Proceedings of the Royal Society B: Biological Sciences

Helminths are widespread parasites that can modulate host immunity, potentially increasing susceptibility to viral infections. However, evidence for these effects varies across systems and environments, and links between laboratory and wild populations remain unclear. We developed a tractable system using wood mice, Heligmosomoides spp. nematodes, and wood mouse herpes virus (WMHV) to bridge this gap. Combining laboratory and field experiments with population modelling, we examined how helminth infection, anthelmintic treatment and diet affect viral dynamics. Across lab and field data, helminth infection consistently increased WMHV risk, with stronger effects at higher worm burdens. Field results showed that anthelmintic treatment reduced viral infection, and laboratory experiments showed that improved nutrition mitigates helminth-induced increases in viral susceptibility. Our population-level modelling suggested that helminth burden-dependent facilitation can generate nonlinear effects on viral spread, dependent on helminth virulence. Our findings highlight the potential importance of helminths as facilitators of viral infections, and suggest that anthelmintic treatment may provide indirect benefits for viral control. We also show the value of integrating lab and field approaches on the same (or closely related) species, in particular the potential offered by the wood mouse - Heligmosomoides - WMHV system, to understand the drivers and consequences of host-helminth-viral interactions.

Within species, individuals of the same age can differ in size. Previously, parental genetics, nutrition, space, and social interactions have been suggested to explain different growth rates. However, direct effects of larger individuals on the physiology and growth of smaller individuals are poorly understood. In this study, we investigated how larger individuals of the marine worm Platynereis dumerilii can impact the growth of smaller conspecifics. Comparing growth distributions in communally and individually reared worms, we show that larger worms suppress the growth of smaller ones. Furthermore, we were able to demonstrate that this suppression is chemically mediated. The chemical cue does not originate from faeces but is water soluble, stable for several days and smaller than 3 kDa. Our findings highlight the importance of non-reproduction related chemical signalling, showing evidence that dominant individuals can chemically suppress the growth of their conspecifics. This study provides new insights into how hierarchy can be established and maintained in a population and is particularly relevant for the growing community studying this model species.

How morphology forms during development and changes during evolution remain major questions in biology. In vertebrates, teeth have long served as model systems to address these questions. In threespine stickleback fish (Gasterosteus aculeatus), repeated and convergent increases in pharyngeal tooth number in derived freshwater sticklebacks occur, suggesting increased tooth number is adaptive in freshwater environments, likely due to a diet of larger prey in freshwater. Whether changes in oral tooth patterning also occur in freshwater sticklebacks was unknown. Here we describe oral tooth number and patterning in a dense developmental time course of lab-reared ancestral marine and derived freshwater fish. We address three major questions. First, is the spatial sequence of early oral tooth formation invariant as we previously described for the pharyngeal dentition? Second, is oral tooth patterning in the upper and lower jaw sexually dimorphic, and if so, when during development does this dimorphism arise? Third, have freshwater fish evolved increases in oral tooth number? We find that (1) unlike the pharyngeal dentition, the oral jaw early spatial sequence is variable, especially in the lower jaw (2) sexual dimorphism in both oral jaws arises at the late juvenile stage with males having more teeth and (3) freshwater fish have evolved more oral teeth similar to the evolved tooth gain in the pharyngeal jaw. Together our morphological descriptions advance the stickleback oral jaw as a model system to study how morphology forms during development and evolves in nature.

Natural populations are often nonlinear and exhibit substantial variability. A central question is how stochasticity interacts with density-dependent regulation to shape population stability. We address this using four long-term time series of Trinidadian guppies and find that their dynamics are well described by a stochastic logistic model with multiplicative environmental noise. The model predicts that stochasticity does not merely add fluctuations around deterministic carrying capacity, but alters the equilibrium structure. Using stochastic bifurcation theory, we show that increasing noise shifts the most-probable population size below the deterministic equilibrium and can push populations closer to a noise-induced bifurcation, even when mean growth rates remain positive. The effects of stochasticity across populations align with known ecological differences among streams, particularly the effects of light level and seasonality. The analysis also identifies populations most sensitive to perturbations, which are not detected by standard early warning indicators. Temporal and spectral analyses further show that intrinsic growth rate governs local recovery, while seasonal variation interacts with density-dependence to shape longer-term population fluctuations. Together, our results show that stochasticity can alter resilience and vulnerability by reshaping ecological stability landscapes.

The presence of multiple discrete color patterns within a species has captivated evolutionary biologists for more than a century, especially when such polymorphism is confined to one sex. The brown anole Anolis sagrei exhibits a female-limited polymorphism in dorsal patterning, which is controlled by allelic variation at the autosomal gene CCDC170. Here, we present and test a threshold model that can explain why the polymorphism is female-limited. We hypothesize that allelic variation at the CCDC170 locus affects only female color pattern because this gene is co-expressed with its neighboring gene ESR1, highly expressed in female, but not male, embryos. By manipulating embryonic estradiol levels, we show that genetic males can be induced to express the polymorphism according to allelic variation at the CCDC170 locus, which is naturally masked by low expression levels of this gene. Inversely, treating genetic females with fadrozole, which depletes estradiol, leads to monomorphic patterns irrespective of genotype, as for natural males. Using RT-qPCR, we demonstrate that these effects are accompanied by a direct influence of estradiol and fadrozole on gene expression levels of CCDC170 and ESR1, thereby validating the threshold model. Our results suggest that the CCDC170-ESR1-locus is part of a mechanistic link between the morph-determining and the sex differentiation systems and provide a causal explanation for the developmental origin of a sex-limited color polymorphism.

When only some hosts are protected from disease vectors, disease spread may be inhibited through a net reduction in vector visits or amplified as vectors redirect their attention to unprotected hosts. Two factors that determine which outcome prevails are host microbiota that alter vector host-seeking behavior and natural enemies that redistribute or suppress vector populations. Because both shape the frequency and distribution of vector visits, they are essential for understanding how individual-level protection scales to population-level disease dynamics. Yet, how these processes interact across scales remains poorly understood. Pea aphids are major virus vectors in pea crops and are commonly managed using parasitoid wasps. Recent evidence suggests that epiphytic bacteria in the genus Pseudomonas can also repel or kill pea aphids, yet whether Pseudomonas complements or undermines parasitoid-based vector control remains unknown. We used a mathematical model to show when and why Pseudomonas complements versus undermines biocontrol of aphid-vectored virus outbreaks. The effect of Pseudomonas on virus outbreaks depends most strongly on how successful parasitoids are at tracking aphid densities: When parasitoids effectively track aphids, Pseudomonas inhibits virus outbreaks by reducing aphid densities. With poor parasitoid tracking of aphids, Pseudomonas-induced aphid mortality generates spatial variability in aphid densities that slows parasitoid population growth. The net result is amplified crowding in plants not protected by Pseudomonas, increasing winged aphid production and accelerating viral spread. Counterintuitively, the more effective Pseudomonas is at killing aphids, the more strongly it generates spatial variability and promotes virus spread. The only other factor that can change the direction of Pseudomonas effects on virus outbreaks is whether the virus starts on a Pseudomonas-protected plant, which can cause Pseudomonas to inhibit virus outbreaks when it would otherwise promote them. Our results show how community and spatial context dictate whether microbiota protective to individual hosts will accelerate viral outbreaks.

O_LITerrestrial insects are vulnerable to desiccation due to their small body size. Because insects lose most water through cuticular evaporation, cuticular traits strongly influence desiccation tolerance. Individuals with greater cuticular melanisation, i.e., darker ones, are hypothesised to tolerate desiccation better than less melanised ones. C_LIO_LIIn many butterflies, pupal colour is plastic - individuals pupating on leaves tend to be greener, while those that pupate away from leaves (off-leaf), such as on tree bark or defoliated twigs, tend to be browner. Brown pupae are hypothesised to have more cuticular melanin and are expected to experience higher desiccation stress than leaf-borne green pupae. Thus, plasticity in pupal melanisation may be an adaptation against desiccation. We tested this in the butterfly Eurema blanda. C_LIO_LIWe demonstrate that individuals pupating on on-leaf substrates are greener than those pupating on off-leaf substrates, and that desiccation stress is higher in the off-leaf substrates, a microenvironment typical of brown pupae, than in typical green pupae. Using Raman spectroscopy, we show that brown, but not green, pupal cuticles contain melanin. C_LIO_LIFollowing this, we obtained greener and browner pupae by manipulating substrate colour. When subjected to desiccation stress, browner pupae survived better than greener ones. There was no correlation between pupal colour and survival in the absence of desiccation stress. Thus, melanisation appears to confer a survival advantage to pupae by increasing desiccation tolerance. C_LIO_LISurvival under desiccating conditions was inversely related to water loss. Interestingly, melanisation did not correlate with water loss, suggesting that melanisation helps tolerate desiccation through physiological mechanisms not directly related to water loss reduction. C_LIO_LIOur findings reveal an additional, crucial, adaptive value of pupal colour plasticity, a trait that has been studied primarily from an anti-predatory perspective. C_LI

Cuticular hydrocarbons (CHCs) are essential for insect waterproofing, yet how they change seasonally in social insects remains poorly understood. Due to its distinct seasonal worker phenotypes (summer and winter bees) and diverse subspecies, the western honey bee (Apis mellifera) is an ideal model to study seasonal CHC plasticity across populations with distinct local adaptations. We performed a common garden experiment to investigate the seasonal plasticity in CHC profiles across five European subspecies (A. m. carnica, A. m. iberiensis, A. m. ligustica, A. m. macedonica, A. m. ruttneri). We compared the CHC composition of workers performing tasks inside ("in-hive") or outside ("out-hive") the colony during summer and winter. Notably, out-hive workers consistently exhibited more waterproofing CHC profiles compared to in-hive workers, regardless of season or subspecies. The persistence of this stereotypical task-related differentiation in long-lived winter bees, which largely lack an age-based division of labor, indicates a robust, age-independent regulatory mechanism linked to the environment faced by the workers rather than a simple response to seasonal desiccation pressure. Moreover, we demonstrate CHC seasonal plasticity for the first time in honey bees. However, these seasonal shifts in hydrocarbon classes and chain length were not uniform; they varied across subspecies and critically depended on the task the workers performed.

The Biodiversity-Ecosystem Functioning (BEF) literature has shown species diversity to be essential for ecosystem functioning and services. Yet although acquiring information through interspecific networks can impact ecosystem functioning, it is unclear how it is modulated by species diversity. Eliciting vocal responses using predator models across a latitudinal gradient, we first show that the species diversity of birds increases public information about predation both in the low-cost system of mobbing and in the higher-cost system of alarm calls. A similar result was also found across a fragment area gradient for mobbing; this system was then used to test how species diversity affects interspecific information flow in mobbing communities. We set up two BEF playback experiments, manipulating the species richness level of the playback sound files by varying the number of species producing mobbing calls (one, two, four, eight species). In an experiment in which the call rate across treatments was held constant, and only heterospecific responses were counted, increasing species richness of the sound files increased the number of species and individuals responding, the number of calls produced and their frequency range, and decreased latency to call. An experiment in which call rate increased with the addition of species in each treatment showed a similar, but stronger pattern. There was little evidence that the signals of one particular species changed responses. This supports the hypothesis that the species diversity of a community is a key component influencing the quantity and quality of information flow inside it.

Mate preferences are often influenced by the magnitude of sexual signals, which are presumed to indicate underlying aspects of signaller quality. Although the perception of these signals depends on sensory processes, the role of perceptual adaptations and constraints in mate assessment is frequently overlooked. Many sensory systems follow Webers law of proportional processing, where discrimination between signals is based upon their proportional, or relative, difference rather than their absolute difference. Because preference strength varies with relative trait magnitude, Webers law could strongly influence sexual selection, changing the coevolution of traits and preferences. Here, we explore the consequences of Webers law for sexual selection using individual-based models, applying Scalar Utility Theory to mate choice. We investigate the coevolution of male ornaments and female preferences under both Fisherian and good genes scenarios, as well as scrutinizing the sexual selection of multiple ornaments and preferences. Including Webers law in these models either reduced ornament exaggeration, or promoted exaggeration and diversification of ornaments and preferences, depending on the costs of choice and how rapidly female survival decreases when preferences evolve away from the naturally selected optimum. These results highlight the importance of perception and cognitive processing in shaping sexual selection and its evolutionary impacts.

Parasites can alter host populations in fundamentally different ways depending on whether exposure results in infection. Yet, most epidemiological and evolutionary inference focuses on established infections, leaving the fitness consequences of parasite exposure comparatively understudied. This gap is consequential because hosts are frequently exposed to diverse parasite genotypes, and these encounters can impose substantial fitness costs even when infection does not occur. Theory predicts that hosts may mitigate these costs when interacting with commonly encountered parasite genotypes, such that exposure to sympatric parasites incurs lower fitness consequences than exposure to novel, allopatric parasites. Here, we examine the fitness consequences of exposure and infection in the first intermediate host of the trophically transmitted tapeworm Schistocephalus solidus, a cyclopoid copepod that serves as the first host in a three-host life cycle. Using sympatric (Vancouver Island, Canada) and allopatric (Norway) host-parasite combinations, we found a striking reciprocal asymmetry. Sympatric parasites were significantly more infective, yet exposure to sympatric parasites imposed weaker fitness costs when infection did not establish. In contrast, allopatric parasites were less infective, but exposed females produced fewer eggs and had lower hatching success than both controls and females exposed to sympatric parasites, indicating substantial genotype-dependent costs of exposure. Moreover, we found that infection was highly virulent across all genotypes: a single parasite caused near-complete reproductive suppression and reduced host survival across all host-parasite pairings, confirming S. solidus as a castrating parasite in copepods. Together, these results demonstrate that exposure, not just infection, acts as a critical ecological filter with potentially large and underappreciated consequences for host population dynamics and parasite transmission.

In modular organisms, where growth and fragmentation blur the boundaries between individuals, the interplay between asexual and sexual reproduction creates complex fitness trade-offs. Life-history theory predicts that resources allocated to one fitness component necessarily reduce investment in others, yet detecting these trade-offs in wild populations of clonal organisms remains challenging. Phenotypic plasticity can enhance survival, yet its influence on reproductive capacity and life history trade-offs remains poorly understood. Using a fully crossed reciprocal transplant design, we tracked 263 colonies of the branching coral Acropora cervicornis across nine reef sites over 42 months, investigating relationships between fragmentation, morphological plasticity, and the capacity for sexual reproduction. Breakage patterns reflected both environmental and genetic factors. Primary branch breaks created a "double negative" effect--simultaneously more than doubling mortality risk and delaying attainment of a validated reproductive size class by [~]40%. Conversely, higher morphological plasticity in surface area-to-volume ratio accelerated sexual maturation up to 6-fold, counteracting the negative effects of fragmentation. In parallel, a simple demographic model parameterized with published fecundity data estimated that primary breakage reduces expected cumulative reproductive output by [~]58%, a result robust across a wide range of parameter assumptions. These results demonstrate a fundamental reproductive trade-off in which asexual reproduction through fragmentation undermines sexual reproductive potential by reducing colony size. Moreover, our findings reveal that fragmentation susceptibility is broadly heritable and subject to selection, and identify a compensatory mechanism through which plasticity enhances fitness beyond immediate survival.

A hosts diet can alter the course of parasite infection. This is especially true of trophic parasites, which a host acquires through feeding. While a large body of work attests to the role of diet in the spread of disease within-hosts, diet can also impact host density and encounter rate with parasites, both of which are expected to modify disease dynamics. When parasites are acquired through feeding, epidemics may be larger and more severe on high-quality diets if these diets support a higher density of hosts that feed more and thus ingest more parasites. Alternately, epidemics may be more severe on low-quality diets if malnourishment decreases hosts ability to resist disease. To differentiate these hypothesized effects of diet on disease, we quantified individual infections and epidemic dynamics for the natural microsporidian parasite Nematocida ironsii infecting its nematode host Caenorhabditis elegans. We measured feeding rate, parasite transmission, and host fitness across three bacterial diets that vary in quality and elicit distinct feeding behaviors in C. elegans. We found that low-quality diets reduced feeding rate, which corresponded to reduced acquisition of parasite spores. However, these diet-mediated differences in parasite acquisition did not directly map onto fitness consequences: hosts eating the poor-quality diet had similar reductions in fitness to those on higher quality diets. During epidemics, a combination of increased parasite acquisition and higher population growth rates resulted in higher parasite abundance for hosts on high-quality diets. Our work underscores the importance of considering both individual- and population-level impacts acting in concert to determine how diet affects the spread of infectious disease.

The basic reproduction number R0 is central to malaria epidemiology, yet it is typically treated as a static quantity derived under memoryless assumptions for mosquito demography. In natural systems, however, mosquito populations are shaped by delayed processes such as larval development and density-dependent feedback, introducing biological memory into vector dynamics. We develop a minimal delay-based framework that incorporates this memory into the Ross-Macdonald model by describing adult mosquito abundance with a retarded differential equation. This formulation induces a time-dependent transmission potential R0(t). Using complex analysis and the argument principle, we derive an explicit stability threshold [Formula], which separates stable from oscillatory transmission regimes. Near this threshold, delayed feedback produces slow relaxation times and sustained transient oscillations, implying that transmission potential may vary intrinsically even in the absence of external forcing. To account for ecological variability, we extend this deterministic condition into a probabilistic framework and define the stability probability as [Formula]. Numerical simulations and global sensitivity analysis show that recruitment and developmental delays are the primary drivers of instability, while adult mortality has a weaker stabilizing effect. These results indicate that malaria interventions may influence not only the magnitude of malaria transmission but also its dynamical stability. By linking delay dynamics, transmission theory, and uncertainty quantification, this framework provides a basis for stability-aware modeling and interpretation of malaria transmission under ecological variability. Author summaryMalaria transmission is often summarized by a single number, R0, treated as a fixed indicator of whether transmission will increase or decline. This assumes mosquito populations respond instantly to environmental conditions. In reality, mosquitoes develop through stages where larval conditions, such as crowding, nutrition, or temperature, affect adult populations only after a delay. This creates biological memory: todays mosquitoes reflect past environments. We show that this memory can fundamentally reshape transmission dynamics. When developmental delays are included, transmission potential is no longer constant but can fluctuate over time, even in stable environments. These fluctuations can persist or amplify depending on the balance between mosquito growth, mortality, and delay. As a result, variability in mosquito abundance or malaria transmission may arise from intrinsic dynamics rather than external drivers alone. Under ecological variability, stability becomes probabilistic, allowing estimation of how likely transmission is to remain stable. Interventions that reduce larval productivity or increase adult mortality may therefore both lower transmission and make it more predictable, improving interpretation and control strategies.

In most species, unmated individuals run the risk of dying with zero fitness. This strong selection on virgin females to mate may also explain why females subsequently remate, despite fitness costs; all that is required is a genetic correlation between virgin and non-virgin mating propensity. Despite being the null model for the evolution and maintenance of polyandry, this hypothesis has received no empirical test. We performed separate quantitative genetic and artificial selection experiments to test the presence of this cross-context genetic correlation in the cow-pea weevil, Callosobruchus maculatus. A quantitative genetic experiment did not find evidence of the hypothesised genetic correlation. However, after 13 generations of artificial selection on virgin mating latency, we found strong evidence for evolutionary divergence in remating latency. Females from lines selected for longer virgin mating latency took approximately twice as long to remate and, were less polyandrous if their virgin mating latency was longer. There was no evidence that females could mate indiscriminately and then trade-up, rather, trading up could only occur if virgin discrimination was present. Selection against virgin death will thus constrain both the evolution of non-virgin discrimination and trading up, increasing rates of polyandry. These findings reveal a genetic correlation between virgin and non-virgin latency to mate suggesting that polyandry may be maintained because of the need to breed.

The evolution of alternative male reproductive strategies represents an intriguing evolutionary phenomenon. Divergent strategies are persistently at risk of local extinction or invasion, depending on the suites of traits expressed within and between morphs; hence, understanding the correlational selection that aligns reproductive strategies with behaviour, morphology and physiology is key to understanding the origin and maintenance of genetic polymorphisms. In the polychromatic painted dragon, Ctenophorus pictus, yellow, orange and red morphs are well characterised, but the blue morph has been historically absent from studied populations. Here we document the local distribution, morphology and male-contest interactions in a population where blue males are relatively common. We find that blue males express head colouration after a reaching a threshold body size, and that small blue males can reside in close proximity to other males; patterns consistent with a novel size-dependent conditional tactic within the suite of genetic strategies seen in this species. Condition-dependent, positively allometric throat bibs were non-randomly distributed among male morphs, implicating variation in correlational selection and the genetic architecture of the polymorphism. We were unable to definitively assign a morph that was superior in male competition but found that within morphs, male size was the determinant of competitive success, whilst between morphs it was not. Furthermore, contests between morphs were resolved with less aggression than contests within morphs, supporting the idea that badges resolve conflict, and that the invasion of new colour morphs may be facilitated by negative frequency dependent benefits to novel colour variants. These findings highlight the divergent phenotypic, genetic and selective environments that lead to the diversity of colour morphs.

Cooperative interactions often unfold in environments that are shaped by collective behavior, yet how knowledge about such changing environments feeds back into evolutionary dynamics remains poorly understood. While network reciprocity explains how spatial structure enables clusters of cooperators to emerge and grow under certain conditions, it typically ignores how individuals respond to environmental change. Here, we integrate stochastic environmental feedback with network reciprocity to examine how knowledge about environmental state shapes the evolution of cooperation in structured populations. We compare regimes in which individuals either condition their behavior on the current state or remain unaware of it. Under weak selection, we derive a simple condition showing that cooperation is favored when the benefit-to-cost ratio exceeds a modified classic reciprocity threshold accounting for the effect of environmental transitions and state knowledge. Environmental shifts can either promote or hinder cooperation depending on accessibility and fidelity of state knowledge. Counterintuitively, greater knowledge does not universally enhance cooperation: for certain transition rules, state awareness raises the critical threshold for cooperation, a phenomenon we term a "knowledge curse". Our results reveal that, in an ever-changing environment, cooperation in structured populations emerges from a subtle interplay between environmental feedback and information availability.

Behaviour is a key yet often overlooked component of animal camouflage and how it evolves alongside colour and morphology remains poorly understood. The repeated evolution of stick-like postures in spiders offers a useful framework for investigating the importance of behaviour for concealment, since matching the environment should rely on specific body forms and postures, not just colouration. We hypothesised that when spiders behaviourally align their body with the background orientation it should influence the shape, posture and colouration that best enhances camouflage. To test this, we used a genetic algorithm and human observers to evolve digital spiders to be harder to find. We evaluated how selection under three behavioural orientation treatments (aligned, random, and evolvable orientation) influenced spider capture time, background match (lightness and colour), posture, and body (cephalothorax and abdomen) dimensions. We found that spiders that behaviourally aligned with the background took substantially longer to find through evolving a better background match, and a more elongated posture and body shape than randomly orientated spiders. Our spiders mirrored the shape and posture adopted by numerous clades, illustrating how behavioural camouflage represents a key concealment strategy in structurally complex habitats, part of an interacting suite of traits that encompass successful concealment.

RDL (Resistance to dieldrin) is a GABA-gated chloride channel that was first described as target of the insecticide dieldrin. Despite dieldrin being discontinued for decades because of its environmental per-sistence and health concerns, Rdl resistance mutations (A296S, A296G) continue at high frequencies in natural populations of the malaria mosquito Anopheles gambiae complex across Africa, suggesting a selective advantage. We have recently shown that RDL acts as a critical modulator of mosquito auditory sensitivity. Because acoustic recognition is essential for mate acquisition in An. gambiae, we hypothesized that these mutations confer a pleiotropic effect on mating success in the field, mediated through altered acoustic sensitivity, with potential consequences for sexual selection. We first provide laboratory evidence that resistance mutations enhance auditory behaviours of An. gambiae and show that the effect of environmental noise on mating success depends on the male Rdl genotype. We then conduct field collections in the city of Bangui (Central African Republic) and surrounding rural areas, revealing the presence of Rdl resistant alleles and their association with the urban environment, and within the city, with the noisiest locations. We also show decreased mating success of susceptible females with increasing noise levels, suggesting detrimental effects. Together, our findings support that Rdl resistance mutations enhance auditory function and mating success in acoustically challenging environments. We propose that this auditory advantage may contribute, together with other selective pressures such as cross-selection by other insecticides, to the persistence of these alleles in nature and may facilitate urban colonization by malaria vectors. Our study reveals, for the first time, an unintended evolutionary consequence of insecticide use, where a resistance mutation has been co-opted to enhance sensory performance and ecological adaptation, with significant implications for vector management strategies.

Conspecific sperm precedence via cryptic female choice is a post-ejaculatory selection process that reduces hybridization, and can be pronounced in sympatric species. In their native Europe, Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) exert conspecific sperm precedence under heterospecific sperm competition, which is at least partially enabled by female reproductive fluid. We examined post-ejaculatory selection of both species in Newfoundland, Canada, where Atlantic salmon evolved in absence of brown trout, but now experience hybridization threats due to anthropogenic introductions. Using split-ejaculate and split-clutch in-vitro fertilizations we evaluated whether allopatric evolution has relaxed this selection in Atlantic salmon, and found that they had no ability to bias paternity towards conspecific males, whereas naturalized brown trout retained a strong ability to do so. Female reproductive fluid influenced this, as when fluid associated with a species eggs was swapped, hybridization increased. In the artificial situation of no female reproductive fluid during sperm competition, paternity changed dramatically, but sperm swimming performance did not predict it. Our findings contribute to understanding the evolution of cryptic female choice and how the mechanisms of reproductive isolation can be reinforced through sympatry, while also highlighting a new potential conservation concern for North American Atlantic salmon.