Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos.

5-methylcytosine is a major epigenetic modification that is sometimes called "the fifth nucleotide." However, our knowledge of how offspring inherit the DNA methylome from parents is limited. We generated nine single-base resolution DNA methylomes, including zebrafish gametes and early embryos. The oocyte methylome is significantly hypomethylated compared to sperm. Strikingly, the paternal DNA methylation pattern is maintained throughout early embryogenesis. The maternal DNA methylation pattern is maintained until the 16-cell stage. Then, the oocyte methylome is gradually discarded through cell division and is progressively reprogrammed to a pattern similar to that of the sperm methylome. The passive demethylation rate and the de novo methylation rate are similar in the maternal DNA. By the midblastula stage, the embryo's methylome is virtually identical to the sperm methylome. Moreover, inheritance of the sperm methylome facilitates the epigenetic regulation of embryogenesis. Therefore, besides DNA sequences, sperm DNA methylome is also inherited in zebrafish early embryos.

Adaptation in Unstable Environments and Global Gene Losses: Small but Stable Gene Networks by the May-Wigner Theory.

Although gene loss is common in evolution, it remains unclear whether it is an adaptive process. In a survey of seven major mangrove clades that are woody plants in the intertidal zones of daily environmental perturbations, we noticed that they generally evolved reduced gene numbers. We then focused on the largest clade of Rhizophoreae and observed the continual gene set reduction in each of the eight species. A great majority of gene losses are concentrated on environmental interaction processes, presumably to cope with the constant fluctuations in the tidal environments. Genes of the general processes for woody plants are largely retained. In particular, fewer gene losses are found in physiological traits such as viviparous seeds, high salinity, and high tannin content. Given the broad and continual genome reductions, we propose the May-Wigner theory (MWT) of system stability as a possible mechanism. In MWT, the most effective solution for buffering continual perturbations is to reduce the size of the system (or to weaken the total genic interactions). Mangroves are unique as immovable inhabitants of the compound environments in the land-sea interface, where environmental gradients (such as salinity) fluctuate constantly, often drastically. Extending MWT to gene regulatory network (GRN), computer simulations and transcriptome analyses support the stabilizing effects of smaller gene sets in mangroves vis-à-vis inland plants. In summary, we show the adaptive significance of gene losses in mangrove plants, including the specific role of promoting phenotype innovation and a general role in stabilizing GRN in unstable environments as predicted by MWT.

Canalization of Phenotypes-When the Transcriptome is Constantly but Weakly Perturbed.

Recent studies have increasingly pointed to microRNAs (miRNAs) as the agent of gene regulatory network (GRN) stabilization as well as developmental canalization against constant but small environmental perturbations. To analyze mild perturbations, we construct a Dicer-1 knockdown line (dcr-1 KD) in Drosophila that modestly reduces all miRNAs by, on average, ∼20%. The defining characteristic of stabilizers is that, when their capacity is compromised, GRNs do not change their short-term behaviors. Indeed, even with such broad reductions across all miRNAs, the changes in the transcriptome are very modest during development in stable environment. By comparison, broad knockdowns of other regulatory genes (esp. transcription factors) by the same method should lead to drastic changes in the GRNs. The consequence of destabilization may thus be in long-term development as postulated by the theory of canalization. Flies with modest miRNA reductions may gradually deviate from the developmental norm, resulting in late-stage failures such as shortened longevity. In the optimal culture condition, the survival to adulthood is indeed normal in the dcr-1 KD line but, importantly, adult longevity is reduced by ∼90%. When flies are stressed by high temperature, dcr-1 KD induces lethality earlier in late pupation and, as the perturbations are shifted earlier, the affected stages are shifted correspondingly. Hence, in late stages of development with deviations piling up, GRN would be increasingly in need of stabilization. In conclusion, miRNAs appear to be a solution to weak but constant environmental perturbations.

Intra- vs. Interhost Evolution of SARS-CoV-2 Driven by Uncorrelated Selection-The Evolution Thwarted.

In viral evolution, a new mutation has to proliferate within the host (Stage I) in order to be transmitted and then compete in the host population (Stage II). We now analyze the intrahost single nucleotide variants (iSNVs) in a set of 79 SARS-CoV-2 infected patients with most transmissions tracked. Here, every mutation has two measures: 1) iSNV frequency within each individual host in Stage I; 2) occurrence among individuals ranging from 1 (private), 2-78 (public), to 79 (global) occurrences in Stage II. In Stage I, a small fraction of nonsynonymous iSNVs are sufficiently advantageous to rise to a high frequency, often 100%. However, such iSNVs usually fail to become public mutations. Thus, the selective forces in the two stages of evolution are uncorrelated and, possibly, antagonistic. For that reason, successful mutants, including many variants of concern, have to avoid being eliminated in Stage I when they first emerge. As a result, they may not have the transmission advantage to outcompete the dominant strains and, hence, are rare in the host population. Few of them could manage to slowly accumulate advantageous mutations to compete in Stage II. When they do, they would appear suddenly as in each of the six successive waves of SARS-CoV-2 strains. In conclusion, Stage I evolution, the gate-keeper, may contravene the long-term viral evolution and should be heeded in viral studies.

The Ecology and Evolution of Cancer: The Ultra-Microevolutionary Process.

Although tumorigenesis has been accepted as an evolutionary process ( 20 , 102 ), many forces may operate differently in cancers than in organisms, as they evolve at vastly different time scales. Among such forces, natural selection, here defined as differential cellular proliferation among distinct somatic cell genotypes, is particularly interesting because its action might be thwarted in multicellular organisms ( 20 , 29 ). In this review, selection is analyzed in two stages of cancer evolution: Stage I is the evolution between tumors and normal tissues, and Stage II is the evolution within tumors. The Cancer Genome Atlas (TCGA) data show a low degree of convergent evolution in Stage I, where genetic changes are not extensively shared among cases. An equally important, albeit much less highlighted, discovery using TCGA data is that there is almost no net selection in cancer evolution. Both positive and negative selection are evident but they neatly cancel each other out, rendering total selection ineffective in the absence of recombination. The efficacy of selection is even lower in Stage II, where neutral (non-Darwinian) evolution is increasingly supported by high-density sampling studies ( 81 , 123 ). Because natural selection is not a strong deterministic force, cancers usually evolve divergently even in similar tissue environments.

Adaptation to a new environment with pre-adaptive genomic features - Evidence from woody plants colonizing the land-sea interface.

Adaptation to new environments is a key evolutionary process which presumably involves complex genomic changes. Mangroves, a collection of approximately 80 woody plants that have independently invaded intertidal zones >20 times, are ideal for studying this process. We assembled near-chromosome-scale genomes of three Xylocarpus species as well as an outgroup species using single-molecule real-time sequencing. Phylogenomic analysis reveals two separate lineages, one with the mangrove Xylocarpus granatum and the other comprising a mangrove Xylocarpus moluccensis and a terrestrial Xylocarpus rumphii. In conjunction with previous studies, we identified several genomic features associated with mangroves: (i) signals of positive selection in genes related to salt tolerance and root development; (ii) genome-wide elevated ratios of non-synonymous to synonymous substitution relative to terrestrial relatives; and (iii) active elimination of long terminal repeats. These features are found in the terrestrial X. rumphii in addition to the two mangroves. These genomic features, not being strictly mangrove-specific, are hence considered pre-adaptive. We infer that the coastal but non-intertidal habitat of X. rumphii may have predisposed the common ancestor to invasion of true mangrove habitats. Other features including the preferential retention of duplicated genes and intolerance to pseudogenization are not found in X. rumphii and are likely true adaptive features in mangroves. In conclusion, by studying adaptive shift and partial shifts among closely related species, we set up a framework to study genomic features that are acquired at different stages of the pre-adaptation and adaptation to new environments.

The Runaway Evolution of SARS-CoV-2 Leading to the Highly Evolved Delta Strain.

In new epidemics after the host shift, the pathogens may experience accelerated evolution driven by novel selective pressures. When the accelerated evolution enters a positive feedback loop with the expanding epidemics, the pathogen's runaway evolution may be triggered. To test this possibility in coronavirus disease 2019 (COVID-19), we analyze the extensive databases and identify five major waves of strains, one replacing the previous one in 2020-2021. The mutations differ entirely between waves and the number of mutations continues to increase, from 3-4 to 21-31. The latest wave in the fall of 2021 is the Delta strain which accrues 31 new mutations to become highly prevalent. Interestingly, these new mutations in Delta strain emerge in multiple stages with each stage driven by 6-12 coding mutations that form a fitness group. In short, the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from the oldest to the youngest wave, and from the earlier to the later stages of the Delta wave, is a process of acceleration with more and more mutations. The global increase in the viral population size (M(t), at time t) and the mutation accumulation (R(t)) may have indeed triggered the runaway evolution in late 2020, leading to the highly evolved Alpha and then Delta strain. To suppress the pandemic, it is crucial to break the positive feedback loop between M(t) and R(t), neither of which has yet to be effectively dampened by late 2021. New waves after Delta, hence, should not be surprising.

Evolution under Spatially Heterogeneous Selection in Solid Tumors.

Spatial genetic and phenotypic diversity within solid tumors has been well documented. Nevertheless, how this heterogeneity affects temporal dynamics of tumorigenesis has not been rigorously examined because solid tumors do not evolve as the standard population genetic model due to the spatial constraint. We therefore, propose a neutral spatial (NS) model whereby the mutation accumulation increases toward the periphery; the genealogical relationship is spatially determined and the selection efficacy is blunted (due to kin competition). In this model, neutral mutations are accrued and spatially distributed in manners different from those of advantageous mutations. Importantly, the distinctions could be blurred in the conventional model. To test the NS model, we performed a three-dimensional multiple microsampling of two hepatocellular carcinomas. Whole-genome sequencing (WGS) revealed a 2-fold increase in mutations going from the center to the periphery. The operation of natural selection can then be tested by examining the spatially determined clonal relationships and the clonal sizes. Due to limited migration, only the expansion of highly advantageous clones can sweep through a large part of the tumor to reveal the selective advantages. Hence, even multiregional sampling can only reveal a fraction of fitness differences in solid tumors. Our results suggest that the NS patterns are crucial for testing the influence of natural selection during tumorigenesis, especially for small solid tumors.

Mutations Beget More Mutations-Rapid Evolution of Mutation Rate in Response to the Risk of Runaway Accumulation.

The rapidity with which the mutation rate evolves could greatly impact evolutionary patterns. Nevertheless, most studies simply assume a constant rate in the time scale of interest (Kimura 1983; Drake 1991; Kumar 2005; Li 2007; Lynch 2010). In contrast, recent studies of somatic mutations suggest that the mutation rate may vary by several orders of magnitude within a lifetime (Kandoth et al. 2013; Lawrence et al. 2013). To resolve the discrepancy, we now propose a runaway model, applicable to both the germline and soma, whereby mutator mutations form a positive-feedback loop. In this loop, any mutator mutation would increase the rate of acquiring the next mutator, thus triggering a runaway escalation in mutation rate. The process can be initiated more readily if there are many weak mutators than a few strong ones. Interestingly, even a small increase in the mutation rate at birth could trigger the runaway process, resulting in unfit progeny. In slowly reproducing species, the need to minimize the risk of this uncontrolled accumulation would thus favor setting the mutation rate low. In comparison, species that starts and ends reproduction sooner do not face the risk and may set the baseline mutation rate higher. The mutation rate would evolve in response to the risk of runaway mutation, in particular, when the generation time changes. A rapidly evolving mutation rate may shed new lights on many evolutionary phenomena (Elango et al. 2006; Thomas et al. 2010, 2018; Langergraber et al. 2012; Besenbacher et al. 2019).

Death of new microRNA genes in Drosophila via gradual loss of fitness advantages.

The prevalence of de novo coding genes is controversial due to length and coding constraints. Noncoding genes, especially small ones, are freer to evolve de novo by comparison. The best examples are microRNAs (miRNAs), a large class of regulatory molecules ∼22 nt in length. Here, we study six de novo miRNAs in Drosophila, which, like most new genes, are testis-specific. We ask how and why de novo genes die because gene death must be sufficiently frequent to balance the many new births. By knocking out each miRNA gene, we analyzed their contributions to the nine components of male fitness (sperm production, length, and competitiveness, among others). To our surprise, the knockout mutants often perform better than the wild type in some components, and slightly worse in others. When two of the younger miRNAs are assayed in long-term laboratory populations, their total fitness contributions are found to be essentially zero. These results collectively suggest that adaptive de novo genes die regularly, not due to the loss of functionality, but due to the canceling out of positive and negative fitness effects, which may be characterized as "quasi-neutrality." Since de novo genes often emerge adaptively and become lost later, they reveal ongoing period-specific adaptations, reminiscent of the "Red-Queen" metaphor for long-term evolution.

Tumorigenesis as the Paradigm of Quasi-neutral Molecular Evolution.

In the absence of both positive and negative selections, coding sequences evolve at a neutral rate (R = 1). Such a high genomic rate is generally not achievable due to the prevalence of negative selection against codon substitutions. Remarkably, somatic evolution exhibits the seemingly neutral rate R ∼ 1 across normal and cancerous tissues. Nevertheless, R ∼ 1 may also mean that positive and negative selections are both strong, but equal in intensity. We refer to this regime as quasi-neutral. Indeed, individual genes in cancer cells often evolve at a much higher, or lower, rate than R ∼ 1. Here, we show that 1) quasi-neutrality is much more likely when populations are small (N < 50); 2) stem-cell populations in single normal tissue niches, from which tumors likely emerge, have a small N (usually <50) but selection at this stage is measurable and strong; 3) when N dips below 50, selection efficacy decreases precipitously; and 4) notably, N is smaller in the stem-cell niche of the small intestine than in the colon. Hence, the ∼70-fold higher rate of phenotypic evolution (observed as cancer risk) in the latter can be explained by the greater efficacy of selection, which then leads to the fixation of more advantageous and fewer deleterious mutations in colon cancers. In conclusion, quasi-neutral evolution sheds a new light on a general evolutionary principle that helps to explain aspects of cancer evolution.

Molecular Evolution in Large Steps-Codon Substitutions under Positive Selection.

Molecular evolution is believed to proceed in small steps. The step size can be defined by a distance reflecting physico-chemical disparities between amino acid (AA) pairs that can be exchanged by single 1-bp mutations. We show that AA substitution rates are strongly and negatively correlated with this distance but only when positive selection is relatively weak. We use the McDonald and Kreitman test to separate the influences of positive and negative selection. While negative selection is indeed stronger on AA substitutions generating larger changes in chemical properties of AAs, positive selection operates by different rules. For 65 of the 75 possible pairs, positive selection is comparable in strength regardless of AA distance. However, the ten pairs under the strongest positive selection all exhibit large leaps in chemical properties. Five of the ten pairs are shared between Drosophila and Hominoids, thus hinting at a common but modest biochemical basis of adaptation across taxa. The hypothesis that adaptive changes often take large functional steps will need to be extensively tested. If validated, molecular models will need to better integrate positive and negative selection in the search for adaptive signal.

Redundant and incoherent regulations of multiple phenotypes suggest microRNAs' role in stability control.

Each microRNA (miRNA) represses a web of target genes and, through them, controls multiple phenotypes. The difficulties inherent in such controls cast doubt on how effective miRNAs are in driving phenotypic changes. A "simple regulation" model posits "one target-one phenotype" control under which most targeting is nonfunctional. In an alternative "coordinate regulation" model, multiple targets are assumed to control the same phenotypes coherently, and most targeting is functional. Both models have some empirical support but pose different conceptual challenges. Here, we concurrently analyze multiple targets and phenotypes associated with the miRNA-310 family (miR310s) of Drosophila Phenotypic rescue in the mir310s knockout background is achieved by promoter-directed RNA interference that restores wild-type expression. For one phenotype (eggshell morphology), we observed redundant regulation, hence rejecting "simple regulation" in favor of the "coordinate regulation" model. For other phenotypes (egg-hatching and male fertility), however, one gene shows full rescue, but three other rescues aggravate the phenotype. Overall, phenotypic controls by miR310s do not support either model. Like a thermostat that controls both heating and cooling elements to regulate temperature, redundancy and incoherence in regulation generally suggest some capacity in stability control. Our results therefore support the published view that miRNAs play a role in the canalization of transcriptome and, hence, phenotypes.

The Genotype-Phenotype Relationships in the Light of Natural Selection.

Although any genotype-phenotype relationships are a result of evolution, little is known about how natural selection and neutral drift, two distinct driving forces of evolution, operate to shape the relationships. By analyzing ∼500 yeast quantitative traits, we reveal a basic "supervisor-worker" gene architecture underlying a trait. Supervisors are often identified by "perturbational" approaches (such as gene deletion), whereas workers, which usually show small and statistically insignificant deletion effects, are tracked primarily by "observational" approaches that examine the correlation between gene activity and trait value across a number of conditions. Accordingly, supervisors provide most of the genetic understandings of the trait whereas workers provide rich mechanistic understandings. Further analyses suggest that most observed supervisor-worker interactions may evolve largely neutrally, resulting in pervasive between-worker epistasis that suppresses the tractability of workers. In contrast, a fraction of supervisors are recruited/maintained by natural selection to build worker co-expression, boosting the tractability of workers. Thus, by revealing a supervisor-worker gene architecture underlying complex traits, the opposite roles of natural selection versus neutral drift in shaping the gene architecture, and the complementary strengths of the perturbational and observational research strategies in characterizing the gene architecture, this study may lay a new conceptual foundation for understanding the molecular basis of complex traits.

On the possibility of death of new genes - evidence from the deletion of de novo microRNAs.

BACKGROUND: New genes are constantly formed, sometimes from non-genic sequences, creating what is referred to as de novo genes. Since the total number of genes remains relatively steady, gene deaths likely balance out new births. In metazoan genomes, microRNAs (miRs) genes, small and non-coding, account for the bulk of functional de novo genes and are particularly suited to the investigation of gene death. RESULTS: In this study, we discover a Drosophila-specific de novo miRNA (mir-977) that may be facing impending death. Strikingly, after this testis-specific gene is deleted from D. melanogaster, most components of male fitness increase, rather than decrease as had been expected. These components include male viability, fertility and males' ability to repress female re-mating. Given that mir-977 has a negative fitness effect in D. melanogaster, this de novo gene with an adaptive history for over 60 Myrs may be facing elimination. In some other species where mir-977 is not found, gene death may have already happened. CONCLUSION: The surprising result suggests that de novo genes, constantly rising and falling during evolution, may often be transiently adaptive and then purged from the genome.

Direct measurement of pervasive weak repression by microRNAs and their role at the network level.

BACKGROUND: A gene regulatory network (GRN) comprises many weak links that are often regulated by microRNAs. Since miRNAs rarely repress their target genes by more than 30%, doubts have been expressed about the biological relevance of such weak effects. These doubts raise the possibility of under-estimation as miRNA repression is usually estimated indirectly from equilibrium expression levels. RESULTS: To measure miRNA repression directly, we inhibited transcript synthesis in Drosophila larvae and collected time-course data on mRNA abundance, the decline of which reflects transcript degradation. The rate of target degradation in the absence of miR310s, a moderately expressed miRNA family, was found to decrease by 5 to 15%. A conventional analysis that does not remove transcript synthesis yields an estimate of 6.5%, within the range of the new estimates. These data permit further examinations of the repression mechanisms by miRNAs including seed matching types, APA (alternative polyadenylation) sites, effects of other highly-expressed miRNAs and the length of 3'UTR. Our direct measurements suggest the latter two factors have a measurable effect on decay rate. CONCLUSION: The direct measurement confirms pervasive weak repression by miRNAs, supporting the conclusions based on indirect assays. The confirmation suggests that this weak repression may indeed be miRNAs' main function. In this context, we discuss the recent proposal that weak repression is "cumulatively powerful" in stabilizing GRNs.

A New Formulation of Random Genetic Drift and Its Application to the Evolution of Cell Populations.

Random genetic drift, or stochastic change in gene frequency, is a fundamental evolutionary force that is usually defined within the ideal Wright-Fisher (WF) population. However, as the theory is increasingly applied to populations that deviate strongly from the ideal model, a paradox of random drift has emerged. When drift is defined by the WF model, it becomes stronger as the population size, N, decreases. However, the intensity of competition decreases when N decreases and, hence, drift might become weaker. To resolve the paradox, we propose that random drift be defined by the variance of "individual output", V(k) [k being the progeny number of each individual with the mean of E(k)], rather than by the WF sampling. If the distribution of k is known for any population, its strength of drift relative to a WF population of the same size, N, can be calculated. Generally, E(k) and V(k) should be density dependent but their relationships are different with or without competition, leading to opposite predictions on the efficiency of random drift as N changes. We apply the "individual output" model to asexual cell populations that are either unregulated (such as tumors) or negatively density-dependent (e.g., bacteria). In such populations, the efficiency of drift could be as low as <10% of that in WF populations. Interestingly, when N is below the carrying capacity, random drift could in fact increase as N increases. Growing asexual populations, especially tumors, may therefore be genetically even more heterogeneous than the high diversity estimated by some conventional models.

Weak Regulation of Many Targets Is Cumulatively Powerful-An Evolutionary Perspective on microRNA Functionality.

Why do microRNAs (miRNAs) weakly repress so many targets such that most targets do not have phenotypic effects? An increasingly accepted view posits that weak targeting has no biological function and each miRNA effectively has only a few functional targets. Here, we review the evolutionary arguments for this postulate and find these arguments seriously flawed. In contrast, from the systems perspective, the power of broad and weak targeting may reside in the cumulative effects of all repressions, which collectively stabilize gene regulatory networks. This view predicts that miRNAs would show little tendency to downsize their target pools. A survey of "twin-miRs" production indeed validates this prediction.

Genome-Wide Convergence during Evolution of Mangroves from Woody Plants.

When living organisms independently invade a new environment, the evolution of similar phenotypic traits is often observed. An interesting but contentious issue is whether the underlying molecular biology also converges in the new habitat. Independent invasions of tropical intertidal zones by woody plants, collectively referred to as mangrove trees, represent some dramatic examples. The high salinity, hypoxia, and other stressors in the new habitat might have affected both genomic features and protein structures. Here, we developed a new method for detecting convergence at conservative Sites (CCS) and applied it to the genomic sequences of mangroves. In simulations, the CCS method drastically reduces random convergence at rapidly evolving sites as well as falsely inferred convergence caused by the misinferences of the ancestral character. In mangrove genomes, we estimated ∼400 genes that have experienced convergence over the background level of convergence in the nonmangrove relatives. The convergent genes are enriched in pathways related to stress response and embryo development, which could be important for mangroves' adaptation to the new habitat.

A Direct Test of Selection in Cell Populations Using the Diversity in Gene Expression within Tumors.

Although intratumor diversity driven by selection has been the prevailing view in cancer biology, recent population genetic analyses have been unable to reject the neutral interpretation. As the power to reject neutrality in tumors is often low, it will be desirable to have an alternative means to test selection directly. Here, we utilize gene expression data as a surrogate for functional significance in intra- and intertumor comparisons. The expression divergence between samples known to be driven by selection (e.g., between tumor and normal tissues) is always higher than the divergence between normal samples, which should be close to the neutral level of divergence. In contrast, the expression differentiation between regions of the same tumor, being lower than the neutral divergence, is incompatible with the hypothesis of selectively driven divergence. To further test the hypothesis of neutral evolution, we select a hepatocellular carcinoma tumor that has large intratumor SNV and CNV (single nucleotide variation and copy number variation, respectively) diversity. This tumor enables us to calibrate the level of expression divergence against that of genetic divergence. We observe that intratumor divergence in gene expression profile lags far behind genetic divergence, indicating insufficient phenotypic differences for selection to operate. All these expression analyses corroborate that natural selection does not operate effectively within tumors, supporting recent interpretations of within-tumor diversity. As the expected level of genetic diversity, hence the potential for drug resistance, would be much higher under neutrality than under selection, the issue is of both theoretical and clinical significance.