Mechanosensitive remodeling of the bacterial flagellar motor is independent of direction of rotation

Motility is important for the survival and dispersal of many bacteria, and it often plays a role during infections. Regulation of bacterial motility by chemical stimuli is well studied, but recent work has added a new dimension to the problem of motility control. The bidirectional flagellar motor of the bacterium Escherichia coli recruits or releases torque-generating units (stator units) in response to changes in load. Here, we show that this mechanosensitive remodeling of the flagellar motor is independent of direction of rotation. Remodeling rate constants in clockwise rotating motors and in counterclockwise rotating motors, measured previously, fall on the same curve if plotted against torque. Increased torque decreases the off rate of stator units from the motor, thereby increasing the number of active stator units at steady state. A simple mathematical model based on observed dynamics provides quantitative insight into the underlying molecular interactions. The torque-dependent remodeling mechanism represents a robust strategy to quickly regulate output (torque) in response to changes in demand (load).

Many bacteria swim through aqueous environments to acquire resources, to disperse progeny, and to infect hosts (1, 2). The rotation of flagella (3, 4), powered by the bidirectional flagellar motor (5–7), drives motility in many bacteria. In Escherichia coli, the flagellar motor consists of over 20 different proteins that self-assemble at the cell wall in varying copy numbers (8–10). Motor structure (Fig. 1A) includes a rotor embedded in the inner cell membrane, a drive shaft, and a flexible hook that transmits torque to the filament (10, 11). The cytoplasmic ring (C ring), which contains copies of the proteins FliG, FliM, and FliN, is mounted on the cytoplasmic face of the rotor and is responsible for directional switching of the motor (12). The rotor is driven by up to 11 ion-powered MotA5B2 stator units (13–16) that surround the rotor and generate torque. MotA engages FliG, whereas MotB is mounted on the rigid framework of the peptidoglycan cell wall (17–20). Motor-bound units exchange with a pool of unbound units in the inner membrane (10, 21).Open in a separate windowFig. 1.Bacterial flagellar motor’s structure and its T-S curve. (A) Schematic representation of the flagellar motor of Gram-negative bacteria. The rotor consists of the MS ring (M for membranous and S for supramembranous) embedded in the inner membrane (IM) and the C ring embedded in the cytoplasm. Stator units (MotA–MotB complexes) that span the inner membrane bind to the peptidoglycan (PG) layer and apply torque on the C ring. The torque is transmitted via a rod (driveshaft) and a flexible hook (universal joint) to the flagellar filament. L and P rings (L for lipopolysaccharide and P for peptidoglycan) are embedded in the outer membrane (OM) and the peptidoglycan, respectively, and act as bushings. Inset shows the outline of an E. coli cell with a square demarcating the region that is represented in detail. (B) T-S curve of the CCW (solid orange) and CW (dashed blue) rotating flagellar motors compared in this study. Data are from refs. 31 and 58. See Materials and Methods for details.Motor function is regulated by inputs from the environment. Detection of specific ligands by chemoreceptors drives a two-component signaling cascade that controls the direction of rotation of the motor (22–24). Upon binding the response regulator CheY-P, the C ring undergoes a concerted conformational change that reverses motor rotation from counterclockwise (CCW) to clockwise (CW), as viewed from outside the cell. This change in the direction of rotation is the basis of run-and-tumble motility in E. coli (CCW = run, CW = tumble). Changes in viscous load trigger remodeling of the stator (25–27), whereby, at high loads, the number of motor-bound stator units increases, and vice versa. Dynamics of stator remodeling have only been quantified in CCW rotating motors, using electrorotation (28) and magnetic tweezers (29, 30). The observed dynamics were rationalized using the CCW torque–speed (T-S) relationship (Fig. 1B) (28). CCW and CW rotating motors have different T-S relationships (Fig. 1B), likely due to differences in stator–rotor interactions (31–33). How the differences in T-S relationship affect stator remodeling in CW motors is unknown. Additionally, the molecular mechanisms underlying the load-dependent remodeling phenomenon remain poorly understood.Here, we use electrorotation to study the dynamics of load-dependent stator remodeling in CW rotating motors. We find that, just like CCW motors, CW rotating flagellar motors release their stator units when the motor torque is low, and recruit stator units when the torque increases again. The rates of stator unit release and recruitment in CW and CCW motors collapse onto a single curve when plotted against torque, despite their dissimilar T-S relationships. The collapse of remodeling data suggests a universal model for torque dependence in the mechanically regulated remodeling of the bacterial flagellar motor. Our in vivo measurements of stator assembly dynamics advance the understanding of a large protein complex with multiple parts.     

Dimensions of invasiveness: Links between local abundance,geographic range size,and habitat breadth in Europe’s alien and native floras

Understanding drivers of success for alien species can inform on potential future invasions. Recent conceptual advances highlight that species may achieve invasiveness via performance along at least three distinct dimensions: 1) local abundance, 2) geographic range size, and 3) habitat breadth in naturalized distributions. Associations among these dimensions and the factors that determine success in each have yet to be assessed at large geographic scales. Here, we combine data from over one million vegetation plots covering the extent of Europe and its habitat diversity with databases on species’ distributions, traits, and historical origins to provide a comprehensive assessment of invasiveness dimensions for the European alien seed plant flora. Invasiveness dimensions are linked in alien distributions, leading to a continuum from overall poor invaders to super invaders—abundant, widespread aliens that invade diverse habitats. This pattern echoes relationships among analogous dimensions measured for native European species. Success along invasiveness dimensions was associated with details of alien species’ introduction histories: earlier introduction dates were positively associated with all three dimensions, and consistent with theory-based expectations, species originating from other continents, particularly acquisitive growth strategists, were among the most successful invaders in Europe. Despite general correlations among invasiveness dimensions, we identified habitats and traits associated with atypical patterns of success in only one or two dimensions—for example, the role of disturbed habitats in facilitating widespread specialists. We conclude that considering invasiveness within a multidimensional framework can provide insights into invasion processes while also informing general understanding of the dynamics of species distributions.

Human socioeconomic activities are altering species’ global distributions, bridging natural dispersal barriers through the accidental and intentional relocation of organisms, and opening opportunities for them to expand into new regions beyond their historic native ranges (1). The outcome of any given introduction event, however, is dependent on ecological and stochastic processes, and many introduced alien species fail to establish and persist (2, 3). Even species that do achieve persistent, self-sustaining populations (i.e., become naturalized sensu ref. 4) show varying degrees of success (i.e., invasiveness) in newly occupied regions. This has been true for natural colonization events throughout Earth’s history [e.g., on islands (5, 6) and during continental biotic interchanges (7–9)] and is certainly the case for the ongoing surge of human-mediated introductions (10–12). Disentangling the factors that lead to invasion success provides an opportunity not only for anticipating and mediating future anthropogenic invasions but also for better understanding the dynamics underlying natural range expansions (13).Quantifying a species’ success in invading the alien range is complex, a fact reflected in the diverse criteria applied by different authorities when deciding whether or not to classify naturalized species as invasive (14). Recent efforts have therefore recognized that invasiveness cannot be captured by a single metric but rather encompasses multiple aspects of ecological success and impact (15, 16). Some proposed metrics, such as spread rate and socioeconomic impacts, are difficult to quantify for large numbers of species (4, 17). However, Rabinowitz’s three-dimensional scheme for characterizing the rarity or commonness of species in their native distributions (18, 19) has been successfully co-opted as a valuable perspective for better understanding the success of alien species (16, 20, 21). Applied in the context of introduced species, this framework recognizes the potential for established aliens to vary along at least three demographic dimensions of invasiveness: 1) in local abundance within the naturalized range, 2) in geographic range size or extent of the naturalized range, and 3) in habitat breadth in the naturalized range (16). We subsequently distinguish these metrics as dimensions of invasiveness when measured in the naturalized distributions of alien species and dimensions of commonness when measured in species native distributions.Considering invasiveness within a multidimensional framework is particularly important if species vary independently among different dimensions (16, 21). Such a scenario opens the possibility for aliens to achieve invasion success in many different ways (Fig. 1). In other words, there could exist different forms of invasiveness, similar to the different forms of rarity or commonness originally proposed by Rabinowitz (19). On the other hand, theoretical concepts and empirical examples suggest correlations between Rabinowitz’s dimensions of commonness among species in their native distributions (6, 22, 23). For example, a positive relationship between local abundance and extent of geographic occurrence or range size has been documented at various scales for numerous taxa (24–26), including plants (24, 27–31), with niche breadth proposed as a linking mechanism (24, 26, 32). If the processes that generate these patterns in native distributions act similarly in species alien distributions, some of the forms of invasiveness outlined in Fig. 1 should be less likely to occur than others. More specifically, if the invasiveness dimensions are correlated, species should vary from excelling (abundant, widespread, generalists; form AWG in Fig. 1) to performing poorly (scarce, restricted, specialists; form 0 in Fig. 1) in all three invasiveness dimensions (33). On the other hand, these macroecological patterns are not without exception, and a recent assessment found little support for correlations among commonness dimensions in Europe’s native flora (34). Alien distributions may further differ because aliens vary in their residence time, and particularly recently introduced species may be in disequilibrium and still increasing along one or more of the invasiveness dimensions (21, 35–37). In line with these alternatives, a continuum from overall poor invaders to species succeeding in all three dimensions has been documented for the regional alien flora of French grassland communities (20), while associations among dimensions were found to be low for the herbaceous alien flora of Southeast Australia (16). The correspondence among different invasiveness dimensions at broader geographic scales has yet to be assessed.Open in a separate windowFig. 1.Conceptual diagram outlining the eight different forms of invasiveness depending on success in zero, one, two, or three dimensions of invasiveness (based on refs. 16, 18, and 20). Forms of invasiveness within the cyan polygon are associated with high naturalized abundance, within the magenta polygon with widespread naturalized geographic extent, and within the yellow polygon with high naturalized habitat breadth. The overlap between magenta and cyan is blue, between cyan and yellow is green, between magenta and yellow is red, and between all three is black. The forms of invasiveness are comparable to analogous forms of commonness used to describe species in their native distributions, and we refer to the same abbreviations in both cases.Functional traits play a role in mediating invasion processes, but efforts to identify characteristics of successful invaders have generally resulted in few or inconsistent associations (38, 39). However, distinguishing between different components of invasiveness may provide additional clarity if each is influenced by different traits or if the same trait has contrasting effects on different dimensions (15, 16, 21, 40, 41). For example, many plant traits are associated with general trade-offs between rapid growth (i.e., acquisitive growth strategies) versus stress tolerance and survival (i.e., conservative growth strategies) (42–44), and one can hypothesize scenarios where these divergent strategies are associated with success in different dimensions of invasiveness (40, 41). Another example are specialized adaptations for long-distance dispersal that may promote rapid range expansion, both in extent and into new habitats, but likely do not provide any advantages that would influence local abundances (45, 46). For habitat specialists, their specific habitat associations may additionally be important for determining whether or not they become widespread (31).A number of hypotheses for invasion success additionally emphasizes the importance of unique ecological dynamics that emerge when species are decoupled from constraints experienced in their native environments (47). For example, because species are able to occupy unfilled niches where introduced [i.e., Darwin’s naturalization hypothesis (48, 49)] or because they leave behind important herbivores, competitors, or pathogens that limit populations in the native distribution [i.e., enemy release (50, 51)]. These mechanisms may be less likely when species expand into areas near the native range, for example, during natural range expansions or intracontinental introductions, as the alien individuals are more likely to encounter conditions similar to those that limited their native distribution compared to species introduced from further abroad (e.g., those with extracontinental origins) (52–54).Here, we combine vegetation plot data covering Europe (55) with databases of alien and native distributions (56, 57), plant traits (58, 59), and historical dates of introduction (60) to provide a comprehensive assessment of multidimensional invasion success for the European alien seed plant flora. First, we test for correlations among local abundance, geographic extent, and habitat breadth of alien species in their naturalized distributions and classify species into one of the eight forms of invasiveness (Fig. 1). We ask whether some forms of invasiveness rarely occur and specifically whether species tend to fit along a continuum ranging from generally poor invaders to super invaders—species excelling in all three dimensions. In addition, we compare relationships among dimensions of invasiveness to those among dimensions of commonness measured for Europe’s native flora, assessing similarities and differences in patterns of distribution between contexts. Next, we explore likely drivers of each invasiveness dimension, testing whether the year of first alien occurrence in Europe, functional traits related to ecological strategies, specialized adaptations for long-distance dispersal, habitat associations, and region of origin explain different forms of invasion success.     

Wolves make roadways safer,generating large economic returns to predator conservation

Recent studies uncover cascading ecological effects resulting from removing and reintroducing predators into a landscape, but little is known about effects on human lives and property. We quantify the effects of restoring wolf populations by evaluating their influence on deer–vehicle collisions (DVCs) in Wisconsin. We show that, for the average county, wolf entry reduced DVCs by 24%, yielding an economic benefit that is 63 times greater than the costs of verified wolf predation on livestock. Most of the reduction is due to a behavioral response of deer to wolves rather than through a deer population decline from wolf predation. This finding supports ecological research emphasizing the role of predators in creating a “landscape of fear.” It suggests wolves control economic damages from overabundant deer in ways that human deer hunters cannot.

Populations of apex predators have declined across the world’s landscapes over the past 200 y due to government bounty programs, hunting pressure, habitat loss, and declines in prey populations (1, 2). Ecologists are beginning to unravel the far-reaching ecological effects of these changes (1–5), but little is known about the economic effects. While many of the costs attributable to predators are salient and quantifiable, such as predation on livestock and pets, estimating and valuing the often subtle and indirect beneficial effects of predators is more difficult (6, 7).^*The recent expansion of the gray wolf (Canis lupus) offers a unique opportunity to concretely measure the cascading benefits of a predator whose reintroduction is controversial. Wolves once ranged over most of the Northern Hemisphere, but humans nearly eradicated the species from the continental United States and Europe by the 1960s (10). Legal protections strengthened during the latter half of the 20th century, and wolf populations returned to 10 coterminous US states and 28 European countries (11, 12). As of 2019, there are about 5,500 wolves in the United States and 11,000 in Europe. More jurisdictions, such as the state of Colorado, are proposing or already planning reintroductions (13).Restoring wolves could benefit humans who enjoy seeing wolves in their natural habitat or who value knowing of their existence (14, 15), but in this study we focus on the potential for wolves to generate indirect benefits by controlling overabundant deer populations. Deer populations have surged in the United States, increasing from about 2 to 4 deer per km² in the precolonial era (16) to 15 to 50 deer per km² in some areas today (17). Overabundant deer populations affect ecosystems by suppressing forest regeneration, altering the composition of tree and herbaceous plant species, and contributing to the spread of invasive species (18–20). Deer also generate economic costs for humans through deer–vehicle collisions (DVCs), Lyme disease (which is transmitted through deer ticks), and damage to agriculture, timber products, and landscaping (21).This study focuses on DVCs, which are the largest known economic cost of deer (SI Appendix, Table S1). About 1 million DVCs occur every year in the United States, causing 29,000 human injuries, 200 human fatalities, and nearly $10 billion in total economic losses (21, 22).^† Europe experiences similar problems, with a lower frequency of collisions with ungulates (such as deer and moose) but a higher rate of fatalities and injuries (23). The problem has worsened over time, with DVCs rapidly increasing since around 1990 (22, 23). If wolves reduce DVCs even modestly, the social and economic benefits could be sizable.We focus on two channels through which wolves could affect DVCs. The first is changes to deer abundance. We hypothesize that larger wolf populations reduce deer abundance directly through predation (18, 24, 25), which in turn reduces DVCs (26–28). The second channel is through changes to deer behavior because wolves create a “landscape of fear” for deer (29–31). Wolves use roads, pipelines, and other linear features as travel corridors, which increases wolves’ travel efficiency and the kill rate of prey near these features (32–36). We hypothesize that wolf presence affects deer movement near these features (34, 37, 38), thereby reducing collision risk for a given number of deer on the landscape. The behavioral effect is important because, through it, wolves can suppress economic damage from deer in ways seasonal human deer hunters cannot.Our empirical analysis focuses on Wisconsin, where wolves began to recolonize naturally around 1975 (Fig. 1) (39). Wisconsin provides an interesting, if not ideal, case study for several reasons. Foremost, the results from Wisconsin should generalize to other settings where wolves are (or could be) allowed to spread naturally into areas of human communities rather than only into wilderness. Much of the prior research on the effects of wolves in the United States focuses on Isle Royale and Yellowstone National Parks; however, the effect of wolves in national parks “would have little relevance to most of wolf range because of overriding anthropogenic influences there on wolves, prey, vegetation, and other parts of the food web” (40). By contrast, the spread of wolves across Wisconsin was natural and unimpeded by wildlife managers. As a result, contiguous wolf range in Wisconsin spans a human-dominated landscape more than 6 times the size of Isle Royale and Yellowstone combined (41). Second, the wolf population in Wisconsin is likely close to ecological carrying capacity (42, 43), which suggests the wolf effects we measure represent a long-term steady state rather than only transitional effects. Finally, if the benefits of a reduction in DVCs outweigh the costs of wolf predation in Wisconsin, then there may be economic justification for allowing wolves to expand not just in the state but also potentially in other states that have suitable wolf habitat and high DVCs (e.g., the northeastern United States) (44). Although verified predation on livestock and pets in Wisconsin is costly (recently, about $174,000 per year, on average) (45), DVC losses are orders of magnitude larger (almost $200 million per year, based on the national average cost per DVC) (22, 46).Open in a separate windowFig. 1.Wolf packs spread across Wisconsin between 1980 and 2010. Wolf packs are concentrated in the forested areas in the northern and central parts of the state. The rest of the state is predominantly nonforested agricultural or urban areas.     

A solution to a sex ratio puzzle in Melittobia wasps

The puzzling sex ratio behavior of Melittobia wasps has long posed one of the greatest questions in the field of sex allocation. Laboratory experiments have found that, in contrast to the predictions of theory and the behavior of numerous other organisms, Melittobia females do not produce fewer female-biased offspring sex ratios when more females lay eggs on a patch. We solve this puzzle by showing that, in nature, females of Melittobia australica have a sophisticated sex ratio behavior, in which their strategy also depends on whether they have dispersed from the patch where they emerged. When females have not dispersed, they lay eggs with close relatives, which keeps local mate competition high even with multiple females, and therefore, they are selected to produce consistently female-biased sex ratios. Laboratory experiments mimic these conditions. In contrast, when females disperse, they interact with nonrelatives, and thus adjust their sex ratio depending on the number of females laying eggs. Consequently, females appear to use dispersal status as an indirect cue of relatedness and whether they should adjust their sex ratio in response to the number of females laying eggs on the patch.

Sex allocation has produced many of the greatest success stories in the study of social behaviors (1–4). Time and time again, relatively simple theory has explained variation in how individuals allocate resources to male and female reproduction. Hamilton’s local mate competition (LMC) theory predicts that when n diploid females lay eggs on a patch and the offspring mate before the females disperse, the evolutionary stable proportion of male offspring (sex ratio) is (n − 1)/2n (Fig. 1) (5). A female-biased sex ratio is favored to reduce competition between sons (brothers) for mates and to provide more mates (daughters) for those sons (6–8). Consistent with this prediction, females of >40 species produce female-biased sex ratios and reduce this female bias when multiple females lay eggs on the same patch (higher n; Fig. 1) (9). The fit of data to theory is so good that the sex ratio under LMC has been exploited as a “model trait” to study the factors that can constrain “perfect adaptation” (4, 10–13).Open in a separate windowFig. 1.LMC. The sex ratio (proportion of sons) is plotted versus the number of females laying eggs on a patch. The bright green dashed line shows the LMC theory prediction for the haplodiploid species (5, 39). A more female-biased sex ratio is favored in haplodiploids because inbreeding increases the relative relatedness of mothers to their daughters (7, 32). Females of many species adjust their offspring sex ratio as predicted by theory, such as the parasitoid Nasonia vitripennis (green diamonds) (82). In contrast, the females of several Melittobia species, such as M. australica, continue to produce extremely female-biased sex ratios, irrespective of the number of females laying eggs on a patch (blue squares) (15).In stark contrast, the sex ratio behavior of Melittobia wasps has long been seen as one of the greatest problems for the field of sex allocation (3, 4, 14–21). The life cycle of Melittobia wasps matches the assumptions of Hamilton’s LMC theory (5, 15, 19, 21). Females lay eggs in the larvae or pupae of solitary wasps and bees, and then after emergence, female offspring mate with the short-winged males, who do not disperse. However, laboratory experiments on four Melittobia species have found that females lay extremely female-biased sex ratios (1 to 5% males) and that these extremely female-biased sex ratios change little with increasing number of females laying eggs on a patch (higher n; Fig. 1) (15, 17–20, 22). A number of hypotheses to explain this lack of sex ratio adjustment have been investigated and rejected, including sex ratio distorters, sex differential mortality, asymmetrical male competition, and reciprocal cooperation (15–18, 20, 22–26).We tested whether Melittobia’s unusual sex ratio behavior can be explained by females being related to the other females laying eggs on the same patch. After mating, some females disperse to find new patches, while some may stay at the natal patch to lay eggs on previously unexploited hosts (Fig. 2). If females do not disperse, they can be related to the other females laying eggs on the same host (27–31). If females laying eggs on a host are related, this increases the extent to which relatives are competing for mates and so can favor an even more female-biased sex ratio (28, 32–35). Although most parasitoid species appear unable to directly assess relatedness, dispersal behavior could provide an indirect cue of whether females are with close relatives (36–38). Consequently, we predict that when females do not disperse and so are more likely to be with closer relatives, they should maintain extremely female-biased sex ratios, even when multiple females lay eggs on a patch (28, 35).Open in a separate windowFig. 2.Host nest and dispersal manners of Melittobia. (A) Photograph of the prepupae of the leaf-cutter bee C. sculpturalis nested in a bamboo cane and (B) a diagram showing two ways that Melittobia females find new hosts. The mothers of C. sculpturalis build nursing nests with pine resin consisting of individual cells in which their offspring develop. If Melittobia wasps parasitize a host in a cell, female offspring that mate with males inside the cell find a different host on the same patch (bamboo cane) or disperse by flying to other patches.We tested whether the sex ratio of Melittobia australica can be explained by dispersal status in a natural population. We examined how the sex ratio produced by females varies with the number of females laying eggs on a patch and whether or not they have dispersed before laying eggs. To match our data to the predictions of theory, we developed a mathematical model tailored to the unique population structure of Melittobia, where dispersal can be a cue of relatedness. We then conducted a laboratory experiment to test whether Melittobia females are able to directly access the relatedness to other females and adjust their sex ratio behavior accordingly. Our results suggest that females are adjusting their sex ratio in response to both the number of females laying eggs on a patch and their relatedness to the other females. However, relatedness is assessed indirectly by whether or not they have dispersed. Consequently, the solution to the puzzling behavior reflects a more-refined sex ratio strategy.     

From the Cover: Datura quids at Pinwheel Cave,California, provide unambiguous confirmation of the ingestion of hallucinogens at a rock art site

While debates have raged over the relationship between trance and rock art, unambiguous evidence of the consumption of hallucinogens has not been reported from any rock art site in the world. A painting possibly representing the flowers of Datura on the ceiling of a Californian rock art site called Pinwheel Cave was discovered alongside fibrous quids in the same ceiling. Even though Native Californians are historically documented to have used Datura to enter trance states, little evidence exists to associate it with rock art. A multianalytical approach to the rock art, the quids, and the archaeological context of this site was undertaken. Liquid chromatography−mass spectrometry (LC-MS) results found hallucinogenic alkaloids scopolamine and atropine in the quids, while scanning electron microscope analysis confirms most to be Datura wrightii. Three-dimensional (3D) analyses of the quids indicate the quids were likely masticated and thus consumed in the cave under the paintings. Archaeological evidence and chronological dating shows the site was well utilized as a temporary residence for a range of activities from Late Prehistory through Colonial Periods. This indicates that Datura was ingested in the cave and that the rock painting represents the plant itself, serving to codify communal rituals involving this powerful entheogen. These results confirm the use of hallucinogens at a rock art site while calling into question previous assumptions concerning trance and rock art imagery.

Since the late 1980s, the role that altered states of consciousness (or ASC) played in the making of rock art has been one of the most contentious questions confronted by rock art researchers across the globe (1–7). The ASC model purports that humans universally experience three distinct visual phases during trance, which are replicated in rock art imagery (1). The ASC model can be induced in a number of ways including the use of hallucinogenic substances (1). However, there remains no clear evidence for the preparation and consumption of hallucinogenic substances directly associated with any rock art site in the world. Indeed, fierce debate has occurred over the last 30 y, with many researchers questioning the validity of the ACS model and the idea of shamanism as a viable explanation for the creation of rock art (2–6). California has been central within this debate (8, 9). Whitley (9) has argued that the many south-central Californian rock paintings were shamanic self-portraits depicting a shaman’s experience during ASC while rock art sites were owned by individual shamans, and avoided by the local populace. In this view, trance, shamanism, and rock art are inextricably linked in their separation from normal activity of the wider populace. However, evidence from systematic archaeological work in south-central California has clearly shown that the majority of rock art sites were integrated into habitation sites, and are not separated from public view (10, 11). Recent analyses also suggest that the pictographs were probably not self-depictions of shamans in trance but, instead, stock iconographic images drawing upon mythology and the personifying of insects, animals, plant, and astronomical elements such as the sun (12, 13).Even so, ethnographic documentation details how hallucinogens played a pivotal role in Native California, especially Datura wrightii (14). A member of the Solanacae family, Datura is distinctive by large white “trumpet” flowers that uncoil in a five-pointed pinwheeling fashion. Datura as a genus can be found across multiple continents, including the Americas, Asia, Europe, and South Africa (15). Its wide availability and hallucinogenic properties, due to the presence of the tropane alkaloids atropine and scopolamine, are behind its use across different cultures (16, 17). The most noted usage of Datura in Native California is in youth initiations where the root was processed into a drink or “tea” known historically as toloache (14, 18–21). Initiates would often be instructed in cultural rules of entering adulthood and how to interpret the visions themselves (14, 19, 21). For some, these ceremonies where highly codified, such as the Chinigchinich religion of Southern California, which ended with the making of a sand painting in which boys learned religious principles (19, 21). The sand paintings did not depict the visions induced by Datura, but, instead, were cosmological maps detailing the ontological principles of the Southern Californian Native societies making them (22). After undergoing the puberty ceremony, Datura could be taken throughout one’s lifetime for a variety of reasons, including to gain supernatural power for doctoring, to counteract negative supernatural events, to ward off ghosts, and to see the future or find lost objects, but, most especially, as a mendicant for a variety of ailments (14, 18, 23). Datura consumption could occur prior to hunting to increase stamina and power (24). Importantly, Datura could be consumed in a variety of ways, including drinking toloache, but also by roasting the roots, eating the flowers or seeds, applying poultices on wounds, or often simply chewing the roots or other parts of the plant (14). For the Tübatulabal, Datura originated as a man named Mo mo ht who subsequently turned into the plant in its present form (25), while, in Chumash mythology, the plant was a prominent supernatural grandmother called Momoy (26). Since Datura, with its psychoactive substances, was used within spiritual, ritual, and mythological contexts, it should be considered as an entheogen (27).Worldwide, different hallucinogens have been suggested as inspiring rock art making, such as mushrooms, Peyote, Datura, San Pedro cactus, Brunsvigia, and others (2, 28–35). Datura is of particular focus in the North American West. Malotki (33) argues that Datura influenced archaic Basketmaker rock art in Arizona, while Boyd’s (32) extensive analyses have shown that Lower Pecos rock art iconography likely relates to mythological narratives concerning Peyote and Datura. Images include hornworms, the larval stages of hawkmoths, Datura’s primary pollinator. Mimbres pottery and Kiva murals also include representations of Datura, plus anthropomorphized versions of the hawkmoth (36, 37). Evidence of Datura alkaloids have been found in ceramics (38), while ancient peyote buttons and Datura seeds have been found in Lower Pecos archaeological deposits, but none have been reported specifically at rock art sites (39).Datura has also been suggested as relating to the making of Chumash rock art in California (40). Found in the Chumash borderlands of interior south-central California, we detail here our investigations at a rock art site called Pinwheel Cave (CA-KER-5836) (Fig. 1) and its associated food processing bedrock mortar (BRM) complex (CA-KER-5837). The name originates from a large red pinwheel motif, which we hypothesized may represent the opening Datura flower (Fig. 1, Bottom Left and Bottom Right). Dozens of fibrous clumps known as quids are also located within crevices in the cave ceiling (41). Quids, usually found in archaeological deposits, are typically made of yucca, agave, tule, or tobacco and are thought to have been chewed to extract nutrients or stimulants (41–44). With the painting likely representing the opening of the Datura flower, and with the very unusual insertion of quids in the ceiling, we investigated the possibility that the quids could contain Datura. We present the results of a multianalytical investigation of the quids and the archaeological context to investigate the potential use of Datura in association with rock art iconography.Open in a separate windowFig. 1.Pinwheel Cave, California. (Top) Interior of cave during laser scanning. (Bottom Left) Pinwheel painting within cave. Image credit: Rick Bury (photographer). (Bottom Right) Unfurling flower of D. wrightii from plant near cave site. Image credit: Melissa Dabulamanzi (photographer).     

Structure,self-assembly,and properties of a truncated reflectin variant

Naturally occurring and recombinant protein-based materials are frequently employed for the study of fundamental biological processes and are often leveraged for applications in areas as diverse as electronics, optics, bioengineering, medicine, and even fashion. Within this context, unique structural proteins known as reflectins have recently attracted substantial attention due to their key roles in the fascinating color-changing capabilities of cephalopods and their technological potential as biophotonic and bioelectronic materials. However, progress toward understanding reflectins has been hindered by their atypical aromatic and charged residue-enriched sequences, extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for aggregation. Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a straightforward mechanical agitation-based methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between the protein’s structural characteristics and intrinsic optical properties. Altogether, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and may inform new research directions across biochemistry, cellular biology, bioengineering, and optics.

Materials from naturally occurring and recombinant proteins are frequently employed for the study of fundamental biological processes and leveraged for applications in fields as diverse as electronics, optics, bioengineering, medicine, and fashion (1–13). Such broad utility is enabled by the numerous advantageous characteristics of protein-based materials, which include sequence modularity, controllable self-assembly, stimuli-responsiveness, straightforward processability, inherent biological compatibility, and customizable functionality (1–13). Within this context, unique structural proteins known as reflectins have recently attracted substantial attention because of their key roles in the fascinating color-changing capabilities of cephalopods, such as the squid shown in Fig. 1A, and have furthermore demonstrated their utility for unconventional biophotonic and bioelectronic technologies (11–40). For example, in vivo, Bragg stack-like ultrastructures from reflectin-based high refractive index lamellae (membrane-enclosed platelets) are responsible for the angle-dependent narrowband reflectance (iridescence) of squid iridophores, as shown in Fig. 1B (15–20). Analogously, folded membranes containing distributed reflectin-based particle arrangements within sheath cells lead to the mechanically actuated iridescence of squid chromatophore organs, as shown in Fig. 1C (15, 16, 21, 22). Moreover, in vitro, films processed from squid reflectins not only exhibit proton conductivities on par with some state-of-the-art artificial materials (23–27) but also support the growth of murine and human neural stem cells (28, 29). Additionally, morphologically variable coatings assembled from different reflectin isoforms can enable the functionality of chemically and electrically actuated color-changing devices, dynamic near-infrared camouflage platforms, and stimuli-responsive photonic architectures (27, 30–34). When considered together, these discoveries and demonstrations constitute compelling motivation for the continued exploration of reflectins as model biomaterials.Open in a separate windowFig. 1.(A) A camera image of a D. pealeii squid for which the skin contains light-reflecting cells called iridophores (bright spots) and pigmented organs called chromatophores (colored spots). Image credit: Roger T. Hanlon (photographer). (B) An illustration of an iridophore (Left), which shows internal Bragg stack-like ultrastructures from reflectin-based lamellae (i.e., membrane-enclosed platelets) (Inset). (C) An illustration of a chromatophore organ (Left), which shows arrangements of reflectin-based particles within the sheath cells (Inset). (D) The logo of the 28-residue-long N-terminal motif (RMN), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (E) The logo of the 28-residue-long internal motif (RMI), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (F) The logo of the 21-residue-long C-terminal motif (RMC), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (G) The amino acid sequence of full-length D. pealeii reflectin A1, which contains a single RMN motif (gray oval) and five RMI motifs (orange ovals). (H) An illustration of the selection of the prototypical truncated reflectin variant (denoted as RfA1TV) from full-length D. pealeii reflectin A1.Given reflectins’ demonstrated significance from both fundamental biology and applications perspectives, some research effort has been devoted to resolving their three-dimensional (3D) structures (30, 31, 35–39). For example, fibers drawn from full-length Euprymna scolopes reflectin 1a and films processed from truncated E. scolopes reflectin 1a were shown to possess secondary structural elements (i.e., α-helices or β-sheets) (30, 31). In addition, precipitated nanoparticles and drop-cast films from full-length Doryteuthis pealeii reflectin A1 have exhibited β-character, which was seemingly associated with their conserved motifs (35, 36). Moreover, nanoparticles assembled from both full-length and truncated Sepia officinalis reflectin 2 variants have demonstrated signatures consistent with β-sheet or α-helical secondary structure, albeit in the presence of surfactants (38). However, such studies were made exceedingly challenging by reflectins’ atypical primary sequences enriched in aromatic and charged residues, documented extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for poorly controlled aggregation (12, 14, 15, 30–32, 34–39). Consequently, the reported efforts have all suffered from multiple drawbacks, including the need for organic solvents or denaturants, the evaluation of only polydisperse or aggregated (rather than monomeric) proteins, a lack of consensus among different experimental techniques, inadequate resolution that precluded molecular-level insight, imperfect agreement between computational predictions and experimental observations, and/or the absence of conclusive correlations between structure and optical functionality. As such, there has emerged an exciting opportunity for investigating reflectins’ molecular structures, which remain poorly understood and the subject of some debate.Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a robust methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between its structural characteristics and optical properties. We first rationally select a prototypical reflectin variant expected to recapitulate the behavior of its parent protein by using a bioinformatics-guided approach. We next map the conformational and energetic landscape accessible to our selected protein by means of all-atom molecular dynamics (MD) simulations. We in turn produce our truncated reflectin variant with and without isotopic labeling, develop solution conditions that maintain the protein in a monomeric state, and characterize the variant’s size and shape with small-angle X-ray scattering (SAXS). We subsequently resolve our protein’s dynamic secondary and tertiary structures and evaluate its backbone conformational fluctuations with NMR spectroscopy. Finally, we demonstrate a straightforward mechanical agitation-based approach to controlling our truncated reflectin variant’s secondary structure, hierarchical self-assembly, and bulk refractive index distribution. Overall, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and appear poised to inform new directions across biochemistry, cellular biology, bioengineering, and optics.     

From the Cover: Neurophysiological coordination of duet singing

Coordination of behavior for cooperative performances often relies on linkages mediated by sensory cues exchanged between participants. How neurophysiological responses to sensory information affect motor programs to coordinate behavior between individuals is not known. We investigated how plain-tailed wrens (Pheugopedius euophrys) use acoustic feedback to coordinate extraordinary duet performances in which females and males rapidly take turns singing. We made simultaneous neurophysiological recordings in a song control area “HVC” in pairs of singing wrens at a field site in Ecuador. HVC is a premotor area that integrates auditory feedback and is necessary for song production. We found that spiking activity of HVC neurons in each sex increased for production of its own syllables. In contrast, hearing sensory feedback produced by the bird’s partner decreased HVC activity during duet singing, potentially coordinating HVC premotor activity in each bird through inhibition. When birds sang alone, HVC neurons in females but not males were inhibited by hearing the partner bird. When birds were anesthetized with urethane, which antagonizes GABAergic (γ-aminobutyric acid) transmission, HVC neurons were excited rather than inhibited, suggesting a role for GABA in the coordination of duet singing. These data suggest that HVC integrates information across partners during duets and that rapid turn taking may be mediated, in part, by inhibition.

Animals routinely rely on sensory feedback for the control of their own behavior. In cooperative performances, such sensory feedback can include cues produced by other participants (1–8). For example, in interactive vocal communication, including human speech, individuals take turns vocalizing. This “turn taking” is a consequence of each participant responding to auditory cues from a partner (4–6, 9, 10). The role of such “heterogenous” (other-generated) feedback in the control of vocal turn taking and other cooperative performances is largely unknown.Plain-tailed wrens (Pheugopedius euophrys) are neotropical songbirds that cooperate to produce extraordinary duet performances but also sing by themselves (Fig. 1A) (4, 10, 11). Singing in plain-tailed wrens is performed by both females and males and used for territorial defense and other functions, including mate guarding and attraction (1, 11–16). During duets, female and male plain-tailed wrens take turns, alternating syllables at a rate of between 2 and 5 Hz (Fig. 1A) (4, 11).Open in a separate windowFig. 1.Neural control of solo and duet singing in plain-tailed wrens. (A) Spectrogram of a singing bout that included male solo syllables (blue line, top) followed by a duet. Solo syllables for both sexes (only male solo syllables are shown here) are sung at lower amplitudes than syllables produced in duets. Note that the smeared appearance of wren syllables in spectrograms reflects the acoustic structure of plain-tailed wren singing. (B and C) Each bird has a motor system that is used to produce song and sensory systems that mediate feedback. (B) During solo singing, the bird hears its own song, which is known as autogenous feedback (orange). (C) During duet singing, each bird hears both its own singing and the singing of its partner, known as heterogenous feedback (green). The key difference between solo and duet singing is heterogenous feedback that couples the neural systems of the two birds. This coupling results in changes in syllable amplitude and timing in both birds.There is a categorical difference between solo and duet singing. In solo singing, the singing bird receives only autogenous (hearing its own vocalization) feedback (Fig. 1B). The partner may hear the solo song if it is nearby, a heterogenous (other-generated) cue. In duet singing, birds receive both heterogenous and autogenous feedback as they alternate syllable production (Fig. 1C). Participants use heterogenous feedback during duet singing for precise timing of syllable production (4, 11). For example, when a male temporarily stops participating in a duet, the duration of intersyllable intervals between female syllables increases (4), showing an effect of heterogenous feedback on the timing of syllable production.How does the brain of each wren integrate heterogenous acoustic cues to coordinate the precise timing of syllable production between individuals during duet performances? To address this question, we examined neurophysiological activity in HVC, a nucleus in the nidopallium [an analogue of mammalian cortex (17, 18)]. HVC is necessary for song learning, production, and timing in species of songbirds that do not perform duets (19–24). Neurons in HVC are active during singing and respond to playback of the bird’s own learned song (25–27). In addition, recent work has shown that HVC is also involved in vocal turn taking (19).To examine the role of heterogenous feedback in the control of duet performances, we compared neurophysiological activity in HVC when female or male wrens sang solo syllables with syllables sung during duets. Neurophysiological recordings were made in awake and anesthetized pairs of wrens at the Yanayacu Biological Station and Center for Creative Studies on the slopes of the Antisana volcano in Ecuador. We found that heterogenous cues inhibited HVC activity during duet performances in both females and males, but inhibition was only observed in females during solo singing.     

Heme-binding protein CYB5D1 is a radial spoke component required for coordinated ciliary beating

Coordinated beating is crucial for the function of multiple cilia. However, the molecular mechanism is poorly understood. Here, we characterize a conserved ciliary protein CYB5D1 with a heme-binding domain and a cordon-bleu ubiquitin-like domain. Mutation or knockdown of Cyb5d1 in zebrafish impaired coordinated ciliary beating in the otic vesicle and olfactory epithelium. Similarly, the two flagella of an insertional mutant of the CYB5D1 ortholog in Chlamydomonas (Crcyb5d1) showed an uncoordinated pattern due to a defect in the cis-flagellum. Biochemical analyses revealed that CrCYB5D1 is a radial spoke stalk protein that binds heme only under oxidizing conditions. Lack of CrCYB5D1 resulted in a reductive shift in flagellar redox state and slowing down of the phototactic response. Treatment of Crcyb5d1 with oxidants restored coordinated flagellar beating. Taken together, these data suggest that CrCYB5D1 may integrate environmental and intraciliary signals and regulate the redox state of cilia, which is crucial for the coordinated beating of multiple cilia.

Cilia and flagella are highly conserved organelles that project from the surface of most eukaryotic cells and perform sensory, secretory, and motile functions (1, 2). The single motile flagellum of spermatozoa propels cell body movement, whereas the two flagella of the green alga Chlamydomonas reinhardtii mediate oriented swimming. Coordinated beating of multiple cilia on epithelial surfaces, for example, in the trachea, oviduct, and brain ventricles of vertebrates, is crucial for mucus clearance in the airway, ovum transport in the oviduct, and cerebrospinal fluid circulation in the brain ventricles (3). Impairment of coordinated beating of these multiple cilia in humans can result in ciliopathies such as chronic respiratory problems, infertility, and hydrocephalus (4).The beating of individual cilia/flagella depends on axonemal dyneins, which are AAA⁺ enzymes that convert ATP (adenosine triphosphate) hydrolysis into mechanical force, resulting in the sliding of doublet microtubules (5). The activity of dyneins is spatiotemporally controlled by various regulatory pathways including changes in Ca²⁺, phosphorylation, and redox state (5). Recent Cryo-EM (cryo-electron microscopy) studies have revealed that these dyneins are generally in a force-balanced state in straight axonemal regions, and that the initiation and formation of a ciliary bend by microtubule sliding may depend on an inhibitory signal that is propagated from the base-to-tip to disrupt the force-balanced state (6). To achieve coordinated beating, the activity of multiple cilia must be synchronized by both intrinsic and extrinsic factors including hydrodynamic coupling, axonemal mechanical feedback, connective structures in the basal body region, and Ca²⁺ signaling (7–10). The hydrodynamic hypothesis is supported by the observation that when two sperm or two micropipette-held somatic cells of Volvox carteri were placed in close proximity under hydrodynamic flow, the beating of two flagella was spontaneously synchronized (8, 10). However, multiple cilia can still maintain coordinated beating when fluid flow disappears (11), suggesting that other mechanisms are also involved.The mechanical feedback system was proposed based on the study of light chain 1 (LCI) from Chlamydomonas. LC1 contains six leucine-rich repeats that form an elongated barrel (12) and binds to the microtubule-binding domain of the γ-HC (γ heavy chain) specifically (13). LC1 mutant proteins expressed in wild type Chlamydomonas resulted in uncoordinated flagellar beating (14). Knockdowns of LC1 in planaria also led to the loss of metachronal synchrony in the ventral cilia (7). As a conformational switch, LC1 may sense changes in axonemal curvature imposed by hydrodynamic flow and thereby modulate the activity of outer dynein arm to coordinate flagellar beating. This model is also supported by recent structural data on the LC1–γ-HC microtubule-binding domain complex (15).Coordinated beating of algal flagella is also achieved by basal coupling through filamentary connections between basal bodies (10). These connectors control basal body alignment and cilia orientation in multiciliated cells and may also participate in coordinated beating by integrating the chemical or mechanical signals among cilia (9). However, the nature of these signals remains unknown. Ca²⁺ is a common second messenger in cilia and may also participate in coordinated beating. Knock-down of the components of the calmodulin-associated complex, such as CaM-IP2, CaM-IP3, PF6, and PCDP1, resulted in uncoordinated flagellar beating in Chlamydomonas (16–18). However, the coordination defect may be a secondary effect since motility-related structures such as the central pair and radial spokes exhibit structural alterations in these mutants. The two flagella of Chlamydomonas respond differently to changes of Ca²⁺ concentration at the low μM range, which alters flagellar coordination and results in phototactic turning (19, 20). Therefore, Ca²⁺ functions in modulating flagellar coordination during these processes.Recent studies have shown that redox state also participates in regulating ciliary/flagellar beating in Chlamydomonas and mammals. The beat frequency of flagella was reduced when Chlamydomonas cells were treated with oxidizing agents (21). Redox poise also regulates the sign of phototaxis in Chlamydomonas (22). When multiple cilia on airway epithelial cells were exposed to oxidants, the mucociliary transport was inhibited (23). In addition, several redox-sensitive components have been identified in flagellar motility-related complexes, such as LC3, LC5, γ-HC, and outer dynein arm-docking complex protein 3 (DC3) in Chlamydomonas (24–26). Disruption of human TXNDC3 and TXNDC6, which contain modules orthologous to the thioredoxin-like LC3 and LC5 proteins in Chlamydomonas, results in respiratory disease (27, 28). Taken together, these findings suggest that redox plays a vital role in coordinating ciliary beating; however, the molecular mechanisms and pathways that mediate the redox response are unknown.Here, we identify a highly conserved ciliary protein cytochrome b5-like/heme-binding domain (CYB5D1), which is required for the coordinated beating of multiple cilia in zebrafish and Chlamydomonas. Furthermore, our results indicate that this heme-binding protein may function in a redox signaling pathway to coordinate ciliary beating.     

Sea spray aerosol concentration modulated by sea surface temperature

Natural aerosols in pristine regions form the baseline used to evaluate the impact of anthropogenic aerosols on climate. Sea spray aerosol (SSA) is a major component of natural aerosols. Despite its importance, the abundance of SSA is poorly constrained. It is generally accepted that wind-driven wave breaking is the principle governing SSA production. This mechanism alone, however, is insufficient to explain the variability of SSA concentration at given wind speed. The role of other parameters, such as sea surface temperature (SST), remains controversial. Here, we show that higher SST promotes SSA mass generation at a wide range of wind speed levels over the remote Pacific and Atlantic Oceans, in addition to demonstrating the wind-driven SSA production mechanism. The results are from a global scale dataset of airborne SSA measurements at 150 to 200 m above the ocean surface during the NASA Atmospheric Tomography Mission. Statistical analysis suggests that accounting for SST greatly enhances the predictability of the observed SSA concentration compared to using wind speed alone. Our results support implementing SST into SSA source functions in global models to better understand the atmospheric burdens of SSA.

Over two-thirds of the Earth is covered by the ocean. The material exchange between the ocean and the atmosphere affects the balance of the Earth’s energy on a global scale (1). Sea spray aerosol (SSA) is the major particulate material directly emitted from the ocean. Studies have shown that SSA dominates the aerosol mass in the marine boundary layer (MBL). Such dominance renders SSA an important player in climate change (2). However, the exact processes by which the SSA is introduced to the atmosphere still remains to be learned, making the SSA budget highly uncertain (3).It is generally established that SSA is produced by mechanical processes (4–6). Wind stress induces breaking waves that entrain bubbles into the surface ocean (7). Film and jet drops formed during bubble bursting are the main sources of SSA particles (8). The wind-driven mechanism is supported by the positive correlation between wind speed and SSA concentration from field observations (9, 10). Therefore, wind speed is used as the sole parameter to characterize SSA in many models (1, 4, 11).In addition to wind speed, sea surface temperature (SST) may play a large role in SSA production (12–15). SST affects the drop formation process by modifying the physical properties of the surface ocean water. An increase of SST reduces the kinematic viscosity and surface tension of the ocean, thereby enhancing the entrainment efficiency and rising speed of bubbles (12, 16). As a result, the number size distribution of the bubbles may change, leading to varying SSA properties (14, 15, 17).Limited laboratory and field studies regarding the effects of SST on SSA production have shown disparate results. Some argue that SSA production is independent of SST (18) or suppressed by increasing SST (14, 15, 19, 20) from 0 to 10 °C, while other laboratory (12, 21–23) and field measurements (3, 5) suggest that SSA production increases monotonically with water temperature. Furthermore, recent observations in the remote Atlantic Ocean shows that increasing SST enhances the modal mean diameter of SSA (24). On the other hand, model simulations have demonstrated that incorporating SST into SSA source functions generally improves the SSA prediction (3, 25, 26). The inconsistency in the previous work suggests that the impacts of SST on SSA formation remain unclear.In this study, we conducted unprecedented aircraft measurements of SSA concentration on a global scale during the Atmospheric Tomography Mission (ATom). These measurements consist of a series of flights spanning three seasons (summer, fall, and winter) over remote oceans (Fig. 1 and SI Appendix, Fig. S1). Our observations again confirm that wind speed is the dominant factor controlling the concentration of SSA. Further, we show that increasing SST enhances the mass concentration of SSA.Open in a separate windowFig. 1.Flight tracks during ATom2. The color indicates the flight altitude. The size of the markers represents the sea salt number fraction. The inset in the bottom right shows the vertical profile of sea salt number fraction. The flight tracks during ATom3 and ATom4 are similar to ATom2.     

A rate threshold mechanism regulates MAPK stress signaling and survival

Cells are exposed to changes in extracellular stimulus concentration that vary as a function of rate. However, how cells integrate information conveyed from stimulation rate along with concentration remains poorly understood. Here, we examined how varying the rate of stress application alters budding yeast mitogen-activated protein kinase (MAPK) signaling and cell behavior at the single-cell level. We show that signaling depends on a rate threshold that operates in conjunction with stimulus concentration to determine the timing of MAPK signaling during rate-varying stimulus treatments. We also discovered that the stimulation rate threshold and stimulation rate-dependent cell survival are sensitive to changes in the expression levels of the Ptp2 phosphatase, but not of another phosphatase that similarly regulates osmostress signaling during switch-like treatments. Our results demonstrate that stimulation rate is a regulated determinant of cell behavior and provide a paradigm to guide the dissection of major stimulation rate dependent mechanisms in other systems.

All cells employ signal transduction pathways to respond to physiologically relevant changes in extracellular stressors, nutrient levels, hormones, morphogens, and other stimuli that vary as functions of both concentration and rate in healthy and diseased states (1–7). Switch-like “instantaneous” changes in the concentrations of stimuli in the extracellular environment have been widely used to show that the strength of signaling and overall cellular response are dependent on the stimulus concentration, which in many cases needs to exceed a certain threshold (8, 9). Previous studies have shown that the rate of stimulation can also influence signaling output in a variety of pathways (10–17) and that stimulation profiles of varying rates can be used to probe underlying signaling pathway circuitry (4, 18, 19). However, it is still not clear how cells integrate information conveyed by changes in both the stimulation rate and concentration in determining signaling output. It is also not clear if cells require stimulation gradients to exceed a certain rate in order to commence signaling.Recent investigations have demonstrated that stimulation rate can be a determining factor in signal transduction. In contrast to switch-like perturbations, which trigger a broad set of stress-response pathways, slow stimulation rates activate a specific response to the stress applied in Bacillus subtilis cells (10). Meanwhile, shallow morphogen gradient stimulation fails to activate developmental pathways in mouse myoblast cells in culture, even when concentrations sufficient for activation during pulsed treatment are delivered (12). These observations raise the possibility that stimulation profiles must exceed a set minimum rate or rate threshold to achieve signaling activation. Although such rate thresholds would help cells decide if and how to respond to dynamic changes in stimulus concentration, the possibility of signaling regulation by a rate threshold has never been directly investigated in any system. Further, no study has experimentally examined how stimulation rate requirements impact cell phenotype or how cells molecularly regulate the stimulation rate required for signaling activation. As such, the biological significance of any existing rate threshold regulation of signaling remains unknown.The budding yeast Saccharomyces cerevisiae high osmolarity glycerol (HOG) pathway provides an ideal model system for addressing these issues (Fig. 1A). The evolutionarily conserved mitogen-activated protein kinase (MAPK) Hog1 serves as the central signaling mediator of this pathway (20–22). It is well established that instantaneous increases in osmotic stress concentration induce Hog1 phosphorylation, activation, and translocation to the nucleus (18, 21, 23–30). Activated Hog1 governs the majority of the cellular osmoadaptation response that enables cells to survive (23, 31, 32). Multiple apparently redundant MAPK phosphatases dephosphorylate and inactivate Hog1, which, along with the termination of upstream signaling after adaptation, results in its return to the cytosol (Fig. 1A) (23, 25, 26, 33–39). Because of this behavior, time-lapse analysis of Hog1 nuclear enrichment in single cells has proven an excellent and sensitive way to monitor signaling responses to dynamic stimulation patterns in real time (18, 27–30, 40, 41). Further, such assays have been readily combined with traditional growth and molecular genetic approaches to link observed signaling responses with cell behavior and signaling pathway architecture (27–29).Open in a separate windowFig. 1.Hog1 signaling and cell survival are sensitive to the rate of preconditioning osmotic stress application. (A) Schematic of the budding yeast HOG response. (B) Preconditioning protection assay workflow indicating the first stress treatments to a final concentration of 0.4 M NaCl (Left), high-stress exposure (Middle), and colony formation readout (Right). (C) High-stress survival as a function of each first treatment relative to the untreated first stress condition. Bars and errors are means and SD from three biological replicates. *Statistically significant by Kolmogorov–Smirnov test (P < 0.05). NS = not significant. (D) Treatment concentration over time. (E) Treatment rate over time for quadratic and pulse treatment. The rate for the pulse is briefly infinite (blue vertical line) before it drops to 0. (F) Hog1 nuclear localization during the treatments depicted in D and E. (Inset) Localization pattern in the quadratic-treated sample. Lines represent means and shaded error represents the SD from three to four biological replicates.Here, we use systematically designed osmotic stress treatments imposed at varying rates of increase to show that a rate threshold condition regulates yeast high-stress survival and Hog1 MAPK signaling. We demonstrate that only stimulus profiles that satisfy both this rate threshold condition and a concentration threshold condition result in robust signaling. We go on to show that the protein tyrosine phosphatase Ptp2, but not the related Ptp3 phosphatase, serves as a major rate threshold regulator. By expressing PTP2 under the control of a series of different enhancer–promoter DNA constructs, we demonstrate that changes in the level of Ptp2 expression can alter the stimulation rate required for signaling induction and survival. These findings establish rate thresholds as a critical and regulated component of signaling biology akin to concentration thresholds.     

The impact of identity by descent on fitness and disease in dogs

Domestic dogs have experienced population bottlenecks, recent inbreeding, and strong artificial selection. These processes have simplified the genetic architecture of complex traits, allowed deleterious variation to persist, and increased both identity-by-descent (IBD) segments and runs of homozygosity (ROH). As such, dogs provide an excellent model for examining how these evolutionary processes influence disease. We assembled a dataset containing 4,414 breed dogs, 327 village dogs, and 380 wolves genotyped at 117,288 markers and data for clinical and morphological phenotypes. Breed dogs have an enrichment of IBD and ROH, relative to both village dogs and wolves, and we use these patterns to show that breed dogs have experienced differing severities of bottlenecks in their recent past. We then found that ROH burden is associated with phenotypes in breed dogs, such as lymphoma. We next test the prediction that breeds with greater ROH have more disease alleles reported in the Online Mendelian Inheritance in Animals (OMIA). Surprisingly, the number of causal variants identified correlates with the popularity of that breed rather than the ROH or IBD burden, suggesting an ascertainment bias in OMIA. Lastly, we use the distribution of ROH across the genome to identify genes with depletions of ROH as potential hotspots for inbreeding depression and find multiple exons where ROH are never observed. Our results suggest that inbreeding has played a large role in shaping genetic and phenotypic variation in dogs and that future work on understudied breeds may reveal new disease-causing variation.

The unique demographic and selective history of dogs has enabled the persistence of deleterious variation, simplified genetic architecture of complex traits, and caused an increase in both runs of homozygosity (ROH) and identity-by-descent (IBD) segments within breeds (1–6). Specifically, the average F_ROH was ∼0.3 in dogs (7), compared to 0.005 in humans, computed from the 1000 Genomes populations (8). The large amount of the genome in ROH in dogs, combined with a wealth of genetic variation and phenotypic data (2, 5, 7, 9–11), allow us to test how ROH and IBD influence complex traits and fitness (Fig. 1). Furthermore, many of the deleterious alleles within dogs likely arose relatively recently within a breed, and dogs tend to share similar disease pathways and genes with humans (4, 12, 13), increasing their relevance for complex traits in humans.Open in a separate windowFig. 1.Potential mechanisms for associations between ROH and phenotypes that depend on recessive mutations. If a recessive deleterious mutation is nonlethal (blue), it may lead to ROH correlating with disease, while lethal (red) recessive mutations will cause a depletion of ROH.Despite IBD segments and ROH being ubiquitous in genomes, the extent to which they affect the architecture of complex traits as well as reproductive fitness has remained elusive. Given that ROH are formed by inheritance of the same ancestral chromosome from both parents, there is a much higher probability of the individual to become homozygous for a deleterious recessive variant (8, 14), leading to a reduction in fitness. This prediction was verified in recent work in nonhuman mammals that has shown that populations suffering from inbreeding depression tend to have an increase in ROH (15, 16). ROH in human populations are enriched for deleterious variants (8, 14, 17). However, the extent to which ROH impact phenotypes remains unclear. For example, several studies have associated an increase in ROH with complex traits in humans (18–23), though some associations remain controversial (24–28). Determining how ROH and IBD influence complex traits and fitness could provide a mechanism for differences in complex-trait architecture across populations that vary in their burden of IBD and ROH.Here, we use IBD segments and ROH from 4,741 breed dogs and village dogs, and 380 wolves to determine the recent demographic history of dogs and wolves and establish a connection between recent inbreeding and deleterious variation associated with both disease and inbreeding depression. This comprehensive dataset contains genotype data from 172 breeds of dog, village dogs from 30 countries, and gray wolves from British Colombia, North America, and Europe. We test for an association with the burden of ROH and case-control status for a variety of complex traits. Remarkably, we also find that the number of disease-associated causal variants identified in a breed is positively correlated with breed popularity rather than burden of IBD or ROH in the genome, suggesting ascertainment biases also exist in databases of dog disease mutations and that many breeds of dog are understudied. Lastly, we identify multiple loci that may be associated with inbreeding depression by examining localized depletions of ROH across dog genomes.     

Developmental influence on evolutionary rates and the origin of placental mammal tooth complexity

Development has often been viewed as a constraining force on morphological adaptation, but its precise influence, especially on evolutionary rates, is poorly understood. Placental mammals provide a classic example of adaptive radiation, but the debate around rate and drivers of early placental evolution remains contentious. A hallmark of early dental evolution in many placental lineages was a transition from a triangular upper molar to a more complex upper molar with a rectangular cusp pattern better specialized for crushing. To examine how development influenced this transition, we simulated dental evolution on “landscapes” built from different parameters of a computational model of tooth morphogenesis. Among the parameters examined, we find that increases in the number of enamel knots, the developmental precursors of the tooth cusps, were primarily influenced by increased self-regulation of the molecular activator (activation), whereas the pattern of knots resulted from changes in both activation and biases in tooth bud growth. In simulations, increased activation facilitated accelerated evolutionary increases in knot number, creating a lateral knot arrangement that evolved at least ten times on placental upper molars. Relatively small increases in activation, superimposed on an ancestral tritubercular molar growth pattern, could recreate key changes leading to a rectangular upper molar cusp pattern. Tinkering with tooth bud geometry varied the way cusps initiated along the posterolingual molar margin, suggesting that small spatial variations in ancestral molar growth may have influenced how placental lineages acquired a hypocone cusp. We suggest that development could have enabled relatively fast higher-level divergence of the placental molar dentition.

Whether developmental processes bias or constrain morphological adaptation is a long-standing question in evolutionary biology (1–4). Many of the distinctive features of a species derive from pattern formation processes that establish the position and number of anatomical structures (5). If developmental processes like pattern formation are biased toward generating only particular kinds of variation, adaptive radiations may often be directed along developmental–genetic “lines of least resistance” (2, 4, 6, 7). Generally, the evolutionary consequences of this developmental bias have been considered largely in terms of how it might influence the pattern of character evolution (e.g., refs. 1, 2, 8–10). But development could also influence evolutionary rates by controlling how much variation is accessible to natural selection in a given generation (11).For mammals, the dentition is often the only morphological system linking living and extinct species (12). Correspondingly, tooth morphology plays a crucial role in elucidating evolutionary relationships, time calibrating phylogenetic trees, and reconstructing adaptive responses to past environmental change (e.g., refs. 13–15). One of the most pervasive features of dental evolution among mammals is an increase in the complexity of the tooth occlusal surface, primarily through the addition of new tooth cusps (16, 17). These increases in tooth complexity are functionally and ecologically significant because they enable more efficient mechanical breakdown of lower-quality foods like plant leaves (18).Placental mammals are the most diverse extant mammalian group, comprising more than 6,000 living species spread across 19 extant orders, and this taxonomic diversity is reflected in their range of tooth shapes and dietary ecologies (12). Many extant placental orders, especially those with omnivorous or herbivorous ecologies (e.g., artiodactyls, proboscideans, rodents, and primates), convergently evolved a rectangular upper molar cusp pattern from a placental ancestor with a more triangular cusp pattern (19–21). This resulted from separate additions in each lineage of a novel posterolingual cusp, the "hypocone'''' [sensu (19)], to the tritubercular upper molar (Fig. 1), either through modification of a posterolingual cingulum (“true” hypocone) or another posterolingual structure, like a metaconule (pseudohypocone) (19). The fossil record suggests that many of the basic steps in the origin of this rectangular cusp pattern occurred during an enigmatic early diversification window associated with the divergence and early radiation of several placental orders (20, 21; Fig. 1). However, there remains debate about the rate and pattern of early placental divergence (22–24). On the one hand, most molecular phylogenies suggest that higher-level placental divergence occurred largely during the Late Cretaceous (25, 26), whereas other molecular phylogenies and paleontological analyses suggest more rapid divergence near the Cretaceous–Paleogene (K–Pg) boundary (21, 24, 27–29). Most studies agree that ecological opportunity created in the aftermath of the K–Pg extinction probably played an important role in ecomorphological diversification within the placental orders (30, 31). But exactly how early placentals acquired the innovations needed to capitalize on ecological opportunity remains unclear. Dental innovations, especially those which facilitated increases in tooth complexity, may have been important because they would have promoted expansion into plant-based dietary ecologies left largely vacant after the K–Pg extinction event (32).Open in a separate windowFig. 1.Placental mammal lineages separately evolved complex upper molar teeth with a rectangular cusp pattern composed of two lateral pairs of cusps from a common ancestor with a simpler, triangular cusp pattern. Many early relatives of the extant placental orders, such as Eritherium, possessed a hypocone cusp and a more rectangular primary cusp pattern. Examples of complex upper molars are the following: Proboscidea, the gomphothere Anancus; Rodentia, the wood mouse Apodemus; and Artiodactyla, the suid Nyanzachoerus.Mammalian tooth cusps form primarily during the “cap” and “bell” stage of dental development, when signaling centers called enamel knots establish the future sites of cusp formation within the inner dental epithelium (33, 34). The enamel knots secrete molecules that promote proliferation and changes in cell–cell adhesion, which facilitates invagination of the dental epithelium into an underlying layer of mesenchymal cells (34, 35). Although a range of genes are involved in tooth cusp patterning (36–38), the basic dynamics can be effectively modeled using reaction–diffusion models with just three diffusible morphogens: an activator, an inhibitor, and a growth factor (39–41). Candidate activator genes in mammalian tooth development include Bmp4, Activin A, Fgf20, and Wnt genes, whereas potential inhibitors include Shh and Sostdc, and Fgf4 and Bmp2 have been hypothesized to act as growth factors (38, 40–43). In computer models of tooth development, activator molecules up-regulated in the underlying mesenchyme stimulate differentiation of overlying epithelium into nondividing enamel knot cells. These in turn secrete molecules that inhibit further differentiation of epithelium into knot cells, while also promoting cell proliferation that creates the topographic relief of the cusp (40). Although many molecular, cellular, and physical processes have the potential to influence cusp formation, and thereby tooth complexity (35, 37), parameters that control the strength and conductance of the activator and inhibitor signals, the core components of the reaction–diffusion cusp patterning mechanism (39, 40) are likely to be especially important.Here, we integrate a previous computer model of tooth morphogenesis called ToothMaker (41), with simulations of trait evolution and data from the fossil record (Fig. 2), to examine the developmental origins of tooth complexity in placental mammals. Specifically, we ask the following: 1) What developmental processes can influence how many cusps form? 2) How might these developmental processes influence the evolution of tooth cusp number, especially rates? And 3) what developmental changes may have been important in the origins of the fourth upper molar cusp, the hypocone, in placental mammal evolution?Open in a separate windowFig. 2.Workflow for simulations of tooth complexity evolution. (A) Tooth shape is varied for five signaling and growth parameters in ToothMaker. (B) From an ancestral state, each parameter is varied in 2.5% increments up to a maximum of ± 50% of the ancestral state. (C) Tooth complexity and enamel knot (EK) pattern were quantified for each parameter combination. Tooth complexity was measured using cusp number/EK number and OPC. ToothMaker and placental upper second molars were classified into categories based on EK/cusp pattern. (D) The parameter space was populated with pattern and tooth complexity datums to build a developmental landscape. (E) Tooth complexity evolution was simulated on each developmental landscape. (F) Resulting diversity and pattern of tooth complexity was compared with placental mammal molar diversity.     

From the Cover: Evidence from South Africa for a protracted end-Permian extinction on land

Earth’s largest biotic crisis occurred during the Permo–Triassic Transition (PTT). On land, this event witnessed a turnover from synapsid- to archosauromorph-dominated assemblages and a restructuring of terrestrial ecosystems. However, understanding extinction patterns has been limited by a lack of high-precision fossil occurrence data to resolve events on submillion-year timescales. We analyzed a unique database of 588 fossil tetrapod specimens from South Africa’s Karoo Basin, spanning ∼4 My, and 13 stratigraphic bin intervals averaging 300,000 y each. Using sample-standardized methods, we characterized faunal assemblage dynamics during the PTT. High regional extinction rates occurred through a protracted interval of ∼1 Ma, initially co-occurring with low origination rates. This resulted in declining diversity up to the acme of extinction near the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary. Regional origination rates increased abruptly above this boundary, co-occurring with high extinction rates to drive rapid turnover and an assemblage of short-lived species symptomatic of ecosystem instability. The “disaster taxon” Lystrosaurus shows a long-term trend of increasing abundance initiated in the latest Permian. Lystrosaurus comprised 54% of all specimens by the onset of mass extinction and 70% in the extinction aftermath. This early Lystrosaurus abundance suggests its expansion was facilitated by environmental changes rather than by ecological opportunity following the extinctions of other species as commonly assumed for disaster taxa. Our findings conservatively place the Karoo extinction interval closer in time, but not coeval with, the more rapid marine event and reveal key differences between the PTT extinctions on land and in the oceans.

Mass extinctions are major perturbations of the biosphere resulting from a wide range of different causes including glaciations and sea level fall (1), large igneous provinces (2), and bolide impacts (3, 4). These events caused permanent changes to Earth’s ecosystems, altering the evolutionary trajectory of life (5). However, links between the broad causal factors of mass extinctions and the biological and ecological disturbances that lead to species extinctions have been difficult to characterize. This is because ecological disturbances unfold on timescales much shorter than the typical resolution of paleontological studies (6), particularly in the terrestrial record (6–8). Coarse-resolution studies have demonstrated key mass extinction phenomena including high extinction rates and lineage turnover (7, 9), changes in species richness (10), ecosystem instability (11), and the occurrence of disaster taxa (12). However, finer time resolutions are central to determining the association and relative timings of these effects, their potential causal factors, and their interrelationships. Achieving these goals represents a key advance in understanding the ecological mechanisms of mass extinctions.The end-Permian mass extinction (ca. 251.9 Ma) was Earth’s largest biotic crisis as measured by taxon last occurrences (13–15). Large outpourings from Siberian Trap volcanism (2) are the likely trigger of calamitous climatic changes, including a runaway greenhouse effect and ocean acidification, which had profound consequences for life on land and in the oceans (16–18). An estimated 81% of marine species (19) and 89% of tetrapod genera became extinct as established Permian ecosystems gave way to those of the Triassic. In the ocean, this included the complete extinction of reef-forming tabulate and rugose corals (20, 21) and significant losses in previously diverse ammonoid, brachiopod, and crinoid families (22). On land, many nonmammalian synapsids became extinct (16), and the glossopterid-dominated floras of Gondwana also disappeared (23). Stratigraphic sequences document a global “coral gap” and “coal gap” (24, 25), suggesting reef and forest ecosystems were rare or absent for up to 5 My after the event (26). Continuous fossil-bearing deposits documenting patterns of turnover across the Permian–Triassic transition (PTT) on land (27) and in the oceans (28) are geographically widespread (29, 30), including marine and continental successions that are known from China (31, 32) and India (33). Continental successions are known from Russia (34), Australia (35), Antarctica (36), and South Africa’s Karoo Basin (Fig. 1 and 37–40), the latter providing arguably the most densely sampled and taxonomically scrutinized (41–43) continental record of the PTT. The main extinction has been proposed to occur at the boundary between two biostratigraphic zones with distinctive faunal assemblages, the Daptocephalus and Lystrosaurus declivis assemblage zones (Fig. 1), which marks the traditional placement of the Permian–Triassic geologic boundary [(37) but see ref. 44]. Considerable research has attempted to understand the anatomy of the PTT in South Africa (38, 39, 45–52) and to place it in the context of biodiversity changes across southern Gondwana (53, 54) and globally (29, 31, 32, 44, 47, 55).Open in a separate windowFig. 1.Map of South Africa depicting the distribution of the four tetrapod fossil assemblage zones (Cistecephalus, Daptocephalus, Lystrosaurus declivis, Cynognathus) and our two study sites where fossils were collected in this study (sites A and B). Regional lithostratigraphy and biostratigraphy within the study interval are shown alongside isotope dilution–thermal ionization mass spectrometry dates retrieved by Rubidge et al., Botha et al., and Gastaldo et al. (37, 44, 80). The traditional (dashed red line) and associated PTB hypotheses for the Karoo Basin (37, 44) are also shown. Although traditionally associated with the PTB, the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary is defined by first appearances of co-occurring tetrapod assemblages, so its position relative to the three PTB hypotheses is unchanged. The Ripplemead member (*) has yet to be formalized by the South African Committee for Stratigraphy.Decades of research have demonstrated the richness of South Africa’s Karoo Basin fossil record, resulting in hundreds of stratigraphically well-documented tetrapod fossils across the PTT (37, 39, 56). This wealth of data has been used qualitatively to identify three extinction phases and an apparent early postextinction recovery phase (39, 45, 51). Furthermore, studies of Karoo community structure and function have elucidated the potential role of the extinction and subsequent recovery in breaking the incumbency of previously dominant clades, including synapsids (11, 57). Nevertheless, understanding patterns of faunal turnover and recovery during the PTT has been limited by the scarcity of quantitative investigations. Previous quantitative studies used coarsely sampled data (i.e., assemblage zone scale, 2 to 3 Ma time intervals) to identify low species richness immediately after the main extinction, potentially associated with multiple “boom and bust” cycles of primary productivity based on δ¹³C variation during the first 5 My of the Triassic (41, 58). However, many details of faunal dynamics in this interval remain unknown. Here, we investigate the dynamics of this major tetrapod extinction at an unprecedented time resolution (on the order of hundreds of thousands of years), using sample-standardized methods to quantify multiple aspects of regional change across the Cistecephalus, Daptocephalus, and Lystrosaurus declivis assemblage zones.     

Paleocene latitude of the Kohistan–Ladakh arc indicates multistage India–Eurasia collision

We report paleomagnetic data showing that an intraoceanic Trans-Tethyan subduction zone existed south of the Eurasian continent and north of the Indian subcontinent until at least Paleocene time. This system was active between 66 and 62 Ma at a paleolatitude of 8.1 ± 5.6 °N, placing it 600–2,300 km south of the contemporaneous Eurasian margin. The first ophiolite obductions onto the northern Indian margin also occurred at this time, demonstrating that collision was a multistage process involving at least two subduction systems. Collisional events began with collision of India and the Trans-Tethyan subduction zone in Late Cretaceous to Early Paleocene time, followed by the collision of India (plus Trans-Tethyan ophiolites) with Eurasia in mid-Eocene time. These data constrain the total postcollisional convergence across the India–Eurasia convergent zone to 1,350–2,150 km and limit the north–south extent of northwestern Greater India to <900 km. These results have broad implications for how collisional processes may affect plate reconfigurations, global climate, and biodiversity.

Classically, the India–Eurasia collision has been considered to be a single-stage event that occurred at 50–55 million years ago (Ma) (1, 2). However, plate reconstructions show thousands of kilometers of separation between India and Eurasia at the inferred time of collision (3, 4). Accordingly, the northern extent of Greater India was thought to have protruded up to 2,000 km relative to present-day India (5, 6) (Fig. 1). Others have suggested that the India–Eurasia collision was a multistage process that involved an east–west trending Trans-Tethyan subduction zone (TTSZ) situated south of the Eurasian margin (7–9) (Fig. 1). Jagoutz et al. (9) concluded that collision between India and the TTSZ occurred at 50–55 Ma, and the final continental collision occurred between the TTSZ and Eurasia at 40 Ma (9, 10). This model reconciles the amount of convergence between India and Eurasia with the observed shortening across the India–Eurasia collision system with the addition of the Kshiroda oceanic plate. Additionally, the presence of two subduction systems can explain the rapid India–Eurasia convergence rates (up to 16 mm a⁻¹) that existed between 135 and 50 Ma (9), as well as variations in global climate in the Cenozoic (11).Open in a separate windowFig. 1.The first panel is an overview map of tectonic structure of the Karakoram–Himalaya–Tibet orogenic system. Blue represents India, red represents Eurasia, and the Kohistan–Ladakh arc (KLA) is shown in gray. The different shades of blue highlight the deformed margin of the Indian plate that has been uplifted to form the Himalayan belt, and the zones of darker red within the Eurasian plate highlight the Eurasian continental arc batholith. Thick black lines denote the suture zones which separate Indian and Eurasian terranes. The tectonic summary panels illustrate the two conflicting collision models and their differing predictions of the location of the Kohistan–Ladakh arc. India is shown in blue, Eurasia is shown in red, and the other nearby continents are shown in gray. Active plate boundaries are shown with black lines, and recently extinct boundaries are shown with gray lines. Subduction zones are shown with triangular tick marks.While the existence of the TTSZ in the Cretaceous is not disputed, the two conflicting collision models make distinct predictions about its paleolatitude in Late Cretaceous to Paleocene time; these can be tested using paleomagnetism. In the single-stage collision model, the TTSZ amalgamated with the Eurasian margin prior to ∼80 Ma (12) at a latitude of ≥20 °N (13, 14). In contrast, in the multistage model, the TTSZ remained near the equator at ≤10 °N, significantly south of Eurasia, until collision with India (9) (Fig. 1).No undisputed paleomagnetic constraints on the location of the TTSZ are available in the central Himalaya (15–17). Westerweel et al. (18) showed that the Burma Terrane, in the eastern Himalaya, was part of the TTSZ and was located near the equator at ∼95 Ma, but they do not constrain the location of the TTSZ in the time period between 50 and 80 Ma, which is required to test the two collision hypotheses. In the western Himalaya, India and Eurasia are separated by the Bela, Khost, and Muslimbagh ophiolites and the 60,000 km² intraoceanic Kohistan Ladakh arc (19, 20) (Fig. 1). These were obducted onto India in the Late Cretaceous to Early Paleocene (19), prior to the closure of the Eocene to Oligocene Katawaz sedimentary basin (20) (Fig. 1). The Kohistan–Ladakh arc contacts the Eurasian Karakoram terrane in the north along the Shyok suture and the Indian plate in the south along the Indus suture (21) (Fig. 1). Previous paleomagnetic studies suggest that the Kohistan–Ladakh arc formed as part of the TTSZ near the equator in the early Cretaceous but provide no information on its location after 80 Ma (22–25). While pioneering, these studies lack robust age constraints, do not appropriately average paleosecular variation of the geodynamo, and do not demonstrate that the measured magnetizations have not been reset during a subsequent metamorphic episode.