the birds in pairs under ALAN or dark nights for three months and then repeated the experiment in the following year with the same pairs under the opposite treatment. The advancement of daily onset under ALAN disappeared over time for city birds, but not for their forest counterparts. For these same birds, we measured their onset of daily activity under constant dim light and shift in activity in response to light pulses and showed that the city/forest difference in behavioural response was not reflected in any clock property measured. This indicates that city and forest birds differ genetically in the downstream behavioural response to ALAN, and not in the circadian clock. Thus, urbanization is unlikely to select for divergence in circadian clock properties in songbirds.

Abstract 1. Biomes are continental-scale vegetation units widely used in ecological and evolutionary research, often together with large-scale species-distribution data. Many conceptually distinct biome schemes exist, differing in the underlying definition, delineation methodology and spatial resolution, yet most studies using biomes default to a few readily available schemes rather than choosing the one best suited to the research question. 2. We present biomes, an R package distributing spatially explicit raster layers of 31 published global biome schemes in a harmonised format at a 10 x 10 km resolution, together with metadata and a concise description. To facilitate their use, biomes provides a user-friendly classification workflow to classify occurrence records into specific biomes and a data-driven ranking algorithm to help researchers to choose the most suitable biome scheme for their data and research question in a transparent, reproducible way. 3. The biomes workflow can be applied to any dataset of georeferenced terrestrial occurrences. It (1) accepts either user-provided occurrence records or records downloaded by the package, (2) ranks the schemes according to their fit to the occurrence data, (3) assigns the records to specific biomes, and (4) summarises the resulting taxon-biome patterns. We demonstrate this workflow using 17,030 occurrence records of 185 species of Bombacoideae (Malvaceae). The analysis identified the vegetation-based biome scheme of Ramankutty and Foley (1999) as the best fit and recovered the forest and savanna biome signal known in the group from the literature, with tropical evergreen woodland being the biome with the largest share of records (46.1%) and highest number of species (162 of 185 species; 87.6%). 4. biomes provides user-friendly access to technically standardised and spatially explicit layers of 31 biome schemes, and thereby enables the transparent, data-driven choice of biome schemes tailored to a specific research data and questions, improving the reproducibility and the specificity of biome use across ecological and evolutionary research.