Natural Selection Research Articles

Population size rescaling significantly biases outcomes of forward-in-time population genetic simulations.

Simulations are an essential tool in all areas of population genetic research, used in tasks such as the validation of theoretical analysis and the study of complex evolutionary models. Forward-in-time simulations are especially flexible, allowing for various types of natural selection, complex genetic architectures, and non-Wright-Fisher dynamics. However, their intense computational requirements can be prohibitive to simulating large populations and genomes. A popular method to alleviate this burden is to scale down the population size by some scaling factor while scaling up the mutation rate, selection coefficients, and recombination rate by the same factor. However, this rescaling approach may in some cases bias simulation results. To investigate the manner and degree to which rescaling impacts simulation outcomes, we carried out simulations with different demographic histories and distributions of fitness effects using several values of the rescaling factor, Ǫ, and compared the deviation of key outcomes (fixation times, allele frequencies, linkage disequilibrium, and the fraction of mutations that fix during the simulation) between the scaled and unscaled simulations. Our results indicate that scaling introduces substantial biases to each of these measured outcomes, even at small values of Ʈ. Moreover, the nature of these effects depends on the evolutionary model and scaling factor being examined. While increasing the scaling factor tends to increase the observed biases, this relationship is not always straightforward, thus it may be difficult to know the impact of scaling on simulation outcomes a priori. However, it appears that for most models, only a small number of replicates was needed to accurately quantify the bias produced by rescaling for a given Ʈ. In summary, while rescaling forward-in-time simulations may be necessary in many cases, researchers should be aware of the rescaling procedure's impact on simulation outcomes and consider investigating its magnitude in smaller scale simulations of the desired model(s) before selecting an appropriate value of Ʈ.

Genome Dynamics of Adaptation and Introgression in Neotropical Palms

ABSTRACTAimThe combined influences of demographic dynamics and gene flow on local adaptation in plants is still poorly understood. Here, we used a genome scan approach on three closely related Neotropical palms, Acrocomia aculeata, A. intumescens and A. totai, to identify the evolutionary processes generating shared and lineage‐specific patterns of differentiation and selection across the genomic landscape.LocationAmazonia, Atlantic Forest, Cerrado and Caatinga ecoregions of Brazil.TaxaArecaceae.MethodsWe used target sequence capture and analysis of climatic correlation, detection of selective sweeps, balancing selection, and spatial and non‐spatial models to identify signatures of natural selection and admixture. We also determined temporal dynamics in spatial distribution and demographic changes.ResultsWe found a higher number of lineage‐specific than shared adaptive sites (SNPs) and no evidence of selective sweeps in shared genes, suggesting lineage specific natural selection across species. Further, evidence of balancing selection in several genes was also identified in the three species. Niche‐based and coalescent models suggest that shifts in spatial range during the Quaternary caused overlapping distributions between species, leading to hybridisation between parapatric localities.Main ConclusionsInterspecific hybridisation may have spread both neutral and adaptive SNPs, which may explain the shared adaptive genes between species. Taken together, we show how genomic adaptation can occur despite introgression, through evolutionary processes that likely drive similar patterns of adaptation in other organisms.

Asymmetric genome merging leads to gene expression novelty through nucleo-cytoplasmic disruptions and transcriptomic shock in Chlamydomonas triploids.

Genome merging is a common phenomenon causing a wide range of consequences on phenotype, adaptation, and gene expression, yet its broader implications are not well-understood. Two consequences of genome merging on gene expression remain particularly poorly understood: dosage effects and evolution of expression. We employed Chlamydomonas reinhardtii as a model to investigate the effects of asymmetric genome merging by crossing a diploid with a haploid strain to create a novel triploid line. Five independent clonal lineages derived from this triploid line were evolved for 425 asexual generations in a laboratory natural selection experiment. Utilizing fitness assays, flow cytometry, and RNA-Seq, we assessed the immediate consequences of genome merging and subsequent evolution. Our findings reveal substantial alterations in genome size, gene expression, protein homeostasis, and cytonuclear stoichiometry. Gene expression exhibited expression-level dominance and transgressivity (i.e. expression level higher or lower than either parent). Ongoing expression-level dominance and a pattern of 'functional dominance' from the haploid parent was observed. Despite major genomic and nucleo-cytoplasmic disruptions, enhanced fitness was detected in the triploid strain. By comparing gene expression across generations, our results indicate that proteostasis restoration is a critical component of rapid adaptation following genome merging in Chlamydomonas reinhardtii and possibly other systems.

The evolutionary landscape of prokaryotic chromosome/plasmid balance

The balance between chromosomal and plasmid DNAs determines the genomic plasticity of prokaryotes. Natural selections, acting on the level of organisms or plasmids, shape the abundances of plasmid DNAs in prokaryotic genomes. Despite the importance of plasmids in health and engineering, there have been rare systematic attempts to quantitatively model and predict the determinants underlying the strength of different selection forces. Here, we develop a metabolic flux model that describes the intracellular resource competition between chromosomal and plasmid-encoded reactions. By coarse graining, this model predicts a landscape of natural selections on chromosome/plasmid balance, which is featured by the tradeoff between phenotypic and non-phenotypic selection pressures. This landscape is further validated by the observed pattern of plasmid distributions in the vast collection of prokaryotic genomes retrieved from the NCBI database. Our results establish a universal paradigm to understand the prokaryotic chromosome/plasmid interplay and provide insights into the evolutionary origin of plasmid diversity.

MicroRNA-mediated network redundancy is constrained by purifying selection and contributes to expression robustness in Drosophila melanogaster

MicroRNAs (miRNAs) are post-transcriptional, non-coding regulatory RNAs that function coordinately with transcription factors (TFs) in gene regulatory networks. TFs and their targets are often co-regulated by miRNAs, forming composite feedforward circuits (cFFCs) with varying degrees of redundancy, primarily mediated by miRNAs. However, the maintenance of miRNA-mediated regulatory redundancy and its impact on gene expression evolution remain elusive. By integrating ChIP-seq data from ENCODE and miRNA targeting from TargetScanFly, we quantified miRNA-mediated cFFC redundancy in Drosophila melanogaster embryos and larvae, revealing more than three quarters of miRNA targets are involved in redundant cFFCs. Higher cFFC redundancy, where more miRNAs target the same gene within a cFFC, is correlated with stronger purifying selection, reduced expression divergence between species, and increased expression stability under heat shock stress. Redundant cFFCs primarily regulate older or broadly expressed young genes. These findings highlight the role of miRNA-mediated cFFC redundancy in enhancing gene expression robustness through natural selection.

Is Clonal Integration a Buffer for the Stress of Resource Acquisition Depletion in Eichhornia crassipes (Pontederiaceae) Ramets?

Natural selection favors the allocation of finite resources to different functions maximizing fitness. In this sense, some functions may decrease whereas others increase when resources are limited in a process called a trade-off. However, a great variety of situations may obscure trade-off detection in clonal plants, such as the ability to generate offspring by clonal growth that represents opportunities for resource uptake. The aim of this work was to evaluate if clonal integration and resource availability mediate biomass allocation patterns in E. crassipes through a greenhouse experiment. We set ramets in clonal and isolated conditions, and with and without leaf blades, and compared the relationship of biomass proportion allocated to each vegetative organ. We found that biomass allocation to vegetative structures in E. crassipes is primarily shaped by resource pools and is enhanced by clonal integration as attached ramets invest more in growth and vegetative structures. In this sense, regarding trade-off patterns in biomass allocation among vegetative organs and under resource depletion, clonal integration may represent a way to stabilize biomass allocation patterns and may decrease trade-off importance. We discuss trade-offs and clonal integration as evolutionary strategies that allow plant persistence and improve plants fitness. These findings may support aquatic plant management and control efforts while highlighting the evolutionary significance of clonal integration for plant life strategies.

The historical transformation of individual concepts into populational ones: an explanatory shift in the gestation of the modern synthesis.

In this paper, I will conduct three interrelated analyses. First, I will develop an analysis of various concepts in the history of biology that used to refer to individual-level phenomena but were then reinterpreted by the Modern Synthesis in terms of populations. Second, a similar situation can be found in contemporary evolutionary theory. While different approaches reflect on the causal role of developing organisms in evolution, proponents of the Modern Synthesis refrain from any substantial change by reinterpreting and explaining individual-level phenomena from a population perspective. Finally, I will approach these historical and contemporary debates by arguing for the statistical reading of natural selection, which holds that explanations by natural selection are statistical. My main conclusion is that the historical conceptual reinterpretations belong to a new explanatory strategy developed by the Modern Synthesis based on population thinking. Adopting the statistical point of view has three advantages for the issues discussed in this paper. First, understanding historical conceptual change as part of an explanatory shift fits with the emergence of population biology as a discipline that employs statistical methods. Second, concerning current debates in evolutionary biology, the statisticalist reading can validate the goal of both sides of the dispute. It ascribes an invaluable role to the population statistical explanation of the MS and also commends the study of developmental and organismal causes of adaptive evolution. Finally, the division of explanatory roles in evolutionary biology, embarrassed by statisticalism, can be related to the different interpretations that important biological concepts have undergone throughout history and contemporary biology, i.e., that the division of explanatory roles allows for a division of conceptual interpretations.

Spread of yellow-bill-color alleles favored by selection in the long-tailed finch hybrid system.

Carotenoid pigments produce the yellow and red colors of birds and other vertebrates. Despite their importance in social signaling and sexual selection, our understanding of how carotenoid ornamentation evolves in nature remains limited. Here, we examine the long-tailed finch Poephila acuticauda, an Australian songbird with a yellow-billed western subspecies acuticauda and a red-billed eastern subspecies hecki, which hybridize where their ranges overlap. We found that yellow bills can be explained by the loss of C(4)-oxidation, thus preventing yellow dietary carotenoids from being converted to red. Combining linked-read genomic sequencing and reflectance spectrophotometry measurements of bill color collected from wild-sampled finches and laboratory crosses, we identify four loci that together explain 53% of variance in this trait. The two loci of largest effect contain the genes CYP2J19, an essential enzyme for producing red carotenoids, and TTC39B, an enhancer of carotenoid metabolism. A paucity of protein-coding changes and an enrichment of associated upstream variants suggest that the loss of C(4)-oxidation results from cis-regulatory evolution. Evolutionary genealogy reconstruction indicates that the red-billed phenotype is ancestral and that yellow alleles at CYP2J19 and TTC39B first arose and fixed in acuticauda approximately 100 kya. Yellow alleles subsequently introgressed into hecki less than 5 kya. Across all color loci, acuticauda-derived variants show evidence of selective sweeps, implying that yellow bill coloration has been favored by natural selection. Our study illustrates how evolutionary transitions between yellow and red coloration can be achieved by successive selective events acting on regulatory changes at a few interacting genes.

Lessons to learn for better safeguarding of genetic resources during tree pandemics: The case of ash dieback in Europe

Ash dieback (ADB) has been threatening populations of European ash (Fraxinus excelsior & F. angustifolia) for more than three decades. Although much knowledge has been gathered in the recent past, practical conservation measures have been mostly implemented at local scale. Since range contraction in both ash species is likely to be exacerbated already in the near future by westward expansion of the emerald ash borer and climate change, systematic conservation frameworks need to be developed to avoid long-term population-genetic consequences and depletion of genomic diversity. In this article, we address the advantages and obstacles of conservation approaches aiming to conserve genetic diversity in situ or ex situ during tree pandemics. We are reviewing 47 studies which were published on ash dieback to unravel three important dimensions of ongoing conservation approaches or perceived conservation problems: i) conservation philosophy (i.e. natural selection, resistance breeding or genetic conservation), ii) the spatial scale (ecosystem, country, continent), and iii) the integration of genetic safety margins in conservation planning. Although nearly equal proportions of the reviewed studies mention breeding or active conservation as possible long-term solutions, only 17 % consider that additional threats exist which may further reduce genetic diversity in both ash species. We also identify and discuss several knowledge gaps and limitations which may have limited the initiation of conservation projects at national and international level so far. Finally, we demonstrate that there is not much time left for filling these gaps, because European-wide forest health monitoring data indicates a significant decline of ash populations in the last 5 years.

Differential predation pressure on island pit vipers shape their niches and defensive behavior

Abstract Animals are naturally exposed to several predators, and, given that a single encounter with a predator may cause prey’s death, natural selection will strongly act upon its defensiveness. Therefore, defensive behavior is strongly shaped by predation pressure. Predation rate in islands tends to be relaxed due to lower abundance of predators. The aim of this study was to test the hypothesis that predation rates upon the two island pit vipers Bothrops alcatraz and B. insularis would be lower than correlated mainland B. jararaca. Additionally, we evaluated the defensive behavior of these island snakes in the field to compare with the mainland B. jararaca. Predation pressure upon the insular species was assessed utilizing plasticine replicas, and we evaluated their defensive behavior to approach. These data were compared to those previously reported for B. jararaca. We found a lower predation pressure on B. insularis (from the smaller area) than on B. alcatraz and B. jararaca from the urban mainland forest fragment (areas with intermediate size), and all of them showed lower predation pressure than B. jararaca from the continuous mainland forested area. In the field, island snakes show narrower defensive repertoire than B. jararaca, since most B. insularis fled, and B. alcatraz either fled or remained immobile on approach. Apparently, the predation pressure to which these island pit vipers are exposed shapes their defensive behavior, as well as the niches exploited by them.

Anthropocentrism and Inheritance in Our Mutual Friend: Return, Recognition, Reanimation

Abstract In decentering “Man,” a category long rhetorically constructed as the privileged species of a divine creator, Charles Darwin’s theory of natural selection necessitated a reexamination of how human subjectivity should be represented in fiction. This article uses the example of Charles Dickens’s Our Mutual Friend (1864) to track and scientifically contextualize how anthropocentrism is constructed and interrogated in fiction after publication of On the Origin of Species (1859). Competing nineteenth-century theories of species transmutation recognized physiological fluidity and variation within species and type, yet unlike Darwin, they sought to define uniquely human qualities of mind. Literary narratives, such as Dickens’s final completed novel, engaged this intellectual project—attempting to stabilize a dominant form of human subjectivity while recognizing shifting varieties of being. Protagonist John Harmon is oddly vacant as an imagined human consciousness; his actions create or resolve conflict within the story, but he fails to demonstrate any stable motivation. Depicting the initial misrecognition, and eventual recognition, of an English heir newly returned from abroad, Our Mutual Friend articulates cultural anxieties about the potential mutability of identity and type and the origins and durability of English dominance. Its post-Darwinian model of consciousness challenges traditional realist valorization of the individualistic, agential subject.

Re-Equilibrating Sex Ratios: Adjustment of Reaction Norms in Species With Temperature-Dependent Sex Determination.

Fisher's general principle for sex allocation holds that population sex ratios are typically balanced because parents producing the rare sex are benefited and the rare sex alternates over time. In species that have temperature-dependent sex determination (TSD), thermal reaction norms need to be adjusted at the population level to avoid extremely biased sex ratios and extinction. Extant species with TSD experienced drastic climatic changes in the geological past and must necessarily have mechanisms of adaptation. I propose here a conceptual framework to explain how TSD curves could be adjusted by means of natural selection, based on Fisher's equilibrium sex-ratio principle. Through a process that alternatively favors mothers that tend to produce the rare sex under new temperatures, sex ratios eventually return toward a theoretical equilibrium. Prerequisites for this model are variability among mothers in the tendency to produce a particular sex at a given temperature (i.e., variability in the thermal reaction norm), inheritance of this trend, and higher fitness of the rare sex. This straightforward mechanism could facilitate thermal adaptation in species with TSD over multiple generations.

A scenario for an evolutionary selection of ageing.

Signs of ageing become apparent only late in life, after organismal development is finalized. Ageing, most notably, decreases an individual's fitness. As such, it is most commonly perceived as a non-adaptive force of evolution and considered a by-product of natural selection. Building upon the evolutionarily conserved age-related Smurf phenotype, we propose a simple mathematical life-history trait model in which an organism is characterized by two core abilities: reproduction and homeostasis. Through the simulation of this model, we observe (1) the convergence of fertility's end with the onset of senescence, (2) the relative success of ageing populations, as compared to non-ageing populations, and (3) the enhanced evolvability (i.e. the generation of genetic variability) of ageing populations. In addition, we formally demonstrate the mathematical convergence observed in (1). We thus theorize that mechanisms that link the timing of fertility and ageing have been selected and fixed over evolutionary history, which, in turn, explains why ageing populations are more evolvable and therefore more successful. Broadly speaking, our work suggests that ageing is an adaptive force of evolution.

Population Genetic Dissection of HLA-DPB1 Amino Acid Polymorphism to Infer Selection

Although allele frequency data for most HLA loci provide strong evidence for balancing selection at the allele level, the DPB1 locus is a notable exception, with allele frequencies compatible with neutral evolution (genetic drift) or directional selection in most populations. This discrepancy is especially interesting as evidence for balancing selection has been seen at the nucleotide and amino acid (AA) sequence levels for DPB1. We describe methods used to examine the global distribution of DPB1 alleles and their constituent AA sequences. These methods allow investigation of the influence of natural selection in shaping DPβ diversity in a hierarchical fashion for DPB1 alleles, all polymorphic DPB1 exon 2-encoded AA positions, as well as all pairs and trios of these AA positions. In addition, we describe how asymmetric linkage disequilibrium for all DPB1 exon 2-encoded AA pairs can be used to complement other methods. Application of these methods provides strong evidence for the operation of balancing selection on AA positions 56, 85–87, 36, 55 and 84 (listed in decreasing order of the strength of selection), but no evidence for balancing selection on DPB1 alleles.

Developmental reaction norms vary among families of lizards in response to multivariate nest environments

Developmental plasticity is the capacity of a single genotype to express multiple phenotypes in response to different early‐life environments. Such responses are defined by reaction norms, which may vary among individuals or populations. Variation in developmental reaction norms allows natural selection to operate on plasticity and is rarely examined in vertebrates. We quantified variation in embryonic developmental plasticity within and between populations using the brown anole lizard. We captured lizards from two islands in the Matanzas River (Florida, USA) and incubated their eggs under one of two multivariate treatments that mimicked the temperature, moisture and substrates of nest sites in either a shaded or open habitat. We measured hatchling morphology, performance, and physiology to quantify variation in family‐level reaction norms. We observed evidence of family‐level variation in reaction norms for morphology but not for performance or physiology, indicating an opportunity for natural selection to shape plasticity in hatchling body size. Overall, the results indicate that multiple abiotic conditions in natural nests combine to increase or reduce phenotypic variation, and that family‐level variation in reaction norms provides a potential for natural selection to shape plasticity.

Performing Dynamic Analysis of the Conceret Structures using Genetic Algorithm and Determining the Optimal Range during a Natural Hazard Crisis (Tempe, Arizona, USA)

The approach employs a binary genetic algorithm alongside natural numbers. Given the differences between accelerometers and scale coefficients, the genetic algorithm introduced herein is a hybrid, featuring two chromosomes. A key factor influencing the accuracy and effectiveness of these programs is the precise estimation of their parameters. When these parameters are determined accurately, the gap between the average response spectrum and the design spectrum is significantly minimized. This program operates with two genetics that run concurrently, yielding results that closely approximate the optimal solution. The program itself is capable of offering users a range of desired coefficients, as well as values for the covariance and mutation of each chromosome. The algorithm detailed in this article can generate a new generation of individuals from an infinite array of ground motion records, utilizing processes such as natural selection, mating, and mutation, and continue this cycle until an individual meets the desired characteristics. This paper presents a novel method for optimally selecting accelerometers and scaling them for dynamic time history analysis, aiming for an average response spectrum that closely aligns with the target spectrum, reflecting the expected seismic activity for the structure in Tempe district in Arizona State. Given the relatively high number of parameters involved, reliance on trial-and-error methods is significantly influenced by the user’s skill set; thus, the hybrid genetic algorithm presented here addresses this limitation.

Comprehensive Analysis of Codon Usage Bias in Human Papillomavirus Type 51.

Human papillomavirus type 51 (HPV-51) is associated with various cancers, including cervical cancer. Examining the codon usage bias of the organism can offer valuable insights into its evolutionary patterns and its relationship with the host. This study comprehensively analyzed codon usage bias in HPV-51 by examining 64 complete genome sequences sourced from the NCBI GenBank database. Our analysis revealed no noteworthy preference for codon usage in HPV-51 overall. However, there was a noticeable bias towards A/T-ending codons, accompanied by GC3s below 32%. Dinucleotide frequency analysis revealed reduced frequencies for ApA, CpG, and TpC dinucleotides, while CpA and TpG dinucleotides were more frequent than others. Relative Synonymous Codon Usage analysis revealed 30 favored codons, primarily concluding with A/T nucleotides. Further analysis using Parity Rule 2, Effective Number of Codons plot, and neutrality plot indicated a balance between mutational pressure and natural selection, with natural selection being the primary force shaping codon usage bias. The Isoacceptor tRNA Pool analysis indicates that HPV-51 has a higher translation efficiency within the human cellular translational system. Moreover, the Codon Adaptation Index and Relative Codon Deoptimization Index analyses suggested a moderate adaptation of HPV-51 to human codon preferences. Our discoveries offer valuable perspectives on how HPV-51 evolves and uses genetic codes, contributing to a deeper comprehension of its endurance and disease-causing potential.

Unlocking nature’s drought resilience: a focus on the parsimonious root phenotype and specialised root metabolism in wild Medicago populations

Abstract Background and aims Crop wild relatives, exposed to strong natural selection, exhibit effective tolerance traits against stresses. While an aggressive root proliferation phenotype has long been considered advantageous for a range of stresses, it appears to be counterproductive under drought due to its high metabolic cost. Recently, a parsimonious root phenotype, metabolically more efficient, has been suggested to be better adapted to semiarid environments, although it is not clear that this phenotype is a trait exhibited by crop wild relatives. Methods Firstly, we analysed the root phenotype and carbon metabolism in four Medicago crop wild relatives adapted to a semiarid environment and compared them with the cultivated M. truncatula Jemalong (A17). Secondly, we exposed the cultivated (probably the least adapted genotype to aridity) and the wild (the most common one in arid zones) M. truncatula genotypes to water deficit. The carbon metabolism response in different parts of their roots was analysed. Results A reduced carbon investment per unit of root length was a common trait in the four wild genotypes, indicative of an evolution towards a parsimonious root phenotype. During the water deficit experiment, the wild M. truncatula showed higher tolerance to drought, along with a superior ability of its taproot to partition sucrose and enhanced capacity of its fibrous roots to maintain sugar homeostasis. Conclusion A parsimonious root phenotype and the spatial specialization of root carbon metabolism represent two important drought tolerance traits. This work provides relevant findings to understand the response of Medicago species roots to water deficit.

Macro- and micro-geographic genetic variation in early fitness traits in populations of maritime pine (Pinus pinaster Ait.).

Assessing adaptive genetic variation and its spatial distribution is crucial to conserve forest genetic resources and manage species' adaptive potential. Macro-environmental gradients commonly exert divergent selective pressures that enhance adaptive genetic divergence among populations. Steep micro-environmental variation might also result in adaptive divergence at finer spatial scales, even under high gene flow, but it is unclear how often this is the case. Here, we assess genetic variation in early fitness traits among distant and nearby maritime pine (Pinus pinaster Ait.) populations, to investigate climatic factors associated with trait divergence, and to examine trait integration during seedling establishment. Open pollinated seeds were collected from seven population pairs across the European species distribution, with paired populations spatially close (between <1 km up to 21 km) but environmentally divergent. Seeds were sown in semi-natural conditions at three environmentally contrasting sites, where we monitored seedling emergence, growth and survival. At large spatial scales, we found significant genetic divergence among populations in all studied traits, with certain traits exhibiting association with temperature and precipitation gradients. Significant trait divergence was also detected between pairs of nearby populations. Besides, we found consistent trait correlations across experimental sites, notably heavier seeds and earlier seedling emergence were both associated with higher seedling survival and fitness over two years in all experimental conditions. We identified mean annual temperature and precipitation seasonality as potential drivers of P. pinaster population divergence in the studied early-life traits. Populations genetically diverge also at local spatial scales, potentially suggesting that divergent natural selection can override gene flow along local-scale ecological gradients. These results suggest the species exhibits substantial adaptive potential that has allowed it to survive and evolve under contrasting environmental conditions.

Measuring undergraduate evolution learning using the CANS: psychometric strengths and limitations

BackgroundEvolution continues to be one of the most difficult biological topics to teach, warranting innovative pedagogical tools and assessment strategies for enhancing evolutionary instruction. A major advance in measuring the evolution knowledge of undergraduate students came with the development of the Conceptual Assessment of Natural Selections (CANS). In this study, we use the CANS to measure knowledge and learning of natural selection in a large (N > 6000) sample of undergraduate students to expand upon prior validity testing of this instrument and advance knowledge of student evolutionary reasoning. We apply the Rasch measurement framework to examine if the CANS productively measures the intended construct and investigate the patterns of knowledge and learning about evolution among students with different backgrounds and demographic characteristics.ResultsWhile a unidimensional Rasch model demonstrated acceptable reliabilities and fit for most of the CANS items, some items showed problematic fit statistics and were resistant to instruction. The instrument items also did not span the full range of student abilities, which suggests relatively low measurement precision. Our large sample also allowed rigorous tests of multidimensionality, revealing the presence of multiple dimensions or constructs, some of which may not be intentional. These results generated specific item-level recommendations for improving this instrument. Using Rasch measures to examine learning patterns, we found that pre-test evolution knowledge was low but that there were high learning gains by the end of the course. However, some concept categories were found to be more difficult than others, suggesting the need for more attention to these areas by instructors. We also identified pre-test disparities in evolutionary knowledge by socially defined race and biological sex, yet students from all groups achieved comparable learning gains at the end of the course.ConclusionThe CANS holds great potential to generate critical insights about student evolutionary reasoning and provide information about which instructional approaches most effectively mitigate the notable knowledge disparities among students. We leverage the findings of this study to propose tangible ways in which this instrument may be improved in order to better achieve both of these goals.