Ecology and Evolution

Body size is a fundamental determinant of ecological performance, and variation in length- weight relationships can provide insight into how populations respond to local environmental conditions. Annual killifishes inhabit highly seasonal wetlands characterized by substantial environmental heterogeneity, yet population-level variation in growth allometry remains poorly understood. We investigated growth allometry in 18 populations of two annual killifish species, Nothobranchius kirki and N. wattersi, distributed across ephemeral habitats in Malawi. Length- weight relationships were analyzed separately for each species using linear mixed-effects models, with locality incorporated as a random effect. Environmental variation among localities was summarized using principal component analysis of water quality variables, and population- specific allometric coefficients were subsequently related to environmental gradients. Significant population-level variation in growth trajectories was detected in both species, indicating divergence in allometric scaling among localities. Divergence was more pronounced in N. wattersi, which exhibited a broader range of population-specific allometric coefficients than N. kirki. Populations also differed significantly in relative body condition after accounting for body length and sex. Environmental gradients explained a significant proportion of variation in allometric slopes in N. kirki, whereas no significant relationship was detected in N. wattersi. Thus, the species exhibiting weaker allometric divergence showed stronger environmental associations, while the species exhibiting greater divergence showed little correspondence with measured environmental variables. These results demonstrate substantial spatial heterogeneity in growth allometry among populations of annual killifishes inhabiting seasonal wetlands. Furthermore, the contrasting environmental associations observed between species suggest that population divergence in growth trajectories may arise through different ecological and evolutionary processes, even among closely related taxa occupying similar habitats.

A comprehensive understanding of life history is vital to successful species conservation and management. When different life history stages are accompanied by considerable morphological or cryptic variation, such as the egg and larval phases exhibited by most fishes, genomic tools are essential for identifying species so that early-life ecology questions can be studied. Genotyping-in-thousands by sequencing (GT-seq) has recently emerged as a targeted and efficient approach for species identification. We leveraged existing genomic and transcriptomic data to develop a GT-seq panel capable of differentiating the members of the Coregonus artedi complex, a radiation of salmonids in the Laurentian Great Lakes whose members are indistinguishable with mitochondrial DNA barcoding loci and are the focus of bi-national conservation initiatives. Our panel of 494 loci was able to assign fishes in the C. artedi complex to species and lake. We examined cross-amplification in other coregonines with overlapping distributions and found that congeneric Lake Whitefish (C. clupeaformis) cross-amplified at 94% of loci and confamilial Round and Pygmy Whitefish (Prosopium spp.) cross-amplified at 42% and 38% of loci, respectively. We adapted bioinformatic probes to account for Prosopium-specific variants including 22 new SNPs and developed a whitelist of 428 SNPs capable of distinguishing these whitefishes. Finally, we demonstrated performance by identifying 3,066 coregonine larvae and juveniles collected in spring 2019-2021 from Lake Superior. These results hold promise for future insights into the species-specific ecology of early life coregonines and demonstrate the flexibility of GT-seq panels, which may cross-amplify hundreds of informative genome-wide loci in related taxa.

Evolution is a foundational framework for understanding biology, yet it remains challenging to teach and learn. Game-based learning may offer one way to support evolution instruction by helping students visualize abstract, dynamic, and difficult-to-observe processes. In this study, we interviewed undergraduate biology instructors to examine how they evaluated video games as potential tools for evolution education, including which topics they perceived as most challenging for students. We found that instructors were broadly open to using video games for evolution instruction, particularly when games could support population-level reasoning, evolutionary mechanisms, speciation and phylogeny, quantitative reasoning, and long time scales. Instructors also emphasized that games must be scientifically accurate, accessible, and aligned with course learning goals. This study contributes an instructor-centered perspective to evolution education and game-based learning research by identifying how instructors connect persistent student learning challenges with potential design priorities for educational video games. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/736808v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@175ce4corg.highwire.dtl.DTLVardef@13ae260org.highwire.dtl.DTLVardef@e44aceorg.highwire.dtl.DTLVardef@1eb1ef1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Many sea anemones reproduce asexually by pedal laceration, shedding fragments of the pedal disk that regenerate into polyps. How feeding affects the amount of this reproduction differs among species, but whether feeding also sets its timing has rarely been examined. We fed the sea anemone Cylista elegans (formerly Sagartia elegans) daily, every second day, every fourth day, or not at all, measured growth, laceration, and survival over 35 days, then reassigned anemones to new schedules to test whether laceration follows the current or previous schedule. Growth rose with feeding but saturated, animals fed daily and every second day growing at similar rates. Total laceration depended on whether an anemone was fed rather than how often. Every fed schedule produced more lacerates than starvation. The timing, by contrast, tracked the schedule closely. Laceration was suppressed for about a day after each meal and recovered before the next, so anemones fed every second or every fourth day lacerated on matching two- and four-day rhythms, while daily-fed animals were arrhythmic. When moved to a new schedule, the period shifted to match it, not the old one. Only starvation caused death. Asexual reproduction in C. elegans is therefore bound closely to feeding, which fuels laceration yet suppresses it during digestion, confining it between meals. This coupling distinguishes C. elegans from anemones with symbiotic microalgae, where starvation rather than feeding drives reproduction, and links it to the feeding-driven clonal proliferation of the invasive sea anemone Diadumene lineata.

Dauer formation and L1 arrest are stress-responsive developmental strategies that enable Caenorhabditis elegans to survive unfavorable conditions. These responses are regulated by environmental cues, including food availability and pheromone signals that communicate population density. However, how dauer entry, L1 arrest, and density-dependent signaling collectively influence long-term population dynamics remains poorly understood. In this study, populations of daf-22 and daf-16 mutants with impaired dauer formation, starvation arrest, and pheromone signaling were compared under control and starvation stress. Measurements of developmental stages were used to evaluate how genotype influenced population growth, developmental stage composition, starvation response, and recovery over time. This population-level approach links individual developmental decisions and inter-organismal communication to broader patterns of persistence and population change. The results show that daf-16 and daf-22 mutant populations differed from wild type in their recovery ability following nutrient deprivation as well as in the stage distributions within the population. These findings suggest that dauer signaling contributes broadly to population persistence by coordinating developmental arrest, reproduction, survival, and recovery. Overall, this work supports the interpretation of dauer formation as a larger population level survival program rather than a single isolated developmental outcome.

Climate change is expected to alter plant populations not only through direct environmental shifts but also via changes in biotic interactions, such as with herbivores and pathogens. As plant species are also expected to differ in their responses to both climate and antagonists, plant responses to both factors are expected to be variable and species-specific. To assess whether interactive effects of climate and antagonists on plant population dynamics are common and whether the strength and direction of plant responses vary across species, we conducted a multi-year field experiment that manipulated realistic climate change and experimentally reduced insect herbivores and fungal pathogens. We measured responses of plant vital rates, such as survivorship, growth, and reproduction across six grassland species. Using Integral Projection Models (IPMs) and Life Table Response Experiments (LTREs), we quantified changes in population growth rate across experimental treatments and the contribution of each vital rate to that observed change. Two of the study species declined so drastically over the course of the experiment that demographic quantification of population growth rates was not possible. From the remaining species, Bromus erectus and Plantago lanceolata show significant interactive responses of climate and antagonist reduction on population growth rates. In contrast, Dianthus carthusianorum and Tragopogon orientalis showed limited responses to experimental treatments. Notably, our results indicate that in some species biotic interactions may amplify the effects of climate change: the presence of plant antagonists exacerbates the negative effects of the future climate treatment on plant population dynamics. Our findings highlight the complexity in predicting plant population responses to climate change and provide insights for grassland management under future environmental conditions.

The Hesperiine genus Atrytonopsis Godman, 1900, occurs broadly across the American Southwest. Atrytonopsis margarita (Skinner, 1913) and Atrytonopsis python (W. H. Edwards, 1882) have look-alike appearances, concurrent flights, and geographic distributions which converge in New Mexico. Their similar wing markings and intertwined taxonomic history has made it challenging to fully understand the identity and occurrence of each. Burns (2015) revealed differences in genitalia, clarifying that they are distinct species. Genomic DNA analysis of more than 100 specimens now illuminates their genetic uniqueness, phylogenetic relationship, field identification challenges and details of their geographic distributions.

Understanding how closely related species coexist in highly seasonal and unpredictable environments is central to studies of ecological differentiation and niche partitioning. We investigated the feeding ecology of sympatric populations of Microcebus murinus and M. griseorufus within a contact zone in Andohahela National Park, southeastern Madagascar, across dry and wet seasons. Using a combination of direct behavioral observations (1,611 feeding records), fecal sample analyses (n = 56), and vegetation phenology surveys, we quantified dietary composition, seasonal shifts in resource use, and habitat-related variation. Seasonal changes in diet were pronounced, with dry-season feeding dominated by exudates and wet-season diets incorporating greater proportions of fruit and flowers, closely tracking phenological patterns at both sites. Diets of both species were dominated by plant resources, but consistent interspecific differences in dietary strategy were evident. Although both species consumed comparable proportions of insect prey, M. murinus showed pronounced wet-season increases in the use of high-sugar, carbohydrate-rich floral resources (15.9%) and hemipteran-associated honeydew (30.6%). In contrast, M. griseorufus relied more consistently on predictable exudates throughout the year. Fecal analyses supported observational data but revealed differences in the detectability of dietary components, with increased representation of invertebrates in the wet season and seeds in the dry season. These results indicate substantial dietary overlap but consistent differences in resource use, suggesting that coexistence is facilitated by fine-scale trophic differentiation within a broadly shared omnivorous niche. Such subtle but persistent differences in feeding strategy likely reduce competitive overlap and enable continued sympatry in a climatically variable and resource-limited system.

Color polymorphism can facilitate local adaptation, maintain intraspecific diversity, or reflect early stages of speciation. Clownfishes (Amphiprion spp.) typically display a simple black, orange, and white pattern, but the saddleback clownfish (Amphiprion polymnus) shows striking variation in melanism and the number of vertical bars, which are thought to play a role in species recognition. In 2024, a revision on iNaturalist split A. polymnus into multiple species based solely on color pattern and geographic range. This raises the question of whether these morphs represent true species or intraspecific polymorphism, which we tested using genomic and image-based data. We sampled 97 individuals from seven populations across the species range and quantified color patterns from standardized photographs. Phenotypic and genomic analyses reveal a complex pattern of divergence. Image analysis identified three distinct phenotypic clusters, with A. polymnus, A. annamensis, and A. laticlavius each showing consistent differences in saddle shape and vertical bar extent. ADMIXTURE resolved three distinct genetic groups corresponding to the morphs. Pairwise FST (0.54-0.71) and dxy indicate extremely high differentiation between A. polymnus and A. annamensis, consistent with species-level divergence, whereas A. laticlavius shows much lower differentiation from A. polymnus (FST 0.09-0.18) and higher differentiation from A. annamensis (FST 0.64-0.66). Overall, phenotypic and genomic data show structured variation, but the status of A. laticlavius remains ambiguous. Our study reveals clear and structured divergence across the full range, yet the taxonomic interpretation of this variation remains inherently challenging. The key question remains: do these patterns reflect a single polymorphic species or a complex of closely related species?

Population dynamics and their component vital rates may be driven by weather, climate teleconnections between sea and air (e.g. ENSO), or biotic interactions. These drivers operate directly or indirectly and on different temporal scales. We used a Bayesian structural equation model to characterize effects among weather, climate, and incidental intraguild predation (IGP) on the butterfly Euphydryas gillettii's vital rate of pre-diapause survival, using an 18 year dataset. IGP was a major determinant of pre-diapause survival, along with direct and indirect effects of weather and spring climate teleconnections. The direction of climate effects was reversed when mediated through IGP. Our analysis illustrates the need for sequential hypotheses to capture the cascading effects of abiotic factors via biotic interactions. Using sequential hypotheses addresses the debate on weather -- climate teleconnection roles by disentangling their contributions from one another. Finally, vital rates must be decomposed to component rates in order to detect their drivers.

Heritable bacterial symbionts are pervasive in terrestrial arthropods, often imposing reproductive manipulations to promote their own spread within host populations. Co-infections are common, potentially allowing symbiont co-infectors to hitchhike through a host population. However, adverse thermal conditions can disrupt these communities, particularly when co-infectors vary in their thermal sensitivity. We used a multi-generation experiment to test whether warm (29 {degrees}C) conditions disrupted spread of heritable symbionts through uninfected populations of the spider, Mermessus fradeorum. We tested two common infection combinations: a single infection with a cytoplasmic incompatibility (CI) inducing Rickettsiella or a feminizing co-infection that included a feminizing Wolbachia, the same Rickettsiella, and up to three apparent hitchhikers (two additional Wolbachia strains and Tisiphia). We initiated replicate populations with 1/3 of one infection type and 2/3 uninfected spiders, evaluating population infection rate over 5 spider generations under different temperature regimes. Under cool (21{degrees}C) conditions, Wolbachia feminization drove co-infection to 88% and Rickettsiella CI drove single infection to 83% of host populations. Vertical transmission for all symbionts was high (97-99%) and hitchhiking symbionts also spread effectively. Under warm conditions, feminization and CI efficacy were reduced, and symbionts suffered variably reduced vertical transmission. Warm conditions ultimately destroyed the co-infecting symbiont consortium and impeded symbiont spread. On its own, though, Rickettsiella was still able to increase, despite reduced strength of CI. We hypothesize that contrasting tensions between feminizing spread of the symbiont consortium versus environmentally driven loss of function and transmission may explain observed patterns of mixed infections in field populations of this spider.

A central goal when assessing patterns of population structure for conservation is to identify populations with unique genetic compositions. The use of genomic sequencing to identify distinct populations has become an increasingly popular method of delineating conservation units. Reduced costs associated with sequencing make it possible to generate larger, more informative datasets to assess genetic diversity within and among populations. In species that exhibit nest-site philopatry, genetic population structure can emerge on much finer scales, particularly in maternally inherited mitochondrial genomes. We demonstrate the feasibility and insight gained by using whole mitochondrial genome sequencing for evaluating population genetic structure and comparing to previous single marker studies in a vulnerable turtle. We used whole mitochondrial genome sequences from diamond-backed terrapin (Malaclemys terrapin) to evaluate whether nest-site philopatry generates fine-scale genetic structure among M. terrapin nesting beaches in western Mobile Bay (Alabama, USA). We then compared haplotype diversity between the Alabama population and M. terrapin populations from the Atlantic and Gulf coasts and evaluated the utility of using whole mitochondrial genomes rather than a subset of loci to characterize unique haplotypic diversity. We found no genetic structure associated with nest-site philopatry within Alabama, but none of the haplotypes in this region were shared with other Gulf Coast sites. This genetic structure is consistent with strong female natal philopatry within western Mobile Bay relative to the Gulf of Mexico and suggests that the Mobile Bay population is genetically unique relative to other M. terrapin populations and merits a unique conservation and management plan.

Littoral mysids facilitate benthic-pelagic coupling through horizontal migration, yet quantitative monitoring in structurally complex habitats remains methodologically challenged where traditional active gears fail. We evaluated the efficacy of standardized light traps for monitoring littoral mysids (Neomysis integer, Praunus flexuosus) and mesopredatory three-spined sticklebacks (Gasterosteus aculeatus) in the northern Baltic proper, Baltic Sea. Using a paired experimental design with predator-exclusion and unmodified traps, alongside concurrent passive benthic trapping, we assessed abiotic drivers affecting catchability, biotic interactions, and statistical power to monitor changes in population size over time. Results indicated significant biotic interference: unmodified traps attracted high densities of sticklebacks, which reduced mysid catches by approximately 85% through predation or behavioural avoidance. Consequently, physical predator exclusion is mandatory for accurate mysid sampling. Generalized Linear Mixed Models (GLMMs) confirmed that catch rates for all taxa were primarily driven by night duration rather than water temperature. While passive benthic trap catches tracked metabolic activity (peaking in warm summer months), light trap efficiency peaked in spring and collapsed during summer, confirming that sampling efficiency was strictly limited by the short duration of the night. Simulation-based power analysis revealed a stark contrast in monitoring utility based on spatial aggregation. For highly aggregated mysids, the method demonstrated low precision (Power < 0.25 to detect a 50% decline), rendering it suitable primarily for detecting substantial population collapses (>90%). In contrast, for less aggregated sticklebacks, the method achieved a more robust statistical power (>0.80 for a 60% decline), validating light traps as a precise tool for monitoring these abundant mesopredators. We conclude that light traps fill a critical methodological gap for winter and early spring monitoring when traditional passive gears underperform. Appropriate abundance indices should be based on statistical models accounting for night duration and strictly employ physical exclusion barriers when targeting mysids.

Inbreeding depression (the reduction in fitness associated with inbreeding) has been demonstrated in a wide range of animals, but despite its ubiquity, is not an inevitable consequence of inbreeding. As a result, there is uncertainty about the extent to which inbreeding depression poses an ongoing risk to endangered species currently experiencing significant demographic recovery. Quantifying inbreeding depression will be critical if we want to understand these risks. A comprehensive quantification of the fitness costs of inbreeding requires detailed individual-based longitudinal data so lifetime impacts can be assessed. Here, we use an extraordinarily detailed long-term dataset on Mauritius kestrels (Falco punctatus) to explore inbreeding depression in a population currently experiencing significant demographic recovery. To do so, we constructed a social pedigree of 1,758 individuals and combined this with 1,240 nest records and 1,411 individual resighting histories to explore lifetime fitness effects over a 30-year period. Inbreeding increased significantly over time as the population recovered before stabilising. Inbred eggs were less likely to survive to fledging. Inbred adult male and female birds had significantly lower annual reproductive success than outbred individuals because of a lower annual egg-to-fledgling survival probability. This resulted in significantly lower lifetime reproductive success in inbred females but not males, which showed a negative trend. Population growth was negative and extinction risk increased slightly at current levels of inbreeding. These impacts will become more severe should inbreeding levels increase in the future, which is highly likely given ongoing genomic erosion. Taken together, our results demonstrate significant fitness costs associated with inbreeding in Mauritius kestrels, which pose an ongoing risk to population viability. This suggests that monitoring and managing inbreeding risks in endangered species will likely be required even in populations that are showing significant demographic recovery in response to conservation interventions.

Sustainable management of commercial and recreational fisheries depends on accurately resolving population connectivity, across both ecological and evolutionary timescales. However, dispersal can vary markedly among life stages, making stock connectivity difficult to resolve using single-method approaches that often differ in spatio-temporal resolution. Here, we integrated population genomics, otolith stable isotope chemistry, and mark-recapture analyses to provide a multi-faceted assessment of stock connectivity in mulloway (Argyrosomus japonicus). Mulloway are a commercially, culturally, and recreationally important estuary associated fish distributed throughout the Indo-Pacific region, including south-eastern Australia where this study was conducted. Genome-wide single nucleotide polymorphism (SNP) analyses revealed significant genetic differentiation between regions influenced by different current systems, but limited structure within regions across distances exceeding 900 km. In contrast, otolith {delta}13C and {delta}18O signatures revealed fine-scale spatial structuring among estuaries, consistent with prolonged occupancy of local habitats. Mark-recapture analyses supported this interpretation, with most fish exhibiting strong estuarine fidelity over extended periods despite occasional long-distance coastal movements. Reconstructed age structures from fish otoliths revealed remarkably similar cohort composition among estuaries, with populations dominated by cohorts originating from a major recruitment pulse centred on 2011-2012, likely associated with a broad-scale flood-driven spawning and recruitment event. Together, our findings indicate that mulloway fisheries function as regionally connected networks of partially independent estuarine assemblages, where strong local residency is periodically offset by dispersive individuals and episodic recruitment events that maintain long-term demographic and genetic connectivity. Consequently, local estuarine populations may be vulnerable to localised depletion despite broader regional connectivity, particularly where sustained fishing pressure coincides with reductions in freshwater flows that constrain spawning and recruitment. More broadly, our study demonstrates the value of integrating complementary approaches to identify biological connections and define meaningful management units in species with complex life histories.

Animals reproduce across a spectrum from oviparity to viviparity. Internal gestation and live birth have inherent advantages despite their costs. However, comparative analyses of key drivers, such as resilience to dehydration, lack taxonomic breadth. In this study, we assessed whether live birth improves the ability to proliferate in environments with limited access to water. To do so, we used the viviparous cockroach, Diploptera punctata, as a model of viviparity to assess dehydration-induced damage during extended periods of water deprivation. During pregnancy, maternal survival did not decline during dry periods compared with non-pregnant individuals, suggesting that pregnancy has minimal impact on resistance to dehydration stress. When mothers are deprived of water for 2 weeks, they show an increase in osmolality after one week, while their embryos show no change in osmolality until after two weeks, at which point abortions occur. Periods of dehydration increased the duration of pregnancy, likely due to a slower production of in utero milk, but there was no change in the size of progeny. After birth, viviparous D. punctata newborns showed increased survival under dry conditions compared to two other ovoviviparous species, which are much smaller and dehydrate more quickly. The combined impact of increased dehydration resistance during pregnancy and during the first independent phase indicates a benefit of prolonged viviparity in Diploptera punctata. This study provides evidence that viviparity in animal systems could enable progeny survival during drought.

Fistulobalanus albicostatus Pilsbry, 1916 (Thoracica: Balanidae) is a tropical to temperate species distributed in the NW Pacific. The previously known northernmost record of this species in Japan was from Aomori Prefecture, at the northern end of Honshu Island, Japan. However, field surveys conducted in 2023 and 2026 confirmed the occurrence of F. albicostatus in Hakodate Bay, the southern end of Hokkaido, Japan, across the Tsugaru Strait, thereby extending the northern limit of its known distribution. A line transect survey conducted in May 2026 recorded seven living individuals within an area of 128 m. F. albicostatus was rare on the mid-high shores, accounting for only 2% of all barnacle individuals in a quadrat survey. The basal diameter of the living individuals ranged from 0.76 to 1.23 cm, and all individuals possessed ovaries. Based on characteristics of both morphological and COI gene, the specimens were identified as F. albicostatus, and belonged to the same haplotype of populations that are present in Honshu Island. The establishment of F. albicostatus in Hokkaido suggests an ongoing northward range shift of this warm-water species, with the potential for further expansion under continued ocean warming.

Phenotypic plasticity, the ability of a single genotype to produce alternative phenotypes in response to environmental cues, is a key driver of evolutionary change. In the water strider Microvelia longipes, males display remarkable continuous variation in hindleg length, a sexually selected trait used as a weapon in male/male contests for access to females. To determine whether DNA methylation mediates this environmentally induced phenotypic variation, we used three inbred lines of M. longipes that differ in mean hindleg length, body size, and allometric coefficients. We performed whole-genome bisulfite sequencing on adult males and females from all lines, and tested the effect of nutritional treatment on DNA methylation patterns. Our analysis identified 12,684,876 CpG 12% of which were methylated. This global level of DNA methylation is among the highest reported in insects. DNA methylation was predominantly concentrated within or near gene bodies (77% of methylated CpGs), consistent with patterns observed in other insects. Unsupervised clustering and principal component analyses revealed that methylation patterns differed significantly between genetic lines but showed minimal differences between sexes, indicating a strong genetic influence. Most surprisingly, despite nutrition having a pronounced effect on leg length, we observed no significant changes in DNA methylation in response to dietary treatment. These results show that in M. longipes, DNA methylation patterns are largely stable across environmental conditions and primarily determined by genetic background. This challenges the common assumption that DNA methylation universally mediates environmentally induced phenotypic plasticity and suggests that other epigenetic mechanisms, such as histone modifications or non-coding RNAs, may play a more direct role in regulating continuous plastic traits. Our study underscores the complexity of epigenetic regulation and highlights the need for broader investigation of molecular pathways to fully understand the molecular basis of phenotypic variation in natural populations.

While emerging diseases pose a global threat to amphibians, the dynamics of understudied vector-borne blood pathogens remain poorly understood. Pathogen occurrence is driven by a combination of environmental, ecological, and phylogenetic factors, yet how these drivers shape blood pathogen communities in tropical amphibians is largely unknown. In this study, we used molecular screening and phylogenetic linear models (PGLMMs) to evaluate how climate and ecomorphology influence the incidence of three blood pathogen groups (Trypanosomatidae, Hepatozoon, and Rickettsia) in wild anurans from a protected area in the Brazilian Atlantic Forest. Among 93 individuals sampled, over 93% were infected with at least one pathogen. Trypanosomatidae was the most common (76.3%), followed by Rickettsia (69.9%) and Hepatozoon (16.1%). Pathogen responses to temperature were contrasting: Hepatozoon occurrence increased in warmer periods, while Trypanosomatidae declined. Furthermore, rheophilic species showed a lower probability of Rickettsia infection, providing the first evidence that microhabitat use influences blood pathogen dynamics in amphibians. Our findings demonstrate that hemoparasites prevalence is driven by a multifaceted interplay of variables, highlighting that conservation strategies must account for these pathogen-specific responses to habitat use and environmental change, even within protected areas.

O_LIInformation on diet composition at both individual and population levels is fundamental to understanding resource use and availability, which influence individual fitness and the population dynamics of both predators and their prey. DNA diet metabarcoding offers a powerful molecular approach to diet analysis, but its application can be limited by methodological biases, particularly in generalist species with highly diverse diets. C_LIO_LIHere, we evaluate the performance of DNA metabarcoding for diet analysis in the lesser black-backed gull (Larus fuscus), a highly generalist mesopredator whose populations have shown complex responses to changes in natural and anthropogenic food resources over the past half-century. We collected faecal and regurgitate samples from pre-fledging chicks at two coastal and inland breeding colonies in northwest England, alongside pharyngeal, stomach, intestinal, and cloacal swabs from adult gull carcasses. Samples were analysed using COI (targeting animal DNA) and 12S (targeting vertebrate DNA) metabarcoding markers, and three blocking primers were developed to reduce host amplification in 12S libraries. C_LIO_LIMetabarcoding performance varied substantially among sample types and primer combinations. Without blocking primers, usable dietary information was recovered from regurgitate and stomach samples but not from intestinal or faecal samples. Blocking primers improved recovery of dietary DNA from faecal samples, but also increased the amplification of contaminants, elevating the risk of false-positive detections. C_LIO_LIAcross all sample types, metabarcoding identified 71 unique species belonging to 61 genera, including earthworms, small mammals, commercial and non-commercial fish species, lapwing, and urban-derived food products originating from livestock species. Dietary profiles revealed distinct clusters of consumed species associated with urban, agricultural, and marine foraging strategies, and demonstrated differences in dietary niche between age and colony cohorts. C_LIO_LIOverall, DNA metabarcoding enabled the detection of a highly diverse diet and revealed differences in resource use among colonies and age cohorts. These findings demonstrate the potential of DNA metabarcoding to advance our understanding of diet in highly generalist species and contribute to ongoing efforts to understand how dietary variation may shape demographic processes in highly dynamic populations. C_LI