Impairment of O-antigen production confers resistance to grazing in a model amoeba–cyanobacterium predator–prey system

The grazing activity of predators on photosynthetic organisms is a major mechanism of mortality and population restructuring in natural environments. Grazing is also one of the primary difficulties in growing cyanobacteria and other microalgae in large, open ponds for the production of biofuels, as contaminants destroy valuable biomass and prevent stable, continuous production of biofuel crops. To address this problem, we have isolated a heterolobosean amoeba, HGG1, that grazes upon unicellular and filamentous freshwater cyanobacterial species. We have established a model predator–prey system using this amoeba and Synechococcus elongatus PCC 7942. Application of amoebae to a library of mutants of S. elongatus led to the identification of a grazer-resistant knockout mutant of the wzm ABC O-antigen transporter gene, SynPCC7942_1126. Mutations in three other genes involved in O-antigen synthesis and transport also prevented the expression of O-antigen and conferred resistance to HGG1. Complementation of these rough mutants returned O-antigen expression and susceptibility to amoebae. Rough mutants are easily identifiable by appearance, are capable of autoflocculation, and do not display growth defects under standard laboratory growth conditions, all of which are desired traits for a biofuel production strain. Thus, preventing the production of O-antigen is a pathway for producing resistance to grazing by certain amoebae.Biofuels derived from algal biomass represent a carbon-neutral and potentially sustainable alternative to fossil fuels that can be developed without massive infrastructure changes for distribution and utilization. The next generation of biofuels is being developed through the mass growth of photosynthetic microorganisms, such as eukaryotic algae and cyanobacteria, in open pond production systems (1, 2). Whereas these systems avoid the high economic costs associated with bioreactor production and maintenance, they are susceptible to contamination with organisms that predate upon or infect the desired production strain, resulting in a “pond crash” that can destroy almost the entire yield of high-density biomass, as occurs with agricultural crops (1, 3, 4). Strategies for crop protection in algal biotechnology must be developed to overcome the impact of grazers and infectious agents that otherwise prevent the growth of biomass for the production of biofuels, nutraceuticals, agricultural feedstocks, waste-water treatment, and other applications (5, 6).In their natural environments, cyanobacteria are subjected to grazing pressure by a variety of organisms, including protistan predators such as ciliates, flagellates, and amoebae (7). Grazing affects both mortality and population structure (8), as has recently been observed in the rapid decline of biomass of Microcystis in a natural pond and in the restructuring of the bloom with a shift from a susceptible Microcystis species to a resistant one upon amoebal grazing (9). Whereas it is known that amoebae graze on cyanobacteria (10–12), these predators have not received much attention. Van Wichelen et al. (2010) attribute this omission to the fact that population densities of amoebae peak for short periods of time and, hence, may be overlooked during monthly or even biweekly samplings of freshwater environments (9).Bacteria have evolved a variety of mechanisms to evade detection, capture, ingestion, and digestion (13), but very little is known about the molecular mechanisms that govern the interactions between cyanobacteria and their protistan predators (14). The cell surface appears to be important as S layers (15), a giant cell-surface protein (16), and lipopolysaccharide (LPS) heterogeneity (17) have been implicated as defenses against nanoflagellates and a dinoflagellate. Xinyao et al. (2006) studied the grazing of an amoeba of the Naegleria genus on a variety of freshwater unicellular and filamentous cyanobacteria and showed that prey morphology such as filament form and aggregation affects ingestion (18). Other undetermined cell properties also affected food selection as some unicellular cyanobacteria were excreted after ingestion.Defenses against amoebae have been characterized in a number of opportunistically pathogenic bacteria, which range from modifications of the cell surface to the production and delivery of cytotoxic compounds. Epitope differences in the O-antigen of otherwise genetically identical serovars of Salmonella enterica are sufficient to account for feeding preferences among intestinal amoebae (19). Legionella pneumophila evades lysosomal digestion in the soil amoeba Acanthamoeba castellani by shedding LPS outer membrane vesicles (20). A type III secretion system confers cytotoxicity of Pseudomonas aeruginosa to A. castellani (21) and of Vibrio parahaemolyticus to both A. castellani and Dictyostelium discoideum (22). A type VI secretion system confers cytotoxicity of Vibrio cholera to D. discoideum (23) through the translocation of an actin cross-linking protein (24). The soil bacterium Pseudomonas fluorescens induces the expression of genes involved in the production of certain extracellular toxins in response to chemical cues from A. castellani (25).As amoebae are known to be a problem in open ponds and little is known about natural mechanisms of amoebal grazing resistance in cyanobacteria, we sought to establish a model system with which we could study the interaction between amoebae and cyanobacteria at the molecular level. Here, we describe the isolation of a heterolobosean amoeba, HGG1, which grazes on the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942 and certain filamentous freshwater cyanobacteria. Through a genetic screen using this amoeba–cyanobacterium model system, we have identified O-antigen synthesis and transport as a major pathway that, when impaired, produces resistance to certain amoebal grazers.     

From the Cover: Kibble–Zurek mechanism in colloidal monolayers

The Kibble–Zurek mechanism describes the evolution of topological defect structures like domain walls, strings, and monopoles when a system is driven through a second-order phase transition. The model is used on very different scales like the Higgs field in the early universe or quantum fluids in condensed matter systems. A defect structure naturally arises during cooling if separated regions are too far apart to communicate (e.g., about their orientation or phase) due to finite signal velocity. This lack of causality results in separated domains with different (degenerated) locally broken symmetry. Within this picture, we investigate the nonequilibrium dynamics in a condensed matter analog, a 2D ensemble of colloidal particles. In equilibrium, it obeys the so-called Kosterlitz–Thouless–Halperin–Nelson–Young (KTHNY) melting scenario with continuous (second order-like) phase transitions. The ensemble is exposed to a set of finite cooling rates covering roughly three orders of magnitude. Along this process, we analyze the defect and domain structure quantitatively via video microscopy and determine the scaling of the corresponding length scales as a function of the cooling rate. We indeed observe the scaling predicted by the Kibble–Zurek mechanism for the KTHNY universality class.In the formalism of gauge theory with spontaneously broken symmetry, Zel''dovich et al. and Kibble postulated a cosmological phase transition during the cooling down of the early universe. This transition leads to degenerated states of vacua below a critical temperature, separated or dispersed by defect structures as domain walls, strings, or monopoles (1–3). In the course of the transition, the vacuum can be described via an N-component, scalar order parameter ϕ (known as the Higgs field) underlying an effective potentialV=aϕ2+b(ϕ2−η02)2,[1]where a is temperature dependent, b is a constant, and η₀ is the modulus of ??? at T = 0. For high temperatures, V has a single minimum at ? = 0 (high symmetry) but develops a minimum “landscape” of degenerated vacua below a critical temperature T_c (e.g., the so-called sombrero shape for N = 2). Cooling down from the high symmetry phase, the system undergoes a phase transition at T_c into an ordered (low symmetry) phase with nonzero ???. For T < T_c it holds〈ϕ〉2=η02(1−T2/Tc2)=η2(T).[2]Caused by thermal fluctuations, one can expect that below T_c, ??? takes different nonzero values in regions that are not connected by causality. The question now arising concerns the determination of the typical length scale ξ_d of these regions and their separation. For a finite cooling rate, ξ_d is limited by the speed of propagating information, which is given by the finite speed of light defining an ultimate event horizon. Independent of the nature of the limiting causality, Kibble argued that as long as the difference in free energy ΔF (of a certain system volume) between its high symmetry state ??? = 0 and a possible finite value of ??? just below T_c is less than k_BT, the volume can jump between both phases. The temperature at which ΔF = k_BT is called the Ginzburg temperature T_G, and the length scale ξ_d of the initial (proto)domains is supposed to be equal to the correlation length at that temperature: ξ_d = ξ(T_G) (2).The geometry of the defect network that separates the uncorrelated domains is given by the topology of the manifold of degenerated states that can exist in the low symmetry phase. Thus, it depends strongly on the dimensionality of the system D and on the dimension N of the order parameter itself. Regarding the square root of Eq. 2, the expectation value of a one-component order parameter (N = 1) can only take two different low symmetry values ??? = ±η(T) (e.g., the magnetization in a 2D or 3D Ising model): the manifold of the possible states is disconnected. This topological constraint has a crucial effect if one considers a mesh of symmetry broken domains where ??? is chosen randomly as either + η or ?η. If two neighboring (but uncorrelated) domains have the same expectation value of ???, they can merge. In contrast, domains with an opposite expectation value will be separated by a domain wall in 3D (or a domain line in 2D). At its center, the domain wall attains a value of ??? = 0, providing a continuous crossover of the expectation value between the domains (Fig. 1A). Consider now N = 2: ??? can take any value on a circle, e.g., ???² = ??_x?² + ??_y?² = η²(T) (all of the order parameter values that are lying on the minimum circle of the sombrero are degenerated). Because the manifold of possible low symmetry states is now connected, ??? can vary smoothly along a path (Fig. 1B). In a network of symmetry broken domains in two dimensions, at least three domains (in Fig. 1B separated by dashed lines) meet at a mutual edge. On a closed path around the edge, the expectation value ??? might be either constant along the path (for a global, uniform ϕ) but can also vary by a multiple of 2π (in analogy to the winding numbers in liquid crystals). In the first case, the closed path can be reduced to a point with ??? ≠ 0, and no defect is built. If the path is shrunk in the second case, the field eventually has to attain ??? = 0 within the path, and one remains with a monopole for D = 2 or a string for D = 3 (2, 3). A condensed matter analog would be a vortex of normal fluid with quantized circulation in superfluid helium. For N = 3 and D = 3, four domains can meet at a mutual point, and the degenerated solutions of the low temperature phase lie on a sphere: ???² = ??_x?² + ??_y?² + ??_z?². If the field now again varies circularly on a spherical path (all field arrows point radially outward), a shrinking of this sphere leads to a monopole in three dimensions (2, 3).Open in a separate windowFig. 1.Emergence of defects in the Higgs field that is illustrated with red vectors (shown in 2D for simplicity). (A) For N = 1 and D = 3, domain walls can appear (strings for D = 2). (B) For N = 2 and D = 3, nontrivial topologies are strings (monopoles for D = 2). The defects are regions where the order parameter ϕ retains the high symmetry phase (??? = 0) to moderate between different degenerated orientations of the symmetry broken field.Zurek extended Kibble’s predictions and transferred his considerations to quantum condensed matter systems. He suggested that ⁴He should intrinsically develop a defect structure when quenched from the normal to the superfluid phase (4, 5). For superfluid ⁴He, the order parameter ψ = |ψ|exp(iΘ) is complex with two independent components: magnitude |ψ| and phase Θ (the superfluid density is given by |ψ|²). A nontrivial, static solution of the equation of state with a Ginzburg–Landau potential yields ψ = ψ₀(r)exp(inφ), where r and φ are cylindrical coordinates, n ∈ ?, and ψ₀(0) = 0. This solution is called a vortex line, which is topologically equivalent to a string for the case N = 2 we discussed before. In the vicinity of the critical temperature during a quench from the normal fluid to the superfluid state, ψ will be chosen randomly in uncorrelated regions, leading to a string network of normal fluid vortices. In condensed matter systems, the role of the limiting speed of light is taken by the sound velocity (in ⁴He, the second sound). This upper boundary leads to a finite speed of the propagation of order parameter fluctuations and sets a “sonic horizon.”Zurek argued that the correlation length is frozen-out close to the transition point or even far before depending on the cooling rate (4, 5). Consider the divergence of the correlation length ξ for a second-order transition, e.g., ξ = ξ₀|?|^?ν, where ? = (T ? T_c)/T_c is the reduced temperature. If the cooling is infinitely slow, the system behaves as in equilibrium: ξ will diverge close to the transition and the system is a monodomain. For an instantaneous quench, the system has minimal time to adapt to its surrounding: ξ will be frozen-out at the beginning of the quench. For second-order phase transitions, the divergence of correlation lengths is accompanied by the divergence of the correlation time τ = τ₀|?|^?μ, which is due to the critical slowing down of order parameter fluctuations. If the time t it takes to reach T_c for a given cooling rate is larger than the correlation time, the system stays in equilibrium and the dynamic is adiabatic. Nonetheless, for every finite but nonzero cooling rate, t eventually becomes smaller than τ, and the system falls out of equilibrium before T_c is reached. The moment when both the correlation time and the time it takes to reach T_c coincide defines the freeze-out time.t^=τ(t^).[3]The frozen-out correlation length ξ^ is then set at the temperature ϵ^ of the corresponding freeze-out time: ξ^=ξ(ϵ^)=ξ(t^). For a linear temperature quench? = (T ? T_c)/T_c = t/τ_q, [4]with the quench time scale τ_q, one observes t^=(τ0τqμ)1/(1+μ) andξ^=ξ(t^)=ξ0(τq/τ0)ν/(1+μ).[5]For the Ginzburg–Landau model (ν = 1/2, μ = 1), one finds the scaling ξ^∼τq1/4, whereas a renormalization group correction (ν = 2/3) leads to ξ^∼τq1/3 (4, 5).A frequently used approximation is that when the adiabatic regime ends at t^ before the transition, the correlation length cannot follow the critical behavior until τ again exceeds the time t when T_c is passed. Given a symmetric divergence of τ around T_c, this is the time t^ after the transition. The period in between is known as the impulse regime, in which the correlation length is assumed not to evolve further. A recent analytical investigation, however, suggests that in this period, the system falls into a regime of critical coarse graining (6). There, the typical length scale of correlated domains continues to grow because local fluctuations are still allowed, and the system is out of equilibrium. On the other side, numerical studies in which dissipative contributions and cooling rates were alternatively varied before and after the transition indicate that the final length scale of the defect and domain network is entirely determined after the transition (7). Several efforts have been made to provide experimental verification of the Kibble–Zurek mechanism in a variety of systems, e.g., in liquid crystals (8) (the transition is weakly first order but the defect network can easily observed with cross polarization microscopy), superfluid ³He (9), superconducting systems (10), convective, intrinsically out of equilibrium systems (11), multiferroics (12), quantum systems (13), ion crystals (14, 15), and Bose–Einstein condensates (16) (the latter two systems contain the effect of inhomogeneities due to, for example, temperature gradients). A detailed review concerning the significance and limitations of these experiments can be found in ref. 17.In this experimental study, we test the validity and applicability of the Kibble–Zurek mechanism in a 2D colloidal model system whose equilibrium thermodynamics follow the microscopically motivated Kosterlitz–Thouless–Halperin–Nelson–Young (KTHNY) theory. This theory predicts a continuous, two-step melting behavior whose dynamics, however, are quantitatively different from phenomenological second-order phase transitions described by the Ginzburg–Landau model. We applied cooling rates over roughly three orders of magnitude, for which we changed the control parameter with high resolution and homogeneously throughout the sample without temperature gradients. Single particle resolution provides a quantitative determination of defect and domain structures during the entire quench procedure, and the precise knowledge of the equilibrium dynamics allows determination of the scaling behavior of corresponding length scales at the freeze-out times. In the following, we validate that the Kibble–Zurek mechanism can be successfully applied to the KTHNY universality class.     

From the Cover: Improving our fundamental understanding of the role of aerosol?cloud interactions in the climate system

The effect of an increase in atmospheric aerosol concentrations on the distribution and radiative properties of Earth’s clouds is the most uncertain component of the overall global radiative forcing from preindustrial time. General circulation models (GCMs) are the tool for predicting future climate, but the treatment of aerosols, clouds, and aerosol−cloud radiative effects carries large uncertainties that directly affect GCM predictions, such as climate sensitivity. Predictions are hampered by the large range of scales of interaction between various components that need to be captured. Observation systems (remote sensing, in situ) are increasingly being used to constrain predictions, but significant challenges exist, to some extent because of the large range of scales and the fact that the various measuring systems tend to address different scales. Fine-scale models represent clouds, aerosols, and aerosol−cloud interactions with high fidelity but do not include interactions with the larger scale and are therefore limited from a climatic point of view. We suggest strategies for improving estimates of aerosol−cloud relationships in climate models, for new remote sensing and in situ measurements, and for quantifying and reducing model uncertainty.     

From the Cover: Efficient replication of Epstein–Barr virus in stratified epithelium in vitro

Epstein–Barr virus is a ubiquitous human herpesvirus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva and must cross the oral mucosal epithelium before infecting B lymphocytes, where it establishes a life-long infection. The latter process is well understood because it can be studied in vitro, but our knowledge of infection of epithelial cells has been limited by the inability to infect epithelial cells readily in vitro or to generate cell lines from EBV-infected epithelial tumors. Because epithelium exists as a stratified tissue in vivo, organotypic cultures may serve as a better model of EBV in epithelium than monolayer cultures. Here, we demonstrate that EBV is able to infect organotypic cultures of epithelial cells to establish a predominantly productive infection in the suprabasal layers of stratified epithelium, similar to that seen with Kaposi’s-associated herpesvirus. These cells did express latency-associated proteins in addition to productive-cycle proteins, but a population of cells that exclusively expressed latency-associated viral proteins could not be detected; however, an inability to infect the basal layer would be unlike other herpesviruses examined in organotypic cultures. Furthermore, infection did not induce cellular proliferation, as it does in B cells, but instead resulted in cytopathic effects more commonly associated with productive viral replication. These data suggest that infection of epithelial cells is an integral part of viral spread, which typically does not result in the immortalization or enhanced growth of infected epithelial cells but rather in efficient production of virus.Although the association between Epstein–Barr virus and epithelial malignancies has been known for more than three decades, the EBV life cycle within the epithelial milieu is still only poorly understood. In contrast, our broad understanding of the biology of EBV within the B-cell compartment has been facilitated by the ability of EBV to infect and immortalize primary B cells in vitro and by the ability of some EBV-positive B-cell tumors to give rise to cell lines that maintain restricted programs of latency gene expression similar to those seen in vivo. Although in primary EBV infection the entire complement of EBV latency-associated nuclear proteins (EBNAs 1, 2, 3A, 3B, 3C, and LP) and membrane proteins (LMPs 1, 2A, and 2B) promote cellular proliferation and survival (Latency III), EBV gene expression must be progressively silenced (Latency II; EBNA1 and LMPs 1 and 2) so that the most restricted program, Latency 0 (in which EBV gene expression is believed to be completely silenced), is achieved in resting memory B cells that serve as the long-term reservoir of latent EBV (1, 2). Because EBNA1 functions to maintain the viral genome during cell division, it alone among the viral protein repertoire is required during periodic proliferation of these memory B cells (Latency I).EBV is strongly associated with subtypes of nasopharyngeal carcinoma and gastric carcinoma, in which it exhibits a Latency I/II program of gene expression (3–5). The presence of latent EBV in epithelial malignancies suggests that EBV might establish a latent infection within primary epithelial cells. In biopsies of oral hairy leukoplakia (OHL), a benign hyperplastic lesion that occurs in immunocompromised individuals, active EBV replication is detected in suprabasal layers of lingual epithelium (6), providing convincing evidence that EBV can infect epithelial cells and undergo productive replication. Similar patterns could be found in biopsies of normal tongue tissue, but in only a small fraction of samples (3 of 217) (7). In contrast, little evidence of latently infected cells is observed, although such cells might be rarer and more difficult to detect. Indeed, possible latently infected epithelial cells are detected in tonsil explants in the presence of acyclovir (an inhibitor of lytic cycle-specific herpesvirus DNA replication), but in less than 0.01% of cells (8).Unfortunately, EBV-infected cell lines cannot be established readily from epithelial tumors, and primary epithelial cells are infected inefficiently in vitro. Nevertheless, EBV infection of primary keratinocytes in monolayer culture results in latency, as evidenced by the expression of Epstein–Barr virus RNA (EBER), with variable expression of EBNA-1 and LMP-1, but the infected cells fail to proliferate (8–10). Although these problems have hindered progress in understanding the life cycle of EBV in epithelial cells, a few EBV-negative epithelial tumor cell lines can be infected with EBV, resulting in Latency II gene expression, and have provided some insight. One notable example is EBV tropism. Although EBV must cross the oral mucosa to gain access to either the B-cell compartment or the oral cavity, whether it infects oral mucosal epithelial cells (with or without a latent infection) to amplify the virus pool or merely crosses the epithelial barrier by transcytosis has been debated (11, 12). However, tropism is controlled during in vitro infection by differences in the glycoprotein profile of virions derived from B versus epithelial cells (13), and virus isolated from the saliva of normal individuals is consistent with that derived from epithelial cells (14). Taken together, these observations suggest that EBV indeed can replicate within the epithelial cell compartment in vivo.To assess better the outcome(s) of EBV infection in an environment more representative of normal epithelium in the host, we examined EBV infection within organotypic (raft) cultures of primary oral keratinocytes. Although these cultures do not contain the complexity of tissues and interactions found in vivo, in all other respects the stratified layers of differentiated tissue generated in vitro resemble the differentiated epithelium, where EBV has been detected in vivo. We demonstrate that EBV is able to infect keratinocytes in raft cultures, and, although we were unable to detect latently infected cells, we readily observed production of virus in the suprabasal layers that was readily disseminated throughout the epithelium, resulting in numerous productively replicating cells. Thus, EBV is able to infect normal epithelial cells and enter the productive cycle to expand the virus pool.     

From the Cover: Antiparallel coiled-coil–mediated dimerization of myosin X

Processive movements of unconventional myosins on actin filaments generally require motor dimerization. A commonly accepted myosin dimerization mechanism is via formation of a parallel coiled-coil dimer by a stretch of amino acid residues immediately carboxyl-terminal to the motor’s lever-arm domain. Here, we discover that the predicted coiled-coil region of myosin X forms a highly stable, antiparallel coiled-coil dimer (anti-CC). Disruption of the anti-CC either by single-point mutations or by replacement of the anti-CC with a parallel coiled coil with a similar length compromised the filopodial induction activity of myosin X. We further show that the anti-CC and the single α-helical domain of myosin X are connected by a semirigid helical linker. The anti-CC–mediated dimerization may enable myosin X to walk on both single and bundled actin filaments.     

From the Cover: Male tolerance and male–male bonds in a multilevel primate society

Male relationships in most species of mammals generally are characterized by intense intrasexual competition, with little bonding among unrelated individuals. In contrast, human societies are characterized by high levels of cooperation and strong bonds among both related and unrelated males. The emergence of cooperative male–male relationships has been linked to the multilevel structure of traditional human societies. Based on an analysis of the patterns of spatial and social interaction in combination with genetic relatedness data of wild Guinea baboons (Papio papio), we show that this species exhibits a multilevel social organization in which males maintain strong bonds and are highly tolerant of each other. Several “units” of males with their associated females form “parties,” which team up as “gangs.” Several gangs of the same “community” use the same home range. Males formed strong bonds predominantly within parties; however, these bonds were not correlated with genetic relatedness. Agonistic interactions were relatively rare and were restricted to a few dyads. Although the social organization of Guinea baboons resembles that of hamadryas baboons, we found stronger male–male affiliation and more elaborate greeting rituals among male Guinea baboons and less aggression toward females. Thus, the social relationships of male Guinea baboons differ markedly from those of other members of the genus, adding valuable comparative data to test hypotheses regarding social evolution. We suggest that this species constitutes an intriguing model to study the predictors and fitness benefits of male bonds, thus contributing to a better understanding of the evolution of this important facet of human social behavior.Traditional human societies typically consist of stable communities comprising several conjugal family groups (1). Sexual relationships are predominantly monogamous, and individuals of both sexes may disperse from their family groups or stay, resulting in coresidence of both brothers and sisters (2). Strikingly, men from different family groups may form long-term alliances within the community, resulting in cooperative relationships among individuals who often are not genetic relatives (3). The advent of such exceptional cooperative relationships within human societies has been linked to their multilevel organization (4). However, what are the evolutionary dynamics that give rise to multilevel systems in the first place, and how do social organization and cooperative tendencies stabilize each other?Evolutionary game theory has been used to model the conditions that favor cooperation among unrelated individuals (5, 6). Such analyses reveal that the evolutionary dynamics have a strong spatial component, in which cooperators prevail against defectors by forming clusters within the social network (7). This theoretical insight is bolstered by empirical studies of the Hadza, a population of hunter-gatherers in Tanzania. In this study, cooperators were found to cluster in physical space, i.e., in the same camp (8). Assortative processes thus may stabilize cooperation, and vice versa.Further empirical evidence to explain key facets of human social evolution comes from comparative studies of nonhuman primates (9–11) and other mammalian species as well. Among mammals, cooperative relationships among unrelated individuals are considered generally rare (12), particularly among males, who—according to sexual selection theory—are predicted to compete with other males over access to females. However, there are notable exceptions, including cooperative hunting and territorial patrols by chimpanzees (Pan troglodytes) (13), joint foraging by male coastal river otters (Lontra canadensis) (14), and the defense of females by male dolphins, Tursiops spp. (15). Such instances of cooperative behavior among unrelated animals can be explained by mutualism, whereby individuals benefit immediately from cooperating, and by reciprocity, whereby one individual experiences a short-term cost by cooperating but obtains a future benefit greater than the initial investment (16, 17).We here show that Guinea baboons (Papio papio) live in a multilevel society with extensive cooperation among unrelated males. Until now, comparatively little attention had been paid to this species, and its social organization was disputed. Although some previous studies, mainly from captivity or short field stints, suggested that Guinea baboon groups, like groups of hamadryas baboons (Papio hamadryas) (18), are comprised of one-male units that aggregate into larger parties (19, 20), other studies suggested a multimale/multifemale organization comparable to that of savanna baboons (21) or one that differs from both the savanna and the hamadryas baboon types (22, 23). However, quantitative data from observations of individually identified animals in the wild were lacking.We used ranging data collected from animals equipped with Global Positioning System (GPS) collars, proximity, and behavioral measures recorded during focal observations from individually identified males, as well as population genetic analyses based on microsatellites to describe the association patterns and individual interactions of Guinea baboons in space and time. Our aim was to contribute to a deeper understanding of the link between social organization and the formation of male bonds and to provide critical empirical evidence for modeling the processes that gave rise to some of the hallmarks in human evolution.     

From the Cover: Solid-to-fluid–like DNA transition in viruses facilitates infection

Releasing the packaged viral DNA into the host cell is an essential process to initiate viral infection. In many double-stranded DNA bacterial viruses and herpesviruses, the tightly packaged genome is hexagonally ordered and stressed in the protein shell, called the capsid. DNA condensed in this state inside viral capsids has been shown to be trapped in a glassy state, with restricted molecular motion in vitro. This limited intracapsid DNA mobility is caused by the sliding friction between closely packaged DNA strands, as a result of the repulsive interactions between the negative charges on the DNA helices. It had been unclear how this rigid crystalline structure of the viral genome rapidly ejects from the capsid, reaching rates of 60,000 bp/s. Through a combination of single-molecule and bulk techniques, we determined how the structure and energy of the encapsidated DNA in phage λ regulates the mobility required for its ejection. Our data show that packaged λ-DNA undergoes a solid-to-fluid–like disordering transition as a function of temperature, resulting locally in less densely packed DNA, reducing DNA–DNA repulsions. This process leads to a significant increase in genome mobility or fluidity, which facilitates genome release at temperatures close to that of viral infection (37 °C), suggesting a remarkable physical adaptation of bacterial viruses to the environment of Escherichia coli cells in a human host.Nucleic acids constitute one of the main components of many viruses by weight. The viral genome is packed tightly into a small volume within a protein shell called the capsid. This is true for most prokaryotic viruses, such as double-stranded DNA (dsDNA) viruses (1–5), as well as many eukaryotic viruses [e.g., herpesviruses (6) and reoviruses (7)]. The length of the ds-genome in these viruses is several hundred times longer than the diameter of the capsid. This tight packaging leads to genome bending stress and strong repulsive interactions, resulting in internal capsid pressures reaching tens of atmospheres. The extreme efficiency of viral replication is associated with a rapid transfer of the genome from the capsid to the host cell. This pressure-driven genome ejection occurs through a single portal opening in the capsid with a cross-section of a few nanometers (8), allowing the passage of one dsDNA chain at a time. The energy and structure of the confined viral genome are closely related and determine the rate of major viral replication steps, such as genome ejection and packaging (9–13). We have shown in vivo that the internal DNA pressure will affect the probability of infecting the cell (11), whereas the rate of packaging is the limiting step for viral assembly (14).Although DNA is always condensed inside the cell (15), it is not condensed to the same extent as inside a viral capsid. Other than in sperm nuclei, in vivo packaging densities range from ∼5–10% by volume (16, 17). DNA confined in viral capsids, on the other hand, is at the extreme end of the packaging scale, where it is confined to 55% by volume, forming a hexagonally ordered structure (2, 3, 10). At only a few angstroms of DNA–DNA surface separation [e.g., 7 Å surface separation in the wild-type (WT) DNA length of 48,500 bp packaged in phage λ] (10), hexagonally ordered DNA has been shown to have very restricted mobility (16, 18). It has been proposed that so-called Coulomb sliding friction between neighboring DNA helices plays a significant role in DNA mobility at high packing densities in the viral capsids (19, 20). Indeed, recently it was shown that interhelical sliding friction leads to a kinetically trapped, glassy DNA state inside the capsid. This high-friction genome state was found to significantly affect the rates of DNA packaging in vitro (21). This occurs from dragging closely packed, negatively charged DNA helices past other helices. Despite decades of investigations of the encapsidated genome structure and its energetics (3, 22), it is not known what provides the required mobility to the hexagonally ordered viral DNA during the initiation of its ultrafast ejection, reaching 60,000 bp/s (9). In this work, we provide an answer to this fundamentally important question.The well-known concept of viral metastability often refers to the viral capsid that must be sufficiently stable to protect the viral genome, and unstable enough to release its genome into the cell (23). In this work, using bacteriophage λ as a model system, we discovered a novel concept of viral metastability attributed to the viral genome. The energetics, structure, and mobility of the encapsidated DNA are studied as a function of temperature, a parameter that rarely is varied in biophysical measurements on viruses but is pertinent to viral replication and survival. This study revealed a remarkable structural transition of dsDNA in phage λ capsids, close to the ideal temperature for infection, i.e., 37 °C. Because phage λ infects Escherichia coli that originate in the human gut, the human body temperature makes phage DNA fluid-like and thus optimized for rapid release into bacterial cells. At the same time, at lower temperatures outside the host, DNA inside the capsid is more restricted or more solid-like when the conditions are less favorable for infection, which helps prevent spontaneous genome release.     

From the Cover: Generation and confirmation of a (100 × 100)-dimensional entangled quantum system

Entangled quantum systems have properties that have fundamentally overthrown the classical worldview. Increasing the complexity of entangled states by expanding their dimensionality allows the implementation of novel fundamental tests of nature, and moreover also enables genuinely new protocols for quantum information processing. Here we present the creation of a (100 × 100)-dimensional entangled quantum system, using spatial modes of photons. For its verification we develop a novel nonlinear criterion which infers entanglement dimensionality of a global state by using only information about its subspace correlations. This allows very practical experimental implementation as well as highly efficient extraction of entanglement dimensionality information. Applications in quantum cryptography and other protocols are very promising.Quantum entanglement of distant particles leads to correlations that cannot be explained in a local realistic way (1–3). To obtain a deeper understanding of entanglement itself, as well as its application in various quantum information tasks, increasing the complexity of entangled systems is important. Essentially, this can be done in two ways. The first method is to increase the number of particles involved in the entanglement (4). The alternative method is to increase the entanglement dimensionality of a system.Here we focus on the latter one, namely on the dimension of the entanglement. The text is structured as follows. After a short review of properties and previous experiments, we present a unique method to verify high-dimensional entanglement. Then we show how we experimentally create our high-dimensional two-photon entangled state. We analyze this state with our method and verify a 100 × 100-dimensional entangled quantum system. We conclude with a short outlook to potential future investigations.High-dimensional entanglement provides a higher information density than conventional two-dimensional (qubit) entangled states, which has important advantages in quantum communication. First, it can be used to increase the channel capacity via superdense coding (5). Second, high-dimensional entanglement enables the implementation of quantum communication tasks in regimes where mere qubit entanglement does not suffice. This involves situations with a high level of noise from the environment (6, 7), or quantum cryptographic systems where an eavesdropper has manipulated the random number generator involved (8). Moreover, the entangled dimensions of the whole Hilbert space also play a very interesting role in quantum computation: high-dimensional systems can be used to simplify the implementation of quantum logic (9). Furthermore, it has been found recently (10) that any continuous measure of entanglement (such as concurrence, entanglement of formation, or negativity) can be very small, while the quantum system still permits an exponential computation speedup over classical machines. This is not the case for the dimension of entanglement—for every quantum computation, it needs to be high (11, 12), which is another hint at the fundamental relevance of the concept.So far, high-dimensional entanglement has been implemented only in photonic systems. There, different multilevel degrees of freedom, such as spatial modes (13), time-energy (14), path (15, 16), as well as continuous variables (17, 18), have been used. Entanglement of spatial modes of photons has especially attracted much attention in recent years (19–28), because it is readily available from optical nonlinear crystals and the number of involved modes of the entanglement can be very high (29).In a recent experiment the nonseparability of a two-photon state was shown, by observing Einstein–Podolsky–Rosen correlations of photon pairs in down-conversion (30) (for a similar experiment, see ref. 31). The authors were able to observe entanglement of ∼2,500 spatial states with a camera. In our experiment we go a step further and not only show nonseparability, but we can also extract information about the dimensionality of the entanglement. Precisely, we experimentally verify 100-dimensional entanglement.One main challenge that remains is the detection and verification of high-dimensional entanglement. For reconstructing the full quantum state via state tomography, the number of required measurements is impractical even for relatively low dimensions because it scales quadratically with the quantum system dimension (24, 27). Even if one had reconstructed the full quantum state, the quantification of the entangled dimensions is a daunting task analytically and even numerically (32). If the full density matrix of the state is not known, it is only possible to give lower bounds of the entangled dimensions. Such methods are usually referred to as a “Schmidt number witness” (33–35).     

From the Cover: Dynamics of a morbillivirus at the domestic–wildlife interface: Canine distemper virus in domestic dogs and lions

Morbilliviruses cause many diseases of medical and veterinary importance, and although some (e.g., measles and rinderpest) have been controlled successfully, others, such as canine distemper virus (CDV), are a growing concern. A propensity for host-switching has resulted in CDV emergence in new species, including endangered wildlife, posing challenges for controlling disease in multispecies communities. CDV is typically associated with domestic dogs, but little is known about its maintenance and transmission in species-rich areas or about the potential role of domestic dog vaccination as a means of reducing disease threats to wildlife. We address these questions by analyzing a long-term serological dataset of CDV in lions and domestic dogs from Tanzania’s Serengeti ecosystem. Using a Bayesian state–space model, we show that dynamics of CDV have changed considerably over the past three decades. Initially, peaks of CDV infection in dogs preceded those in lions, suggesting that spill-over from dogs was the main driver of infection in wildlife. However, despite dog-to-lion transmission dominating cross-species transmission models, infection peaks in lions became more frequent and asynchronous from those in dogs, suggesting that other wildlife species may play a role in a potentially complex maintenance community. Widespread mass vaccination of domestic dogs reduced the probability of infection in dogs and the size of outbreaks but did not prevent transmission to or peaks of infection in lions. This study demonstrates the complexity of CDV dynamics in natural ecosystems and the value of long-term, large-scale datasets for investigating transmission patterns and evaluating disease control strategies.The genus Morbillivirus includes highly contagious, and often fatal, RNA viruses that cause diseases of great public health, economic, and conservation concern, such as measles, rinderpest, and canine distemper. Canine distemper virus (CDV) is distributed worldwide and affects an expanding range of host species, including domestic and wild canids (1, 2), marine mammals (3), felids (2, 4, 5), procyonids and ursids (6), and nonhuman primates (7–9). The propensity of CDV for host-switching has raised concerns about both potential risks for humans (10) and extinction threats to endangered wildlife (11–13).Although previously thought to be nonpathogenic in cats, outbreaks among large captive felids in the 1990s drew attention to CDV as a potential conservation threat to felids (2). The best-studied example of CDV infection in free-ranging felids comes from Tanzania’s Serengeti ecosystem (Fig. 1A), where a CDV epidemic in 1994 killed ∼30% of lions (Panthera leo) and affected several other carnivore species within the Serengeti National Park (SNP) (14). The close similarity of viruses recovered from wild carnivores and domestic dogs (Canis lupus familiaris) (15) indicated that a single dog variant was responsible for the die-off in lions. Prolonged viral circulation during 1992–1994 was documented in higher-density dog populations adjacent to the northwestern boundaries of the SNP with more sporadic exposure detected in lower-density dog populations to the east (exposed in 1991 and 1994 but not in 1992–1993). The high-density dog populations therefore were considered the most likely source of infection for wildlife (Fig. 1A) (16).Open in a separate windowFig. 1.Map of the Serengeti ecosystem (Tanzania). Circles represent human settlements (gray) surrounding the Serengeti National Park, villages/households from which domestic dogs were sampled (dark blue), locations where lions were sampled (black), and villages included in domestic dog vaccination campaigns that were not sampled (pale blue). (A) Arrows indicate the direction of the spread of CDV during the 1994 epidemic as reconstructed by Cleaveland et al. (16). (B) Small-scale domestic dog vaccination campaigns conducted during 1996–2002. (C) Expanded domestic dog vaccination program implemented during 2003–2012.Similar to other morbilliviruses [e.g., measles virus (17–19)], the acute, highly immunizing nature of CDV infection suggests that large populations of susceptible hosts are required for persistence. In smaller populations, more or less regular epidemics are typically followed by “fade-outs” during which infection disappears until reintroduced from outside (20). However, many questions remain regarding population size thresholds and determinants of CDV persistence in natural ecosystems comprising a wide range of susceptible hosts.In the Serengeti ecosystem, earlier studies indicate that CDV was unlikely to have been maintained by lion populations or other wildlife (e.g., combined lion, hyena, and jackal populations) in the SNP before the 1994 epidemic (21, 22), further implicating higher-density domestic dogs as a more likely reservoir of infection (16). However, observations from other ecosystems do not support this hypothesis. In northern Kenya, for example, domestic dog populations adjacent to wildlife protected areas show patterns of exposure consistent with reoccurring outbreaks rather than persistent infection (23), suggesting that the dog population is insufficiently large to maintain CDV and that infection needs to be reintroduced from outside sources (e.g., other domestic dog or wildlife communities). In other large, protected areas such as the Yellowstone National Park, the periodic nature of CDV occurrence in wild carnivore communities and the small size of the dog population around the park suggest disease persistence in the wild populations themselves (24).Knowledge of the mechanisms of long-term CDV maintenance is essential to optimize disease management (25). Mass vaccination of domestic dogs has been proposed as a strategy for protecting endangered wildlife in many areas (26, 27), but concerns arise over its cost-effectiveness as a conservation tool within a potentially complex system, especially when the contribution of dogs to disease maintenance is uncertain (28). The implementation of mass dog vaccination programs in the Serengeti ecosystem since 1996, although driven largely by the need to control rabies (29), provides an opportunity to evaluate the impact of dog vaccination on CDV infection in both dog and wildlife populations.In this study we analyze the most comprehensive available multihost dataset on CDV in Africa, including domestic dog and lion serology data from the Serengeti ecosystem spanning almost three decades as well as data from mass dog vaccination interventions. These data are analyzed using a Bayesian state–space modeling approach to examine long-term patterns of infection in a large, multihost ecosystem. We first determine the role of domestic dogs and lions in maintaining CDV by assessing the within- and between-species dynamics and subsequently investigate the impact of small- (Fig. 1B) and large-scale (Fig. 1C) vaccination programs on infection dynamics.     

From the Cover: Dynamics and mechanism of ultrafast water–protein interactions

Protein hydration is essential to its structure, dynamics, and function, but water–protein interactions have not been directly observed in real time at physiological temperature to our awareness. By using a tryptophan scan with femtosecond spectroscopy, we simultaneously measured the hydration water dynamics and protein side-chain motions with temperature dependence. We observed the heterogeneous hydration dynamics around the global protein surface with two types of coupled motions, collective water/side-chain reorientation in a few picoseconds and cooperative water/side-chain restructuring in tens of picoseconds. The ultrafast dynamics in hundreds of femtoseconds is from the outer-layer, bulk-type mobile water molecules in the hydration shell. We also found that the hydration water dynamics are always faster than protein side-chain relaxations but with the same energy barriers, indicating hydration shell fluctuations driving protein side-chain motions on the picosecond time scales and thus elucidating their ultimate relationship.Water–protein interactions are critical to protein structural stability and flexibility, functional dynamics, and biological activities (1, 2). Various methods such as neutron scattering (3), NMR (4), laser spectroscopy (5, 6), and molecular dynamics (MD) simulations (7) have been used to reveal protein surface hydration and coupled water–protein dynamics on different time and length scales. Hydration water molecules often participate in various protein functions and their motions even directly “control” protein fluctuations (2, 8). Frauenfelder et al. recently proposed a unified model for protein dynamics (8): large-scale protein motions are slaved to the fluctuations of bulk solvent and controlled by solvent viscosity while internal protein motions are slaved to the fluctuations of the hydration shell and controlled by hydration water. However, direct measurements of such coupled fluctuations at physiological temperature are challenging as a result of the ultrafast nature of water motions, and therefore most studies are indirect or at low temperature (3, 4). Here, we used a tryptophan (W) scan to probe global surface hydration (9) and used femtosecond spectroscopy to follow hydration water motions and local side-chain fluctuations in real time. With temperature dependence, we systematically measured their dynamics and thus finally elucidate their ultimate relationship.     

From the Cover: Bacteriophage adhering to mucus provide a non–host-derived immunity

Mucosal surfaces are a main entry point for pathogens and the principal sites of defense against infection. Both bacteria and phage are associated with this mucus. Here we show that phage-to-bacteria ratios were increased, relative to the adjacent environment, on all mucosal surfaces sampled, ranging from cnidarians to humans. In vitro studies of tissue culture cells with and without surface mucus demonstrated that this increase in phage abundance is mucus dependent and protects the underlying epithelium from bacterial infection. Enrichment of phage in mucus occurs via binding interactions between mucin glycoproteins and Ig-like protein domains exposed on phage capsids. In particular, phage Ig-like domains bind variable glycan residues that coat the mucin glycoprotein component of mucus. Metagenomic analysis found these Ig-like proteins present in the phages sampled from many environments, particularly from locations adjacent to mucosal surfaces. Based on these observations, we present the bacteriophage adherence to mucus model that provides a ubiquitous, but non–host-derived, immunity applicable to mucosal surfaces. The model suggests that metazoan mucosal surfaces and phage coevolve to maintain phage adherence. This benefits the metazoan host by limiting mucosal bacteria, and benefits the phage through more frequent interactions with bacterial hosts. The relationships shown here suggest a symbiotic relationship between phage and metazoan hosts that provides a previously unrecognized antimicrobial defense that actively protects mucosal surfaces.     

From the Cover: Evidence of Lévy walk foraging patterns in human hunter–gatherers

When searching for food, many organisms adopt a superdiffusive, scale-free movement pattern called a Lévy walk, which is considered optimal when foraging for heterogeneously located resources with little prior knowledge of distribution patterns [Viswanathan GM, da Luz MGE, Raposo EP, Stanley HE (2011) The Physics of Foraging: An Introduction to Random Searches and Biological Encounters]. Although memory of food locations and higher cognition may limit the benefits of random walk strategies, no studies to date have fully explored search patterns in human foraging. Here, we show that human hunter–gatherers, the Hadza of northern Tanzania, perform Lévy walks in nearly one-half of all foraging bouts. Lévy walks occur when searching for a wide variety of foods from animal prey to underground tubers, suggesting that, even in the most cognitively complex forager on Earth, such patterns are essential to understanding elementary foraging mechanisms. This movement pattern may be fundamental to how humans experience and interact with the world across a wide range of ecological contexts, and it may be adaptive to food distribution patterns on the landscape, which previous studies suggested for organisms with more limited cognition. Additionally, Lévy walks may have become common early in our genus when hunting and gathering arose as a major foraging strategy, playing an important role in the evolution of human mobility.Over the last decade, researchers have applied sophisticated analytical techniques to explore the movement patterns of a wide variety of organisms from insects to mammals (1–8). Many of these taxa seem to use a similar movement pattern during foraging, where the length of move steps (distance traveled between two points marked by either a pause or a change in direction) is distributed according to a power law function with a heavy tail:, where l is move step length and μ is the power law exponent with 1 < μ ≤ 3 (1). In this distribution, termed a Lévy walk, groups of short step lengths are interspersed with longer movements between them, and this pattern is repeated across all scales (i.e., the distribution is scale-free) (9). Modeling studies have shown that this step length distribution is advantageous when searching for resources that are patchily distributed and can be profitably revisited (i.e., resources are not depleted after a given visit) (1, 10, 11). In these cases, the optimal Lévy strategy has μ ∼ 2, because the rare long steps minimize oversampling a given patch and take organisms to new food patches without requiring memory or high levels of cognition (1, 10, 11).When similar analytical techniques are applied to human movements, researchers have found some support for Lévy walks in our own species (12–15). In most cases, patterns found in urban-dwelling humans are attributed to the requirements of life (work, shopping, etc.) in a human-designed landscape (15) rather than an evolved search strategy as suggested for other organisms (1, 16). One previous study found evidence of Lévy walks in Ju/’hoansi hunter–gatherers of Botswana and Namibia, suggesting that random walk searches may be advantageous to humans living more traditional lifestyles (13, 17). However, this study examined the distribution of distances between residential camp locations [which are largely tied to the locations of permanent waterholes (18)] rather than the steps taken during actual foraging bouts (13). Thus, we are left with the question of whether Lévy walk patterns occur in cognitively complex foragers.In this study, we examined individual movement patterns among Hadza hunter–gatherers of northern Tanzania to determine whether the most cognitively complex foragers on Earth perform Lévy walks while foraging. The Hadza hunter–gatherers who we worked with adhered to a traditional hunting and gathering lifestyle—foraging for wild plant foods and game on foot with simple tools (bow and arrow, digging sticks, and axes) and without any modern technologies or agriculture (19). We recruited 44 Hadza subjects from two camps to wear global positioning system (GPS) units during foraging bouts (Table S1). Individuals wore GPS units on multiple days, and we collected data from camps during different seasons (SI Methods). We define a foraging bout as a round trip taken by a subject from and back to his or her residential camp. In addition to full foraging bouts, we examined the outbound leg of foraging bouts separately (from camp to the farthest point from camp in a given bout). We analyzed individual subject’s movement data from the longest foraging bout for each day separately (n = 342 total bouts) (analyses of all bouts for all individuals are in SI Methods). Step lengths are defined as the distance traveled between two points followed by either a pause or a change in direction (defined by a turning angle). The definition for a change in direction is generally arbitrary among various studies (15). Here, we ran our full dataset (Dataset S1) through multiple analyses, where the definition of directional change was altered in 10° increments from 0° to 180° (SI Methods).For each foraging bout and all step length definitions, we used maximum likelihood methods to fit six models (power law, truncated power law, exponential function, and three composite exponential models) to our data, and we used the Akaike Information Criterion (AIC) for model selection (20). Because humans and other animals are limited by physiology and time of day to some maximum step length, the truncated power law is generally thought to better represent movement patterns in nature (3) given by the following probability density function: f(l) = (μ − 1)(a^{1 − μ} − b^{1 − μ})⁻¹l^−μ, where a and b are the minimum and maximum values of l, respectively, for which a distribution is valid (21). In practice, these values are the minimum and maximum step lengths observed in a dataset. We also tested an exponential model to represent Brownian motion, a classic alternative movement strategy to Lévy walks (20): f(l) = . Finally, we tested three composite exponential models (composite Brownian walks) using information in the work by Jansen et al. (22): , where k is the number of exponential models mixed and p_j is the proportional contribution of the jth model to the overall distribution of steps. We tested models that included two, three, and four exponential functions. We performed Kolmogorov–Smirnov tests to determine the significance of model fits chosen by AIC (23). This method, which directly compares fits of power law models with the alternative exponential models, is considered the most statistically robust for determining the presence of a power law in movement data (20, 23).     

From the Cover: Genetic erosion in maize’s center of origin

Crop genetic diversity is an indispensable resource for farmers and professional breeders responding to changing climate, pests, and diseases. Anecdotal appraisals in centers of crop origin have suggested serious threats to this diversity for over half a century. However, a nationwide inventory recently found all maize races previously described for Mexico, including some formerly considered nearly extinct. A flurry of social studies seems to confirm that farmers maintain considerable diversity. Here, we compare estimates of maize diversity from case studies over the past 15 y with nationally and regionally representative matched longitudinal data from farmers across rural Mexico. Our findings reveal an increasing bias in inferences based on case study results and widespread loss of diversity. Cross-sectional, case study data suggest that farm-level richness has increased by 0.04 y⁻¹ nationwide; however, direct estimates using matched longitudinal data reveal that richness dropped −0.04 y⁻¹ between 2002 and 2007, from 1.43 to 1.22 varieties per farm. Varietal losses occurred across regions and altitudinal zones, and regardless of farm turnover within the sector. Extinction of local maize populations may not have resulted in an immediate loss of alleles, but low varietal richness and changes in maize’s metapopulation dynamics may prevent farmers from accessing germplasm suitable to a rapidly changing climate. Declining yields could then lead farmers to leave the sector and result in a further loss of diversity. Similarities in research approaches across crops suggest that methodological biases could conceal a loss of diversity at other centers of crop origin.A decade ago, crop scientists considered maize (Zea mays L.) diversity in danger across wide areas of Mexico, its center of origin and diversity: seven races at risk for extinction and many others under threat (1, 2). However, those appraisals, like others before them (3, 4), were anecdotal. In 2011, the Global Project on Native Maize—a 3-y effort involving 55 institutions and 138 researchers—reported encouraging findings from its first nationwide inventory: all 59 races previously described for Mexico were recorded, including those formerly considered nearly extinct (5). Scientists also found unexpectedly high diversity of races endemic to northern Mexico, several maize types in Michoacán that could represent new races, and new records for some locations (including Vandeño in Sonora and four Guatemalan races). Contrary to previous appraisals, only two races (Palomero Toluqueño and Chapalote) were listed under threat based on small population sizes. Leading experts remain cautious nevertheless (6).Crop genetic erosion has been a constant concern since the late 1940s, when conservation efforts began in earnest, but it has never been demonstrated by longitudinal data across environments for any major crop in its center of diversity (3, 4, 7–10). Inconsistencies in the classification of infraspecific diversity have been a serious hurdle. Utilitarian rather than natural, taxonomies reflect large disciplinary biases: crop scientists favor racial groupings, whereas social scientists prefer folk taxonomies (1–6, 9–14). Phenotypic variation across races is indeed remarkable (1, 2, 5, 6, 15, 16), yet racial groups account for only 2–3% of genetic variation in maize (6). Moreover, races are not discrete entities (13–15). Farmers recognize, value, and maintain unique traits in innumerable racial variants and mixtures—known as farmer varieties or landraces—exerting an influence on maize’s genetic structure (12–14, 17). Seed exchange presumably explains why 91% of isoenzymatic variation in maize landraces occurs within populations, whereas individual teosinte (wild Z. mays) populations remain genetically distinct (6, 18). The low genetic diversity of some accessions also has been attributed to human factors—i.e., small field sizes (or few ears used for seed) for specialty varieties (1). In fact, most maize alleles are very rare (frequencies <0.01), and many are found in single accessions that presumably correspond with farmer fields (1). Rather than segregated into discrete races, maize diversity may be spread continuously across thousands of populations (i.e., fields) in rural Mexico (13, 14, 17, 18). Accounting for maize’s metapopulation structure is difficult because of farmers’ extensive control of crop population dynamics (11, 13). Although maize demography can be modeled on management practices, the data required remain critically scarce (11, 19). Numerous statistics have been reported, but only average varietal richness per farm is estimated consistently across studies. This is considered the key statistic for diversity conservation in crops (10).We compare farm richness estimates based on cross-sectional case study data and longitudinal survey data from a representative sample of rural farms to assess the state of maize conservation in Mexico. Our findings reveal significant changes in maize diversity between 2002 and 2007 that are not evident in case study data. This represents the first (to our knowledge) formal assessment of genetic erosion in a center of crop diversity. A social perspective on maize diversity allows us to explore possible reasons for recent changes and their potential implications.     

From the Cover: In vitro and in vivo reconstitution of the cadherin–catenin–actin complex from Caenorhabditis elegans

The ternary complex of cadherin, β-catenin, and α-catenin regulates actin-dependent cell–cell adhesion. α-Catenin can bind β-catenin and F-actin, but in mammals α-catenin either binds β-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of α-catenin is evolutionarily conserved. The Caenorhabditis elegans α-catenin homolog HMP-1 is essential for actin-dependent epidermal enclosure and embryo elongation. Here we show that HMP-1 is a monomer with a functional C-terminal F-actin binding domain. However, neither full-length HMP-1 nor a ternary complex of HMP-1–HMP-2(β-catenin)–HMR-1(cadherin) bind F-actin in vitro, suggesting that HMP-1 is auto-inhibited. Truncation of either the F-actin or HMP-2 binding domain of HMP-1 disrupts C. elegans development, indicating that HMP-1 must be able to bind F-actin and HMP-2 to function in vivo. Our study defines evolutionarily conserved properties of α-catenin and suggests that multiple mechanisms regulate α-catenin binding to F-actin.     

From the Cover: Self-organization of axial polarity,inside-out layer pattern,and species-specific progenitor dynamics in human ES cell–derived neocortex

Here, using further optimized 3D culture that allows highly selective induction and long-term growth of human ES cell (hESC)-derived cortical neuroepithelium, we demonstrate unique aspects of self-organization in human neocorticogenesis. Self-organized cortical tissue spontaneously forms a polarity along the dorsocaudal-ventrorostral axis and undergoes region-specific rolling morphogenesis that generates a semispherical structure. The neuroepithelium self-forms a multilayered structure including three neuronal zones (subplate, cortical plate, and Cajal-Retzius cell zones) and three progenitor zones (ventricular, subventricular, and intermediate zones) in the same apical-basal order as seen in the human fetal cortex in the early second trimester. In the cortical plate, late-born neurons tend to localize more basally to early-born neurons, consistent with the inside-out pattern seen in vivo. Furthermore, the outer subventricular zone contains basal progenitors that share characteristics with outer radial glia abundantly found in the human, but not mouse, fetal brain. Thus, human neocorticogenesis involves intrinsic programs that enable the emergence of complex neocortical features.The mammalian neocortex has a multilayered structure (layers I–VI) (1). The neocortex arises from the neuroepithelium (NE) of the dorsal telencephalon, which evaginates to form a semispherical brain vesicle (Fig. S1A) (2). The dorsocaudal side of the neocortex is flanked by the cortical hem, whereas its ventrorostral side is neighbored by the lateral ganglionic eminence (LGE; striatum anlage) and septum. Layer I [its fetal primordium is called the marginal zone (MZ); Fig. S1B] is qualitatively different from other layers, as this superficial-most layer is mainly composed of Reelin⁺ Cajal-Retzius (CR) cells, which are largely derived from neighboring tissues such as the cortical hem and septum (3) (in the case of human cortex, some Reelin⁺ cells also arise directly from neocortical NE) (4). The rest of the cortical layers are generated with the “inside-out” pattern: the deeper the layer, the earlier the neurons are born from cortical progenitors (Fig. S1B) (5, 6).A detailed understanding of early human corticogenesis remains elusive because of the limited access to fetal brain tissues. We previously established a 3D culture of mouse and human ES cell (hESC) aggregates that recapitulates early steps of corticogenesis [or serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq)] (7–9). This method has been also applied to human induced pluripotent stem (iPS) cell culture (10). In this self-organization culture, large domains of cortical NE self-form within a floating hESC aggregate and spontaneously develop ventricular zone (VZ), cortical plate (CP) (mostly deep-layer neurons), and MZ by culture day 40–45. This cortical NE was still immature, mimicking human corticogenesis during the first trimester (Fig. S1C) (7).Here, using an optimized culture, we revealed unique self-organizing aspects of human corticogenesis. Moreover, the optimized culture generates species-specific progenitors in the outer subventricular zone (oSVZ), called outer radial glia (oRG), which are abundantly present in the human neocortex (11, 12) but rare in the mouse cortex (13, 14). Thus, an unexpectedly wide range of self-organizing events is internally programmed within the cortical NE.