Journal of Heredity

The hybrid grouper 'Yushuban' (Cromileptes altivelis [female] x Epinephelus fuscoguttatus [male]) has shown promising performance in aquaculture trials, yet no complete reference genome has been available for this hybrid. We generated two gapless, telomere-to-telomere (T2T) haplotype-resolved genome assemblies of a single individual by integrating short-read, PacBio HiFi, Oxford Nanopore ultralong-read and Hi-C data. The maternal (Yushu_Calti_1.0) and paternal (Yushu_Efusc_1.0) haplotypes are 1,077.33 Mb and 1,083.36 Mb in length, with N50 values of 45.46 Mb and 45.55 Mb, respectively. Across all platforms, over 99.4% of reads mapped to each assembly, and Merqury-based quality values reached 57.7413 and 59.2068 (PacBio). BUSCO recovered 99.78% and 99.75% of complete core genes. We predicted 27,248 and 27,566 protein-coding genes, of which 98.52% and 99.23% were functionally annotated, and annotated telomeric motifs and repetitive elements (46.12% and 47.18% of each genome). These assemblies fill a gap in grouper genomic resources and provide a foundation for genetic dissection, parental genome comparison and molecular breeding of this hybrid.

Eucalyptus recurva (Mongarlowe Mallee) is Critically Endangered, with only six known adult individuals persisting across two sites in the Southern Tablelands of New South Wales, Australia. Its extreme rarity, uniquely long lifespan, and limited reproductive output make it a priority for conservation genomics. Here we report the first genome assembly of E. recurva: a haplotype-resolved, gapless, telomere-to-telomere (T2T) assembly produced from Oxford Nanopore Technologies (ONT) long-read sequencing. Both haplotypes span 11 chromosomes (consistent with the conserved Eucalyptus karyotype of 2n = 22), with assembly sizes of 521.1 Mb (Hap 1) and 501.5 Mb (Hap 2), BUSCO completeness >99.6%, and quality values of >QV 62. Comparative analyses place E. recurva within section Maidenaria as sister to Eucalyptus viminalis and reveal high synteny between the two species. Inter-haplotype comparison identified 3.6 million SNPs and modest structural variation, suggesting that, despite extreme demographic bottlenecking, E. recurva retains meaningful genomic heterozygosity. We also characterise the chloroplast genome, which exhibits heteroplasmy, and report an apparently bipartite mitochondrial genome. This reference genome provides an essential resource for conservation management, population genetics, and the study of eucalypt genome evolution.

We present draft genome assemblies for two poneroid ant species with distinctive life history traits: Platythyrea mocquerysi Emery, 1899 (Ponerinae) and Stigmatomma oregonense Wheeler, 1915 (Amblyoponinae). Platythyrea mocquerysi is a Malagasy ant in which larvae produce secretions from a ventral tubercle that are consumed by adults, representing an unusual form of larva-to-adult trophallaxis. Stigmatomma oregonense, a North American "dracula ant," feeds non-lethally on larval haemolymph. Using PacBio HiFi long-read sequencing, we assembled the P. mocquerysi genome (187 Mb, 307 scaffolds, N50 = 5.26 Mb, BUSCO 99.2% complete) and the S. oregonense genome (262 Mb, 1,189 scaffolds, N50 = 0.84 Mb, BUSCO 98.3% complete). These genomes encode 10,860 and 12,354 protein-coding genes, respectively. Repeat content is higher in S. oregonense than in P. mocquerysi, 54.3% against a lower bound of 15.7%. Most of the difference is in unclassified repeats, which we show to be diverged transposable elements. Comparative analysis with two additional poneroid genomes from closely related species reveals that genome size variation across poneroid ants is largely explained by transposable element dynamics, with notable expansions in several gene families including carboxylesterases, serine proteases, and lipid metabolism genes in Amblyoponinae. These genomes provide essential resources for investigating the molecular basis of unconventional brood management strategies in ants.

Arctic ecosystems are undergoing rapid climate warming, providing an opportunity to examine molecular evolution under environmental stress. Here, we used whole-genome resequencing to assess population structure, genetic diversity, and recent demographic history in the snowy owl (Bubo scandiacus), a highly mobile Arctic apex predator classified as Vulnerable by the IUCN. We analysed genome-wide data from individuals sampled across the species' circumpolar range using a chromosome-level reference genome. Population genomic analyses revealed an absence of detectable population structure, indicating extensive gene flow and near-panmixia across continents. Genome-wide nucleotide diversity was low ({pi}= 2.47 x 10^(-4)), while runs of homozygosity were dominated by short tracts, indicating limited standing genetic variation despite little evidence for recent close inbreeding. Our demographic inference revealed a sustained decline in effective population size over the past ~1,600 years, a pronounced reduction coinciding with the Medieval Warm Period followed by a partial recovery during the subsequent cooler interval. This direct, measurable effect of past climate warming on effective population size raises concerns for the long-term survival of the snowy owl. Our results demonstrate that high connectivity and low levels of recent inbreeding have not necessarily buffered this species against climate-driven demographic erosion of genetic diversity. This study highlights the value of using whole-genome population genomics to inform genomic monitoring and conservation decision-making in rapidly changing environments.

Understanding the demographic processes shaping population connectivity is essential for conservation. Yet disentangling the effects of dispersal and local effective density remains challenging because traditional genetic approaches integrate signals over evolutionary timescales. Identity-by-descent (IBD) segments provide opportunities to reconstruct recent demographic connectivity over timescales relevant to contemporary conservation challenges, but their reliability under realistic ecological conditions remains poorly evaluated. Here, we assess the ability of two promising spatiall IBD-based methods, IBD-Analysis and MAPS, to estimate effective density and dispersal variance. Using a simulation-inference framework combining spatially explicit simulations, IBD segment extraction, and demographic inference, we evaluated both methods across a wide range of demographic, ecological and methodological scenarios. Both approaches reliably recovered demographic parameters under homogeneous landscapes, with IBD-Analysis providing accurate estimates of global effective density and dispersal across a broad range of conditions. Spatial heterogeneity emerged as the main driver of inference performance. When demographic parameters varied across space, IBD-Analysis integrated local variation into biased global estimates, whereas MAPS captured spatial patterns but showed substantial variability and generated spurious spatial structure in homogeneous landscapes. Methodological factors had comparatively weaker effects than ecological complexity, highlighting the importance of accounting for landscape structure when applying IBD-based demographic inference. These results highlight the potential of IBD-based approaches to move beyond descriptive patterns of genetic connectivity toward mechanistic inference of demographic processes. By distinguishing the contributions of dispersal and density, these methods can improve conservation decisions by linking genomic patterns to key demographic parameters relevant for conservation.

Polyandry, multiple mating by females, both benefits females by increases genetic variation within families and provides an opportunity for sexual selection through sperm competition and cryptic female choice. Estimating polyandry in species with large clutches is challenging because genotyping all progeny individually can be impractical. We present a likelihood-based framework for estimating the number of sires and their relative contributions from the maternal genotype and pooled sequencing of entire progeny pools. The method evaluates the probability of observed pooled offspring read counts under alternative paternal genotype mixtures, accounting for Mendelian segregation and sampling variation from finite sequencing depth. We implement this framework for both biallelic SNPs and short-read mini-haplotype markers constructed from closely linked SNPs, which provide multi-allelic information comparable to traditional microsatellite markers. Simulations show that this approach accurately recovers sire number and sire contributions across a range of conditions. We then applied the method to 275 wild-caught Drosophila melanogaster females and their offspring from a Kansas population using 3,053 biallelic SNPs and 239 four-allele mini-haplotype markers. Estimates from the two marker sets were highly concordant, and combined analyses indicate that 57% of families were sired by a single male. These results demonstrate that pooled progeny sequencing provides an efficient genomic approach for estimating polyandry in species with large families.

Marine species with broad distributions often exhibit complex patterns of genetic connectivity, shaped by historical and contemporary processes. Understanding these patterns is critical for conserving taxa threatened by ongoing and future environmental change. Here, we use reduced-representation and whole-genome sequencing to investigate population genetic patterns in three co-occurring sea snake species (Hydrophis stokesii, Hydrophis major, and Aipysurus laevis) across northern Australia and New Caledonia. Broadscale analyses revealed major genetic breaks across all species between: (1) Australia and New Caledonia, (2) northwestern and northeastern Australia, and (3) the offshore Timor Sea reefs and adjacent northwestern continental shelf of Australia. More subtle genetic differentiation was observed between Shark Bay in Western Australia and northern localities. All species showed relatively large effective population sizes and broadscale connectivity across ~1,300 km of Australia's northwestern continental shelf. This likely reflects substantial historical range expansion over late Quaternary timescales, which may have contributed to the observed genetic connectivity across the region. However, subtle patterns of isolation by distance emphasised the contribution of species-specific traits in shaping connectivity. The isolated offshore reef populations are genetically distinct and potentially threatened by environmental change. Disjunct populations in Shark Bay and the Timor Sea maintain similar levels of genetic diversity to the extensive continental shelf populations, while New Caledonian populations exhibit reduced diversity. Our results support the delineation of spatial units for sea snake conservation based on shared phylogeographic structure, while also finding important population- and species-specific differences in connectivity at finer spatial and temporal scales.

At large scales, our ability to compare genetic diversity across species is often challenged by the difficulty of extracting range-wide sequence data from online databases. Using mitochondrial DNA from amphibians and reptiles spanning across six regions: Brazilian Atlantic Forest, Iberian Peninsula, northwestern Africa, Arabian Peninsula, Madagascar and Australian Monsoonal Tropics, we develop and implement a framework to overcome common limitations in macrogenetics. We compiled and manually curated a spatially explicit genetic dataset from public repositories, quantified the spatial representativeness of genetic sampling across species' ranges and developed a novel interpolation framework to estimate and map species-specific patterns of intraspecific genetic diversity (ISD) while accounting for heterogeneous spatial sampling. We estimated mean nucleotide diversity across species to produce regional maps for each genetic marker. Our final dataset comprised 182 amphibian and 239 reptile species, encompassing 184 genera from 54 families. Range representativeness of genetic sampling was generally high, despite some variation across study regions. Spatial patterns of ISD revealed geographic heterogeneity, and were consistent across the best-sampled mitochondrial markers. By combining automated and manual georeferencing, quantifying range representativeness, and accounting for bias in spatial interpolations, our pipeline is marker-agnostic and directly applicable to nuclear and genomic data as they become available.

A complete reference genome for species with chromosomally-determined separate sexes should contain scaffolds for all sex chromosome homologs. However, sex chromosomes present distinct computational challenges compared to autosomes. Here we present a k-mer based analysis that utilizes whole-genome sequencing of a few sex-identified isolates: Cytogenetics-By-Sequencing (CBS) tools. Unlike other approaches that typically address one aspect of the sex chromosomes, CBS-tools strives to guide users from the discovery of the heterogametic sex through identifying the sex-determination region (SDR). The core of CBS-tools is automated quantification of sex-specific k-mers in order to predict the heterogametic sex. Using publicly-available datasets, CBS-tools correctly identified the known sex-system of the 31 species tested. Additionally, we used these k-mers to verify and correct phasing of sex chromosomes between haplotypes in species representing different sex-systems. Finally, we used these k-mers to delimit the SDR boundary using an interactive web platform. CBS-tools was developed with previously unexplored sex chromosome systems in mind, but is also suitable for well-examined sex chromosome pairs.

Extreme climatic events (ECEs) could potentially induce episodes of rapid adaptive evolution, altering the evolutionary trajectories and viability of wild populations experiencing increasingly stochastic environments. Adaptive potential stems substantially from additive genetic variance (V) in survival. However, the degree to which ECEs impact this variance, and the resulting evolutionary implications, have not been fully conceptualised or quantified in nature. These aims are highly challenging because underlying genetic and environmental variances have fundamentally non-linear effects on the dichotomous phenotypic outcome of survival versus death, while substantial data are required for estimation. We present a conceptual and analytical framework that formalises biological hypotheses for how V depends on environmental severity, how such dependences are reshaped by non-linear genotype-environment-phenotype relationships and demographic structure, and the implications for evolutionary responses. We then use 25 years of survival and pedigree data from European shags (Gulosus aristotelis) to decompose variance in adult survival across diverse environmental conditions including ECEs. Using a capture-recapture animal model, we demonstrate non-negligible V underlying adult survival across all years combined, implying notable adaptive potential. Yet, partitioning by environmental severity revealed substantial V in good years with high survival but negligible V in bad years with ECE-induced low survival, implying a genotype-by-environment interaction that constrains adaptive potential under severe conditions. However, scale transformations showed how the non-linear genotype-environment-phenotype relationship can distort or even reverse such patterns, by reducing the extent to which V is phenotypically expressed in good conditions with high mean survival and amplifying it in more severe conditions with lower mean survival. Evolutionary responses to severe conditions could consequently be non-negligible even given small underlying V. We thereby demonstrate how ECEs can reshape adaptive potential, highlighting the need to integrate impacts of environmental severity on genetic variation in fitness components into predictions of population resilience under climate change.

Thrips are known for being pests of many agricultural crops, as well as vectors of plant viruses. Yet despite their economic importance, genomic resources for this group remain limited, and no chromosome-level genome has been available for the genus Scirtothrips. One such species is the chilli thrips, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae). This invasive and highly polyphagous pest is established across much of the world and transmits several tospoviruses. Genomic resources would provide a new means to understand the traits that make thrips difficult to manage. Here, we present a chromosome-level genome assembly for S. dorsalis. The assembly is 266.5 Mb, with 96.9% of the sequence anchored in 16 chromosome-scale scaffolds. It contains 20,557 genes, and repetitive elements account for 28.8% of the sequence, which is comparable to those of other sequenced Thripidae. A species tree based on 3,019 single copy orthologs placed S. dorsalis as sister to Scirtothrips hansoni, with Scirtothrips most closely related to Frankliniella, Megalurothrips, and Odontothrips. The mitochondrial genome is a single circular molecule of 15,207 bp in which the sequence of the published mini circle is integrated, in contrast to the bipartite mitogenome previously reported for the same cryptic species.

Demographic history can profoundly shape genomic variation by altering levels of inbreeding, the strength of genetic drift, and the efficacy of purifying selection, thereby influencing both the accumulation and expression of deleterious variation. Yet the extent to which these genomic consequences translate into organismal performance remains poorly understood. Here, we investigated how contrasting demographic histories shape genomic diversity, inbreeding, genetic load, and performance across mainland and insular populations of the Italian wall lizard, Podarcis siculus. Using whole-genome sequence data from 263 wild-caught individuals and field-based performance measurements from 134 lizards, we characterised population structure and demographic history, quantified genomic diversity, runs of homozygosity, and deleterious variation, and tested associations between genomic inbreeding and performance traits. Island populations showed reduced genetic diversity, increased autozygosity, and elevated genetic load relative to mainland populations, consistent with stronger genetic drift and reduced efficacy of purifying selection. Patterns of deleterious variation were consistent with purging of strongly, but not mildly, deleterious variants. Greater genomic autozygosity was associated with reduced bite force in island lizards, a pattern consistent with inbreeding depression, whereas sprint speed, sperm quality, immunocompetence, and spatial cognition showed no significant association with inbreeding. Genome-wide mapping of runs of homozygosity further identified multiple genomic regions associated with reduced bite performance. Together, these results show how demographic history shapes genomic diversity, homozygosity, and deleterious variation in wild vertebrate populations, while revealing that phenotypic consequences can be trait-specific, a pattern that may reflect differences in genetic architecture and the extent of environmental influence on trait variation.

Background Comparisons of single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) data have been described in some mammalian tissues and, subsequently, in Drosophila, but remain unexplored in most invertebrate lineages. The xenacoelomorphs occupy key phylogenetic positions, yet they differ anatomically from mammals. They have a reduced extracellular matrix, high-salt body fluid, and no circulatory system. Despite these differences, they possess a well-developed nervous system. One of the xenacoelomorphs, the photosymbiotic acoel (Praesagittifera naikaiensis) also harbours symbiotic Tetraselmis algae, whose RNA can be co-captured with host RNA. Results We compared scRNA-seq and snRNA-seq data from whole P. naikaiensis specimens. Both methods yielded high-quality data with comparable gene detection but a larger share of scRNA-seq reads derived from symbiotic algae. Gene-level analyses revealed that neural genes were enriched in snRNA-seq relative to non-neural genes. Cross-method label transfer and integration-based validation identified six snRNA-seq clusters, including some neural populations, that lacked a clear scRNA-seq counterpart. In contrast, three cell populations, including muscle and metabolically active clusters, were reciprocally validated as captured by both methods. Conclusions Our results show that key snRNA-seq advantages, particularly the enhanced recovery of neural transcripts, are recapitulated in our dataset, which is consistent with previous reports in mammals. Furthermore, snRNA-seq reduces symbiont-derived reads and recovers several cell populations underrepresented in scRNA-seq. These findings provide practical guidance for cell atlas construction in non-model, symbiotic invertebrates.

Demographic inference based on sequentially Markovian coalescent (SMC) model is widely used for disentangling the evolutionary history of species and populations. However, the genetic effects of self-fertilization, including the expected reduction of population-level genetic diversity and hence effective population size (Ne), are often ignored in demographic inference, despite the fact that selfing is a common reproductive mode in both invertebrates and plants. To investigate how self-fertilization could bias demographic inference, we apply eSMC2, a selfing-aware demographic inference method, to Hawaiian populations of Caenorhabditis elegans, a predominantly self-fertilizing nematode. Using an expanded dataset that contains more Hawaiian strains than in previous studies, we recovered previously reported genetic groups, including one largely consisting of strains from the Big Island. Population substructure is also detected among these Big Island subpopulations, highlighting the importance of identifying subgroups to avoid misinterpretations of demography. For different Big Island subpopulations, we estimate high selfing rates compatible with previous studies and infer consistent population declines. Population sizes inferred using eSMC2 are smaller than those inferred using PSMC', which does not account for selfing. However, rescaling PSMC' parameters to account for self-fertilization produces a similar demographic trajectory as eSMC2 inference. Our study thus demonstrates how one can account for effects of selfing when using SMC-based methods. It also indicates that the Hawaiian group shows population declines similar to those previously found outside Hawaii.

Self-fertilization results in reduction of heterozygosity over time, accelerated by small population sizes, restricted ranges, and fragmented habitats. Yet some species maintain heterozygosity despite these factors. One is Corallorhiza bentleyi, a threatened, mycoheterotrophic, self-pollinating orchid restricted to Virginia and West Virginia, USA. Reduced representation sequencing was employed across five of the ~12 known localities of C. bentleyi, including representatives of sister species C. involuta from Mexico, to quantify patterns of population structure and genomic diversity. Surveys of C. bentleyi revealed a predominance of the cleistogamous (closed-flowered) growth form. Differentiation was observed between the two species but with weak population structure in C. bentleyi. Surprisingly, excess heterozygosity and negative inbreeding were recovered in both species, counter to expectations. Analyses provided evidence against bioinformatic artifacts (collapsed paralogs), small-sample bias (Robertson effect), and accumulation of mutations along chromosomes due to low recombination associated with selfing (Meselson effect) as explanations for excess heterozygosity. Demographic analyses favored a model of post-glacial divergence among C. bentleyi and C. involuta lacking bottlenecks. Finally, simulations under alternative selection scenarios favored models of overdominance vs. those of negative frequency dependent selection. It was concluded that overdominance, namely selection against selfed homozygotes, is the most likely explanation for elevated heterozygosity in C. bentleyi. Conservation concerns are discussed in light of these findings and the threats of extinction for one of the most vulnerable orchid species in North America.

Recombination shapes allelic combinations across the genome and plays a central role in adaptation and speciation. While recombination rates are known to vary and evolve, how recombination landscapes diverge between populations and which processes drive this divergence remains poorly understood. Here, we use the Population Recombination Divergence Index (PRDI) to quantify divergence in effective recombination landscapes between seven pairs of populations and species from four percomorph fish lineages (cichlids, sticklebacks, killifish, and clownfish). While accounting for stochasticity and confounders of linkage disequilibrium (LD) -based recombination rate estimates, we find that recombination landscapes diverged substantially in all comparisons. This divergence generally increases with genetic differentiation and divergence time, but the amount of PRDI accumulated per unit of genetic divergence (FST), net nucleotide divergence (da), or divergence time varies markedly among population and species pairs. Comparisons across systems suggest that PRDI accumulation tends to be faster (per unit of FST, da, or divergence time) in pairs with higher inferred gene flow. We also introduce a local version of PRDI (_l) to better characterize genomic heterogeneity in effective recombination divergence. We find that higher inferred gene flow between populations tends to be associated with stronger intragenomic correlation between local FST and _l. Finally, we propose a conceptual framework for investigating how divergent selection and gene flow may interact to shape effective recombination landscape divergence. Our work highlights the heterogeneous dynamics of effective recombination landscape evolution, and motivates further theoretical and empirical efforts to integrate recombination landscape evolution into studies of adaptation and speciation.

The evolution of genome size and eukaryotic genome organisation has puzzled evolutionary biologists for decades. Are differences in DNA content and protein-coding capacity of eukaryotic genomes a result of natural selection, or neutral processes? If they were shaped by natural selection, which features might be connected with these differences: cell volume, ecological strategy, availability of nutrients, etc.? Kinetoplastids are protists with compact nuclear genomes that almost entirely lack introns, but whose mitochondrial DNA content (kDNA) can rival that of their nuclei. We hypothesised that the reduction in non-protein coding nuclear DNA content during the evolution of kinetoplastids is connected with an increase in kDNA content: species with larger fractions of total cellular DNA that is allocated to kDNA will have concomitantly smaller fractions of non-coding DNA in the nuclear genome. We developed a framework for estimation of DNA content in both the mitochondria and nuclei from quantitative fluorescence microscopy data, and inferred the fraction of non-coding DNA in nuclear genomes based on the combination of transcriptomic and fluorescence imaging data. Across 10 genetically distant isolates from diverse free-living kinetoplastid lineages, there is indeed a trend towards a lower proportion of non-protein-coding nuclear DNA when kDNA proportion is higher relative to the total DNA in the cell. This observation is consistent with the hypothesis that in free-living kinetoplastids, mitochondrial and nuclear genomes might co-evolve in terms of their DNA content. This finding raises the possibility that natural selection acts on genome size evolution in kinetoplastids, and contributes evidence and methodology to address this question more broadly in eukaryotes.

Populations that have detectable additive genetic variance for fitness (VA(W)) have the capacity for increase in their mean fitness through natural selection. The extent to which the magnitude of VA(W) in wild populations is predictive of the rate of adaptation is not well known, because few empirical field-based studies have been designed to address this question. Here, we report a two-year field experiment with pedigreed seeds embedded in four natural populations of the annual wildflower Baby Blue Eyes (Nemophila menziesii H. & A.), a species native to western North America. We first estimated VA(W) in a parental generation and then evaluated whether that estimate accurately predicted the adaptive response observed in the subsequent offspring generation grown under natural field conditions. We achieved this by directly comparing the mean fitnesses of the parental generation and their progeny grown under the same environmental conditions. We detected VA(W) and evidence of adaptation in all populations despite inter-annual differences in environmental conditions that directly affected fitness. Genotype-by-year interactions in fitness indicate that the fitness ranking of families differed between years, implying that the selective environment shifted over time. Additionally, although the environment in the second year reduced population mean fitness in three sites, the offspring generation maintained mean fitness at or above replacement, suggesting that adaptive evolution may have buffered the effects of environmental change. Overall, our results suggest that the four populations harbored sufficient VA(W) for adaptation through natural selection to the environmental conditions observed during the two years of this study.

Structural variants (SVs) can strongly influence evolutionary processes by suppressing recombination to create jointly inherited regions and by altering gene expression and the 3D organization of DNA. Recent improvements in sequencing technologies have led to an influx of high-resolution genomic data, bolstering research on SVs. When detecting and characterizing SVs, researchers face trade-offs in data and methods concerning scale, resolution, and computational intensity. Here, we evaluate such trade-offs by quantifying how data type, analytical approach, and SV attributes affect the ability to detect and characterize SVs in Timema cristinae stick insects, with a focus on inversions. We specifically evaluate inversions characterized by (i) a population genomic approach based on patterns of local population structure inferred from genotyping-by-sequencing data, (ii) pairwise alignments of eight de novo genome assemblies and SV calling with SyRI, and (iii) a pangenome constructed from the alignment of the eight genome assemblies. Our results suggest that while broad genomic regions harboring SVs can be detected by any of the approaches, the approaches differ in how they characterize inversions, especially small or complex ones. The local population structure approach is exploratory and generally only detects the largest inversions. Pairwise comparative alignments identify the greatest number of inversions, but downstream analyses are influenced by identification of shared inversions across genome pairs. Pangenome approaches are scalable and produce network graphs that describe complex SVs but require a conceptual shift in visualization and interpretation. The collective results highlight limitations and benefits of different approaches in a burgeoning research field.

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.