Robust folding of a de novo designed ideal protein even with most of the core mutated to valine

Protein design provides a stringent test for our understanding of protein folding. We previously described principles for designing ideal protein structures stabilized by consistent local and nonlocal interactions, based on a set of rules relating local backbone structures to tertiary packing motifs. The principles have made possible the design of protein structures having various topologies with high thermal stability. Whereas nonlocal interactions such as tight hydrophobic core packing have traditionally been considered to be crucial for protein folding and stability, the rules proposed by our previous studies suggest the importance of local backbone structures to protein folding. In this study, we investigated the robustness of folding of de novo designed proteins to the reduction of the hydrophobic core, by extensive mutation of large hydrophobic residues (Leu, Ile) to smaller ones (Val) for one of the designs. Surprisingly, even after 10 Leu and Ile residues were mutated to Val, this mutant with the core mostly filled with Val was found to not be in a molten globule state and fold into the same backbone structure as the original design, with high stability. These results indicate the importance of local backbone structures to the folding ability and high thermal stability of designed proteins and suggest a method for engineering thermally stabilized natural proteins.

The de novo design of protein structures, starting from pioneering work (1, 2), has been achieved in tandem with our understanding of how amino acid sequences determine folded structures (3–16). A breakthrough in protein design methodology was a finding of principles for encoding funnel-shaped energy landscapes into amino acid sequences (7, 10, 17, 18). Based on studies of protein folding, it had been suggested that naturally occurring proteins have evolved to have funnel-shaped energy landscapes toward their folded structures (19–23). However, complicated structures of naturally occurring proteins with nonideal features for folding—for example, kinked α-helices, bulged β-strands, long or strained loops, and buried polar groups—make it difficult to understand how the funnels are encoded in amino acid sequences. By focusing on protein structures without such nonideal features, we proposed principles for designing ideal protein structures stabilized by completely consistent local and nonlocal interactions (24), based on a set of rules relating local backbone structures to preferred tertiary motifs (7, 10). These design rules describe the relation of the lengths or torsion patterns of two secondary structure elements and the connecting loop to favorable packing geometries (SI Appendix, Fig. S1A). The design principles enable to encode strongly funneled energy landscapes into amino acid sequences, by the stabilization of folded structures (positive design) and by the destabilization of nonnative conformations (negative design) due to the restriction of folding conformational space by the rules (SI Appendix, Fig. S1C). In the design procedure, backbone structures for a target topology are generated based on a blueprint (SI Appendix, Fig. S1B), in which either the lengths or backbone torsion patterns of the secondary structures and loops are determined using the rules so that the tertiary motifs present in the target topology are favored, and then amino acid sequences stabilizing the generated backbone structures are designed. The designed amino acid sequences stabilize their folded structures both with nonlocal interactions such as hydrophobic core packing and with local interactions favoring the secondary structures and loops specified in the blueprint, which destabilize a myriad of nonnative topologies through local backbone strain captured by the rules, thereby resulting in funnel-shaped energy landscapes (SI Appendix, Fig. S1C). The principles have enabled the de novo design of ideal protein structures for various topologies with atomic-level accuracy (Fig. 1) (6, 7, 10, 13).Open in a separate windowFig. 1.In silico energy landscapes and far-UV circular dichroism (CD) spectra for 10 de novo designed ideal proteins. (A–E) Five designs by Koga et al. in 2012 (7). (F–I) Four designs by Lin et al. in 2015 (10). (J) Top7 by Kuhlman et al. in 2003 (6). (Top) Design models. (Middle) Energy landscapes obtained from Rosetta ab initio structure prediction simulations (41). Red points represent the lowest energy structures obtained in independent Monte Carlo structure prediction trajectories starting from an extended chain for each sequence; the y axis is the Rosetta all-atom energy; the x axis is the Cα root-mean-square deviation (RMSD) to the design model. Green points represent the lowest energy structures obtained in trajectories starting from the design model. (Bottom) The far-UV CD spectra during thermal denaturation with the melting temperature T_m, which is obtained by fitting to the denaturation curves shown in SI Appendix, Fig. S2.Interestingly, the de novo designs exhibit prominent characteristics in terms of thermal stability when compared with naturally occurring proteins. The circular dichroism (CD) measurements up to 170 °C conducted in this study revealed the melting temperature (T_m), which was above 100 °C for most of the designs (Fig. 1) (6, 7, 10). Therefore, the designs have great potential for use as scaffolds to engineer proteins with specific functions of interest. Indeed, miniprotein structures (∼40 residues) designed de novo according to the rules were applied as scaffolds for creating protein binders specific for influenza hemagglutinin and botulinum neurotoxin, displaying high thermal stability (>70 °C) despite the small size (25).The rules in the principles described above emphasize the importance of local backbone structures not the details of amino acid side chains to protein folding, which is also supported by studies using simple calculations with the hydrophobic-polar lattice model or the snake-cube model (26, 27). On the other hand, it is known that hydrophobic interactions are the dominant driving force for folding (28, 29) and the cores of naturally occurring proteins are tightly packed with hydrophobic amino acid residues (30, 31) like a jigsaw puzzle. Indeed, in our design principles, protein cores were designed to be tightly packed and as “fat” as possible with larger hydrophobic residues so that energy landscapes were sculpted to be deeply funneled into a target topology by lowering its energy (SI Appendix, Fig. S1C).Which factor, the local backbone structures encoded by the rules or the tight core packing with fat hydrophobic residues, contributes more to the generation of funnels in the designs? Here, we studied the contribution of hydrophobic core packing to folding ability and thermal stability by investigating the robustness of folding against the reduction of packing, using the design with the highest thermal stability among our nine de novo designs (Fig. 1, except Top7), Rsmn2x2_5_6 (10). We started to study single-residue mutants from Leu or Ile to Val that prune one carbon atom from the aliphatic side chain, which lose the tight packing like a jigsaw puzzle and decrease the hydrophobicity, and then, we combined the mutations. Consequently, we found that a mutant with 10 residue substitutions of Leu or Ile with Val still has the folding ability and high thermal stability despite its reduced and loosened hydrophobic core packing. This result suggests the importance of the local backbone structures for the folding ability and stability of the de novo designs.     

Structure of the Dietzia Mrp complex reveals molecular mechanism of this giant bacterial sodium proton pump

Multiple resistance and pH adaptation (Mrp) complexes are sophisticated cation/proton exchangers found in a vast variety of alkaliphilic and/or halophilic microorganisms, and are critical for their survival in highly challenging environments. This family of antiporters is likely to represent the ancestor of cation pumps found in many redox-driven transporter complexes, including the complex I of the respiratory chain. Here, we present the three-dimensional structure of the Mrp complex from a Dietzia sp. strain solved at 3.0-Å resolution using the single-particle cryoelectron microscopy method. Our structure-based mutagenesis and functional analyses suggest that the substrate translocation pathways for the driving substance protons and the substrate sodium ions are separated in two modules and that symmetry-restrained conformational change underlies the functional cycle of the transporter. Our findings shed light on mechanisms of redox-driven primary active transporters, and explain how driving substances of different electric charges may drive similar transport processes.

In all life kingdoms, homeostasis of Na⁺ and H⁺ is essential for many aspects of cell physiology, including maintaining appropriate osmotic pressure, intracellular pH, and electrostatic membrane potential (ΔΨ) (1). A variety of Na⁺/H⁺ exchangers (also called antiporters) are directly involved in regulating the homeostasis of Na⁺ and H⁺, and form a major part of the superfamily of monovalent cation/proton antiporters (CPA) (2, 3). Members of the CPA superfamily have been identified and characterized in fungi, plants, and mammals (4). Usually, Na⁺/H⁺ antiporters utilize the electrochemical potential of protons, commonly called proton motive force (PMF), to catalyze efflux of Na⁺ as well as other monovalent cations. The stoichiometric ratio of protons to sodium ions may vary from transporter to transporter, primarily depending on the strength of PMF in which the transporter has been evolved to function optimally (see discussion in SI Appendix, Supplementary Material). One family of Na⁺/H⁺ antiporters, namely, the NhaA-like single-subunit antiporters, has been well studied (5, 6), and was shown to represent the canonical type of secondary active transporters, in which the proton influx and the Na⁺ efflux most likely share the same pathway. In contrast, multiple resistance and pH adaptation (Mrp) complexes are the most sophisticated known secondary active transporters. They alone form a unique family of atypical antiporters, termed CPA3 (7–10), in which protons and Na⁺ ions are likely to flow through distinct physical paths. Under physiological conditions, the driving force for Mrp transport can only come from protons but not Na⁺ ions (SI Appendix, Supplementary Material); therefore, protons are referred to as the driving substance. Moreover, Mrp complexes are essential for alkaliphilic and/or halophilic microorganisms to adapt to their extreme environments (11).A member of the Mrp family usually contains one copy of each of seven subunits called MrpA-F, all of which contribute to a total of ∼50 predicted transmembrane helices (TMs) (SI Appendix, Fig. S1). In the so-called group II Mrp complexes, subunit MrpB is fused to the C terminus of MrpA while keeping other subunits essentially unchanged, and the remaining nonfused complexes are termed group I (12). Amino acid sequence analysis showed that MrpA and MrpD are homologous (13), and their corresponding three-dimensional (3D) folding pattern is referred to as Mrp antiporter/pump folding. In addition, every subunit of the Mrp complex was shown to be important for the Na⁺/H⁺ antiporter activity (14). This notion raises an interesting question as to why a seemingly simple task requires such a complexed protein machinery.Furthermore, a number of important energy-converting protein complexes, including those from bacteria (15), plants (16), and mammalian animals (17), are phylogenetically related to the Mrp complex. For instance, components of the respiratory chain complex I (henceforth called complex I) are highly homologous to MrpA and MrpD subunits. In the membrane arm of complex I, three Mrp pump subunits are aligned in a head-to-tail fashion, and presumably carry out proton efflux, and their driving forces come from the redox reaction between NADH (nicotinamide adenine dinucleotide) and quinone (18). In some anaerobic archaea lacking complex I, the membrane-bound hydrogenase (MBH) complex replaces the functional role of complex I (19). In this type of ion-pumping complex, a subcomplex containing Mrp homologous subunits functions as a Na⁺ pump, and the driving force for this pump comes from the redox reaction between reduced ferredoxin and protons, subsequently generating hydrogen gas. In complex I, MBH complex, as well as other related redox-driven transporters, cellular redox energy (i.e., differential redox potential between electron donors and acceptors) is converted to transmembrane electrochemical potential of either protons or Na⁺; thus, these primary active transporters play essential roles in cellular energy homeostasis (20). It is generally accepted that, in these redox-driven ion pumps, electrostatic interactions between the transferred electrons and pumps drive the conformational changes that are required for the cation export (21, 22). While a number of 3D structures have been reported in recent years for such redox-driven transporters (15, 16, 23), the detailed mechanisms responsible for the energy conversion remain under debate. Taken together, the Mrp complex likely shares a common ancestor with the ion-pumping module(s) in a variety of redox-driven transporters, and thus understanding of the mechanism of Mrp antiporter complex should shed light on the energy-coupling mechanisms of all Mrp antiporter-containing transporters.Here, we report the cryoelectron microscopy (cryo-EM) structure of the group II Mrp complex from Dietzia sp. DQ12-45-1b, a strain of Gram⁺ bacteria isolated from the production water of a deep subterranean oil reservoir and which has also been found in a number of high-salinity environments (24). Our results from structural and functional analyses show that this Mrp complex contains two modules responsible for proton transport and Na⁺ pumping, respectively. On the basis of these findings, we propose a hitherto unknown mechanism for energy coupling between the two modules.     

Inhibition of 3-phosphoinositide–dependent protein kinase 1 (PDK1) can revert cellular senescence in human dermal fibroblasts

Cellular senescence is defined as a stable, persistent arrest of cell proliferation. Here, we examine whether senescent cells can lose senescence hallmarks and reenter a reversible state of cell-cycle arrest (quiescence). We constructed a molecular regulatory network of cellular senescence based on previous experimental evidence. To infer the regulatory logic of the network, we performed phosphoprotein array experiments with normal human dermal fibroblasts and used the data to optimize the regulatory relationships between molecules with an evolutionary algorithm. From ensemble analysis of network models, we identified 3-phosphoinositide–dependent protein kinase 1 (PDK1) as a promising target for inhibitors to convert the senescent state to the quiescent state. We showed that inhibition of PDK1 in senescent human dermal fibroblasts eradicates senescence hallmarks and restores entry into the cell cycle by suppressing both nuclear factor κB and mTOR signaling, resulting in restored skin regeneration capacity. Our findings provide insight into a potential therapeutic strategy to treat age-related diseases associated with the accumulation of senescent cells.

Cellular senescence is defined as a stable, persistent exit from the cell cycle in response to stresses such as telomere shortening, oxidative stress, oncogene activation, and DNA damage (1, 2). A benefit of cellular senescence is prevention of tumorigenesis by blocking proliferation of damaged cells that may undergo malignant transformation (2, 3). However, senescent cells accumulate in tissues during aging and secrete proinflammatory cytokines, which can contribute to aging and age-related diseases, including cancer (2, 3). In studies with animal models, elimination of senescent cells prevents, alleviates, or reverses symptoms of aging (4, 5) and various age-related diseases (6, 7), such as osteoarthritis and atherosclerosis.Cell-cycle arrest alone is not cellular senescence; cellular senescence requires additional signals that convert transient cell-cycle arrest into persistent exit from the cell cycle so that the cells fail to proliferate in response to growth signals, a process called geroconversion (8, 9). Terminally differentiated, nonmitotic cells can also undergo senescence; thus, cell-cycle exit is only one aspect of the senescent phenotype. Cellular senescence is a complex biological mechanism regulated by various signaling pathways (10, 11). Signaling pathways that mediate cellular senescence can be divided into three major categories. The first category includes the pathways that cause cell-cycle arrest in response to DNA damage, such as p53/p21^CIP1 and p16^INK4a/pRb pathways (11–14). The second category consists of the pathways mediating cell growth and energy metabolism, such as PI3K/AKT/mTOR and SIRT1/AMPK pathways (15–18). Activation of mTOR in cells arrested by persistent DNA damage represents a second stimulus that can convert transiently arrested cells into senescent cells that exhibit hypertrophy and an expanded lysosomal compartment (19). The last category consists of the pathways mediating the senescence-associated secretory phenotype (SASP) (3, 10, 20). The SASP is a characteristic feature of senescent cells and reflects their secretion of proinflammatory cytokines and chemokines. These cytokines and chemokines maintain cellular senescence through positive autoregulatory feedbacks, affect nonsenescent nearby cells, and promote aging and age-related diseases, including cancer (3, 21). Nuclear factor κB (NF-κB) activity is important for SASP, and suppression of NF-κB prevents age-related diseases and delays aging in mice (22, 23).Spontaneous reversion from senescence to proliferation is extremely rare, but the reversion through manipulations is not. Some studies reported that senescent cells can reenter the cell cycle (24). The current understanding of senescence is as a dynamic multistep process that is reversible under some conditions (25). About 70 to 90% of cells with low p16^INK4a levels in replicative senescence, which is senescence related to the finite number of divisions a cell can perform before telomeres become too short, resume proliferation following p53 inactivation (26). Inactivation of p53 also enables cells to escape from therapy-induced senescence, caused by the chemotherapeutic agent Adriamycin (27). Cells with oncogene-induced senescence can also escape from the senescent state. For instance, about 50% of mouse embryo fibroblasts with high Ras levels reenter the cell cycle upon inactivation of all three Rb family members (28), and about 70% of the fibroblasts reenter upon activation of H3K9 demethylases (29).Here, we applied a systems biology approach to identify mechanisms underlying cell-cycle arrest, cell growth, and the SASP with the goal of finding inhibitable targets to convert the senescent state to the quiescent state. We studied normal human dermal fibroblasts (NHDFs), which can be experimentally induced into the senescent state (8). We constructed a molecular signaling network of cellular senescence using information in the literature and network databases to identify the relevant molecules, experimental data from time series of phosphorylated proteins in NHDFs to define the input–output relationships that reflect cellular states upon each input condition, and an evolutionary algorithm to determine the regulatory logic of the network (SI Appendix, Fig. S1 A–C). By analyzing the regulatory signaling network, we predicted that PDK1 was an inhibitor target that can convert senescent fibroblasts to quiescent fibroblasts (SI Appendix, Fig. S1D). To validate this prediction, we conducted experiments with NHDFs exposed to PDK1 inhibitors (SI Appendix, Fig. S1E), which eliminated hallmarks of cellular senescence, restored the proliferation of the cells in response to growth factors, and restored skin regeneration capacity in two-dimensional (2D) culture and a three-dimensional (3D) skin equivalent model. Our findings provide insight into a potential therapeutic strategy to treat aging and age-related diseases.     

Human-relevant near-organ neuromodulation of the immune system via the splenic nerve

Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve–induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.

The inflammatory status of the body is monitored and regulated through the neuroimmune axis, connecting the brain to the immune system via both humoral and neural pathways (1–3). In particular, the inflammatory reflex (3) controls systemic immune responses; detection of inflammatory stimuli in the periphery is communicated to the brain that activates outflow of neural signals to promote peripheral immune responses proportional to the threat. Studies in rodent models have identified the cholinergic anti-inflammatory pathway (CAIP) as the brain’s efferent response to infection and inflammation through peripheral neurotransmitters released in lymphoid organs, mainly the spleen (4, 5). Within this pathway, the peripheral connection between the vagus nerve (VN), the splenic nerve (SpN), and its terminal release of noradrenaline (NA) into the spleen have been identified as crucial components of this neural circuit (6–8) (SI Appendix, Fig. S1A).Importantly, the CAIP can be harnessed to promote immune control. Activation of the cervical VN by electrical stimulation (vagus nerve stimulation—VNS; SI Appendix, Fig. S1A) has been shown to be effective in reducing lipopolysaccharide (LPS)-induced levels of tumor necrosis factor alpha (TNF-α) (4, 6, 7) and in preclinical rodent models of chronic inflammatory diseases (9, 10). Murine models have generally been used to demonstrate biological proof of concepts of novel neuromodulation therapies in this and other contexts. However, the development of clinical bioelectronic medicines requires the accurate estimation and validation of stimulation parameters in a histologically, surgically, and anatomically relevant model to define device and therapy requirements. The translation of stimulation parameters from rodent to human is hampered by anatomical (e.g., size of nerves), histological (e.g., number of axons, connective tissue thickness, proportion of adipose tissue), and physiological (e.g., immunological) differences. Therefore, it is suggested that the use of large animal models, human tissues, and in silico modeling are more appropriate for the optimization and scaling of human-relevant parameters (11, 12).Although early clinical feasibility studies have provided preliminary evidence of immunomodulatory effects of VNS in patients (13, 14), clear demonstration of the translation of the splenic anti-inflammatory pathway in clinically relevant species is currently lacking in the literature. The VN has a functionally and anatomically complex composition. In animals and humans, the VN contains both afferent and efferent axons of varying size (large, medium, and small) and degree of myelination (heavily myelinated, lightly myelinated, and unmyelinated axons) innervating multiple organs and muscles (15). As a consequence, currently used VNS results in activation of off-target circuits (SI Appendix, Fig. S1A) that can cause dysphonia, coughing, hoarseness, pain, and dyspnea (16–18); in some patients, these can be managed and can also improve over time (18). Further, it remains unclear which axons (efferent versus afferent, myelinated versus unmyelinated) within the VN relay immunomodulatory signals to peripheral organs (19, 20). As a result, it is difficult to optimize the stimulation parameters necessary to activate axons within the VN which carry signals to the spleen. Typically, clinical parameters are selected based on the individual patient’s tolerance of off-target effects (13, 21) without direct evidence of activation of the anti-inflammatory pathway because of a lack of an organ-specific biomarker. Since the SpN directly transmits neural signals to the spleen and is the fundamental nodal circuit in mediating the anti-inflammatory response (22), SpN stimulation (SpNS) may represent an alternative modality providing the opportunity for near-organ modulation of the immune system (SI Appendix, Fig. S1 B and C). Proof of concept experiments in rodents have shown that immune responses can indeed be modulated by stimulation of the SpN with comparable cytokine suppressive effects to VNS (7, 8, 23).Here, we anatomically, histologically, and functionally compared the mouse, rat, pig, and human SpN, demonstrating the superiority of the pig as a translational model of the human SpN. We then performed functional in vivo pig electrophysiological studies to identify organ-specific physiological biomarkers that can be used to assess nerve engagement and to refine stimulation parameters. Finally, we assessed the large animal translation of the spleen-dependent anti-inflammatory pathway in the pig using in vitro splenocyte preparations together with two in vivo models of acute inflammation.     

From the Cover: Aquatic biodiversity enhances multiple nutritional benefits to humans

Humanity depends on biodiversity for health, well-being, and a stable environment. As biodiversity change accelerates, we are still discovering the full range of consequences for human health and well-being. Here, we test the hypothesis—derived from biodiversity–ecosystem functioning theory—that species richness and ecological functional diversity allow seafood diets to fulfill multiple nutritional requirements, a condition necessary for human health. We analyzed a newly synthesized dataset of 7,245 observations of nutrient and contaminant concentrations in 801 aquatic animal taxa and found that species with different ecological traits have distinct and complementary micronutrient profiles but little difference in protein content. The same complementarity mechanisms that generate positive biodiversity effects on ecosystem functioning in terrestrial ecosystems also operate in seafood assemblages, allowing more diverse diets to yield increased nutritional benefits independent of total biomass consumed. Notably, nutritional metrics that capture multiple micronutrients and fatty acids essential for human well-being depend more strongly on biodiversity than common ecological measures of function such as productivity, typically reported for grasslands and forests. Furthermore, we found that increasing species richness did not increase the amount of protein in seafood diets and also increased concentrations of toxic metal contaminants in the diet. Seafood-derived micronutrients and fatty acids are important for human health and are a pillar of global food and nutrition security. By drawing upon biodiversity–ecosystem functioning theory, we demonstrate that ecological concepts of biodiversity can deepen our understanding of nature’s benefits to people and unite sustainability goals for biodiversity and human well-being.

Species losses and range shifts because of climate change, harvesting, and other human activities are altering aquatic biodiversity locally and globally (1–5). In aquatic ecosystems, not only are some species severely depleted because of overfishing or habitat loss (3, 6–8), the ecosystem-level dimensions of biodiversity such as the total number of species and their functional diversity have also changed (9). Beyond the loss of particular species, changes in ecosystem-level dimensions of biodiversity threaten numerous ecosystem services to humans, which include the cultural, economic, or health benefits people derive from nature (10–13). In many regions, such as tropical coastal systems, the cumulative impacts of human activities are severe and associated with strong declines in taxonomic and ecological functional diversity (6) and coincide with regions with a high dependence of people upon wild-caught seafood for food and nutrition (14). In temperate regions, where some coastal communities depend on local wild seafood harvests to meet their nutritional needs (15, 16), species richness may be increasing as species recover from exploitation and warmer oceans allow species to expand their ranges into new territory (1, 2, 17).There is growing concern that biodiversity change leads to changes in human health and well-being (10, 13, 18). Specific and quantitative links between aquatic biodiversity and human health that distinguish contributions of species diversity from those of biomass, as predicted by biodiversity–ecosystem functioning theory, have not been established. At a time of unprecedented global change and increasing reliance on seafood to meet nutritional demands (19), there is an urgent need to understand how changing aquatic ecosystem structure may alter the provisioning of seafood-derived human nutrition.Seafood, consisting of wild-caught marine and freshwater finfish and invertebrates, provides an important source of protein and calories to humans. Additionally, unlike staple foods such as rice or other grains, seafood can address multiple dimensions of food and nutritional security simultaneously by providing essential micronutrients, such as vitamins, minerals, and polyunsaturated essential fatty acids critical to human health (19–22). Given the multiple attributes of seafood that are valuable to human health, it is possible that the diversity of an aquatic assemblage, distinct from the inclusion of any particularly nutritious species, could support human well-being consistent with a large body of evidence for biodiversity’s major contributions to ecological functions (11, 23–26). Dietary diversity is a basic tenet of a nutritious diet (27) and it is widely appreciated that diets composed of more food groups and more species are more nutritious (28–31). Ecological measures of dietary diversity (diet diversity, species richness, functional diversity, and Simpson’s index of evenness) have been associated with the nutritional value of diets in a range of contexts (27, 29, 32–38). These studies rely on relationships between species included in the diet (or other food intake measures) and nutritional adequacy of reported diets. However, a simple correlation between dietary diversity and a measure of dietary benefits provides only partial support for a claim that biodiversity benefits human well-being, consistent with the same ecological processes by which biodiversity supports numerous ecosystem functions and services (23, 26). We build upon this foundation of empirical relationships between diet diversity and diet quality by placing this question in the quantitative ecological theoretical framework that relates biodiversity to function (24, 25), thereby laying the groundwork for additional development of links between biodiversity science and our understanding of human well-being.Ecological theory predicts that biodiversity can be ecologically and economically important, apart from the importance of total biomass or the presence of particular species (23, 39). According to theory and over 500 explicit experimental tests (23, 40, 41), diversity in ecological communities and agricultural systems enhances ecosystem functioning by two mechanisms: 1) more diverse assemblages may outperform less diverse assemblages of the same density or biomass of individuals because more diverse assemblages will include more of the possible species and are therefore more likely to include high-performing species, assuming random processes of including species from the species pool (a selection effect), or 2) more diverse assemblages of a given density (or biomass) contain species with complementary functional traits, allowing them to function more efficiently (a complementarity effect) (25, 39). For aquatic animals, increased diversity enhances productivity of fish biomass (42) and also enhances temporal stability of biomass production and total yields (43, 44), providing economic and nutritional benefits to humans related to increased stability of harvests and production of biomass for consumption (43). However, when considering aquatic species from the perspective of human nutrition, functions other than biomass production become relevant because total seafood biomass consumption is not predictive of micronutrient benefits from seafood (45, 46).Here, we test a hypothesis central to ecological theory in the 21st century: whether biodiversity per se (species richness and ecological functional diversity), distinct from the identities and abundance of species, enhances human well-being (Fig. 1). We chose a measure of human well-being distinct from provision of protein, calories, or total yields—the micronutrient and essential fatty acid benefits of seafood. For increasing biodiversity per se (as opposed to increasing total seafood consumption) to enhance nutritional benefits as predicted by biodiversity–ecosystem functioning theory (25, 47), the amounts of various nutrients within edible tissues must differ among species, and furthermore, nutrient concentrations must trade off among species, such that species that have relatively high concentrations of some nutrients also have relatively low concentrations of others (25). Specifically, a “biodiversity effect” (sensu ref. 25) on nutritional benefits requires that concentrations of multiple nutrients are negatively correlated with each other, or uncorrelated, when compared among species, creating a complementary distribution of nutrients across species. In contrast, if nutrient concentrations in edible tissue are positively correlated for multiple nutrients across species such that, for example, a species containing high amounts of iron also has a high essential fatty acid concentration, thereby containing multiple nutrients in high concentrations simultaneously, seafood species or ecological functional diversity in the diet would not be important. In the case of positive correlations among nutrient concentrations, the ecosystem service of nutritional benefits would be enhanced by consuming more fish biomass or by selecting a few highly nutritious species, without considering species richness or ecological functional diversity.Open in a separate windowFig. 1.Aquatic biodiversity increases human well-being because edible species have distinct and complementary multinutrient profiles (A) and differ in mean micro- and macronutrient content (shown here relative to 10 and 25% thresholds of recommended dietary allowance, RDA, guidelines) for representative finfish (Abramis brama, Mullus surmuletus), mollusc (Mytilus galloprovincialis), and crustacean species (Nephrops norvegicus). Biodiversity–ecosystem functioning theory predicts that nutritional benefits, including the number of nutrient RDA targets met per 100 g portion (NT; i, iii) and minimum portion size (P_min; ii, iv) (B and E), are enhanced with increasing seafood species richness. Orange dots in B and E correspond to potential diets of high and low biodiversity levels. Seafood consumers with limited access to seafood each day may not reach RDA targets if diets are low in diversity (D–F versus A–C; gray shading indicates proportion of population that meets nutrient requirements). DHA: docosahexaenoic acid, EPA: eicosapentaenoic acid.We aimed to bridge two distinct theoretical frameworks—the biodiversity–ecosystem functioning theory and human nutrition science—by quantitatively testing for effects of aquatic species richness and ecological functional diversity (48, 49) in seafood diets on nutritional benefits via complementarity or selection effects. We used the public health measure of recommended dietary allowance (RDA) index to quantify nutritional benefits. RDAs are nutrient-based reference values that indicate the average daily dietary intake level that is sufficient to meet the nutrient requirement of nearly all (97 to 98%) healthy individuals in a particular life stage and gender group (50). Here, we used the RDA for females aged 19 to 50 y (SI Appendix, Tables S1 and S2; see SI Appendix, Table S1 for definitions of key terms). We measured nutritional value in terms of concentrations relative to RDAs, and we refer to these recommended amounts (or portions thereof) as “RDA targets” (SI Appendix, Tables S1 and S2 and Metrics). We quantified nutritional value in two ways: 1) the minimum amount of seafood tissue (in grams) required to meet given RDA targets (either for a single nutrient or the five micronutrients and fatty acids simultaneously; referred to as “minimum portion size required,” P_min [SI Appendix, Table S1, Eq. A1, and Metrics]) and 2) the number of nutrients that meet an RDA target in a single 100 g seafood portion (NT, SI Appendix, Table S1, Eq. A2). By considering nutritional value per unit biomass in both metrics, we avoided confounding diversity of seafood consumed with the total amount consumed (Metrics). We first tested two hypotheses: 1) seafood species richness increases NT because of complementarity in nutrient concentrations among species, and 2) seafood species richness increases the nutritional value of a 100 g edible portion of seafood, thereby lowering the minimum portion size, P_min, and improving the efficiency with which seafood consumers reach nutritional targets (Fig. 1). Following biodiversity–ecosystem functioning theory, we predicted that increased species richness is correlated with ecological functional diversity (51) in potential seafood diets and that ecological functional diversity is related to diversity in the concentration of essential elements and fatty acids that have nutritional value to human consumers, such that species and ecological functional diversity yields increased nutritional benefits. We also tested the hypothesis that seafood diversity increases total intake of heavy metal contaminants because some aquatic animals are known to bioaccumulate toxic metals in their tissues. For this reason, variation in bioaccumulation among species could lead to a biodiversity effect on contaminant intake that is detrimental to human health.In a global analysis of over 5,040 observations of nutrient concentrations in 547 aquatic species (SI Appendix, Fig. S1), we considered the provision of nutritional benefits to human consumers. To assess whether the relationships between biodiversity and human nutrition benefits depend on the geographic extent (global or local) over which seafood are harvested or accessed (11), we tested whether seafood species richness is associated with higher nutritional value at local scales (versus global scale) in traditional Indigenous seafood diets in North America (SI Appendix, Methods 1.4). Seafood is critical for Indigenous groups, who on average consume seafood at a rate that is 15 times higher than the global average per capita consumption rate (16). To test our hypotheses at the geographic scale of local consumer communities, we complemented our global analysis with additional analyses of 25 to 57 species in 14 geographically constrained groups of species consumed together as part of traditional Indigenous diets (SI Appendix, Methods 1.4).     

A new sea-level record for the Neogene/Quaternary boundary reveals transition to a more stable East Antarctic Ice Sheet

Sea-level rise resulting from the instability of polar continental ice sheets represents a major socioeconomic hazard arising from anthropogenic warming, but the response of the largest component of Earth’s cryosphere, the East Antarctic Ice Sheet (EAIS), to global warming is poorly understood. Here we present a detailed record of North Atlantic deep-ocean temperature, global sea-level, and ice-volume change for ∼2.75 to 2.4 Ma ago, when atmospheric partial pressure of carbon dioxide (pCO₂) ranged from present-day (>400 parts per million volume, ppmv) to preindustrial (<280 ppmv) values. Our data reveal clear glacial–interglacial cycles in global ice volume and sea level largely driven by the growth and decay of ice sheets in the Northern Hemisphere. Yet, sea-level values during Marine Isotope Stage (MIS) 101 (∼2.55 Ma) also signal substantial melting of the EAIS, and peak sea levels during MIS G7 (∼2.75 Ma) and, perhaps, MIS G1 (∼2.63 Ma) are also suggestive of EAIS instability. During the succeeding glacial–interglacial cycles (MIS 100 to 95), sea levels were distinctly lower than before, strongly suggesting a link between greater stability of the EAIS and increased land-ice volumes in the Northern Hemisphere. We propose that lower sea levels driven by ice-sheet growth in the Northern Hemisphere decreased EAIS susceptibility to ocean melting. Our findings have implications for future EAIS vulnerability to a rapidly warming world.

The instability of polar continental ice sheets in a warmer future is an issue of major societal concern (1–5). Based on linear extrapolation of recent sea-level rise (2), mean global sea level could increase by 65 ± 12 cm by 2100 relative to the 2005 baseline, consistent with Intergovernmental Panel on Climate Change projections (1) of a ∼30- to 100-cm increase by 2100. Further, satellite observations (4) document substantial mass loss of both the Greenland Ice Sheet (GIS) and the West Antarctic Ice Sheet (WAIS) over the past decade—the two ice sheets that are most susceptible to global warming because of rapidly rising Arctic air temperatures (1) (GIS) and vulnerability to ocean-atmospheric warming (5, 6) (WAIS). The mass balance of the much larger EAIS and its contribution to ongoing sea-level change, however, remain poorly constrained (1).The role of atmospheric partial pressure of carbon dioxide (pCO₂) as a driver of long-term changes in ice volume and sea level over the Cenozoic Era (past ∼66 My) is widely documented (7–9) and there is compelling evidence (6, 10–12) of East Antarctic Ice Sheet (EAIS) retreat during warm intervals of the Pliocene epoch between ∼5.3 and 3.3 Ma when pCO₂ levels (13, 14) last reached values close to the present day (∼400 parts per million volume [ppmv]; Fig. 1 A and B and see SI Appendix, section S1). However, there is disagreement over EAIS behavior under pCO₂ levels (13) similar to those of preindustrial Quaternary times (<280 ppmv). A compilation of marine geochemical paleo-sea-level and pCO₂ records suggests that the EAIS was stable under these conditions (7). In contrast, while the amplitudes of change are controversial (15) (SI Appendix, section S2), sea-level reconstructions from paleoshorelines (16) and benthic geochemical data (9, 17, 18) (Fig. 2) imply EAIS melting during the Quaternary “super-interglacials” of Marine Isotope Stage (MIS) 11 (∼400 ka) and 31 (∼1.07 Ma) under relatively low pCO₂ conditions. Supporting evidence for EAIS retreat during the most recent “super-interglacial” MIS 11 comes from isotope measurements in mineral deposits recording past changes in subglacial East Antarctic waters (19), as well as records of ice-rafted debris (IRD) and detrital sediment neodymium isotopes from offshore the Wilkes Subglacial Basin (20). The latter records (20) also indicate EAIS retreat during the last interglacial MIS 5e (∼120 ka). Melting of the EAIS as inferred in the late Quaternary was likely driven by ocean–atmosphere warming around Antarctica and grounding-line retreat in response to ice–ocean interactions (19, 20).Open in a separate windowFig. 1.Neogene to Quaternary climate and sea-level evolution. (A) LR04 stack (21) for the past 5 My; arrow indicates the iNHG (∼3.6 to 2.4 Ma) and its culmination (thick-arrowed interval) (22); green line indicates the benthic δ¹⁸O level associated with MIS 101. (B) Atmospheric pCO₂ estimates of refs. 13 (blue) and 23 (purple) for the past 5 My; the late Quaternary glacial–interglacial pCO₂ range (1) is indicated as preindustrial pCO₂ band. Yellow shading in A and B highlights the study interval (∼2.75 to 2.4 Ma). (C and D) Site U1313 benthic δ¹⁸O and Mg/Ca raw data, respectively. (E) Site U1313 deep-sea temperature. (F) Site U1313 δ¹⁸O_sw-based sea level relative to present (black line); blue shading: 95% probability interval from Monte Carlo simulations (2σ); red line: threshold (11.6 msle) above which a smaller-than-present EAIS is signaled (24–26); m = marine part of EAIS, t = terrestrial part of EAIS. Glacials are highlighted in gray.Open in a separate windowFig. 2.Implication of different sea-level-δ¹⁸O_sw conversions for estimates of interglacial ice-volume loss. y axis shows lower-than-modern δ¹⁸O_sw values (∆δ¹⁸O_sw) and the x axis (log-scale) the corresponding sea-level increase for commonly used conversion factors (27–29) (0.011 [black], 0.010 [purple], and 0.008 ‰⋅m⁻¹ [red]) and those for Antarctica only (11) (0.014 ‰⋅m⁻¹) ignoring (yellow) and incorporating (brown) the impact of its marine-based ice sheets. Stars mark ∆δ¹⁸O_sw for interglacials of this study and corresponding sea-level equivalents in dependence of the conversion applied. Orange, blue, and purple diamonds show the same for MIS 31, 11 (18), and 5e (17), respectively. Vertical lines indicate the sea-level increase resulting from complete melting of the GIS (+7.3 m), WAIS (+4.3 m), and EAIS (+53.3 m) (24–26).To further investigate past EAIS response to climate forcing we studied the Neogene/Quaternary transition when mean pCO₂ (13, 23) fell from levels similar to the anthropogenically perturbed values of today into the Quaternary range, leading to progressive high-latitude cooling and the intensification of Northern Hemisphere Glaciation (21, 30–33) (iNHG; Fig. 1 A and B). Our approach is based on a simple approximation that, once estimated past global sea level exceeds 11.6 m sea-level equivalent (msle) above modern, which corresponds to the complete melting of the present-day GIS [7.3 msle (24, 25)] and the marine- and land-based WAIS [3.4 and 0.9 msle (25, 26), respectively], EAIS instability (i.e., a retreat from its present-day size) can be inferred (see SI Appendix, section S4.1 for details). We quantified sea-level and ice-volume changes for the interval ∼2.75 to 2.4 Ma (MIS G7 to 95) by measuring the oxygen-isotope composition (δ¹⁸O) and Mg/Ca ratio in well-preserved benthic foraminiferal calcite (Oridorsalis umbonatus) from Integrated Ocean Drilling Program (IODP) Site U1313 [41°0′N, 32°57′W; 3,426-m water depth (34)] in the North Atlantic Ocean (Fig. 1 C and D). Using this approach we reconstructed changes in seawater δ¹⁸O (δ¹⁸O_sw), a proxy for global sea level and continental ice volume (35). This was done by 1) calculating bottom-water temperatures (BWT) derived from Mg/Ca (36) (Fig. 1E), 2) combining Mg/Ca-derived BWTs with δ¹⁸O to determine δ¹⁸O_sw (37) (Fig. 1F), and 3) converting δ¹⁸O_sw to sea level using a relationship between changes in sea level and δ¹⁸O_sw of 0.011 ‰⋅m⁻¹ (27) (Materials and Methods and Fig. 1F). Ninety-five percent probability intervals calculated through Monte Carlo simulations for individual sea-level data points yield an average uncertainty for our sea-level estimates of ± 28 m (∼2σ [SD]) (Materials and Methods and Fig. 1F), roughly equivalent to the decay/growth of ice four times greater than the GIS. Our approach was validated by reconstructing δ¹⁸O_sw for the recent (∼0 to 7 ka) at IODP Site U1313 and for late Holocene core-top (multicorer) samples from a neighboring site (MSM58) which are indistinguishable from the observed modern-day values (see Materials and Methods and SI Appendix, section S4.2.8 for details).     

From the Cover: Invasiveness is linked to greater commercial success in the global pet trade

The pet trade has become a multibillion-dollar global business, with tens of millions of animals traded annually. Pets are sometimes released by their owners or escape, and can become introduced outside of their native range, threatening biodiversity, agriculture, and health. So far, a comprehensive analysis of invasive species traded as pets is lacking. Here, using a unique dataset of 7,522 traded vertebrate species, we show that invasive species are strongly overrepresented in trade across mammals, birds, reptiles, amphibians, and fish. However, it is unclear whether this occurs because, over time, pet species had more opportunities to become invasive, or because invasive species have a greater commercial success. To test this, we focused on the emergent pet trade in ants, which is too recent to be responsible for any invasions so far. Nevertheless, invasive ants were similarly overrepresented, demonstrating that the pet trade specifically favors invasive species. We show that ant species with the greatest commercial success tend to have larger spatial distributions and more generalist habitat requirements, both of which are also associated with invasiveness. Our findings call for an increased risk awareness regarding the international trade of wildlife species as pets.

The extraordinary movement of our own species through migration, colonization, and travel has driven the geographic expansion of countless other species since prehistoric times (1). Humans have deliberately introduced a diverse range of species, in particular domesticated crops and animals that have contributed to our success (1). Today, however, the trade in live organisms for nonutilitarian reasons has rocketed (2–4). In the last decade alone, billions of plants and animals comprising thousands of species were traded annually, fueling a multibillion-dollar global business (2, 3, 5, 6). In particular, the demand for nontraditional (also known as “exotic”) ornamentals and pets, i.e., organisms without a long history of domestication, has grown (2). These species are sometimes released into the wild or escape and may survive and reproduce (2, 7–9). Species with populations that have established outside of their native range are referred to as invasive species hereafter (see Table 1 for terminology). Some invasive species can have severe impacts on global biodiversity (10–13) and impose tremendous costs on society by damaging physical infrastructure, agriculture, forestry, and human health (14, 15). However, even though it is undisputed that the trade in pets and ornamentals contributes to the global movement of invasive animals (16–19), it is still unclear whether this trade specifically favors invasive species.Table 1.Glossary

Superelastic oxide micropillars enabled by surface tension–modulated 90° domain switching with excellent fatigue resistance

Superelastic materials capable of recovering large nonlinear strains are ideal for a variety of applications in morphing structures, reconfigurable systems, and robots. However, making oxide materials superelastic has been a long-standing challenge due to their intrinsic brittleness. Here, we fabricate ferroelectric BaTiO₃ (BTO) micropillars that not only are superelastic but also possess excellent fatigue resistance, lasting over 1 million cycles without accumulating residual strains or noticeable variation in stress–strain curves. Phase field simulations reveal that the large recoverable strains of BTO micropillars arise from surface tension–modulated 90° domain switching and thus are size dependent, while the small energy barrier and ultralow energy dissipation are responsible for their unprecedented cyclic stability among superelastic materials. This work demonstrates a general strategy to realize superelastic and fatigue-resistant domain switching in ferroelectric oxides for many potential applications.

Superelastic materials are capable of recovering large amount of nonlinear “plastic” strains, way beyond their linear elastic regimes (1–4). They are ideal for a variety of applications from morphing structures, reconfigurable systems, to robots (5–8). The effects have traditionally been associated with macroscopically compliant/ductile rubbers (2) or microscopically phase-transforming shape memory alloys (SMAs) (7–11). The only macroscopically brittle oxide recently discovered to be superelastic is ZrO₂-based micropillars or particles (12–20), which is realized via austenite-martensite phase transformation similar to SMAs. Although ultimate strengths approaching the theoretical limit have been demonstrated in nanoscale samples (21, 22), long fatigue life is elusive, which is arguably more important for most applications. As a matter of fact, poor fatigue life has been a long-standing challenge for oxide ceramics in general (23, 24). Even for ductile SMAs that enjoy excellent fatigue life, irrecoverable residual strains gradually accumulate over cycling, leading to substantial variations in stress–strain curves at different cycles (9, 10, 25). We overcome these difficulties by reporting superelastic barium titanate (BaTiO₃ [BTO]) micropillars enabled by surface tension–modulated 90° domain switching, which exhibit excellent fatigue resistance, while bulk BTO crystals or ceramics are rather brittle. The demonstration of over one million cycles of loading and unloading without accumulating residual strains or noticeable variation in stress–strain curves is unprecedented among superelastic materials.BTO is a ferroelectric oxide exhibiting modest piezoelectric strains around 0.1 to 0.2% (26) and fracture toughness of ∼1 MPa ⋅ m^1/2, and thus it is quite brittle (27). Considerable research efforts have been devoted to enhancing its electric field–induced strain via 90° ferroelectric domain switching (28–30). However, the process is often irreversible, and external mechanisms such as restoring force (28, 29) and internal mechanisms such as defect pinning (30) have to be invoked to make the electrostrain recoverable. Nevertheless, it hints at the possibility of BTO being made superelastic by taking advantage of the stress-induced 90° domain switching (6). Earlier works suggest that surface tension induces an in-plane compressive stress that favors the axial polarization in one-dimensional ferroelectrics at small size (31, 32), which may provide the necessary restoring mechanism for the stress-switched domains. Thus, if a compressive axial force is applied, reversible domain switching may occur during unloading, leading to superelasticity. To verify this hypothesis, we fabricated single-crystalline BTO micropillars from [001]-oriented bulk crystals (SI Appendix, Fig. S1A) via focused ion beam (FIB), as detailed in Materials and Methods and SI Appendix, Fig. S1B. The diameters (Φ) of the micropillars range from 0.5 μm to 5 μm, with their height to diameter ratio fixed at 3. No visible defects can be seen from the scanning electron microscopy (SEM) images of these micropillars shown in Fig. 1 A–D, and their surfaces appear to be quite smooth, suggesting that no apparent damages are induced by FIB.Open in a separate windowFig. 1.Superelastic BTO micropillars below a critical size. (A–D) SEM images of the micropillars with Φ = 5, 3, 2, and 0.5 μm. (E–G) The first and second cycles of stress–strain curves for BTO micropillars with Φ = 5, 2, and 0.5 μm. (H) S_r/S_max and ΔW/W_max during the first cycle for BTO micropillars of different diameters. Here, S_r and S_max denote the residual strain and the maximum strain (SI Appendix, Fig. S6A), while ΔW and W_max are energy dissipated and stored in the first cycle, respectively (SI Appendix, Fig. S6F).     

From the Cover: Ancient noeggerathialean reveals the seed plant sister group diversified alongside the primary seed plant radiation

Noeggerathiales are enigmatic plants that existed during Carboniferous and Permian times, ∼323 to 252 Mya. Although their morphology, diversity, and distribution are well known, their systematic affinity remained enigmatic because their anatomy was unknown. Here, we report from a 298-My-old volcanic ash deposit, an in situ, complete, anatomically preserved noeggerathialean. The plant resolves the group’s affinity and places it in a key evolutionary position within the seed plant sister group. Paratingia wuhaia sp. nov. is a small tree producing gymnospermous wood with a crown of pinnate, compound megaphyllous leaves and fertile shoots each with Ω-shaped vascular bundles. The heterosporous (containing both microspores and megaspores), bisporangiate fertile shoots appear cylindrical and cone-like, but their bilateral vasculature demonstrates that they are complex, three-dimensional sporophylls, representing leaf homologs that are unique to Noeggerathiales. The combination of heterospory and gymnospermous wood confirms that Paratingia, and thus the Noeggerathiales, are progymnosperms. Progymnosperms constitute the seed plant stem group, and Paratingia extends their range 60 My, to the end of the Permian. Cladistic analysis resolves the position of the Noeggerathiales as the most derived members of a heterosporous progymnosperm clade that are the seed plant sister group, altering our understanding of the relationships within the seed plant stem lineage and the transition from pteridophytic spore-based reproduction to the seed. Permian Noeggerathiales show that the heterosporous progymnosperm sister group to seed plants diversified alongside the primary radiation of seed plants for ∼110 My, independently evolving sophisticated cone-like fertile organs from modified leaves.

The origin of the seed in the Late Devonian, ∼365 Mya, represents a key innovation in land plant evolution. Seeds provided a fundamentally new reproductive strategy that overcame the limitations of free-sporing, pteridophytic reproduction and enabled colonization of drier habitats (1‒3). Progymnosperms, the evolutionary stem group leading to seed plants, display a mosaic of evolutionary characters combining free-sporing reproduction with production of secondary xylem (wood) through a bifacial vascular cambium characteristic of seed plants (2‒4). Although not representing a monophyletic evolutionary group, progymnosperms are important for our present understanding of the origin of the seed and represent intermediates between pteridophytes and seed plants (1, 2, 4). Progymnosperms include the basally divergent homosporous Aneurophytales, as well as the more derived heterosporous Archaeopteridales, Protopityales (1, 4), and the enigmatic Carboniferous cone Cecropsis (5). The transition to seed plants requires multiple character state transitions from known progymnosperm sister groups comprising either Archaeopteris (6, 7) or Archaeopteris + Cecropsis (e.g., ref. 8). For each of these cases, viable intermediates are absent from the fossil record.In contrast, Noeggerathiales (9) have at times been proposed as progymnosperms (9–12), but this has been controversial. Comprising ∼20 genera and 50 species, Noeggerathiales are known from the late Carboniferous–Permian (323 to 251 Ma) tropical floras in North America, Europe, and East Asia (10), where they are identified as a related group based on shared features of heterospory, adaxial sporangial attachment to “sporophylls,” longitudinal sporangial dehiscence, plagiotropic pinnule attachment to the rachis, and once-pinnate compound megaphylls (9–12). However, their systematic position remained uncertain because details of their stem anatomy were unknown. In the absence of this anatomical information, they have been postulated as close relatives of the progymnosperms, leptosporangiate ferns, sphenopsids, the extant fern Tmesipteris, or as a distinct class of their own (see ref. 9 for summary). Recently, leaves of Plagiozamites oblongifolius that were interpreted as noeggerathialean (9) have also been interpreted as cycads (13). Wang et al. (9) considered the Noeggerathiales to be putative progymnosperms, but as their specimens lacked wood, a more confident assignment was not possible. This uncertainty meant that even when considered as progymnosperms, their relationship to other progymnosperms and seed plants has remained speculative.Here, we report a species that we name Paratingia wuhaia J. Wang et al. sp. nov. and ascribe to the Noeggerathiales. The fossils were collected from a single 66-cm-thick volcanic ash bed in the Chinese “vegetational Pompeii” from the Taiyuan Formation at Wuda open coalmine, Inner Mongolia (14, 15) (SI Appendix, Geological Information, Materials). The ash preserved in situ the morphology and anatomy of plants in exquisite detail (Fig. 1 A–N) and has been dated to 298.34 ± 0.09 Ma during the Asselian stage of the early Permian (16).Open in a separate windowFig. 1.P. wuhaia J. Wang et al. sp. nov. from the early Permian Taiyuan Formation of Wuda Coalfield, Inner Mongolia. (A) Holotype with an entire crown consisting of pseudostrobili and leaves. (B) Once-pinnate compound leaf with both large and small pinnules visible. Reprinted with permission from ref. 15. (C) Cross-section of a crown illustrating pseudostrobili around the stem. (D) Cross-section of pseudostrobilus with microsporangia around the axis with bilateral, inversed Ω-shaped vascular bundle. (E) Cross-section of a leaf rachis showing the same form of vascular bundle as that of pseudostrobili axes. (F–H) Partial cross, radial, and tangential sections of the stem showing the secondary xylem (wood). (I) Tangential section of pseudostrobilus showing sporangial arrangement with single line of megasporangia along with the axis (middle-right pseudostrobilus of the fragmental crown illustrated SI Appendix, Fig. S8; specimen PB22132; magnified in SI Appendix, Fig. S21C.) (J) Radial section of pseudostrobilus showing adaxial sporangia and axis lacking nodes (upmost-left pseudostrobilus of the fragmental crown in SI Appendix, Fig. S8; magnified in SI Appendix, Fig. S21A). (K) Tangential section showing adaxial sporangia and a single line of megasporangia along with the axis (SI Appendix, Fig. S7; specimen PB22131, second up-right pseudostrobilus). (L) Tangential section through same specimen as K showing megasporangial arrangement (magnified in SI Appendix, Figs. S21B and S23A). (M) Detail of the middle part of L showing the megasporangia and microsporangia. (N) Single spore macerated from the holotype. (Scale bars: A, 10 cm; B, 3 cm; C–E, 1 cm; F, 100 μm; G and H, 200 μm; I–L, 5 mm; M, 2 mm; N, 10 μm.)Isolated noeggerathialean fertile shoots (SI Appendix, Figs. S3–S8) and megaphyllous leaves (SI Appendix, Figs. S9‒S14) are common in the assemblage. The species P. wuhaia is based on >200 specimens, including >10 specimens that were preserved with intact crowns containing both leaves and fertile shoots in organic attachment to the stems, demonstrating that they belong to a single species (Fig. 1A and SI Appendix, Figs S2‒S4).     

The evolution and changing ecology of the African hominid oral microbiome

The oral microbiome plays key roles in human biology, health, and disease, but little is known about the global diversity, variation, or evolution of this microbial community. To better understand the evolution and changing ecology of the human oral microbiome, we analyzed 124 dental biofilm metagenomes from humans, including Neanderthals and Late Pleistocene to present-day modern humans, chimpanzees, and gorillas, as well as New World howler monkeys for comparison. We find that a core microbiome of primarily biofilm structural taxa has been maintained throughout African hominid evolution, and these microbial groups are also shared with howler monkeys, suggesting that they have been important oral members since before the catarrhine–platyrrhine split ca. 40 Mya. However, community structure and individual microbial phylogenies do not closely reflect host relationships, and the dental biofilms of Homo and chimpanzees are distinguished by major taxonomic and functional differences. Reconstructing oral metagenomes from up to 100 thousand years ago, we show that the microbial profiles of both Neanderthals and modern humans are highly similar, sharing functional adaptations in nutrient metabolism. These include an apparent Homo-specific acquisition of salivary amylase-binding capability by oral streptococci, suggesting microbial coadaptation with host diet. We additionally find evidence of shared genetic diversity in the oral bacteria of Neanderthal and Upper Paleolithic modern humans that is not observed in later modern human populations. Differences in the oral microbiomes of African hominids provide insights into human evolution, the ancestral state of the human microbiome, and a temporal framework for understanding microbial health and disease.

The oral cavity is colonized by one of the most diverse sets of microbial communities of the human body, currently estimated at over 600 prevalent taxa (1). Dental diseases, such as caries and periodontitis, remain health burdens in all human populations despite hygiene interventions (2, 3), and oral microbes are often implicated in extraoral inflammatory diseases (4, 5). To date, most oral microbiome research has focused on clinical samples obtained from industrialized populations that have daily oral hygiene routines and access to antibiotics (1, 6), but far less is known about the global diversity of the oral microbiome, especially from diverse past and present nonindustrialized societies (7). The oral cavity contains at least six distinct habitats, but dental biofilms, including both supra- and subgingival dental plaque, are among the most diverse and clinically important (1, 6, 8). During life, these dental biofilms naturally and repeatedly calcify, forming dental calculus (tooth tartar) (9), a robust, long-term record of the oral microbiome (10). Archaeological dental calculus has been shown to preserve authentic oral bacterial metagenomes in a wide range of historic and prehistoric populations and up to 50 thousand years ago (ka) (10–13). As such, dental calculus presents an opportunity to directly investigate the evolution of the hominid microbiome and to reconstruct ancestral states of the modern human oral microbiome. In addition, because research has shown that evolutionary traits, diet, and cultural behaviors shape modern human microbiome structure and function at other body sites, such as the gut and skin microbiomes (14–18), investigating ancient oral metagenomes has the potential to reveal valuable information about major events in modern human evolution and prehistory, such as predicted dietary changes during the speciation of Homo (19–21) and the direct interaction of Neanderthals and modern humans during the Late Pleistocene (22).To better understand the evolutionary ecology of the African hominid microbiome, we generated and analyzed 109 dental calculus metagenomes from present-day modern humans (n = 8), gorillas (Gorilla, n = 29), chimpanzees (Pan, n = 20), Neanderthals (n = 13), and two groups of archaeological modern humans associated with major lifestyle transitions (preagricultural, n = 20; preantibiotic, n = 14), as well as New World howler monkeys (n = 5) for comparison (SI Appendix, Fig. S1). To account for potential sampling biases, we analyzed multiple subspecies and populations of each African great ape genus, which were obtained from C20^th or C21^st-collected museum collections, and for modern humans we sampled multiple populations from both Africa and Europe. To this, we added previously published microbiome data from chimpanzees (n = 1) (13), Neanderthals (n = 4) (13), and present-day modern humans (n = 10) (23), for a total dataset of 124 individuals (Fig. 1A, SI Appendix, Table S1, and Dataset S1). We also generated eight new radiocarbon dates for archaeological individuals, for a total of 44 directly or indirectly dated ancient individuals in this study (Dataset S1).Open in a separate windowFig. 1.Sample locations and oral microbiome authentication of ancient dental calculus. (A) Sample locations. (B) PCoA comparing euclidean distances of microbial genera of well-preserved ancient and present-day dental calculus to environmental proxy controls (degraded archaeological bone) and present-day dental plaque and feces. Ancient dental calculus is distinct from gut and archaeological bone but overlaps with present-day dental plaque. (C) Representative DNA damage patterns for Neanderthals and ancient and present-day modern humans for four oral-specific bacterial species. The Neanderthal and upper Paleolithic modern human individuals show expected damage patterns consistent with authentic aDNA, whereas the present-day individual does not. See also SI Appendix, Fig. S4.Here, we investigate the structure, function, and core microbial members of the human oral microbiome within an evolutionary framework, seeking to determine whether a core microbiome can be defined for each African hominid group, whether the core is phylogenetically coherent, and whether some members of the core are specific to certain host groups. We test whether the oral microbiome of hominids reflects host phylogeny, finding that African hominid oral microbiota are distinguished by major taxonomic and functional differences that only weakly reflect host relationships and are likely influenced by other physiological, dietary, or behavioral factors. We compare the microbial profiles of Neanderthals and modern humans and, contrary to expectations (12, 13), find a high consistency of oral microbiome structure within Homo, regardless of geography, time period, or diet/lifestyle. We detect the persistence of shared genetic diversity in core taxa between Neanderthals and Upper Paleolithic humans prior to 14 ka, supporting a growing body of evidence for earlier admixture and interaction in Ice Age Europe (24, 25). Finally, we explore possible implications of our findings on Homo-associated encephalization (19, 26) and the role of dietary starch in human evolution (20, 21) by investigating the evolutionary history of amylase-binding capability by oral streptococci. We find that amylase binding is an apparent Homo-specific trait, suggestive of microbial coadaptation to starch-rich diets early in human evolution.     

Stage-specific overcompensation,the hydra effect,and the failure to eradicate an invasive predator

As biological invasions continue to increase globally, eradication programs have been undertaken at significant cost, often without consideration of relevant ecological theory. Theoretical fisheries models have shown that harvest can actually increase the equilibrium size of a population, and uncontrolled studies and anecdotal reports have documented population increases in response to invasive species removal (akin to fisheries harvest). Both findings may be driven by high levels of juvenile survival associated with low adult abundance, often referred to as overcompensation. Here we show that in a coastal marine ecosystem, an eradication program resulted in stage-specific overcompensation and a 30-fold, single-year increase in the population of an introduced predator. Data collected concurrently from four adjacent regional bays without eradication efforts showed no similar population increase, indicating a local and not a regional increase. Specifically, the eradication program had inadvertently reduced the control of recruitment by adults via cannibalism, thereby facilitating the population explosion. Mesocosm experiments confirmed that adult cannibalism of recruits was size-dependent and could control recruitment. Genomic data show substantial isolation of this population and implicate internal population dynamics for the increase, rather than recruitment from other locations. More broadly, this controlled experimental demonstration of stage-specific overcompensation in an aquatic system provides an important cautionary message for eradication efforts of species with limited connectivity and similar life histories.

Theoretical population models can produce counterintuitive predictions regarding the consequences of harvest or removal of predatory species. These models show that for simple predator-prey systems, there can be positive population responses to predator mortality resulting from harvest for fisheries or population management, which can create an increased equilibrium level of that predator species (1–5). Among these mortality processes is the “hydra effect,” named after the mythical multi-headed serpent that grew two new heads for each one that was removed (6, 7). This counterintuitive outcome can be driven by a density-dependent process known as overcompensation. The hydra effect typically refers to higher equilibrium or time-averaged densities in response to increased mortality, typically involving consumer populations undergoing population cycles. Population increases in response to mortality can be the result of stage-specific overcompensation, which involves an increase in a specific life history stage or a size class following increased mortality. The first analysis of overcompensatory responses to mortality did not depend on stage specificity and was applied initially to fisheries harvests (1). Subsequent models have included stage specificity and have been applied to a broad range of systems in which species have been harvested for consumption or removed for population control of non-native species (4, 5, 8–15).Theory suggests that overcompensation in response to harvest or removal can occur for a variety of reasons, including 1) reduced competition for resources and increased adult reproduction rates, 2) faster rates of juvenile maturation or greater success in reaching the adult stage, and 3) increased juvenile or adult survival rates (1–7). An increase in reproductive output in response to reduced adult density can be the result of a reduction in resource competition (SI Appendix, Fig. S1).While there is substantial evidence that conditions that could produce density-dependent overcompensation occur frequently, evidence for overcompensation in natural populations is rare. For only a few populations do we have the long-term demographic data collected over a sufficiently long duration and for population densities over a wide enough range to detect this effect. Unfortunately, recent reviews of population increases in response to increased mortality do not include field studies with explicit controls for removals (13–17).There are examples of density-dependent overcompensation from field populations (4, 13–15), as well as a larger number of studies from the laboratory and greenhouse typically involving plant and insect populations (18–22). Among the field examples is a population control program for smallmouth bass in a lake in upstate New York, which paradoxically resulted in greater bass abundance, primarily of juveniles, after 7 y of removal efforts (23, 24). Another field study in the United Kingdom showed that perch populations responded similarly when an unidentified pathogen decimated adults (25). Other programs that attempted to remove invasive fishes, including pikeperch in England (26), brook trout in Idaho (27), and Tilapia in Australia (28), showed similar results. However, although many of these examples involved well-executed studies with substantial field data, none had explicit controls for removal, such as comparable populations without harvest (or disease). Thus, despite the support of current theory in these studies, the contribution of external factors to observed population responses to harvest remains uncertain. To date, we are unaware of any experimental studies with comparable controls in a field population that demonstrates overcompensation in a single species (13–15).     

Transfer-printed,tandem microscale light-emitting diodes for full-color displays

Inorganic semiconductor-based microscale light-emitting diodes (micro-LEDs) have been widely considered the key solution to next-generation, ubiquitous lighting and display systems, with their efficiency, brightness, contrast, stability, and dynamic response superior to liquid crystal or organic-based counterparts. However, the reduction of micro-LED sizes leads to the deteriorated device performance and increased difficulties in manufacturing. Here, we report a tandem device scheme based on stacked red, green, and blue (RGB) micro-LEDs, for the realization of full-color lighting and displays. Thin-film micro-LEDs (size ∼100 μm, thickness ∼5 μm) based on III–V compound semiconductors are vertically assembled via epitaxial liftoff and transfer printing. A thin-film dielectric-based optical filter serves as a wavelength-selective interface for performance enhancement. Furthermore, we prototype arrays of tandem RGB micro-LEDs and demonstrate display capabilities. These materials and device strategies provide a viable path to advanced lighting and display systems.

Microscale light-emitting diodes (micro-LEDs) based on inorganic semiconductors have been widely regarded as one of the most promising solutions to the next generation of emissive display technologies for versatile applications, from televisions, smartphones and wearable watches, to advanced virtual, augmented, and mixed realities (1–4). Constructed from single-crystalline–compound semiconductors like gallium arsenide (GaAs), gallium phosphide (GaP), and gallium nitride (GaN), these micro-LEDs present significant benefits over conventional liquid crystal displays (LCDs) (5), organic LEDs (OLEDs) (6–9), and more recent quantum dot (10, 11) and perovskite-based LEDs (12), in terms of their efficiencies, brightness, contrast, dynamic response, and long-time stability. High-resolution displays rely on arrays of polychromatic (red, green, and blue [RGB]) emissive elements with dimensions of less than 100 μm or even 10 μm, which are massively and heterogeneously assembled onto silicon, glass, or plastic substrates (13–17). This scaling down of device sizes enhances the display resolution and reduces the raw material cost; however, it is also accompanied by significant challenges. First, reducing LED size causes dramatic efficiency decreases for all types of LEDs (18–21), particularly GaAs- or GaP-based red LEDs that are more susceptible to sidewall defects (SI Appendix, Fig. S1). Second, device shrinkage also results in deteriorated uniformity, which influences the reliability and performance of display systems. Third, fabricating and transferring smaller devices demand higher accuracies for alignment and positioning (22), imposing greatly increased manufacturing expenses (SI Appendix, Fig. S2).Alternatively, device arrays with a single-pixel, spectrally tunable LED (23) or vertically stacked tandem LEDs (24) relieve the above constraints associated with conventional laterally arranged RGB micro-LEDs. However, color-tunable LEDs based on voltage-dependent spectral change can neither achieve full-range tunability nor obtain independent color/intensity controllability (25, 26). On the other hand, the current state-of-the-art wafer-bonding–based assembly methods only realize double-layer stacking with the capability of dual-color tuning (green/blue or red/blue) (22, 27, 28). Although there are some reports on full-color stacked inorganic and organic RGB LEDs (24, 29–31), these demonstrations are only limited to a few of chip-scale, large or thick LEDs for lighting purposes.In this paper, we report a tandem thin-film RGB micro-LED architecture with full-range color tunability to overcome the limitations of previously explored technologies. Based on the epitaxial liftoff and transfer printing method, arrays of thin-film, high-performance, inorganic RGB micro-LEDs made of different III–V compounds are assembled in a vertical stack. Embedded into the stacked structure, a thin-film dielectric filter serves as a wavelength selective interlayer to improve the LED light output. Independently addressable active arrays show full-color electroluminescent patterns, demonstrating the display capability of the tandem micro-LEDs.     

Structural basis of Chikungunya virus inhibition by monoclonal antibodies

Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.

Chikungunya virus (CHIKV), a single-stranded positive-sense RNA envelope virus, is an emerging alphavirus transmitted to humans by Aedes species mosquitoes (1, 2). CHIKV consists of three related genotypes: Asian, East/Central/South African (ECSA), and West African (3). According to the Centers for Disease Control and Prevention, there have been millions of cases reported in approximately 100 countries. CHIKV infection causes an acute febrile illness accompanied by musculoskeletal disease (4, 5). A subset of cases (∼30%) showed that chronic arthritis can develop and persist for months to years (6, 7).The 12-kb positive-sense RNA genome is packaged within an icosahedral nuclear capsid core composed of 240 copies of capsid proteins, which is surrounded by a host-derived lipid bilayer. The surface of the mature CHIKV particle (diameter ∼700 Å) has 80 trimeric envelope E1-E2 heterodimer protein spikes anchored on the lipid bilayer membrane (SI Appendix, Fig. S1A) arranged in T = 4 icosahedral symmetry. E1 and E2 protein ectodomains each consist of three domains: E1-DI; E1-DII and E1-DIII; and E2-DA, E2-DB, and E2-DC (SI Appendix, Fig. S1B). The fusion loop on the distal end of E1-DII mediates endosomal membrane fusion. The groove formed by E2-DA and E2-DB shields the fusion loop of E1 protein from premature membrane fusion at neutral pH (8). Multiple attachment factors have been implicated in CHIKV entry of cells (9), and E2-DB reportedly contains receptor-binding sites (10, 11). Mxra8, a recently identified alphavirus receptor (12), recognizes an epitope spanning both the E1 and E2 proteins (13, 14).The virus infection cycle starts with the E1-E2 proteins binding to the cell-surface receptors (12). The virion is then internalized into the endosome (15, 16). The acidic condition of the endosome causes E1-E2 heterodimers to undergo conformational changes, exposure of the E1 fusion loop for insertion into the endosomal membrane, and subsequent reorganization of the E1 protein into E1 trimers to allow endosomal membrane fusion (17, 18). After fusion, the capsid and RNA genome are released into the cytoplasm (19) to allow translation and replication of the viral genome. The newly synthesized virus buds at the plasma membrane (20).Currently, there exist no licensed CHIKV vaccine or therapeutics. Neutralizing antibodies have been shown to confer both prophylactic and therapeutic protection in animal models (21–28). Here we show the potencies of two CHIKV antibodies, CHK-124 and CHK-263, in vivo and demonstrate that they inhibit multiple steps in the virus infection cycle in vitro. We also determined the cryogenic electron microscopy (cryo-EM) structures of their Fab fragments complexed with CHIKV to 4- to 5-Å resolution. For CHK-124, the predominant neutralization mechanisms are aggregation of virus particles and inhibition of receptor binding. For CHK-263, the mechanism is the inhibition of fusion by locking E1 and E2 proteins together. Altogether, our study provides a structural understanding as to how potent antibodies block CHIKV infection.     

Deep time diversity and the early radiations of birds

Reconstructing the history of biodiversity has been hindered by often-separate analyses of stem and crown groups of the clades in question that are not easily understood within the same unified evolutionary framework. Here, we investigate the evolutionary history of birds by analyzing three supertrees that combine published phylogenies of both stem and crown birds. Our analyses reveal three distinct large-scale increases in the diversification rate across bird evolutionary history. The first increase, which began between 160 and 170 Ma and reached its peak between 130 and 135 Ma, corresponds to an accelerated morphological evolutionary rate associated with the locomotory systems among early stem birds. This radiation resulted in morphospace occupation that is larger and different from their close dinosaurian relatives, demonstrating the occurrence of a radiation among early stem birds. The second increase, which started ∼90 Ma and reached its peak between 65 and 55 Ma, is associated with rapid evolution of the cranial skeleton among early crown birds, driven differently from the first radiation. The third increase, which occurred after ∼40 to 45 Ma, has yet to be supported by quantitative morphological data but gains some support from the fossil record. Our analyses indicate that the bird biodiversity evolution was influenced mainly by long-term climatic changes and also by major paleobiological events such as the Cretaceous–Paleogene (K–Pg) extinction.

The evolution of global biodiversity is a focal area of study in both evolutionary biology and paleontology, but its examination has been approached in different ways. Neontological studies reconstruct the history of biodiversity mainly by analyzing the tempo and mode of diversification based on molecular timetrees composed of extant species (1). By contrast, paleontologists normally measure past biodiversity by investigating morphological evolution and taxic diversity from the fossil record (2). This dichotomy in both methodology and data sources is best exemplified by recent studies on the evolution of bird biodiversity (the vernacular term “birds” is equivalent to the phylogenetic taxon “Avialae” in the present paper; see Methods and SI Appendix, Supplemental Text A). For example, the time-calibrated phylogeny of extant birds and related diversification rate analyses have revealed a rapid diversification of crown birds (equivalent to the phylogenetic taxon “Aves”) near the Cretaceous–Paleogene (K–Pg) boundary followed by a period of low-level net diversification rates starting about 50 Ma (3–5). Paleontological studies have revealed high morphological evolutionary rates both prior to and after the origin of Avialae (6–14), and an increased taxonomic diversity in the Early Cretaceous based on the known Mesozoic fossil record (15) (SI Appendix, Supplemental Text B). However, these results are not directly comparable and are difficult to be evaluated within the same evolutionary framework given that they are based on different evaluation parameters of bird diversity.