A test of Darwin's naturalization hypothesis in the thistle tribe shows that close relatives make bad neighbors

Invasive species have great ecological and economic impacts and are difficult to control once established, making the ability to understand and predict invasive behavior highly desirable. Preemptive measures to prevent potential invasive species from reaching new habitats are the most economically and environmentally efficient form of management. Darwin’s naturalization hypothesis predicts that invaders less related to native flora are more likely to be successful than those that are closely related to natives. Here we test this hypothesis, using the weed-rich thistle tribe, Cardueae, in the California Floristic Province, a biodiversity hotspot, as our study system. An exhaustive molecular phylogenetic approach was used, generating and examining more than 100,000 likely phylogenies of the tribe based on nuclear and chloroplast DNA markers, representing the most in-depth reconstruction of the clade to date. Branch lengths separating invasive and noninvasive introduced taxa from native California taxa were used to represent phylogenetic distances between these groups and were compared at multiple biogeographical scales to ascertain whether invasive thistles are more or less closely related to natives than noninvasive introduced thistles are. Patterns within this highly supported clade show that not only are introduced thistles more closely related to natives more likely to be invasive, but these invasive species are also evolutionarily closer to native flora than by chance. This suggests that preadaptive traits are important in determining an invader’s success. Such rigorous molecular phylogenetic analyses may prove a fruitful means for furthering our understanding of biological invasions and developing predictive frameworks for screening potential invasive taxa.Spread beyond their native ranges, some species have become numerically and ecologically dominant in new regions (1) and become of great interest and concern to scientists, policymakers, and the public. Such invasive species, sensu Colautti and MacIsaac (2), affect biodiversity, ecosystem function, and human health (3) and have ecological and economic impacts that cannot be ignored (4, 5), making the ability to understand and predict the invasiveness of species of great importance. Also, once exotic species become established in a new region, they are often extremely difficult to control (6, 7). Identifying and preventing new potentially invasive exotic species from reaching ground zero is, by far, the most economically and environmentally efficient management method (8). Hence, there is great need for early warning systems to determine the probability that a given species will become invasive (9–12).The number of plant species introduced into the United States far exceeds that of other groups of organisms (13). However, although many case studies have illuminated various aspects of plant invasions (14–22), it has proven difficult to quantify and/or make generalizations about traits, characteristics, and circumstances that contribute to plant invasiveness across multiple geographic scales and ecological systems (10, 23–28). The difficulty of devising a framework to predict the behavior of exotic plants following dislocation and the challenges of designing effective control strategies for the ones that have become invasive result from the uniqueness of the organisms involved in each case, as well as the complexity of interactions between invaders and native communities (29). Few studies have provided a practical means of addressing these issues (30, 31).Quantifiable measures that can provide robust predictions are therefore required (32), and phylogenetic relationships between native and introduced taxa may reveal patterns that invoke testable hypotheses that could not be derived from examining species traits alone (33). Distinct sets of traits evolve in response to environmental conditions, which in turn reflect past and present selection pressures and are therefore expected to differ not only among geographic regions and local communities (34), but also among evolutionary lineages. Hence species’ responses are not statistically independent from their shared evolutionary histories (35–37), and phylogeny may affect a species’ biotic interactions when it is introduced to a novel environment (38). As Darwin (39) observed, this results in a link between the evolutionary relatedness of organisms in a community, their characteristics, and the ecological processes that determine their distributions and abundance. Darwin’s naturalization hypothesis posits that invaders that are closely related to native taxa are less likely to be successful than those that are not. Assuming evolutionary relatedness is correlated with ecological similarity, such a pattern might emerge as a result of niche overlap and competitive exclusion between introduced taxa and their native relatives, in addition to being subject to the same predators and pathogens (40, 41). The enemy escape hypothesis also supports this view (42, 43). An alternative, opposing hypothesis is that relatedness to native taxa may convey degrees of preadaptation to the conditions of the invaded environment, rendering close relatives more likely to succeed once introduced (32, 39, 44).Previous studies have been equivocal, finding evidence both for (30, 31, 38, 45–50), and against (24, 32, 41, 44, 51–55) Darwin’s hypothesis. However, few have used a strict phylogenetic approach based on evolutionary divergence, instead predominantly relying on taxonomic ranks (e.g., refs. 45 and 56, reviewed in refs. 57 and 58), which are highly subjective as measures of relatedness (59). In instances where phylogenetic trees were used, some have used supertrees compiled from multiple studies (46, 60, 61), with estimated branch lengths that may not accurately reflect the evolutionary distances between taxa. Other studies have been based on community phylogenetic trees (47, 60, 62). This approach may be problematic, as communities are not necessarily monophyletic groups, but collections of co-occurring species whatever their evolutionary relationships may be (63), and sampling in such studies is unlikely to include adequate representation of all lineages present, whereas phylogenetic analyses assume monophyly of the ingroup and their accuracy is dependent on sampling that is representative of the diversity within (64, 65).This study sought to address theoretical and methodological issues that may have limited progress toward resolving Darwin’s naturalization conundrum (56) by using phylogenies based on molecular markers to assess the evolutionary distances between native and nonnative taxa in a strongly supported monophyletic group, the thistle tribe (Cardueae, Asteraceae), in a well-defined biogeographic area, the California Floristic Province (CAFP) (66, 67). The thistles of California offer an ideal opportunity to test Darwin’s hypothesis. The tribe, which boasts an impressive list of Mediterranean and temperate invaders, is most prolific in Mediterranean climate regions, which not only rank among the most biodiversity-rich biomes on the planet (68, 69), but among the most imperiled as well (70–74). The CAFP is one of such biodiversity hotspots, as defined by Conservation International, and is often recognized as a biogeological entity (75, 76). Also, programs and legislation regarding invasive taxa are usually state specific, making the study of invasive Cardueae in California both politically and biologically meaningful.Phylogenies of Cardueae based on sequences from three genomic regions commonly used in phylogenetic studies of angiosperms were generated with taxon sampling representing the full lineage diversity of the tribe, including all species native and naturalized in the CAFP. Phylogenetic trees were constructed using parsimony, maximum likelihood, and Bayesian approaches. Phylogenetic distances of invasive and noninvasive introduced species from natives were compared using a comprehensive set of metrics and statistical tests to assess the utility of phylogenetic distance from natives as a predictor of invasive behavior. This study finds evidence contrary to Darwin’s hypothesis and demonstrates the robustness of such metrics, which should be more informative and meaningful than taxonomic groupings (30, 46, 77–79), especially when supported by a well-resolved molecular phylogeny.     

Antibiotics induce redox-related physiological alterations as part of their lethality

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H₂O₂ sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H₂O₂. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.The increasing incidence of antibiotic-resistant infections coupled with a declining antibiotic pipeline has created a global public health threat (1–6). Therefore there is a pressing need to expand our conceptual understanding of how antibiotics act and to use insights gained from such efforts to enhance our antibiotic arsenal. It has been proposed that different classes of bactericidal antibiotics, regardless of their drug–target interactions, generate varying levels of deleterious reactive oxygen species (ROS) that contribute to cell killing (7, 8). This unanticipated notion, built upon important prior work (9–11), has been extended and supported by multiple laboratories investigating wide-ranging drug classes (e.g., β-lactams, aminoglycosides, and fluoroquinolones) and bacterial species (e.g., Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepecia, Streptococcus pneumonia, Enterococcus faecalis) using independent lines of evidence (12–39). Importantly, these ongoing efforts have served to refine aspects of the initial model and show that antibiotic-induced ROS generation is a more complex process than originally suggested, likely involving additional means of production.The redox stress component of antibiotic lethality is hypothesized to derive from alterations to multiple core aspects of cellular physiology and stress response activation. Specifically, this component includes alterations to central metabolism, cellular respiration, and iron metabolism initiated by drug-mediated disruptions of target-specific processes and resulting cellular damage (Fig. 1A). Important support for this hypothesis can be found in pathogenic clinical isolates whose drug tolerance involves mutations in oxidative stress response and defense genes and not exclusively in drug target mutagenesis (40–48).Open in a separate windowFig. 1.Bactericidal antibiotics promote the generation of toxic reactive species. (A) Bactericidal antibiotics of different classes are capable of inducing cell death by interfering with their primary targets and corrupting target-specific processes, resulting in lethal cellular damage. Target-specific interactions trigger stress responses that induce redox-related physiological alterations resulting in the formation of toxic reactive species, including ROS, which further contribute to cellular damage and death. (B) Treatment of wild-type E. coli with ampicillin (Amp, 5 μg/mL), gentamicin (Gent, 5 μg/mL), or norfloxacin (Nor, 250 ng/mL) induces ROS, detectable by several chemically diverse fluorescent dyes with ranging specificity. One-way ANOVA was performed to determine statistical significance against the no-dye autofluorescence control. The dyes used were 5/6-carboxy-2'',7''-dichlorodihydrofluorescein diacetate (Carboxy-H₂DCFDA); 5/6-chloromethyl-2'',7''-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA); 4-amino-5-methylamino-2´,7´-diflurorescein diacetate (DAF-FM); 2'',7''-dichlorodihydrofluorescein diacetate (H₂DCFDA); 3′-(p-hydroxyphenyl) fluorescein (HPF); OxyBURST Green (Oxyburst); and Peroxy-Fluor 2 (PF2). (C) Treatment of a quinolone-resistant strain (gyrA17) with norfloxacin does not produce detectable ROS. Data shown reflect mean ± SEM of three or more technical replicates. Where appropriate, statistical significance is shown and computed against the no-treatment control or no-dye control (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).Recent critiques of this evolving model have misinterpreted an essential aspect of the hypothesis. Specifically, these recent studies (49–51) are predicated on the notion that ROS are the sole arbiters of antibiotic lethality, thereby implying that the model suggests that antibiotics do not kill by disrupting their well-established, target-specific processes. However, the evolving model is completely consistent with the literature indicating that bactericidal antibiotics are capable of inducing lethal cellular damage via interference with target-specific processes, ultimately resulting in cell death. Rather than refute this traditional view of antibiotic action, the hypothesis extends it by suggesting that an additional component of toxicity results from ROS, which are generated as a downstream physiological consequence of antibiotics interacting with their traditional targets. In this respect, reactive species are thought to contribute causatively to drug lethality.However, an important gap exists in our general understanding of how bacteria respond physiologically to antibiotic–target interactions on a system-wide level, how these responses contribute to antibiotic killing, and how the extracellular environment protects or exacerbates the intracellular contributions to cell death. Here, we use a multidisciplinary set of biochemical, enzymatic, biophysical, and genetic assays to address these issues and expand our understanding of antibiotic-induced physiological responses and factors contributing to antibiotic lethality. Data from the present work indicate that antibiotic lethality is accompanied by ROS generation and that such reactive species causatively contribute to antibiotic lethality.     

From the Cover: Why the proportion of transmission during early-stage HIV infection does not predict the long-term impact of treatment on HIV incidence

Antiretroviral therapy (ART) reduces the infectiousness of HIV-infected persons, but only after testing, linkage to care, and successful viral suppression. Thus, a large proportion of HIV transmission during a period of high infectiousness in the first few months after infection (“early transmission”) is perceived as a threat to the impact of HIV “treatment-as-prevention” strategies. We created a mathematical model of a heterosexual HIV epidemic to investigate how the proportion of early transmission affects the impact of ART on reducing HIV incidence. The model includes stages of HIV infection, flexible sexual mixing, and changes in risk behavior over the epidemic. The model was calibrated to HIV prevalence data from South Africa using a Bayesian framework. Immediately after ART was introduced, more early transmission was associated with a smaller reduction in HIV incidence rate—consistent with the concern that a large amount of early transmission reduces the impact of treatment on incidence. However, the proportion of early transmission was not strongly related to the long-term reduction in incidence. This was because more early transmission resulted in a shorter generation time, in which case lower values for the basic reproductive number (R₀) are consistent with observed epidemic growth, and R₀ was negatively correlated with long-term intervention impact. The fraction of early transmission depends on biological factors, behavioral patterns, and epidemic stage and alone does not predict long-term intervention impacts. However, early transmission may be an important determinant in the outcome of short-term trials and evaluation of programs.Recent studies have confirmed that effective antiretroviral therapy (ART) reduces the transmission of HIV among stable heterosexual couples (1–3). This finding has generated interest in understanding the population-level impact of HIV treatment on reducing the rate of new HIV infections in generalized epidemic settings (4). Research, including mathematical modeling (5–10), implementation research (11), and major randomized controlled trials (12–14), are focused on how ART provision might be expanded strategically to maximize its public health benefits (15, 16).One concern is that if a large fraction of HIV transmission occurs shortly after a person becomes infected, before the person can be diagnosed and initiated on ART, this will limit the potential impact of HIV treatment on reducing HIV incidence (9, 17, 18). Data suggest that persons are more infectious during a short period of “early infection” after becoming infected with HIV (19–22), although there is debate about the extent, duration, and determinants of elevated infectiousness (18, 23). The amount of transmission that occurs also will depend on patterns of sexual behavior and sexual networks (17, 24–27). There have been estimates for the contribution of early infection to transmission from mathematical models (7, 17, 21, 24–26) and phylogenetic analyses (28–31), but these vary widely, from 5% to above 50% (23).In this study, we use a mathematical model to quantify how the proportion of transmission that comes from persons who have been infected recently affects the impact of treatment scale-up on HIV incidence. The model is calibrated to longitudinal HIV prevalence data from South Africa using a Bayesian framework. Thus, the model accounts for not only the early epidemic growth rate highlighted in previous research (5, 9, 18), but also the heterogeneity and sexual behavior change to explain the peak and decline in HIV incidence observed in sub-Saharan African HIV epidemics (32, 33).The model calibration allows uncertainty about factors that determine the amount of early transmission, including the relative infectiousness during early infection, heterogeneity in propensity for sexual risk behavior, assortativity in sexual partner selection, reduction in risk propensity over the life course, and population-wide reductions in risk behavior in response to the epidemic (32, 33). This results in multiple combinations of parameter values that are consistent with the observed epidemic and variation in the amount of early transmission. We simulated the impact of a treatment intervention and report how the proportion of early transmission correlates with the reduction in HIV incidence from the intervention over the short- and long-term.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

The extent of functional redundancy changes as species’ roles shift in different environments

Assessing the ecological impacts of environmental change requires knowledge of the relationship between biodiversity and ecosystem functioning. The exact nature of this relationship can differ considerably between ecosystems, with consequences for the efficacy of species diversity as a buffer against environmental change. Using a microbial model system, we show that the relationship can vary depending on environmental conditions. Shapes suggesting functional redundancy in one environment can change, suggesting functional differences in another environment. We find that this change is due to shifting species roles and interactions. Species that are functionally redundant in one environment may become pivotal in another. Thus, caution is advised in drawing conclusions about functional redundancy based on a single environmental situation. It also implies that species richness is important because it provides a pool of species with potentially relevant traits. These species may turn out to be essential performers or partners in new interspecific interactions after environmental change. Therefore, our results challenge the generality of functional redundancy.Many ecosystems are currently in decline due to rapid loss of biodiversity and immense changes in species compositions (1). The impact of these changes on important ecosystem processes is still unclear (2, 3). Ecosystem functioning (e.g., in terms of biomass production) is often positively correlated with species richness (4–8). This correlation is typically explained by two types of mechanisms that are not mutually exclusive: selection and complementarity (9, 10). The former refers to the rising probability for species with particular traits (e.g., highly productive or resource-efficient species) to be included and achieve high abundance in species-rich communities. The latter refers to the rising potential for particular processes to operate in species-rich communities, such as niche partitioning (e.g., species use different resources or exploit limiting resources in different ways) or positive interactions (e.g., a species facilitates growth of another species). Complementarity can thus lead to species-rich communities performing better than any of the included species in monoculture (transgressive overyielding) (11, 12). As a consequence of these mechanisms, the biodiversity-ecosystem functioning (BEF) relationship is affected by the number and identities of species, their evenness within the community, their functional traits, and their interactions (13–17).The BEF relationship also reflects whether species are functionally redundant and perform similar roles in given communities. Functioning increasing with species richness points to functional differences among the species (i.e., the added species have unique roles for ecosystem functioning). Saturation in functioning points to functional redundancy (i.e., the roles of the added species are already performed by other species) (18–20). Hence, stepwise inclusion of one species after another to experimental communities and observation of the associated functioning (e.g., in terms of biomass production) indicate the effect of each newly included species on the functioning in these particular communities.Another factor may have an impact on the BEF relationship as well: the environmental context (3). The environmental context is important because contemporary ecosystems are facing multiple environmental changes of increasing intensity. To date, BEF relationships have mainly been investigated under a single environmental scenario leaving the effects of potential environmental changes unexplored. Only a few studies have tested the impact of disturbances (21), pathogens (22), drought (17), resource richness (23), and temperature fluctuations (24) on the BEF relationship. Thus, knowledge on the functioning of ecosystems under different environmental conditions after anticipated change is rare but urgently required (25).Microbial model systems provide a formidable tool with which to address such theoretical ecological questions (26, 27). They offer various advantages: High numbers of experiments can be carried out in small laboratory microcosms, all external factors can be controlled and easily manipulated, and many generations of the focal organisms can be tested (26, 28–31). In contrast to such model systems, investigating natural microbial ecosystems may pose challenges, such as the difficulties in defining and estimating the vast number of bacterial species (32, 33). However, these limitations do not apply to laboratory systems with defined artificial communities containing well-characterized species.In this study, we used microbial model systems to investigate how species richness, species identities, and potential species interactions affect ecosystem functioning under a different set of environmental conditions. We systematically assembled more than 800 artificial communities, with each containing between one and 12 bacterial species. As an indicator of ecosystem functioning, we observed the communities’ biomass production in three different abiotic environments of increasing abiotic stresses: low benzoate concentration (1 g/L, “environment 1”), high benzoate concentration (6 g/L, “environment 2”), and high benzoate concentration (6 g/L) with NaCl (15 g/L, “environment 3”). Each environment led to a differently shaped BEF relationship, and thus different levels of functional redundancy as well. Further, for analyzing the effects of particular compositions of species, we developed the concept of “minimal communities.” This concept allowed us to go beyond the BEF relationship curves and identify the roles of particular species and potential species interactions for ecosystem functioning.     

From the Cover: Genome of a SAR116 bacteriophage shows the prevalence of this phage type in the oceans

The abundance, genetic diversity, and crucial ecological and evolutionary roles of marine phages have prompted a large number of metagenomic studies. However, obtaining a thorough understanding of marine phages has been hampered by the low number of phage isolates infecting major bacterial groups other than cyanophages and pelagiphages. Therefore, there is an urgent requirement for the isolation of phages that infect abundant marine bacterial groups. In this study, we isolated and characterized HMO-2011, a phage infecting a bacterium of the SAR116 clade, one of the most abundant marine bacterial lineages. HMO-2011, which infects “Candidatus Puniceispirillum marinum” strain IMCC1322, has an ∼55-kb dsDNA genome that harbors many genes with novel features rarely found in cultured organisms, including genes encoding a DNA polymerase with a partial DnaJ central domain and an atypical methanesulfonate monooxygenase. Furthermore, homologs of nearly all HMO-2011 genes were predominantly found in marine metagenomes rather than cultured organisms, suggesting the novelty of HMO-2011 and the prevalence of this phage type in the oceans. A significant number of the viral metagenome sequences obtained from the ocean surface were best assigned to the HMO-2011 genome. The number of reads assigned to HMO-2011 accounted for 10.3%–25.3% of the total reads assigned to viruses in seven viromes from the Pacific and Indian Oceans, making the HMO-2011 genome the most or second-most frequently assigned viral genome. Given its ability to infect the abundant SAR116 clade and its widespread distribution, Puniceispirillum phage HMO-2011 could be an important resource for marine virus research.Viruses are the most abundant biological entities in diverse marine environments, as revealed by electron microscopy, epifluorescence microscopy, and flow cytometry studies (1–3). The average number of virus-like particles in surface seawater is ∼10⁷ per mL, and it typically exceeds the number of prokaryotes by an order of magnitude. Marine viruses play an important role in nutrient cycles by mediating a significant proportion of bacterial mortality. This so-called “viral shunt” diverts organic matter from particulate forms to dissolved forms, influencing the overall biogeochemistry of various elements (4). Viruses affect the community composition and genetic diversity of marine organisms by selectively infecting susceptible hosts (5, 6), which also increases the genetic diversity of the viruses themselves through mechanisms such as antagonistic coevolution (6, 7).Recent metagenomic studies of the oceans have revealed the numerous novel genetic repertoires of marine viromes (8–14). However, most genome fragments in these viromes cannot be categorized into any known viral group (8, 9, 13, 14). Because most marine viruses are believed to be phages (15), a more thorough understanding of the ecological and evolutionary roles of marine viruses and a better interpretation of the rapidly increasing virome sequences require the isolation and genomic analysis of individual viruses, especially phages infecting diverse marine bacterioplankton.The importance of phage isolation is exemplified by studies on marine cyanophages and pelagiphages. The isolation and characterization of cyanophages have shown that they have auxiliary metabolic genes (16), including photosynthesis-related genes that are expressed during infection and modulate host metabolism toward successful infection (17–21). Cyanophages are important in the diversification of hosts and horizontal gene transfer and are extensively used to interpret marine metagenome sequences (6, 22, 23). Very recently, four pelagiphages, viruses infecting the SAR11 clade, were isolated (24). Pelagiphage genome sequences have been found to be crucial for the interpretation of viromes from the Pacific Ocean (24). However, there are many abundant marine bacterial groups, including SAR86, SAR116, and Bacteroidetes, for which few phages have been isolated or characterized. Given the poor assignment of virome sequences into specific viral groups and the presence of difficult-to-cultivate or unculturable bacterial groups in the ocean, cultivating these bacteria and isolating the phages infecting them are prerequisites for understanding phage diversity.The SAR116 clade is one of the most abundant groups of heterotrophic bacteria inhabiting the surface of the ocean. Since its initial discovery through the cloning of 16S rRNA genes from the Sargasso Sea (25), many culture-independent studies have shown that the SAR116 clade contributes significantly—more than 10% in some cases—to the bacterial assemblages of the marine euphotic zone (26–33). The genome sequences of two bacteria in this clade, HIMB100 and “Candidatus Puniceispirillum marinum” IMCC1322, have recently been reported (34, 35). Both strains have genes for proteorhodopsin-based photoheterotrophy, carbon monoxide dehydrogenase, and dimethylsulfoniopropionate demethylase, suggesting the diverse metabolic potential and biogeochemical importance of the SAR116 clade. Considering the widespread distribution and ecologically meaningful genome characteristics of this clade, their phages are likely to be abundant in marine environments and may provide useful references for functional and phylogenetic annotation of marine viromes.Here, we report the isolation and genomic characterization of HMO-2011, a phage that infects strain IMCC1322 of the SAR116 clade. The HMO-2011 genome harbors many novel genes, such as a gene encoding a DNA polymerase with a DnaJ central domain. The genome of HMO-2011 was a notable resource for the identification of unknown marine viral gene fragments, as HMO-2011 accounted for 10.3%–25.3% of viral metagenome reads assigned to viruses.     

Generation of reactive oxygen species by lethal attacks from competing microbes

Whether antibiotics induce the production of reactive oxygen species (ROS) that contribute to cell death is an important yet controversial topic. Here, we report that lethal attacks from bacterial and viral species also result in ROS production in target cells. Using soxS as an ROS reporter, we found soxS was highly induced in Escherichia coli exposed to various forms of attacks mediated by the type VI secretion system (T6SS), P1vir phage, and polymyxin B. Using a fluorescence ROS probe, we found enhanced ROS levels correlate with induced soxS in E. coli expressing a toxic T6SS antibacterial effector and in E. coli treated with P1vir phage or polymyxin B. We conclude that both contact-dependent and contact-independent interactions with aggressive competing bacterial species and viruses can induce production of ROS in E. coli target cells.Microbial species exist predominantly in complex communities in the natural environment and animal hosts. To survive in a multispecies environment, bacteria have developed various strategies to compete with other species. For example, some bacteria can exert long-range inhibitory effects by secreting diffusible molecules, such as antibiotics, bacteriocins, and H₂O₂ (1), whereas others require direct cell-to-cell contact to kill nearby organisms (2, 3). One such contact-dependent inhibitory system is the type VI secretion system (T6SS), a protein translocating nanomachine expressed by many Gram-negative bacterial pathogens that can kill both bacterial and eukaryotic cells (3–5). Structurally analogous to an inverted bacteriophage tail, the T6SS delivers effectors into target cells by using a contractile sheath to propel an inner tube out of the producer cell and into nearby target cells. The inner tube (composed of Hcp protein) is thought to carry toxic effector proteins within its lumen or on its tip, which is decorated with VgrG and PAAR proteins (4, 6, 7). Given that some cells can detect T6SS attack but not suffer any measurable loss in viability (8, 9), it would seem that cell killing is likely due to the toxicity of effectors rather than membrane disruptions caused by insertion of the spear-like VgrG/PAAR/Hcp tube complex. T6SS-dependent effectors can attack a number of essential cellular targets, including the cell wall (10, 11), membranes (11, 12), and nucleic acids (13), and thus can mimic the actions of antibiotics and bacteriocins. As a model prey or target organism, Escherichia coli can be killed by the T6SS activities of a number of bacteria including Vibrio cholerae (14), Pseudomonas aeruginosa (10, 15), and Acinetobacter baylyi ADP1 (7).Collins and coworkers (16–18) have reported that antibiotic treatment of E. coli elicits the production of reactive oxygen species (ROS) resulting from a series of events involving perturbation of the central metabolic pathway, NADPH depletion, and the Fenton reaction. ROS can cause lethal damage to DNA, lipid, and proteins (19, 20) and thus can contribute to cell death in combination with the deleterious effects of antibiotics on their primary targets. The idea that antibiotics kill bacterial cells, in part, through the action of ROS has been supported by a number of follow-up studies (18, 21–23) but has also been challenged by others as a result of observations contradictory to a model where ROS is the sole mediator of antibiotic lethality (24–26). These observations include the fact that antibiotics kill under anaerobic conditions, oxidation of the hydroxyphenyl fluorescein fluorescence dye used to measure ROS levels is nonspecific, and the extracellular level of H₂O₂ is not elevated by antibiotic treatment (24, 26). To address these concerns, Dwyer et al. (27) used a panel of ROS-detection fluorescence dyes, a defined growth medium under stringent anaerobic conditions, and an in vivo H₂O₂ enzymatic assay to study the effects of antibiotics on cells. The results further support that antibiotics induce ROS generation, which contributes to the efficacy of antibiotics in addition to their primary lethal actions (18, 27, 28).     

Species formation by host shifting in avian malaria parasites

The malaria parasites (Apicomplexa: Haemosporida) of birds are believed to have diversified across the avian host phylogeny well after the origin of most major host lineages. Although many symbionts with direct transmission codiversify with their hosts, mechanisms of species formation in vector-borne parasites, including the role of host shifting, are poorly understood. Here, we examine the hosts of sister lineages in a phylogeny of 181 putative species of malaria parasites of New World terrestrial birds to determine the role of shifts between host taxa in the formation of new parasite species. We find that host shifting, often across host genera and families, is the rule. Sympatric speciation by host shifting would require local reproductive isolation as a prerequisite to divergent selection, but this mechanism is not supported by the generalized host-biting behavior of most vectors of avian malaria parasites. Instead, the geographic distribution of individual parasite lineages in diverse hosts suggests that species formation is predominantly allopatric and involves host expansion followed by local host–pathogen coevolution and secondary sympatry, resulting in local shifting of parasite lineages across hosts.Cospeciation of symbionts with their hosts has been recognized in parasites with strong vertical transmission (1, 2), viruses that spread by direct contact (3), and bacterial and viral symbionts passed from mother to offspring through the egg (4). Species formation in parasites that are transmitted between hosts by vectors is less well-understood (5, 6). Poor matching between the phylogenetic trees of vector-borne hemosporidian (malaria) parasites and their North American avian hosts suggests a predominance of host shifting compared with cospeciation (7) (reviewed in a broader context in ref. 8). Whether host shifting occurs primarily between closely related hosts and in geographic sympatry, and whether rates of host shifting followed by species formation are sufficient to explain the contemporary diversity of hemosporidian parasites, have not been determined.Many species of hemosporidian parasites have been described and named based primarily on the microscopic morphology of meronts and gametocytes in blood smears (9). The more recent discovery of hundreds of lineages based on DNA sequence variation in the mitochondrial cytochrome b gene (cyt b) (5, 10, 11) requires, however, a different species concept based on analysis of independent components of the genome (12–16). Recent estimates of the rate of molecular evolution in hemosporidian mitochondrial genes imply that the contemporary malaria parasites of vertebrates might have descended from a common ancestor within the past 20 (17) or 40 Ma (18) or, perhaps, a longer time period (19). Although an appropriate calibration for the rate of hemosporidian evolution remains unsettled (20, 21), host shifting almost certainly has been frequent, likely across great host distances at times, over the recent history of the group.Speciation in sympatry (i.e., in the absence of geographic barriers to gene flow through local host specialization) might follow host shifting if mating between parasites was assortative with respect to vertebrate host or if different hosts imposed strong disruptive selection on parasites (22). However, despite some documented feeding preferences (23–25), dipteran vectors of avian malaria parasites do not seem to be sufficiently specialized to isolate populations of parasites on different hosts (26–29). In addition, many parasite species and many parasite lineages distinguished by DNA sequence variation occur locally across broad ranges of hosts without apparent differentiation, at least in the mitochondrial cytochrome b gene (30–32) and several nuclear markers (12, 14). Alternatively, host shifting in one allopatric population of a parasite species could be followed, after sufficient host–pathogen coevolution and evolutionary differentiation to produce reproductive incompatibility, by secondary sympatry, thereby increasing local parasite diversity.Here, we examine recent nodes in an mtDNA-based phylogeny of New World hemosporidian parasites to determine the degree to which lineage formation is associated with host shifting. Although our phylogenetic reconstruction is based on a single mitochondrial gene (cyt b), phylogenies based on genes from the mitochondrial, nuclear, and apicoplast genomes are broadly consistent for the relatively recent nodes considered in this analysis (6, 12–14, 33–35). In addition, analyses of avian hemosporidian parasites based on multiple independent markers have distinguished mtDNA-defined lineages on the basis of significant linkage disequilibrium (13).We distinguish as species the lineages that differ in their mtDNA cytochrome b gene sequence (by as few as 2 nt) and, for the most part, occur in either different hosts in the same local area or the same or different hosts in different geographic areas (32, 36). In some cases, closely related lineages occur in the same host locally. Sister lineages in this analysis differ by an average of about 1% sequence divergence, although some sequences separated by as little as a single nucleotide can exhibit consistent host or geographic differences. Inference concerning the mode of species formation is based primarily on host and geographic distributions of these hemosporidian mtDNA lineages. However, the correspondence between lineages and reproductively isolated species is poorly resolved (13, 37, 38). Each node was designated as either sympatric or allopatric depending on whether the descendant lineages occurred on the same West Indian islands or in the same regions within larger continental areas. The status of closely related parasite lineages occurring locally in the same host species is ambiguous, but these lineages might reflect genetic variation within a parasite species.Previous analyses have suggested that host shifting, rather than codivergence, predominates species formation in the hemosporidian parasites of birds (5, 7). We find this most frequently to be the case in this analysis, and we discuss whether species formation by host shifting occurs primarily in sympatry or allopatry.     

Fenton chemistry at aqueous interfaces

In a fundamental process throughout nature, reduced iron unleashes the oxidative power of hydrogen peroxide into reactive intermediates. However, notwithstanding much work, the mechanism by which Fe²⁺ catalyzes H₂O₂ oxidations and the identity of the participating intermediates remain controversial. Here we report the prompt formation of O=Fe^IVCl₃⁻ and chloride-bridged di-iron O=Fe^IV·Cl·Fe^IICl₄⁻ and O=Fe^IV·Cl·Fe^IIICl₅⁻ ferryl species, in addition to Fe^IIICl₄⁻, on the surface of aqueous FeCl₂ microjets exposed to gaseous H₂O₂ or O₃ beams for <50 μs. The unambiguous identification of such species in situ via online electrospray mass spectrometry let us investigate their individual dependences on Fe²⁺, H₂O₂, O₃, and H⁺ concentrations, and their responses to tert-butanol (an ·OH scavenger) and DMSO (an O-atom acceptor) cosolutes. We found that (i) mass spectra are not affected by excess tert-butanol, i.e., the detected species are primary products whose formation does not involve ·OH radicals, and (ii) the di-iron ferryls, but not O=Fe^IVCl₃⁻, can be fully quenched by DMSO under present conditions. We infer that interfacial Fe(H₂O)_n²⁺ ions react with H₂O₂ and O₃ >10³ times faster than Fe(H₂O)₆²⁺ in bulk water via a process that favors inner-sphere two-electron O-atom over outer-sphere one-electron transfers. The higher reactivity of di-iron ferryls vs. O=Fe^IVCl₃⁻ as O-atom donors implicates the electronic coupling of mixed-valence iron centers in the weakening of the Fe^IV–O bond in poly-iron ferryl species.High-valent Fe^IV=O (ferryl) species participate in a wide range of key chemical and biological oxidations (1–4). Such species, along with ·OH radicals, have long been deemed putative intermediates in the oxidation of Fe^II by H₂O₂ (Fenton’s reaction) (5, 6), O₃, or HOCl (7, 8). The widespread availability of Fe^II and peroxides in vivo (9–12), in natural waters and soils (13), and in the atmosphere (14–18) makes Fenton chemistry and Fe^IV=O groups ubiquitous features in diverse systems (19). A lingering issue regarding Fenton’s reaction is how the relative yields of ferryls vs. ·OH radicals depend on the medium. For example, by assuming unitary ·OH radical yields, some estimates suggest that Fenton’s reaction might account for ∼30% of the ·OH radical production in fog droplets (20). Conversely, if Fenton’s reaction mostly led to Fe^IV=O species, atmospheric chemistry models predict that their steady-state concentrations would be ∼10⁴ times larger than [·OH], thereby drastically affecting the rates and course of oxidative chemistry in such media (20). Fe^IV=O centers are responsible for the versatility of the family of cytochrome P450 enzymes in catalyzing the oxidative degradation of a vast range of xenobiotics in vivo (21–28), and the selective functionalization of saturated hydrocarbons (29). The bactericidal action of antibiotics has been linked to their ability to induce Fenton chemistry in vivo (9, 30–34). Oxidative damage from exogenous Fenton chemistry likely is responsible for acute and chronic pathologies of the respiratory tract (35–38).Despite its obvious importance, the mechanism of Fenton’s reaction is not fully understood. What is at stake is how the coordination sphere of Fe²⁺ (39–46) under specific conditions affects the competition between the one-electron transfer producing ·OH radicals (the Haber–Weiss mechanism) (47), reaction R1, and the two-electron oxidation via O-atom transfer (the Bray–Gorin mechanism) into Fe^IVO²⁺, reaction R2 (6, 23, 26, 27, 45, 48–51):Ozone reacts with Fe²⁺ via analogous pathways leading to (formally) the same intermediates, reactions R3a, R3b, and R4 (8, 49, 52, 53):At present, experimental evidence about these reactions is indirect, being largely based on the analysis of reaction products in bulk water in conjunction with various assumptions. Given the complex speciation of aqueous Fe²⁺/Fe³⁺ solutions, which includes diverse poly-iron species both as reagents and products, it is not surprising that classical studies based on the identification of reaction intermediates and products via UV-absorption spectra and the use of specific scavengers have fallen short of fully unraveling the mechanism of Fenton’s reaction. Herein we address these issues, focusing particularly on the critically important interfacial Fenton chemistry that takes place at boundaries between aqueous and hydrophobic media, such as those present in atmospheric clouds (16), living tissues, biomembranes, bio-microenvironments (38, 54, 55), and nanoparticles (56, 57).We exploited the high sensitivity, surface selectivity, and unambiguous identification capabilities of a newly developed instrument based on online electrospray mass spectrometry (ES-MS) (58–62) to identify the primary products of reactions R1–R4 on aqueous FeCl₂ microjets exposed to gaseous H₂O₂ and O₃ beams under ambient conditions [in N₂(g) at 1 atm at 293 ± 2 K]. Our experiments are conducted by intersecting the continuously refreshed, uncontaminated surfaces of free-flowing aqueous microjets with reactive gas beams for τ ∼10–50 μs, immediately followed (within 100 μs; see below) by in situ detection of primary interfacial anionic products and intermediates via ES-MS (Methods, SI Text, and Figs. S1 and S2). We have previously demonstrated that online mass spectrometric sampling of liquid microjets under ambient conditions is a surface-sensitive technique (58, 62–67).     

Commonness and rarity in the marine biosphere

Explaining patterns of commonness and rarity is fundamental for understanding and managing biodiversity. Consequently, a key test of biodiversity theory has been how well ecological models reproduce empirical distributions of species abundances. However, ecological models with very different assumptions can predict similar species abundance distributions, whereas models with similar assumptions may generate very different predictions. This complicates inferring processes driving community structure from model fits to data. Here, we use an approximation that captures common features of “neutral” biodiversity models—which assume ecological equivalence of species—to test whether neutrality is consistent with patterns of commonness and rarity in the marine biosphere. We do this by analyzing 1,185 species abundance distributions from 14 marine ecosystems ranging from intertidal habitats to abyssal depths, and from the tropics to polar regions. Neutrality performs substantially worse than a classical nonneutral alternative: empirical data consistently show greater heterogeneity of species abundances than expected under neutrality. Poor performance of neutral theory is driven by its consistent inability to capture the dominance of the communities’ most-abundant species. Previous tests showing poor performance of a neutral model for a particular system often have been followed by controversy about whether an alternative formulation of neutral theory could explain the data after all. However, our approach focuses on common features of neutral models, revealing discrepancies with a broad range of empirical abundance distributions. These findings highlight the need for biodiversity theory in which ecological differences among species, such as niche differences and demographic trade-offs, play a central role.Determining how biodiversity is maintained in ecological communities is a long-standing ecological problem. In species-poor communities, niche and demographic differences between species can often be estimated directly and used to infer the importance of alternative mechanisms of species coexistence (1–3). However, the “curse of dimensionality” prevents the application of such species-by-species approaches to high-diversity assemblages: the number of parameters in community dynamics models increases more rapidly than the amount of data, as species richness increases. Moreover, most species in high-diversity assemblages are very rare, further complicating the estimation of strengths of ecological interactions among species, or covariation in different species’ responses to environmental fluctuations. Consequently, ecologists have focused instead on making assumptions about the overall distribution of demographic rates, niche sizes, or other characteristics of an assemblage, and then deriving the aggregate assemblage properties implied by those assumptions (4–8). One of the most commonly investigated of these assemblage-level properties is the species abundance distribution (SAD)—the pattern of commonness and rarity among species (9–11). Ecologists have long sought to identify mechanisms that can explain common features of, and systematic differences among, the shapes of such distributions, and have used the ability to reproduce empirical SADs as a key test of biodiversity theory in species-rich systems (4, 6, 11–14).Over the last decade, one of the most prevalent and influential approaches to explaining the structure of high-diversity assemblages has been neutral theory of biodiversity (12, 15, 16). Neutral models assume that individuals are demographically and ecologically equivalent, regardless of species. Thus, variation in relative abundance among species arises purely from demographic stochasticity: chance variation in the fates of individuals (i.e., birth, death, immigration, and speciation events). Most studies investigating neutral theory aim to determine whether community structure in nature is consistent with the theory’s core species equivalence assumption. This is typically done by assessing the fit of a neutral model to empirical data, sometimes relative to a putatively nonneutral alternative (17–20). However, although all neutral models share the species equivalence assumption, they differ with respect to auxiliary assumptions, such as the mode of speciation assumed, leading to different predictions for SADs and other ecological patterns. Indeed, attempts to draw conclusions from tests of neutral theory are almost invariably disputed, largely due to arguments about the extent to which alternative auxiliary assumptions can materially alter neutral theory’s ability to explain the data (11, 12, 18, 21).An alternative, potentially more robust approach to evaluating neutral theory was proposed by Pueyo (22), based on approximating neutral and nonneutral dynamics as successively higher-order perturbations of a model for the idealized case of pure random drift in abundances. This approach predicts that a gamma distribution should approximate the distribution of species abundances for small departures for random drift, whereas assemblages exhibiting greater departures from neutrality should be better approximated by a lognormal distribution. This raises the possibility that a comparison of gamma and lognormal SADs could offer a robust test for the signature of nonneutrality in species abundance data, provided that the gamma distribution provides a sufficiently close approximation to SADs produced by neutral models.Here, we evaluate Pueyo’s framework and apply it to patterns of commonness and rarity in 1,185 SADs from 14 marine ecosystems ranging from shallow reef platforms to abyssal depths, and from the tropics to polar regions (Fig. 1 and Tables S1 and S2). First, we test the gamma neutral approximation against several models of community dynamics that share the core neutrality assumption of species equivalence, but make different assumptions about the speciation process, spatial structure of the metacommunity, and the nature of competition between individuals. Then, we analyze the marine species abundance data, to evaluate whether they are consistent with the hypothesis that marine assemblages are neutrally structured. Finally, we ask whether patterns of commonness and rarity deviate from neutral expectation in idiosyncratic ways, or whether there are particular features of real SADs that cannot be captured by neutral models.Open in a separate windowFig. 1.Sampling locations of SADs. Color and symbol combinations correspond to particular ecosystems. These symbols are reproduced in the surrounding panels, which show observed and fitted SADs for the site-level data (averaged across sites) of the corresponding ecosystem. The bars represent the mean proportion of species at each site in different octave classes of abundance, across all sites in the corresponding dataset [the first bar represents species with abundance 1, then abundances 2–3, abundances 4–7, etc. (10)]. The blue and red lines show the mean of fitted values from site-by-site fits of the Poisson-gamma and Poisson-lognormal distributions to the data, respectively.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

A legacy of contrasting spatial genetic structure on either side of the Atlantic–Mediterranean transition zone in a marine protist

The mechanisms that underpin the varied spatial genetic structures exhibited by free-living marine microorganisms remain controversial, with most studies emphasizing a high dispersal capability that should redistribute genetic diversity in contrast to most macroorganisms whose populations often retain a genetic signature of demographic response to historic climate fluctuations. We quantified the European phylogeographic structure of the marine flagellate Oxyrrhis marina and found a marked difference in spatial genetic structure, population demography, and genetic diversity between the northwest Atlantic and Mediterranean Sea that reflects the persistent separation of these regions as well as context-dependent population responses to contrasting environments. We found similar geographic variation in the level of genetic diversity in the sister species Oxyrrhis maritima. Because the capacity for wide dispersal is not always realized, historic genetic footprints of range expansion and contraction persist in contemporary populations of marine microbes, as they do in larger species. Indeed, the well-described genetic effects of climatic variation on macroorganisms provide clear, testable hypotheses about the processes that drive genetic divergence in marine microbes and thus about the response to future environmental change.Free-living protists (eukaryotic microbes) are key components of marine ecosystems, driving productivity and biogeochemical cycles (1), but we lack understanding of the processes that underpin the contemporary distribution of genetic diversity in such organisms. A persistent paradigm emphasizes the importance of organism size, with microorganisms (<1 mm) such as protists having huge population sizes and being widely dispersed (2, 3). The corollary is that, compared with most macroorganisms, protists have little opportunity for allopatric divergence and will not exhibit pronounced spatial genetic structure. This paradigm is thought to be typical in the marine environment where the few obvious barriers to planktonic dispersal should permit extensive gene flow, and the abundance of cryptic marine microbial species (4, 5) is hypothesized to reflect sympatric divergence (6, 7). An opposing view is that there is no clear micro/macroorganism dichotomy, and that many free-living protists have restricted distributions, low rates of dispersal, and thus exhibit parapatric or allopatric divergence (8). Resolving these dichotomous viewpoints is essential to understand the processes that drive differentiation among populations of free-living protists and, ultimately, adaptation and speciation.An increasing number of studies therefore have evaluated the degree of population structure in free-living marine protists. A complex picture has emerged, with reports of homogeneity at regional (9, 10) and global scales (11), as well as substantial genetic divergence that occurs locally (12), regionally (9, 13–16), or globally (17–19). It is clear that marine protist populations vary considerably in the scale over which significant genetic structure occurs. However, most studies interpret patterns in terms of putative dispersal ability in response to contemporary seascape features such as the structure of ocean basins, currents, and/or fronts. This approach overlooks the legacy of historic climate change that has shaped the present-day distribution of genetic diversity in many macroscopic taxa as a consequence of geographic range shifts in response to periodic cycles of glaciation and warming throughout the Quaternary (2.59 Mya to present) (20).Although species vary in their ability to track suitable habitat during periods of climate change, the general patterns exhibited by European marine macroorganisms (21–23) provide an insight into processes that may have affected marine protists. First, most northern populations exhibit a shallow population structure and lack genetic diversity because of recent (post-Last Glacial Maximum, 25–18 kya) expansion into formerly inhospitable glaciated areas from southern refugia, albeit with alternative genetic patterns indicating that species persisted in northern periglacial refugia (22, 24). Second, marked divergence between Atlantic and Mediterranean populations often is interpreted as vicariance associated with the oscillations in sea level that isolated the Mediterranean Sea from the Atlantic at the Strait of Gibraltar; however, this pattern is not universal, and genetic congruence between the Atlantic and Mediterranean has been reported for some pelagic species (21). Finally, contemporary Mediterranean marine fauna bear a genetic legacy resulting from an extensive period of desiccation between 5.59 and 5.3 Mya [the Messinian Salinity Crisis (25)] that extirpated most species from the region or reduced species’ distributions to small, isolated eastern refugia. Subsequent flooding of the Mediterranean basin from the Atlantic was followed by episodic changes in sea level associated with glacial cycles. Successive periods of colonization and isolation left longitudinal gradients in genetic diversity across the Mediterranean and in some cases resulted in greatest genetic diversity in the central Mediterranean as a result of bidirectional colonization and secondary contact (26).No study has yet sought to identify the contribution of historical legacies in shaping microbial population structures, and thus patterns of colonization, spatial variation in population demography, and the occurrence of potential refugia remain undocumented for any free-living marine protist. This lack is surprising, given the evidence for complex population structures and biogeographic patterns (8, 27) in marine microbial populations, which support the more general role for divergence in geographically distinct locations, as opposed to sympatry, as hypothesized by Foissner (8). Although two studies on marine protists have dated periods of diversification to the Pleistocene (19, 28), we still do not know the extent to which contemporary marine protist populations retain a genetic legacy of historic dispersal and divergence.The free-living marine flagellate Oxyrrhis marina (Dinoflagellata: Oxyrrhinales) is common in coastal habitats (29) and thus is a model of marine protists that have wide geographic distributions, large population sizes, and putative high dispersal capabilities. The genus Oxyrrhis comprises two sibling species (5, 30). One, O. marina, is common and widely distributed; the other, Oxyrrhis maritima, has a more limited distribution (29). By quantifying spatial genetic structure in these species across the European seascape, we tested the hypothesis that contemporary populations of free-living marine protists do not redistribute genetic diversity rapidly but rather retain the distinct genetic signatures of population responses to historic changes in climate.     

Macroevolutionary speciation rates are decoupled from the evolution of intrinsic reproductive isolation in Drosophila and birds

The rate at which speciation occurs varies greatly among different kinds of organisms and is frequently assumed to result from species- or clade-specific factors that influence the rate at which populations acquire reproductive isolation. This premise leads to a fundamental prediction that has never been tested: Organisms that quickly evolve prezygotic or postzygotic reproductive isolation should have faster rates of speciation than organisms that slowly acquire reproductive isolation. We combined phylogenetic estimates of speciation rates from Drosophila and birds with a method for analyzing interspecific hybridization data to test whether the rate at which individual lineages evolve reproductive isolation predicts their macroevolutionary rate of species formation. We find that some lineages evolve reproductive isolation much more quickly than others, but this variation is decoupled from rates of speciation as measured on phylogenetic trees. For the clades examined here, reproductive isolation—especially intrinsic, postzygotic isolation—does not seem to be the rate-limiting control on macroevolutionary diversification dynamics. These results suggest that factors associated with intrinsic reproductive isolation may have less to do with the tremendous variation in species diversity across the evolutionary tree of life than is generally assumed.A central challenge at the interface between macroevolution and microevolution is to explain the population-level processes that contribute to biological variation in diversification rates and species richness (1). Phylogenetic evidence for biological variation in the rate of species diversification is widespread (2, 3), and numerous studies have now linked specific traits to the dynamics of speciation and extinction as realized over macroevolutionary timescales (2, 4). At the population level, a microevolutionary research program on the biology of speciation has focused on the factors that lead to various forms of reproductive isolation (RI) between populations (5, 6). Explaining how and why RI evolves is generally considered to be the central and defining challenge in the study of speciation (2, 7–9), and recent studies have made great progress toward explaining the genetic and ecological basis for various forms of RI (7, 8, 10).Most microevolutionary research on speciation implicitly assumes that RI is the defining and rate-limiting step in the speciation process (7, 8, 11), but the evolution of RI need not bear any predictive relationship to rates of species diversification as realized over macroevolutionary timescales (12). This has long been recognized by the paleontological community, where “successful” speciation is believed to entail not only the evolution of reproductive isolation but also the persistence of incipient species (13–15). For example, speciation might be limited primarily by the rate at which lineages form allopatric isolates (6, 16) or by the capacity for geographic range expansion (17, 18). Likewise, speciation might be limited more by factors that influence the temporal persistence of reproductively isolated populations (15, 19) than by the rate at which RI itself evolves. Finally, macroevolutionary diversity dynamics might be regulated primarily by factors that influence extinction rates (4). These factors need not be independent of reproductive isolation. For example, reproductive barriers can reduce the probability of population fusion following secondary contact between nascent species, thus exerting a direct effect on population persistence (2). Likewise, RI can influence the dynamics of geographic range evolution, which may in turn have secondary consequences for rates of demographic extinction and allospecies formation (17, 18).Here, we provide a direct test of the relationship between reproductive isolation and macroevolutionary diversification. If the widespread variation observed in macroevolutionary diversification rates (20, 21) is attributable to factors that cause RI (2, 22, 23), then it must also be true that lineage-specific differences in the rate at which RI evolves will influence large-scale patterns of species diversification. In this context, a species with a “fast” rate of RI evolution will, all else being equal, form more reproductively isolated lineages than a species with a “slow” rate of RI evolution.We use data from the two groups of animals for which the most extensive multispecies RI data have been compiled and for which we could derive phylogeny-based estimates of species diversification rates. The first dataset is an update of Coyne and Orr’s (2) seminal work on the relationship between genetic distance, geographic status, and reproductive isolation in Drosophila (24) and contains estimates of both premating and intrinsic postzygotic RI. The second dataset includes measurements of intrinsic postyzygotic isolation from interspecies hybridizations in birds (25, 26). We developed a modeling framework for estimating species- and clade-specific differences in the rate of evolution of RI and applied it to both birds and flies to test whether lineages that quickly evolve RI are characterized by fast rates of macroevolutionary diversification. This framework can be used to assess whether any components of reproductive isolation are associated with speciation rates as measured at macroevolutionary scales. We find significant heterogeneity within both flies and birds in the rate at which lineages evolve intrinsic postzygotic isolation. However, this variation is uncorrelated with macroevolutionary rates of species diversification. These results suggest that patterns of biological diversity may have less to do with reproductive isolation than generally assumed.     

Cytonuclear interactions affect adaptive traits of the annual plant Arabidopsis thaliana in the field

Although the contribution of cytonuclear interactions to plant fitness variation is relatively well documented at the interspecific level, the prevalence of cytonuclear interactions at the intraspecific level remains poorly investigated. In this study, we set up a field experiment to explore the range of effects that cytonuclear interactions have on fitness-related traits in Arabidopsis thaliana. To do so, we created a unique series of 56 cytolines resulting from cytoplasmic substitutions among eight natural accessions reflecting within-species genetic diversity. An assessment of these cytolines and their parental lines scored for 28 adaptive whole-organism phenotypes showed that a large proportion of phenotypic traits (23 of 28) were affected by cytonuclear interactions. The effects of these interactions varied from slight but frequent across cytolines to strong in some specific parental pairs. Two parental pairs accounted for half of the significant pairwise interactions. In one parental pair, Ct-1/Sha, we observed symmetrical phenotypic responses between the two nuclear backgrounds when combined with specific cytoplasms, suggesting nuclear differentiation at loci involved in cytonuclear epistasis. In contrast, asymmetrical phenotypic responses were observed in another parental pair, Cvi-0/Sha. In the Cvi-0 nuclear background, fecundity and phenology-related traits were strongly affected by the Sha cytoplasm, leading to a modified reproductive strategy without penalizing total seed production. These results indicate that natural variation in cytoplasmic and nuclear genomes interact to shape integrative traits that contribute to adaptation, thereby suggesting that cytonuclear interactions can play a major role in the evolutionary dynamics of A. thaliana.The genomes of eukaryotes originate from ancient endosymbiotic associations that eventually led to energy-harnessing organelles: mitochondria, common to all eukaryotes, and chloroplasts in the “green” lineage. The evolution of endosymbionts into cellular organelles was accompanied by massive gene loss, with a large proportion being transferred to the nucleus (1, 2). Nevertheless, mitochondria and chloroplasts retained a few (30–80) protein-encoding genes that play crucial roles in energy metabolism (respiration and photosynthesis). Mitochondrion and chloroplast metabolisms rely on the proper interaction of nuclear-encoded proteins and their counterparts encoded in the organelle genome. Consequently, the genes in nuclear and organellar compartments are expected to be coadapted (3).Cytonuclear coadaptation has been demonstrated by altered phenotypes observed on interspecific exchanges of cytoplasm between related species in mammals (4), yeast (5), arthropods (6), and plants, whose interspecific crosses are frequently successful (7). These alterations affect organelle function and even the organism phenotype, indicating epistasis between nuclear and cytoplasmic genes. Although cytonuclear coadaptation is generally studied at the interspecific level, the existence of intraspecific genetic diversity in organelle genomes suggests a potential for genomic coadaptation within species. A few studies have reported phenotypic effects of intraspecific cytonuclear epistasis in nonplant species (8–11). In plants, many studies have focused on cytoplasmic male sterility (CMS), an impairment of pollen production governed by nucleo-mitochondrial interactions in some hermaphroditic species (12), in particular in crops and their relatives (13). The phenotypic effects of intraspecific cytonuclear epistasis other than CMS have been reported in only a limited number of plant systems (14–17), with evidence that cytoplasmic variation contributes to local adaptation (18, 19).In recent years, several studies using reciprocal segregating populations of the model plant Arabidopsis thaliana have investigated the effect of cytonuclear epistasis on a number of laboratory-measured phenotypes such as the metabolome, defense chemistry and growth (17, 20, 21), water-use efficiency (22, 23), and seed germination (24, 25). Although some studies have reported significant effects of cytonuclear epistasis (17, 20, 21, 23, 25), others have found additive cytoplasmic effects but with weak or no cytonuclear epistasis (22). Each of these studies (with the exception of ref. 25) was, however, based on a single reciprocal cross between two natural accessions, thereby preventing the estimation of the prevalence of cytonuclear epistasis in this species. In addition, although these reports involve adaptive traits (26–30), the investigation of the effect of cytonuclear epistasis on adaptive phenotypes in field conditions is, at best, scarce in A. thaliana.Here, following the modern standards of ecological genomics (31), we explored the prevalence of cytonuclear interactions on adaptive whole-organism traits in the model plant A. thaliana in a field experiment. To do so, based on eight natural accessions of a core collection that covers a significant part of the species’ cytoplasmic and nuclear genetic diversity in A. thaliana (25, 32), we created eight series of seven cytolines. Cytolines are genotypes that combine the nuclear genome from one parent with the organelle genomes of another (33). We examined the cytolines and their parental accessions for effects of cytonuclear interactions on 28 field-measured traits related to germination, phenology, resource acquisition, plant architecture and seed dispersal, fecundity, and survival.     

From the Cover: Threat of plastic pollution to seabirds is global,pervasive, and increasing

Plastic pollution in the ocean is a global concern; concentrations reach 580,000 pieces per km² and production is increasing exponentially. Although a large number of empirical studies provide emerging evidence of impacts to wildlife, there has been little systematic assessment of risk. We performed a spatial risk analysis using predicted debris distributions and ranges for 186 seabird species to model debris exposure. We adjusted the model using published data on plastic ingestion by seabirds. Eighty of 135 (59%) species with studies reported in the literature between 1962 and 2012 had ingested plastic, and, within those studies, on average 29% of individuals had plastic in their gut. Standardizing the data for time and species, we estimate the ingestion rate would reach 90% of individuals if these studies were conducted today. Using these results from the literature, we tuned our risk model and were able to capture 71% of the variation in plastic ingestion based on a model including exposure, time, study method, and body size. We used this tuned model to predict risk across seabird species at the global scale. The highest area of expected impact occurs at the Southern Ocean boundary in the Tasman Sea between Australia and New Zealand, which contrasts with previous work identifying this area as having low anthropogenic pressures and concentrations of marine debris. We predict that plastics ingestion is increasing in seabirds, that it will reach 99% of all species by 2050, and that effective waste management can reduce this threat.Introduction of plastic waste into the marine environment is a global concern. Plastic production is rapidly rising, with a doubling of production every 11 y since commercial production began in the 1950s (1). This growth in production has been accompanied by a corresponding increase in the concentration of plastics in the marine environment although it has been suggested that marine organisms may be a major sink reducing this increase (2–4). The durability of plastic implies that it is retained for years to centuries, in some cases failing to degrade at all if it is not exposed to bacterial activity or UV radiation (5).Plastic fragments can be found throughout the world’s oceans, with observed concentrations up to 580,000 plastic pieces per square kilometer (2, 3, 6). Modeling studies, validated by global sampling efforts, demonstrate that plastics are ubiquitous, with high concentrations in all five subtropical convergence zones and along the coastal margins near human population centers (3, 6, 7).In addition to the evidence of its prevalence, there is emerging evidence of the threats plastics pose to wildlife, and indirectly to human health. Plastic waste affects wildlife via two means: entanglement and ingestion (8). A recent review for the United Nations Convention on Biological Diversity documented over 600 species, ranging from microorganisms to whales, affected by marine plastic waste, largely through ingestion (9). Ingestion is known to have many effects, ranging from physical gut blockage (10) to organ damage from leaching toxins (11). Recent experimental studies have also demonstrated transmission and toxicological effects of plastics, or adsorbed chemicals, at environmentally relevant concentrations in higher vertebrates (11–13).The effect of plastic ingestion on seabirds in particular has been of concern. This concern is due to the frequency with which seabirds ingest plastic (12) and because of emerging evidence of both impacts on body condition and transmission of toxic chemicals, which could result in changes in mortality or reproduction (13–16). Understanding the contribution of this threat is particularly pressing because half of all seabird species are in decline, a higher fraction than other comparable taxa (17). Despite a recent extensive review of the threats to seabirds by a globally recognized authority (17), however, pollution has been identified only in a coastal context, and there is little mention of the impact of plastic ingestion, particularly on the high seas where the most threatened seabirds forage (17).We predict the extent of plastics exposure for 186 pelagic seabird species worldwide, excluding coastal taxa such as shorebirds, sea ducks, and gulls and species for which distribution data were not available (SI Appendix, Table S1). We compare our predictions with diet studies published over the last 40 y and incorporate additional factors such as foraging strategy, body size, and sampling method that may affect the relationship between exposure and ingestion. Based on this adjusted model of risk, we map the global distribution of plastic ingestion risk for seabirds and highlight global areas of concern.     

The transition between the niche and neutral regimes in ecology

An ongoing debate in ecology concerns the impacts of ecological drift and selection on community assembly. Here, we show that there is a transition in diverse ecological communities between a selection-dominated regime (the niche phase) and a drift-dominated regime (the neutral phase). Simulations and analytic arguments show that the niche phase is favored in communities with large population sizes and relatively constant environments, whereas the neutral phase is favored in communities with small population sizes and fluctuating environments. Our results demonstrate how apparently neutral populations may arise even in communities inhabited by species with varying traits.The success of the neutral theory of biodiversity and biogeography (1, 2) at explaining patterns in biodiversity has resulted in a vigorous debate on the processes underlying the assembly, dynamics, and structure of ecological communities (1, 3–12). Starting with the pioneering work of MacArthur (13–15), ecologists have emphasized the roles of interspecific competition and environmental interactions in community assembly and dynamics. These niche-based models emphasize ecological selection as the driving force of community assembly, whereas neutral models of biodiversity assume a functional equivalence between species and emphasize the role of ecological drift (i.e., stochasticity) in community dynamics (1, 2, 16, 17). The success of both types of models at explaining ecological data highlights the crucial need for understanding the impacts of ecological drift and selection in community ecology (18).     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.