Single-cell transcriptomics reveals evolutionary reconfiguration of embryonic cell fate specification in the sea urchin Heliocidaris erythrogramma.

Altered regulatory interactions during development likely underlie a large fraction of phenotypic diversity within and between species, yet identifying specific evolutionary changes remains challenging. Analysis of single-cell developmental transcriptomes from multiple species provides a powerful framework for unbiased identification of evolutionary changes in developmental mechanisms. Here, we leverage a "natural experiment" in developmental evolution in sea urchins, where a major life history switch recently evolved in the lineage leading to Heliocidaris erythrogramma , precipitating extensive changes in early development. Comparative analyses of scRNA-seq developmental time courses from H. erythrogramma and Lytechinus variegatus (representing the derived and ancestral states respectively) reveals numerous evolutionary changes in embryonic patterning. The earliest cell fate specification events, and the primary signaling center are co-localized in the ancestral dGRN but remarkably, in H. erythrogramma they are spatially and temporally separate. Fate specification and differentiation are delayed in most embryonic cell lineages, although in some cases, these processes are conserved or even accelerated. Comparative analysis of regulator-target gene co-expression is consistent with many specific interactions being preserved but delayed in H. erythrogramma , while some otherwise widely conserved interactions have likely been lost. Finally, specific patterning events are directly correlated with evolutionary changes in larval morphology, suggesting that they are directly tied to the life history shift. Together, these findings demonstrate that comparative scRNA-seq developmental time courses can reveal a diverse set of evolutionary changes in embryonic patterning and provide an efficient way to identify likely candidate regulatory interactions for subsequent experimental validation.

A human-specific enhancer fine-tunes radial glia potency and corticogenesis.

Humans evolved an extraordinarily expanded and complex cerebral cortex, associated with developmental and gene regulatory modifications 1-3 . Human accelerated regions (HARs) are highly conserved genomic sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to human-specific cortical development is largely unknown 4,5 . HARE5 is a HAR transcriptional enhancer of the WNT signaling receptor Frizzled8 (FZD8) active during brain development 6 . Here, using genome-edited mouse and primate models, we demonstrate that human (Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacity of neural progenitor cells (NPCs). Hs-HARE5 knock-in mice have significantly enlarged neocortices containing more neurons. By measuring neural dynamics in vivo we show these anatomical features correlate with increased functional independence between cortical regions. To understand the underlying developmental mechanisms, we assess progenitor fate using live imaging, lineage analysis, and single-cell RNA sequencing. This reveals Hs-HARE5 modifies radial glial progenitor behavior, with increased self-renewal at early developmental stages followed by expanded neurogenic potential. We use genome-edited human and chimpanzee (Pt) NPCs and cortical organoids to assess the relative enhancer activity and function of Hs-HARE5 and Pt-HARE5. Using these orthogonal strategies we show four human-specific variants in HARE5 drive increased enhancer activity which promotes progenitor proliferation. These findings illustrate how small changes in regulatory DNA can directly impact critical signaling pathways and brain development. Our study uncovers new functions for HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex.

Genomic insights into Plasmodium vivax population structure and diversity in central Africa.

BACKGROUND: Though Plasmodium vivax is the second most common malaria species to infect humans, it has not traditionally been considered a major human health concern in central Africa given the high prevalence of the human Duffy-negative phenotype that is believed to prevent infection. Increasing reports of asymptomatic and symptomatic infections in Duffy-negative individuals throughout Africa raise the possibility that P. vivax is evolving to evade host resistance, but there are few parasite samples with genomic data available from this part of the world. METHODS: Whole genome sequencing of one new P. vivax isolate from the Democratic Republic of the Congo (DRC) was performed and used in population genomics analyses to assess how this central African isolate fits into the global context of this species. RESULTS: Plasmodium vivax from DRC is similar to other African populations and is not closely related to the non-human primate parasite P. vivax-like. Evidence is found for a duplication of the gene PvDBP and a single copy of PvDBP2. CONCLUSION: These results suggest an endemic P. vivax population is present in central Africa. Intentional sampling of P. vivax across Africa would further contextualize this sample within African P. vivax diversity and shed light on the mechanisms of infection in Duffy negative individuals. These results are limited by the uncertainty of how representative this single sample is of the larger population of P. vivax in central Africa.

Hybrid Epigenomes Reveal Extensive Local Genetic Changes to Chromatin Accessibility Contribute to Divergence in Embryonic Gene Expression Between Species.

Chromatin accessibility plays an important role in shaping gene expression, yet little is known about the genetic and molecular mechanisms that influence the evolution of chromatin configuration. Both local (cis) and distant (trans) genetic influences can in principle influence chromatin accessibility and are based on distinct molecular mechanisms. We, therefore, sought to characterize the role that each of these plays in altering chromatin accessibility in 2 closely related sea urchin species. Using hybrids of Heliocidaris erythrogramma and Heliocidaris tuberculata, and adapting a statistical framework previously developed for the analysis of cis and trans influences on the transcriptome, we examined how these mechanisms shape the regulatory landscape at 3 important developmental stages, and compared our results to similar analyses of the transcriptome. We found extensive cis- and trans-based influences on evolutionary changes in chromatin, with cis effects generally larger in effect. Evolutionary changes in accessibility and gene expression are correlated, especially when expression has a local genetic basis. Maternal influences appear to have more of an effect on chromatin accessibility than on gene expression, persisting well past the maternal-to-zygotic transition. Chromatin accessibility near gene regulatory network genes appears to be distinctly regulated, with trans factors appearing to play an outsized role in the configuration of chromatin near these genes. Together, our results represent the first attempt to quantify cis and trans influences on evolutionary divergence in chromatin configuration in an outbred natural study system and suggest that chromatin regulation is more genetically complex than was previously appreciated.

Near-Chromosomal-Level Genome Assembly of the Sea Urchin Echinometra lucunter, a Model for Speciation in the Sea.

Echinometra lucunter, the rock-boring sea urchin, is a widely distributed echinoid and a model for ecological studies of reproduction, responses to climate change, and speciation. We present a near chromosome-level genome assembly of E. lucunter, including 21 scaffolds larger than 10 Mb predicted to represent each of the chromosomes of the species. The 760.4 Mb assembly includes a scaffold N50 of 30.0 Mb and BUSCO (benchmarking universal single-copy orthologue) single copy and a duplicated score of 95.8% and 1.4%, respectively. Ab-initio gene model prediction and annotation with transcriptomic data constructed 33,989 gene models composing 50.4% of the assembly, including 37,036 transcripts. Repetitive elements make up approximately 39.6% of the assembly, and unresolved gap sequences are estimated to be 0.65%. Whole genome alignment with Echinometra sp. EZ revealed high synteny and conservation between the two species, further bolstering Echinometra as an emerging genus for comparative genomics studies. This genome assembly represents a high-quality genomic resource for future evolutionary and developmental studies of this species and more broadly of echinoderms.

Feedback circuits are numerous in embryonic gene regulatory networks and offer a stabilizing influence on evolution of those networks.

The developmental gene regulatory networks (dGRNs) of two sea urchin species, Lytechinus variegatus (Lv) and Strongylocentrotus purpuratus (Sp), have remained remarkably similar despite about 50 million years since a common ancestor. Hundreds of parallel experimental perturbations of transcription factors with similar outcomes support this conclusion. A recent scRNA-seq analysis suggested that the earliest expression of several genes within the dGRNs differs between Lv and Sp. Here, we present a careful reanalysis of the dGRNs in these two species, paying close attention to timing of first expression. We find that initial expression of genes critical for cell fate specification occurs during several compressed time periods in both species. Previously unrecognized feedback circuits are inferred from the temporally corrected dGRNs. Although many of these feedbacks differ in location within the respective GRNs, the overall number is similar between species. We identify several prominent differences in timing of first expression for key developmental regulatory genes; comparison with a third species indicates that these heterochronies likely originated in an unbiased manner with respect to embryonic cell lineage and evolutionary branch. Together, these results suggest that interactions can evolve even within highly conserved dGRNs and that feedback circuits may buffer the effects of heterochronies in the expression of key regulatory genes.

Hybrid epigenomes reveal extensive local genetic changes to chromatin accessibility contribute to divergence in embryonic gene expression between species.

Chromatin accessibility plays an important role in shaping gene expression patterns across development and evolution; however, little is known about the genetic and molecular mechanisms that influence chromatin configuration itself. Because cis and trans influences can both theoretically influence the accessibility of the epigenome, we sought to better characterize the role that both of these mechanisms play in altering chromatin accessibility in two closely related sea urchin species. Using hybrids of the two species, and adapting a statistical framework previously developed for the analysis of cis and trans influences on the transcriptome, we examined how these mechanisms shape the regulatory landscape at three important developmental stages, and compared our results to similar patterns in the transcriptome. We found extensive cis- and trans-based influences on evolutionary changes in chromatin, with cis effects slightly more numerous and larger in effect. Genetic mechanisms influencing gene expression and chromatin configuration are correlated, but differ in several important ways. Maternal influences also appear to have more of an effect on chromatin accessibility than on gene expression, persisting well past the maternal-to-zygotic transition. Furthermore, chromatin accessibility near GRN genes appears to be regulated differently than the rest of the epigenome, and indicates that trans factors may play an outsized role in the configuration of chromatin near these genes. Together, our results represent the first attempt to quantify cis and trans influences on evolutionary divergence in chromatin configuration in an outbred natural study system, and suggest that the regulation of chromatin is more genetically complex than was previously appreciated.

Recent reconfiguration of an ancient developmental gene regulatory network in Heliocidaris sea urchins.

Changes in developmental gene regulatory networks (dGRNs) underlie much of the diversity of life, but the evolutionary mechanisms that operate on regulatory interactions remain poorly understood. Closely related species with extreme phenotypic divergence provide a valuable window into the genetic and molecular basis for changes in dGRNs and their relationship to adaptive changes in organismal traits. Here we analyse genomes, epigenomes and transcriptomes during early development in two Heliocidaris sea urchin species that exhibit highly divergent life histories and in an outgroup species. Positive selection and chromatin accessibility modifications within putative regulatory elements are enriched on the branch leading to the derived life history, particularly near dGRN genes. Single-cell transcriptomes reveal a dramatic delay in cell fate specification in the derived state, which also has far fewer open chromatin regions, especially near conserved cell fate specification genes. Experimentally perturbing key transcription factors reveals profound evolutionary changes to early embryonic patterning events, disrupting regulatory interactions previously conserved for ~225 million years. These results demonstrate that natural selection can rapidly reshape developmental gene expression on a broad scale when selective regimes abruptly change. More broadly, even highly conserved dGRNs and patterning mechanisms in the early embryo remain evolvable under appropriate ecological circumstances.

A Chromosome-level Genome Assembly of the Highly Heterozygous Sea Urchin Echinometra sp. EZ Reveals Adaptation in the Regulatory Regions of Stress Response Genes.

Echinometra is the most widespread genus of sea urchin and has been the focus of a wide range of studies in ecology, speciation, and reproduction. However, available genetic data for this genus are generally limited to a few select loci. Here, we present a chromosome-level genome assembly based on 10x Genomics, PacBio, and Hi-C sequencing for Echinometra sp. EZ from the Persian/Arabian Gulf. The genome is assembled into 210 scaffolds totaling 817.8 Mb with an N50 of 39.5 Mb. From this assembly, we determined that the E. sp. EZ genome consists of 2n = 42 chromosomes. BUSCO analysis showed that 95.3% of BUSCO genes were complete. Ab initio and transcript-informed gene modeling and annotation identified 29,405 genes, including a conserved Hox cluster. E. sp. EZ can be found in high-temperature and high-salinity environments, and we therefore compared E. sp. EZ gene families and transcription factors associated with environmental stress response ("defensome") with other echinoid species with similar high-quality genomic resources. While the number of defensome genes was broadly similar for all species, we identified strong signatures of positive selection in E. sp. EZ noncoding elements near genes involved in environmental response pathways as well as losses of transcription factors important for environmental response. These data provide key insights into the biology of E. sp. EZ as well as the diversification of Echinometra more widely and will serve as a useful tool for the community to explore questions in this taxonomic group and beyond.

Evolutionary Changes in the Chromatin Landscape Contribute to Reorganization of a Developmental Gene Network During Rapid Life History Evolution in Sea Urchins.

Chromatin configuration is highly dynamic during embryonic development in animals, exerting an important point of control in transcriptional regulation. Yet there exists remarkably little information about the role of evolutionary changes in chromatin configuration to the evolution of gene expression and organismal traits. Genome-wide assays of chromatin configuration, coupled with whole-genome alignments, can help address this gap in knowledge in several ways. In this study we present a comparative analysis of regulatory element sequences and accessibility throughout embryogenesis in three sea urchin species with divergent life histories: a lecithotroph Heliocidaris erythrogramma, a closely related planktotroph H. tuberculata, and a distantly related planktotroph Lytechinus variegatus. We identified distinct epigenetic and mutational signatures of evolutionary modifications to the function of putative cis-regulatory elements in H. erythrogramma that have accumulated nonuniformly throughout the genome, suggesting selection, rather than drift, underlies many modifications associated with the derived life history. Specifically, regulatory elements composing the sea urchin developmental gene regulatory network are enriched for signatures of positive selection and accessibility changes which may function to alter binding affinity and access of developmental transcription factors to these sites. Furthermore, regulatory element changes often correlate with divergent expression patterns of genes involved in cell type specification, morphogenesis, and development of other derived traits, suggesting these evolutionary modifications have been consequential for phenotypic evolution in H. erythrogramma. Collectively, our results demonstrate that selective pressures imposed by changes in developmental life history rapidly reshape the cis-regulatory landscape of core developmental genes to generate novel traits and embryonic programs.

Extreme phenotypic divergence and the evolution of development.

As analyses of developmental mechanisms extend to ever more species, it becomes important to understand not just what is conserved or altered during evolution, but why. Closely related species that exhibit extreme phenotypic divergence can be uniquely informative in this regard. A case in point is the sea urchin genus Heliocidaris, which contains species that recently evolved a life history involving nonfeeding larvae following nearly half a billion years of prior evolution with feeding larvae. The resulting shift in selective regimes produced rapid and surprisingly extensive changes in developmental mechanisms that are otherwise highly conserved among echinoderm species. The magnitude and extent of these changes challenges the notion that conservation of early development in echinoderms is largely due to internal constraints that prohibit modification and instead suggests that natural selection actively maintains stability of inherently malleable trait developmental mechanisms over immense time periods. Knowing how and why natural selection changed during the evolution of nonfeeding larvae can also reveal why developmental mechanisms do and do not change in particular ways.

Developmental single-cell transcriptomics in the Lytechinus variegatus sea urchin embryo.

Using scRNA-seq coupled with computational approaches, we studied transcriptional changes in cell states of sea urchin embryos during development to the larval stage. Eighteen closely spaced time points were taken during the first 24 h of development of Lytechinus variegatus (Lv). Developmental trajectories were constructed using Waddington-OT, a computational approach to 'stitch' together developmental time points. Skeletogenic and primordial germ cell trajectories diverged early in cleavage. Ectodermal progenitors were distinct from other lineages by the 6th cleavage, although a small percentage of ectoderm cells briefly co-expressed endoderm markers that indicated an early ecto-endoderm cell state, likely in cells originating from the equatorial region of the egg. Endomesoderm cells also originated at the 6th cleavage and this state persisted for more than two cleavages, then diverged into distinct endoderm and mesoderm fates asynchronously, with some cells retaining an intermediate specification status until gastrulation. Seventy-nine out of 80 genes (99%) examined, and included in published developmental gene regulatory networks (dGRNs), are present in the Lv-scRNA-seq dataset and are expressed in the correct lineages in which the dGRN circuits operate.

The epidemiology of Plasmodium vivax among adults in the Democratic Republic of the Congo.

Reports of P. vivax infections among Duffy-negative hosts have accumulated throughout sub-Saharan Africa. Despite this growing body of evidence, no nationally representative epidemiological surveys of P. vivax in sub-Saharan Africa have been performed. To overcome this gap in knowledge, we screened over 17,000 adults in the Democratic Republic of the Congo (DRC) for P. vivax using samples from the 2013-2014 Demographic Health Survey. Overall, we found a 2.97% (95% CI: 2.28%, 3.65%) prevalence of P. vivax infections across the DRC. Infections were associated with few risk-factors and demonstrated a relatively flat distribution of prevalence across space with focal regions of relatively higher prevalence in the north and northeast. Mitochondrial genomes suggested that DRC P. vivax were distinct from circulating non-human ape strains and an ancestral European P. vivax strain, and instead may be part of a separate contemporary clade. Our findings suggest P. vivax is diffusely spread across the DRC at a low prevalence, which may be associated with long-term carriage of low parasitemia, frequent relapses, or a general pool of infections with limited forward propagation.

Microbiome reduction and endosymbiont gain from a switch in sea urchin life history.

Animal gastrointestinal tracts harbor a microbiome that is integral to host function, yet species from diverse phyla have evolved a reduced digestive system or lost it completely. Whether such changes are associated with alterations in the diversity and/or abundance of the microbiome remains an untested hypothesis in evolutionary symbiosis. Here, using the life history transition from planktotrophy (feeding) to lecithotrophy (nonfeeding) in the sea urchin Heliocidaris, we demonstrate that the lack of a functional gut corresponds with a reduction in microbial community diversity and abundance as well as the association with a diet-specific microbiome. We also determine that the lecithotroph vertically transmits a Rickettsiales that may complement host nutrition through amino acid biosynthesis and influence host reproduction. Our results indicate that the evolutionary loss of a functional gut correlates with a reduction in the microbiome and the association with an endosymbiont. Symbiotic transitions can therefore accompany life history transitions in the evolution of developmental strategies.

An early cell shape transition drives evolutionary expansion of the human forebrain.

The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla, and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that neuroepithelial differentiation is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition, associated with differences in interkinetic nuclear migration and cell cycle length. Comparative RNA sequencing (RNA-seq) reveals differences in expression dynamics of cell morphogenesis factors, including ZEB2, a known epithelial-mesenchymal transition regulator. We show that ZEB2 promotes neuroepithelial transition, and its manipulation and downstream signaling leads to acquisition of nonhuman ape architecture in the human context and vice versa, establishing an important role for neuroepithelial cell shape in human brain expansion.

The hard clam genome reveals massive expansion and diversification of inhibitors of apoptosis in Bivalvia.

BACKGROUND: Inhibitors of apoptosis (IAPs) are critical regulators of programmed cell death that are essential for development, oncogenesis, and immune and stress responses. However, available knowledge regarding IAP is largely biased toward humans and model species, while the distribution, function, and evolutionary novelties of this gene family remain poorly understood in many taxa, including Mollusca, the second most speciose phylum of Metazoa. RESULTS: Here, we present a chromosome-level genome assembly of an economically significant bivalve, the hard clam Mercenaria mercenaria, which reveals an unexpected and dramatic expansion of the IAP gene family to 159 members, the largest IAP gene repertoire observed in any metazoan. Comparative genome analysis reveals that this massive expansion is characteristic of bivalves more generally. Reconstruction of the evolutionary history of molluscan IAP genes indicates that most originated in early metazoans and greatly expanded in Bivalvia through both lineage-specific tandem duplication and retroposition, with 37.1% of hard clam IAPs located on a single chromosome. The expanded IAPs have been subjected to frequent domain shuffling, which has in turn shaped their architectural diversity. Further, we observed that extant IAPs exhibit dynamic and orchestrated expression patterns among tissues and in response to different environmental stressors. CONCLUSIONS: Our results suggest that sophisticated regulation of apoptosis enabled by the massive expansion and diversification of IAPs has been crucial for the evolutionary success of hard clam and other molluscan lineages, allowing them to cope with local environmental stresses. This study broadens our understanding of IAP proteins and expression diversity and provides novel resources for studying molluscan biology and IAP function and evolution.

Methodologies for Following EMT In Vivo at Single Cell Resolution.

An epithelial-mesenchymal transition (EMT) occurs in almost every metazoan embryo at the time mesoderm begins to differentiate. Several embryos have a long record as models for studying an EMT given that a known population of cells enters the EMT at a known time thereby enabling a detailed study of the process. Often, however, it is difficult to learn the molecular details of these model EMT systems because the transitioning cells are a minority of the population of cells in the embryo and in most cases there is an inability to isolate that population. Here we provide a method that enables an examination of genes expressed before, during, and after the EMT with a focus on just the cells that undergo the transition. Single cell RNA-seq (scRNA-seq) has advanced as a technology making it feasible to study the trajectory of gene expression specifically in the cells of interest, in vivo, and without the background noise of other cell populations. The sea urchin skeletogenic cells constitute only 5% of the total number of cells in the embryo yet with scRNA-seq it is possible to study the genes expressed by these cells without background noise. This approach, though not perfect, adds a new tool for uncovering the mechanism of EMT in this cell type.

TBX5-encoded T-box transcription factor 5 variant T223M is associated with long QT syndrome and pediatric sudden cardiac death.

Long QT syndrome (LQTS) is a genetic disease resulting in a prolonged QT interval on a resting electrocardiogram, predisposing affected individuals to polymorphic ventricular tachycardia and sudden death. Although a number of genes have been implicated in this disease, nearly one in four individuals exhibiting the LQTS phenotype are genotype-negative. Whole-exome sequencing identified a missense T223M variant in TBX5 that cosegregates with prolonged QT interval in a family with otherwise genotype-negative LQTS and sudden death. The TBX5-T223M variant was absent among large ostensibly healthy populations (gnomAD) and predicted to be pathogenic by in silico modeling based on Panther, PolyPhen-2, Provean, SIFT, SNAP2, and PredictSNP prediction tools. The variant was located in a highly conserved region of TBX5 predicted to be part of the DNA-binding interface. A luciferase assay identified a 57.5% reduction in the ability of TBX5-T223M to drive expression at the atrial natriuretic factor promotor compared to wildtype TBX5 in vitro. We conclude that the variant is pathogenic in this family, and we put TBX5 forward as a disease susceptibility allele for genotype-negative LQTS. The identification of this familial variant may serve as a basis for the identification of previously unknown mechanisms of LQTS with broader implications for cardiac electrophysiology.

Ocean acidification induces distinct transcriptomic responses across life history stages of the sea urchin Heliocidaris erythrogramma.

Ocean acidification (OA) from seawater uptake of rising carbon dioxide emissions impairs development in marine invertebrates, particularly in calcifying species. Plasticity in gene expression is thought to mediate many of these physiological effects, but how these responses change across life history stages remains unclear. The abbreviated lecithotrophic development of the sea urchin Heliocidaris erythrogramma provides a valuable opportunity to analyse gene expression responses across a wide range of life history stages, including the benthic, post-metamorphic juvenile. We measured the transcriptional response to OA in H. erythrogramma at three stages of the life cycle (embryo, larva, and juvenile) in a controlled breeding design. The results reveal a broad range of strikingly stage-specific impacts of OA on transcription, including changes in the number and identity of affected genes; the magnitude, sign, and variance of their expression response; and the developmental trajectory of expression. The impact of OA on transcription was notably modest in relation to gene expression changes during unperturbed development and much smaller than genetic contributions from parentage. The latter result suggests that natural populations may provide an extensive genetic reservoir of resilience to OA. Taken together, these results highlight the complexity of the molecular response to OA, its substantial life history stage specificity, and the importance of contextualizing the transcriptional response to pH stress in light of normal development and standing genetic variation to better understand the capacity for marine invertebrates to adapt to OA.

Positive selection within the genomes of SARS-CoV-2 and other Coronaviruses independent of impact on protein function.

BACKGROUND: The emergence of a novel coronavirus (SARS-CoV-2) associated with severe acute respiratory disease (COVID-19) has prompted efforts to understand the genetic basis for its unique characteristics and its jump from non-primate hosts to humans. Tests for positive selection can identify apparently nonrandom patterns of mutation accumulation within genomes, highlighting regions where molecular function may have changed during the origin of a species. Several recent studies of the SARS-CoV-2 genome have identified signals of conservation and positive selection within the gene encoding Spike protein based on the ratio of synonymous to nonsynonymous substitution. Such tests cannot, however, detect changes in the function of RNA molecules. METHODS: Here we apply a test for branch-specific oversubstitution of mutations within narrow windows of the genome without reference to the genetic code. RESULTS: We recapitulate the finding that the gene encoding Spike protein has been a target of both purifying and positive selection. In addition, we find other likely targets of positive selection within the genome of SARS-CoV-2, specifically within the genes encoding Nsp4 and Nsp16. Homology-directed modeling indicates no change in either Nsp4 or Nsp16 protein structure relative to the most recent common ancestor. These SARS-CoV-2-specific mutations may affect molecular processes mediated by the positive or negative RNA molecules, including transcription, translation, RNA stability, and evasion of the host innate immune system. Our results highlight the importance of considering mutations in viral genomes not only from the perspective of their impact on protein structure, but also how they may impact other molecular processes critical to the viral life cycle.