The Number and Pattern of Viral Genomic Reassortments are not Necessarily Identifiable from Segment Trees.

Reassortment is an evolutionary process common in viruses with segmented genomes. These viruses can swap whole genomic segments during cellular co-infection, giving rise to novel progeny formed from the mixture of parental segments. Since large-scale genome rearrangements have the potential to generate new phenotypes, reassortment is important to both evolutionary biology and public health research. However, statistical inference of the pattern of reassortment events from phylogenetic data is exceptionally difficult, potentially involving inference of general graphs in which individual segment trees are embedded. In this paper, we argue that, in general, the number and pattern of reassortment events are not identifiable from segment trees alone, even with theoretically ideal data. We call this fact the fundamental problem of reassortment, which we illustrate using the concept of the "first-infection tree," a potentially counterfactual genealogy that would have been observed in the segment trees had no reassortment occurred. Further, we illustrate four additional problems that can arise logically in the inference of reassortment events and show, using simulated data, that these problems are not rare and can potentially distort our observation of reassortment even in small data sets. Finally, we discuss how existing methods can be augmented or adapted to account for not only the fundamental problem of reassortment, but also the four additional situations that can complicate the inference of reassortment.

Inferring Viral Transmission Time from Phylogenies for Known Transmission Pairs.

When the time of an HIV transmission event is unknown, methods to identify it from virus genetic data can reveal the circumstances that enable transmission. We developed a single-parameter Markov model to infer transmission time from an HIV phylogeny constructed of multiple virus sequences from people in a transmission pair. Our method finds the statistical support for transmission occurring in different possible time slices. We compared our time-slice model results to previously described methods: a tree-based logical transmission interval, a simple parsimony-like rules-based method, and a more complex coalescent model. Across simulations with multiple transmitted lineages, different transmission times relative to the source's infection, and different sampling times relative to transmission, we found that overall our time-slice model provided accurate and narrower estimates of the time of transmission. We also identified situations when transmission time or direction was difficult to estimate by any method, particularly when transmission occurred long after the source was infected and when sampling occurred long after transmission. Applying our model to real HIV transmission pairs showed some agreement with facts known from the case investigations. We also found, however, that uncertainty on the inferred transmission time was driven more by uncertainty from time calibration of the phylogeny than from the model inference itself. Encouragingly, comparable performance of the Markov time-slice model and the coalescent model-which make use of different information within a tree-suggests that a new method remains to be described that will make full use of the topology and node times for improved transmission time inference.

Modeling the evolution of segment trees reveals deficiencies in current inferential methods for genomic reassortment.

Reassortment is an evolutionary process common in viruses with segmented genomes. These viruses can swap whole genomic segments during cellular co-infection, giving rise to new viral variants. Large-scale genome rearrangements, such as reassortment, have the potential to quickly generate new phenotypes, making the understanding of viral reassortment important to both evolutionary biology and public health research. In this paper, we argue that reassortment cannot be reliably inferred from incongruities between segment phylogenies using the established remove-and-rejoin or coalescent approaches. We instead show that reassortment must be considered in the context of a broader population process that includes the dynamics of the infected hosts. Using illustrative examples and simulation we identify four types of evolutionary events that are difficult or impossible to reconstruct with incongruence-based methods. Further, we show that these specific situations are very common and will likely occur even in small samples. Finally, we argue that existing methods can be augmented or modified to account for all the problematic situations that we identify in this paper. Robust assessment of the role of reassortment in viral evolution is difficult, and we hope to provide conceptual clarity on some important methodological issues that can arise in the development of the next generation of tools for studying reassortment.

Inferring viral transmission time from phylogenies for known transmission pairs.

When the time of an HIV transmission event is unknown, methods to identify it from virus genetic data can reveal the circumstances that enable transmission. We developed a single-parameter Markov model to infer transmission time from an HIV phylogeny constructed of multiple virus sequences from people in a transmission pair. Our method finds the statistical support for transmission occurring in different possible time slices. We compared our time-slice model results to previously-described methods: a tree-based logical transmission interval, a simple parsimony-like rules-based method, and a more complex coalescent model. Across simulations with multiple transmitted lineages, different transmission times relative to the source's infection, and different sampling times relative to transmission, we found that overall our time-slice model provided accurate and narrower estimates of the time of transmission. We also identified situations when transmission time or direction was difficult to estimate by any method, particularly when transmission occurred long after the source was infected and when sampling occurred long after transmission. Applying our model to real HIV transmission pairs showed some agreement with facts known from the case investigations. We also found, however, that uncertainty on the inferred transmission time was driven more by uncertainty from time-calibration of the phylogeny than from the model inference itself. Encouragingly, comparable performance of the Markov time-slice model and the coalescent model-which make use of different information within a tree-suggests that a new method remains to be described that will make full use of the topology and node times for improved transmission time inference.

Swift and extensive Omicron outbreak in China after sudden exit from 'zero-COVID' policy.

In late 2022, China transitioned from a strict 'zero-COVID' policy to rapidly abandoning nearly all interventions and data reporting. This raised great concern about the presumably-rapid but unreported spread of the SARS-CoV-2 Omicron variant in a very large population of very low pre-existing immunity. By modeling a combination of case count and survey data, we show that Omicron spread extremely rapidly, at a rate of 0.42/day (95% credibility interval: [0.35, 0.51]/day), translating to an epidemic doubling time of 1.6 days ([1.6, 2.0] days) after the full exit from zero-COVID on Dec. 7, 2022. Consequently, we estimate that the vast majority of the population (97% [95%, 99%], sensitivity analysis lower limit of 90%) was infected during December, with the nation-wide epidemic peaking on Dec. 23. Overall, our results highlight the extremely high transmissibility of the variant and the importance of proper design of intervention exit strategies to avoid large infection waves.

Hypoxia Resistance Is an Inherent Phenotype of the Mouse Flexor Digitorum Brevis Skeletal Muscle.

The various functions of skeletal muscle (movement, respiration, thermogenesis, etc.) require the presence of oxygen (O2). Inadequate O2 bioavailability (ie, hypoxia) is detrimental to muscle function and, in chronic cases, can result in muscle wasting. Current therapeutic interventions have proven largely ineffective to rescue skeletal muscle from hypoxic damage. However, our lab has identified a mammalian skeletal muscle that maintains proper physiological function in an environment depleted of O2. Using mouse models of in vivo hindlimb ischemia and ex vivo anoxia exposure, we observed the preservation of force production in the flexor digitorum brevis (FDB), while in contrast the extensor digitorum longus (EDL) and soleus muscles suffered loss of force output. Unlike other muscles, we found that the FDB phenotype is not dependent on mitochondria, which partially explains the hypoxia resistance. Muscle proteomes were interrogated using a discovery-based approach, which identified significantly greater expression of the transmembrane glucose transporter GLUT1 in the FDB as compared to the EDL and soleus. Through loss-and-gain-of-function approaches, we determined that GLUT1 is necessary for the FDB to survive hypoxia, but overexpression of GLUT1 was insufficient to rescue other skeletal muscles from hypoxic damage. Collectively, the data demonstrate that the FDB is uniquely resistant to hypoxic insults. Defining the mechanisms that explain the phenotype may provide insight towards developing approaches for preventing hypoxia-induced tissue damage.

Global estimates of the fitness advantage of SARS-CoV-2 variant Omicron.

New variants of SARS-CoV-2 show remarkable heterogeneity in their relative fitness over both time and space. In this paper we extend the tools available for estimating the selection strength for new SARS-CoV-2 variants to a hierarchical, mixed-effects, renewal equation model. This formulation allows us to estimate selection effects at the global level while incorporating both measured and unmeasured heterogeneity among countries. Applying this model to the spread of Omicron in forty countries, we find evidence for very strong but very heterogeneous selection effects. To test whether this heterogeneity is explained by differences in the immune landscape, we considered several measures of vaccination rates and recent population-level infection as covariates, finding moderately strong, statistically significant effects. We also found a significant positive correlation between the selection advantage of Delta and Omicron at the country level, suggesting that other region-specific explanatory variables of fitness differences do exist. Our method is implemented in the Stan programming language, can be run on standard consumer-grade computing resources, and will be straightforward to apply to future variants.

Effects of Plasticity on Elevational Range Size and Species Richness.

AbstractElevational ranges within many taxa are greater in the north temperate region than the tropics. Two hypotheses to explain the pattern are, first, that large elevational ranges in the temperate region arise because species have evolved broad tolerance curves in response to seasonality and, second, that a low diversification rate in the temperate region (speciation minus extinction) has led to relatively few species, each of which occupies a large elevational range in the absence of competitors (character release). We build a quantitative genetic model of selection on a phenotypic trait, whereby increased tolerance is modeled as arising from plasticity in the trait. We show that broad tolerances result in evolution of large elevational ranges because they induce shallower genotypic clines and hence reduced maladaptive gene flow. The evolution of large elevational ranges results in relatively few competing species arranged along the elevational gradient at a species carrying capacity. In such saturated communities, species have much elevational overlap. In contrast, in similar-sized communities that could accommodate many more species, the resulting character release is associated with smaller elevational overlaps. Empirical assessment of these predictions should contribute to assessing any role for ecological limits in driving the latitudinal diversity gradient in species richness.

Global estimates of the fitness advantage of SARS-CoV-2 variant Omicron.

New variants of SARS-CoV-2 show remarkable heterogeneity in their relative fitness both over time and space. In this paper we extend a previously published model for estimating the selection strength for new SARS-CoV-2 variants to a hierarchical, mixed-effects, renewal equation model. This formulation allows us to globally estimate selection effects at different spatial levels while controlling for complex patterns of transmission and jointly inferring the effects of unit-level covariates in the spatial heterogeneity of SARS-CoV-2 selection effects. Applying this model to the spread of Omicron in 40 counties finding evidence for very strong (64%) but very heterogeneous selection effects at the country level. We further considered different measures of vaccination levels and measures of recent population-level infection as possible explanations. However, none of those variables were found to explain a significant proportion of the heterogeneity in country-level selection effects. We did find a significant positive correlation between the selection advantage of Delta and Omicron at the country level, suggesting that region-specific explanatory variables of fitness differences do exist. Our method is implemented in the Stan programming language, can be run on standard commercial-grade computing resources, and should be straightforward to apply to future variants.

Estimating the strength of selection for new SARS-CoV-2 variants.

Controlling the SARS-CoV-2 pandemic becomes increasingly challenging as the virus adapts to human hosts through the continual emergence of more transmissible variants. Simply observing that a variant is increasing in frequency is relatively straightforward, but more sophisticated methodology is needed to determine whether a new variant is a global threat and the magnitude of its selective advantage. We present two models for quantifying the strength of selection for new and emerging variants of SARS-CoV-2 relative to the background of contemporaneous variants. These methods range from a detailed model of dynamics within one country to a broad analysis across all countries, and they include alternative explanations such as migration and drift. We find evidence for strong selection favoring the D614G spike mutation and B.1.1.7 (Alpha), weaker selection favoring B.1.351 (Beta), and no advantage of R.1 after it spreads beyond Japan. Cutting back data to earlier time horizons reveals that uncertainty is large very soon after emergence, but that estimates of selection stabilize after several weeks. Our results also show substantial heterogeneity among countries, demonstrating the need for a truly global perspective on the molecular epidemiology of SARS-CoV-2.

Estimating the strength of selection for new SARS-CoV-2 variants.

Controlling the SARS-CoV-2 pandemic becomes increasingly challenging as the virus adapts to human hosts through the continual emergence of more transmissible variants. Simply observing that a variant is increasing in frequency is relatively straightforward, but more sophisticated methodology is needed to determine whether a new variant is a global threat and the magnitude of its selective advantage. We present three methods for quantifying the strength of selection for new and emerging variants of SARS-CoV-2 relative to the background of contemporaneous variants. These methods range from a detailed model of dynamics within one country to a broad analysis across all countries, and they include alternative explanations such as migration and drift. We find evidence for strong selection favoring the D614G spike mutation and B.1.1.7 (Alpha), weaker selection favoring B.1.351 (Beta), and no advantage of R.1 after it spreads beyond Japan. Cutting back data to earlier time horizons reveals large uncertainty very soon after emergence, but that estimates of selection stabilize after several weeks. Our results also show substantial heterogeneity among countries, demonstrating the need for a truly global perspective on the molecular epidemiology of SARS-CoV-2.

Racial differences in the limb skeletal muscle transcriptional programs of patients with critical limb ischemia.

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease (PAD) and is characterized by high rates of morbidity and mortality. As with most severe cardiovascular disease manifestations, Black individuals disproportionately present with CLI. Accordingly, there remains a clear need to better understand the reasons for this discrepancy and to facilitate personalized therapeutic options specific for this population. Gastrocnemius muscle was obtained from White and Black healthy adult volunteers and patients with CLI for whole transcriptome shotgun sequencing (WTSS) and enrichment analysis was performed to identify alterations in specific Reactome pathways. When compared to their race-matched healthy controls, both White and Black patients with CLI demonstrated similar reductions in nuclear and mitochondrial encoded genes and mitochondrial oxygen consumption across multiple substrates, indicating a common bioenergetic paradigm associated with amputation outcomes regardless of race. Direct comparisons between tissues of White and Black patients with CLI revealed hemostasis, extracellular matrix organization, platelet regulation, and vascular wall interactions to be uniquely altered in limb muscles of Black individuals. Among traditional vascular growth factor signaling targets, WTSS revealed only Tie1 to be significantly altered from White levels in Black limb muscle tissues. Quantitative reverse transcription polymerase chain reaction validation of select identified targets verified WTSS directional changes and supports reductions in MMP9 and increases in NUDT4P1 and GRIK2 as unique to limb muscles of Black patients with CLI. This represents a critical first step in better understanding the transcriptional program similarities and differences between Black and White patients in the setting of amputations related to CLI and provides a promising start for therapeutic development in this population.

Diversification or Collapse of Self-Incompatibility Haplotypes as a Rescue Process.

AbstractIn angiosperm self-incompatibility systems, pollen with an allele matching the pollen recipient at the self-incompatibility locus is rejected. Extreme allelic polymorphism is maintained by frequency-dependent selection favoring rare alleles. However, two challenges result in a chicken-or-egg problem for the spread of a new allele (a tightly linked haplotype in this case) under the widespread "collaborative non-self-recognition" mechanism. A novel pollen function mutation alone would merely grant compatibility with a nonexistent style function allele: a neutral change at best. A novel pistil function mutation alone could be fertilized only by pollen with a nonexistent pollen function allele: a deleterious change that would reduce seed set to zero. However, a pistil function mutation complementary to a previously neutral pollen mutation may spread if it restores self-incompatibility to a self-compatible intermediate. We show that novel haplotypes can also drive elimination of existing ones with fewer siring opportunities. We calculate relative probabilities of increase and collapse in haplotype number given the initial collection of incompatibility haplotypes and the population gene conversion rate. Expansion in haplotype number is possible when population gene conversion rate is large, but large contractions are likely otherwise. A Markov chain model derived from these expansion and collapse probabilities generates a stable haplotype number distribution in the realistic range of 10-40 under plausible parameters. However, smaller populations might lose many haplotypes beyond those lost by chance during bottlenecks.

PFKFB3-mediated glycolysis rescues myopathic outcomes in the ischemic limb.

Compromised muscle mitochondrial metabolism is a hallmark of peripheral arterial disease, especially in patients with the most severe clinical manifestation - critical limb ischemia (CLI). We asked whether inflexibility in metabolism is critical for the development of myopathy in ischemic limb muscles. Using Polg mtDNA mutator (D257A) mice, we reveal remarkable protection from hind limb ischemia (HLI) due to a unique and beneficial adaptive enhancement of glycolytic metabolism and elevated ischemic muscle PFKFB3. Similar to the relationship between mitochondria from CLI and claudicating patient muscles, BALB/c muscle mitochondria are uniquely dysfunctional after HLI onset as compared with the C57BL/6 (BL6) parental strain. AAV-mediated overexpression of PFKFB3 in BALB/c limb muscles improved muscle contractile function and limb blood flow following HLI. Enrichment analysis of RNA sequencing data on muscle from CLI patients revealed a unique deficit in the glucose metabolism Reactome. Muscles from these patients express lower PFKFB3 protein, and their muscle progenitor cells possess decreased glycolytic flux capacity in vitro. Here, we show supplementary glycolytic flux as sufficient to protect against ischemic myopathy in instances where reduced blood flow-related mitochondrial function is compromised preclinically. Additionally, our data reveal reduced glycolytic flux as a common characteristic of the failing CLI patient limb skeletal muscle.

Heterogeneity in the rate of molecular sequence evolution substantially impacts the accuracy of detecting shifts in diversification rates.

As species richness varies along the tree of life, there is a great interest in identifying factors that affect the rates by which lineages speciate or go extinct. To this end, theoretical biologists have developed a suite of phylogenetic comparative methods that aim to identify where shifts in diversification rates had occurred along a phylogeny and whether they are associated with some traits. Using these methods, numerous studies have predicted that speciation and extinction rates vary across the tree of life. In this study, we show that asymmetric rates of sequence evolution lead to systematic biases in the inferred phylogeny, which in turn lead to erroneous inferences regarding lineage diversification patterns. The results demonstrate that as the asymmetry in sequence evolution rates increases, so does the tendency to select more complicated models that include the possibility of diversification rate shifts. These results thus suggest that any inference regarding shifts in diversification pattern should be treated with great caution, at least until any biases regarding the molecular substitution rate have been ruled out.

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia.

Stored muscle carbohydrate supply and energetic efficiency constrain muscle functional capacity during exercise and are influenced by common physiological variables (e.g. age, diet, and physical activity level). Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute hypoxia is not known. We interrogated skeletal muscle contractile properties in two anatomically distinct rodent hindlimb muscles that have well characterized differences in energetic efficiency (locomotory- extensor digitorum longus (EDL) and postural- soleus muscles) following a 24 hour fasting period that resulted in substantially reduced muscle carbohydrate supply. 180 mins of acute hypoxia resulted in complete energetic failure in all muscles tested, indicated by: loss of force production, substantial reductions in total adenosine nucleotide pool intermediates, and increased adenosine nucleotide degradation product-inosine monophosphate (IMP). These changes occurred in the absence of apparent myofiber structural damage assessed histologically by both transverse section and whole mount. Fasting and the associated reduction of the available intracellular carbohydrate pool (~50% decrease in skeletal muscle) did not significantly alter the timing to muscle functional impairment or affect the overall force/work capacities of either muscle type. Fasting resulted in greater passive tension development in both muscle types, which may have implications for the design of pre-clinical studies involving optimal timing of reperfusion or administration of precision therapeutics.

Memory in Trait Macroevolution.

The history of a trait within a lineage may influence its future evolutionary trajectory, but macroevolutionary theory of this process is not well developed. For example, consider the simplified binary trait of living in cave versus surface habitat. The longer a species has been cave dwelling, the more accumulated loss of vision, pigmentation, and defense may restrict future adaptation if the species encounters the surface environment. However, the Markov model of discrete trait evolution that is widely adopted in phylogenetics does not allow the rate of cave-to-surface transition to decrease with longer duration as a cave dweller. Here we describe three models of evolution that remove this memoryless constraint, using a renewal process to generalize beyond the typical Poisson process of discrete trait macroevolution. We then show how the two-state renewal process can be used for inference, and we investigate the potential of phylogenetic comparative data to reveal different influences of trait duration, or memory in trait evolution. We hope that such approaches may open new avenues for modeling trait evolution and for broad comparative tests of hypotheses that some traits become entrenched.

Interaction among ploidy, breeding system and lineage diversification.

If particular traits consistently affect rates of speciation and extinction, broad macroevolutionary patterns can be interpreted as consequences of selection at high levels of the biological hierarchy. Identifying traits associated with diversification rates is difficult because of the wide variety of characters under consideration and the statistical challenges of testing for associations from comparative phylogenetic data. Ploidy (diploid vs polyploid states) and breeding system (self-incompatible vs self-compatible states) are both thought to be drivers of differential diversification in angiosperms. We fit 29 diversification models to extensive trait and phylogenetic data in Solanaceae and investigate how speciation and extinction rate differences are associated with ploidy, breeding system, and the interaction between these traits. We show that diversification patterns in Solanaceae are better explained by breeding system and an additional unobserved factor, rather than by ploidy. We also find that the most common evolutionary pathway to polyploidy in Solanaceae occurs via direct breakdown of self-incompatibility by whole genome duplication, rather than indirectly via breakdown followed by polyploidization. Comparing multiple stochastic diversification models that include complex trait interactions alongside hidden states enhances our understanding of the macroevolutionary patterns in plant phylogenies.

Temporal Association Between Ischemic Muscle Perfusion Recovery and the Restoration of Muscle Contractile Function After Hindlimb Ischemia.

During incomplete skeletal muscle recovery from ischemia, such as that occurs with critical limb ischemia, the temporal relationship between recovery of muscle capillary perfusion and contractile function is poorly defined. We examined this relationship in BALB/cJ mice (N = 24) following unilateral hindlimb ischemia (HLI), which pre-clinically mimics the myopathy observed in critical limb ischemia patients. Specifically, we examined this relationship in two phenotypically distinct muscles (i.e., "oxidative" soleus - Sol and "glycolytic" extensor digitorum longus - EDL) 14- or 56-days after HLI. Although overall limb blood flow (LDPI) reached its' recovery peak (48% of control) by HLI d14, the capillary networks in both the Sol and EDL (whole mount confocal imaging) were disrupted and competent muscle capillary perfusion (perfused lectin+µm2/muscle µm2) remained reduced. Interestingly, both Sol and EDL muscles recovered their distinct capillary structures and perfusion (Con Sol; 0.056 ± 0.02 lectin+µm2/muscle µm2, and Con EDL; 0.039 ± 0.005 lectin+µm2/muscle µm2) by HLI d56 (Sol; 0.062 ± 0.011 lectin+µm2/muscle µm2 and EDL; 0.0035 ± 0.005 lectin+µm2/muscle µm2), despite no further improvement in limb blood flow (LDPI). Both muscles suffered severe myopathy, indicated by loss of dystrophin positive immunostaining and the absence of stimulation induced isometric force production at HLI d14. Dystrophin immunofluorescence returned at HLI d56, although neither myofiber CSA (µm2) nor isometric force production (58 and 28% sustained deficits, Sol and EDL, respectively) recovered completely in either muscle. In summary, we reveal that the temporal relationship between the restoration of muscle capillary perfusion and functional ischemic skeletal muscle regeneration favors competent muscle capillary perfusion recovery in BALB/c mice in a phenotypically non-distinct manner.