In situ three-dimensional spider web construction and mechanics

Spiders are nature’s engineers that build lightweight and high-performance web architectures often several times their size and with very few supports; however, little is known about web mechanics and geometries throughout construction, especially for three-dimensional (3D) spider webs. In this work, we investigate the structure and mechanics for a Tidarren sisyphoides spider web at varying stages of construction. This is accomplished by imaging, modeling, and simulations throughout the web-building process to capture changes in the natural web geometry and the mechanical properties. We show that the foundation of the web geometry, strength, and functionality is created during the first 2 d of construction, after which the spider reinforces the existing network with limited expansion of the structure within the frame. A better understanding of the biological and mechanical performance of the 3D spider web under construction could inspire sustainable robust and resilient fiber networks, complex materials, structures, scaffolding, and self-assembly strategies for hierarchical structures and inspire additive manufacturing methods such as 3D printing as well as inspire artistic and architectural and engineering applications.

Spiders are among nature’s most efficient and prolific engineers: they design, build, use, and maintain high-performance and lightweight silk webs. They have inspired applications in the field of biomedicine (1), structural engineering (2), electronics (3), optics (4), art (5, 6), and music (7). Spiders produce silk fibers with outstanding mechanical and biological properties; they are strong and extensible while also being biodegradable and biocompatible. Their superior mechanical properties are rooted in their hierarchical organization that spans from protein amino acid chains to silk fibers to spider webs (8–10). They have survived and prospered through millions of years of evolution owing to their superb adaptation skills (11). Indeed, spiders can spin up to eight different types of silks with different properties and functions and create numerous web architectures that range from simple T webs and typical two-dimensional (2D) orb webs to complex three-dimensional (3D) webs such as funnel, cob, and tangle webs (12). Not only do they produce, build, and tune highly functional silks and large-scale web structures, they also monitor, repair, and recycle webs. Moreover, the complex structures seen in spider webs have been compared to other natural structures such as galaxies (13, 14).Spiders use vibrational information to locate and identify potential mates, prey, predators, and defects on their webs (15–18). Webs avoid catastrophic failure because the interplay between the nonlinear behavior of dragline silk and complex spider web architecture localizes defects, making quick repairs possible (19). This makes the complex of spider, silk, and web a self-sufficient, self-monitored, and self-repairable system. This system can inspire sustainable and high-performance complex new materials, structural designs, and construction procedures (20). Spiders build web structures several times their size with very few external supports, using only silk fibers. Comparing them to traditional construction at the human scale, such a large structure would require bulky scaffolding, large construction equipment, and many workers. Understanding the construction stages of a spider web could lead to more efficient and sustainable construction.Gaining such understanding can begin by analyzing the 2D orb web geometry and construction process by facing a camera to the plane of the web. Orb webs are composed of stiff dragline silk radial threads and extensible and sticky viscid silk spiral threads. Orb web construction starts with the spider building the frame of the web after exploring the site. Orb-weaver spiders build the hub with radial threads and then place the spiral threads using spiral scaffolding as a guide. Compared to 2D orb webs, 3D spider webs are more complicated to describe because of their complex fiber architecture and their nanoscale fibers. Su et al. (12) have reported a method to automatically quantify 3D spider web geometry by taking high-resolution images of slices of the web that are illuminated by a sliding sheet laser. Image-processing algorithms were then used to translate the sequence of 2D images into the fibers and nodes of the 3D web network (12). The scanned spider webs were built on top of a water container, necessary to deter the spider’s escape (21), and then brought to the experimental setup for scanning (12). The original manual scanning and spider web method was developed by Saraceno et al. (5, 22–26). Similarly, Yablonina (27) approximated 3D spider web architecture using infrared cameras and a sliding laser and investigated the importance of supporting threads, reinforcing threads, and joints between threads by recording spider movement during web construction. Arachnologists have studied 3D spider web construction through meticulous behavioral observations and video recordings. Jörger and Eberhard (28) characterized three stages of the construction of Achaearanea tesselata spider webs: exploration, construction of anchor lines and tangle web, and alternating construction of sheet and tangle webs. Other species follow a similar web construction process. For example, Tidarren sisyphoides spiders explore the surroundings, anchor the retreat, build the web scaffolding, and then construct the dome-shaped sheet, horizontal sheet, and upper tangle web (29). Steatoda triangulosa comb-footed spiders explore and then build a 3D supporting structure and gumfooted lines that connect the 3D structure and the substrate (30). Web construction observations have already inspired new automation methods (25) and algorithms (31) for fiber network construction. Other work (32) has recently investigated the mechanical behavior of Cyrtophora citricola 3D spider webs under uniaxial stretching and projectile impact. The interplay between the nonlinear behavior of dragline silk and the complex redundant architecture of the webs leads to robust and resilient webs under uniaxial stretching. The tangle web plays a crucial role in the functionality of the spider webs: it filters in prey and protects the spider from predators.While completed 3D web-construction patterns and topologies have been investigated, little is known about the structure and mechanics of 3D spider webs during construction. We provide insights into these heretofore unknown processes in this paper. In the current study, we will investigate the mechanical behavior of the different construction stages of a T. sisyphoides spider web and determine whether the web can carry out its biological functions during construction. We use image processing and computational simulation methods to quantify and validate what has been observed in nature.Our paper is organized as follows. First, we present the different topological and mechanical properties of the web under construction. Second, we conclude with a summary and investigate future applications of web-inspired structures and construction processes. Finally, we describe the experimental and computational methods to obtain the topology of a 3D spider web during construction. We also describe the mechanical model and simulation setup of the 3D spider web during this time.     

Critical role of minor eggcase silk component in promoting spidroin chain alignment and strong fiber formation

Natural spider silk with extraordinary mechanical properties is typically spun from more than one type of spidroin. Although the main components of various spider silks have been widely studied, little is known about the molecular role of the minor silk components in spidroin self-assembly and fiber formation. Here, we show that the minor component of spider eggcase silk, TuSp2, not only accelerates self-assembly but remarkably promotes molecular chain alignment of spidroins upon physical shearing. NMR structure of the repetitive domain of TuSp2 reveals that its dimeric structure with unique charged surface serves as a platform to recruit different domains of the main eggcase component TuSp1. Artificial fiber spun from the complex between TuSp1 and TuSp2 minispidroins exhibits considerably higher strength and Young’s modulus than its native counterpart. These results create a framework for rationally designing silk biomaterials based on distinct roles of silk components.

Spider silks are semicrystalline protein polymers and are spun through the assembly of different spider fibroins (spidroins) (1, 2). Orb-weaving spiders can produce up to six different types of silks and one silk glue (3). Each silk displays a unique combination of mechanical properties, such as high strength, long extension, and high toughness (4, 5). Studies on spider silk genes have revealed that most spidroins (or silk proteins) contain multiple repetitive motifs or domains, flanked by two nonrepetitive terminal domains or unstructured tails on both ends (6–9). The amino acid compositions from different spidroin repeats (RPs) are highly variable, contributing to the distinctive physical and functional properties of each silk type (10, 11). The N-terminal domain (NTD) and the C-terminal domain (CTD) play important roles in the regulation of spidroin self-assembly and protein storage (12–18). Most full-length spidroins have high molecular weights (MWs), ranging from ∼150 to ∼1,380 kDa (6, 7, 19–21). The large size of spidroins has been proven to be one of the key determinants of the silk’s extraordinary mechanical properties (22–24). Depending on silk types and spider species, each spider silk is characteristically made from more than one type of spidroin (8, 9, 25–30). Spidroins in each silk gland can be classified into main and minor components based on their abundance (9, 31–33). Many main silk components have been identified, such as major ampullate spidroin 1 and 2 (MaSp1 and MaSp2), minor ampullate spidroin 1 and 2 (MiSp1 and MiSp2), tubuliform spidroin 1 (TuSp1), aciniform spidroin 1 (AcSp1), flagelliform spidroin 1 (FlSp1), pyriform spidroin 1 (PySp1), and aggregate spidroin 1 and 2 (AgSp1 and AgSp2) (19, 31, 34–36). Minor silk components typically show limited sequence homology to other spidroin families and may not contain conserved terminal domains (9, 28). Spiders adopt unique recipes of spinning dope comprising main and minor components for production of different silks for various purposes. However, very little is known about how minor silk components contribute to spidroins self-assembly and fiber formation.Among all types of spider silks, eggcase or tubuliform silk is distinctive due to its high serine and low glycine content and its exclusive role in the construction of protective cocoons, where spider eggs can hatch for up to several months (37). Eggcase silk is much more robust to resist different threats over such an extended period compared to other types of silks. TuSp1 was first identified as the main component in tubuliform glands (32, 38–40). Structural and functional studies on TuSp1 individual domains from the orb-web spider Nephila antipodiana (N.an.) indicated that interactions among the terminal domains of TuSp1 can initiate TuSp1 assembly to form micelle-like structures through hydrophobic interactions among the terminal domains (41, 42). TuSp1-RP domains linked to the terminal domain are also required to ensure the formation of micelles, which is one of the crucial intermediates during natural silk processing (43, 44). The second constituent of tubuliform spidroins (TuSp2) was later identified as a minor component in eggcase silk from the complementary DNA (cDNA) library constructed by using the seven types of N.an. silk glands. Although TuSp2 also contains multiple repetitive domains, it lacks typical spidroin terminal domains. Instead, it contains a short C-terminal tail of 37 amino acid residues, and the sequence of its N-terminal tail is currently unknown. Neither the RP nor the C-terminal tail of TuSp2 shows homology to TuSp1 or other known spidroins. The MW of TuSp2 is estimated to be >200 kDa based on Northern blotting, but the exact number of its RP domain is not clear because of the unavailability of TuSp2 full-length sequence (9). Similar to TuSp1, TuSp2 is also specifically expressed in the tubuliform gland. However, the mechanism of TuSp2 involved in the spinning of eggcase silk remains completely unknown.Herein, we investigated the role of TuSp2 in eggcase silk formation. We found that TuSp2-RP significantly promotes spidroins self-assembly and chain alignment in the long axis of the fibers upon shear force. We determined the NMR structure of TuSp2-RP dimer and provided evidence that the dimeric structure and its unique charged surface of TuSp2-RP are essential for its interaction with TuSp1. We further demonstrate that artificial silk spun from complexes between TuSp1 and TuSp2 minispidroins with low MWs can exhibit much higher strength and Young’s modulus compared to its native counterpart.     

Outsourced hearing in an orb-weaving spider that uses its web as an auditory sensor

Hearing is a fundamental sense of many animals, including all mammals, birds, some reptiles, amphibians, fish, and arthropods. The auditory organs of these animals are extremely diverse in anatomy after hundreds of millions of years of evolution, yet all are made up of cellular tissue and are morphologically part of the bodies of animals. Here, we show that hearing in the orb-weaving spider Larinioides sclopetarius is not constrained by the organism’s body but is extended through outsourcing hearing to its extended phenotype, the proteinaceous, self-manufactured orb web. We find that the wispy, wheel-shaped orb web acts as a hyperacute acoustic antenna to capture the sound-induced air particle movements that approach the maximum physical efficiency better than the acoustic responsivity of all previously known eardrums. By sensing the motion of web threads, the spider remotely detects and localizes the source of an incoming airborne acoustic wave, such as those emitted by approaching prey or predators. By outsourcing its acoustic sensors to its web, the spider is released from body size constraints and permits the araneid spider to increase its sound-sensitive surface area enormously, up to 10,000 times greater than the spider itself. The spider also enables the flexibility to functionally adjust and regularly regenerate its external "eardrum" according to its needs. The outsourcing and supersizing of auditory function in spiders provides unique features for studying extended and regenerative sensing and designing novel acoustic flow detectors for precise fluid dynamic measurement and manipulation.

During the water-to-land transition, animals have gone through dramatic challenges in aerial hearing (1, 2). To effectively detect weak, distant airborne sound, terrestrial vertebrates and some invertebrates have evolved tympanic eardrums, which are very sensitive to the pressure component of sound (1, 3). Alternatively, some arthropods, especially those of miniscule size, have evolved pendulum-like, long wispy filaments to detect the velocity component of sound (4–6). While the auditory organs of different animals are extremely diverse in anatomy after hundreds of millions of years of evolution (7, 8), they are all organs of cellular origin and are morphologically parts of the bodies of animals.Spiders are among the oldest land animals, with a fossil record dating back to the Devonian Period (around 380 Mya) (9). All spiders produce silk, a biomaterial that can be stronger than steel in strength-to-weight ratio yet extremely flexible (10), owing to its exceptional material properties. When woven into a broad latticework, a web can serve as a net for capturing prey that fly or walk into it (11–13). We previously showed that a single strand of nano-dimensional spider silk can move with a velocity very close to that of the surrounding air particle movements, with the maximum physical efficiency from infrasound to ultrasound despite the low viscosity and low density of air (14). Here, we show that the highly responsive aerodynamic property of silk fibers is woven and stretched into a diaphanous orb web that can function as a huge acoustic “antenna”, which allows the spider to efficiently detect faint airborne sound from a distant source. This outsourced orb web “eardrum” as an extended phenotype beyond the body operates on a very different principle from the much smaller auditory organs of all other animals.     

Sensing surface morphology of biofibers by decorating spider silk and cellulosic filaments with nematic microdroplets

Probing the surface morphology of microthin fibers such as naturally occurring biofibers is essential for understanding their structural properties, biological function, and mechanical performance. The state-of-the-art methods for studying the surfaces of biofibers are atomic force microscopy imaging and scanning electron microscopy, which well characterize surface geometry of the fibers but provide little information on the local interaction potential of the fibers with the surrounding material. In contrast, complex nematic fluids respond very well to external fields and change their optical properties upon such stimuli. Here we demonstrate that liquid crystal droplets deposited on microthin biofibers—including spider silk and cellulosic fibers—reveal characteristics of the fibers’ surface, performing as simple but sensitive surface sensors. By combining experiments and numerical modeling, different types of fibers are identified through the fiber-to-nematic droplet interactions, including perpendicular and axial or helicoidal planar molecular alignment. Spider silks align nematic molecules parallel to fibers or perpendicular to them, whereas cellulose aligns the molecules unidirectionally or helicoidally along the fibers, indicating notably different surface interactions. The nematic droplets as sensors thus directly reveal chirality of cellulosic fibers. Different fiber entanglements can be identified by depositing droplets exactly at the fiber crossings. More generally, the presented method can be used as a simple but powerful approach for probing the surface properties of small-size bioobjects, opening a route to their precise characterization.Natural microfilaments produced by plants, insects, or spiders are fascinating materials not just because of their specific properties such as wear resistance, elasticity, tensile strength, and toughness (1–5) but also because of their microorganization (6–9). Their macroscopic properties can match properties of materials like kevlar but are at the same time biocompatible and biodegradable (10). These fascinating macroscopic properties actually originate from bulk and surface properties of the fibers (1). The chemical composition of the threads combined with their morphology determines the final properties of the material (11–13). The mechanical properties of the spider fibers are determined by the existence of a lyotropic liquid crystalline phase, from which the threads are drawn (14). Such silks are known to include nanoscale networks of defects and cavities that yield surface structures notably dependent on the spider species (3). These differences do not affect much the mechanical performance of the fibers (1, 3, 5). From a technological perspective, many attempts have been made to reproduce these natural bionetworks (15–17). In fact cellulose-based fibers with few micrometers of diameter, produced by electrospinning, can also acquire different morphologies depending upon the processing conditions, giving diverse features of the final threads and mats (18). Therefore, probing the surface structure of the microfibers is crucial for a complete understanding of their individual and interthreaded properties.From another perspective, nematic complex fluids are materials which are inherently responsive to diverse external stimuli, notably including diverse surface interactions which in the literature are known as the surface anchoring (19). Being effectively elastic materials, the orientational order of nematics responds on long, typically micrometer scales (20–22), which results in a spatially varying birefringence that can be optically detected (23). Recently, it was demonstrated that glass fibers induce numerous defects in a well-aligned nematic liquid crystal cell and thus provide a simple illustration of topological phenomena (24). It is also known that liquid crystal droplets can considerably change their structure by the action of otherwise imperceptibly small external stimuli (21). Pierced nematic and chiral nematic droplets develop defects that can be controlled by the liquid crystal elasticity, chirality, and surface boundary conditions (25, 26) indicating exceptional sensitivity. Therefore, to generalize, putting nematics into contact with diverse surfaces (18, 27) can be used as a simple but very powerful technique to detect the surface properties of microobjects such as biological fibers.In this paper we demonstrate the surface morphology sensing of biorelevant fibers, including spider silk and cellulosic microfibers, by nematic droplets that are sprayed onto the fibers. Specifically, we explore the chiral and achiral nature of the fiber’s surface and the in-plane or perpendicular alignment fields the fibers impose on the nematic. Droplets with degenerate in-plane and perpendicular alignment of the nematic at their free surfaces are explored, combining experiments and numerical modeling, to allow for tuning of the sensing precision. Further, the entanglement sites of the fiber webs are explored, with the droplets deposited at the sites clearly revealing contact, noncontact, and entangled morphologies.     

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

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

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

Physical evidence of predatory behavior in Tyrannosaurus rex

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

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

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

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

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

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

Assessing the origins of the European Plagues following the Black Death: A synthesis of genomic,historical, and ecological information

The second plague pandemic started in Europe with the Black Death in 1346 and lasted until the 19th century. Based on ancient DNA studies, there is a scientific disagreement over whether the bacterium, Yersinia pestis, came into Europe once (Hypothesis 1) or repeatedly over the following four centuries (Hypothesis 2). Here, we synthesize the most updated phylogeny together with historical, archeological, evolutionary, and ecological information. On the basis of this holistic view, we conclude that Hypothesis 2 is the most plausible. We also suggest that Y. pestis lineages might have developed attenuated virulence during transmission, which can explain the convergent evolutionary signals, including pla decay, that appeared at the end of the pandemics.

Researchers agree that the second plague pandemic was caused by Yersinia pestis (1–9), which arrived in Europe from Caffa transported by Genoese galleys on the Black Sea at the beginning of the Black Death (10). However, there is no consensus among researchers as to the origins of plague epidemics in Europe following the Black Death and ravaging Europe until the 19th century, as attested by historical documents (11).The two main theories are that one or more plague reservoirs remained in Western Europe during the entire second plague pandemic (referred to in the following as Hypothesis 1) (3, 4, 8, 12) or the bacteria repeatedly invaded Europe from non–Western European reservoir(s) during the same period (referred to in the following as Hypothesis 2) (6, 7, 9, 11, 13). Here, we assess these two hypotheses using a broad spectrum of evidence, including historical and archeological, genetic, evolutionary, and ecological information.     

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

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

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

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

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

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

Ancestral polymorphisms shape the adaptive radiation of Metrosideros across the Hawaiian Islands

Some of the most spectacular adaptive radiations begin with founder populations on remote islands. How genetically limited founder populations give rise to the striking phenotypic and ecological diversity characteristic of adaptive radiations is a paradox of evolutionary biology. We conducted an evolutionary genomics analysis of genus Metrosideros, a landscape-dominant, incipient adaptive radiation of woody plants that spans a striking range of phenotypes and environments across the Hawaiian Islands. Using nanopore-sequencing, we created a chromosome-level genome assembly for Metrosideros polymorpha var. incana and analyzed whole-genome sequences of 131 individuals from 11 taxa sampled across the islands. Demographic modeling and population genomics analyses suggested that Hawaiian Metrosideros originated from a single colonization event and subsequently spread across the archipelago following the formation of new islands. The evolutionary history of Hawaiian Metrosideros shows evidence of extensive reticulation associated with significant sharing of ancestral variation between taxa and secondarily with admixture. Taking advantage of the highly contiguous genome assembly, we investigated the genomic architecture underlying the adaptive radiation and discovered that divergent selection drove the formation of differentiation outliers in paired taxa representing early stages of speciation/divergence. Analysis of the evolutionary origins of the outlier single nucleotide polymorphisms (SNPs) showed enrichment for ancestral variations under divergent selection. Our findings suggest that Hawaiian Metrosideros possesses an unexpectedly rich pool of ancestral genetic variation, and the reassortment of these variations has fueled the island adaptive radiation.

Adaptive radiations exhibit extraordinary levels of morphological and ecological diversity (1). Although definitions of adaptive radiation vary (2–7), all center on ecological opportunity as a driver of adaptation and, ultimately, diversification (2, 8–10). Divergent selection, the primary mechanism underlying adaptive radiations, favors extreme phenotypes (11) and selects alleles that confer adaptation to unoccupied or under-utilized ecological niches. Differential adaptation results in divergence and, ultimately, reproductive isolation between populations (12). Adaptive radiations demonstrate the remarkable power of natural selection as a driver of biological diversity and provide excellent systems for studying evolutionary processes involved in diversification and speciation (13).Adaptive radiations on remote oceanic islands are especially interesting, as colonization of remote islands is expected to involve population bottlenecks that restrict genetic variation (14). Adaptive radiations in such settings are especially impressive and even paradoxical, given the generation of high species richness from an initially limited gene pool (15). Several classic examples of adaptive radiation occur on oceanic islands, such as Darwin’s finches from the Galapagos islands (16), anole lizards from the Caribbean islands (9), Hawaiian Drosophilids (17), and Hawaiian silverswords (18), to name a few.Recent advances in genome sequencing and analyses have greatly improved our ability to examine the genetics of speciation and adaptive radiation. By examining sequences of multiple individuals from their natural environment, it has become possible to “catch in the act” the speciation processes between incipient lineages (19). Genomic studies of early stage speciation show that differentiation accumulates in genomic regions that restrict the homogenizing effects of gene flow between incipient species (20). The number, size, and distribution of these genomic regions can shed light on evolutionary factors involved in speciation (19). Regions of high genomic differentiation can also form from evolutionary factors unrelated to speciation, such as linkage associated with recurrent background selection or selective sweeps on shared genomic features (21, 22).Genomic studies of lineages undergoing rapid ecological diversification have begun to reveal the evolutionary mechanisms underlying adaptive radiations. Importantly, these studies highlight the pivotal role of hybridization between populations and the consequent exchange of adaptive alleles that facilitates rapid speciation and the colonization of diverse niches (23–25). Most genomic studies of adaptive radiation involve animal systems, however, in particular, birds and fishes. In plants, genomic studies of adaptive radiation are sparse (26–28), and all examine continent-wide radiations. There are no genomics studies of plant adaptive radiations in geographically restricted systems such as remote islands. Because the eco-evolutionary scenarios associated with adaptive radiations are diverse (5, 29), whether commonalities identified in adaptive radiations in animals (23, 30) are applicable to plants is an open question. For example, the genetic architecture of animal adaptive radiations typically involves differentiation at a small number of genomic regions (31–33). In contrast, the limited insights available for plants suggest a more complex genetic architecture (26).We investigated the evolutionary genomics of adaptive radiation in Metrosideros Banks ex Gaertn. (Myrtaceae) across the Hawaiian Islands. Hawaiian Metrosideros is a landscape-dominant, hypervariable, and highly dispersible group of long-lived (possibly >650 y) (34) woody taxa that are nonrandomly distributed across Hawaii’s heterogeneous landscape, including cooled lava flows, wet forests and bogs, subalpine zones, and riparian zones (35, 36). About 25 taxa or morphotypes are distinguished by vegetative characters ranging from prostate plants that flower a few centimeters above ground to 30-m-tall trees, and leaves range dramatically in size, shape, pubescence, color, and rugosity (35, 37, 38); a majority of these forms are intraspecific varieties or races (provisional varieties) of the abundant species, Metrosideros polymorpha (35, 36, 38). Variation in leaf mass per area within the four Metrosideros taxa on Hawaii Island alone matches that observed for woody species globally (39). Common garden experiments (38, 40–44) and parent–offspring analysis (45) demonstrate heritability of taxon-diagnostic vegetative traits, indicating that taxa are distinct genetic groups and not the result of phenotypic plasticity. Metrosideros taxa display evidence of local adaptation to contrasting environments (46, 47), suggesting ecological divergent selection is responsible for diversification within the group (48). This diversification, which spans the past ∼3.1 to 3.9 million years (49, 50), has occurred despite the group’s high capacity for gene flow by way of showy bird-pollinated flowers and tiny wind-dispersed seeds (36, 51). Lastly, the presence of partial reproductive isolating barriers between taxa is consistent with the early stages of speciation (52). Here, we generated several genomic resources for Hawaiian Metrosideros and used these in population genomics analyses to gain deeper insights into the genomic architecture and evolutionary processes underlying this island adaptive radiation.     

Radiation and hybridization underpin the spread of the fire ant social supergene

Supergenes are clusters of tightly linked genes that jointly produce complex phenotypes. Although widespread in nature, how such genomic elements are formed and how they spread are in most cases unclear. In the fire ant Solenopsis invicta and closely related species, a “social supergene controls whether a colony maintains one or multiple queens. Here, we show that the three inversions constituting the Social b (Sb) supergene emerged sequentially during the separation of the ancestral lineages of S. invicta and Solenopsis richteri. The two first inversions arose in the ancestral population of both species, while the third one arose in the S. richteri lineage. Once completely assembled in the S. richteri lineage, the supergene first introgressed into S. invicta, and from there into the other species of the socially polymorphic group of South American fire ant species. Surprisingly, the introgression of this large and important genomic element occurred despite recent hybridization being uncommon between several of the species. These results highlight how supergenes can readily move across species boundaries, possibly because of fitness benefits they provide and/or expression of selfish properties favoring their transmission.

Understanding how complex traits requiring multiple novel mutations arise and are maintained in populations is a long-standing question in evolutionary biology (1–3). Supergenes are clusters of multiple, tightly linked genes that collectively produce complex phenotypes (2). They have evolved independently in many taxa and are responsible for intraspecific polymorphism in several important morphological and behavioral traits. The most prominent examples of supergenes are heteromorphic sex chromosomes, which drive the alternate development of males or females. Other examples of supergenes underpinning alternate phenotypes include Batesian mimicry in numerous butterfly species (4–7), self-incompatibility and floral heteromorphy in plants (S locus) (8–11), male meiotic drive (sperm killers) (12), bird plumage color polymorphism (13), and alternative social organization in ant colonies (14–17). In most known cases, the structural integrity of the supergene results from chromosomal rearrangements that suppress local recombination and thereby prevent dissociation of the genetic elements responsible for the integrated expression of complex character suites (5, 10, 13, 16).The first supergene producing alternative social organization was identified in the fire ant Solenopsis invicta (16). In this species, colonies contain either a single egg-laying queen (monogyne social form) or multiple queens (polygyne social form), a fundamental distinction associated with a suite of important individual- and colony-level phenotypic differences (15, 18–21). Studies of invasive populations in the United States have revealed that this supergene comprises two haplotypes, the Social b (Sb) and the Social B (SB), which regulate the polymorphism (16, 22). In S. invicta, monogyne colonies invariably have a single homozygous SB/SB queen and only SB/SB workers, while polygyne colonies always have multiple heterozygous (SB/Sb) queens as well as workers of all three genotypes (15). Moreover, in invasive US populations, the Sb haplotype is responsible for a selfish “green beard” effect whereby SB/Sb (polygyne) workers recognize the cuticular chemical profiles of SB/SB queens and selectively eliminate them as they mature sexually and initiate reproduction (23, 24). Recent studies revealed that social organization also is regulated by the presence/absence of workers with the Sb haplotype in six closely related species of the Solenopsis saevissima species group (i.e., Solenopsis richteri, Solenopsis megergates, Solenopsis macdonaghi, Solenopsis quinquecuspis, the undescribed Solenopsis AdRX, and the undescribed Solenopsis near interrupta) (22, 25, 26). These species collectively are referred to as the socially polymorphic South American fire ants (22).The extant Sb haplotype invariably consists of three inversions that together span a region of ∼11.4 Mb on chromosome 16 containing >476 described genes. As a result, recombination is greatly reduced between the Sb and the SB haplotypes (16, 22, 27). The largest inversion, In(16)1, spans 9.48 Mb of chromosome 16. A second inversion, In(16)2, spans 0.84 Mb between In(16)1 and a third inversion, In(16)3. In(16)2 likely emerged after In(16)1, given that a small fragment of In(16)1—telomeric in SB but central in Sb—is inverted again in the In(16)2 Sb haplotype (22). In(16)3 (1.07 Mb) is located ∼25 kb distant from In(16)2 in a pericentric region of chromosome 16 (22, 28). Previous analyses of the divergence between the Sb and SB haplotypes within species suggested that the three inversions arose over an evolutionarily short time span (22).Reconstructing the evolutionary history of supergene evolution is essential to understanding how such remarkable genomic elements originate and, possibly, the sequence of incorporation of key phenotypic elements into complex traits. The socially polymorphic species are hypothesized to have diverged from the outgroup species S. saevissima and Solenopsis metallica, ∼0.75 to 1.75 million y ago and then radiated within the past ∼500,000 y (22, 29). While a single origin of the Sb haplotype is robustly supported (30, 31), it remains unclear whether this unique element originated in the ancestral population before the radiation, or whether it has invaded the known socially polymorphic species through introgression events (22, 29, 32). Multiple studies have documented recent hybridization involving three of the socially polymorphic Solenopsis species in both the native and introduced ranges (33–37), highlighting a demographic context conducive to such genomic invasion in at least some cases.Here, we present a comprehensive analysis of the history of the fire ant supergene intended to shed light on when and in what order each inversion occurred, and whether introgression of the Sb haplotype between species best explains observed patterns in the comparative genomic data. Using complementary approaches, we conclude that In(16)3 is the oldest inversion and that it likely emerged in the common ancestor of S. richteri and the S. invicta/AdRX lineage. In(16)1 emerged next, during the divergence between the S. richteri and S. invicta/AdRX lineages. Finally, the youngest [In(16)2] inversion emerged in the S. richteri lineage. The supergene comprising all three inversions thus emerged in S. richteri, only to spread to the five most closely related species and confer the social polymorphism to them.     

Fog and rain in the Amazon

The diurnal and seasonal water cycles in the Amazon remain poorly simulated in general circulation models, exhibiting peak evapotranspiration in the wrong season and rain too early in the day. We show that those biases are not present in cloud-resolving simulations with parameterized large-scale circulation. The difference is attributed to the representation of the morning fog layer, and to more accurate characterization of convection and its coupling with large-scale circulation. The morning fog layer, present in the wet season but absent in the dry season, dramatically increases cloud albedo, which reduces evapotranspiration through its modulation of the surface energy budget. These results highlight the importance of the coupling between the energy and hydrological cycles and the key role of cloud albedo feedback for climates over tropical continents.Tropical forests, and the Amazon in particular, are the biggest terrestrial CO₂ sinks on the planet, accounting for about 30% of the total net primary productivity in terrestrial ecosystems. Hence, the climate of the Amazon is of particular importance for the fate of global CO₂ concentration in the atmosphere (1). Besides the difficulty of estimating carbon pools (1–3), our incapacity to correctly predict CO₂ fluxes in the continental tropics largely results from inaccurate simulation of the tropical climate (1, 2, 4, 5). More frequent and more intense droughts in particular are expected to affect the future health of the Amazon and its capacity to act as a major carbon sink (6–8). The land surface is not isolated, however, but interacts with the weather and climate through a series of land−atmosphere feedback loops, which couple the energy, carbon, and water cycles through stomata regulation and boundary layer mediation (9).Current General Circulation Models (GCMs) fail to correctly represent some of the key features of the Amazon climate. In particular, they (i) underestimate the precipitation in the region (10, 11), (ii) do not reproduce the seasonality of either precipitation (10, 11) or surface fluxes such as evapotranspiration (12), and (iii) produce errors in the diurnal cycle and intensity of precipitation, with a tendency to rain too little and too early in the day (13, 14). In the more humid Western part of the basin, surface incoming radiation, evapotranspiration, and photosynthesis all tend to peak in the dry season (15–17), whereas GCMs simulate peaks of those fluxes in the wet season (10, 11). Those issues might be related to the representation of convection (1, 2, 4, 5, 13, 14) and vegetation water stress (6–8, 15–17) in GCMs.We here show that we can represent the Amazonian climate using a strategy opposite to GCMs in which we resolve convection and parameterize the large-scale circulation (Methods). The simulations lack many of the biases observed in GCMs and more accurately capture the differences between the dry and wet season of the Amazon in surface heat fluxes and precipitation. Besides top-of-the-atmosphere insolation, the simulations require the monthly mean temperature profile as an input. We demonstrate that this profile, whose seasonal cycle itself is a product of the coupled ocean−land−atmosphere dynamics, mediates the seasonality of the Amazonian climate by modulating the vertical structure of the large-scale circulation in such a way that thermal energy is less effectively ventilated in the rainy season.     

Evidence that dimethyl sulfide facilitates a tritrophic mutualism between marine primary producers and top predators

Tritrophic mutualistic interactions have been best studied in plant–insect systems. During these interactions, plants release volatiles in response to herbivore damage, which, in turn, facilitates predation on primary consumers or benefits the primary producer by providing nutrients. Here we explore a similar interaction in the Southern Ocean food web, where soluble iron limits primary productivity. Dimethyl sulfide has been studied in the context of global climate regulation and is an established foraging cue for marine top predators. We present evidence that procellariiform seabird species that use dimethyl sulfide as a foraging cue selectively forage on phytoplankton grazers. Their contribution of beneficial iron recycled to marine phytoplankton via excretion suggests a chemically mediated link between marine top predators and oceanic primary production.Many plant species interact with carnivores to gain protection from herbivory. Such mutualistic tritrophic interactions have been studied extensively in plant–insect systems, and are frequently mediated by plant volatiles released in response to insect feeding (1). One example that has received detailed study is the interaction between the phytophagous two-spotted spider mite Tetranychus urticae, the lima bean plant Phaseolus lunatus, and the predatory mite Phytoseiulus persimilis (2, 3). In this model system, grazing by the herbivorous spider mite has been demonstrated to elicit a cascade of biochemical reactions within the afflicted plants, stimulating the release of a suite of volatile terpenoids such as (E)-4,8-dimethyl-l,3,7-nonatriene, (E)-β-ocimene, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (3). These volatiles attract olfactory-searching P. persimilis that prey upon herbivorous spider mites.The possibility of tritrophic mutualisms involving plant volatiles has received considerable attention in terrestrial communities (2–5); however, similar interactions have rarely been suggested for marine systems (6). Dimethyl sulfide (DMS) and its precursor dimethylsulfoniopropionate (DMSP) are well-established infochemicals in the marine environment, and as such are good candidate molecules for mediating tritrophic interactions between phytoplankton and carnivores (7–10). DMS arises as a catabolic breakdown product of DMSP, and has been studied extensively for its putative role as a global climate regulator (11). DMSP is produced by marine algae, where it has been proposed to function as an osmolyte (12) and a cryoprotectant (13). When algal cells lyse, due to biotic or abiotic stress, one of the fates of DMSP is catabolism by the enzyme DMSP lyase to DMS and acrylic acid (14–16). This process may also occur during autocatalytic cell death (17). It has been proposed that acrylic acid is the biologically salient product of this reaction due to its antimicrobial properties (18).DMS production has also been shown to increase during zooplankton grazing (14). It has been previously proposed that this phytoplankton-derived odorant is an important infochemical for marine apex predators including whale sharks (19), harbor seals (20), penguins (21–23), and procellariiform (tube-nosed) seabirds (24). Procellariiform seabirds have been the best-studied in this regard, and many species have been shown to detect and respond to biogenic concentrations of DMS in foraging contexts (24, 25). Members of this order share highly pelagic lifestyles and are central-place foragers associated with land only during incubation and chick rearing (26). Procellariiformes routinely range thousands of kilometers to forage (27) and have large olfactory bulbs compared with other avian clades (28), and some species have been shown to track their prey using their sense of smell (29). Some procellariiform species are attracted to DMS, whereas others are not (24, 30) (Fig. 1); however, the relationship between DMS behavioral sensitivity and the consumption of herbivorous crustacea has not previously been shown.Open in a separate windowFig. 1.Phylogenetic relationships between the species included in the meta-analysis, mapped with DMS responsiveness. DMS responsiveness is thought to be ancestral in this lineage (30). Certain species in the outgroup, sphenisciformes (penguins), have also been shown to be responsive to DMS (21–23).The Southern Ocean is the largest marine ecosystem in the world, with the polar front forming a distinct northern boundary to this ecoregion (31). Our rationale for using this system is twofold: (i) A majority of the world’s procellariiform species breed or forage in the Southern Ocean (32), and (ii) food web relationships are relatively simple by comparison with other marine systems. Phaeocystis antarctica and several siliceous diatom species are the dominant DMS-producing phytoplankton species in this ecosystem, and Antarctic krill (Euphasia superba) and other small crustaceans (copepods, decapods, amphipods, etc.) are their major consumers.Here we take advantage of a 50-y dietary database of Southern Ocean seabirds (33) to explore whether DMS mediates a mutualistic tritrophic interaction in the Southern Ocean pelagic ecosystem. If this is the case, then we predict that (i) carnivorous species, such as seabirds, that are attracted to this infochemical should specialize on primary consumers, such as crustaceans, and (ii) primary producers should gain some benefit from this interaction.