Rapid genome reshaping by multiple-gene loss after whole-genome duplication in teleost fish suggested by mathematical modeling

Whole-genome duplication (WGD) is believed to be a significant source of major evolutionary innovation. Redundant genes resulting from WGD are thought to be lost or acquire new functions. However, the rates of gene loss and thus temporal process of genome reshaping after WGD remain unclear. The WGD shared by all teleost fish, one-half of all jawed vertebrates, was more recent than the two ancient WGDs that occurred before the origin of jawed vertebrates, and thus lends itself to analysis of gene loss and genome reshaping. Using a newly developed orthology identification pipeline, we inferred the post–teleost-specific WGD evolutionary histories of 6,892 protein-coding genes from nine phylogenetically representative teleost genomes on a time-calibrated tree. We found that rapid gene loss did occur in the first 60 My, with a loss of more than 70–80% of duplicated genes, and produced similar genomic gene arrangements within teleosts in that relatively short time. Mathematical modeling suggests that rapid gene loss occurred mainly by events involving simultaneous loss of multiple genes. We found that the subsequent 250 My were characterized by slow and steady loss of individual genes. Our pipeline also identified about 1,100 shared single-copy genes that are inferred to have become singletons before the divergence of clupeocephalan teleosts. Therefore, our comparative genome analysis suggests that rapid gene loss just after the WGD reshaped teleost genomes before the major divergence, and provides a useful set of marker genes for future phylogenetic analysis.The recent rapid growth of genome data has made it possible to clarify major evolutionary events that have shaped eukaryote genomes, such as gene duplication, chromosomal rearrangement, and whole-genome duplication (WGD) (1). In particular, WGD events, known to have occurred in several major lineages of flowering plants (2), budding yeasts (3), and vertebrates (4) (Fig. 1A), are considered to have had a major impact on genomic architecture and consequently organismal features.Open in a separate windowFig. 1.Inferred spatiotemporal process of gene loss and persistence after TGD in teleost ancestors. (A) The estimated numbers of gene loss events in the teleost phylogeny, time-scaled tree of vertebrates (11, 41) with the timing of genome duplication events at the base of vertebrates (VGD1/2) and teleosts (TGD), and the number of extant species (26). Species used in this study are connected by solid branches. The numbers were parsimoniously inferred from the presence or absence of TGD-derived gene lineage pairs belonging to 6,892 orthogroups and mapped onto the time points of TGD (306 Mya), nodes a–g (a: 245 Mya; b: 158; c: 120; d: 105; e: 41; f: 164; g: 86) (11), and h (74 Mya) (28). On the left side of the tree, ortholog arrangements are compared between representatives (connected by bold branches in the tree) by CIRCOS (circos.ca) using orthology information for 5,655 orthogroups belonging to the 1to1 category (Fig. S2). (B) Definition of terms relating to WGD events. An orthogroup is a monophyletic group containing WGD-derived paralogs (gene lineages) of all focal species (Sp1) and orthologs of their sister species (Sp2), ignoring lineage-specific gene duplications (GeneA-1′ and -1″) or gene loss (GeneA-1″). (C) Approximation of the pattern of the number of gene loss and persistence events associated with TGD. The estimated number of retained paired gene lineages at nodes a to h and current teleosts (Ca, Ze, Co, Ti, Pl, Me, St, Te, and Fu) were used to compare the fit of the one-phase [αe^–2_μt (14)] and two-phase models. (D) Region of C detailing the recent pattern of gene loss. The solid and dashed curves have been corrected upward to remove the bias expected to result from parsimony analysis. These approximations are effectively insensitive to fluctuations in the estimated numbers of gene lineage pairs and times for the TGD event and ancestral nodes (a to h) (SI Text). The evolutionary scenario is essentially unchanged if the number of gene lineage pairs estimated without the BS 70% criterion or the divergence times estimated by nuclear gene (28)/mitochondrial genome (42) data were used. Note that the two-phase model can be roughly approximated by a double-exponential curve.Duplicate genes generated by WGD are typically assumed to be redundant and therefore subsequently lost in a stochastic manner. Comparative genome studies have suggested that 90% of duplicate genes were rapidly lost (5) by a neutral process (6) after WGD in budding yeast, but 20–30% of them were retained in human (7) even after several hundred million years. However, few genome-wide studies have addressed the temporal pattern of gene loss or persistence after WGD with reference to a reliable timescale (but see refs. 6 and 8). Such examination is indispensable for understanding when duplicate genes were lost and, consequently, genome structures were reshaped, during vertebrate diversification after the WGD (Fig. 1).To examine the detailed process of duplicate gene loss after WGD, one needs to estimate the number (proportion) of remaining duplicates in extant and ancestral species. For this purpose, both (i) reliably time-calibrated phylogenetic trees of species and (ii) well-annotated genomes are required. These two requirements have been met for several vertebrate lineages, including some teleost fishes. Given this, the next step should be to accurately estimate orthology and paralogy relationships of all of the genes that experienced WGD. For the analysis of gene orthology and paralogy, a homology search- or synteny-based approach has usually been used (9). In addition to the homology search-based approach (e.g., COGs and OrthoDB), a phylogenetic tree-based approach has also been introduced (e.g., Ensembl and PhylomeDB) (9). Recent developments of tree search algorithms and increased computing power allow a sophisticated tree-based approach, comparing each gene tree with the species tree. Such an approach is indispensable for the effective analysis of gene orthology and paralogy across many species, providing us with a powerful opportunity to investigate genome evolution after WGD.Here, we aim to investigate the gene loss/persistence pattern using genome-wide data, focusing on what is known as the teleost genome duplication (TGD). TGD is estimated to have occurred in an ancestor of teleosts (Fig. 1A) but after the divergence of tetrapods and teleosts (10). Thus, it is a relatively recent WGD shared by a large vertebrate group, i.e., the Teleostei. For teleosts, reliably time-calibrated phylogenies, including phylogenetic position and timing of the TGD event, are available (e.g., ref. 11). In addition, well-annotated whole-genome data from at least nine phylogenetically representative teleost species (cave fish, zebrafish, cod, tilapia, platyfish, medaka, stickleback, Tetraodon, and fugu) are now available from Ensembl (12). In the present study, we inferred the timing of rapid genome reshaping through gene loss after TGD by estimating the temporal and genomic positional (spatiotemporal) loss/persistence pattern of TGD-derived gene lineage pairs (Fig. 1B) over the past several hundred million years, using accurate tree-based orthology estimation (Fig. S1) and a reliable time-calibrated teleost tree. We investigated the mechanism of rapid gene loss after TGD by fitting a newly developed model for the observed temporal pattern of gene loss. This new model is necessary because standard models, based upon random and independent loss of duplicate genes, fail to fit our data. Our model analysis explicitly includes both the possibility of the loss of multiple genes in single events, and also the known phylogeny of the relevant species. The significance of the inclusion of events that result in the loss of multiple genes is that it reproduces the two phases of loss. The inclusion of known phylogeny allows us to correct for the bias associated with parsimony analysis.     

Little evidence for enhanced phenotypic evolution in early teleosts relative to their living fossil sister group

Since Darwin, biologists have been struck by the extraordinary diversity of teleost fishes, particularly in contrast to their closest “living fossil” holostean relatives. Hypothesized drivers of teleost success include innovations in jaw mechanics, reproductive biology and, particularly at present, genomic architecture, yet all scenarios presuppose enhanced phenotypic diversification in teleosts. We test this key assumption by quantifying evolutionary rate and capacity for innovation in size and shape for the first 160 million y (Permian–Early Cretaceous) of evolution in neopterygian fishes (the more extensive clade containing teleosts and holosteans). We find that early teleosts do not show enhanced phenotypic evolution relative to holosteans. Instead, holostean rates and innovation often match or can even exceed those of stem-, crown-, and total-group teleosts, belying the living fossil reputation of their extant representatives. In addition, we find some evidence for heterogeneity within the teleost lineage. Although stem teleosts excel at discovering new body shapes, early crown-group taxa commonly display higher rates of shape evolution. However, the latter reflects low rates of shape evolution in stem teleosts relative to all other neopterygian taxa, rather than an exceptional feature of early crown teleosts. These results complement those emerging from studies of both extant teleosts as a whole and their sublineages, which generally fail to detect an association between genome duplication and significant shifts in rates of lineage diversification.Numbering ∼29,000 species, teleost fishes account for half of modern vertebrate richness. In contrast, their holostean sister group, consisting of gars and the bowfin, represents a mere eight species restricted to the freshwaters of eastern North America (1). This stark contrast between teleosts and Darwin''s original “living fossils” (2) provides the basis for assertions of teleost evolutionary superiority that are central to textbook scenarios (3, 4). Classic explanations for teleost success include key innovations in feeding (3, 5) (e.g., protrusible jaws and pharyngeal jaws) and reproduction (6, 7). More recent work implicates the duplicate genomes of teleosts (8–10) as the driver of their prolific phenotypic diversification (8, 11–13), concordant with the more general hypothesis that increased morphological complexity and innovation is an expected consequence of genome duplication (14, 15).Most arguments for enhanced phenotypic evolution in teleosts have been asserted rather than demonstrated (8, 11, 12, 15, 16; but see ref. 17), and draw heavily on the snapshot of taxonomic and phenotypic imbalance apparent between living holosteans and teleosts. The fossil record challenges this neontological narrative by revealing the remarkable taxonomic richness and morphological diversity of extinct holosteans (Fig. 1) (18, 19) and highlights geological intervals when holostean taxonomic richness exceeded that of teleosts (20). This paleontological view has an extensive pedigree. Darwin (2) invoked a long interval of cryptic teleost evolution preceding the late Mesozoic diversification of the modern radiation, a view subsequently supported by the implicit (18) or explicit (19) association of Triassic–Jurassic species previously recognized as “holostean ganoids” with the base of teleost phylogeny. This perspective became enshrined in mid-20th century treatments of actinopterygian evolution, which recognized an early-mid Mesozoic phase dominated by holosteans sensu lato and a later interval, extending to the modern day, dominated by teleosts (4, 20, 21). Contemporary paleontological accounts echo the classic interpretation of modest teleost origins (22–24), despite a systematic framework that substantially revises the classifications upon which older scenarios were based (22–25). Identification of explosive lineage diversification in nested teleost subclades like otophysans and percomorphs, rather than across the group as a whole, provides some circumstantial neontological support for this narrative (26).Open in a separate windowFig. 1.Phenotypic variation in early crown neopterygians. (A) Total-group holosteans. (B) Stem-group teleosts. (C) Crown-group teleosts. Taxa illustrated to scale.In contrast to quantified taxonomic patterns (20, 23, 24, 27), phenotypic evolution in early neopterygians has only been discussed in qualitative terms. The implicit paleontological model of morphological conservatism among early teleosts contrasts with the observation that clades aligned with the teleost stem lineage include some of the most divergent early neopterygians in terms of both size and shape (Fig. 1) (see, for example, refs. 28 and 29). These discrepancies point to considerable ambiguity in initial patterns of phenotypic diversification that lead to a striking contrast in the vertebrate tree of life, and underpins one of the most successful radiations of backboned animals.Here we tackle this uncertainty by quantifying rates of phenotypic evolution and capacity for evolutionary innovation for the first 160 million y of the crown neopterygian radiation. This late Permian (Wuchiapingian, ca. 260 Ma) to Cretaceous (Albian, ca. 100 Ma) sampling interval permits incorporation of diverse fossil holosteans and stem teleosts alongside early diverging crown teleost taxa (Figs. 1 and ​and2A2A and Figs. S1 and ​andS2),S2), resulting in a dataset of 483 nominal species-level lineages roughly divided between the holostean and teleost total groups (Fig. 2B and Fig. S2). Although genera are widely used as the currency in paleobiological studies of fossil fishes (30; but see ref. 31), we sampled at the species level to circumvent problems associated with representing geological age and morphology for multiple congeneric lineages. We gathered size [both log-transformed standard length (SL) and centroid size (CS); results from both are highly comparable (Figs. S3 and ​andS4);S4); SL results are reported in the main text] and shape data (the first three morphospace axes arising from a geometric morphometric analysis) (Fig. 2A and Figs. S1) from species where possible. To place these data within a phylogenetic context, we assembled a supertree based on published hypotheses of relationships. We assigned branch durations to a collection of trees under two scenarios for the timescale of neopterygian diversification based on molecular clock and paleontological estimates. Together, these scenarios bracket a range of plausible evolutionary timelines for this radiation (Fig. 2B). We used the samples of trees in conjunction with our morphological datasets to test for contrasts in rates of, and capacity for, phenotypic change between different partitions of the neopterygian Tree of Life (crown-, total-, and stem-group teleosts, total-group holosteans, and neopterygians minus crown-group teleosts), and the sensitivity of these conclusions to uncertainty in both relationships and evolutionary timescale. Critically, these include comparisons of phenotypic evolution in early crown-group teleosts—those species that are known with certainty to possess duplicate genomes—with rates in taxa characterized largely (neopterygians minus crown teleosts) or exclusively (holosteans) by unduplicated genomes. By restricting our scope to early diverging crown teleost lineages, we avoid potentially confounding signals from highly nested radiations that substantially postdate both genome duplication and the origin of crown teleosts (26, 32). This approach provides a test of widely held assumptions about the nature of morphological evolution in teleosts and their holostean sister lineage.Open in a separate windowFig. 2.(A) Morphospace of Permian–Early Cretaceous crown Neopterygii. (B) One supertree subjected to our paleontological (Upper) and molecular (Lower) timescaling procedures to illustrate contrasts in the range of evolutionary timescales considered. Colors of points (A) and branches (B) indicate membership in major partitions of neopterygian phylogeny. Topologies are given in Datasets S4 and S5. See Dataset S6 for source trees.Open in a separate windowFig. S1.Morphospace of 398 Permian–Early Cretaceous Neopterygii. Three major axes of shape variation are presented. Silhouettes and accompanying arrows illustrate the main anatomical correlates of these principal axes, as described in Open in a separate windowFig. S2.Morphospace of 398 Permian–Early Cretaceous Neopterygii, illustrating the major clades of (A) teleosts and (B) holosteans.Open in a separate windowFig. S3.Comparisons of size rates between (A) holosteans and teleosts, (B) crown teleosts and all other neopterygians, (C) crown teleosts and stem teleosts, (D) crown teleosts and holosteans, and (E) stem teleosts and holosteans. Comparisons were made using the full-size SL dataset, a CS dataset, and a smaller SL dataset pruned to exactly match the taxon sampling of the CS dataset. Identical taxon sampling leads the CS and pruned SL datasets to yield near identical results. Although the larger SL dataset results often differ slightly, the overall conclusion from each pairwise comparison (i.e., which outcome is the most likely in an overall majority of trees) is identical in all but one comparison (E, under molecular timescales).Open in a separate windowFig. S4.Comparisons of size innovation between (A) holosteans and teleosts, (B) crown teleosts and all other neopterygians, (C) crown teleosts and stem teleosts, (D) crown teleosts and holosteans, and (E) stem teleosts and holosteans. Comparisons were made using the full-size SL dataset, a CS dataset, and a smaller SL dataset pruned to exactly match the taxon sampling of the CS dataset. Comparisons of size innovation are presented for K value distributions of the three datasets resemble each other closely.     

The rise of biting during the Cenozoic fueled reef fish body shape diversification

Diversity of feeding mechanisms is a hallmark of reef fishes, but the history of this variation is not fully understood. Here, we explore the emergence and proliferation of a biting mode of feeding, which enables fishes to feed on attached benthic prey. We find that feeding modes other than suction, including biting, ram biting, and an intermediate group that uses both biting and suction, were nearly absent among the lineages of teleost fishes inhabiting reefs prior to the end-Cretaceous mass extinction, but benthic biting has rapidly increased in frequency since then, accounting for about 40% of reef species today. Further, we measured the impact of feeding mode on body shape diversification in reef fishes. We fit a model of multivariate character evolution to a dataset comprising three-dimensional body shape of 1,530 species of teleost reef fishes across 111 families. Dedicated biters have accumulated over half of the body shape variation that suction feeders have in just 18% of the evolutionary time by evolving body shape ∼1.7 times faster than suction feeders. As a possible response to the ecological and functional diversity of attached prey, biters have dynamically evolved both into shapes that resemble suction feeders as well as novel body forms characterized by lateral compression and small jaws. The ascendance of species that use biting mechanisms to feed on attached prey reshaped modern reef fish assemblages and has been a major contributor to their ecological and phenotypic diversification.

Reef habitats are renowned for high biodiversity (1–5). Often, this pattern is attributed to the structural complexity of reefs, as complex habitats provide increased opportunity for microhabitat-related adaptations and niche partitioning (6–8). Among reef fishes, many major drivers of phenotypic and ecological diversity have been recognized at a range of phylogenetic scales (9–13), but we still lack a clear understanding of the processes and mechanisms that have made reef fish faunas the most diverse in modern oceans.One striking axis of diversity that distinguishes reef fish communities from those in other marine habitats is the variety of feeding modes used to capture prey. Fishes can employ a direct biting mechanism to remove attached prey from hard substrates or can use suction feeding, which relies on the density and viscosity of an aquatic medium to pull in water and prey via rapid expansion of the head. Suction feeding, which is most effectively used to capture mobile prey (14–19), is both ancestral for teleost fishes (20) and well represented on modern reefs (21). However, direct biting feeding mechanisms characterize many iconic reef fish groups, including parrotfishes, butterflyfishes, surgeonfishes, and triggerfishes. The evolution of biting has allowed fishes to exploit a variety of benthic prey that are firmly attached to reef surfaces and thus, resist suction, including molluscs, echinoderms, cnidarians, sponges, algae, and other primary producers (22–27). The ecosystem importance of this functional breakthrough in trophic habits is perhaps best represented by the many benthic biting herbivores and detritivores (27–32) that play a central role in energy transfer through reefs and regulating the composition of benthic communities (33–37).Benthic biting has been a major facet of the trophic diversity of reef fishes since at least the Eocene. Herbivores were well established in the Monte Bolca lagerstätte (∼50 Ma), marking the first evidence that teleosts could graze upon the reef surface and signaling a major shift in reef community functions (38–40). These herbivores appear to have risen to dominance within reef ecosystems globally through expansion and colonization following the split of the Tethys Sea and the increased availability of reef flat habitats in the Late Cenozoic (40–44), although the implications of biting for phenotypic diversification of reef fishes remain unknown (40). Use of a biting feeding mode prior to the Eocene appears to be primarily the domain of nonteleost fishes. As long ago as the Devonian, lungfishes and some arthrodire placoderms captured and crushed hard prey with their jaws (45–48). Several lineages of early-branching ray-finned fishes used biting for prey capture throughout the Mesozoic, including pycnodonts, macrosemiids, and semionotids (38, 49–51); of these, pycnodonts persisted until the Eocene (51). The striking lack of biting teleosts prior to the Eocene (38) may be due to a 20-My gap in major deposits of spiny-rayed (Acanthomorph) fishes from the Late Campanian (∼75 Ma) to the Late Paleocene (∼55 Ma) (52), during which biting by teleost fishes most likely proliferated to its Eocene prominence. The ambiguity regarding the origins of the expansion of biting among teleosts and its role in morphological diversification presents an opportunity for comparative phylogenetics to provide insight into the history of modern reef fishes.In this study, we explored the evolutionary history of benthic biting feeding mechanisms in reef fishes and the impact this novelty had on their phenotypic diversification. We compared benthic biting with three other feeding modes: suction feeding, an intermediate group using a mix of both suction and biting, and an uncommon feeding mode we refer to as “ram biting.” To determine how the prevalence of benthic biting has changed through time, we reconstructed the history of feeding modes among reef-dwelling teleosts using stochastic mapping on a time-calibrated phylogeny. We then measured the effect of feeding mode on rates of body shape evolution across a broad sample of 1,530 species of reef fishes spanning 111 families of extant teleosts. If biting feeding modes have been a significant stimulus to the diversification of modern reef fishes, we expect to see differences in body shape occupation of morphospace and phenotypic diversification when comparing biters with fish that employ other feeding modes. Our results provide insight into the evolutionary mechanisms underlying the vast phenotypic and ecological diversity of reef fishes.     

Structural changes in bacteriophage T7 upon receptor-induced genome ejection

Many tailed bacteriophages assemble ejection proteins and a portal–tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal–tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near–atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane.

Many double-stranded DNA (dsDNA) viruses, including tailed bacteriophages and herpesviruses, have a portal attached to a unique pentameric vertex of their icosahedral capsid shell (1–3). The portal is a dodecameric channel for viral DNA packaging and ejection. The tailed bacteriophages and herpesviruses encapsidate DNA in the capsid shell through the portal channel (4–10), and the last packaged DNA is held by tunnel loops (or β-hairpins for herpesviruses) in the portal (11–16). The last packaged DNA in most of the tailed bacteriophages and herpesvirus is the first to be ejected during the genome delivery (17). In tailed bacteriophages, the portal connects to a tail, which serves to recognize host cell receptors and deliver the genome into the cytoplasm (18). Gram-negative bacteriophage in Podoviridae initiate infection through a specific interaction of its receptor-binding protein with the receptor lipopolysaccharide (LPS) on the host cell surface. The phages in Podoviridae have a noncontractile tail that is too short to span the gram-negative bacteria envelope that comprises the outer membrane, the cytoplasmic membrane, and the peptidoglycan layer in the hydrophilic periplasm in between (19). After adsorption, a signal is transmitted for the release of internal ejection proteins to form a channel that extends the tail across the cell envelope and that allows for subsequent genome ejection into the infected cell (20–23). In many previous studies, structural analyses have been performed at resolutions of 9 to 40 Å on this highly coordinated dynamic infection process (21–26). These studies have provided insights on structural changes of phage particles that accompany the infection steps before and after the genome ejection. However, these studies did not resolve structures of the internal ejection proteins. Furthermore, the relative low resolutions cannot clarify the dynamic genome ejection process orchestrated by the ejection proteins, portal, and tail.Escherichia coli bacteriophage T7, a member of the Podoviridae family, has been used as a model for understanding the DNA packaging and delivery mechanism that are common to tailed phages and related dsDNA viruses (10, 21, 27–33). T7 has an icosahedral capsid shell formed by gene product 10 (gp10). The 12-fold portal (gp8) shares a very similar topology with those in other phages and herpesviurses (14–16, 30, 34). The tail comprises a 12-fold adaptor protein gp11 assembly, a sixfold nozzle protein gp12 assembly, and six subunits of trimeric tail fiber gp17 (21, 30). These tail fibers are responsible for bacterial receptor recognition and adsorption (21, 33). On top of the portal within the capsid shell is a hollow cylinder-shaped core structure (10, 28) formed by the ejection proteins (core proteins) gp14, gp15, and gp16, which have been suggested to form a transenvelope channel for the genome delivery into the infected cell (20, 35, 36). The gp16 harbors lytic transglycosylase (LTase) activity, which allows for penetration into the bacterial peptidoglycan layer (37).In this study, we present the structure of the mature bacteriophage T7 with internal core proteins at near-atomic resolution and the structures of the full and empty T7 particles in complex with their cell receptor at subnanometer and near-atomic resolutions, respectively. Our reconstruction reveals that the core in the mature T7 is formed by a fourfold gp16 ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The putative gp14 structures mediate the core–portal interaction. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a LTase domain. When the T7 phage interacts with the LPS, the tail nozzle opens. Six copies of gp14 anchor to the sixfold tail channel, extending the tail across the LPS lipid bilayer. A conformational change in the portal then triggers the genome ejection. Our structures reveal the structural changes of the phage genome-delivery molecular machines after the genome delivery.     

Recombinant transfer in the basic genome of Escherichia coli

An approximation to the ∼4-Mbp basic genome shared by 32 strains of Escherichia coli representing six evolutionary groups has been derived and analyzed computationally. A multiple alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ∼90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single base-pair mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly between genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome pairs have one or two recombinant transfers of length ∼40–115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4–1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kilobase pairs. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. Most recombinant transfers seem likely to be due to generalized transduction by coevolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.The increasing availability of complete genome sequences of many different bacterial and archaeal species, as well as metagenomic sequencing of mixed populations from natural environments, has stimulated theoretical and computational approaches to understand mechanisms of speciation and how prokaryotic species should be defined (1–8). Much genome analysis and comparison has been at the level of gene content, identifying core genomes (the set of genes found in most or all genomes in a group) and the continually expanding pan-genome. Population genomics of Escherichia coli has been particularly well studied because of its long history in laboratory research and because many pathogenic strains have been isolated and completely sequenced (9–14). Proposed models of how related groups or species form and evolve include isolation by ecological niche (7–9, 11, 15), decreased homologous recombination as divergence between isolated populations increases (2–4, 8, 14, 16), and coevolving phage and bacterial populations (6).E. coli genomes are highly variable, containing an array of phage-related mobile elements integrated at many different sites (17), random insertions of multiple transposable elements (18), and idiosyncratic genome rearrangements that include inversions, translocations, duplications, and deletions. Although E. coli grows by binary cell division, genetic exchange by homologous recombination has come to be recognized as a significant factor in adaptation and genome evolution (9, 10, 19). Of particular interest has been the relative contribution to genome variability of random mutations (single base-pair differences referred to as SNPs) and replacement of genome regions by homologous recombination with fragments imported from other genomes (here referred to as recombinant transfers or transferred regions). Estimates of the rate, extent, and average lengths of recombinant transfers in the core genome vary widely, as do methods for detecting transferred regions and assessing their impact on phylogenetic relationships (12–14, 20, 21).In a previous comparison of complete genome sequences of the K-12 reference strain MG1655 and the reconstructed genome of the B strain of Delbrück and Luria referred to here as B-DL, we observed that SNPs are not randomly distributed among 3,620 perfectly matched pairs of coding sequences but rather have two distinct regimes: sharply decreasing numbers of genes having 0, 1, 2, or 3 SNPs, and an abrupt transition to a much broader exponential distribution in which decreasing numbers of genes contain increasing numbers of SNPs from 4 to 102 SNPs per gene (22). Genes in the two regimes of the distribution are interspersed in clusters of variable lengths throughout what we referred to as the basic genome, namely, the ∼4 Mbp shared by the two genomes after eliminating mobile elements. We speculated that genes having 0 to 3 SNPs may primarily have been inherited clonally from the last common ancestor, whereas genes comprising the exponential tail may primarily have been acquired by horizontal transfer from diverged members of the population.The current study was undertaken to extend these observations to a diverse set of 32 completely sequenced E. coli genomes and to analyze how SNP distributions in the basic genome change as a function of evolutionary divergence between the 496 pairs of strains in this set. We have taken a simpler approach than those of Touchon et al. (13), Didelot et al. (14), and McNally et al. (21), who previously analyzed multiple alignments of complete genomes of E. coli strains. The appreciably larger basic genome derived here is not restricted to protein-coding sequences and retains positional information.     

The impact of paleoclimatic changes on body size evolution in marine fishes

Body size is an important species trait, correlating with life span, fecundity, and other ecological factors. Over Earth’s geological history, climate shifts have occurred, potentially shaping body size evolution in many clades. General rules attempting to summarize body size evolution include Bergmann’s rule, which states that species reach larger sizes in cooler environments and smaller sizes in warmer environments, and Cope’s rule, which poses that lineages tend to increase in size over evolutionary time. Tetraodontiform fishes (including pufferfishes, boxfishes, and ocean sunfishes) provide an extraordinary clade to test these rules in ectotherms owing to their exemplary fossil record and the great disparity in body size observed among extant and fossil species. We examined Bergmann’s and Cope’s rules in this group by combining phylogenomic data (1,103 exon loci from 185 extant species) with 210 anatomical characters coded from both fossil and extant species. We aggregated data layers on paleoclimate and body size from the species examined, and inferred a set of time-calibrated phylogenies using tip-dating approaches for downstream comparative analyses of body size evolution by implementing models that incorporate paleoclimatic information. We found strong support for a temperature-driven model in which increasing body size over time is correlated with decreasing oceanic temperatures. On average, extant tetraodontiforms are two to three times larger than their fossil counterparts, which otherwise evolved during periods of warmer ocean temperatures. These results provide strong support for both Bergmann’s and Cope’s rules, trends that are less studied in marine fishes compared to terrestrial vertebrates and marine invertebrates.

Paleoclimatic changes are recognized as strong factors affecting the macroevolutionary dynamics of clades, including their distribution, ecology, and diversification (1). Throughout the course of Earth’s geological history, several large, dynamic climatic shifts have occurred, such as the end-Permian extinction event (ca. 252 Ma), the Cretaceous–Paleogene extinction event (ca. 66 Ma), and the Paleocene-Eocene Thermal Maximum (ca. 55.6 Ma) (2, 3). These periods are often marked by large changes in temperature, ocean acidification, and anoxia, as well as increases in volcanic activity (3). These environmental shifts have led to mass extinction events in fishes (4) and changes in rates and magnitude of body size evolution in amphibians, birds, and mammals (5, 6), among others. Morphological responses to paleoclimate change can be directly observed from the fossil record. As body size correlates with many aspects of a species’ biology, physiology, and ecology, its evolution should be associated with shifts in climate (5, 7, 8).Bergmann’s rule attempts to summarize body size responses to climatic changes, stating that species within a clade (or populations within a species) tend to grow to larger sizes in cooler environments and smaller sizes in warmer environments. While Bergmann’s rule can apply at multiple evolutionary scales (9), from an interspecific viewpoint it can be defined as an ecogeographical trend where species’ body size varies as a negative function of temperature. Originally studied in mammals, this trend has now been identified in a range of animals such as crustaceans, amphibians, and ray-finned fishes (8, 10–12). Various explanations have been proposed for Bergmann’s rule, from heat conservation in endotherms to oxygen availability in ectotherms (13, 14). Another broad hypothesis summarizing body size patterns is Cope’s rule, stating that species tend to increase in size over evolutionary time. Explanations for Cope’s rule are thought to be linked to fitness advantages at larger body sizes or an increase in size variance as lineages diversify from a smaller ancestor following a passive trend (15, 16). Cope’s rule could simply be an evolutionary or temporal manifestation of Bergmann’s rule if lineages evolve larger body sizes during periods of climatic cooling (8). This idea, termed the Cope–Bergmann hypothesis by Hunt and Roy (8), has received considerably less attention than studies that examine body size trends relating to Cope’s and Bergmann’s rules separately (but see 8, 17).Species’ responses to climate will vary, but ectotherms that rely on their environment for temperature regulation are likely more susceptible than endotherms to climatic changes (18). Temperature controls a variety of aspects of ectotherm biology and is strongly linked with an organism’s fitness, affecting growth rates and overall body size (14, 18). Understanding ectotherm morphological responses to global paleoclimate change may benefit greatly from examining clades with a rich fossil record. Ectothermic invertebrates have been studied in great detail, particularly brachiopods and marine arthropods (8, 19–21), due to their exceptional fossil record (22). Among ectothermic vertebrates, Cope’s and Bergmann’s rules have been tested in amphibians and reptiles (5, 23), but comparatively less in teleost fishes (but see 7, 24).Fishes in the order Tetraodontiformes provide a model clade to test patterns of body size evolution in relation to paleoclimate events, owing to their exemplary and well-studied fossil record and extraordinary morphological diversity (4, 25). They constitute a circumglobally distributed taxonomic order of mostly marine, subtropical/tropical dwelling fishes, represented by ca. 450 living species, including the charismatic pufferfishes, triggerfishes, and ocean sunfishes. Tetraodontiforms exhibit a diverse array of body shapes, from nearly square (boxfishes) to globose (pufferfishes) and laterally compressed (filefishes). Species in this order also feature remarkable variation in adult body size, ranging from just 25 mm total length (TL) (e.g., Rudarius excelsus, Carinotetraodon salivator) to 3.4 m TL (e.g., Mola, Masturus lanceolatus). The tetraodontiform fossil record extends to the Late Cretaceous with representatives from 12 exclusively fossil families; all 10 extant families are also present in the paleontological record, and on average, body size is smaller among fossil taxa (25). Their morphological diversity, coupled with a robust fossil record, provides a unique system to test the Cope–Bergmann hypothesis in ectotherms.This study aimed to investigate patterns of body size evolution in relation to paleoclimate events in ectotherms using tetraodontiform fishes as a model clade. We addressed the following questions: 1) Are paleoclimatic changes correlated with changes in tetraodontiform body size? and 2) If a correlation between paleoclimate and body size is observed, do tetraodontiforms follow the Cope–Bergmann hypothesis? That is, does their body size evolution correlate with a paleotemperature curve where tetraodontiforms are evolving toward a larger body size during periods of climate cooling? To address these questions, we estimated a time-calibrated phylogeny for tetraodontiforms using total-evidence dating approaches that combine genome-wide data from extant species with a morphological matrix coded from both fossil and extant species. We also incorporated body length data and paleotemperature records spanning the past 100 Ma into a series of evolutionary model fitting analyses. We hypothesize that tetraodontiform evolution has been driven by past temperature changes and that body size is strongly linked to past climate.     

A rapidly reversible mutation generates subclonal genetic diversity and unstable drug resistance

Most genetic changes have negligible reversion rates. As most mutations that confer resistance to an adverse condition (e.g., drug treatment) also confer a growth defect in its absence, it is challenging for cells to genetically adapt to transient environmental changes. Here, we identify a set of rapidly reversible drug-resistance mutations in Schizosaccharomyces pombe that are caused by microhomology-mediated tandem duplication (MTD) and reversion back to the wild-type sequence. Using 10,000× coverage whole-genome sequencing, we identify nearly 6,000 subclonal MTDs in a single clonal population and determine, using machine learning, how MTD frequency is encoded in the genome. We find that sequences with the highest-predicted MTD rates tend to generate insertions that maintain the correct reading frame, suggesting that MTD formation has shaped the evolution of coding sequences. Our study reveals a common mechanism of reversible genetic variation that is beneficial for adaptation to environmental fluctuations and facilitates evolutionary divergence.

Different mechanisms of adaptation have different timescales. Epigenetic changes are often rapid and reversible, while most genetic changes have nearly negligible rates of reversion (1). This poses a challenge for genetic adaptation to transient conditions such as drug treatment; mutations that confer drug resistance are often deleterious in the absence of drug, and the second-site suppressor mutations are required to restore fitness (2, 3). Preexisting tandem repeats (satellite DNA) undergo frequent expansion and contraction (4–6). While repeats are rare inside of most coding sequences and functional elements, there is some evidence for conserved repetitive regions that undergo expansion and contraction to regulate protein functions or expression (6–8). RNA interference– or Chromatin-based epigenetic states have been associated with transient drug resistance in fungi (9) and cancer cells (10, 11), and transient resistant states have been characterized by differences in organelle state, growth rate, and gene expression in budding yeast (12, 13). In bacteria and in fungi, copy-number gain and subsequent loss can result in reversible drug resistance (14–18). However, all genetic systems developed so far for studying unstable genotypes rely on reporter genes and thus investigate only one genetic locus and only one type of genetic change.Unbiased, next-generation sequencing-based approaches could give a more global view, allowing us to understand the rules that govern unstable genotypes at a genome-wide scale. However, genetic changes with high rates of reversion tend to remain subclonal (19–21), and it is challenging to distinguish most types of low-frequency mutations from sequencing errors (22), especially in complex genomes with large amount of repetitive DNA or de novo duplicated genes. Thus, fast-growing organisms with relatively small and simple genomes are particularly well suited for determining whether transient mutations exist, for the genome-wide characterization of such mutations, and for identification of the underlying mechanisms.     

New Age of Fishes initiated by the Cretaceous?Paleogene mass extinction

Ray-finned fishes (Actinopterygii) comprise nearly half of all modern vertebrate diversity, and are an ecologically and numerically dominant megafauna in most aquatic environments. Crown teleost fishes diversified relatively recently, during the Late Cretaceous and early Paleogene, although the exact timing and cause of their radiation and rise to ecological dominance is poorly constrained. Here we use microfossil teeth and shark dermal scales (ichthyoliths) preserved in deep-sea sediments to study the changes in the pelagic fish community in the latest Cretaceous and early Paleogene. We find that the Cretaceous−Paleogene (K/Pg) extinction event marked a profound change in the structure of ichthyolith communities around the globe: Whereas shark denticles outnumber ray-finned fish teeth in Cretaceous deep-sea sediments around the world, there is a dramatic increase in the proportion of ray-finned fish teeth to shark denticles in the Paleocene. There is also an increase in size and numerical abundance of ray-finned fish teeth at the boundary. These changes are sustained through at least the first 24 million years of the Cenozoic. This new fish community structure began at the K/Pg mass extinction, suggesting the extinction event played an important role in initiating the modern “age of fishes.”Ray-finned fishes are a dominant and exceptionally diverse member of modern pelagic ecosystems; however, both the fossil record and molecular clocks suggest that the vast majority of living ray-finned fishes developed only recently, during the last 100 million years (1–3). It has been proposed that the explosion in actinoptygerian diversity in the Late Mesozoic and Early Cenozoic represents a new “age of fishes” in contrast to the initial diversification of fish clades in the Devonian (2, 3). However, the mechanisms and timing of this Mesozoic−Cenozoic radiation and rise to dominance by ray-finned fishes are not well constrained in current molecular phylogenies or from the relatively sparse fossil record. While the Cretaceous−Paleogene (K/Pg) mass extinction occurred ∼66 million years ago (Ma), in the middle of this radiation, there is little clear phylogenetic evidence linking any changes in fish diversity directly to this event (1), although a recent phylogenetic study on pelagic fish families suggested that open ocean fishes radiated during the early Paleogene following the extinction (4).The K/Pg extinction had a dramatic effect on open ocean marine ecosystems (5–7), although the severity of the extinction varied around the globe (7–9). Major groups at both the base and top of the food web were decimated (5, 6, 10). While the traditional model of mass extinction due to primary productivity collapse (11) has been generally discredited due to the continued productivity of select consumer groups (12, 13), it is likely that upheaval among primary producers reverberated up the food web to cause extinctions at higher trophic levels. In the open ocean, calcifying plankton such as foraminifera and calcareous nannofossils suffered >90% species-level extinctions (9, 14). These changes in the structure of the base of the food web likely helped to cause the extinctions of pelagic consumers such as ammonites and marine reptiles (10). The trophic link between the plankton and large consumers in pelagic ecosystems is small pelagic fish, which would be expected to be similarly decimated by changes in food web structure. However, recent work has shown that while there was a collapse of small pelagic fish production in the Tethys Sea, in the Pacific Ocean, these midlevel consumers maintained Cretaceous-like or higher levels of production in the earliest Danian (15).Changes in abundance do not tell the whole story of how pelagic fishes responded to the extinction event. Indeed, despite dramatic levels of extinction, a few species of planktonic foraminifera thrived in the postextinction oceans, reaching abundances in the ∼500,000 y following the event that far exceed those of typical high-diversity Cretaceous assemblages (7). This foraminifer response shows that taxonomic diversity and biological production can be decoupled in postdisaster ecosystems like those of the earliest Danian. Fishes are highly diverse and occupy a range of ecological niches, from the smallest plankton feeders through predatory sharks. This means that different groups could exhibit differential responses to the extinction (16). Work on well-preserved body fossils has found that there was a selective extinction of shallow marine predatory fishes at the K/Pg extinction, and a radiation during the early Cenozoic (17, 18). Additionally, a low level of extinction (<33%) of sharks and rays has been inferred across the event (19, 20). However, the magnitude of pelagic fish extinction is poorly known, although a relatively modest ∼12% extinction has been documented for fish tooth morphotypes between the Late Cretaceous and the early Paleocene (21).Here we use ichthyoliths, the isolated teeth and dermal scales (denticles) of sharks and ray-finned fishes found in deep-sea sediments, to investigate the response of sharks and fishes to the K/Pg extinction. Calcium phosphate ichthyoliths are found in nearly all marine sediments, even red clays (22), where other microfossils have been dissolved by corrosive bottom water conditions. Therefore, ichthyoliths are relatively unaffected by the preservation biases typically found in other microfossil groups. Teeth and denticles are reasonably common, with 10s to 100s found in a few grams of sediment, allowing studies of the fish community rather than isolated individuals. The abundance of ichthyoliths also allows for high-temporal resolution sampling similar to other microfossils. The well-resolved ichthyolith records stand in sharp contrast to those for the comparatively rare body fossil record, and can provide a complimentary analysis of abrupt biotic events such as mass extinctions or transient climate changes. In addition, the abundance, assemblage, and morphological composition of ichthyoliths record the productivity and biodiversity of the pelagic fish community.We investigate how the pelagic fish community responded to the K/Pg extinction at six deep-sea sites in the Pacific, Atlantic, and Tethys Oceans. We use ichthyolith community metrics, including the relative abundance of microfossils from sharks and ray-finned fishes, and the size structure of the tooth assemblage to assess the changes in the pelagic fish community across the K/Pg mass extinction around the world. This represents, to our knowledge, the first geographically comprehensive, high-resolution study of pelagic marine vertebrate communities across the extinction.     

DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.DNA double-strand breaks (DSBs) are toxic lesions that can be repaired by two major pathways in eukaryotes: nonhomologous end-joining (NHEJ) and homologous recombination (HR) (1). Although HR repairs DSBs in a template-dependent, high-fidelity manner, NHEJ functions to ligate DSB ends together using no or very short (1–4 bp) homology. Recently, a new pathway was identified in eukaryotes, which uses microhomologies (MHs) to repair a DSB and does not require the central proteins used in HR (Rad51, Rad52) or NHEJ (Ku70–Ku80) (2–5). In mammalian cells, this pathway of repair is known as alternative end-joining (Alt-EJ) and is often but not always associated with MHs, whereas in budding yeast, the commensurate pathway, MH-mediated end-joining (MMEJ), will typically use 5–25 bp of MH (6, 7). These pathways are associated with genomic rearrangements, and cancer genomes show evidence of MH-mediated rearrangements (8–12). In addition, eukaryotic genomes contain many dispersed repetitive elements that can lead to genome rearrangements when recombination occurs between them (13–16). Therefore, controlling DSB repair in the human genome, which features a variety of repeats, is especially important given the fact that recombination between repetitive elements has been implicated in genomic instability associated with disease (17–20).The original characterization of Alt-EJ in mammalian cells suggested it did not represent a significant DNA repair pathway and only operated in the absence of functional HR and NHEJ pathways. More recent analyses demonstrate a physiological role of Alt-EJ during DNA repair in the presence of active HR and NHEJ pathways (2, 12, 21, 22). Furthermore, examination of I-SceI–induced translocation junctions in mammalian cells revealed the frequent presence of MHs (23, 24). NHEJ-deficient and p53-null mice develop pro–B-cell lymphomas, and nonreciprocal translocations characterized by small MHs are found at their break point junctions (25–28). Similarly, in human cancers, many translocation break point junctions contain MHs, suggesting a role for Alt-EJ in cancer development (29–31) and resistance to chemotherapy and genetic disease (32–36). Hence, the presence of many short repetitive sequences in the human genome is likely to increase rearrangements mediated by MHs following the creation of a DSB.MMEJ is a distinct DSB repair pathway that operates in the presence of functional NHEJ and HR pathways (10, 37). The genetic requirements of MMEJ are being studied in the model eukaryote Saccharomyces cerevisiae and involve components traditionally considered specific to the NHEJ (Pol λ) and HR (Rad1–Rad10, Rad59, and Mre11–Rad50–Xrs2) pathways (4, 5, 10, 38). Although being clearly independent of the central NHEJ factor Ku70–Ku80 heterodimer (10, 37), the involvement of the key HR factor Rad52 in MMEJ remains uncertain. It has been reported that Rad52 is required for MMEJ repair (4, 10, 38), whereas in another assay system Rad52 suppresses MMEJ repair (37). More recently, it has been proposed that the replication protein A (RPA) regulates pathway choice between HR and MMEJ (37). In addition, several models have been proposed that identify specific pathways that may use MHs for the repair of DNA damage (39–41). Despite current advancements in our understanding of MMEJ, the precise involvement of DNA polymerases in supporting the repair of DSBs using MHs remains poorly understood. DNA polymerase λ (also called Pol4 in budding yeast) and its human homolog Pol λ are considered to be the primary candidates for the DNA polymerases working in NHEJ and MMEJ (4, 5, 42–46). Both genetic and biochemical evidence shows that Pol δ is recruited during HR to extend Rad51-dependent recombination intermediates (47–50). Recent analysis using pol32 mutants (5, 10) implicated the Pol32 subunit of Pol δ in MMEJ. Pol32 and Pol31 were also identified as subunits of the DNA polymerase zeta complex (Pol ζ) (51, 52), but previous analysis showed no effect of rev3 mutants in MMEJ (10). REV3 encodes the catalytic subunit of Pol ζ. However, an involvement of Pol δ had not been demonstrated directly before, and it is possible that Pol32 could act in conjunction with yet another DNA polymerase.Here, we report the development of a series of interchromosomal MMEJ assays in diploid S. cerevisiae to assess the mechanisms underlying the repair of DSBs using varying MHs. We focus on diploid cells, as they represent the natural state of budding yeast, which is a diplontic organism (53). The yeast mating-type switching system represents a mechanism to return haploid yeast as efficiently as possible to diploidy (54). Using a combination of genetic, molecular, and in vivo chromatin immunoprecipitation (ChIP) experiments, we provide compelling evidence for a direct involvement of Pol δ in coordinating with Pol λ in MMEJ in budding yeast.     

Polynucleobacter necessarius,a model for genome reduction in both free-living and symbiotic bacteria

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius (Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss.Symbiosis, defined as a close relationship between organisms belonging to different species (1), is a ubiquitous, diverse, and important mechanism in ecology and evolution (e.g., refs. 2–4). In extreme cases, through the establishment of symbiotic relationships, quite unrelated lineages can functionally combine their genomes and generate advantageous emergent features or initiate parasite/host arms races. Ciliates, common unicellular protists of the phylum Ciliophora, are extraordinary receptacles for prokaryotic ecto- and endosymbionts (5, 6) that provide varied examples of biodiversity and ecological roles (6). Nevertheless, most of these symbionts are understudied, partially owing to the scarcity of available molecular data and the absence of sequenced genomes. Yet, thanks to their various biologies and the ease of sampling and cultivating their protist hosts, they are excellent potential models for symbioses between bacteria and heterotrophic eukaryotes. Until recently this field was dominated by studies on endosymbionts of invertebrates, especially insects (e.g., ref. 7), although unicellular systems like amoebas (e.g., refs. 8 and 9) have been shown to be suitable models.Polynucleobacter necessarius was first described as a cytoplasmic endosymbiont of the ciliate Euplotes aediculatus (10, 11). Further surveys detected its presence in a monophyletic group of fresh and brackish water Euplotes species (12, 13). All of the investigated strains of these species die soon after being cured of the endosymbiont (10, 12, 13). In the few cases in which P. necessarius is not present, a different and rarer bacterium apparently supplies the same function (12, 14). No attempt to grow symbiotic P. necessarius outside their hosts has yet been successful (15), strongly suggesting that the relationship is obligate for both partners, in contrast to most other known prokaryote/ciliate symbioses (6).Thus, the findings of many environmental 16S rRNA gene sequences similar to that of the symbiotic P. necessarius (16) but belonging to free-living freshwater bacteria came as a surprise. These free-living strains, which have been isolated and cultivated (17), are ubiquitous and abundant in the plankton of lentic environments (17, 18). They are smaller and do not show the most prominent morphological feature of the symbiotic form: the presence of multiple nucleoids, each containing one copy of the genome (10, 11). It is clear that free-living and endosymbiotic P. necessarius are not different life stages of the same organism (15). Nevertheless, these strikingly different bacteria, occupying separate ecological niches, exhibit >99% 16S rRNA gene sequence identity, and phylogenetic analyses fail to separate them into two distinct groups (15). Rather, several lines of evidence point to multiple, recent origins of symbiotic strains from the free-living bacterial pool (14, 15).Thus, the Euplotes–Polynucleobacter symbiosis provides a promising system for the study of changes promoting or caused by the shift to an intracellular lifestyle. The remarkably small (2.16 Mbp) genome of the free-living strain QLW-P1DMWA-1 has been sequenced and studied, especially for features that would explain the success of this lineage in freshwater systems worldwide (19, 20). Phylogenies based on the 16S rRNA gene (13, 14) and multiple-gene analyses (19, 21, 22) consistently cluster Polynucleobacter with bacteria of the family Burkholderiaceae (Betaproteobacteria), either in a basal position or as the sister group of Ralstonia and Cupriavidus.Here we provide the complete genomic sequence of a symbiotic P. necessarius harbored in the cytoplasm of E. aediculatus and present a comparative analysis of the two sequenced Polynucleobacter genomes, addressing the possible biological basis of the Euplotes–Polynucleobacter symbiosis. We also provide insights into the evolution of the unique two-step genome reduction in this bacterial species: the first step involving streamlining in a free-living ancestor and the second a more recent period of genome erosion confined to the symbiotic lineage.     

Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)–distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg²⁺ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg²⁺-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

CRISPR-Cas, a prokaryotic adaptive immune system, is a revolutionary tool for genome editing (1–6). Among the various types of the Cas systems, Cas12a (also known as Cpf1), class 2 type V-A CRISPR-Cas system, catalyzes double-stranded DNA (dsDNA) targets by utilizing single CRISPR RNA (crRNA) (7–10). The Cas12a-crRNA ribonucleoprotein (RNP) complex first identifies the dsDNA target via a T-rich protospacer-adjacent motif (PAM). Upon binding with cognate DNA, the Cas12a RNP unwinds the DNA via the formation of a crRNA-target strand (TS) heteroduplex and the simultaneous displacement of the nontarget strand (NTS) (a so-called R-loop structure) (11). Then, Cas12a generates double-strand DNA breaks with sticky ends by using a single RuvC nuclease domain in a sequential manner. Furthermore, in contrast to Cas9, Cas12a exhibits distinct features of pre-crRNA processing and indiscriminate single-stranded DNA cleavage activity (7, 12, 13). Owing to these unique features, CRISPR/Cas12a has been extensively utilized for the detection of nucleic acids as well as programmable genome editing (13–21).Meanwhile, recently reported base and prime editors, which accomplish targeted edits in a highly efficient manner, utilized a nickase form of CRISPR/Cas9 to reduce the frequency of undesired insertions and deletions (22–24). However, the distinct feature by which both strands of target DNA are cleaved by a single catalytic site of Cas12a has hampered the development of engineered Cas12a RNPs including an efficient nickase, resulting in a limited range of Cas12a application (25–27). Given the advantages of Cas12a, including its multiplexing capability using the intrinsic crRNA processing activity and fewer off-target effects compared to Cas9 (14, 15, 17, 28), the development of various engineered Cas12a RNPs is necessary to improve genome editing techniques. Although recently several studies have suggested the nickase form of Cas12a RNPs using alterations of crRNA (29) or mutations of protein residues (30, 31), existing nickase variants still have much room for enhancement of the nicking activity. In this regard, thorough understanding of the mechanisms that regulate the sequential cleavage reaction of dsDNA, beginning with the NTS and proceeding to the TS, by a single catalytic site in the Cas12a RuvC domain, is required. However, despite many recent biochemical and structural studies (30–40), a detailed mechanistic understanding of the way in which Cas12a uses its single catalytic site to completely break the double strand of the target DNA is still lacking.Here we perform single-molecule fluorescence assay to monitor conformational dynamics of the Cas12a ternary complex during TS cleavage following the initial cleavage of NTS of DNA. Recently, several groups have utilized similar methodological approaches to monitor the molecular interaction between Cas12a RNP and target DNA by using labeled target DNA and crRNA (35–37) and the interdomain dynamics of Cas12a protein by using labeled Cas12a (31, 41). Using this assay, here we identified the features of intermediates that form during conformational rearrangements in the TS cleavage reaction to complete dsDNA cleavage and revealed its underlying mechanism based on a kinetic analysis of the conformational dynamics. The results of our study suggest that Mg²⁺-mediated local DNA unwinding in the PAM-distal region is an essential prerequisite for the regulation of the sequential dsDNA cleavage reaction. This allosteric mechanism provides molecular insight into Cas12a engineering toward the development of Cas12a nickase.