Phylogenomic analyses reveal convergent patterns of adaptive evolution in elephant and human ancestries

Specific sets of brain-expressed genes, such as aerobic energy metabolism genes, evolved adaptively in the ancestry of humans and may have evolved adaptively in the ancestry of other large-brained mammals. The recent addition of genomes from two afrotherians (elephant and tenrec) to the expanding set of publically available sequenced mammalian genomes provided an opportunity to test this hypothesis. Elephants resemble humans by having large brains and long life spans; tenrecs, in contrast, have small brains and short life spans. Thus, we investigated whether the phylogenomic patterns of adaptive evolution are more similar between elephant and human than between either elephant and tenrec lineages or human and mouse lineages, and whether aerobic energy metabolism genes are especially well represented in the elephant and human patterns. Our analyses encompassed ≈6,000 genes in each of these lineages with each gene yielding extensive coding sequence matches in interordinal comparisons. Each gene''s nonsynonymous and synonymous nucleotide substitution rates and dN/dS ratios were determined. Then, from gene ontology information on genes with the higher dN/dS ratios, we identified the more prevalent sets of genes that belong to specific functional categories and that evolved adaptively. Elephant and human lineages showed much slower nucleotide substitution rates than tenrec and mouse lineages but more adaptively evolved genes. In correlation with absolute brain size and brain oxygen consumption being largest in elephants and next largest in humans, adaptively evolved aerobic energy metabolism genes were most evident in the elephant lineage and next most evident in the human lineage.     

Diploid-dominant life cycles characterize the early evolution of Fungi

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane– and cell-wall–associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

Fungi and Metazoa evolved from a common protist-like ancestor, yet the two kingdoms have diverged in ways that make their kinship as Opisthokonts barely recognizable. Fungi grow on or within their food and feed by external digestion (osmotrophy), while animals mostly eat things smaller than themselves via ingestion. This difference is the basis for massive changes in morphology, including loss of motility during feeding and polarized cell growth in fungi (1, 2). The two kingdoms are also considered intrinsically different in life cycles, because fungi are characterized as being haplontic (haploid-dominant life cycle) while animals are diplontic (diploid-dominant). However, this textbook difference is inaccurate in two ways. First, the subkingdom Dikarya, with the majority of fungal species diversity, comprises lineages that spend some or most of their life cycles in a dikaryotic phase wherein two haploid nuclei undergo conjugate division, a cell type genetically analogous to a diploid (3). Further, life cycles have not been carefully investigated in most early-diverging fungal lineages (EDF), which include many phyla outside of Dikarya (e.g., non-Dikarya fungi). EDF have retained ancestral traits also retained in Metazoa, such as flagellation, actin structures used for crawling, presence of cholesterol in cell membranes, vitamin dependencies, and cell-cycle genes (4–8). However, life-cycle transitions between the Opisthokont ancestor and the extant Fungi are shrouded due to a lack of information on the genetic characteristics of EDF and the undersampling of their genomic diversity (9–11). The goal of this paper is to provide a robust and comprehensive phylogeny of the Fungi, emphasizing zoosporic taxa, to reassess the evolution of life-cycle and cellular characters during early fungal diversification using genomic data.Although fungi are often considered to have haploid-dominant life cycles, there are many variations observed (Fig. 1). In a haplontic life cycle, mitosis is restricted to the haploid phase, and meiosis ensues immediately following sex and nuclear fusion (Fig. 1A). In contrast, in the diplontic life cycle that generally characterizes Metazoa, mitosis is restricted to diploid cells (Fig. 1B). The alternation between haploid and diploid mitotic cycles, which generally characterizes plants, is documented, albeit rarely, in fungi, such as baker’s yeast and the water mold Allomyces (Fig. 1C). Despite this general avoidance of diploid mitosis in fungi, many Dikarya show a distinctive dikaryotic life cycle wherein, following mating, haploid nuclei of the two partners remain paired and undergo synchronous mitoses (Fig. 1D). This life cycle is analogous to diploidy with respect to genetic dominance (12) and would provide some of the proposed advantages of diploidy, such as buffering against somatic mutation (13). Overall, although we appreciate that fungal life cycles have great potential to vary, we have a poor understanding of life cycles of the EDF which represent the majority of the phylogenetic diversity of Fungi.Open in a separate windowFig. 1.Illustrated life cycles observed in fungi. (A) In haplontic life cycles mitosis is limited to the haploid phase, with plasmogamy of gametes followed by meiosis. (B) In diplontic life cycles, mitosis only occurs in the diploid phase with haploid cells only functioning as gametes. (C) Life cycles may alternative between haploid and diploid mitotic phases and may show morphological differences between ploidies as in Allomyces. (D) The dikaryotic life cycle is an alternative to alternation of haploid and diploid generations which lacks diploid mitosis and instead has a phase with two nuclear genotypes undergoing synchronous division.We consider EDF to comprise 11 phyla, including 8 zoosporic phyla that reproduce with swimming spores and form a contentious paraphyletic grade along the backbone of the fungal tree (9, 10, 14–16). The deeply diverging phyla, Rozellomycota/Cryptomycota and Aphelidiomycota, are endoparasites that have the ability to phagocytize, which enables them to ingest host cytoplasm, a trait presumably retained from the most recent common ancestor (MRCA) of Opisthokonta (17, 18). The remaining free-living zoosporic phyla have microscopic vegetative thalli that may be unicellular or mycelial (SI Appendix, Fig. S1), and the greatest species diversity is found in the Chytridiomycota, which has an estimated 14 orders (9). Chytridiomycota is united with the phyla Monoblepharidomycota + Neocallimastigomycota in subkingdom Chytridiomyceta, though the branching order of the three phyla is uncertain (14, 15, 19).Blastocladiomycota is an enigmatic group with a life cycle alternating between morphologically distinctive haploid and diploid thalli (Fig. 1C) (20, 21). Members include the water mold, Allomyces, that has been used as a model system for genetics and physiology (22) and a genus of obligate fatal parasites, Coelomomyces, that has a haploid phase in copepods and a diploid phase in mosquitoes (23). The precise phylogenetic placement of the Blastocladiomycota has been controversial (10, 15, 19, 24), with nearly equal support for the Blastocladiomycota diverging before the Chytridiomyceta or after the Chytridiomyceta. Several traits of Blastocladiomycota ally them with the terrestrial fungi (here defined as the nonzoosporic phyla Mucoromycota and Zoopagomycota and subkingdom Dikarya): closed mitosis, the presence of a Spitzenkörper, beta 1,3 glucans in the cell wall, and true mycelial growth in some members (22, 25). The detection of mating types in Coelomomyces (26), which have only been otherwise documented in terrestrial fungi, may be indicative that Blastocladiomycota is more closely related to Dikarya than Chytridiomyceta.Mating and sexuality are poorly described in zoosporic fungi beyond the well-characterized water mold model Allomyces. According to mycological textbooks, life cycles of Chytridiomycota are characterized as being haplontic with zygotic meiosis (27–29), but the majority of assumptions of meiotic stages are unconfirmed by cytology. Moreover, the requisite genetic studies using molecular markers to confirm ploidy cycling have not been accomplished for these presumably sexual species. Importantly, the best-studied chytrid fungus, Batrachochytrium dendrobatidis, has a life cycle that appears to be dominated by asexually reproducing diploid, or aneuploid, thalli (30). More recently, additional studies have indicated that non-Dikarya phyla have heterozygosity indicative of diploidy or higher ploidy (31–34), suggesting that the assumption of haplontic life cycles for the Chytridiomyceta and other EDF may be false.Current sequencing technologies now create the potential for leveraging genomic sequencing to broadly sample fungal genomes for estimating ploidy and other cellular traits in a robust phylogenomic framework. Here, we sampled 69 zoosporic fungal genomes using both culture and single-cell approaches. Our genome analyses provide a strongly supported phylogeny for understanding taxonomy and the evolution of ploidy and other traits which had previously been held to be distinctive between Fungi and Metazoa. These data bolster the growing picture that many traits including motility, feeding modes, and life cycles changed gradually during the early diversification of fungi. The high levels of heterozygosity estimated from genomes analyzed in this study reveal that somatic diploidy is much more common in Fungi than previously appreciated.     

Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution

To what extent are evolutionary outcomes determined by a population''s recent environment, and to what extent do they depend on historical contingency and random chance? Here we apply a unique experimental system to investigate evolutionary reproducibility and path dependence at the protein level. We combined phage-assisted continuous evolution with high-throughput sequencing to analyze evolving protein populations as they adapted to divergent and then convergent selection pressures over hundreds of generations. Independent populations of T7 RNA polymerase genes were subjected to one of two selection histories (“pathways”) demanding recognition of distinct intermediate promoters followed by a common final promoter. We observed distinct classes of solutions with unequal phenotypic activity and evolutionary potential evolve from the two pathways, as well as from replicate populations exposed to identical selection conditions. Mutational analysis revealed specific epistatic interactions that explained the observed path dependence and irreproducibility. Our results reveal in molecular detail how protein adaptation to different environments, as well as stochasticity among populations evolved in the same environment, can both generate evolutionary outcomes that preclude subsequent convergence.     

Phylogenetic analyses reveal the shady history of C4 grasses

Grasslands cover more than 20% of the Earth''s terrestrial surface, and their rise to dominance is one of the most dramatic events of biome evolution in Earth history. Grasses possess two main photosynthetic pathways: the C₃ pathway that is typical of most plants and a specialized C₄ pathway that minimizes photorespiration and thus increases photosynthetic performance in high-temperature and/or low-CO₂ environments. C₄ grasses dominate tropical and subtropical grasslands and savannas, and C₃ grasses dominate the world''s cooler temperate grassland regions. This striking pattern has been attributed to C₄ physiology, with the implication that the evolution of the pathway enabled C₄ grasses to persist in warmer climates than their C₃ relatives. We combined geospatial and molecular sequence data from two public archives to produce a 1,230-taxon phylogeny of the grasses with accompanying climate data for all species, extracted from more than 1.1 million herbarium specimens. Here we show that grasses are ancestrally a warm-adapted clade and that C₄ evolution was not correlated with shifts between temperate and tropical biomes. Instead, 18 of 20 inferred C₄ origins were correlated with marked reductions in mean annual precipitation. These changes are consistent with a shift out of tropical forest environments and into tropical woodland/savanna systems. We conclude that C₄ evolution in grasses coincided largely with migration out of the understory and into open-canopy environments. Furthermore, we argue that the evolution of cold tolerance in certain C₃ lineages is an overlooked innovation that has profoundly influenced the patterning of grassland communities across the globe.     

Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli

The role of historical contingency in evolution has been much debated, but rarely tested. Twelve initially identical populations of Escherichia coli were founded in 1988 to investigate this issue. They have since evolved in a glucose-limited medium that also contains citrate, which E. coli cannot use as a carbon source under oxic conditions. No population evolved the capacity to exploit citrate for >30,000 generations, although each population tested billions of mutations. A citrate-using (Cit+) variant finally evolved in one population by 31,500 generations, causing an increase in population size and diversity. The long-delayed and unique evolution of this function might indicate the involvement of some extremely rare mutation. Alternately, it may involve an ordinary mutation, but one whose physical occurrence or phenotypic expression is contingent on prior mutations in that population. We tested these hypotheses in experiments that "replayed" evolution from different points in that population's history. We observed no Cit+ mutants among 8.4 x 10(12) ancestral cells, nor among 9 x 10(12) cells from 60 clones sampled in the first 15,000 generations. However, we observed a significantly greater tendency for later clones to evolve Cit+, indicating that some potentiating mutation arose by 20,000 generations. This potentiating change increased the mutation rate to Cit+ but did not cause generalized hypermutability. Thus, the evolution of this phenotype was contingent on the particular history of that population. More generally, we suggest that historical contingency is especially important when it facilitates the evolution of key innovations that are not easily evolved by gradual, cumulative selection.     

Gregor Johann Mendel and Modern Evolutionary Biology: Genetics of adaptation

The rediscovery of Mendel’s work showing that the heredity of phenotypes is controlled by discrete genes was followed by the reconciliation of Mendelian genetics with evolution by natural selection in the middle of the last century with the Modern Synthesis. In the past two decades, dramatic advances in genomic methods have facilitated the identification of the loci, genes, and even individual mutations that underlie phenotypic variants that are the putative targets of natural selection. Moreover, these methods have also changed how we can study adaptation by flipping the problem around, allowing us to first examine what loci show evidence of having been under selection, and then connecting these genetic variants to phenotypic variation. As a result, we now have an expanding list of actual genetic changes that underlie potentially adaptive phenotypic variation. Here, we synthesize how considering the effects of these adaptive loci in the context of cellular environments, genomes, organisms, and populations has provided new insights to the genetic architecture of adaptation.     

Testing hypotheses on the rate of molecular evolution in relation to gene expression using microRNAs

There exists an inverse relationship between the rate of molecular evolution and the level of gene expression. Among the many explanations, the "toxic-error" hypothesis is a most general one, which posits that processing errors may often be toxic to the cells. However, toxic errors that constrain the evolution of highly expressed genes are often difficult to measure. In this study, we test the toxic-error hypothesis by using microRNA (miRNA) genes because their processing errors can be directly measured by deep sequencing. A miRNA gene consists of a small mature product (≈22 nt long) and a "backbone." Our analysis shows that (i) like the mature miRNA, the backbone is highly conserved; (ii) the rate of sequence evolution in the backbone is negatively correlated with expression; and (iii) although conserved between distantly related species, the error rate in miRNA processing is also negatively correlated with the expression level. The observations suggest that, as a miRNA gene becomes more highly (or more ubiquitously) expressed, its sequence evolves toward a structure that minimizes processing errors.     

Rapid evolution of stability and productivity at the origin of a microbial mutualism

Mutualistic interactions are taxonomically and functionally diverse. Despite their ubiquity, however, the basic ecological and evolutionary processes underlying their origin and maintenance are poorly understood. A major reason for this is the lack of an experimentally tractable model system. We examine the evolution of an experimentally imposed obligate mutualism between sulfate-reducing and methanogenic microorganisms that have no known history of previous interaction. Twenty-four independent pairings (cocultures) of the bacterium Desulfovibrio vulgaris and the archaeon Methanococcus maripaludis were established and followed for 300 community doublings in two environments, one allowing for the development of a heterogeneous distribution of resources and the other not. Evolved cocultures grew up to 80% faster and were up to 30% more productive (biomass yield per mole of substrate) than the ancestors. The evolutionary process was marked by periods of significant instability leading to extinction of two of the cocultures, but it resulted in more stable, efficient, and productive mutualisms for most replicated pairings. Comparisons of evolved cocultures with those assembled from one evolved mutualist and one ancestral mutualist showed that evolution of both species contributed to improved productivity. Surprisingly, however, overall improvements in growth rate and yield were less than the sum of the individual contributions, suggesting antagonistic interactions between mutations from the coevolved populations. Physical constraints on the transfer of metabolites in the evolution environment affected the evolution of M. maripaludis, but not of D. vulgaris. Together, these results demonstrate that challenges can imperil nascent obligate mutualisms and demonstrate the evolutionary responses that enable their persistence and future evolution.     

From the Cover: Comparative analyses of animal-tracking data reveal ecological significance of endothermy in fishes

Despite long evolutionary separations, several sharks and tunas share the ability to maintain slow-twitch, aerobic red muscle (RM) warmer than ambient water. Proximate causes of RM endothermy are well understood, but ultimate causes are unclear. Two advantages often proposed are thermal niche expansion and elevated cruising speeds. The thermal niche hypothesis is generally supported, because fishes with RM endothermy often exhibit greater tolerance to broad temperature ranges. In contrast, whether fishes with RM endothermy cruise faster, and achieve any ecological benefits from doing so, remains unclear. Here, we compiled data recorded by modern animal-tracking tools for a variety of free-swimming marine vertebrates. Using phylogenetically informed allometry, we show that both cruising speeds and maximum annual migration ranges of fishes with RM endothermy are 2–3 times greater than fishes without it, and comparable to nonfish endotherms (i.e., penguins and marine mammals). The estimated cost of transport of fishes with RM endothermy is twice that of fishes without it. We suggest that the high energetic cost of RM endothermy in fishes is offset by the benefit of elevated cruising speeds, which not only increase prey encounter rates, but also enable larger-scale annual migrations and potentially greater access to seasonally available resources.In 1835, the British physician John Davy reported that skipjack tuna have body temperatures 10 °C higher than ambient waters and considered this fish an exception to the general rule that fishes are cold-blooded (1). It is currently known that at least 14 species of tuna (family Scombridae) and five species of shark (four species in the family Lamnidae and one species in the family Alopiidae) have the ability to retain metabolic heat via vascular countercurrent heat exchangers, and to maintain the temperature of slow-twitch, aerobic red muscle (hereafter denoted RM) significantly above that of the ambient water (2–7). This “RM endothermy” (see SI Materials and Methods for terminology) in fishes represents a remarkable example of convergent evolution, because bony fishes and cartilaginous fishes diverged as long as 450 million years ago (8). In addition to elevated RM temperature, tunas and endothermic sharks share a number of morphological (e.g., medially located RM), physiological (e.g., high metabolic rates), and ecological (e.g., highly mobile and predatory lifestyle) characteristics (9).RM endothermy is an energetically expensive thermal strategy (9), and its convergent evolution indicates that the extra energetic costs incurred by RM endothermy can be outweighed by some ecological advantages. This topic has been discussed intensively, and two primary, nonmutually exclusive hypotheses have been proposed: expansion of the thermal niche and elevated cruising speeds (2). The thermal niche hypothesis states that fishes with RM endothermy can tolerate a broader range of water temperatures and, thus, can expand their geographic niche. An increasing suite of evidence supports this hypothesis; tunas and endothermic sharks often range widely and dive well beneath the thermocline and, consequently, experience a broad temperature range (e.g., more than 20 °C in some species; refs. 10 and 11). However, some ectothermic species (e.g., blue shark) experience similar temperature ranges by diving deep (11, 12), suggesting that other factors may also affect the thermal preference and tolerance of pelagic fishes.The elevated cruising speed hypothesis states that elevated RM temperature enhances the power output of RM and, thereby, increases cruising speed of the fishes (2). This hypothesis is reasonable, because the contraction speed and power output of the isolated RM (13) and the sustained swim speed of ectothermic fishes in captivity (14) all increase with temperature within a species, at least within their normal temperature range. Surprisingly, however, a previous laboratory study found no differences in the sustained swim speeds between two Scombridae species with and without RM endothermy (15). As a result, evidence for the hypothesis is still lacking.If fishes with RM endothermy are shown to cruise faster in nature, what ecological benefits could they achieve from doing so? Fishes can increase prey encounter rates and, thus, potential energy gains by cruising faster (16); however, this benefit may be counteracted if energetic costs incurred by cruising faster and being endothermic are high. It is therefore important to examine whether the cost of transport (i.e., the energy needed to move a unit body mass over a unit distance) at their cruising speeds is higher for fishes with RM endothermy.In addition to the benefit of increased prey encounter rates, fishes with RM endothermy may be able to move greater distances in a given time period, such as a year, because of their fast cruising speed. Annual migrations are common in fishes, often between foraging grounds and reproductive habitats (10, 11, 17); therefore, it is hypothesized that fishes with RM endothermy exhibit annual migrations over larger spatial scales than fishes without it. If such difference is observed, large-scale migration could be an ecological advantage, because it allows the fishes with RM endothermy to better exploit seasonal peaks of resource abundance and avoid seasonal resource depression (18).Because of the rapid development and improvement of various data-recording or transmitting tags, information on fish movements in the wild is increasingly available, both from fine-scale (e.g., recording swim speed; ref. 19) and long-term (e.g., recording migration path; ref. 11) animal-tracking studies. Such information has provided much insight into the ecology of many species; however, no previous studies have examined the possible differences in the movement patterns or swimming energetics in nature between fishes with and without RM endothermy. In this study, therefore, we compiled data on cruising swim speed and migration range of fishes, recorded by various animal-tracking tools, both from the literature and our own fieldwork. We also estimated the cost of transport for each species swimming at each cruising speed. Using phylogenetically informed allometry, we examined whether fishes with RM endothermy (i) swim faster, (ii) have higher cost of transport, and (iii) exhibit larger-scale annual migrations.     

Comparative genomics reveal the mechanism of the parallel evolution of O157 and non-O157 enterohemorrhagic Escherichia coli

Among the various pathogenic Escherichia coli strains, enterohemorrhagic E. coli (EHEC) is the most devastating. Although serotype O157:H7 strains are the most prevalent, strains of different serotypes also possess similar pathogenic potential. Here, we present the results of a genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains. All EHECs have much larger genomes (5.5–5.9 Mb) than the other strains and contain surprisingly large numbers of prophages and integrative elements (IEs). The gene contents of the 4 EHECs do not follow the phylogenetic relationships of the strains, and they share virulence genes for Shiga toxins and many other factors. We found many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EHEC. Particularly interesting is the evolution of the type III secretion system (T3SS). We found that the T3SS of EHECs is composed of genes that were introduced by 3 different types of genetic elements: an IE referred to as the locus of enterocyte effacement, which encodes a central part of the T3SS; SpLE3-like IEs; and lambdoid phages carrying numerous T3SS effector genes and other T3SS-related genes. Our data demonstrate how E. coli strains of different phylogenies can independently evolve into EHECs, providing unique insights into the mechanisms underlying the parallel evolution of complex virulence systems in bacteria.     

From the Cover: Ribonucleotide reductases reveal novel viral diversity and predict biological and ecological features of unknown marine viruses

Virioplankton play a crucial role in aquatic ecosystems as top-down regulators of bacterial populations and agents of horizontal gene transfer and nutrient cycling. However, the biology and ecology of virioplankton populations in the environment remain poorly understood. Ribonucleotide reductases (RNRs) are ancient enzymes that reduce ribonucleotides to deoxyribonucleotides and thus prime DNA synthesis. Composed of three classes according to O₂ reactivity, RNRs can be predictive of the physiological conditions surrounding DNA synthesis. RNRs are universal among cellular life, common within viral genomes and virioplankton shotgun metagenomes (viromes), and estimated to occur within >90% of the dsDNA virioplankton sampled in this study. RNRs occur across diverse viral groups, including all three morphological families of tailed phages, making these genes attractive for studies of viral diversity. Differing patterns in virioplankton diversity were clear from RNRs sampled across a broad oceanic transect. The most abundant RNRs belonged to novel lineages of podoviruses infecting α-proteobacteria, a bacterial class critical to oceanic carbon cycling. RNR class was predictive of phage morphology among cyanophages and RNR distribution frequencies among cyanophages were largely consistent with the predictions of the “kill the winner–cost of resistance” model. RNRs were also identified for the first time to our knowledge within ssDNA viromes. These data indicate that RNR polymorphism provides a means of connecting the biological and ecological features of virioplankton populations.Viruses are key players in biogeochemical cycling and energy flow and help shape the composition of aquatic microbial communities (1–3). Additionally, viruses influence microbial metabolism through horizontal gene transfer and expression of auxiliary metabolic genes during infection (4). Despite their impact, we understand little about the specific biological features and ecological strategies of viral populations within natural ecosystems. Constraining these second-order issues is critical to building better quantitative models of how viral processes affect ecosystems (5).Methodological limitations have hindered efforts to understand viral ecology. Viruses lack a universally conserved phylogenetic marker, akin to the 16S rRNA gene in cells, which can broadly assay viral distributions and diversity. Marker genes used as proxies of environmental viral diversity are typically limited to specific viral taxa. Furthermore, PCR-based approaches can fail to detect prominent and biologically important viral populations owing to the potential for low nucleotide similarity between homologous genes. Recent work examining the diversity of viral DNA polymerase A genes within virioplankton metagenomic (virome) sequence data revealed that low-efficiency DNA polymerases, undetected by PCR, were predominant within virioplankton (6). That work also highlighted the unique ability of DNA polA sequences to provide insights into the biological features of unknown phages within the virioplankton. In general, the ability to connect biological features with sequence diversity in marker genes—including those widely used in ecological studies, such as the 16S rRNA gene—can be tenuous (7).Ideally, a marker gene of viral diversity should (i) be widely distributed among diverse viral lineages and, therefore, evolutionarily ancient; (ii) be abundant within environmental viral assemblages; (iii) play an important role in viral biology; (iv) have a single evolutionary origin and not be replaceable through nonorthologous gene displacement; (v) be phylogenetically informative; and (vi) be well represented in reference databases. Ribonucleotide reductase (RNR) gene products fulfill these criteria. Nucleotide metabolism pathways, including biosynthesis, are among the most represented within the virioplankton (8, 9). RNRs are the only known enzymes capable of reducing ribonucleotides to deoxyribonucleotides (10), an essential step for DNA synthesis. As such, RNRs are key to nucleotide biosynthesis, under stringent evolutionary selection pressure, and among the most abundant annotated genes in marine virome libraries (11). Importantly, RNR genes are present in all three families of tailed phages in the order Caudovirales and have been identified in viruses infecting hosts within all three domains of life (10). RNRs are strongly tied to lytic marine phages (12), which significantly influence nutrient cycles within the global ocean (5). Therefore, RNRs easily fit the criteria of being functionally nonredundant, abundant, and widely distributed.In addition, RNRs are biologically informative and form three physiological classes according to reactivity with O₂. Class I RNRs are O₂-dependent. Class II RNRs are O₂-independent and rely upon adenosylcobalamin (vitamin B₁₂). Class III RNRs are sensitive to O₂. All three classes share a common catalytic center and use similar radical-based chemistry (13). Therefore, all three modern classes of RNR likely evolved from a single common ancestor (14). This study focused on the catalytic (alpha) subunit of the holoenzyme identified in virome libraries spanning a broad oceanic transect. Subsequently these data were used to examine the biological and ecological features of lytic phage populations within the Caudovirales. The outcomes of these analyses were interpreted within the context of known viral diversity and the “kill the winner–cost of resistance” (KTW–COR) model for viral–host interactions (15). Overall, these data show that RNR sequence diversity within the virioplankton connects broadly with phage morphological groups and can be predictive of the ecological strategies within the virioplankton.     

Adaptive differentiation and rapid evolution of a soil bacterium along a climate gradient

Microbial community responses to environmental change are largely associated with ecological processes; however, the potential for microbes to rapidly evolve and adapt remains relatively unexplored in natural environments. To assess how ecological and evolutionary processes simultaneously alter the genetic diversity of a microbiome, we conducted two concurrent experiments in the leaf litter layer of soil over 18 mo across a climate gradient in Southern California. In the first experiment, we reciprocally transplanted microbial communities from five sites to test whether ecological shifts in ecotypes of the abundant bacterium, Curtobacterium, corresponded to past adaptive differentiation. In the transplanted communities, ecotypes converged toward that of the native communities growing on a common litter substrate. Moreover, these shifts were correlated with community-weighted mean trait values of the Curtobacterium ecotypes, indicating that some of the trait variation among ecotypes could be explained by local adaptation to climate conditions. In the second experiment, we transplanted an isogenic Curtobacterium strain and tracked genomic mutations associated with the sites across the same climate gradient. Using a combination of genomic and metagenomic approaches, we identified a variety of nonrandom, parallel mutations associated with transplantation, including mutations in genes related to nutrient acquisition, stress response, and exopolysaccharide production. Together, the field experiments demonstrate how both demographic shifts of previously adapted ecotypes and contemporary evolution can alter the diversity of a soil microbiome on the same timescale.

Microbial communities respond quickly to environmental change (1, 2). These responses are typically associated with ecological processes; however, the potential for microbes to evolve and adapt to changes in the environment on ecological timescales remains largely unexplored in natural ecosystems. While evolutionary processes are typically considered over longer timescales, the short generation times, large populations, and high mutation rates indicative of microorganisms may allow for rapid adaptation. Laboratory studies have repeatedly demonstrated rapid evolution of bacterial populations (3) with consequences for organismal physiology (4), yet it remains unclear how these in vitro studies extend to in situ communities (5).Both ecological and evolutionary processes likely contribute simultaneously (6, 7) to the response of a microbiome to changing environmental conditions (8). However, separating these processes for bacteria can be difficult as they occur along a continuum of temporal and genetic scales. In terms of ecological processes, microbiome composition can respond demographically, as selective forces promote the growth and survival of differentially adapted taxa within the bacterial community. Certainly, many studies have observed such shifts in taxonomic composition of 16S ribosomal RNA (rRNA)–defined taxa in response to simulated global changes (9), and these responses are considered an ecological process (e.g., species sorting). Few examples, however, link these responses to trait differences among bacterial taxa (10–13), precluding direct insights into whether these ecological shifts are due to adaptive differentiation among taxa as a result of past evolutionary divergence. Concurrently, the same selective forces can also shift the abundance of conspecific strains and alter the allele frequencies of preexisting genetic variation, which at this genetic scale is defined as an evolutionary process (14). Finally, evolution through de novo mutation can provide a new source of genetic variation that may allow for further adaptation to environmental change.In this study, we aimed to capture this continuum of ecological and evolutionary processes that together produce the response of a microbiome’s diversity to environmental change (Fig. 1A). Studying evolution in microbial communities in situ, however, is challenging. For one, variation in highly conserved marker genes used in many microbiome studies (e.g., 16S rRNA) represents distant evolutionary divergences, and thus these regions are too conserved to detect locally adapted lineages (11, 12, 15), let alone recent evolutionary change within communities (16). To overcome this limitation, studies have leveraged shotgun metagenomic data (17, 18) and genome sequences of co-occurring, closely related strains (19, 20) to characterize evolutionary processes (e.g., recombination and gene flow) structuring the genetic diversity of bacterial lineages. However, these studies are also limited by an inherent challenge in microbiome research: delineating population boundaries, the fundamental unit of evolution. While progress has been made in defining microbial species (21–23), the high genetic heterogeneity within diverse microbial communities, such as soils, convolute the boundaries of the fine-scale patterns of genetic diversity within microbial taxonomic units (12). For instance, metagenome-assembled genomes are often composed of a composite of strains forming a large population of mosaic genomes (24) that may not fully capture the diversity of the local population (25). As such, it remains difficult to study evolutionary rates within microbial communities (however, see refs. 26, 27), and the extent and time scale at which evolutionary processes contribute to both standing and new genetic variation relative to ecological processes.Open in a separate windowFig. 1.Microbial community transplant experiment. (A) Changes in microbial community composition can be due to a continuum of ecological and evolutionary processes. For instance, shifts in standing genetic variation can be attributed to both ecological and evolutionary processes depending on the level of biological resolution, while de novo mutations can be a result from evolutionary adaptation. (B) A schematic of the two parallel transplant experiments at the community and strain level. Inoculated litterbags were transplanted to all sites along an elevation gradient that covaried in temperature and precipitation. Site codes: D=Desert; Sc=Scrubland; G=Grassland; P=Pine-Oak; S=Subalpine.Here, we asked the following question: can we characterize the ecological and evolutionary processes that are contributing concurrently to the response of a soil bacterial community to a changing environment? To answer this question, we utilized a field-based experimental approach to quantify the influence of both ecological and evolutionary processes on one focal soil bacterium in its natural environment, the genus Curtobacterium (28). Specifically, we transplanted the bacterium across an elevation gradient on a common resource (leaf litter) substrate (29) to assess its response to new climates in two parallel experiments over the same 18 mo time period (Fig. 1B). In both experiments, we used microbial cages [nylon mesh bags that allow for nutrient transport (30)] to manipulate microbial composition while restricting microbial migration to eliminate the introduction of new alleles and/or variants from dispersal (31). A reciprocal transplant design allowed for direct testing of microbial adaptation to abiotic conditions (i.e., moisture and temperature) in a natural setting.In the first experiment, we conducted a reciprocal transplant of the entire microbial community (32) and tracked the ecological response of Curtobacterium ecotypes (33). A bacterial ecotype is defined as highly clustered genotypic and phenotypic strains occupying the same ecological niche, somewhat equivalent to a eukaryotic species (34). To test the hypothesis that Curtobacterium ecotypes are locally adapted to their climate conditions, we assessed the convergence of ecotype composition in the transplanted communities to that of control communities (those that remained in their native environment; Fig. 1B). We further hypothesized that the demographic shifts were due to differential adaptation to local climates as a result of trait variation among the ecotypes. Thus, we expected that the climate gradient would select for a strong trait–environment relationship (assessed by community-weighted mean (CWM) trait values) as typically observed in plant communities (35, 36).In parallel, we conducted an in situ evolution experiment by transplanting an isogenic Curtobacterium strain across the same gradient to investigate the potential for rapid evolution on the same timescales. We hypothesized that a variety of genomic mutations would be associated with adaptation to local climate conditions. Therefore, we expected fewer genetic changes when the strain was transplanted to its original environment, the midelevation Grassland site, while the extreme sites of the gradient would impose stronger selective pressures resulting in greater genetic changes. We further expected to observe parallel mutations among replicates within a site, which would be indicative of adaptive events (37). Variation in such mutations across sites would suggest selection differences across the climate gradient. Together, the two experiments capture the simultaneous effects of both ecological and evolutionary processes on the response of a soil bacterium to new climates in the field.     

From the Cover: Recursive genomewide recombination and sequencing reveals a key refinement step in the evolution of a metabolic innovation in Escherichia coli

Evolutionary innovations often arise from complex genetic and ecological interactions, which can make it challenging to understand retrospectively how a novel trait arose. In a long-term experiment, Escherichia coli gained the ability to use abundant citrate (Cit⁺) in the growth medium after ∼31,500 generations of evolution. Exploiting this previously untapped resource was highly beneficial: later Cit⁺ variants achieve a much higher population density in this environment. All Cit⁺ individuals share a mutation that activates aerobic expression of the citT citrate transporter, but this mutation confers only an extremely weak Cit⁺ phenotype on its own. To determine which of the other >70 mutations in early Cit⁺ clones were needed to take full advantage of citrate, we developed a recursive genomewide recombination and sequencing method (REGRES) and performed genetic backcrosses to purge mutations not required for Cit⁺ from an evolved strain. We discovered a mutation that increased expression of the dctA C₄-dicarboxylate transporter greatly enhanced the Cit⁺ phenotype after it evolved. Surprisingly, strains containing just the citT and dctA mutations fully use citrate, indicating that earlier mutations thought to have potentiated the initial evolution of Cit⁺ are not required for expression of the refined version of this trait. Instead, this metabolic innovation may be contingent on a genetic background, and possibly ecological context, that enabled citT mutants to persist among competitors long enough to obtain dctA or equivalent mutations that conferred an overwhelming advantage. More generally, refinement of an emergent trait from a rudimentary form may be crucial to its evolutionary success.Key innovations in the history of life are often caused by the acquisition of a qualitatively new trait that is “an evolutionary novelty which allows the exploitation of new resources or habitats and thus triggers an adaptive radiation” (1). Such innovations are typically rare and difficult to predict because they result from complex nonadditive (i.e., epistatic) genetic interactions or ecological interactions, within or between species, that develop only over the course of long evolutionary trajectories (2). Evolution of a new trait can be conceptually divided into three steps: potentiation, actualization, and refinement (3). First, one or more potentiating events may be necessary to generate a genetic background or environmental conditions that make a new trait accessible to evolution. Genetic potentiation, for example, may involve a period of nonadaptive genetic drift wherein a phenotype stays constant or the accumulation of mutations that are immediately advantageous for reasons unrelated to the new trait (4, 5). Then, a keystone actualizing mutation or environmental shift may lead to expression of the new trait, possibly by coopting latent changes in a cellular network or physical structure for a new use (6–8). Finally, there may be many subsequent opportunities for further refinement mutations that improve an emergent trait so that a newly colonized niche can be fully exploited (1).The appearance of citrate utilization in a >25-y long-term evolution experiment (LTEE) with Escherichia coli provides an opportunity to study a deep historical record leading to a key metabolic innovation (3, 9, 10). E. coli cannot ordinarily grow on citrate as a sole carbon source under aerobic conditions (11, 12), a phenotype that has been used to define it as a species (13). Just 1 of 12 replicate LTEE populations gained the ability to aerobically use citrate (Cit⁺), and this rare innovation happened only after ∼31,500 generations of growth in glucose-limited media, despite the presence of an excess of citrate as an untapped carbon source all along (10). The actualizing event for the Cit⁺ trait is known: tandem amplifications of a chromosomal region that place a copy of an aerobically active promoter upstream of a citrate transporter (citT) are present in all Cit⁺ isolates from this population (3).However, the Cit⁺ trait was surprisingly weak when it first appeared. The earliest individuals with the citT mutation exhibit little or no growth on citrate as a sole carbon source and appear to have derived only a small benefit from this mutation under the conditions of the LTEE (3). In fact, a majority of the population remained Cit^– for at least 1,500 generations (225 d) after the citT mutation evolved, and these initial Cit⁺ individuals were only detected retrospectively in historical samples of the population by using a sensitive indicator agar test for citrate utilization and allowing days to weeks for a positive result (10). Adding a high-copy plasmid with a module containing the new promoter configuration and citT gene to the ancestral strain of the LTEE leads to a phenotype similar to that of the early Cit⁺ clones, indicating that this mutation is, at least qualitatively, sufficient on its own for this rudimentary version of the new trait (3).Shortly after ∼33,000 generations, this LTEE population experienced a massive increase in the final cell density it reached at the end of each daily growth cycle (10). This population expansion was due to the evolution of new Cit⁺ variants that fully use the abundant citrate in the media after glucose depletion. We call this strong phenotype Cit⁺⁺, to differentiate it from the weak Cit⁺ phenotype of earlier isolates with just the citT mutation. Cit⁺⁺ cells contain one or more additional refinement mutations that make robust growth on citrate as a sole carbon source possible. Strains with the Cit⁺⁺ trait can be readily distinguished from Cit⁺ strains by their ability to form colonies within 48 h on minimal agar containing citrate as the only carbon source.Experiments that replayed the evolution of a Cit⁺⁺ phenotype many times from Cit^– isolates taken at different generational time points from the focal LTEE population, found evidence that one or more as-yet-unknown potentiating mutations accumulated in the lineage leading to Cit⁺ that made later strains more likely to access this metabolic innovation (10). Determining what evolved alleles are required for efficient citrate utilization would shed light on the evolutionary pathway that led to the appearance and refinement of this trait. However, identifying these mutations is confounded by the presence of >70 evolved alleles in the earliest strains that have the Cit⁺⁺ phenotype, most of which likely improved growth on glucose by mechanisms that were not relevant for the emergence of efficient citrate utilization.Finding the minimal set of mutations required for Cit⁺⁺, as well as many other complex evolved phenotypes, is currently a daunting task in asexual microbes. Methods such as genome shuffling (14), multiplex automated genome engineering (MAGE) (15), and array-based discovery of adaptive mutations (ADAM) (16) can be used to create and test of libraries consisting of many genetic variants to dissect complex traits. However, no current technique is suitable in a bacterium like E. coli for concurrently (i) capturing the structure of epistatic networks involving multiple alleles spread across the entire chromosome, (ii) introducing point mutations without associated genetic markers, (iii) broad compatibility with different strain backgrounds, and (iv) reconstructing complex chromosomal rearrangements, mobile element insertions, or large genomic duplications, which are common features of these evolved strains (17, 18). To overcome these limitations, we developed recursive genomewide recombination and sequencing (REGRES), a method that uses conjugative chromosomal transfer, phenotypic selection, and whole-genome sequencing to identify the alleles required for an evolved trait.Qualitative traits, including evolutionary innovations, may be the result of all-or-none epistatic interactions, where no subset of mutations is sufficient for expression of a new phenotype (5). This absolute genetic interdependence will lead to a sudden and discrete change in a trait when an actualizing or refinement mutation occurs, as observed with the two steps in the evolution of citrate utilization in the LTEE. In accordance with this model, we expected REGRES with selection for the Cit⁺⁺ phenotype to preserve the citT amplification and some combination of unknown potentiation and refinement mutations. We found that a single refinement mutation affecting the dctA gene was the only evolved allele other than citT conserved across REGRES genomes. These two mutations were sufficient for qualitatively reproducing full utilization of citrate, suggesting a physiological mechanism for refinement that involved activation of a second transporter that makes citrate import by the CitT transporter independent of the production of a cosubstrate by central metabolism. Finally, we discuss an alternative to the all-or-none epistasis hypothesis for how mutations could potentiate the evolution of the strong Cit⁺⁺ trait without being strictly required for its expression.     

From the Cover: Dynamic evolutionary change in post-Paleozoic echinoids and the importance of scale when interpreting changes in rates of evolution

How ecological and morphological diversity accrues over geological time has been much debated by paleobiologists. Evidence from the fossil record suggests that many clades reach maximal diversity early in their evolutionary history, followed by a decline in evolutionary rates as ecological space fills or due to internal constraints. Here, we apply recently developed methods for estimating rates of morphological evolution during the post-Paleozoic history of a major invertebrate clade, the Echinoidea. Contrary to expectation, rates of evolution were lowest during the initial phase of diversification following the Permo-Triassic mass extinction and increased over time. Furthermore, although several subclades show high initial rates and net decreases in rates of evolution, consistent with “early bursts” of morphological diversification, at more inclusive taxonomic levels, these bursts appear as episodic peaks. Peak rates coincided with major shifts in ecological morphology, primarily associated with innovations in feeding strategies. Despite having similar numbers of species in today’s oceans, regular echinoids have accrued far less morphological diversity than irregular echinoids due to lower intrinsic rates of morphological evolution and less morphological innovation, the latter indicative of constrained or bounded evolution. These results indicate that rates of evolution are extremely heterogenous through time and their interpretation depends on the temporal and taxonomic scale of analysis.Assessing how rates of morphological evolution have changed over geological time has been a major research goal of evolutionary paleobiologists since Westoll’s classic study of lungfish evolution (1). A common pattern to emerge from the fossil record is that many clades reach maximal morphological diversity early in their evolutionary history (2–4). This sort of pattern could be the result of an “early burst” of morphological diversification as taxa diverge followed by a slow-down in rates as ecological space becomes filled (5, 6). Internal constraint or long-term selective pressures could also limit overall disparity, leading to a slowdown in the rate of new trait acquisition over time (7, 8). However, only a small proportion of fossil disparity studies have also assessed changes in rates of evolution within lineages (e.g., along phylogenetic branches) thereby providing a more nuanced understanding of how this disparity came about (e.g., refs. 9–13). Simultaneously, decreasing rates in trait evolution have been difficult to detect using phylogenetic comparative data of extant taxa, because of low statistical power (14, 15), loss of signal through extinction (16), and inaccuracies in reconstructing ancestral nodes (17). Here we take advantage of recently developed methods for directly estimating per-lineage-million-year rates of evolution from phylogenies with both fossil and living taxa to test whether declining rates characterize the evolutionary history of a major clade of marine invertebrates, the echinoids.Since originating some 265 million years ago (18, 19), crown group echinoids have evolved to become ecologically and morphologically diverse in today’s oceans, and are an important component of both past and present marine ecosystems (e.g., refs. 20–22). However, analysis of how this diversity arose has either been based on taxonomic counts (e.g., ref. 23) or has adopted a morphometric approach where the requirement of a homologous set of landmarks limits taxonomic, temporal, and geographic scope (e.g., ref. 24). We use a discrete-character-based approach and a recent taxonomically comprehensive analysis of post-Paleozoic echinoids as our phylogenetic framework (25). This tree is almost entirely resolved (SI Appendix, Fig. S1) and branches may be scaled using the first appearance of each taxon in the fossil record (SI Appendix, Table S1). We tabulated the number of character state changes that occurred along each branch within ∼10-million-year time intervals spanning the Permian and post-Paleozoic (SI Appendix, Table S2), and divided this by the summed duration of branch lengths to compute a time series of per-lineage-million-year rates of morphological evolution. We accounted for uncertainty in phylogenetic structure, uncertainty in the timing of the first appearance of taxa, and uncertainty in the timing of character changes along each branch using a randomization approach (12). We also estimated rates within subclades, corroborating our findings by using likelihoods tests to determine whether some branches had higher rates than expected given rates across the entire tree. Finally, we compared rates of evolution through time with the structure of diversification within a character-defined morphospace, and looked for evidence of differences in evolutionary modes among subclades. The pattern that emerges is one of dynamic evolutionary change through time: Both rates and patterns of evolution vary temporally and across subclades, such that the overall pattern depends highly on the temporal and taxonomic scale of the analysis.     

Information-based fitness and the emergence of criticality in living systems

Empirical evidence suggesting that living systems might operate in the vicinity of critical points, at the borderline between order and disorder, has proliferated in recent years, with examples ranging from spontaneous brain activity to flock dynamics. However, a well-founded theory for understanding how and why interacting living systems could dynamically tune themselves to be poised in the vicinity of a critical point is lacking. Here we use tools from statistical mechanics and information theory to show that complex adaptive or evolutionary systems can be much more efficient in coping with diverse heterogeneous environmental conditions when operating at criticality. Analytical as well as computational evolutionary and adaptive models vividly illustrate that a community of such systems dynamically self-tunes close to a critical state as the complexity of the environment increases while they remain noncritical for simple and predictable environments. A more robust convergence to criticality emerges in coevolutionary and coadaptive setups in which individuals aim to represent other agents in the community with fidelity, thereby creating a collective critical ensemble and providing the best possible tradeoff between accuracy and flexibility. Our approach provides a parsimonious and general mechanism for the emergence of critical-like behavior in living systems needing to cope with complex environments or trying to efficiently coordinate themselves as an ensemble.Physical systems undergo phase transitions from ordered to disordered states on changing control parameters (1, 2). Critical points, with all their remarkable properties (1, 2), are only observed upon parameter fine tuning. This is in sharp contrast to the ubiquity of critical-like behavior in complex living matter. Indeed, empirical evidence has proliferated that living systems might operate at criticality (3)—i.e. at the borderline between order and disorder—with examples ranging from spontaneous brain behavior (4) to gene expression patterns (5), cell growth (6), morphogenesis (7), bacterial clustering (8), and flock dynamics (9). Even if none of these examples is fully conclusive and even if the meaning of “criticality” varies across these works, the criticality hypothesis—as a general strategy for the organization of living matter—is a tantalizing idea worthy of further investigation.Here we present a framework for understanding how self-tuning to criticality can arise in living systems. Unlike models of self-organized criticality in which some inanimate systems are found to become critical in a mechanistic way (10), our focus here is on general adaptive or evolutionary mechanisms, specific to biological systems. We suggest that the drive to criticality arises from functional advantages of being poised in the vicinity of a critical point.However, why is a living system fitter when it is critical? Living systems need to perceive and respond to environmental cues and to interact with other similar entities. Indeed, biological systems constantly try to encapsulate the essential features of the huge variety of detailed information from their surrounding complex and changing environment into manageable internal representations, and they use these as a basis for their actions and responses. The successful construction of these representations, which extract, summarize, and integrate relevant information (11), provides a crucial competitive advantage, which can eventually make the difference between survival and extinction. We suggest here that criticality is an optimal strategy to effectively represent the intrinsically complex and variable external world in a parsimonious manner. This is in line with the hypothesis that living systems benefit from having attributes akin to criticality—either statistical or dynamical (3)—such as a large repertoire of dynamical responses, optimal transmission and storage of information, and exquisite sensitivity to environmental changes (2, 5, 12–16).As conjectured long ago, the capability to perform complex computations, which turns out to be the fingerprint of living systems, is enhanced in “machines” operating near a critical point (17–19), i.e., at the border between two distinct phases: a disordered phase, in which perturbations and noise propagate unboundedly—thereby corrupting information transmission and storage—and an ordered phase where changes are rapidly erased, hindering flexibility and plasticity. The marginal, critical situation provides a delicate compromise between these two impractical tendencies, an excellent tradeoff between reproducibility and flexibility (12, 13, 16) and, on larger time scales, between robustness and evolvability (20). A specific example of this general framework is genetic regulatory networks (19, 21). Cells ranging from those in complex organisms to single-celled microbes such as bacteria respond to signals in the environment by modifying the expression of their genes. Any given genetic regulatory network, formed by the genes (nodes) and their interactions (edges) (22), can be tightly controlled to robustly converge to a fixed almost-deterministic attractor—i.e. a fixed “phenotype”—or it can be configured to be highly sensitive to tiny fluctuations in input signals, leading to many different attractors, i.e., to large phenotypic variability (23). These two situations correspond to the ordered and disordered phases, respectively. The optimal way for genetic regulatory networks to reconcile controllability and sensitivity to environmental cues is to operate somewhere in between the two limiting and impractical limits alluded to above (19) as has been confirmed in different experimental setups (5, 7, 24). Still, it is not clear how such tuning to criticality comes about.Our goal here is to exploit general ideas from statistical mechanics and information theory to construct a quantitative framework showing that self-tuning to criticality is a convenient strategy adopted by living systems to effectively cope with the intrinsically complex external world in an efficient manner, thereby providing an excellent compromise between accuracy and flexibility. To provide some further intuition, we use genetic regulatory networks as a convenient guiding example, but one could equally well consider neural networks, models for the immune response, groups of animals exhibiting collective behavior, etc., with each specific realization requiring a more detailed modeling of its special attributes.We uncover coevolutionary and coadaptive mechanisms by which communities of living systems, even in the absence of other forms of environmental complexity, converge to be almost critical in the process of understanding each other and creating a “collective entity.” The main result is that criticality is an evolutionary/adaptive stable solution reached by living systems in their striving to cope with complex heterogeneous environments or when trying to efficiently coordinate themselves as an ensemble.     

Evolutionary and phylogenetic insights from a nuclear genome sequence of the extinct,giant, “subfossil” koala lemur Megaladapis edwardsi

No endemic Madagascar animal with body mass >10 kg survived a relatively recent wave of extinction on the island. From morphological and isotopic analyses of skeletal “subfossil” remains we can reconstruct some of the biology and behavioral ecology of giant lemurs (primates; up to ∼160 kg) and other extraordinary Malagasy megafauna that survived into the past millennium. Yet, much about the evolutionary biology of these now-extinct species remains unknown, along with persistent phylogenetic uncertainty in some cases. Thankfully, despite the challenges of DNA preservation in tropical and subtropical environments, technical advances have enabled the recovery of ancient DNA from some Malagasy subfossil specimens. Here, we present a nuclear genome sequence (∼2× coverage) for one of the largest extinct lemurs, the koala lemur Megaladapis edwardsi (∼85 kg). To support the testing of key phylogenetic and evolutionary hypotheses, we also generated high-coverage nuclear genomes for two extant lemurs, Eulemur rufifrons and Lepilemur mustelinus, and we aligned these sequences with previously published genomes for three other extant lemurs and 47 nonlemur vertebrates. Our phylogenetic results confirm that Megaladapis is most closely related to the extant Lemuridae (typified in our analysis by E. rufifrons) to the exclusion of L. mustelinus, which contradicts morphology-based phylogenies. Our evolutionary analyses identified significant convergent evolution between M. edwardsi and an extant folivore (a colobine monkey) and an herbivore (horse) in genes encoding proteins that function in plant toxin biodegradation and nutrient absorption. These results suggest that koala lemurs were highly adapted to a leaf-based diet, which may also explain their convergent craniodental morphology with the small-bodied folivore Lepilemur.

Madagascar is exceptionally biodiverse today. Yet, the island’s endemic diversity was even greater in the relatively recent past. Specifically, there is an extensive “subfossil” record of now-extinct Malagasy fauna, with some of these species persisting until at least ∼500 y B.P. (1). The Late Holocene extinction pattern in Madagascar resembles other “megafaunal extinction” patterns in that it is strikingly body-mass structured, with the majority of extinct subfossil taxa substantially larger than their surviving counterparts. For example, the average adult body mass of the largest of the ∼100 extant lemur (primates) species is 6.8 kg (2), well below that of the 17 described extinct subfossil lemur taxa, for which estimated adult body masses ranged from ∼11 kg to an incredible ∼160 kg (3).Despite a tropical and subtropical environment in which nucleotide (nt) strands rapidly degrade, in a select subset of Malagasy subfossil samples, ancient DNA (aDNA) is sufficiently preserved for paleogenomic analysis (4–10). In our group’s previous study (6), we reconstructed complete or near-complete mitochondrial genomes from five subfossil lemur species, with population-level data in two cases. As part of that work, we identified one Megaladapis edwardsi (body mass ∼85 kg) (3, 11) sample with an especially high proportion of endogenous aDNA. We have subsequently performed additional rounds of extraction and sequencing of this sample to amass sufficient data for studying the M. edwardsi nuclear genome.In this study, we analyzed the M. edwardsi nuclear genome to help reconstruct subfossil lemur behavioral ecology and evolutionary biology. Our approach included an unbiased search across the genome for Megaladapis-specific signatures of positive selection at the individual gene level. We also searched for striking patterns of genomic convergence with a set of biologically diverse extant mammals across sets of functionally annotated genes. The results from these analyses may serve to extend current hypotheses or to offer potentially unexpected insights into the evolutionary biology of Megaladapis.Additionally, we aimed to resolve lingering uncertainty over Megaladapis phylogenetic relationships with other lemurs. At one point, a sister taxon relationship between Megaladapis and extant sportive lemurs (genus Lepilemur) was inferred based on craniodental similarities (3, 12). A different phylogeny was estimated, however, following the successful recovery of several hundred base pairs (bp) of the Megaladapis mitochondrial genome in several early aDNA studies (4, 5). Specifically, Megaladapis and the extant Lemuridae (genera Eulemur, Lemur, Varecia, Prolemur, and Hapalemur) formed a clade to the exclusion of Lepilemur. Our more recent aDNA study (6) resolved a similar phylogeny but with greater confidence (e.g., 87% bootstrap support) given the near-complete recovery of the Megaladapis mitochondrial genome (16,714 bp). Still, the mitochondrial genome is a single, nonrecombining locus; in certain cases, true species-level phylogenies are not reconstructed accurately from mitochondrial DNA alone (13). Most recently, Herrera and Dávalos (14) estimated a “total evidence” phylogeny by analyzing the combination of both morphological and genetic characters. Their result was dissimilar to each of the above phylogenies, instead supporting an early divergence of the Megaladapis lineage from all other non-Daubentonia (aye-aye) lemurs.Because the nuclear genome is comprised of thousands of effectively independent markers of ancestry, we expected to achieve a more definitive phylogenetic result with our Megaladapis paleogenome sequence. To distinguish among competing phylogenetic hypotheses, we also needed to generate genome data for representatives of the extant Lemuridae and Lepilemur lineages, which we did for Eulemur rufifrons (red-fronted lemur) and Lepilemur mustelinus (greater sportive lemur), respectively. We aligned the three lemur genome sequences with those previously published for extant lemurs Daubentonia madagascariensis (aye-aye) (15), Microcebus murinus (gray mouse lemur) (16), and Propithecus diadema (diademed sifaka) (17), and with 47 nonlemur outgroup species, for phylogenetic and evolutionary analyses.     

Targeted vaccination and the speed of SARS-CoV-2 adaptation

The limited supply of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) raises the question of targeted vaccination. Many countries have opted to vaccinate older and more sensitive hosts first to minimize the disease burden. However, what are the evolutionary consequences of targeted vaccination? We clarify the consequences of different vaccination strategies through the analysis of the speed of viral adaptation measured as the rate of change of the frequency of a vaccine-adapted variant. We show that such a variant is expected to spread faster if vaccination targets individuals who are likely to be involved in a higher number of contacts. We also discuss the pros and cons of dose-sparing strategies. Because delaying the second dose increases the proportion of the population vaccinated with a single dose, this strategy can both speed up the spread of the vaccine-adapted variant and reduce the cumulative number of deaths. Hence, strategies that are most effective at slowing viral adaptation may not always be epidemiologically optimal. A careful assessment of both the epidemiological and evolutionary consequences of alternative vaccination strategies is required to determine which individuals should be vaccinated first.

The development of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) raises hope regarding the possibility of eventually halting the ongoing pandemic. However, vaccine supply shortages have sparked a debate about the optimal distribution of vaccination among different categories of individuals. Typically, infections with SARS-CoV-2 are far more deadly in older individuals than in younger ones (1). Prioritizing vaccination for older classes may thus provide a direct benefit in terms of mortality (2, 3). Yet, younger individuals are usually more active, and consequently, they may contribute more to the spread of the epidemic. Prioritizing vaccination for younger and more active individuals may thus provide an indirect benefit through a reduction of the epidemic size (4, 5). Earlier studies have compared alternative ways to deploy vaccination in heterogeneous host populations and showed that recommendation varies with the choice of the quantity one is trying to minimize (e.g., the cumulative number of deaths, the remaining life expectancy, or the number of infections) (3, 6, 7). The recommendation also varies with the properties of the pathogen and the efficacy of the vaccine (3, 4, 8). For SARS-CoV-2, the increase in mortality with age is such that the direct benefit associated with vaccinating more vulnerable individuals tends to overwhelm the indirect benefits obtained from vaccinating more active individuals (2, 3, 9, 10). However, some studies challenge this view and identified specific conditions where vaccinating younger and more active classes could be optimal (5, 7, 11, 12). A similar debate emerges over the possibility to delay the second vaccination dose to maximize the number of partially vaccinated individuals. A quantitative exploration of alternative vaccination strategies can help provide useful recommendations: a two-dose strategy is recommended when the level of protection obtained after the first dose is low and/or when vaccine supply is large (13–16).Vaccine-driven evolution, however, could erode the benefit of vaccination and alter the above recommendations which are based solely on the analysis of epidemiological dynamics. Given that hosts differ both in their sensitivity to the disease and in their contribution to transmission, who should we vaccinate first if we want to minimize the spread of vaccine-adapted variants? The effect of alternative vaccination strategies on the speed of pathogen adaptation remains unclear. Previous studies of adaptation to vaccines focused on long-term evolutionary outcomes (17, 18). These analyses are not entirely relevant for the ongoing pandemic because what we want to understand first is the short-term consequence of different vaccination strategies (19). A few studies have discussed the possibility of SARS-CoV-2 adaptation following different targeted vaccination strategies but did not explicitly account for evolutionary dynamics (12, 20). A recent simulation study explored the effect of a combination of vaccination and social distancing strategies on the probability of vaccine-driven adaptation (21). This model, however, did not study the impact of targeted vaccination strategies on the speed of adaptation.Here we develop a theoretical framework based on the analysis of the deterministic dynamics of multiple variants after they successfully managed to reach a density at which they are no longer affected by the action of demographic stochasticity. We study the impact of different vaccination strategies on the rate of change of the frequency of a novel variant, which allows us to quantify the speed of virus adaptation to vaccines. Numerical simulations tailored to the epidemiology of SARS-CoV-2 confirm the validity of our approximation of the strength of selection for vaccine-adapted variants.     

Exploring the likelihood and mechanism of a climate-change-induced dieback of the Amazon rainforest

We examine the evidence for the possibility that 21st-century climate change may cause a large-scale “dieback” or degradation of Amazonian rainforest. We employ a new framework for evaluating the rainfall regime of tropical forests and from this deduce precipitation-based boundaries for current forest viability. We then examine climate simulations by 19 global climate models (GCMs) in this context and find that most tend to underestimate current rainfall. GCMs also vary greatly in their projections of future climate change in Amazonia. We attempt to take into account the differences between GCM-simulated and observed rainfall regimes in the 20th century. Our analysis suggests that dry-season water stress is likely to increase in E. Amazonia over the 21st century, but the region tends toward a climate more appropriate to seasonal forest than to savanna. These seasonal forests may be resilient to seasonal drought but are likely to face intensified water stress caused by higher temperatures and to be vulnerable to fires, which are at present naturally rare in much of Amazonia. The spread of fire ignition associated with advancing deforestation, logging, and fragmentation may act as nucleation points that trigger the transition of these seasonal forests into fire-dominated, low biomass forests. Conversely, deliberate limitation of deforestation and fire may be an effective intervention to maintain Amazonian forest resilience in the face of imposed 21st-century climate change. Such intervention may be enough to navigate E. Amazonia away from a possible “tipping point,” beyond which extensive rainforest would become unsustainable.