From the Cover: Canine sexual dimorphism in Ardipithecus ramidus was nearly human-like

Body and canine size dimorphism in fossils inform sociobehavioral hypotheses on human evolution and have been of interest since Darwin’s famous reflections on the subject. Here, we assemble a large dataset of fossil canines of the human clade, including all available Ardipithecus ramidus fossils recovered from the Middle Awash and Gona research areas in Ethiopia, and systematically examine canine dimorphism through evolutionary time. In particular, we apply a Bayesian probabilistic method that reduces bias when estimating weak and moderate levels of dimorphism. Our results show that Ar. ramidus canine dimorphism was significantly weaker than in the bonobo, the least dimorphic and behaviorally least aggressive among extant great apes. Average male-to-female size ratios of the canine in Ar. ramidus are estimated as 1.06 and 1.13 in the upper and lower canines, respectively, within modern human population ranges of variation. The slightly greater magnitude of canine size dimorphism in the lower than in the upper canines of Ar. ramidus appears to be shared with early Australopithecus, suggesting that male canine reduction was initially more advanced in the behaviorally important upper canine. The available fossil evidence suggests a drastic size reduction of the male canine prior to Ar. ramidus and the earliest known members of the human clade, with little change in canine dimorphism levels thereafter. This evolutionary pattern indicates a profound behavioral shift associated with comparatively weak levels of male aggression early in human evolution, a pattern that was subsequently shared by Australopithecus and Homo.

A small canine tooth with little sexual dimorphism is a well-known hallmark of the human condition. The small and relatively nonprojecting deciduous canine of the first known fossil of Australopithecus, the Taung child skull, was a key feature used by Raymond Dart for his inference that the fossil represented an early stage of human evolution (1). However, recovery of additional Australopithecus fossils led to the canine of Australopithecus africanus to be characterized as large (compared to that of humans or “robust australopithecines”) and its morphology primitive, based on a projecting main cusp and crown structures lacking or hardly expressed in Homo (2). Later, the perception of a large and primitive canine was enhanced by the discovery and recognition of Australopithecus afarensis and Australopithecus anamensis (3–8), the latter species extending back in time to 4.2 million years ago (Ma). Although assessments of canine size variation and sexual dimorphism in Au. afarensis were hampered by limited sample sizes (9, 10), some suggested that the species had a more dimorphic canine than do humans, equivalent in degree to the bonobo (11) or to chimpanzees and orangutans (12). Initially, Au. anamensis was suggested to express greater canine dimorphism than did Au. afarensis (13, 14). However, based on a somewhat larger sample size, this is now considered to be the case with the tooth root but not necessarily its crown (15–17).Throughout the 1990s and 2000s, a pre-Australopithecus record of fossils spanning >6.0 to 4.4 Ma revealed that the canines of these earlier forms did not necessarily exceed those of Au. afarensis or Au. anamensis in general size (18–28). However, all these taxa apparently possessed canine crowns on average about 30% larger than in modern humans, which makes moderately high levels of sexual dimorphism potentially possible. Canine sexual dimorphism, combined with features such as body size dimorphism, inform sociobehavioral and ecological adaptations of past and present primates, and therefore have been of considerable interest since Darwin’s 1871 considerations (29–57). In particular, the relationship of canine size dimorphism (and/or male and female relative canine sizes) with reproductive strategies and aggression/competition levels in primate species have been a continued focus of interest (14, 33, 35–45, 49–56). Conspecific-directed agonistic behavior in primates related to mate and/or resource competition can be particularly intense among males both within and between groups (14, 44, 57). It is widely recognized that a large canine functions as a weapon in intra- and intergroup incidences of occasional lethal aggression (45, 58–61), and a large, tall canine has been shown or inferred to significantly enhance male fitness (50, 56). Hence, canine size and dimorphism levels in fossil species provide otherwise unavailable insights into their adaptive strategies.Here, we apply a recently developed method of estimating sexual size dimorphism from fossil assemblages of unknown sex compositions, the posterior density peak (pdPeak) method (62), and reexamine canine sexual dimorphism in Ardipithecus ramidus at ∼4.5 Ma. We include newly available fossils recovered from the Middle Awash and Gona paleoanthropological research areas in the Afar Rift, Ethiopia (26, 63, 64) in order to obtain the most reliable dimorphism estimates currently possible. We apply the same method to Australopithecus, Homo, and selected fossil apes, and evaluate canine sexual dimorphism through evolutionary time.We operationally define canine sexual dimorphism as the ratio between male and female means of basal canine crown diameters (the m/f ratio). Because the canines of Ar. ramidus, Au. anamensis, and extant and fossil apes are variably asymmetric in crown shape, we examine the maximum basal dimension of the crown. This can be either the mesiodistal crown diameter or a maximum diameter taken from the distolingual to mesiobuccal crown base (7, 27, 65). In the chronologically later Au. afarensis and all other species of Australopithecus sensu lato and Homo, we examine the more widely available conventional metric of buccolingual breadth, which corresponds to or approximates the maximum basal crown diameter. In anthropoid primates, canine height is more informative than basal canine diameter as a functional indicator of aggression and/or related display (14, 41–44). We therefore also examine available unworn and minimally worn fossil canines with reliable crown heights.     

Probing exoplanet clouds with optical phase curves

Kepler-7b is to date the only exoplanet for which clouds have been inferred from the optical phase curve—from visible-wavelength whole-disk brightness measurements as a function of orbital phase. Added to this, the fact that the phase curve appears dominated by reflected starlight makes this close-in giant planet a unique study case. Here we investigate the information on coverage and optical properties of the planet clouds contained in the measured phase curve. We generate cloud maps of Kepler-7b and use a multiple-scattering approach to create synthetic phase curves, thus connecting postulated clouds with measurements. We show that optical phase curves can help constrain the composition and size of the cloud particles. Indeed, model fitting for Kepler-7b requires poorly absorbing particles that scatter with low-to-moderate anisotropic efficiency, conclusions consistent with condensates of silicates, perovskite, and silica of submicron radii. We also show that we are limited in our ability to pin down the extent and location of the clouds. These considerations are relevant to the interpretation of optical phase curves with general circulation models. Finally, we estimate that the spherical albedo of Kepler-7b over the Kepler passband is in the range 0.4–0.5.Phase curves provide unique insight into the atmosphere of a planet, a fact well known and tested in solar system exploration (1–3). Disentangling the information encoded in a phase curve is a complex process however, and interpretations can be faced with degeneracies. The potential of phase curves to characterize exoplanet atmospheres, particularly in combination with other techniques, is tantalizing. Phase curves observed over all orbital phases (OPs) are available for a few close-in planets in the optical (passband central wavelengths λ < 0.8 μm) (4–15) and the infrared (1 μm ≤ λ ≤ 24 μm) (16–19). At infrared wavelengths the measured flux from hot planets is typically dominated by thermal emission. In the optical, both thermal emission and reflected starlight contribute, with the relative size of the contributions dependent on the measurement wavelength as well as on the temperature of the atmosphere and the occurrence of condensates (20–25).Kepler-7b (26) is one of the ∼1,000 planets discovered by the Kepler mission. Its inferred mass M_p (= 0.44 M_J; J for Jupiter) and radius R_p (= 1.61 R_J) result in an unusually low bulk density (0.14 g⋅cm⁻³) that is inconsistent with current models of giant planet interiors (27, 28). Kepler-7b orbits a quiet G-type star of effective temperature T_? = 5,933 K every 4.89 d (orbital distance a = 0.062 astronomical units) (6, 7), and tidal forces have likely synchronized its orbit and spin motions. Taken together these set a planet equilibrium temperature T_eq ≤  1,935 K.Kepler photometry (0.4–0.9 μm) of the star–planet system has enabled the optical study of Kepler-7b (4–7, 10, 14). The inferred geometric albedo, A_g = 0.25–0.38 (4, 6, 7, 10, 14), reveals a planet of reflectivity comparable to the solar system giants (A_g = 0.4–0.5), which is unexpectedly high for a close-in gas planet. Theory indeed predicts that the strong stellar irradiation that a planet in such an orbit experiences strips off reflective clouds, rendering the planet dark (A_g <  0.1) (22, 25). The prediction is largely consistent with empirical evidence, and dark planets dominate the sample of known close-in giant planets (8, 13, 21, 29, 30). Exceptions exist, and other planets [51 Peg b, A_g = 0.5  ×  (1.9/(R_p/R_J))² at 0.38–0.69 μm (31); HD 189733b, A_g = 0.40 ± 0.12 at 0.29–0.45 μm (32); and KOI-196b, A_g = 0.30 ± 0.08 at 0.4–0.9 μm (33)] with elevated albedos suggest that we are beginning to sample the diversity of exoplanet atmospheres. Potentially compensating for strong stellar irradiation, Kepler-7b’s low surface gravity (417 cm s⁻²) may help sustain reflective condensates lofted in the upper atmosphere that would increase the planet albedo (25).Brightness temperatures for Kepler-7b inferred from occultations at 3.6 μm and 4.5 μm with Spitzer [<1,700 K and 1,840 K, respectively (7)] are well below the equivalent brightness temperature deduced from Kepler data (∼2,600 K). This key constraint, placed in the framework of heat recirculation in the atmospheres of close-in giants, is evidence that the Kepler optical phase curve is dominated by reflected starlight rather than by thermal emission (7, 21, 34). Interestingly, the peak of the optical phase curve occurs after secondary eclipse (OP >  0.5), when the planet as viewed from Earth is not fully illuminated and longitudes westward of the substellar point are preferentially probed. This asymmetry hints at a spatial structure in Kepler-7b’s envelope caused by horizontally inhomogenous clouds (7, 21, 34). Subsequent investigations have identified other planets that show similar offset between occultation and peak brightness (4, 10). However, the lack of infrared measurements for these means that it has not been possible to rule out contamination in the optical by a thermal component as the cause of the asymmetry.Recent work has used the optical phase curve of Kepler-7b to build brightness maps (7, 34), investigate the prevalence of reflected starlight over thermal emission (34), and explore plausible cloud configurations (35). No previous study has systematically connected the extent, location, and optical thickness of the cloud, or the composition and size of the suspended particles, to the measured phase curve. That exercise is the objective of this paper.     

Physical evidence of predatory behavior in Tyrannosaurus rex

Feeding strategies of the large theropod, Tyrannosaurus rex, either as a predator or a scavenger, have been a topic of debate previously compromised by lack of definitive physical evidence. Tooth drag and bone puncture marks have been documented on suggested prey items, but are often difficult to attribute to a specific theropod. Further, postmortem damage cannot be distinguished from intravital occurrences, unless evidence of healing is present. Here we report definitive evidence of predation by T. rex: a tooth crown embedded in a hadrosaurid caudal centrum, surrounded by healed bone growth. This indicates that the prey escaped and lived for some time after the injury, providing direct evidence of predatory behavior by T. rex. The two traumatically fused hadrosaur vertebrae partially enclosing a T. rex tooth were discovered in the Hell Creek Formation of South Dakota.One of the most daunting tasks of paleontology is inferring the behavior and feeding habits of extinct organisms. Accurate reconstruction of the lifestyle of extinct animals is dependent on the fossil evidence and its interpretation is most confidently predicated on analogy with modern counterparts (1–6). This challenge to understanding the lifestyle of extinct animals is exemplified by the controversy over the feeding behavior of the Late Cretaceous theropod Tyrannosaurus rex (3, 7–17). Although predation and scavenging have often been suggested as distinct feeding behavior alternatives (3, 7–9, 11–17), these terms merit semantic clarification. In this study, predation is considered a subset of feeding behavior, by which any species kills what it eats. Although the term “predator” is used to distinguish such animals from obligate scavengers, it does not imply that the animal did not also scavenge.Ancient diets can be readily reconstructed on the basis of the available evidence, although their derivation (e.g., predation or scavenging behavior) often remains elusive. Speculation as to dinosaur predation has ranged from inferences based on skeletal morphology, ichnofossils such as bite marks, coprolites, stomach contents, and trackways and, by more rarely, direct predator–prey skeletal associations (3, 4, 18–23).Direct evidence of predation in nonavian dinosaurs other than tyrannosaurids has been observed in rare instances, such as the Deinonychus–Tenontosaurus kill site of the Cloverly Formation where the remains of both were found in close association along with shed teeth (9, 24), and the “fighting dinosaurs” from the Gobi Desert, in which a Velociraptor and Protoceratops were found locked in mortal combat (9, 17). The evidence on tyrannosaurids is more limited. Putative stomach contents, such as partially digested juvenile hadrosaur bones, have been reported in association with tyrannosaurid remains (3, 12, 18). This latter instance only represents physical evidence of the last items consumed before the animal’s death, an indicator of diet but not behavior.Mass death assemblages of ornithischians frequently preserve shed theropod teeth (6, 22, 24). Lockley et al. (23) suggest such shed teeth are evidence of scavenging behavior. It is widely argued that T. rex procured food through obligate scavenging rather than hunting (11, 14, 25–27) despite the fact that there is currently no modern analog for such a large bodied obligate scavenger (26). Horner (25) argued that T. rex was too slow to pursue and capture prey items (14) and that large theropods procured food solely through scavenging, rather than hunting (11, 25). Horner also suggested that the enlarged olfactory lobes in T.rex were characteristic of scavengers (25). More recent studies (28, 29) determined the olfactory lobes of modern birds are “poorly developed,” inferring that enlarged olfactory lobes in T. rex are actually a secondary adaptation for predation navigation “to track mobile, dispersed prey” (30). T. rex has a calculated bite force stronger than that of any other terrestrial predator (7), between 35,000 and 57,000 Newtons (30, 31), and possible ambulatory speeds between 20 and 40 kph (7, 15, 16), documenting that it had the capability to pursue and kill prey items.Healed injuries on potential prey animals provide the most unequivocal evidence of survival of a traumatic event (e.g., predation attempt) (3, 32, 33), and several reports attribute such damage to T. rex (4, 17, 19, 20). These include broken and healed proximal caudal vertebral dorsal spines in Edmontosaurus (17) and healed cranial lesions in Triceratops (4, 19). Although the presence of healed injuries demonstrates that an animal lived long enough after the attack to create new bone at the site of the damage (a rare occurrence in the fossil record) (19), the healing usually obliterates any clear signature linking the injury to a specific predator. Bite traces (e.g., raking tooth marks on bone and puncture wounds in the bones of possible prey animals) attributed to T. rex (2, 4, 19) are ambiguous, because the damage inflicted upon an animal during and after a successful hunt mirrors feeding during scavenging. This makes distinction between the two modes of food acquisition virtually impossible with such evidence (3, 34–38).Tooth marks, reported from dinosaur bone-bearing strata worldwide (e.g., 2–4, 8, 19, 20, 39, 40), are further direct evidence of theropod feeding behavior, attributed by some to specific theropod groups (2, 4, 19, 20). Happ (19) and Carpenter (17) identified theropods to family and genus by matching spaces to parallel marks (traces) with intertooth distance. Happ (19) described opposing conical depressions on a left supraorbital Triceratops horn that was missing its distal third (tip), attributing them to a bite by either a T. rex or a crocodilian. Happ (19) stated that the spacing of the parallel marks present on the left squamosal of the same individual matched the intertooth distance of tyrannosaurids. The presence of periosteal reaction documents healing. This contrasts with the report by Farlow and Holtz (3) and again by Hone and Rauhut (20) of the same Hypacrosaurus fibula containing a superficially embedded theropod tooth. Absence of bone reaction precludes confident attribution to predation.Two coalesced hadrosaur (compare with Edmontosaurus annectens) caudal vertebrae were discovered in the Hell Creek Formation of Harding County, South Dakota (40). Archosaur fauna identified in this site include crocodiles, dinosaurs, and birds (41). Physical evidence of dental penetration and extensive infection (osteomylitis) of the fused vertebral centra and healing (bone overgrowth) document an unsuccessful attack by a large predator. A tooth crown was discovered within the wound, permitting identification of the predator as T. rex. This is unambiguous evidence that T. rex was an active predator, fulfilling the criteria that Farlow and Holtz (3) advanced. As T. rex comprises between 1% and 16% of the Upper Cretaceous dinosaurian fauna in Western North America (41–45), its status as a predator or obligate scavenger is nontrivial and could have significant implications for paleoecological reconstructions of that time period. The present contribution provides unique information demonstrating the ecological role for T. rex as that of an active predator. Despite this documentation of predatory behavior by T. rex, we do not make the argument that T. rex was an obligate predator. Like most modern large predators (27, 45) it almost certainly did also scavenge carcasses (9, 16).     

Flight performance of the largest volant bird

Pelagornithidae is an extinct clade of birds characterized by bizarre tooth-like bony projections of the jaws. Here, the flight capabilities of pelagornithids are explored based on data from a species with the largest reported wingspan among birds. Pelagornis sandersi sp. nov. is represented by a skull and substantial postcranial material. Conservative wingspan estimates (∼6.4 m) exceed theoretical maximums based on extant soaring birds. Modeled flight properties indicate that lift:drag ratios and glide ratios for P. sandersi were near the upper limit observed in extant birds and suggest that pelagornithids were highly efficient gliders, exploiting a long-range soaring ecology.Flight ability in birds is largely governed by scaling effects, leading to a tradeoff between the benefits of physiological efficiencies and the drawbacks of decreasing power margin (ratio of available muscle power to required mechanical power) as size increases (1–4). Over their ∼150-My history, birds evolved to span at least four orders of magnitude in size (2) and achieve a wide variety of flight styles, such a flap-gliding, soaring, and hovering. Today, the largest directly measured wild individuals of volant birds reach wingspans of ∼3.5 m (Royal Albatross Diomedea exulans) and masses of ∼19 kg (Great Bustard Otis tarda and Kori Bustard Ardeotis kori) (5). In the past, the extinct terrestrial teratorn Argentavis magnificens (6) and the extinct soaring Pelagornithidae (7–10) greatly exceeded these sizes. Given the challenges of flight at large size, there has been much debate over potential upper size limits for different styles of flight in vertebrates (1, 2, 11–13), and the evolution of specialized taxa, like teratorns and pelagornithids, has been tied to environmental factors, such as wind patterns (6, 14–16).A well-preserved associated skeleton representing the largest known volant bird provides the basis for the present study of flight properties in the remarkable extinct clade Pelagornithidae. Pelagornithidae appeared in the Paleocene (9) and attained a global distribution before going extinct in the Pliocene (8, 17). Skeletal characteristics, including pseudoteeth (spike-like protrusions of the jaw bones), a hinged mandible, and specialized wing bones (7–10, 18, 19), have raised questions about the paleoecology and phylogenetic affinities of Pelagornithidae. Although Pelagornithidae has historically been linked to the Pelecaniformes and Procellariiformes (7, 8, 20), more recent phylogenetic analyses suggest that this extinct clade is the sister taxon to Anseriformes (waterfowl) (9) or Galloanserae (waterfowl and landfowl) (21).Regardless of their affinities, Pelagornithidae evolved such highly modified skeletal morphologies that it would not be plausible to make meaningful inferences about their flight style and ecology based on extant relatives. Therefore, computer modeling provides the best path toward understanding the flight capabilities of these remarkable birds. Flight 1.25 (2), a program designed to model avian flight under user-specified conditions, is ideally suited to this task, because it models both flapping and gliding flight performance. In this study, Flight 1.25 is applied to infer flight parameters in the largest known pelagornithid species. As with all research on fossil organisms, estimating variables, such as mass and feather length, requires additional extrapolation, and these uncertainties are taken into account with 24 iterative analyses based on combinations of viable mass estimates, aspect ratios, and wingspans as described in Methods.     

Highly sensitive detection of nanoparticles with a self-referenced and self-heterodyned whispering-gallery Raman microlaser

Optical whispering-gallery-mode resonators (WGMRs) have emerged as promising platforms for label-free detection of nano-objects. The ultimate sensitivity of WGMRs is determined by the strength of the light–matter interaction quantified by quality factor/mode volume, Q/V, and the resolution is determined by Q. To date, to improve sensitivity and precision of detection either WGMRs have been doped with rare-earth ions to compensate losses and increase Q or plasmonic resonances have been exploited for their superior field confinement and lower V. Here, we demonstrate, for the first time to our knowledge, enhanced detection of single-nanoparticle-induced mode splitting in a silica WGMR via Raman gain-assisted loss compensation and WGM Raman microlaser. In particular, the use of the Raman microlaser provides a dopant-free, self-referenced, and self-heterodyned scheme with a detection limit ultimately determined by the thermorefractive noise. Notably, we detected and counted individual nanoparticles with polarizabilities down to 3.82 × 10⁻⁶ μm³ by monitoring a heterodyne beatnote signal. This level of sensitivity is achieved without exploiting plasmonic effects, external references, or active stabilization and frequency locking. Single nanoparticles are detected one at a time; however, their characterization by size or polarizability requires ensemble measurements and statistical averaging. This dopant-free scheme retains the inherited biocompatibility of silica and could find widespread use for sensing in biological media. The Raman laser and operation band of the sensor can be tailored for the specific sensing environment and the properties of the targeted materials by changing the pump laser wavelength. This scheme also opens the possibility of using intrinsic Raman or parametric gain for loss compensation in other systems where dissipation hinders progress and limits applications.There is an increasing demand for new technologies to detect small molecules, nanoparticles, and airborne species (1–4). In the past decade we have witnessed a boost in the number of label-free detection techniques with varying levels of sensitivities. Techniques relying on electrical conductance (5), light scattering and interferometry (6–8), surface and localized plasmon resonance (9, 10), nanomechanical resonators (11, 12), and optical resonances (13–17) have been demonstrated.Whispering-gallery-mode (WGM) microresonators with their high quality factor, Q, and small mode volume, V, are known to enhance light–matter interactions and have extraordinary sensitivities to changes and perturbations in their structure or proximity (18, 19). They have been of great interest for sensing biomarkers, DNA, and medium-size proteins at low concentrations, as well as for detecting viruses and nanoparticles at single-particle resolution (19–31). A particle or molecule entering the mode volume of a resonator or binding onto its surface induces a net change in the polarizability of the resonator-surrounding system and perturbs its optical properties (19). This manifests itself as a shift of the resonance frequency, broadening of the resonance linewidth, or formation of a doublet via mode splitting depending on the interaction strength and the scattering and absorption properties of the binding particle or the molecule (14, 15, 17, 32, 33).In WGM sensors, the fundamental limit of sensitivity is determined by Q/V, which quantifies the strength of the interaction between the particle and the cavity field. Thus, it can be improved by decreasing V or increasing Q. One can increase Q by compensating the losses and decrease V by shrinking the size of the WGM resonator (WGMR). However, decreasing the resonator size below a critical value inevitably increases bending losses and eventually decreases Q. Instead, hybrid systems combining high-Q WGMs with highly confined (small-V) localized plasmons have been demonstrated (21, 22, 24, 27, 34), achieving detection of single proteins and very small viruses. Q enhancement of WGM resonances by compensating losses via optical gain has also been demonstrated (15, 35, 36) in silica microtoroids doped with rare-earth ions such as erbium (Er³⁺) and ytterbium (Yb³⁺). Resonators with optical gain are referred to as active resonators (36–38). When such a WGMR is optically pumped above lasing threshold, the resultant laser has a narrower linewidth than the cold cavity and thereby improves the detection limit and sensitivity beyond what can be achieved by the passive (no optical gain-providing mechanism) or by the active resonator below lasing threshold (15, 35, 38). However, fabricating WGM–plasmon hybrids and active WGMRs with dopants introduces additional processing steps and costs. For example, WGM–plasmon hybrids require preparation and adsorption of plasmonic nanostructures onto the resonator surface, and active resonators suffer from the fact that most rare-earth ions are not biocompatible and that for each different wavelength band of operation a different rare-earth ion and a different pump laser should be used.     

A single mutation weakens symbiont-induced reproductive manipulation through reductions in deubiquitylation efficiency

Animals interact with microbes that affect their performance and fitness, including endosymbionts that reside inside their cells. Maternally transmitted Wolbachia bacteria are the most common known endosymbionts, in large part because of their manipulation of host reproduction. For example, many Wolbachia cause cytoplasmic incompatibility (CI) that reduces host embryonic viability when Wolbachia-modified sperm fertilize uninfected eggs. Operons termed cifs control CI, and a single factor (cifA) rescues it, providing Wolbachia-infected females a fitness advantage. Despite CI’s prevalence in nature, theory indicates that natural selection does not act to maintain CI, which varies widely in strength. Here, we investigate the genetic and functional basis of CI-strength variation observed among sister Wolbachia that infect Drosophila melanogaster subgroup hosts. We cloned, Sanger sequenced, and expressed cif repertoires from weak CI–causing wYak in Drosophila yakuba, revealing mutations suspected to weaken CI relative to model wMel in D. melanogaster. A single valine-to-leucine mutation within the deubiquitylating (DUB) domain of the wYak cifB homolog (cidB) ablates a CI-like phenotype in yeast. The same mutation reduces both DUB efficiency in vitro and transgenic CI strength in the fly, each by about twofold. Our results map hypomorphic transgenic CI to reduced DUB activity and indicate that deubiquitylation is central to CI induction in cid systems. We also characterize effects of other genetic variation distinguishing wMel-like cifs. Importantly, CI strength determines Wolbachia prevalence in natural systems and directly influences the efficacy of Wolbachia biocontrol strategies in transinfected mosquito systems. These approaches rely on strong CI to reduce human disease.

Many endosymbionts spread through host populations by manipulating their reproduction. For example, Rickettsiella (1), Mesenet (2), Cardinium (3), and Wolbachia (4) all cause cytoplasmic incompatibility (CI) that reduces the viability of uninfected host embryos fertilized by symbiont-modified sperm (5–8). CI is common among Wolbachia bacterial strains, being observed in at least 10 arthropod orders (6). CI strength influences Wolbachia prevalence, with stronger CI producing higher Wolbachia infection frequencies in host populations (8, 9). Indeed, CI contributes significantly to Wolbachia’s status as the most-common known endosymbionts in nature (10).CI strength directly influences the efficacy of Wolbachia biocontrol programs, with vector-control groups relying on strong CI to either suppress mosquito populations (11, 12) or to transform them with pathogen-blocking Wolbachia like wMel that naturally infects Drosophila melanogaster (13–15). The World Health Organization recommends further developing these programs (16), which are currently protecting seven million people from disease with a goal of protecting half a billion by 2030 (14, 17).Operons generally termed cifs control CI (cifA/B) (5, 18–22), and CI induction can be rescued by one factor (cifA) (5, 19, 23). Theory indicates that natural selection does not act to increase or maintain CI (24), which varies considerably among even very closely related Wolbachia (25–27), potentially due to mutational erosion of cifs (28). For example, CI strength differs significantly among model wMel from Drosophila melanogaster and closely related wMel-like Wolbachia in the Drosophila yakuba clade (wYak, wSan, and wTei) that wMel diverged from in only the last 30,000 y (25, 27, 29, 30). We sought to determine how much and why naturally observed mutations in wMel-like cifs influence CI strength.     

From the Cover: Predicting overfishing and extinction threats in multispecies fisheries

Threats to species from commercial fishing are rarely identified until species have suffered large population declines, by which time remedial actions can have severe economic consequences, such as closure of fisheries. Many of the species most threatened by fishing are caught in multispecies fisheries, which can remain profitable even as populations of some species collapse. Here we show for multispecies fisheries that the biological and socioeconomic conditions that would eventually cause species to be severely depleted or even driven extinct can be identified decades before those species experience high harvest rates or marked population declines. Because fishing effort imposes a common source of mortality on all species in a fishery, the long-term impact of a fishery on a species is predicted by measuring its loss rate relative to that of species that influence the fishery’s maximal effort. We tested our approach on eight Pacific tuna and billfish populations, four of which have been identified recently as in decline and threatened with overfishing. The severe depletion of all four populations could have been predicted in the 1950s, using our approach. Our results demonstrate that species threatened by human harvesting can be identified much earlier, providing time for adjustments in harvesting practices before consequences become severe and fishery closures or other socioeconomically disruptive interventions are required to protect species.Marine fisheries are an important global source of food and livelihoods (1–4), but there are concerns that current fishing practices threaten some marine species with severe depletion or eventual extinction (2–5). Many of the largest commercial fishing methods, such as trawling, longlining, and seining, unavoidably catch multiple species simultaneously (6–9). Multispecies fisheries pose a particular threat of extinction or severe depletion because fishing can remain profitable as long as some valuable species remain abundant, even while others collapse (6–11). In contrast, in a single-species fishery profits tend to fall as the target population declines, thereby removing the incentive to fish before extinction occurs (10). Multispecies fisheries pose a threat to two types of species or stocks (populations): (i) commercially valued species, called “weak stocks”, which are more vulnerable to overharvesting than are other commercially valuable species (6), and (ii) by-catch species, which are caught accidentally and create little economic incentive to cease fishing as their populations collapse because they have little or no commercial value (7–9).Failure to prevent collapse of weak stocks and by-catch species can impose substantial long-term environmental and economic costs. Slow-growing populations are most likely to collapse, but can take several decades to recover (5). Recovery often requires long-term fishery closures or reductions in effort, having substantial economic and social consequences (3, 5). Moreover, population declines caused by one fishery can diminish yields and profits in other commercial or artisanal fisheries that depend on the same species (e.g., ref. 12).Despite these costs, species threatened by fishing have rarely been identified until after their populations have declined substantially (2–5, 7, 8). Assessments of fishery impacts on species mostly focus on estimating current exploitation rates or past population trends (13–15), which identifies already declining species rather than predicting future declines. Data limitations have made empirical prediction of future threats from fishing challenging, particularly for weak stocks and by-catch species. Oceans are difficult to sample extensively, and few economic incentives exist to gather data on species other than the most commercially valued species (7, 8). Some predictive models (e.g., ref. 16) have been developed to forecast the impacts of some fisheries, but these are often data intensive. Some of the characteristics that make a population susceptible to overfishing are well known—for example, low population growth rates (3–11, 17, 18), high value and/or low fishing costs (10, 11, 17–19), and schooling behavior (18). Recently, some correlative approaches based on these characteristics have been developed for assessing likely relative threats to data-poor species (4, 20–22). However, predicting the severity of future threats in absolute terms with this type of approach can be challenging.Here, we present a mechanistic approach that uses readily available data to predict the potential of current fishing practices, if maintained, to eventually cause a population to be driven extinct or “overfished”, here defined as depletion below its maximum sustainable yield (MSY) abundance (N_MSY) (3). Our approach identifies combinations of biological and socioeconomic conditions that are likely to eventually lead to high mortality rates and population declines. As we show, these conditions can be identified long before either occurs.We test the predictive power of our approach on eight tuna and billfish populations of the Western and Central Pacific Ocean fisheries. High-seas tuna and billfish have elicited recent conservation concern due to significant population declines and range contractions found in many species (17, 23, 24). Three of the populations in our study, bigeye tuna (Thunnus obesus) and both the northern and the southern striped marlin (Tetrapturus audax) populations, have been recently identified as experiencing overfishing—meaning their exploitation rates have exceeded the MSY exploitation rate (F_MSY) (24–27). A fourth, blue marlin (Makaira nigricans), whose overfishing status has been subject to considerable uncertainty (28), has undergone a significant population decline and range contraction (13, 23, 28). We determine whether our approach could have predicted threats to these four populations, using data from as early as the 1950s, and assess the threats predicted by the latest available data to all populations.     

Coulomb interaction,phonons, and superconductivity in twisted bilayer graphene

The polarizability of twisted bilayer graphene, due to the combined effect of electron–hole pairs, plasmons, and acoustic phonons, is analyzed. The screened Coulomb interaction allows for the formation of Cooper pairs and superconductivity in a significant range of twist angles and fillings. The tendency toward superconductivity is enhanced by the coupling between longitudinal phonons and electron–hole pairs. Scattering processes involving large momentum transfers, Umklapp processes, play a crucial role in the formation of Cooper pairs. The magnitude of the superconducting gap changes among the different pockets of the Fermi surface.

Twisted bilayer graphene (TBG) shows a complex phase diagram which combines superconducting and insulating phases (1, 2) and resembles strongly correlated materials previously encountered in condensed matter physics (3–6). On the other hand, superconductivity seems more prevalent in TBG (7–11), while in other strongly correlated materials magnetic phases are dominant.The pairing interaction responsible for superconductivity in TBG has been intensively studied. Among other possible pairing mechanisms, the effect of phonons (12–19) (see also ref. 20), the proximity of the chemical potential to a van Hove singularity in the density of states (DOS) (21–25) and excitations of insulating phases (26–28) (see also refs. 29–31), and the role of electronic screening (32–35) have been considered.In the following, we analyze how the screened Coulomb interaction induces pairing in TBG. The calculation is based on the Kohn–Luttinger formalism (36) for the study of anisotropic superconductivity via repulsive interactions. The screening includes electron–hole pairs (37), plasmons (38), and phonons (note that acoustic phonons overlap with the electron–hole continuum in TBG). Our results show that the repulsive Coulomb interaction, screened by plasmons and electron–hole pairs only, leads to anisotropic superconductivity, although with critical temperatures of order T_c ∼ 10⁻³ to 10⁻² K. The inclusion of phonons in the screening function substantially enhances the critical temperature, to T_c ∼ 1 to 10 K.     

Direct numerical simulations of aeolian sand ripples

Aeolian sand beds exhibit regular patterns of ripples resulting from the interaction between topography and sediment transport. Their characteristics have been so far related to reptation transport caused by the impacts on the ground of grains entrained by the wind into saltation. By means of direct numerical simulations of grains interacting with a wind flow, we show that the instability turns out to be driven by resonant grain trajectories, whose length is close to a ripple wavelength and whose splash leads to a mass displacement toward the ripple crests. The pattern selection results from a compromise between this destabilizing mechanism and a diffusive downslope transport which stabilizes small wavelengths. The initial wavelength is set by the ratio of the sediment flux and the erosion/deposition rate, a ratio which increases linearly with the wind velocity. We show that this scaling law, in agreement with experiments, originates from an interfacial layer separating the saltation zone from the static sand bed, where momentum transfers are dominated by midair collisions. Finally, we provide quantitative support for the use of the propagation of these ripples as a proxy for remote measurements of sediment transport.Observers have long recognized that wind ripples (1, 2) do not form via the same dynamical mechanism as dunes (3). Current explanations ascribe their emergence to a geometrical effect of solid angle acting on sediment transport. The motion of grains transported in saltation is composed of a series of asymmetric trajectories (4–7) during which they are accelerated by the wind. These grains, in turn, decelerate the airflow inside the transport layer (1, 7–12). On hitting the sand bed, they release a splash-like shower of ejected grains that make small hops from the point of impact (1, 13, 14). This process is called reptation. Previous wind ripple models assume that saltation is insensitive to the sand bed topography and forms a homogeneous rain of grains approaching the bed at a constant oblique angle (15–20). Upwind-sloping portions of the bed would then be submitted to a higher impacting flux than downslopes (1). With a number of ejecta proportional to the number of impacting grains, this effect would produce a screening instability with an emergent wavelength λ determined by the typical distance over which ejected grains are transported (15–17), a few grain diameters d. However, observed sand ripple wavelengths are about 1,000 times larger than d, on Earth. The discrepancy is even more pronounced on Mars, where regular ripples are 20–40 times larger than those on a typical Earth sand dune (21, 22). Moreover, the screening scenario predicts a wavelength independent of the wind shear velocity u_?, in contradiction with field and wind tunnel measurements that exhibit a linear dependence of λ with u_? (23–25).     

Microscopic origins of the crystallographically preferred growth in evaporation-induced colloidal crystals

Unlike crystalline atomic and ionic solids, texture development due to crystallographically preferred growth in colloidal crystals is less studied. Here we investigate the underlying mechanisms of the texture evolution in an evaporation-induced colloidal assembly process through experiments, modeling, and theoretical analysis. In this widely used approach to obtain large-area colloidal crystals, the colloidal particles are driven to the meniscus via the evaporation of a solvent or matrix precursor solution where they close-pack to form a face-centered cubic colloidal assembly. Via two-dimensional large-area crystallographic mapping, we show that the initial crystal orientation is dominated by the interaction of particles with the meniscus, resulting in the expected coalignment of the close-packed direction with the local meniscus geometry. By combining with crystal structure analysis at a single-particle level, we further reveal that, at the later stage of self-assembly, however, the colloidal crystal undergoes a gradual rotation facilitated by geometrically necessary dislocations (GNDs) and achieves a large-area uniform crystallographic orientation with the close-packed direction perpendicular to the meniscus and parallel to the growth direction. Classical slip analysis, finite element-based mechanical simulation, computational colloidal assembly modeling, and continuum theory unequivocally show that these GNDs result from the tensile stress field along the meniscus direction due to the constrained shrinkage of the colloidal crystal during drying. The generation of GNDs with specific slip systems within individual grains leads to crystallographic rotation to accommodate the mechanical stress. The mechanistic understanding reported here can be utilized to control crystallographic features of colloidal assemblies, and may provide further insights into crystallographically preferred growth in synthetic, biological, and geological crystals.

As an analogy to atomic crystals, colloidal crystals are highly ordered structures formed by colloidal particles with sizes ranging from 100 nm to several micrometers (1–6). In addition to engineering applications such as photonics, sensing, and catalysis (4, 5, 7, 8), colloidal crystals have also been used as model systems to study some fundamental processes in statistical mechanics and mechanical behavior of crystalline solids (9–14). Depending on the nature of interparticle interactions, many equilibrium and nonequilibrium colloidal self-assembly processes have been explored and developed (1, 4). Among them, the evaporation-induced colloidal self-assembly presents a number of advantages, such as large-size fabrication, versatility, and cost and time efficiency (3–5, 15–18). In a typical synthesis where a substrate is immersed vertically or at an angle into a colloidal suspension, the colloidal particles are driven to the meniscus by the evaporation-induced fluid flow and subsequently self-assemble to form a colloidal crystal with the face-centered cubic (fcc) lattice structure and the close-packed {111} plane parallel to the substrate (2, 3, 19–23) (see Fig. 1A for a schematic diagram of the synthetic setup).Open in a separate windowFig. 1.Evaporation-induced coassembly of colloidal crystals. (A) Schematic diagram of the evaporation-induced colloidal coassembly process. “G”, “M”, and “N” refer to “growth,” “meniscus,” and “normal” directions, respectively. The reaction solution contains silica matrix precursor (tetraethyl orthosilicate, TEOS) in addition to colloids. (B) Schematic diagram of the crystallographic system and orientations used in this work. (C and D) Optical image (Top Left) and scanning electron micrograph (SEM) (Bottom Left) of a typical large-area colloidal crystal film before (C) and after (D) calcination. (Right) SEM images of select areas (yellow rectangles) at different magnifications. Corresponding fast-Fourier transform (see Inset in Middle in C) shows the single-crystalline nature of the assembled structure. (E) The 3D reconstruction of the colloidal crystal (left) based on FIB tomography data and (right) after particle detection. (F) Top-view SEM image of the colloidal crystal with crystallographic orientations indicated.While previous research has focused on utilizing the assembled colloidal structures for different applications (4, 5, 7, 8), considerably less effort is directed to understand the self-assembly mechanism itself in this process (17, 24). In particular, despite using the term “colloidal crystals” to highlight the microstructures’ long-range order, an analogy to atomic crystals, little is known regarding the crystallographic evolution of colloidal crystals in relation to the self-assembly process (3, 22, 25). The underlying mechanisms for the puzzling—yet commonly observed—phenomenon of the preferred growth along the close-packed <110> direction in evaporation-induced colloidal crystals are currently not understood (3, 25–29). The <110> growth direction has been observed in a number of processes with a variety of particle chemistries, evaporation rates, and matrix materials (3, 25–28, 30), hinting at a universal underlying mechanism. This behavior is particularly intriguing as the colloidal particles are expected to close-pack parallel to the meniscus, which should lead to the growth along the <112> direction and perpendicular to the <110> direction (16, 26, 31)^*.Preferred growth along specific crystallographic orientations, also known as texture development, is commonly observed in crystalline atomic solids in synthetic systems, biominerals, and geological crystals. While current knowledge recognizes mechanisms such as the oriented nucleation that defines the future crystallographic orientation of the growing crystals and competitive growth in atomic crystals (32–34), the underlying principles for texture development in colloidal crystals remain elusive. Previous hypotheses based on orientation-dependent growth speed and solvent flow resistance are inadequate to provide a universal explanation for different evaporation-induced colloidal self-assembly processes (3, 25–29). A better understanding of the crystallographically preferred growth in colloidal self-assembly processes may shed new light on the crystal growth in atomic, ionic, and molecular systems (35–37). Moreover, mechanistic understanding of the self-assembly processes will allow more precise control of the lattice types, crystallography, and defects to improve the performance and functionality of colloidal assembly structures (38–40).     

Precise spatial structure impacts antimicrobial susceptibility of S. aureus in polymicrobial wound infections

A hallmark of microbial ecology is that interactions between members of a community shape community function. This includes microbial communities in human infections, such as chronic wounds, where interactions can result in more severe diseases. Staphylococcus aureus is the most common organism isolated from human chronic wound infections and has been shown to have both cooperative and competitive interactions with Pseudomonas aeruginosa. Still, despite considerable study, most interactions between these microbes have been characterized using in vitro well-mixed systems, which do not recapitulate the infection environment. Here, we characterized interactions between S. aureus and P. aeruginosa in chronic murine wounds, focusing on the role that both macro- and micro-scale spatial structures play in disease. We discovered that S. aureus and P. aeruginosa coexist at high cell densities in murine wounds. High-resolution imaging revealed that these microbes establish a patchy distribution, only occupying 5 to 25% of the wound volume. Using a quantitative framework, we identified a precise spatial structure at both the macro (mm)- and micro (µm)-scales, which was largely mediated by P. aeruginosa production of the antimicrobial 2-heptyl-4-hydroxyquinoline N-oxide, while the antimicrobial pyocyanin had no impact. Finally, we discovered that this precise spatial structure enhances S. aureus tolerance to aminoglycoside antibiotics but not vancomycin. Our results provide mechanistic insights into the biogeography of S. aureus and P. aeruginosa coinfected wounds and implicate spatial structure as a key determinant of antimicrobial tolerance in wound infections.

Polymicrobial human infections are a major burden on human health. These infections are often more tolerant to antibiotics and have worse clinical outcomes compared to their single-microbe counterparts (1–7). Properties specific to polymicrobial infections are often attributed to interactions occurring between microbes, and much work has been done to identify and mechanistically understand these interactions (8–12). Recent evidence using preclinical infection models has shown that interactions between microbes impact the micron-scale spatial structure of the infecting community (13–16), implicating the spatial structure as a key component controlling community function, and thus infection outcomes (17). However, most of our understanding of polymicrobial interactions is derived from studies using in vitro models (13, 14). Hence, key elements of infection dynamics and the role of host factors are often overlooked.Pseudomonas aeruginosa and Staphylococcus aureus are commonly used to study microbe–microbe interactions, both in vitro and in vivo (11, 13, 18–24). These microbes cooccur in several polymicrobial human infections, including chronic wounds and in the lungs of people with cystic fibrosis (1–3, 5, 25–29). There is conflicting evidence regarding the impact of coinfection on human disease outcomes, with some studies concluding that P. aeruginosa alone has worse outcomes (32–34) while others conclude that P. aeruginosa–S. aureus coinfections lead to more severe diseases (35–37). The experimental data are clearer in murine models of infection, which have shown that coinfection can result in increased antibiotic tolerance and worse infection outcomes (8, 10, 20, 28). While the mechanisms controlling these synergistic interactions are largely unknown in vivo, it has been hypothesized that P. aeruginosa and S. aureus occupy distinct regions in human chronic wounds (28), suggesting that biogeography may play a role in mediating polymicrobial wound infection outcomes.Here, we collected more than 100 high-resolution confocal images of mouse chronic wounds infected with P. aeruginosa and S. aureus in mono- and co-infection. Using these images, we quantified the 3-dimensional macro- and micron-scale spatial structure of P. aeruginosa and S. aureus communities in vivo and defined the role of known P. aeruginosa extracellular antimicrobials on the spatial structure. We discovered that S. aureus and P. aeruginosa coexist in mouse wound infections at high bacterial densities, but their distribution is patchy. In addition, we discovered and quantified a precise, micron-scale spatial structure dependent on the P. aeruginosa-secreted small-molecule 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) and that this spatial structure is different at the healing edge versus the center of the wound. Finally, we show that the community spatial structure has clinically important outcomes, including altered antibiotic tolerance.     

miR-182 targeting reprograms tumor-associated macrophages and limits breast cancer progression

The protumor roles of alternatively activated (M2) tumor-associated macrophages (TAMs) have been well established, and macrophage reprogramming is an important therapeutic goal. However, the mechanisms of TAM polarization remain incompletely understood, and effective strategies for macrophage targeting are lacking. Here, we show that miR-182 in macrophages mediates tumor-induced M2 polarization and can be targeted for therapeutic macrophage reprogramming. Constitutive miR-182 knockout in host mice and conditional knockout in macrophages impair M2-like TAMs and breast tumor development. Targeted depletion of macrophages in mice blocks the effect of miR-182 deficiency in tumor progression while reconstitution of miR-182-expressing macrophages promotes tumor growth. Mechanistically, cancer cells induce miR-182 expression in macrophages by TGFβ signaling, and miR-182 directly suppresses TLR4, leading to NFκb inactivation and M2 polarization of TAMs. Importantly, therapeutic delivery of antagomiR-182 with cationized mannan-modified extracellular vesicles effectively targets macrophages, leading to miR-182 inhibition, macrophage reprogramming, and tumor suppression in multiple breast cancer models of mice. Overall, our findings reveal a crucial TGFβ/miR-182/TLR4 axis for TAM polarization and provide rationale for RNA-based therapeutics of TAM targeting in cancer.

It is well known that the nonmalignant stromal components in tumors play pivotal roles in tumor progression and therapeutic responses (1, 2). Macrophages are a major component of tumor microenvironment and display considerable phenotypic plasticity in their effects toward tumor progression (3–5). Classically activated (M1) macrophages often exert direct tumor cytotoxic effects or induce antitumor immune responses by helping present tumor-related antigens (6, 7). In contrast, tumoral cues can polarize macrophages toward alternative activation with immunosuppressive M2 properties (6–8). Numerous studies have firmly established the protumor effects of M2-like tumor-associated macrophages (TAMs) and the association of TAMs with poor prognosis of human cancer (9–11). However, how tumors induce the coordinated molecular and phenotypic changes in TAMs for M2 polarization remains incompletely understood, impeding the designing of TAM-targeting strategies for cancer intervention. In addition, drug delivery also represents a hurdle for therapeutic macrophage reprogramming.Noncoding RNAs, including microRNAs, have been shown to play vital roles in various pathological processes of cancer (12). The microRNA miR-182 has been implicated in various developmental processes and disease conditions (13–15). Particularly, it receives extensive attention in cancer studies. Prevalent chromosomal amplification of miR-182 locus and up-regulation of its expression in tumors have been observed in numerous cancer types including breast cancer, gastric cancer, lung adenocarcinoma, colorectal adenocarcinoma, ovarian carcinoma, and melanoma (16–21). miR-182 expression is also linked to higher risk of metastasis and shorter survival of patients (20, 22–24). Functional studies showed that miR-182 expression in cancer cells plays vital roles in various aspects of cancer malignancy, including tumor proliferation (25–29), migration (30, 31), invasion (16, 32, 33), epithelial-mesenchymal transition (34–36), metastasis (21, 37, 38), stemness (30, 39, 40), and therapy resistance (41, 42). A number of target genes, including FOXO1/3 (18, 21, 43–45), CYLD (46), CADM1 (47), BRCA1 (27, 48), MTSS1 (34), PDK4 (49), and SMAD7 (35), were reported to be suppressed by miR-182 in cancer cells. Our previous work also proved that tumoral miR-182 regulates lipogenesis in lung adenocarcinoma and promotes metastasis of breast cancer (34, 35, 49). Although miR-182 was established as an important regulator of cancer cell malignancy, previous studies were limited, with analyses of miR-182 in cultured cancer cells and transplanted tumors. Thus, the consequences of miR-182 regulation in physiologically relevant tumor models, such as genetically modified mice, have not been shown. More importantly, whether miR-182 also plays a role in tumor microenvironmental cell components is unknown.In this study, we show that miR-182 expression in macrophages can be induced by breast cancer cells and regulates TAM polarization in various tumor models of mice. In addition, miR-182 inhibition with TAM-targeting exosomes demonstrates promising efficacy for cancer treatment.     

From the Cover: A single macrolichen constitutes hundreds of unrecognized species

The number of Fungi is estimated at between 1.5 and 3 million. Lichenized species are thought to make up a comparatively small portion of this figure, with unrecognized species richness hidden among little-studied, tropical microlichens. Recent findings, however, suggest that some macrolichens contain a large number of unrecognized taxa, increasing known species richness by an order of magnitude or more. Here we report the existence of at least 126 species in what until recently was believed to be a single taxon: the basidiolichen fungus Dictyonema glabratum, also known as Cora pavonia. Notably, these species are not cryptic but morphologically distinct. A predictive model suggests an even larger number, with more than 400 species. These results call into question species concepts in presumably well-known macrolichens and demonstrate the need for accurately documenting such species richness, given the importance of these lichens in endangered ecosystems such as paramos and the alarming potential for species losses throughout the tropics.Fungi make up the second largest kingdom, with an estimated number of 1.5–3 million species (1–3). Lichenization plays an important role in fungal evolution, particularly in the Ascomycota, where lichens make up 30% of the currently recognized species (4–6). Transition toward a lichenized lifestyle appears to have taken place at least 10 times in the Ascomycota and 5 times in the Basidiomycota (7–9), but the distribution of lichen formers favors the Ascomycota, with the Basidiomycota accounting for less than 0.3% of all lichenized Fungi (7, 10). Altogether, ∼18,000 lichenized species are currently accepted, but estimates suggest that this represents only 50–65% of the true species richness (4, 6).Global species richness of lichenized Basidiomycota appears to be especially underestimated. The Dictyonema clade, which includes some of the best-known basidiolichens, until recently was considered to represent five species in a single genus, Dictyonema (11, 12). Subsequent taxonomic and molecular phylogenetic studies suggested that this concept did not reflect the true diversity in this clade (7, 12, 13). Currently, a total of 43 species are recognized in five genera (14, 15). Two genera, Cora and Corella, are foliose macrolichens, with a total of 16 species, corresponding to what was considered a single species, Dictyonema glabratum (11, 12, 16). This name is well known in the scientific community and even among nonspecialists and is included in the Listing of Interesting Plants of the World (17). The 16-fold increase in the number of species now recognized is a striking figure that even surpasses recent findings reported from the large macrolichens Lobariella and Sticta in the Ascomycota (18, 19). The dramatic change in the taxonomic concept of these basidiolichens has important implications for recognizing their role in ecosystem function and as model organisms. Species of Cora abound in tropical montane regions and, with their cyanobacterial photobionts capable of fixing atmospheric nitrogen, serve as biological fertilizers (20). Cora is also one of the best studied lichens in terms of ecomorphology, ecophysiology, and biochemistry (10, 21–28).     

Metabolism drives demography in an experimental field test

Metabolism should drive demography by determining the rates of both biological work and resource demand. Long-standing “rules” for how metabolism should covary with demography permeate biology, from predicting the impacts of climate change to managing fisheries. Evidence for these rules is almost exclusively indirect and in the form of among-species comparisons, while direct evidence is exceptionally rare. In a manipulative field experiment on a sessile marine invertebrate, we created experimental populations that varied in population size (density) and metabolic rate, but not body size. We then tested key theoretical predictions regarding relationships between metabolism and demography by parameterizing population models with lifetime performance data from our field experiment. We found that populations with higher metabolisms had greater intrinsic rates of increase and lower carrying capacities, in qualitative accordance with classic theory. We also found important departures from theory—in particular, carrying capacity declined less steeply than predicted, such that energy use at equilibrium increased with metabolic rate, violating the long-standing axiom of energy equivalence. Theory holds that energy equivalence emerges because resource supply is assumed to be independent of metabolic rate. We find this assumption to be violated under real-world conditions, with potentially far-reaching consequences for the management of biological systems.

Metabolism is thought to drive demography by setting the rate of biological work and resource consumption. For instance, theory predicts that metabolic rate should determine a population’s carrying capacity by setting per capita resource demands (1–5). The idea that carrying capacity—the density at which a population stops growing—is linked to metabolism has intuitive appeal. Because higher metabolic rates are associated with higher resource demands, a population’s carrying capacity (in terms of number of individuals) should be inversely proportional to metabolic rate (MR). In other words, carrying capacity (K) should scale at an exponent of MR⁻¹ (1, 3, 4). Similarly, because metabolism powers the biological work of production and time to first reproduction, organisms with higher mass-specific metabolic rates should be able to replicate themselves faster. Accordingly, theory predicts that the rate at which populations grow should be proportional to mass-specific metabolic rate—that is, the intrinsic rate of increase (r) of populations should scale at an exponent of MR¹ (6, 7). The idea that metabolic rate mechanistically determines population processes is well accepted (5), providing an explanation for biogeographical patterns and informing projections of the impacts of global change (8). Yet empirical support for relationships between metabolic rate and demography is largely indirect, mostly in the form of among-species comparisons of organisms that also differ in body size and temperature range. In contrast, these relationships have rarely been tested directly within species.Among species, metabolic rate strongly covaries with body size, which in turn covaries with demography (2, 9). There is a long history of studies that examine how body size covaries with population density at equilibrium and whether this covariance matches predictions from metabolic theory (1, 3, 8, 10). For example, because larger species have higher absolute metabolic rates, population density at carrying capacity should covary negatively with body size—and among species, they often do (1, 3, 8, 10). Furthermore, because metabolic rate scales hypo-allometrically with body size (4, 11), larger species have lower mass-specific metabolic rates, such that per unit body mass, larger species have lower energy demands. Note that classic metabolic theory focuses on resource use alone—factors such as predation risk, while undoubtedly demographically important (12), do not feature. Consequently, the total biomass of a population at carrying capacity should covary positively with body size (13, 14). For example, because metabolic rate scales with body size at an exponent of around 0.75 in mammals (4), mammal population density should scale with body size at −0.75, and total population biomass should scale with body mass at around 0.25 (5). Meanwhile, the total energy consumption of a population at equilibrium should be mass independent—that is, populations consisting of individuals that differ in their mean body size (but share the same cumulative biomass) should have equivalent energy consumption rates, known as the “energy equivalence rule” (1, 3).Among-species comparisons of mass and population density have long been used as indirect (albeit compelling) evidence for the negative relationship between energy consumption and density (1, 3, 8, 10). Despite the often-remarkable congruence between predicted and observed relationships between body size and population density among species, these patterns do not directly test the link between metabolic rate and demography. Yet the energy equivalence rule and links between body size and population processes are explicitly mechanistic—body size should affect population processes because of differences in absolute and mass-specific metabolic rates. However, species of different body sizes also differ in myriad other life-history traits such as growth, longevity, and reproduction, all of which also affect demography (2, 10). Thus, interspecific covariances between body size and demography could be driven by metabolic rate but could also be driven by other covarying factors (10, 15).A more direct test of the link between metabolic rate and demography is to manipulate metabolic rate within species, independently of body size. In a rare and elegant example, Bernhardt, Sunday, and O''Connor (16) manipulated metabolic rate by growing phytoplankton under different thermal regimes. Their results were consistent with theoretical expectations based on the temperature-dependence of metabolic rate—population density at carrying capacity decreased with increasing metabolic rates at higher temperatures (refer also to ref. 17). While strongly suggestive of a link between metabolic rate and demography, other effects of temperature on biological processes cannot be excluded. Few studies vary metabolic rate independently of body size or temperature, particularly under field conditions. We would argue that such tests are critical, given that alternative hypotheses imply different expectations about how metabolic rate should affect energy acquisition and demography (18–20).Since its inception, discussions regarding the “energy equivalence rule” and other metabolic theories at the population level have focused on the special case in which the rate of resource delivery into a population is assumed to be independent of metabolic rate and body size (13, 21). On the other hand, studies of individuals often find that metabolic rate affects access to resources (18–20). The increased intake hypothesis posits that because individuals with a higher metabolic rate have faster physiologies, they may forage more voraciously or effectively, such that they can extract more resources from their environment (12, 18–20). Thus, resource access could positively covary with metabolic rate at the level of populations—a possibility that contradicts the assumptions of energy equivalence—but too few studies have explored the extent to which such covariance occurs, particularly under field conditions.In a manipulative field experiment, we tested theoretical predications regarding metabolic rate and demography using the sessile, filter-feeding marine bryozoan, Bugula neritina. We generated 172 experimental populations that differed in population size (density) and per capita metabolic demands, while holding mean body size constant (SI Appendix, Appendix S1). We measured several performance components (i.e., survival, growth, and reproduction) for all individuals (n = 1,028) in these experimental populations across their entire lifetime. We then parameterized integral projection models (IPMs) (22) with our experimental data to determine how metabolic rate and population size alters the asymptotic growth rate (λ) of populations. From these projections of λ, we were then able to estimate several demographic parameters—intrinsic rate of increase (r), carrying capacity (K), and total energy use at equilibrium (ΣE). Consequently, our combined experimental and modeling approach allowed us to provide direct field estimates of the covariance between metabolic rate and fundamental parameters of population dynamics predicted by classic metabolic theories.     

Cryo-EM structure of the needle filament tip complex of the Salmonella type III secretion injectisome

Type III secretion systems are multiprotein molecular machines required for the virulence of several important bacterial pathogens. The central element of these machines is the injectisome, a ∼5-Md multiprotein structure that mediates the delivery of bacterially encoded proteins into eukaryotic target cells. The injectisome is composed of a cytoplasmic sorting platform, and a membrane-embedded needle complex, which is made up of a multiring base and a needle-like filament that extends several nanometers from the bacterial surface. The needle filament is capped at its distal end by another substructure known as the tip complex, which is crucial for the translocation of effector proteins through the eukaryotic cell plasma membrane. Here we report the cryo-EM structure of the Salmonella Typhimurium needle tip complex docked onto the needle filament tip. Combined with a detailed analysis of structurally guided mutants, this study provides major insight into the assembly and function of this essential component of the type III secretion protein injection machine.

Many pathogenic or symbiotic bacteria for plants or animals have evolved specialized molecular machines known as type III protein secretion systems (T3SSs) (1–3). These machines inject bacterially encoded effector proteins into target eukaryotic cells to modulate cellular processes and ensure the survival and replication of the pathogens or symbionts that encode them (4–6). Although the structural organization of this secretion machine has been largely derived from studies of the T3SSs of the bacterial pathogens Salmonella Typhimurium and Shigella flexneri (7–15), given the conservation of its core components, it is predicted that the T3SSs in other bacteria exhibit a similar architecture. The secretion machine itself is composed of a ∼5-Md multiprotein assembly known as the injectisome (1, 7, 14, 16, 17). This core structure consists of two large substructures, an envelope-associated needle complex (7, 16, 18, 19) and a large cytoplasmic complex known as the “sorting platform” (14, 15, 20).The needle complex is composed of a multiring hollow base, which anchors the injectisome to the bacterial envelope (18). The base encloses the export apparatus, a helical structure that serves as a conduit for the passage through the inner membrane of the proteins destined to transit this secretion pathway (21). The base is also linked to a needle-like filament, which protrudes several nanometers from the bacterial surface and is formed by a single protein arranged in a helical fashion (17, 22). The needle filament, which is traversed in its entire length by a central channel ∼3 nm in diameter, is linked to the base through a structure known as the inner rod (11) and is capped at its distal end by another substructure known as the “tip complex” (23, 24).The sorting platform is located in its entirety within the cytoplasm (14, 15, 20). It is made of six pods that form a cage-like enclosure, which is capped on its cytoplasmic side by a wheel-like structure that holds a hexameric ATPase. Also harbored within the sorting platform cage is the large cytoplasmic domain of one of the components of the export apparatus, which is arranged as two concentric rings and forms a conduit for the secreted substrates to reach the entrance of the export channel (25).A distinctive feature of the T3SSs is that their activation requires contact with the target eukaryotic cell (26, 27). The activation of the T3SS is followed by the deployment of the translocon substructure, which firmly anchors the injectisome to the target cell and serves as the passageway for the effector proteins across the eukaryotic cell membrane. Although little is known about the activation process, it is thought that sensing of the target cell by the tip complex initiates a signaling event that is transduced to the secretion machine by the needle filament itself (26–31). Activation of the secretion machine is then followed by the deployment of the translocon on the target cell membrane, which along with the tip complex and the needle filament, form a continuous passageway through which effector proteins transit from the bacterial cytoplasm to the cytosol of the eukaryotic cell (23, 32, 33). The composition of the tip complex has been the subject of some controversy. While it has been proposed that in the T3SSs of Yersinia spp., Pseudomonas aeruginosa, and Salmonella spp. the tip structure is made up of a single protein, LcrV (24), PcrV (34), and SipD (35), respectively, in the case of Shigella spp., it has been alternatively proposed to be composed of two proteins, IpaB and IpaD (36), or just IpaD (37, 38). The crystal structures of monomeric SipD and close homologs show that these proteins are arranged in three domains: an N-terminal α-helical hairpin, a central coiled-coil, and a mixed α/β carboxyl-terminal domain (39–42). It has been proposed that the N-terminal α-helical hairpin domain functions as a self-chaperone that prevents the self-oligomerization and/or the premature interaction of the tip protein with the needle filament subunit within the bacterial cytoplasm (39). A current hypothesis is that during assembly at the tip of the needle, the N-terminal α-helical hairpin of SipD/IpaD is displaced to allow other domains to interact with the needle. However, there is no structural information of the fully assembled tip complex to support this hypothesis. How the tip protein assembles into the tip complex, and how it is anchored at the distal end of the needle filament, is currently unknown in large part because of the absence of a high-resolution structure of this complex. Understanding of the events that lead to the activation of the secretion machine requires detailed knowledge not only of the structure of the tip complex that caps the needle filament but, importantly, its interface with the needle filament itself.Advances in cryoelectron microscopy (cryo-EM) have allowed the visualization of most components of the T3SS machine at high resolution, both in isolation as well as in situ (7–15). However, the tip structure has eluded high-resolution visualization, in part because existing needle complex isolation protocols result in the dissociation of the tip complex from the needle filament. Here we report the visualization at high resolution by cryo-EM of the tip structure of the needle complex of the S. Typhimurium T3SS encoded within its pathogenicity island 1. Combined with functional analysis, the structure provides major insight into the potential mechanisms of injectisome assembly and activation and fills one of the remaining gaps in the quest for the high-resolution visualization of the entire T3SS injectisome.