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Neutral theory of molecular evolution

The neutral theory of molecular evolution posits that the majority of genetic variations and evolutionary changes observed at the molecular level result from the random fixation of selectively neutral mutations through genetic drift, rather than from natural selection acting on advantageous or deleterious variants.^[1] Proposed by Japanese population geneticist Motoo Kimura in 1968, the theory emerged in response to empirical data showing unexpectedly high rates of nucleotide substitutions and protein evolution across species, which could not be adequately explained by adaptive processes alone.^[1]^[2] At its core, the neutral theory distinguishes between neutral mutations—those with no significant effect on an organism's fitness—and the rarer adaptive mutations that drive phenotypic evolution.^[2] Kimura argued that in sufficiently large populations, the rate of molecular evolution equals the neutral mutation rate, leading to a relatively constant "molecular clock" of evolutionary change, independent of organismal generation time or ecological pressures.^[3] This framework predicts that most fixed differences between species at the DNA or protein level are selectively neutral, with purifying selection eliminating harmful mutations and genetic drift governing the fate of the rest.^[4] Empirical support for the theory includes observations of higher substitution rates in noncoding DNA regions, pseudogenes, and synonymous codon positions compared to functional sites, consistent with neutrality in unconstrained sequences.^[2] Genomic studies, such as those comparing polymorphism levels in Drosophila and mammals, have further validated patterns where neutral processes dominate molecular variation, though positive selection is evident in specific genes like those involved in immunity.^[5] The theory sparked the neutralist-selectionist debate in the 1970s and 1980s, highlighting the complementary roles of drift and selection in evolution.^[4] Subsequent developments, such as Tomoko Ohta's nearly neutral theory in the 1970s, extended Kimura's ideas by incorporating slightly deleterious mutations whose effective neutrality depends on effective population size, providing a more nuanced view for small or bottlenecked populations.^[4] As of 2025, proposals like adaptive tracking with antagonistic pleiotropy suggest that adaptive processes can produce patterns resembling neutral evolution due to frequent environmental changes and pleiotropic effects.^[6] Today, the neutral theory serves as a null hypothesis for interpreting genomic data, influencing fields from phylogenetics to conservation genetics.^[2]

Introduction and Fundamentals

Definition and Principles

The neutral theory of molecular evolution posits that the majority of evolutionary changes at the molecular level arise from the random fixation of selectively neutral mutations through genetic drift, rather than from adaptive natural selection.^[1] Molecular evolution refers to the accumulation of changes in DNA, RNA, or protein sequences over generations, driven by processes such as mutation, genetic drift, and selection.^[2] Under this framework, neutral mutations are those that have little or no effect on an organism's fitness, allowing them to spread or be lost stochastically in populations without selective pressure.^[3] A key principle is the distinction between synonymous and nonsynonymous substitutions: synonymous changes alter the DNA sequence but not the encoded amino acid, often being neutral, while nonsynonymous changes modify the amino acid and are more likely to affect function and thus subject to selection.^[7] The theory emphasizes that the rate of molecular evolution is primarily determined by the neutral mutation rate (μ), where the substitution rate equals μ for neutral alleles in the absence of selection.^[1] This contrasts with phenotypic evolution, where selection plays a dominant role, and positions neutrality as the null hypothesis for molecular changes.^[2] Introduced by Motoo Kimura in 1968, the theory addressed the unexpectedly high levels of molecular polymorphism and substitution rates observed in early protein sequence data, which classical selection-based models struggled to explain without invoking excessive adaptive fixes.^[1] By attributing most fixed differences between species to neutral drift, it provides a parsimonious explanation for the observed uniformity in evolutionary rates across lineages.^[3]

Neutral Mutations and Genetic Drift

In the neutral theory of molecular evolution, mutations are classified based on their impact on organismal fitness: neutral mutations confer no selective advantage or disadvantage, deleterious mutations reduce fitness and are typically purged by natural selection, and advantageous mutations enhance fitness and can spread rapidly. Neutral mutations, in particular, include silent or synonymous substitutions in protein-coding genes, where changes in the DNA sequence do not alter the amino acid encoded by a codon due to the degeneracy of the genetic code; such mutations are estimated to account for approximately 20% of nucleotide replacements and serve as a key example of selectively neutral variation. Genetic drift refers to the random fluctuations in allele frequencies within finite populations, driven by stochastic sampling of gametes across generations rather than deterministic forces like selection. For a new neutral mutation arising as a single copy in a diploid population, the probability of eventual fixation by drift is \frac{1}{2N_e}, where N_e is the effective population size, reflecting the equal chance among all alleles to become the sole variant through random processes. In molecular evolution, genetic drift predominates for neutral variants, especially in populations with small effective sizes, where it can lead to the fixation or loss of alleles independent of their functional effects, resulting in stochastic changes in genetic composition over time. This mechanism underscores how neutral evolution proceeds via random walks in allele space, contrasting with the directional trajectories imposed by selection on non-neutral variants. Representative examples of neutrally evolving sequences include pseudogenes, which are non-functional duplicates of genes that accumulate mutations without selective constraint, evolving at rates consistent with drift alone. Similarly, many non-coding DNA regions, such as introns or intergenic spacers, exhibit neutral evolution, where mutations fix primarily through genetic drift, providing a baseline for comparing selective pressures elsewhere in the genome.

Historical Origins

Precursors and Early Ideas

The foundations of the neutral theory of molecular evolution trace back to early 20th-century developments in population genetics, where key figures grappled with the roles of mutation, selection, and random processes in evolutionary change. Ronald Fisher, in his 1930 book The Genetical Theory of Natural Selection, explored the evolution of dominance in mutations, arguing that most genetic variants arise as recessive deleterious changes but could influence evolutionary dynamics through selective pressures on modifiers of dominance.^[8] Sewall Wright's shifting balance theory, formalized in 1932, emphasized genetic drift as a mechanism allowing small, semi-isolated populations to randomly shift allele frequencies across adaptive valleys toward higher fitness peaks, complementing natural selection in complex landscapes. These ideas highlighted drift's potential alongside selection, setting the stage for later considerations of non-adaptive molecular changes.^[9] A notable precursor emerged in the study of microbial genetics, where Ernst Freese proposed in 1962 that variations in DNA base composition evolve primarily through neutral mutation pressure rather than selection, informed by his analyses of mutagenesis patterns induced by chemical agents like hydroxylamine.^[10] Independently, Noboru Sueoka suggested in the same year that differences in base composition across species arise from biased neutral mutations, challenging selectionist explanations for molecular variation.^[11] These suggestions aligned with observations of high mutational variability in simple organisms and implied that many molecular changes could be selectively neutral. Meanwhile, J.B.S. Haldane's 1957 analysis of the "cost of natural selection" posed a paradox: the substantial genetic load required to fix multiple adaptive substitutions under selection could not easily explain the high levels of polymorphism observed in populations, implying that many variants might persist without selective cost.^[12] The mid-20th century marked a pivotal shift from organismal to molecular perspectives on evolution, catalyzed by the 1953 discovery of DNA's double-helix structure by James Watson and Francis Crick, which illuminated the genetic material's capacity for precise replication and variation. Advances in protein sequencing during the 1950s and 1960s, pioneered by Frederick Sanger's determination of insulin's amino acid sequence in 1951–1952 and Pehr Edman's degradative method from 1956, enabled direct comparisons of molecular structures across species.^[13] These tools revealed unexpectedly rapid and roughly constant rates of protein evolution, as noted by Émile Zuckerkandl and Linus Pauling in their 1962 and 1965 works, where hemoglobin sequence differences suggested a clock-like accumulation of changes uncorrelated with adaptive needs.^[14]^[15] By the 1960s, debates intensified over whether such molecular alterations were primarily adaptive or driven by random fixation, fueled by protein electrophoresis studies like those of Richard Lewontin and John Hubby in 1966, which uncovered extensive silent genetic variation unexplained by classical selectionist models.^[16]

Kimura's Formal Proposal

In 1968, Motoo Kimura, a population geneticist at Japan's National Institute of Genetics in Mishima, formally proposed the neutral theory of molecular evolution in his seminal paper published in Nature.^[1] There, he argued that the majority of evolutionary changes observed at the molecular level—such as nucleotide substitutions in DNA and amino acid replacements in proteins—are not driven by Darwinian natural selection but by the random fixation of selectively neutral mutations through genetic drift.^[1] Kimura posited that in a population, the rate of fixation for these neutral alleles equals the neutral mutation rate per gamete per generation, denoted as k = \mu, where k is the substitution rate and \mu is the mutation rate.^[1] This formulation provided a mathematical foundation for understanding molecular evolution as a stochastic process largely independent of adaptive pressures.^[1] Kimura's proposal was partly motivated by empirical observations of unexpectedly high levels of genetic polymorphism within populations, as documented by researchers like Richard Lewontin, which posed challenges to traditional selectionist explanations that emphasized adaptive maintenance of variation.^[17] By invoking neutrality, Kimura resolved this apparent paradox: most polymorphisms could persist transiently due to drift before fixation or loss, without requiring balancing selection.^[17] His work built on earlier conceptual precursors, such as ideas about neutral mutations in biochemical contexts, but formalized them within a rigorous population genetic framework.^[18] A key innovation in Kimura's 1968 paper was the application of diffusion equations—derived from the works of Sewall Wright and Ronald Fisher—to approximate the probability distribution of allele frequencies under genetic drift in finite populations.^[1] This integration allowed precise predictions about fixation probabilities and rates, leading to the expectation of approximately constant evolutionary rates at the molecular level across lineages, a concept that underpinned the molecular clock hypothesis.^[1] Kimura's insights were shaped by his long-term research environment at the National Institute of Genetics, where collaborations with colleagues like Tomoko Ohta and others facilitated the development of these ideas through ongoing discussions on stochastic processes in evolution.^[19]^[17] Independently of Kimura, Jack L. King and Thomas H. Jukes advanced a similar viewpoint in their 1969 Science paper titled "Non-Darwinian Evolution," emphasizing that protein evolution proceeds primarily through neutral mutations fixed by drift rather than adaptive changes.^[7] They highlighted the abundance of synonymous codon changes and the evolution of non-functional DNA sequences, such as pseudogenes, as evidence for non-adaptive processes dominating at the molecular scale.^[7] This concurrent proposal reinforced Kimura's framework, shifting focus from phenotypic adaptation to the neutral dynamics of genetic material.^[17]

Core Theoretical Components

Functional Constraints

In the neutral theory of molecular evolution, functional constraints refer to the selective pressures imposed by the biological roles of genetic regions, which determine the proportion of mutations that are selectively neutral. Regions under strong functional constraint, such as active sites in enzymes or critical protein domains, experience intense purifying selection that eliminates most deleterious mutations, resulting in a low rate of neutral substitutions.^[4] In contrast, less constrained areas, like non-coding introns or certain linker peptides, tolerate a higher fraction of neutral mutations, leading to faster evolutionary rates.^[20] This variability arises because the theory posits that the evolutionary rate is proportional to the neutral mutation rate, modulated by the degree of constraint rather than solely by genetic drift.^[21] Classic examples illustrate these differences in constraint levels. Fibrinopeptides, short peptides cleaved from fibrinogen during blood clotting, exhibit minimal functional importance after cleavage and thus evolve rapidly, with substitution rates as high as 9.0 per amino acid site per 10^9 years, consistent with near-neutral evolution.^[21] Conversely, histones, which are essential for DNA packaging and highly conserved across eukaryotes, face stringent constraints, evolving slowly at 0.006 substitutions per site per 10^9 years, though synonymous changes at the third codon position occur more freely.^[21] The ratio of nonsynonymous to synonymous substitutions (dN/dS) serves as a key metric to quantify these constraints: values below 1 indicate purifying selection due to functional importance, while dN/dS approaching 1 suggests relaxed constraint and neutrality.^[22] The implications of functional constraints within the neutral theory are profound, predicting heterogeneous evolutionary rates across the genome driven by varying levels of constraint, independent of population size or environmental factors.^[23] For instance, highly constrained coding regions accumulate substitutions at rates close to the neutral mutation rate, while unconstrained regions evolve faster, explaining observed molecular clock variations without invoking adaptive selection.^[24] This framework underscores that most genomic evolution proceeds neutrally in permissible spaces, with constraints shaping the landscape of permissible mutations. Functional constraints are inferred through comparative genomics by analyzing substitution patterns in aligned sequences across species. Under neutral theory, slower divergence in functional regions relative to neutrally evolving sites signals purifying selection, quantified via phylogenetic models that estimate branch lengths and substitution rates.^[25] For example, multi-species alignments reveal conserved blocks where the substitution rate is significantly below the neutral expectation, directly attributing reduced evolution to constraint.^[26] This approach has become foundational for mapping constraint landscapes without prior functional annotations.^[27]

Quantitative Models

The quantitative models of the neutral theory provide a mathematical framework to describe the dynamics of neutral mutations under genetic drift in finite populations, assuming no natural selection acts on them. These models rely on several key assumptions: an infinite number of possible sites in the genome where mutations can occur, neutrality of mutations (i.e., no effect on fitness), constant population size, random mating, and a constant mutation rate μ per site per generation.^[1] The population is typically modeled as diploid with effective size N_e, often denoted as 2N for 2N gene copies, and mutations arise at rate μ per gene copy per generation. A central result is the fixation rate of neutral mutations, which determines the rate of molecular evolution. For a neutral allele arising as a single mutant in a population of 2N gene copies, the probability of ultimate fixation by drift is 1/(2N), as derived from diffusion theory. The total number of new mutations per generation across the population is 2Nμ, so the rate of substitution (fixations per generation) λ equals (2Nμ) × (1/(2N)) = μ.^[1] Thus, the evolutionary rate k, measured in substitutions per site per year, equals the neutral mutation rate μ, independent of population size.^[1] This implies that neutral evolution proceeds at a pace set solely by mutation input, with drift determining which variants fix. The infinite alleles model, developed to analyze genetic diversity at loci where each mutation produces a novel allele, further elucidates neutral dynamics. In this model, mutations are irreversible and create infinitely many possible alleles, with no back-mutations to prior states. Under neutrality and drift, the probability that a new neutral mutation fixes is again 1/(2N), leading to an equilibrium where the rate of allele substitutions equals μ. The model predicts the expected number of alleles in a population sample and the average heterozygosity, both functions of the population mutation parameter θ = 4Nμ, providing a basis for measuring neutral diversity without selection. The infinite sites model (ISM) extends this to nucleotide sequences, assuming an infinite number of sites in the genome such that each mutation occurs at a unique site, precluding multiple hits or back-mutations at the same position. This model predicts a constant rate of neutral evolution, manifesting as the molecular clock: the number of substitutions d between two lineages diverges linearly with time t since their common ancestor, given by d = 2μt. For a population, the expected number of segregating sites in a sample scales with θ = 4Nμ, allowing inference of demographic history under neutrality. Diffusion approximations, pioneered by Wright and extended by Kimura, mathematically describe the stochastic change in allele frequencies due to drift in continuous time. For neutral alleles, the diffusion equation for the probability density φ(p, t) of frequency p at time t is \frac{\partial \phi}{\partial t} = \frac{1}{4N} \frac{\partial^2}{\partial p^2} \left[ p(1-p) \phi \right] reflecting variance in frequency change of p(1-p)/(2N) per generation. Solutions yield the fixation probability and mean time to fixation ≈ 4N generations for neutral mutants that fix. These approximations also predict variance in substitution rates across lineages, with relative variance 2t / T (where T is total divergence time), due to fluctuating drift effects.

Theoretical Extensions

Nearly Neutral Theory

The nearly neutral theory of molecular evolution, proposed by Tomoko Ohta in 1973, extends the strict neutral theory by incorporating mutations with small selection coefficients (|s|), where these mutations behave nearly neutral when |s| < 1/(2N_e) and genetic drift dominates their fate, with N_e denoting the effective population size.^[28] In this framework, slightly deleterious mutations can accumulate and fix more readily in populations with smaller N_e, as selection becomes ineffective against them, contrasting with the strict neutral model's assumption that all fixed mutations have zero selective effect.^[28]^[29] A primary distinction from the strict neutral theory lies in the dependence of effective neutrality on N_e: in large populations, even weakly deleterious mutations are purged by selection, reducing substitution rates, whereas in small populations, drift allows fixation of such alleles, leading to higher rates of molecular change.^[29] This population-size effect implies that evolutionary rates vary across lineages based on fluctuations in N_e, such as during population bottlenecks or expansions.^[29] The theory predicts variable substitution rates among lineages due to changes in N_e, with smaller populations exhibiting faster accumulation of slightly deleterious mutations, and lower levels of polymorphism in large populations where selection more efficiently purges weakly deleterious variants at selected sites.^[29] For instance, species with larger N_e, like certain Drosophila populations, show reduced polymorphism compared to those with smaller N_e, as selection more efficiently removes variants.^[29] Applications of the nearly neutral theory include explanations for codon usage bias, where in organisms with large N_e such as bacteria, weak selection favors optimal codons, while in small-N_e mammals, mutation pressure dominates, resulting in less bias.^[29] It also accounts for the rapid evolution of introns, which experience minimal functional constraints and thus accumulate nearly neutral changes at rates around 5 × 10^{-9} substitutions per site per year.^[29] Furthermore, the theory integrates with analyses of site frequency spectra, revealing patterns of polymorphism in genomes like Drosophila, where locus-specific variations reflect the interplay of drift and weak selection.^[29]^[30]

Constructive Neutral Evolution

Constructive neutral evolution (CNE) posits that neutral genetic changes can progressively build biological complexity by creating interdependencies that become essential over time, without requiring initial adaptive selection. Proposed by Arlin Stoltzfus in 1999, this framework extends neutral theory by emphasizing how non-adaptive processes, driven by genetic drift and biased variation, lead to the emergence of novel structures and functions. In CNE, redundant or gratuitous capacities in biological systems allow neutral mutations to accumulate, fostering epistatic interactions that lock in complexity, effectively ratcheting up molecular elaboration.^[31] Key mechanisms include gene duplication followed by neutral divergence, where duplicated genes initially provide redundancy, permitting deleterious mutations in one copy that impair its original function while the other compensates. Over time, these mutations fix via drift, creating mutual dependencies between the duplicates, as seen in subfunctionalization models. Another mechanism is the neutral coalescence of pathways, where independent components with excess capacity merge into interdependent networks; for instance, chaperone proteins can evolve to stabilize proteins harboring neutral destabilizing mutations, making the chaperone indispensable despite no initial selective advantage. These processes rely on the interplay of neutral drift with intrinsic biases, such as higher rates of duplication or mutation in certain genomic regions, to directionally increase complexity.^[31]^[32] Representative examples illustrate CNE's role in molecular systems. The evolution of spliceosomes likely arose from neutral incorporation and fragmentation of self-splicing introns, where gratuitous reassociation capacities allowed stepwise addition of snRNAs and proteins, transforming a simple RNA-based mechanism into a complex ribonucleoprotein machine essential for eukaryotic gene expression. Similarly, mitochondrial protein import systems have diversified through neutral evolution, with components like the TIM22 complex accruing subunits via duplication and drift, enhancing carrier protein insertion without adaptive pressure. In chaperone systems, neutral mutations reducing protein stability can spread if buffered by existing chaperones, leading to obligatory interactions that elevate cellular complexity.^[31]^[32]^[33] By demonstrating that neutrality can act as a creative force, CNE challenges adaptationist paradigms that attribute all complexity to natural selection, suggesting instead that drift-driven interdependencies provide a substrate upon which selection may later act. This perspective reframes molecular evolution as a balance of neutral construction and selective refinement, highlighting non-adaptive origins for many elaborate traits.^[32]^[33]

The Neutralist-Selectionist Debate

Neutralist Perspectives

Neutralists argue that the high levels of silent (synonymous) polymorphisms observed in coding regions are primarily governed by random genetic drift rather than natural selection, as these mutations do not alter protein function and accumulate at rates predicted by neutral expectations. This pattern aligns with Motoo Kimura's foundational emphasis on non-adaptive molecular changes, where the majority of evolutionary substitutions at the molecular level result from neutral mutations fixed by drift in finite populations. Similarly, the uniformity of the molecular clock—evidenced by roughly constant rates of nucleotide substitution across diverse species—supports the view that most molecular evolution proceeds independently of adaptive pressures, as selection would introduce irregular rate variations. In response to critics questioning the persistence of extensive polymorphisms without imposing a heavy selective load, neutralists contend that the vast majority of mutations are either neutral or slightly deleterious transients that do not contribute to long-term fixation, thereby resolving the polymorphism-selection paradox without invoking widespread balancing selection.^[34] Tomoko Ohta extended this perspective by highlighting how the efficiency of natural selection is constrained by effective population size (N_e), such that in smaller populations, slightly deleterious mutations behave more neutrally and fix more readily via drift, amplifying non-adaptive evolution. This population-size dependence underscores why neutral processes dominate molecular change even when weak selection operates. Statistical tests like Tajima's D are used to test for departures from neutral expectations in molecular evolution. Values close to zero are consistent with the neutral hypothesis of mutation-drift balance, while significant deviations—such as negative values indicating an excess of rare variants—may reflect demographic changes like population expansion or the effects of purifying or positive selection.^[35] Such tests provide a framework for assessing the prevalence of neutral processes across genomes, though interpretations must account for potential confounding factors like demography.

Selectionist Counterarguments

Selectionists have challenged the neutral theory by highlighting evidence of positive selection driving molecular evolution, particularly in genes involved in immune responses and adaptations to environmental pressures. For instance, analyses of major histocompatibility complex (MHC) class I genes reveal patterns of nucleotide substitution consistent with overdominant selection, where nonsynonymous changes in antigen recognition sites exceed synonymous ones, indicating adaptive diversification to counter pathogen evolution. Similarly, the McDonald-Kreitman test, applied to loci like alcohol dehydrogenase in Drosophila, demonstrates an excess of fixed amino acid replacement substitutions relative to polymorphisms, suggesting a significant role for positive selection in interspecies divergence beyond what genetic drift alone would predict.^[36] Critics argue that the neutral theory underestimates the prevalence of adaptive or nearly adaptive polymorphisms, proposing instead that most molecular variation arises from weak but pervasive selection pressures rather than random drift. John Gillespie, a prominent selectionist, contended that molecular evolution is better explained by his shifting balance theory, where fluctuating environmental selection leads to episodic adaptive fixations, challenging the constant rate predicted by neutrality.^[37] Additionally, selective hitchhiking—where neutral variants linked to beneficial mutations are swept to fixation—can mimic the signatures of drift, such as reduced polymorphism levels, thereby confounding neutralist interpretations of genomic data. This tension persists in modern analyses, where ratios of nonsynonymous to synonymous substitutions (dN/dS > 1) at specific loci, including immune-related genes, signal ongoing positive selection that neutral models struggle to accommodate without ad hoc adjustments. Furthermore, epistatic interactions among mutations complicate neutrality assessments, as the fitness effects of variants depend on genetic background, potentially masking weak selection and inflating apparent neutral evolution rates.^[38] These critiques underscore selectionists' view that adaptive processes dominate molecular change, contrasting with neutralist emphasis on stochastic fixation. Key figures in the debate included neutralists like Jack Lester King and Thomas Jukes, who emphasized non-Darwinian evolution at the molecular level, and selectionists like John Maynard Smith, who advocated for the pervasive role of natural selection. As of 2024, the debate continues to shape the field, with genomic evidence supporting neutral processes as the null hypothesis complemented by selection at functional sites.^[5]

Empirical Support and Evidence

Classical Evidence

The concept of the molecular clock, proposed by Émile Zuckerkandl and Linus Pauling in 1965, posited that molecular evolution proceeds at a relatively constant rate over time, akin to the ticking of a clock, due to the accumulation of neutral mutations.^[39] This idea was supported by observations of steady substitution rates in specific proteins across mammalian lineages; for instance, hemoglobin evolved slowly with approximately one amino acid replacement every 14 million years per chain, while fibrinopeptides changed more rapidly at about 100 replacements per 100 million years, yet both exhibited consistent tempos when calibrated against fossil records.^[40] These patterns aligned with neutral theory's prediction of divergence rates equaling the mutation rate μ for unconstrained sites, providing early empirical validation without invoking adaptive selection as the primary driver.^[41] Pioneering studies on protein polymorphism further bolstered neutral expectations by revealing unexpectedly high levels of genetic variation in natural populations. In 1966, Richard Lewontin and John Hubby employed starch gel electrophoresis to survey 18 enzyme loci in Drosophila pseudoobscura, uncovering that approximately 30% of the loci were polymorphic, with an average heterozygosity of about 12% per locus. This elevated heterozygosity matched the neutral model's anticipated value of 4N_e μ, where N_e is the effective population size and μ the mutation rate, suggesting that much of the observed variation arose from random genetic drift rather than balancing selection.^[42] Such findings challenged classical population genetics views of low variability under strict selective constraints and highlighted the role of neutral processes in maintaining polymorphism. Observations of substitution rates in duplicated genes and pseudogenes provided additional classical support for neutral divergence. Susumu Ohno's 1970 analysis of gene duplications argued that redundant copies could accumulate mutations freely post-duplication, evolving at the neutral rate without immediate functional penalty, as seen in the rapid divergence of paralogous genes like those in the globin family. Complementing this, early examinations of pseudogenes—non-functional gene relics—demonstrated evolution at the baseline mutation rate μ, unhindered by purifying selection; for example, the human β-globin pseudogene showed synonymous substitution rates comparable to those in unconstrained non-coding regions, consistent with neutral fixation.^[43] These patterns underscored how neutral processes facilitated genomic innovation through relaxed constraints after duplication events. Early comparative studies between humans and chimpanzees also revealed neutral-like divergence patterns, particularly in less constrained regions. In their 1975 work, Mary-Claire King and Allan Wilson compared protein sequences and immunological distances, finding only about 1% amino acid differences despite profound phenotypic disparities, which they attributed to regulatory changes in non-coding DNA evolving under minimal selective pressure. This implied that much of the genomic divergence, estimated at around 1-2% via early DNA hybridization techniques, proceeded neutrally in non-coding sequences, aligning with the theory's emphasis on drift-dominated evolution in functional slack areas.^[44]

Modern Genomic Evidence

The advent of next-generation sequencing (NGS) technologies has revolutionized the study of molecular evolution by enabling whole-genome analyses across diverse organisms, revealing patterns consistent with the neutral theory in both microbial and eukaryotic genomes. These methods have facilitated the detection of polymorphisms at unprecedented scales, showing that the majority of genetic variation arises through neutral processes rather than adaptive selection. For instance, NGS has uncovered extensive neutral divergence in non-coding regions of eukaryotic genomes, where substitution rates align with expectations of genetic drift rather than functional constraints.^[45] In human population genomics, projects like the 1000 Genomes Project have demonstrated that a substantial portion of genomic variation—estimated at around 80-90% in unconstrained regions—evolves neutrally, as evidenced by the cataloging of millions of single-nucleotide polymorphisms (SNPs) that show no signatures of selection. Site frequency spectra (SFS) derived from these datasets frequently match predictions from neutral drift models, with allele frequencies distributed according to coalescent theory under constant population size, indicating that most variants fix or segregate via random genetic drift rather than directional selection. This alignment holds particularly for synonymous sites and intergenic regions, underscoring the prevalence of neutral evolution across the human genome.^[46]^[47] Recent experimental approaches using CRISPR-Cas9, developed post-2012, have provided direct evidence for neutral mutation fixation in cell lines. Studies introducing targeted mutations into cancer cell lines, such as basal breast cancer models, observed no significant fitness effects for many variants, allowing them to drift and fix neutrally in evolving populations, consistent with neutral theory predictions. Complementing this, analyses of ancient DNA have confirmed the molecular clock's regularity, as seen in Neanderthal genomes where neutral substitution rates remain constant over tens of thousands of years, supporting drift-dominated evolution in archaic human lineages.^[48]^[49] Big data integrations, powered by NGS, have leveraged machine learning to identify neutral evolution in vast genomic datasets. Convolutional neural networks, such as those in ImaGene, classify genomic windows as neutral by training on simulated drift patterns versus selection signals, revealing that large swaths of microbial and eukaryotic genomes lack adaptive footprints. In viral evolution, SARS-CoV-2 provides a stark example: genomic analyses show nearly neutral evolution in many lineages, with neutral sweeps dominating intra-host dynamics and early population spread, where mutations fix primarily through drift rather than strong positive selection.^[50]^[51] Contemporary challenges integrate these findings with adaptive processes, such as in cancer genomics, where neutral mutations accumulate alongside selection hotspots driving tumor evolution. 2020s studies using dN/dS ratios in non-model organisms, like the fungal pathogen Pyricularia oryzae, resolve ongoing debates by showing overall dN/dS values below 1 indicative of purifying selection on functional sites but neutral dominance (dN/dS ≈ 1) in non-essential regions, with localized positive selection (dN/dS > 1) in adaptation hotspots. These insights affirm neutral theory's core tenet that most molecular changes are selectively neutral, while highlighting nuanced interactions with selection in complex genomes.^[52]

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A Nearly Neutral Model of Molecular Signatures of Natural Selection ...
Abstract. The nearly neutral theory is a common framework to describe natural selection at the molecular level. This theory emphasizes the importance of sl.
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On the Possibility of Constructive Neutral Evolution
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Constructive neutral evolution: exploring evolutionary theory's ...
Oct 13, 2012 · Constructive neutral evolution (CNE) suggests that neutral evolution may follow a stepwise path to extravagance.
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Constructive Neutral Evolution 20 Years Later | Journal of Molecular ...
Feb 19, 2021 · The theory of Constructive Neutral Evolution has been around for more than two decades. Despite this, few molecular and evolutionary biologists ...
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Aug 24, 2023 · Kimura (1976) argued that the nearly neutral theory accentuated the difference between neutralists and selectionists. He wrote (p. 249): 'If we ...
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Epistasis Increases the Rate of Conditionally Neutral Substitution in ...
Kimura observed that the rate of neutral substitution should equal the neutral mutation rate. This classic result is central to our understanding of molecular ...
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Frequency spectrum: Two commonly studied properties of a sample of neutral diversity at a locus are the expected number of segregating sites in a sample of ...
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Aug 5, 2014 · This phylogeny was constructed with the BEAST software using 59 cAMHs, 21 aAMHs, and 5 archaic Neanderthals. The topology presented is well ...
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ImaGene: a convolutional neural network to quantify natural ...
Nov 22, 2019 · ImaGene to apply convolutional neural networks on population genomic data for the detection and quantification of natural selection.
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Natural selection in the evolution of SARS-CoV-2 in bats created a ...
Here we assess the types of natural selection taking place in Sarbecoviruses in horseshoe bats versus the early SARS-CoV-2 evolution in humans.
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Genomic profiles of Pyricularia oryzae in Sub-Saharan Africa
Oct 6, 2025 · 00128431, which had highly variable dN/dS ratios. Collectively, these results highlight the dual forces of purifying. selection and localized ...