Three-dimensionally preserved minute larva of a great-appendage arthropod from the early Cambrian Chengjiang biota

A three-dimensionally preserved 2-mm-long larva of the arthropod Leanchoilia illecebrosa from the 520-million-year-old early Cambrian Chengjiang biota of China represents the first evidence, to our knowledge, of such an early developmental stage in a short-great-appendage (SGA) arthropod. The larva possesses a pair of three-fingered great appendages, a hypostome, and four pairs of well-developed biramous appendages. More posteriorly, a series of rudimentary limb Anlagen revealed by X-ray microcomputed tomography shows a gradient of decreasing differentiation toward the rear. This, and postembryonic segment addition at the putative growth zone, are features of late-stage metanauplii of eucrustaceans. L. illecebrosa and other SGA arthropods, however, are considered representative of early chelicerates or part of the stem lineage of all euarthropods. The larva of an early Cambrian SGA arthropod with a small number of anterior segments and their respective appendages suggests that posthatching segment addition occurred in the ancestor of Euarthropoda.Evolutionary developmental biology (evo-devo) explains evolutionary changes in different organisms by investigating their developmental processes (1). Paleontology contributes to evo-devo by providing information that is only available in fossil organisms (2). Studies of evolutionary development in fossil arthropods, which have dominated faunas from the early Cambrian (∼520 million years ago) to the present, have focused on trilobites (3), “Orsten”-type fossil crustaceans (4–6), and Mesozoic malacostracan crustaceans (7). Due to their small size and low preservation potential, fossil evidence of the appendages of early developmental stages of arthropods are rare, and known mainly from those with the special “Orsten” type of preservation (8), i.e., with the cuticle secondarily phosphatized, from the mid-Cambrian (500–497 million years ago) (9).Here we describe an exceptionally preserved early developmental stage of a Cambrian arthropod from the Chengjiang biota of China. The specimen is only 2 mm long and is three-dimensionally preserved (Fig. 1, Insets). We interpret this specimen as a representative of the short-great-appendage (SGA) arthropod Leanchoilia illecebrosa—the most abundant SGA arthropod from this biota (10). SGA arthropods form a distinct early group characterized by prominent anteriormost appendages specialized for sensory (11) or feeding purposes (11, 12). Thus far, knowledge of L. illecebrosa is based mainly on adult specimens with a body length ranging from 20 to 46 mm (13) (Fig. 1). Specimens smaller than 20 mm are rare—only two examples, both 8 mm long, have been reported (8, 12) (Fig. S1B).Open in a separate windowFig. 1.L. illecebrosa from the Chengjiang biota. Macrophotographs of an adult (specimen YKLP 11087) and the minute larva (Insets; specimen YKLP 11088a, b). cs, cephalic shield; rs, rostrum; sga, short great appendage; ts1 and ts11, trunk segments 1 and 11; te, telson. Insets are to the same scale as main image. (Scale bar: 5 mm.)Open in a separate windowFig. S1.Two larval stages of L. illecebrosa. (A) The 2-mm-long larva described here (specimen YKLP 11088a, b). (B) An 8-mm-long larva previously reported in ref. 12 (specimen YKLP 11084a, b; reprinted with permission from ref. 12). (Scale bar: 2 mm.)     

From the Cover: Amplifying the response of soft actuators by harnessing snap-through instabilities

Soft, inflatable segments are the active elements responsible for the actuation of soft machines and robots. Although current designs of fluidic actuators achieve motion with large amplitudes, they require large amounts of supplied volume, limiting their speed and compactness. To circumvent these limitations, here we embrace instabilities and show that they can be exploited to amplify the response of the system. By combining experimental and numerical tools we design and construct fluidic actuators in which snap-through instabilities are harnessed to generate large motion, high forces, and fast actuation at constant volume. Our study opens avenues for the design of the next generation of soft actuators and robots in which small amounts of volume are sufficient to achieve significant ranges of motion.The ability of elastomeric materials to undergo large deformation has recently enabled the design of actuators that are inexpensive, easy to fabricate, and only require a single source of pressure for their actuation, and still achieve complex motion (1–5). These unique characteristics have allowed for a variety of innovative applications in areas as diverse as medical devices (6, 7), search and rescue systems (8), and adaptive robots (9–11). However, existing fluidic soft actuators typically show a continuous, quasi-monotonic relation between input and output, so they rely on large amounts of fluid to generate large deformations or exert high forces.By contrast, it is well known that a variety of elastic instabilities can be triggered in elastomeric films, resulting in sudden and significant geometric changes (12, 13). Such instabilities have traditionally been avoided as they often represent mechanical failure. However, a new trend is emerging in which instabilities are harnessed to enable new functionalities. For example, it has been reported that buckling can be instrumental in the design of stretchable soft electronics (14, 15), and tunable metamaterials (16–18). Moreover, snap-through transitions have been shown to result in instantaneous giant voltage-triggered deformation (19, 20).Here, we introduce a class of soft actuators comprised of interconnected fluidic segments, and show that snap-through instabilities in these systems can be harnessed to instantaneously trigger large changes in internal pressure, extension, shape, and exerted force. By combining experiments and numerical tools, we developed an approach that enables the design of customizable fluidic actuators for which a small increment in supplied volume (input) is sufficient to trigger large deformations or high forces (output).Our work is inspired by the well-known two-balloon experiment, in which two identical balloons, inflated to different diameters, are connected to freely exchange air. Instead of the balloons becoming equal in size, for most cases the smaller balloon becomes even smaller and the balloon with the larger diameter further increases in volume (Movie S1). This unexpected behavior originates from the balloons’ nonlinear relation between pressure and volume, characterized by a pronounced pressure peak (21, 22). Interestingly, for certain combinations of interconnected balloons, such nonlinear response can result in snap-through instabilities at constant volume, which lead to significant and sudden changes of the membranes’ diameters (Figs. S1 and ​andS2).S2). It is straightforward to show analytically that these instabilities can be triggered only if the pressure–volume relation of at least one of the membranes is characterized by (i) a pronounced initial peak in pressure, (ii) subsequent softening, and (iii) a final steep increase in pressure (Analytical Exploration: Response of Interconnected Spherical Membranes Upon Inflation).Open in a separate windowFig. S1.Relation between the pressure and volume for three different hyperelastic spherical membranes upon inflation.Open in a separate windowFig. S2.Response of two interconnected spherical membranes upon inflation. The response of the individual membranes is shown in Fig. S1. The equilibrium states and their stability have been obtained numerically. The pressure–volume relation and the relation between the volume of the individual membranes are shown for systems comprising (A) membranes a and b, (B) membranes b and c, and (C) membranes a and c.     

Anomalous scaling law of strength and toughness of cellulose nanopaper

The quest for both strength and toughness is perpetual in advanced material design; unfortunately, these two mechanical properties are generally mutually exclusive. So far there exists only limited success of attaining both strength and toughness, which often needs material-specific, complicated, or expensive synthesis processes and thus can hardly be applicable to other materials. A general mechanism to address the conflict between strength and toughness still remains elusive. Here we report a first-of-its-kind study of the dependence of strength and toughness of cellulose nanopaper on the size of the constituent cellulose fibers. Surprisingly, we find that both the strength and toughness of cellulose nanopaper increase simultaneously (40 and 130 times, respectively) as the size of the constituent cellulose fibers decreases (from a mean diameter of 27 μm to 11 nm), revealing an anomalous but highly desirable scaling law of the mechanical properties of cellulose nanopaper: the smaller, the stronger and the tougher. Further fundamental mechanistic studies reveal that reduced intrinsic defect size and facile (re)formation of strong hydrogen bonding among cellulose molecular chains is the underlying key to this new scaling law of mechanical properties. These mechanistic findings are generally applicable to other material building blocks, and therefore open up abundant opportunities to use the fundamental bottom-up strategy to design a new class of functional materials that are both strong and tough.The need for engineering materials that are both strong and tough is ubiquitous. However, the design of strong and tough materials is often inevitably a compromise as these two properties generally contradict each other (1). Toughness requires a material’s ability of dissipating local high stress by enduring deformation. Consequently, hard materials tend to be brittle (less tough); lower-strength materials, which can deform more readily, tend to be tougher (2, 3). For example, the toughness of metals and alloys is usually inversely proportional to their strength (4). Acknowledging such a necessary compromise, one would expect that research on advanced material design would be focused on achieving an optimum combination of these two properties. Indeed much research effort is focused on pursuing higher strength, with rather limited corresponding regard for toughness (5–10). One example is the enthusiasm sparked by the discovery of carbon nanotubes (CNTs), which exhibit remarkably high strength. However, it still remains uncertain how such a strong material can be incorporated with bulk materials to benefit from its high strength without sacrificing toughness.There have been tremendous efforts recently to develop materials with higher strength using smaller material structures. For example, by decreasing the grain size of metals, dislocation motions (thus plasticity) are more restricted, leading to a higher strength (5–10). However, such treatments also minimize possible mechanisms (e.g., crack-tip blunting) to relieve local high stress, resulting in lower toughness. The atomic scale origins of high strength of a material, e.g., strong directional bonding and limited dislocation mobility, are also essentially the roots for brittleness of the material. In short, the well-recognized scaling law of “the smaller, the stronger” comes at a price of sacrificing toughness (Fig. 1).Open in a separate windowFig. 1.An anomalous but desirable scaling law of mechanical properties requires defeating the conventional conflict between strength and toughness.The prevailing toughening mechanisms can be categorized into two types: intrinsic and extrinsic. Intrinsic toughening operates ahead of a crack tip to suppress its propagation; it is primarily related to plasticity, and thus the primary source of fracture toughness in ductile materials. Recent progress involves introducing high-density nanotwin boundaries in metals to achieve high strength and toughness (11–15). Intrinsic toughening mechanisms are essentially ineffective with brittle materials, e.g., ceramics, which invariably must rely on extrinsic toughening (2). Extrinsic toughening acts mainly behind the crack tip to effectively reduce the crack-driving force by microstructural mechanisms, e.g., crack bridging and meandering and crack surface sliding (16–18). A counterintuitive but successful example is the development of bulk metallic glass (BMG)-based composites, in which a crystalline dendrite second phase is introduced into the BMG matrix to promote the formation of multiple shear bands, leading to a strong and also tough material (3, 9, 16, 19–21). Intrinsic and extrinsic toughening mechanisms are also found to be effective in natural materials (e.g., bones and nacres), which often involve the hierarchical structure and/or a “brick-and-mortar” hybrid microstructure of the material (22–26). Nature-inspired toughening mechanisms are also used to synthesize biomimetic structural materials. Nonetheless, so far, there exists only rather limited success in attaining both strength and toughness, which often involve material-specific, complicated (e.g., growing high density nanotwins), or expensive (e.g., BMG-dendrite composites) synthesis processes and thus are hardly applicable to other materials. A general and feasible mechanism to address the conflict between strength and toughness still remains elusive.Aiming to shed insight on the long-sought strategy addressing the conflict between strength and toughness, we rationally design cellulose-based nanopaper and investigate the dependence of their mechanical properties on constituent cellulose fiber size. Surprisingly, we find that both the strength and toughness of the nanopaper increase simultaneously (40 and 130 times, respectively) as the size of the constituent cellulose building blocks decreases (from a mean diameter of 27 µm to 11 nm). These stimulating results suggest the promising potential toward a new and highly desirable scaling law: the smaller, the stronger and the tougher (Fig. 1). Though the increasing strength as the diameter of cellulose fiber decreases can be attributed to reduced intrinsic defect size, and the dependence is well captured by a continuum fracture mechanics model, our atomistic simulations reveal that facile formation and reformation of strong hydrogen bonding among cellulose chains is the key to the simultaneously increasing toughness. These mechanistic findings that underpin the highly desirable scaling law of mechanical properties suggest a fundamental bottom-up material design strategy generally applicable to other material building blocks as well, and therefore open up abundant opportunities toward a novel class of engineering materials that are both strong and tough.Cellulose is the most abundant biopolymer on Earth and has long been used as the sustainable building block for conventional paper. Cellulose has appealing mechanical properties, with specific modulus [∼100 GPa/(g/cm³)] and specific strength [∼4 GPa/(g/cm³)] higher than most metals and composites, and many ceramics, making it as a promising building block for functional and structural materials (27). Wood fibers are the main natural source of cellulose and have an intrinsically hierarchical structure (Fig. 2). A 20- to ∼50-µm-thick native wood fiber comprises thousands of nanofibrillated cellulose (CNF) fibers (5–50 nm in diameter), each of which can be disintegrated into finer elementary fibrils consisting of cellulose molecular chains (27–36). Cellulose molecule is a linear chain of ringed glucose molecules, with a repeat unit (Fig. S1) comprising two anhydroglucose rings (C₆H₁₀O₅) linked through C–O–C covalent bond. Rich hydroxyl groups in cellulose molecule (six in each repeat unit) enable facile formation of hydrogen bonds, both intrachain and interchain (Fig. 2). Whereas the intrachain hydrogen bonding stabilizes the linkage and results in the linear configuration of the cellulose chain, interchain hydrogen bonding among neighboring cellulose molecules plays a pivotal role in the deformation and failure behaviors of cellulose-based materials.Open in a separate windowFig. 2.Hierarchical structure of wood fibers and the characteristic of cellulose fibrils. Note the rich interchain hydrogen bonds among neighboring cellulose molecular chains.Open in a separate windowFig. S1.Atomic structure of a cellulose chain repeat unit. Note the six hydroxyl groups (red circles) in each repeat unit.In this study, cellulose fibers of different mean diameters [27 μm (native fiber), 28 nm, and 11 nm, respectively] are isolated from wood cell walls using a top-down approach and characterized (SI Text and Figs. S2 and ​andS3).S3). Cellulose nanopaper is made of a highly entangled random network of CNF fibers (Fig. 3A; Materials and Methods). Regular paper made of 27-μm native cellulose fibers with the same mass per area as the nanopaper is also fabricated as the control. The mechanical properties of both the cellulose nanopaper and regular paper are measured according to ASTM Standard D638 (details in SI Text).Open in a separate windowFig. 3.An anomalous scaling law of strength and toughness of cellulose nanopaper. (A) Schematic of cellulose nanopaper, made of a random network of CNF fibers. (Inset) High-resolution transmission electron microscopy (HRTEM) image of an ∼11-nm CNF fiber. (B) Stress–strain curves of cellulose paper made of cellulose fibers of various mean diameters. As the cellulose fiber diameter decreases from micrometer scale to nanometer scale, both tensile strength and ductility of the cellulose paper increases significantly, leading to an anomalous scaling law (C): the smaller, the stronger and the tougher. (D) Reveals that the ultimate tensile strength scales inversely with the square root of cellulose fiber diameter.Open in a separate windowFig. S2.(A) Optical microscope image of native cellulose fiber with a mean diameter of 27 μm. (B) Size distribution histogram. (C) AFM image of cellulose fibers with mean diameters of 28 nm. (D) Size distribution histogram. (E) HRTEM crystalline lattice image of fiber with a mean diameter of 11 nm. (F) Size distribution histogram.Open in a separate windowFig. S3.(A) A picture of a transparent cellulose nanopaper (made of CNF fibers of a mean diameter of 11 nm) on the university logo (Left). A schematic of fibrous nanostructure of the nanopaper is also shown (Right). (B) Optical transmittance of transparent cellulose nanopaper in visible and near-infrared range. (C) AFM image of cellulose nanopaper made of CNF fibers of a mean diameter of 28 nm. (D) AFM image and height scan of cellulose nanopaper made of CNF fibers of a mean diameter of 11 nm, showing rms at 1 × 1-μm scan size is 1.5 nm.     

Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

Leiomodin (Lmod) is a class of potent tandem-G-actin–binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of high-resolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin–Lmod2_162–495 nucleus. The structure contains two actin subunits connected by one Lmod2_162–495 molecule in a non–filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin–binding nucleators.In response to environmental or cellular signals, eukaryotic cells use actin nucleators to convert globular actin monomers (G-actin) into actin oligomers (actin nuclei), which then quickly lead to actin filaments (F-actin). Actin-related protein 2/3 (Arp2/3), formins, and tandem-G-actin–binding proteins are the three classes of known actin nucleators in nonmuscle cells (1–7). Arp2/3-mediated actin nucleation produces branched actin networks, whereas formins and tandem-G-actin–binding nucleators result in long, unbranched actin filaments (1–7). In muscle cells, the specific mechanisms for actin nucleation and maintenance in sarcomeres were poorly understood (8). Recent studies have uncovered actin nucleation activities of the nebulin–N-WASP complex (9) and of formin proteins FHOD3 (10–12), mDia2, DAAM, FMNL1, and FMNL2 (13, 14) in sarcomeres. In particular, leiomodin (Lmod) has been identified as a class of potent tandem-G-actin–binding nucleators in muscle cells (15, 16); Lmod1 is found in smooth muscle of many human tissues, and Lmod2 and Lmod3 are found in cardiac and skeletal muscle (17). Lmod2 knockdown severely compromises sarcomere organization and assembly in muscle cells (15), whereas mutations, deletions (18), or instability (19) in Lmod3 underlies severe, often lethal, human nemaline myopathy.Full-length human Lmod2 is predicted to have 547 residues with two regions of low sequence complexity, an acidic region between residues 97–138 and a polyproline (polyP) region between residues 421–448 (Fig. S1A). Probably because low-complexity regions tend to be intrinsically disordered, previous studies of human Lmod2 used a protein construct that deleted residues 99–130 in the acidic region and residues 421–440 in the polyP region, resulting in Lmod2_1–495 (15, 16). Another study on chicken Lmod2 removed 12 residues in the polyP region (20). In all cases, Lmod2 remained fully functional (15, 16, 20). Therefore, in the present study we focused on the human Lmod2_1–495 construct as previously used (Fig. S1A) (15, 16).Open in a separate windowFig. S1.Lmod sequences. (A) Sequence alignment of human Lmod2_1–547 and Lmod2_1–495. (B) Sequence alignment of human Tmod1 (hTmod1), human Lmod (hLmod), and mouse Lmod (mLmod) isoforms 1–3. The human Lmod2_1–495 sequence is at the top, and the residues that were tested by mutagenesis in this study are indicated by arrows.Human Lmod2_1–495 has three actin-binding sites (15). The first ∼340 residues are about 45% identical to the pointed-end capping protein tropomodulin 1 (Tmod1) (21) and contain a tropomyosin-binding helix (TM-h) and two actin-binding sites [an actin-binding helix (A-h) and a leucine-rich repeat (LRR) domain] (Fig. 1A and Figs. S1 and S2A). The C-terminal ∼150-residue extension of Lmod2 includes two predicted short helices (h1 and h2), a basic segment (B) harboring the nuclear localization sequence (16), and a Wiskott–Aldrich syndrome protein-homology 2 (W) domain (Fig. 1A and Figs. S1 and S2A). Thus, Lmod2 has the capacity to bind three actin subunits and one tropomyosin (15). Unexpectedly, tropomyosin promoted Lmod2-mediated actin nucleation only weakly (15). In sharp contrast, tropomyosin substantially enhanced the binding of Lmod2 to the pointed end of preformed actin filament for controlled elongation in cardiac muscle (16, 20). In the absence of high-resolution structures of the actin–Lmod complex, however, rationalization of these seemingly contradictory findings is difficult.Open in a separate windowFig. 1.Structure of actin–Lmod2. (A) Domain organization of human Lmod2_1–495 and constructs used in this study. (B) Pyrene-based activity assay of Lmod2_1–495 and its various constructs. a.u., arbitrary units. (C) The crystal structure of actin–Lmod2_162–495(B-GS). All residues are visualized except an internal flexible region (residues 339–388) between LRR and polyP, the extreme four N-terminal residues (162–165), and five C-terminal residues (491–495). AMPPNP is shown as ball-and-stick models, and the Mg²⁺ ions are shown as purple spheres. (D) The modeled structure of actin–Lmod2_1–495 in which the actin(A-h)–A-h complex structure was borrowed from the Tmod1 structure (PDB ID code: 4PKG) and combined with our crystal structure of actin–Lmod2_162–495(B-GS). See also Movies S1–S3.Open in a separate windowFig. S2.Structure of actin–Lmod2_162–495(B-GS). (A) Comparison of domain organization of human Lmod2_1–495 and Tmod1. (B) The structure of actin–Lmod2_162–495(B-GS) as observed in the asymmetric unit. It contains two actin subunits (in green and cyan) bound with one Lmod2_162–495(B-GS) (in magenta) and one extra LRR domain from degradation (in yellow). (C) The modeled actin–Tmod1 structure is superimposed on subunits n+1 and n of the pointed end of the actin filament. (D) Superposition of our actin–Lmod2_162–495(B-GS) structure with the modeled actin–Tmod1 structure at the actin(LRR)–LRR. Lmod2 is shown in magenta, and the interacting actin(LRR) and actin(W) are in green and cyan, respectively. Tmod1 is in orange, and its interacting actin(A-h) and actin(LRR) are in blue and pink, respectively.Historically study of the crystallographic structure of the complexes of actin nucleators with oligomeric actin or of F-actin–binding proteins with F-actin was difficult because actin dimers and trimers are kinetically unstable, and actin tetramers rapidly polymerize into F-actin that is refractory to crystallization (22). Indeed, although Arp2/3 has been subjected to intensive structural studies (23–25), the crystal structure of the actin–Arp2/3 complex has eluded investigation so far. Before our study (26), the only available crystal structure of this kind was the yeast formin Bni1p FH2 domain that binds to two crystallographically related tetramethylrhodamine-modified actin (TMR–actin) subunits in a pseudo short-pitch fashion (27). However, the large size of TMR likely interferes with its interaction with actin and with actin function, thus limiting the use of TMR–actin in investigations of crystal structure.To enable crystallographic studies of biological actin complexes, our group recently has developed a double-mutant strategy in which actin-binding proteins and two types of nonpolymerizable actin mutants are combined to form stable complexes amenable to crystallization (26). This strategy made possible the rapid determination of the first two crystal structures of oligomeric actin with tandem-G-actin–binding nucleator complexes: a mammalian nucleator Cordon-bleu (Cobl) (26) and a bacterial effector Vibrio parahaemolyticus protein L (VopL) (28). Importantly, the observed non–filament-like conformation in actin–Cobl and the filament-like conformation in actin–VopL together suggest that both types of conformation are fully accessible to an actin complex obtained via the double-mutant strategy; thus the observed structure most likely reflects its native functional state.Here we report the crystal structure of the actin–Lmod2 nucleus and complementary functional studies. Our data not only unraveled the atomic mechanisms of Lmod’s essential functions in muscle cells but also suggested a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin–binding nucleators.     

Emerging single-phase state in small manganite nanodisks

In complex oxides systems such as manganites, electronic phase separation (EPS), a consequence of strong electronic correlations, dictates the exotic electrical and magnetic properties of these materials. A fundamental yet unresolved issue is how EPS responds to spatial confinement; will EPS just scale with size of an object, or will the one of the phases be pinned? Understanding this behavior is critical for future oxides electronics and spintronics because scaling down of the system is unavoidable for these applications. In this work, we use La_0.325Pr_0.3Ca_0.375MnO₃ (LPCMO) single crystalline disks to study the effect of spatial confinement on EPS. The EPS state featuring coexistence of ferromagnetic metallic and charge order insulating phases appears to be the low-temperature ground state in bulk, thin films, and large disks, a previously unidentified ground state (i.e., a single ferromagnetic phase state emerges in smaller disks). The critical size is between 500 nm and 800 nm, which is similar to the characteristic length scale of EPS in the LPCMO system. The ability to create a pure ferromagnetic phase in manganite nanodisks is highly desirable for spintronic applications.Owing to strong coupling between spin, charge, orbital, and lattice (1, 2), different electronic phases often coexist spatially in strongly correlated materials known as electronic phase separation (EPS) (3, 4). For colossal magnetoresistance (CMR) manganites, EPS has been observed to have strong influence on the global magnetic and transport properties (5, 6). Regarding the physical origin of EPS, it has been shown theoretically that quenched disorder can lead to inhomogeneous states in manganites (1, 3, 7). Once long-range effects such as coulombic forces (8), cooperative oxygen octahedral distortions (9), or strain effects (10) are included, calculations show infinitesimal disorder (8, 11) or even no explicit disorder (10) may lead to EPS. Within a phenomenological Ginzburg–Landau theory, it has been shown that EPS is intrinsic in complex systems as a thermodynamic equilibrium state (12).Although the details of the origin of the EPS remain as a matter of dispute, its very existence as a new form of electronic state has been well accepted. The length scale of the EPS has been observed to vary widely from nanometers to micrometers depending on many parameters that can affect the competition between different electronic phases (13–20). It is thus of great interest to examine whether the EPS state still exists as the system is scaled down, especially when the spatial dimension of the system is smaller than the length scale of the EPS domains.In this work, we use La_0.325Pr_0.3Ca_0.375MnO₃ (LPCMO) as a prototype system to show a spatial confinement-induced transition from the EPS state to a single ferromagnetic phase state. The LPCMO system is chosen because of its well-known large length scale of EPS domains (approximately a micrometer) (21), which allows us to conveniently fabricate LPCMO epitaxial thin films into disks with diameters that are smaller than the EPS domain size. In LPCMO bulk (21) and thin films (6, 22), the EPS state was observed to be the low-temperature ground state. Using magnetic force microscope, we observe that the EPS state remains to be the ground state in disks with the size of 800 nm in diameter or larger but vanishes in the 500-nm-diameter disks whose size is distinctly smaller than the characteristic length scale of the EPS domains. In the 500-nm disks, only the ferromagnetic phase can be observed at all temperatures below Curie temperature T_c, indicating that the system is in a single-phase state rather than a EPS state. Our results further indicate that the large length scale EPS in the LPCMO system does not cost extra Coulomb energy, which otherwise should lead to a scaling down of EPS with decreasing size of the LPCMO disks (23, 24).LPCMO films with 60-nm thickness were epitaxially grown on SrTiO₃(001) substrates by pulsed-laser deposition. The substrates were kept at 780 °C in oxygen atmosphere of 5 × 10⁻³ millibars during growth. Unit cell by unit cell growth was achieved as indicated by oscillations of intensity of reflection high-energy electron diffraction (RHEED). The films were postannealed to 950 °C for 3 h in flowing oxygen to reduce oxygen vacancy and make sure that the films have the same magnetic properties as the bulk. The LPCMO disks with diameters from 500 nm to 20 μm were fabricated from the epitaxial thin films by electron beam lithography with a negative tone resist (for details, see the sample fabrication method and Fig. S1 in the Supporting Information). Magnetic properties of the LPCMO disk arrays were carried out using superconducting quantum interference device (SQUID) and magnetic force microscope (MFM) measurements.Open in a separate windowFig. S1.(A and B) Schematics of LPCMO disks samples for magnetic property measurement (A) and MFM mapping (B). (C) Optical microscopic image of LPCMO disk array with specific diameter. (D) SEM image of LPCMO disk sample for MFM imaging.A distinct signature of the EPS state in the LPCMO system is the thermal hysteresis for temperature-dependent magnetic and transport properties. Fig. 1 A–D shows temperature dependent magnetic properties of LPCMO disks with different diameters. To enhance the measuring signal for SQUID, we fabricate disk arrays for each selected diameter (the optical microscopic image shown in Fig. 1B, Inset for the 1-μm disk array). Fig. 1 A–D shows temperature-dependent magnetization measured under 1,000 Oe in-plane field for 7-μm, 1-μm, 800-nm, and 500-nm disk arrays, respectively. Thermal hysteresis can be observed for disk arrays with size down to 800 nm, reflecting the fact that ferromagnetic metallic (FMM) and charge order insulating (COI) phases coexist during the first-order phase transition (7, 25). For the 500-nm disk array, however, no thermal hysteresis can be observed. This observation implies that the EPS state may no longer exist in the system (7, 26).Open in a separate windowFig. 1.Temperature dependence of magnetization (black lines for cooling and red lines for warming) under 1,000 Oe (A–D) and initial magnetization (red lines) and hysteresis loop (black lines) (E–H) at 5 K of arrays of LPCMO disks with sizes of 7 μm (A and E), 1 μm (B and F), 800 nm (C and G), and 500 nm (D and H) in diameter and an area of 3 mm × 3 mm. (B, Inset) The optical microscopic image of d = 1 μm array. (C and D, Insets) Zoomed-in M vs. T loop around the thermal hysteresis region.The lack of EPS state in the 500-nm disk array is supported by the field-dependent magnetization measurements. Fig. 1 E–H shows in-plane initial magnetization curves and magnetic hysteresis loops (M-H loops) for the disk arrays measured at 5 K after zero-field cooling. For 800-nm or larger disk arrays, there is a clear difference between the initial magnetization curves and the corresponding M-H loops due to the coexistence of FMM and COI phases. When the magnetic field is applied from the initial state, the magnetization of the FMM phase first quickly aligns along the field direction, leading to the low field fast rise of the initial magnetization curve. With increasing field, the COI phase is melted and transits into the FMM phase. Once transited, the FMM phase will mostly stay even if the field is reduced, giving rise to the difference between initial magnetization curve and the M-H loop. The difference, however, becomes smaller with decreasing size. For the 500-nm disk array, the initial magnetization curve and the M-H loop virtually superimpose each other, indicating no melting of COI phase occurs. Both the temperature- and field-dependent magnetization measurements show a transition from the EPS state to a single FMM state with decreasing size of the disk, and the critical size should be between 500 nm and 800 nm.The transition from the EPS state to a single FMM state can be seen in MFM images shown in Fig. 2 (for MFM imaging details, see micromagnetic mapping method in Supporting Information). Fig. 2A shows morphological appearance of LPCMO disks with different sizes acquired by atomic force microscope (AFM). Fig. 2 B–D shows the corresponding MFM images of the LPCMO disks acquired at different temperatures under a perpendicular magnetic field of 1T. Here, the perpendicular magnetic field is applied to yield some perpendicular magnetization components for MFM imaging because the easy magnetization axis is in the plane. In the present color scale, the contrast below zero (red or black) represents FMM phase, whereas the contrast above zero (green or blue) represents nonferromagnetic phase [i.e., COI phase based on previous knowledge of the LPCMO system (21, 22)]. Apparently, except the 500-nm disk, all other disks show distinct features of the EPS state (i.e., the coexistence of the FMM and COI phases). Although the portion of FMM phase increases noticeably with decreasing temperature, the system stays in the EPS state even at 10 K. The typical length scale of the EPS domains is around a micrometer, which is consistent with previous reports (21, 27).Open in a separate windowFig. 2.(A) AFM images of LPCMO disks with sizes of 500 nm, 1 μm, 2 μm, 3.8 μm, 5 μm, and 7 μm in diameter. (B–D) The MFM images of LPCMO disks under 1T field (external magnetic field direction is pointing perpendicularly to the sample surface plane) taken at 10 K (B), 100 K (C), and 180 K (D). The sizes of disks in MFM images are adjusted and corrected to have same scales for each size with the help of scanning electron microscope (SEM) images (shown in Fig. S2) and dash lines show the approximate physical boundary of disks. The negative value in MFM image indicates attractive force and positive value indicates repulsive force.In stark contrast to the larger disks, the 500-nm disk does not exhibit any features of EPS in Fig. 2. Instead, the whole disk is in a ferromagnetic phase with a magnetization profile peaking in the center. To ensure that the EPS state is not diminished by the magnetic field applied during MFM imaging, we took MFM images of the 500-nm disk at 10 K under different perpendicular magnetic fields from 0T to 1T, as shown in Fig. 3A. At 0T, signals with opposite sign can only be seen on two sides of the disk along the marked line (MFM images of 4 disks shown in Fig. S3). This pattern is a typical MFM image for an in-plane ferromagnetic single domain, because only the two ends of an in-plane magnetic dipole yield perpendicular field gradient (with opposite signs) for the MFM tip to detect. Once a perpendicular field of 0.15T is applied, the in-plane magnetization is driven out of plane, leading to a center peaked MFM contour. The MFM signal increases with increasing field, as shown in Fig. 3B by the marked line profiles extracted from Fig. 3A.Open in a separate windowFig. 3.(A) MFM images of 500-nm disks at 10 K under different magnetic field. The lines show the path the line profile extracted. (B) Line profiles extracted from MFM images partly shown in Fig. 3A. (C) Simulated results corresponding to line-profiles in B. (D and E) The simulated Z-component of stray field 100 nm above sample disks and magnetic structure of 500-nm disks under a different magnetic field. In E, the direction and size of arrows show the direction and relative value of in-plane component of magnetization, and the color of disk indicates the value of Z-component of magnetization presented by ratio of Z-component to total magnetization, as shown in the color bar.Open in a separate windowFig. S3.MFM images of 500-nm disks taken at 10 K under zero field after zero-field cooling.The field-dependent behavior of the MFM contrast of the 500-nm disk is in qualitative agreement with micromagnetic simulations. Based on the MFM observation, the 500-nm disk is in an in-plane, single-domain state. Using this model as input, we performed micromagnetic simulation and obtained the Z-component of magnetic stray field distribution at 100 nm above sample surface (Fig. 3D; for details, see the micromagnetic simulation method in the Supporting Information), which is virtually the signal detected by MFM tip. The corresponding magnetic structures under different magnetic fields are shown in Fig. 3E. The marked line profiles extracted from simulation (Fig. 3D) are shown in Fig. 3C alongside with the experimental MFM line profiles (Fig. 3B). The subtle differences between Fig. 3B and Fig. 3C are likely caused by the fact that experimental MFM images are convoluted from signals of both the LPCMO disks and the MFM tips (∼100 nm in size). The consistency of MFM images and simulation confirms that the 500-nm disk is in a ferromagnetic single-domain state with an in-plane easy magnetization axis.Finally, we show that the 500-nm disk is in a single FMM state at all temperatures. Fig. 4 shows MFM images of the 500-nm disk acquired every 20 K, from 20 K to 200 K under 1,000 Oe. Other than the center-peaked FMM phase, no traces of COI phase can be observed. The MFM signal decreases with increasing temperature, which is consistent with the behavior of the temperature-dependent magnetization shown in Fig. 1. Considering the fact that we have never observed pure COI phase in the 500-nm disks, we believe this phenomenon may be caused by the existence of the ferromagnetic metallic edge state in the LPCMO system (22), which assists the 500-nm disk to be in pure ferromagnetic state when a single state is energetically preferred in the 500-nm disk.Open in a separate windowFig. 4.MFM images of 500-nm disks taken every 20 K, from 20 K to 200 K.In summary, we discovered a spatial confinement-induced transition from a EPS state featuring coexistence of FMM and COI phases to a single FMM state in the LPCMO system. The critical size for the transition is between 500 nm and 800 nm, which is similar to the characteristic length scale of the EPS state in the LPCMO system. Combining the MFM data and the micromagnetic simulation, we conclude that the 500-nm LPCMO disk is in a single-domain ferromagnetic state at all temperatures below T_c. A similar conclusion can be reached for 300-nm LPCMO disks (shown in Fig. S4), although it needs to be studied further whether a new state would emerge if the disk size becomes a few tens of nanometers or smaller. Our work opens a way to control EPS without external field or introducing strain and disorder, which is potentially useful to design electronic and spintronic devices in complex oxides systems.Open in a separate windowFig. S4.MFM images of 300-nm disks taken at different temperatures and magnetic fields.     

Suburbanization,estrogen contamination,and sex ratio in wild amphibian populations

Research on endocrine disruption in frog populations, such as shifts in sex ratios and feminization of males, has predominantly focused on agricultural pesticides. Recent evidence suggests that suburban landscapes harbor amphibian populations exhibiting similar levels of endocrine disruption; however the endocrine disrupting chemical (EDC) sources are unknown. Here, we show that sex ratios of metamorphosing frogs become increasingly female-dominated along a suburbanization gradient. We further show that suburban ponds are frequently contaminated by the classical estrogen estrone and a variety of EDCs produced by plants (phytoestrogens), and that the diversity of organic EDCs is correlated with the extent of developed land use and cultivated lawn and gardens around a pond. Our work also raises the possibility that trace-element contamination associated with human land use around suburban ponds may be contributing to the estrogenic load within suburban freshwaters and constitutes another source of estrogenic exposure for wildlife. These data suggest novel, unexplored pathways of EDC contamination in human-altered environments. In particular, we propose that vegetation changes associated with suburban neighborhoods (e.g., from forests to lawns and ornamental plants) increase the distribution of phytoestrogens in surface waters. The result of frog sex ratios varying as a function of human land use implicates a role for environmental modulation of sexual differentiation in amphibians, which are assumed to only have genetic sex determination. Overall, we show that endocrine disruption is widespread in suburban frog populations and that the causes are likely diverse.Amphibians are a model animal system for studying endocrine disruption in nature (1). Wild amphibians show evidence (such as testicular oocytes) of feminizing endocrine disruption in human-modified environments (2–7). With few exceptions (6, 7), research on amphibians has focused on agricultural landscapes (2, 4, 5) and the effects of pesticides such as atrazine (2, 8). In parallel, endocrine disruption in fish has been observed in the context of chemical gradients associated with sources of endocrine disrupting chemicals (EDCs) such as municipal wastewater treatment facility discharges (9–11). These studies imply that endocrine disruption in aquatic systems is caused predominantly by industrial- or municipal-scale activities. However, recent work has revealed that suburban neighborhoods also may be hotspots for endocrine disruption, and green frogs [Rana (Lithobates) clamitans] from “backyard ponds” have been reported (6, 7) to exhibit higher frequencies of feminizing endocrine disruption (testicular oocytes in males) than frogs from other land uses (forest, agricultural, and urban). Endocrine disruption appears to be a ubiquitous phenomenon in suburban ponds, with testicular oocytes observed in male green frogs at all 34 suburban ponds studied to date (6, 7). The causes of endocrine disruption in suburban landscapes have not yet been studied. Although some sources of EDCs are obvious, as with agricultural landscapes and wastewater treatment facilities, EDC sources in suburban neighborhoods are less evident. Suburban ponds may be influenced by a variety of potential sources relative to undeveloped forested ponds (Fig. 1).Open in a separate windowFig. 1.Conceptual framework of potential endocrine-disrupting chemical sources in a forested pond (Left) and a developed suburban pond (Right). Sources include (a) lawn plants or forest vegetation (e.g., phytoestrogens), (b) atmospheric transport of pollutants (e.g., alkylphenol in precipitation), (c) biogenic hormone inputs from animals and plants (e.g., estrone excretion), (d) wastewater from domestic sewer lines or septic tanks (e.g., pharmaceuticals), (e) runoff from roads (e.g., antimony), (f) runoff from lawns (e.g., pesticides and fertilizers), (g) runoff from buildings (e.g., copper), and (h) altered surface water/groundwater redox states resulting in mobilization of trace elements (e.g., manganese). Figure courtesy of Corrine Edwards (Illustrator).We compare ponds in landscapes ranging from highly developed suburban backyards to undeveloped forests (Fig. S1), and focused on sex ratios of green frogs (R. clamitans), which inhabit both environments and were previously evaluated for evidence of endocrine disruption (6, 7). Offspring sex ratio is an established metric of endocrine disruption (8, 12) and is ecologically relevant (13). Here, we address whether sex ratio in cohorts of metamorphosing green frogs varies with landscape structure surrounding ponds.Open in a separate windowFig. S1.Location of the study area in southern Connecticut (Inset) and the locations of forested and suburban ponds. All ponds are within 35 km of New Haven, Connecticut. Suburban ponds are broken down into sewer and septic sites. The background colors indicate land cover classifications. Green colors represent forested cover, yellow colors represent suburban vegetation (predominantly lawn and turf grass cover), and red colors represent developed land use (e.g., impervious surfaces).Human modification of the environment alters chemical sources and exposure pathways, providing a link between landscape structure and frog developmental biology. In parallel with frog sampling, we tested water from ponds to develop a profile of EDCs (both organic and inorganic) across suburban and forested landscapes. We hypothesized that the landscape structure surrounding study ponds would be related to patterns of detected EDCs, and explored the extent to which proportions of different land cover categories (Fig. 2) were associated with metamorph sex ratios and pond EDCs. Forested ponds are surrounded entirely by undeveloped forest vegetation. The landscape surrounding suburban ponds is composed of a heterogeneous mix of human residences, roads, sidewalks, and other structures (hereafter denoted “Developed Land Use”) as well as lawns and other plantings (hereafter “Suburban Vegetation”) in addition to small amounts of natural vegetation.Open in a separate windowFig. 2.Examples of an undeveloped forested pond (Left) and a developed suburban pond (Right). Images at top illustrate differences in land cover between the two categories. Forested ponds were composed of 100% forest (coniferous, deciduous, mixed forest, scrub shrub, and forested wetland land-cover types). Suburban ponds were a composite of at least 70% Suburban Vegetation (“Open Spaces Developed;” lawns and landscape plantings) and Developed Land Use (composed of “High-Intensity Developed,” “Medium-Intensity Developed,” and “Low-Intensity Developed”).Here we show that sex ratios of frog metamorphs are sensitive to suburbanization intensity surrounding ponds, and also that increasing suburbanization is correlated with higher diversity of detected EDCs including biogenic steroidal hormones, phytoestrogens, and metalloestrogens. Without discounting potential contributions of pesticides or pharmaceuticals, which have been the focus of past studies on frog endocrine disruption and environmental EDCs (2, 3), results presented here expand our understanding of endocrine disruption and suggest that human land uses expose wildlife to EDCs via previously unexplored pathways (e.g., vegetation biosynthesis and trace element contamination). Our results connect landscape structure with amphibian sex ratios, implying environmental modulation of sex differentiation in a taxon typically presumed to have only genetic sex determination (14).     

Dietary options and behavior suggested by plant biomarker evidence in an early human habitat

The availability of plants and freshwater shapes the diets and social behavior of chimpanzees, our closest living relative. However, limited evidence about the spatial relationships shared between ancestral human (hominin) remains, edible resources, refuge, and freshwater leaves the influence of local resources on our species’ evolution open to debate. Exceptionally well-preserved organic geochemical fossils—biomarkers—preserved in a soil horizon resolve different plant communities at meter scales across a contiguous 25,000 m² archaeological land surface at Olduvai Gorge from about 2 Ma. Biomarkers reveal hominins had access to aquatic plants and protective woods in a patchwork landscape, which included a spring-fed wetland near a woodland that both were surrounded by open grassland. Numerous cut-marked animal bones are located within the wooded area, and within meters of wetland vegetation delineated by biomarkers for ferns and sedges. Taken together, plant biomarkers, clustered bone debris, and hominin remains define a clear spatial pattern that places animal butchery amid the refuge of an isolated forest patch and near freshwater with diverse edible resources.Spatial patterns in archaeological remains provide a glimpse into the lives of our ancestors (1–5). Although many early hominin environments are interpreted as grassy or open woodlands (6–8), fossil bones and plant remains are rarely preserved together in the same settings. As a result, associated landscape reconstructions commonly lack coexisting fossil evidence for hominins and local-scale habitat (microhabitat) that defined the distribution of plant foods, refuge, and water (7). This problem is exacerbated by the discontinuous nature and low time resolution often available across ancient soil (paleosol) horizons, including hominin archaeological localities. One notable exception is well-time-correlated 1.8-million-y-old paleosol horizons exposed at Olduvai Gorge. Associated horizons contain exceptionally preserved plant biomarkers along with many artifacts and fossilized bones. Plant biomarkers, which previously revealed temporal patterns in vegetation and water (8), are well preserved in the paleosol horizon and document plant-type spatial distributions that provide an ecosystem context (9, 10) for resources that likely affected the diets and behavior of hominin inhabitants.Plant biomarkers are delivered by litter to soils and can distinguish plant functional type differences in standing biomass over scales of 1–1,000 m² (11). Trees, grasses, and other terrestrial plants produce leaf waxes that include long-chain n-alkanes such as hentriacontane (nC₃₁), whereas aquatic plants and phytoplankton produce midchain homologs (e.g., nC₂₃) (12, 13). The ratio of shorter- versus long-chain n-alkane abundances distinguish relative organic matter inputs from aquatic versus terrestrial plants to sediments (13):P_aq = (nC₂₃ + nC₂₅)/(nC₂₃ + nC₂₅ + nC₂₉ + nC₃₁).Sedges and ferns are prolific in many tropical ecosystems (14). These plants both have variable and therefore nondiagnostic n-alkane profiles. However, sedges produce distinctive phenolic compounds [e.g., 5-n-tricosylresorcinol (ⁿR₂₃)] and ferns produce distinctive midchain diols [e.g., 1,13-dotriacontanediol (C₃₂-diol)] (SI Discussion).Lignin monomers provide evidence for woody and nonwoody plants. This refractory biopolymer occurs in both leaves and wood, serves as a structural tissue, and accounts for up to half of the total organic carbon in modern vegetation (11). Lignin is composed of three phenolic monomer types that show distinctive distributions in woody and herbaceous plant tissues. Woody tissues from dicotyledonous trees and shrubs contain syringyl (S) and vanillyl (V) phenols (12), whereas cinnamyl (C) phenols are exclusively found in herbaceous tissues (12). The relative abundance of C versus V phenols (C/V) is widely used to distinguish between woody and herbaceous inputs to sedimentary and soil organic matter (15).Plant biomarker ¹³C/¹²C ratios (expressed as δ¹³C values) are sensitive indicators of community composition, ecosystem structure, and climate conditions (8). Most woody plants and forbs in eastern Africa use C₃ photosynthesis (6), whereas arid-adapted grasses use C₄ photosynthesis (8, 14). These two pathways discriminate differently against ¹³C during photosynthesis, resulting in characteristic δ¹³C values for leaf waxes derived from C₃ (about –36.0‰) and C₄ (–21.0‰) plants (16). Carbon isotopic abundances of phenolic monomers of lignin amplify the C₃–C₄ difference and range between ca. –34.0‰ (C₃) and –14.0‰ (C₄) in tropical ecosystems (15). Terrestrial C₃ plant δ¹³C values decrease with increased exposure to water, respired CO₂, and shade (8), with lowest values observed in moist regions with dense canopy (17). Although concentration and δ¹³C values of atmospheric CO₂ can affect C₃ plant δ¹³C values (17), this influence is not relevant to our work here, which focuses on a single time window (SI Discussion). The large differences in leaf-wax δ¹³C values between closed C₃ forest to open C₄ grassland are consistent with soil organic carbon isotope gradients across canopy-shaded ground surfaces (6) and serve as a quantitative proxy for woody cover (f_woody) in savannas (8).As is observed for nonhuman primates, hominin dietary choices were likely shaped by ecosystem characteristics over habitat scales of 1–1,000 m² (3–5). To evaluate plant distributions at this small spatial scale (9), we excavated 71 paleosol samples from close-correlated trenches across a ∼25,000-m² area that included FLK Zinjanthropus Level 22 (FLK Zinj) at Olduvai Gorge (Fig. 1). Recent excavations (18–21) at multiple trenches at four sites (FLK^NN, FLK^N, FLK, and FLK^S, Fig. 1D) exposed a traceable thin (5–50 cm), waxy green to olive-brown clay horizon developed by pedogenic alterations of playa lake margin alluvium (22). Weak stratification and irregular redox stains suggest initial soil development occurred during playa lake regression (18, 22), around 1.848 Ma (ref. 23 and SI Discussion). To date, craniodental remains from at least three hominin individuals (18–20), including preadolescent early Homo and Paranthropus boisei, were recovered from FLK Zinj. Fossils and artifacts embedded in the paleosol horizon often protrude into an overlying airfall tuff (18, 19), which suggests fossil remains were catastrophically buried in situ under volcanic ash. Rapid burial likely fostered the exceptional preservation of both macrofossils (10) and plant biomarkers across the FLK Zinj land surface.Open in a separate windowFig. 1.Location and map of FLK Zinj paleosol excavations. (A and B) Location of FLK Zinj as referenced to reconstructed depositional environments at Olduvai Gorge during the early Pleistocene (18, 22) and the modern gorge walls. The perennial lake contained shallow saline–alkaline waters that frequently flooded the surrounding playa margin (i.e., floodplain) flats. (C) Outline of FLK Zinj paleosol excavation sites used for our spatial biomarker reconstructions. (D) Concentric (5 m) gridded distribution map of FLK Zinj paleosol excavations relative to previous archaeological trenches (18–21). Major aggregate complexes (FLK^NN, FLK^N, FLK, and FLK^S) are color-coded to show excavation-site associations.Plant biomarker signatures reveal distinct types of vegetation juxtaposed across the FLK Zinj land surface (Figs. 2–4 and Fig. S1). In the northwest, FLK^NN trenches show high nC₂₃ δ¹³C values (Fig. 2B) as well as high C/V and P_aq values (Figs. 3 and ​and4A).4A). They indicate floating or submerged aquatic plants (macrophytes) in standing freshwater (13), a finding that is consistent with nearby low-temperature freshwater carbonates (tufa), interpreted to be deposited from spring waters (22). Adjacent FLK^N trenches have lower P_aq values (Fig. 4A) with occurrences of fern-derived C₃₂-diol and sedge-derived ⁿR₂₃ (Fig. 2 C and D). These biomarker distributions indicate an abrupt (around 10 m) transition from aquatic to wetland vegetation. Less than 100 m away (Fig. 1C), low nC₃₁ δ¹³C values (Fig. 2A) and low C/V and very low P_aq values (Figs. 3 and ​and4A)4A) collectively indicate dense woody cover (Fig. 4B). In the farthest southeastern (FLK^S) trenches, high C/V values and high δ¹³C values for C lignin phenols (Fig. 3) indicate open C₄ grassland.Open in a separate windowFig. 2.Spatial distributions and δ¹³C values for plant biomarkers across FLK Zinj. Measured and modeled δ¹³C values (large and smaller circles, respectively) are shown for (A) nC₃₁ from terrestrial plants, (B) nC₂₃ from (semi)aquatic plants, (C) C₃₂-diol from ferns, and (D) ⁿR₂₃ from sedges (see refs. 12 and 13 and SI Discussion). Modeled values [inverse distance-weighted (9)] account for spatial autocorrelation (15-m radius) in standing biomass (35) over scales of soil organic matter accumulation (11). Black dots represent paleosols with insufficient plant biomarker concentrations for isotopic analysis.Open in a separate windowFig. 3.Molecular and isotopic signatures for lignin phenols across FLK Zinj. Bivariate plots are shown for diagnostic lignin compositional parameters (see refs. 12 and 15 and Fig. 1C). Symbols are colored according to respective δ¹³C values for the C lignin phenol, p-coumaric acid. FLK symbols are uncolored due to insufficient p-coumaric acid concentrations for isotopic analysis. Representative lignin compositional parameters (12, 15) are shown for monocotyledonous herbaceous tissues (G), dicotyledonous herbaceous tissues (H), cryptogams (N), and dicotyledonous woody tissues (W).Open in a separate windowFig. 4.Spatial relationships shared between local plant resources and hominin remains. Measured and modeled values (large and smaller circles, respectively) are shown for (A) P_aq (13) and (B) f_woody (8). Modeled values [inverse distance-weighted (9)] account for spatial autocorrelation (15-m radius) in standing biomass (35) over scales of soil organic matter accumulation (11). (C) Kernel density map of cut-marked bones (18–21) across the FLK Zinj land surface (Fig. S4). High estimator values indicate hotspots of hominin butchery (Fig. S5). A shaded rectangle captures the area (ca. 0.68 probability mass) with highest cut-marked bone densities and is shown in A and B for reference.Open in a separate windowFig. S1.Total ion chromatograms for saturated hydrocarbons in representative paleosols at (A) FLK^NN, (B) FLK^N, (C) FLK, and (D) FLK^S. C₂₃, tricosane; C₂₅, pentacosane; C₂₉ nonacosane; C₃₁, hentriacontane.Biomarkers define a heterogeneous landscape at Olduvai and suggest an influence of local resources on hominin diets and behavior. It is recognized (2, 24–26) that early Homo species and P. boisei had similar physiological characteristics. These similarities in physical attributes suggest behavioral differences were what allowed for overlapping ranges and local coexistence (sympatry) of both hominins. For instance, differences in seasonal subsistence strategies or different behavior during periods of drought and limited food could have reduced local hominin competition and fostered diversification via niche specialization (27–29).Physical and isotopic properties of fossil teeth indicate P. boisei was more water-dependent [low enamel δ¹⁸O values (24)] and consumed larger quantities of abrasive, ¹³C-enriched foodstuffs [flat-worn surfaces (25) and high enamel δ¹³C values (26)] than coexisting early Homo species. Although ¹³C-enriched enamel is commonly attributed to consumption of C₄ grasses or meat from grazers (14), this was not likely, because P. boisei craniodental features are inconsistent with contemporary gramnivores (24, 25) or extensive uncooked flesh mastication (26). Numerous scholars have proposed the nutritious underground storage organs (USOs) of C₄ sedges were a staple of hominin diets (14, 24, 26, 27). Consistent with this suggestion, occurrences of ⁿR₂₃ attest to the presence of sedges at FLK^NN and FLK^N (Fig. 2D). However, the low δ¹³C values measured for ⁿR₂₃ at these same sites (Fig. 2D and Fig. S2) indicate C₃ photosynthesis (12, 16), a trait common in modern sedges that grow in alkaline wetlands and lakes (30) (Fig. S3). Thus, biomarker signatures support the presence of C₃ sedges in the wetland area of FLK Zinj.Open in a separate windowFig. S2.Total ion chromatogram [TIC (A)] and selected ion chromatograms for derivatized 5-n-alkylresorcinols [m/z 268 (●)] and midchain diols [m/z 369 (○)] from a representative paleosol at FLK^N. Also shown are δ¹³C values for homologous (B) 5-n-alkylresorcinols and (C) midchain diols. C₃₂-diol, dotriacontanediol; ⁿR₂₃, tricosylresorcinol.Open in a separate windowFig. S3.Summary phyogenetic consensus tree of Cyperaceae (sedges) based on nucleotide (rcbL and ETS1f) sequence data (50–54, 95, 96). Important taxonomic distinctions discussed in SI Discussion, Fern Alkyldiols are shown explicitly. Triangle-enclosed digits represent the number of additional branches at different levels of taxonomic classification. CEFA, Cypereae Eleocharideae Fuireneae Abildgaardieae; CSD, Cariceae Scirpeae Dulichieae.Alternative foodstuffs with abrasive, ¹³C-enriched biomass include seedless vascular plants (cryptogams), such as ferns and lycophytes [e.g., quillworts (27, 30)]. Ferns are widely distributed throughout eastern Africa in moist and shaded microhabitats (31) and are often found near dependable sources of drinking water (32). Today, ferns serve as a dietary resource for humans and nonhuman primates alike (27), and fiddlehead consumption is consistent with the inferred digestive physiology [salivary proteins (33)] and the microwear on molars (34) of P. boisei in eastern Africa (25, 26). Ferns were present at FLK^N, based on measurements of C₃₂-diol (Fig. 2D). Further, the high δ¹³C values measured for these compounds are consistent with significant fern consumption by P. boisei at Olduvai Gorge.Ferns and grasses were not the only plant foods present during the time window documented by FLK Zinj. Further, the exclusive reliance on a couple of dietary resources was improbable for P. boisei, because its fossils occur in diverse localities (24–26). Aquatic plants are an additional candidate substrate, as evidenced by high P_aq values at FLK^NN and FLK^N (Fig. 4A). Floating and submerged plants proliferate in wetlands throughout eastern Africa today (13, 14), and many produce nutritious leaves and rootstock all year long (27, 28). Although C₄ photosynthesis is rare among modern macrophytes (30), they can assimilate bicarbonate under alkaline conditions, which results in C₄-like isotope signatures in their biomass (30). Their leaf waxes, such as nC₂₃ (13), are both present and carry ¹³C-enriched signatures at FLK^NN and FLK^N (Fig. 2B). It is also likely that aquatic macrophytes sustained invertebrates and fish with comparably ¹³C-enriched biomass, as they do in modern systems (14), and we suggest aquatic animal foods could have been important in P. boisei diets (27, 28).Biomarkers across the FLK Zinj soil horizon resolve clear patterns in the distribution of plants and water and suggest critical resources that shaped hominin existence at Olduvai Gorge. The behavioral implications of local conditions require understanding of regional climate and biogeography (3–5, 7), because hominin species likely had home ranges much larger than the extent of excavated sites at FLK Zinj. Lake sediments at Olduvai Gorge include numerous stacked tuffs with precise radiometric age constraints (23). These tephrostratigraphic correlations (21) tie the FLK Zinj landscape horizon to published records of plant biomarkers in lake sediments that record climate cycles and catchment-scale variations in ecology. Correlative lake sediment data indicate the wet and wooded microhabitats of FLK Zinj sat within a catchment dominated by arid C₄ grassland (8). Under similarly arid conditions today, only a small fraction of landscape area (ca. 0.05) occurs within 5 km of either forest or standing freshwater (35). Given a paucity of shaded refuge and potable water in the catchment, the concentration of hominin butchery debris (18–21) exclusively within the forest microhabitat and adjacent to a freshwater wetland (Fig. 4) is notable. We suggest the spatial patterns defined by both macro- and molecular fossils reflect hominins engaged in social transport of resources (1–5), such as bringing animal carcasses and freshwater-sourced foods from surrounding grassy or wetland habitats to a wooded patch that provided both physical protection and access to water.     

Nectar vs. pollen loading affects the tradeoff between flight stability and maneuverability in bumblebees

Bumblebee foragers spend a significant portion of their lives transporting nectar and pollen, often carrying loads equivalent to more than half their body mass. Whereas nectar is stored in the abdomen near the bee’s center of mass, pollen is carried on the hind legs, farther from the center of mass. We examine how load position changes the rotational moment of inertia in bumblebees and whether this affects their flight maneuverability and/or stability. We applied simulated pollen or nectar loads of equal mass to Bombus impatiens bumblebees and examined flight performance in a wind tunnel under three conditions: flight in unsteady flow, tracking an oscillating flower in smooth flow, and flower tracking in unsteady flow. Using an inertial model, we estimated that carrying a load on the legs rather than in the abdomen increases a bee’s moment of inertia about the roll and yaw axes but not the pitch axis. Consistent with these predictions, we found that bees carrying a load on their legs displayed slower rotations about their roll and yaw axes, regardless of whether these rotations were driven by external perturbations or self-initiated steering maneuvers. This allowed pollen-loaded bees to maintain a more stable body orientation and higher median flight speed in unsteady flow but reduced their performance when tracking a moving flower, supporting the concept of a tradeoff between stability and maneuverability. These results demonstrate that the types of resources collected by bees affect their flight performance and energetics and suggest that wind conditions may influence resource selection.The ability to carry external loads is an essential component of resource acquisition in many flying insects, including central place foragers that regularly transport provisions from the field back to their hive. Bumblebees, like all eusocial bees, rely exclusively on floral nectar and pollen to meet the energetic and nutritional demands of their colony, and much of a bumblebee forager’s life is spent gathering food resources. Bombus impatiens foragers spend an average of 7.5 h/d collecting and transporting food from flower patches to their hive over the course of 3–15 roundtrip flights (1), and the loads the bees carry can be quite substantial. Goulson et al. (2) measured an average load mass (pollen and/or nectar) equal to 23% of unladen body mass, and a maximum load of 77% body mass, in Bombus terrestris foragers. Free (3) measured Bombus sylvarum foragers ingesting nectar quantities ranging from 23% to 91% of their unladen body mass.Because load carriage requires an increase in lift production to support the additional mass, and thus an increase in induced power output (4, 5), it is generally assumed that carrying extra weight should adversely affect flight performance in some way. However, the physiological and behavioral consequences of this common flight challenge have rarely been examined, and the few studies addressing the metabolic cost of load carriage have produced conflicting results. Some have found that load carriage has no effect on metabolic rates of honey bees (6, 7), nor does it alter their foraging behavior or efficiency (8), whereas others have found that metabolic rates do increase with load carriage (9, 10). The effect of load carriage on many other important aspects of insect flight performance, including stability and maneuverability, has rarely been explored.Animal locomotion is often thought to involve a tradeoff between stability and maneuverability [although some animals appear to be both stable and highly maneuverable; thus, these traits are not always mutually exclusive (11–13)], but this tradeoff has rarely been tested empirically, particularly in flying insects. Stability is typically defined as the capacity to both resist and recover from disturbances to an intended trajectory (13). Maneuverability has been defined in various ways but, in the most general sense, is recognized as the ability to voluntarily change trajectory (13, 14). In the case of insect flight, both stability and maneuverability are thought to depend on the animal’s mass moment of inertia, because this property determines the torque required to create rotational body accelerations (15, 16), which result in reorientation of the aerodynamic force vector (17). The applied body torque may either be imposed upon the insect by external perturbations (relevant to stability) or generated by the insect as a steering behavior (relevant to maneuverability). Thus, a body with a lower moment of inertia requires less torque to initiate rotations, which may be beneficial for maneuverability but potentially disadvantageous for stability. This mechanical tradeoff is suggested by the flight dynamics of many insects, which are often the least stable to external perturbations about the roll axis—the axis with the lowest moment of inertia—and simultaneously display a propensity for maneuvering via rolling and lateral acceleration (14, 17–19).The presumed dependency of maneuverability and stability on a body’s moment of inertia raises the interesting possibility that nectar and pollen loads may differentially affect insect flight performance, because they are carried at different locations on the body. Whereas nectar is stored in a specialized stomach within the abdominal cavity, near the bee’s center of mass (COM), pollen is carried externally and more distally, packed into shallow cavities on the hind legs. To explore this hypothesis, we performed experiments on Bombus impatiens bumblebees in which we simulated pollen or nectar loads of equal mass, and examined flight performance in a wind tunnel under three different flight conditions: (i) unsteady flow in a von Kármán vortex street (VORT), designed to test flight stability; (ii) laminar airflow with a laterally oscillating flower (FLR), designed to test flight maneuverability; and (iii) unsteady vortex street flow combined with a laterally oscillating flower (VORT/FLR), designed to simultaneously test both stability and maneuverability (Movies S1–S3). We simulated pollen or nectar loads by attaching a small ball bearing to the corbicula (pollen basket) of each hind leg or a pair of ball bearings to the dorsal anterior region of the abdomen, respectively (Fig. 1A and Fig. S1). The combined mass of the ball bearings (∼25 mg) was ∼15% of the body mass of an average bee used in this study, which is well within the range of a typical pollen or nectar load for a bumblebee forager (2). We built a simple inertial model of a bee body (Fig. S2) to estimate the effects of our two load types on the body moment of inertia about the roll, pitch, and yaw axes and used these values to predict how each load treatment would affect flight stability and maneuverability (Fig. 1B).Open in a separate windowFig. 1.Load treatments and their predicted effects on body moment of inertia, maneuverability, and stability. (A) Load treatments consisted of either attaching a pair of small steel ball bearings to the dorsal surface of the anterior-most plate of the abdomen, simulating a nectar load (blue) or attaching a single ball bearing to the corbicula (“pollen basket”) on the outer face of each hind tibia, simulating a pollen load (red). Yellow stars indicate the approximate location of the COM, around which the body rotates, based on results of the inertial model illustrated in Fig. S1. (B) Estimated change in moment of inertia (I) around the three rotational axes when a load is carried on the legs vs. on the abdomen. Values are derived from an inertial model of a bumblebee body subject to each load treatment (see Open in a separate windowFig. S1.Still frames from high-speed videos of a bumblebee subjected to the abdominal load treatment (A) and the leg load treatment (B).Open in a separate windowFig. S2.Model of a bumblebee body used to estimate moments of inertia, consisting of separate ellipsoids representing the head, thorax, abdomen, and two hind legs. Dimensions and masses for each body segment were taken from measurements of dissected bumblebees. (A) Unloaded body, with no additional weights attached. (B) Abdominal (simulated nectar) load treatment, with two blue spheres representing ball bearings as they were positioned on bees in our experiments (i.e., attached to the anterior plate of the abdomen). In the model, the ball bearings are not in direct contact with the abdominal segment because of limitations on the model’s abdominal angle imposed by the parallel axis theorem, as described in Materials and Methods. (C) Leg (simulated pollen) load treatment, with two red spheres representing ball bearings attached to the corbiculae on the hind legs. Estimated MOI values for each treatment are reported in 20). We calculated body rotation rates around the roll, pitch, and yaw axes (Fig. 2), as well as median velocity along the bee’s flight path. To further explore how loading affects maneuverability, we examined how well bees were able to track the moving flower under each load treatment. For the two flight conditions with an oscillating flower, we calculated the phase lag and normalized correlation between the lateral position of the bee and the flower. We also calculated flight path sinuosity, which reflects how much a bee “overshoots” in its attempts to track the flower’s directional changes.Open in a separate windowFig. 2.Representative paired flight trials under each load treatment (abdominal load in blue, leg load in red), for the three flight conditions. (A) Unsteady vortex street flow with a stationary flower (VORT). (B) Laminar airflow with a laterally oscillating flower (FLR). (C) Unsteady vortex street flow with a laterally oscillating flower (VORT/FLR). For each flight condition, we show the 3D trajectories (top row), lateral position of the bee relative to the flower (indicated by a black dashed line) through time (middle row), and a subsection of body roll rate through time (bottom row).     

Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide,the key lipid for skin permeability barrier formation

A skin permeability barrier is essential for terrestrial animals, and its impairment causes several cutaneous disorders such as ichthyosis and atopic dermatitis. Although acylceramide is an important lipid for the skin permeability barrier, details of its production have yet to be determined, leaving the molecular mechanism of skin permeability barrier formation unclear. Here we identified the cytochrome P450 gene CYP4F22 (cytochrome P450, family 4, subfamily F, polypeptide 22) as the long-sought fatty acid ω-hydroxylase gene required for acylceramide production. CYP4F22 has been identified as one of the autosomal recessive congenital ichthyosis-causative genes. Ichthyosis-mutant proteins exhibited reduced enzyme activity, indicating correlation between activity and pathology. Furthermore, lipid analysis of a patient with ichthyosis showed a drastic decrease in acylceramide production. We determined that CYP4F22 was a type I membrane protein that locates in the endoplasmic reticulum (ER), suggesting that the ω-hydroxylation occurs on the cytoplasmic side of the ER. The preferred substrate of the CYP4F22 was fatty acids with a carbon chain length of 28 or more (≥C28). In conclusion, our findings demonstrate that CYP4F22 is an ultra-long-chain fatty acid ω-hydroxylase responsible for acylceramide production and provide important insights into the molecular mechanisms of skin permeability barrier formation. Furthermore, based on the results obtained here, we proposed a detailed reaction series for acylceramide production.A skin permeability barrier protects terrestrial animals from water loss from inside the body, penetration of external soluble materials, and infection by pathogenetic organisms. In the stratum corneum, the outermost cell layer of the epidermis, multiple lipid layers (lipid lamellae) play a pivotal function in barrier formation (Fig. S1) (1–3). Impairment of the skin permeability barrier leads to several cutaneous disorders, such as ichthyosis, atopic dermatitis, and infectious diseases.Open in a separate windowFig. S1.Structures of the epidermis, the stratum corneum, acylceramide, and protein-bound ceramide. Acylceramides are produced mainly in the stratum granulosum and partly in the stratum spinosum and are stored in lamellar bodies as glucosylated forms (acyl glucosylceramides). At the interface of the stratum granulosum and stratum corneum, the lamellar bodies fuse with the plasma membrane and release their contents into the extracellular space, where acyl glucosylceramides are converted to acylceramides. Thus, released acylceramides, FAs, and cholesterol form lipid lamellae in the stratum corneum. Some acylceramide is hydrolyzed to ω-hydroxyceramide, followed by covalent binding to corneocyte surface proteins to create corneocyte lipid envelopes. Acylceramide contains ULCFAs with carbon chain lengths of C28–C36. The FA elongase ELOVL1 produces VLCFAs, which are further elongated to ULCFAs by ELOVL4 (29). The ceramide synthase CERS3 creates an amide bond between ULCFA and LCB (17). ω-Hydroxylation of ULCFA is required for acylceramide production. However, the responsible ω-hydroxylase had not been identified previously; its identification is the subject of this research.The major components of the lipid lamellae are ceramide (the sphingolipid backbone), cholesterol, and free fatty acid (FA). In most tissues, ceramide consists of a long-chain base (LCB; usually sphingosine) and an amide-linked FA with a chain length of 16–24 (C16–C24) (4, 5). On the other hand, ceramide species in the epidermis are strikingly unique (Fig. S2A). For example, epidermal ceramides contain specialized LCBs (phytosphingosine and 6-hydroxysphingosine) and/or FAs with α- or ω-hydroxylation (1–3). In addition, substantial amounts of epidermal ceramides have ultra-long-chain FAs (ULCFAs) with chain lengths of 26 or more (≥C26) (4, 5). Unique epidermal ceramides are acylceramides having C28–C36 ULCFAs, which are ω-hydroxylated and esterified with linoleic acid [EOS in Fig. S1; EODS, EOS, EOP, and EOH in Fig. S2A; EOS stands for a combination of an esterified ω-hydroxy FA (EO) and sphingosine (S); DS, dihydrosphingosine; P, phytosphingosine; H, 6-hydroxysphingosine] (1–3, 6, 7). These characteristic molecules may be important to increase the hydrophobicity of lipid lamellae and/or to stabilize the multiple lipid layers. Linoleic acid is one of the essential FAs, and its deficiency causes ichthyosis symptoms resulting from a failure to form normal acylceramide (8). Ichthyosis is a cutaneous disorder accompanied by dry, thickened, and scaly skin; it is caused by a barrier abnormality. In patients who have atopic dermatitis, both total ceramide levels and the chain length of ceramides are decreased, and ceramide composition is altered also (9–11).Open in a separate windowFig. S2.Structure and synthetic pathways of ceramides in mammals. (A) Structure and nomenclature of epidermal ceramides. Epidermal ceramides are classified into 12 classes depending on their differences in the LCB and FA moieties. N-type and A-type ceramides contain C16–C30 FAs (n = 1–15), whereas EO-type ceramides contain C28–C36 FAs (n = 13–21) (6, 7). (B) FA elongation and ceramide synthesis in mammals. The FA elongation pathways of saturated and monounsaturated FAs and the ceramide-synthetic pathways are illustrated. E1–E7 and C1–C6 indicate the ELOVL (ELOVL1–7) and CERS (CERS1–6) isozymes involved in each step, respectively. The differences in the letter size of E1–E7 reflect their enzyme activities in each FA elongation reaction. Cer, ceramide; MUFA, monounsaturated FA; SFA, saturated FA.In addition to its essential function in the formation of lipid lamellae, acylceramide also is important as a precursor of protein-bound ceramide, which functions to connect lipid lamellae and corneocytes (Fig. S1) (12, 13). After the removal of linoleic acid, the exposed ω-hydroxyl group of acylceramide is covalently bound to corneocyte proteins, forming a corneocyte lipid envelope. Acylceramides and protein-bound ceramides are important in epidermal barrier formation, and mutations in the genes involved in their synthesis, including the ceramide synthase CERS3, the 12(R)-lipoxygenase ALOX12B, and the epidermal lipoxygenase-3 ALOXE3, can cause nonsyndromic, autosomal recessive congenital ichthyosis (ARCI) (3, 14–16). CERS3 catalyzes the amide bond formation between an LCB and ULCFA, producing ULC-ceramide, which is the precursor of acylceramide (Fig. S1 and Fig. S2B) (17). ALOX12B and ALOXE3 are required for the formation of protein-bound ceramides (13, 18). Other ARCI genes include the ATP-binding cassette (ABC) transporter ABCA12, the transglutaminase TGM1, NIPAL4 (NIPA-like domain containing 4)/ICHTHYIN, CYP4F22/FLJ39501, LIPN (lipase, family member N), and PNPLA1 (patatin-like phospholipase domain containing 1) (16, 19). The exact functions of NIPAL4, LIPN, and PNPLA1 are currently unclear. Causative genes of syndromic forms of ichthyosis also include a gene required for acylceramide synthesis: the FA elongase ELOVL4, which produces ULCFA-CoAs, the substrate of CERS3 (20).Although acylceramide is essential for the epidermal barrier function, the mechanism behind acylceramide production is still poorly understood, leaving the molecular mechanisms behind epidermal barrier formation unclear. For example, acylceramide production requires ω-hydroxylation of the FA moiety of ceramide. However, the ω-hydroxylase responsible for this reaction was unidentified heretofore (Fig. S1). Here, we identified the cytochrome P450, family 4, subfamily F, polypeptide 22 (CYP4F22), also known as “FLJ39501,” as this missing FA ω-hydroxylase required for acylceramide production. CYP4F22 had been identified as one of the ARCI genes (21), although its function in epidermal barrier formation remained unsolved. Our findings clearly demonstrate a relationship between ARCI pathology, acylceramide levels, and ω-hydroxylase activity.     

Insect nicotinic receptor interactions in vivo with neonicotinoid,organophosphorus, and methylcarbamate insecticides and a synergist

The nicotinic acetylcholine (ACh) receptor (nAChR) is the principal insecticide target. Nearly half of the insecticides by number and world market value are neonicotinoids acting as nAChR agonists or organophosphorus (OP) and methylcarbamate (MC) acetylcholinesterase (AChE) inhibitors. There was no previous evidence for in vivo interactions of the nAChR agonists and AChE inhibitors. The nitromethyleneimidazole (NMI) analog of imidacloprid, a highly potent neonicotinoid, was used here as a radioligand, uniquely allowing for direct measurements of house fly (Musca domestica) head nAChR in vivo interactions with various nicotinic agents. Nine neonicotinoids inhibited house fly brain nAChR [³H]NMI binding in vivo, corresponding to their in vitro potency and the poisoning signs or toxicity they produced in intrathoracically treated house flies. Interestingly, nine topically applied OP or MC insecticides or analogs also gave similar results relative to in vivo nAChR binding inhibition and toxicity, but now also correlating with in vivo brain AChE inhibition, indicating that ACh is the ultimate OP- or MC-induced nAChR active agent. These findings on [³H]NMI binding in house fly brain membranes validate the nAChR in vivo target for the neonicotinoids, OPs and MCs. As an exception, the remarkably potent OP neonicotinoid synergist, O-propyl O-(2-propynyl) phenylphosphonate, inhibited nAChR in vivo without the corresponding AChE inhibition, possibly via a reactive ketene metabolite reacting with a critical nucleophile in the cytochrome P450 active site and the nAChR NMI binding site.The nicotinic nervous system has two principal sites of insecticide action, the nicotinic receptor (nAChR) activated by acetylcholine (ACh) and neonicotinoid agonists (1–6), and acetylcholinesterase (AChE) inhibited by organophosphorus (OP) and methylcarbamate (MC) compounds to generate and maintain localized toxic ACh levels (Fig. 1) (7). The nAChR and AChE targets have been identified in insects by multiple techniques but not by direct assays of the ACh binding site in the brain of poisoned insects. Here we use the outstanding insecticidal potency of the nitromethyleneimidazole (NMI) analog of imidacloprid (IMI) (8) as a radioligand (9), designated [³H]NMI, to directly measure the house fly (Musca domestica) nAChR not only in vitro but also in vivo, allowing us to validate by a previously undescribed method the neonicotinoid direct and OP/MC indirect nAChR targets (Fig. 2). This approach also helped solve the intriguing mechanism by which an O-(2-propynyl) phosphorus compound strongly synergizes neonicotinoid insecticidal activity (10) by dual inhibition of cytochrome P450 (CYP) (11–13) and the nAChR agonist site (described herein). Insecticide disruption at the insect nAChR can now be readily studied in vitro and in vivo with a single radioligand allowing better understanding of the action of several principal insecticide chemotypes (Fig. 3).Open in a separate windowFig. 1.The insect nicotinic receptor is the direct or indirect target for neonicotinoids, organophosphorus compounds and methylcarbamates, which make up about 45% of the insecticides by number and world market value (2, 7).Open in a separate windowFig. 2.In this study, Musca nicotinic receptor in vivo interactions with major insecticide chemotypes are revealed by a [³H]NMI radioligand reporter assay. *Position of tritium label.Open in a separate windowFig. 3.Two neonicotinoid nicotinic agonists and two anticholinesterase insecticides.     

From the Cover: Ammonite habitat revealed via isotopic composition and comparisons with co-occurring benthic and planktonic organisms

Ammonites are among the best-known fossils of the Phanerozoic, yet their habitat is poorly understood. Three common ammonite families (Baculitidae, Scaphitidae, and Sphenodiscidae) co-occur with well-preserved planktonic and benthic organisms at the type locality of the upper Maastrichtian Owl Creek Formation, offering an excellent opportunity to constrain their depth habitats through isotopic comparisons among taxa. Based on sedimentary evidence and the micro- and macrofauna at this site, we infer that the 9-m-thick sequence was deposited at a paleodepth of 70–150 m. Taxa present throughout the sequence include a diverse assemblage of ammonites, bivalves, and gastropods, abundant benthic foraminifera, and rare planktonic foraminifera. No stratigraphic trends are observed in the isotopic data of any taxon, and thus all of the data from each taxon are considered as replicates. Oxygen isotope-based temperature estimates from the baculites and scaphites overlap with those of the benthos and are distinct from those of the plankton. In contrast, sphenodiscid temperature estimates span a range that includes estimates of the planktonic foraminifera and of the warmer half of the benthic values. These results suggest baculites and scaphites lived close to the seafloor, whereas sphenodiscids sometimes inhabited the upper water column and/or lived closer to shore. In fact, the rarity and poorer preservation of the sphenodiscids relative to the baculites and scaphites suggests that the sphenodiscid shells may have only reached the Owl Creek locality by drifting seaward after death.Ammonites have constituted a primary data source for the fields of evolution, paleoceanography, biostratigraphy, and paleoecology for more than a century; their ubiquity, diversity, occurrence in a wide variety of marine environments, and readily preservable shell account for their utility in both paleontological and geological studies. Ammonites have been used extensively in studies of heterochrony because their shells preserve distinct ontogenetic changes that can be tracked in evolving lineages (1, 2); they are valued in paleoceanographic research because, like most mollusks, they are inferred to have precipitated their aragonitic shells in isotopic equilibrium with the surrounding seawater (3, 4). Thus, shell chemistry may record temperature, via oxygen isotopes (δ¹⁸O) (5), and water mass properties, such as strontium isotopes (⁸⁷Sr/⁸⁶Sr), which are used to estimate numerical age (6). Ammonites are also a textbook example of an index fossil; besides being abundant and widespread, they evolved rapidly, making them the dominant Mesozoic tool for relative dating and correlation of shallow water strata. For example, the 35-My-long stratigraphic record of Upper Cretaceous deposits in the US Western Interior Seaway (WIS) has been partitioned into 66 ammonite zones (7). Finally, ammonites underwent a spectacular extinction at the close of the Mesozoic. Explanations for why the ammonites, which were flourishing immediately before the Cretaceous–Paleogene (K–Pg) mass extinction (8), died out, whereas their relatives the nautiloids survived (9), have been used to understand the selectivity of marine microfossil groups across the K–Pg event (10), highlighting the importance of ammonites in understanding extinction mechanisms.Despite the utility of ammonites to many disciplines, their ecology remains poorly known. A challenge in reconstructing their habitat(s) is establishing if ammonites lived at the site from which they are recovered. Ammonite tissues could drop out after death, and the shell might float to the surface buoyed by relict air contained within the phragmocone (11). Empty shells of Nautilus are found on beaches at remote distances from their actual habitat, documenting the potential for postmortem drift of positively buoyant shells (12). Similarly, Tanabe (13) mapped the distribution of Turonian ammonites along an onshore–offshore transect, and noted that their postmortem distribution was broader than the settings they inhabited during life. Uncertainty in ammonites’ preferred habitat is especially concerning for temperature reconstructions based on their occurrence or isotopes because temperature varies both with depth and with distance to the shoreline.A variety of studies have attempted to determine the ecology of ammonites based on analogies with living relatives, shell morphology, facies distribution, faunal associations, and isotopic composition. However, these studies have had limited success for both biological and geological reasons. Ammonites are extinct, and their closest living relatives, the octopods, squids, cuttlefish, and Nautilus, are all in different orders/subclasses (14). Even among living cephalopods, a variety of behaviors are observed, including vertical and lateral migrations (15). Other studies have sought to reconstruct ammonite habitat by comparing the isotopes recorded in their shells to those of co-occurring, or nearly co-occurring, taxa of known depth habitats. A powerful approach in theory, these studies have been limited in practice because ammonites are only rarely recovered with the planktonic and benthic organisms needed to establish a temperature-depth profile. Further, many studies were undertaken in the WIS, where the water mass properties are poorly understood and controversial (16, 17). We expand upon previous isotopic studies by using exceptionally well-preserved ammonites from the Owl Creek Formation (fm.) type locality in northern Mississippi (Fig. S1). Ammonites are abundant at this site and co-occur with bivalves, gastropods, and planktonic and benthic foraminifera (Fig. 1 and Fig. S2), thus providing an excellent opportunity to reconstruct a water column profile and establish where the ammonites fall within it.Open in a separate windowFig. 1.Representative mollusks from the Owl Creek fm. (A) Left lateral and ventral views of a Discoscaphites iris macroconch AMNH 91329; (B) right lateral and ventral views of a D. iris microconch AMNH 91335; (C) right lateral view of D. iris, showing a healed injury AMNH 77461; (D) ventral and right lateral views of Eubaculites latecarinatus AMNH 91330; (E) ventral and right lateral views of Eubaculites carinatus AMNH 91334; (F) left lateral view of Sphenodiscus pleurisepta; sutures are visible because most of the shell is missing AMNH 91520; (G) Gyrodes crenata AMNH 91333; (H) Nucula percrassa AMNH 91331.Open in a separate windowFig. S1.(A) Map of the modern coastline of the United States; gray line shows the Cretaceous coastline (from National Oceanic and Atmospheric Administration Coastal Resource Management website); circle is the Owl Creek fm. type locality; (B) inset map; (C) stratigraphic column of the Owl Creek fm. type section. St., stage.Open in a separate windowFig. S2.Representative Owl Creek fm. foraminifera. (A) Planoheterohelix globulosa; (B) SEM of test wall of A; (C) Rugoglobigerina rugosa; (D) SEM of test wall of C; (E) Lingulogavelinella sp.     

Conjugated polymers based on metalla-aromatic building blocks

Conjugated polymers usually require strategies to expand the range of wavelengths absorbed and increase solubility. Developing effective strategies to enhance both properties remains challenging. Herein, we report syntheses of conjugated polymers based on a family of metalla-aromatic building blocks via a polymerization method involving consecutive carbyne shuttling processes. The involvement of metal d orbitals in aromatic systems efficiently reduces band gaps and enriches the electron transition pathways of the chromogenic repeat unit. These enable metalla-aromatic conjugated polymers to exhibit broad and strong ultraviolet–visible (UV–Vis) absorption bands. Bulky ligands on the metal suppress π–π stacking of polymer chains and thus increase solubility. These conjugated polymers show robust stability toward light, heat, water, and air. Kinetic studies using NMR experiments and UV–Vis spectroscopy, coupled with the isolation of well-defined model oligomers, revealed the polymerization mechanism.

Conjugated polymers are macromolecules usually featuring a backbone chain with alternating double and single bonds (1–3). These characteristics allow the overlapping p-orbitals to form a system with highly delocalized π-electrons, thereby giving rise to intriguing chemical and physical properties (4–6). They have exhibited many applications in organic light-emitting diodes, organic thin film transistors, organic photovoltaic cells, chemical sensors, bioimaging and therapies, photocatalysis, and other technologies (7–10). To facilitate the use of solar energy, tremendous efforts have been devoted in recent decades to developing previously unidentified conjugated polymers exhibiting broad and strong absorption bands (11–13). The common strategies for increasing absorption involve extending π-conjugation by incorporating conjugated cyclic moieties, especially fused rings; modulating the strength of intramolecular charge transfer between donor and acceptor units (D–A effect); increasing the coplanarity of π conjugation through weak intramolecular interactions (e.g., hydrogen bonds); and introducing heteroatoms or heavy atoms into the repeat units of conjugated polymers (11–16). Additionally, appropriate solubility is a prerequisite for processing and using polymers and is usually achieved with the aid of long alkyl or alkoxy side chains (12, 17).Aromatic rings are among the most important building blocks for conjugated polymers. In addition to aromatic hydrocarbons, a variety of aromatic heterocycles composed of main-group elements have been used as fundamental components. These heteroatom-containing conjugated polymers show unique optical and electronic properties (4–10). However, while metalla-aromatic systems bearing a transition metal have been known since 1979 due to the pioneering work by Thorn and Hoffmann (18), none of them have been used as building blocks for conjugated polymers. The HOMO–LUMO gaps (E_g) of metalla-aromatics are generally narrower (Fig. 1) than those of their organic counterparts (19–22). We reasoned that this feature should broaden the absorption window if polymers stemming from metalla-aromatics are achievable.Open in a separate windowFig. 1.Comparison of traditional organic skeletons with metalla-aromatic building blocks (the computed energies are in eV). (A) HOMO–LUMO gaps of classic aromatic skeletons. (B) Carbolong frameworks as potential building blocks for novel conjugated polymers with broad absorption bands and improved solubility.In recent years, we have reported a series of readily accessible metal-bridged bicyclic/polycyclic aromatics, namely carbolong complexes, which are stable in air and moisture (23–25). The addition of osmium carbynes (in carbolong complexes) and alkynes gave rise to an intriguing family of d_π–p_π conjugated systems, which function as excellent electron transport layer materials in organic solar cells (26, 27). These observations raised the following question: Can this efficient addition reaction be used to access metalla-aromatic conjugated polymers? It is noteworthy that incorporation of metalla-aromatic units into conjugated polymers is hitherto unknown. In this contribution, we disclose a polymerization reaction involving M≡C analogs of C≡C bonds, which involves a unique carbyne shuttling strategy (Fig. 2A). This led to examples of metalla-aromatic conjugated polymers (polycarbolongs) featuring metal carbyne units in the main chain. On the other hand, the development of polymerization reactions plays a crucial role in involving certain building blocks in conjugated polymers (28–32). These efficient, specific, and feasible polymerizations could open an avenue for the synthesis of conjugated polymers.Open in a separate windowFig. 2.Design of polymers and synthesis of monomers. (A) Schematic illustration of the polymerization strategy. (B) Preparation of carbolong monomers. Insert: X-ray molecular structure for the cations of complex 3. Ellipsoids are shown at the 50% probability level; phenyl groups in PPh₃ are omitted for clarity.     

Early human use of anadromous salmon in North America at 11,500 y ago

Salmon represented a critical resource for prehistoric foragers along the North Pacific Rim, and continue to be economically and culturally important; however, the origins of salmon exploitation remain unresolved. Here we report 11,500-y-old salmon associated with a cooking hearth and human burials from the Upward Sun River Site, near the modern extreme edge of salmon habitat in central Alaska. This represents the earliest known human use of salmon in North America. Ancient DNA analyses establish the species as Oncorhynchus keta (chum salmon), and stable isotope analyses indicate anadromy, suggesting that salmon runs were established by at least the terminal Pleistocene. The early use of this resource has important implications for Paleoindian land use, economy, and expansions into northwest North America.Each year along the Pacific coast of North America, millions of salmon migrate from the ocean to spawn and die in their natal rivers and lakes; however, during the last Ice Age, many of the rivers that today support salmon were blocked by glacial ice, severely restricting salmon ranges (1). A potential glacial refugium for salmon was Beringia, the mostly ice-free landmass that bridged northeast Asia and Alaska (1–3). Evidence for such a refugium comes from studies of present-day diversity and distributions of Pacific salmon (1, 2), but there is little direct evidence of the antiquity of salmon spawning runs in North America. Here we confirm the presence of an anadromous salmon species, Oncorhynchus keta (chum salmon) through ancient DNA (aDNA) and stable isotope analyses of fish remains at the Upward Sun River site located deep in the interior of Alaska, about 50 km downstream from the modern limit of major spawning areas (Fig. 1). These specimens, dating to the terminal Pleistocene, represent the oldest genetically confirmed Pacific salmon species in an archaeological context in North America. These data are important for testing competing models of subsistence strategies and diet breadths of Paleoindian populations in the New World (4, 5), as well as for understanding Beringian ecosystem biodiversity.Open in a separate windowFig. 1.Location of Upward Sun River Site, course of the Tanana-Yukon River and possible course across the Bering Shelf during lower sea level, and modern chum salmon fall spawning limit along the Tanana River. Details are provided in SI Text.Oncorhynchus is a salmonid genus that includes several Pacific salmon and Pacific trout species; some species of this genus occur as both freshwater resident and anadromous forms, migrating from the sea to freshwater to spawn (6). The spawning behavior of anadromous Pacific salmon results in massive and predictable runs in freshwater streams over a short period, making these fish a potentially valuable human food resource (7). Salmon are also ecologically important because they transport rich marine-derived nutrients into relatively unproductive interior riparian areas (8). Five species of Pacific salmon and one species of Pacific trout presently occur in central Alaskan waters, including Chinook (Oncorhynchus tshawytscha), coho (Oncorhynchus kisutch), chum (O. keta), sockeye (Oncorhynchus nerka), pink salmon (Oncorhynchus gorbuscha), and rainbow/steelhead trout (Oncorhynchus mykiss) (1).Pleistocene-aged remains of Pacific salmon from North America are extremely rare in both paleontological and archaeological contexts. This scarcity is related in part to the spawning habitat of most salmon species, leading to death in river gravels where remains are unlikely to be preserved (9) and to the fragility of fish skeletal elements and their small size (inhibiting recovery), resulting in their underrepresentation in the archaeological record (10). Paleontological specimens of Pacific salmon have been recovered from middle Pleistocene sediments in the Skokomish Valley, Washington (United States) (9), and from late Pleistocene sediments at Kamloops Lake, British Columbia (Canada) (11). Although the remains from both of these locales were morphologically identified as O. nerka, carbon stable isotope analysis of specimens from Kamloops Lake suggests that the fish were likely the landlocked form of O. nerka, known as kokanee (11). Other late Pleistocene paleontological fish remains assigned to Pacific salmon derive from two additional sites in British Columbia, including Courtenay (Vancouver Island) and Gaadu Din 1 cave (Haida Gwaii) (12, 13). Specimens from the latter site were genetically identified as “salmon,” but the details of the aDNA analysis were not reported (13).The only report of Oncorhynchus remains from a Pleistocene-age archaeological site in North America comes from Upward Sun River, located adjacent to the Tanana River (a major tributary of the Yukon River) in central Alaska (14) (see also SI Text) (Fig. 1). Here, 308 Oncorhynchus specimens were recovered from the central hearth of a residential feature, also associated with a cremated 3-y-old child (15). A double infant burial with associated grave goods was located directly below (40 cm) this hearth. Radiocarbon and contextual data suggest near contemporaneity between the hearth and the burial pit, with a mean pooled age of 9,970 ± 30 B.P. (11,600–11,270 cal B.P.); thus, these represent the oldest known human remains in the North American Arctic/Subarctic (Fig. 2 and SI Text). A total of 29 additional Oncorhynchus specimens were found within the pit fill. The fish remains were mostly fragmentary and over 90% were burned and calcined. The component and burials are culturally affiliated with the Denali Complex, which was widespread in Eastern Beringia from ∼12,700 cal B.P. to the early Holocene (3, 16).Open in a separate windowFig. 2.Upward Sun River stratigraphy, chronology, and aDNA and stable isotope bone samples. Details provided in SI Text and 17) (Fig. 2). Based on overall size and occupation season, based on other fauna, the vertebrae resembled O. keta (Fig. S1 and 18, 19). Species distinctions are critical to separate salmon from trout and other salmonids, because although some other members of this family are anadromous, they do not typically form the extensive and massive spawning runs that make salmon such an exceptional resource (6, 20). Additionally, salmon species differ in habitat requirements, run timing and abundance, and body size and fat content, all of which have implications for understanding past human land use and subsistence strategies (18, 19, 21, 22). aDNA analysis provides more accurate identifications of fish remains, and has recently been successfully applied to fish assemblages from Holocene archaeological sites in the Pacific Northwest of North America (18, 19, 23, 24).Open in a separate windowFig. S1.USR osteometric values compared with various genetically identified Oncorhynchus datasets. (A) Fused vertebra (type III) measurements [data from Huber et al. (21)]. (B) Vertebral transverse diameter measurements of genetically identified salmon species [summary data from Moss et al. (18); original data: “1” Grier et al. (19), “2” Orchard and Szpak (20), “3” Moss et al. (18), and “4” Yang et al. (22)].

Table S1.

USR fish vertebrae samples subjected to aDNA analysis

From the Cover: Eleventh-century shift in timber procurement areas for the great houses of Chaco Canyon

An enduring mystery from the great houses of Chaco Canyon is the origin of more than 240,000 construction timbers. We evaluate probable timber procurement areas for seven great houses by applying tree-ring width-based sourcing to a set of 170 timbers. To our knowledge, this is the first use of tree rings to assess timber origins in the southwestern United States. We found that the Chuska and Zuni Mountains (>75 km distant) were the most likely sources, accounting for 70% of timbers. Most notably, procurement areas changed through time. Before 1020 Common Era (CE) nearly all timbers originated from the Zunis (a previously unrecognized source), but by 1060 CE the Chuskas eclipsed the Zuni area in total wood imports. This shift occurred at the onset of Chaco florescence in the 11th century, a time with substantial expansion of existing great houses and the addition of seven new great houses in the Chaco Core area. It also coincides with the proliferation of Chuskan stone tools and pottery in the archaeological record of Chaco Canyon, further underscoring the link between land use and occupation in the Chuska area and the peak of great house construction. Our findings, based on the most temporally specific and replicated evidence of Chacoan resource procurement obtained to date, corroborate the long-standing but recently challenged interpretation that large numbers of timbers were harvested and transported from distant mountain ranges to build the great houses at Chaco Canyon.The high desert landscape of Chaco Canyon, New Mexico was the locale of a remarkable cultural development of Ancestral Puebloan peoples, including the construction of some of the largest pre-Columbian buildings in North America (1) (Fig. 1). The monumental “great houses” of Chaco Canyon reflect an elaborate socioecological system that spanned much of the 12,000-km² San Juan Basin from 850 to 1140 Common Era (CE) (2). These massive stone masonry structures required a wealth of resources to erect, including an estimated 240,000 trees incorporated as roof beams, door and window lintels, and other building elements (3). The incongruity of the great houses located in a nearly treeless landscape has led archaeologists and paleoecologists to investigate the origins of timbers used in construction (4–9). Beyond the simple curiosity driving this question, the answer has important implications for understanding the complexities of human–environmental interactions, the sociopolitical organization, and the economic structure of Chacoan society (10–12).Fig. 1.Aerial view of Pueblo Bonito, the largest of the Chaco Canyon great houses. Image courtesy of Adriel Heisey.The first excavators of the great houses in the early 20th century speculated that construction timbers were harvested locally, perhaps resulting in deforestation of the surrounding landscape (13). Paleoecological studies conducted during the late 1970s and early 1980s, however, showed that ponderosa pine (Pinus ponderosa), the primary tree species used in construction, was not abundant enough at the relatively low elevations (1,800–2,000 m above sea level) of Chaco Canyon and nearby mesas to support timber demand (14–16). Spruce (Picea spp.) and fir (Abies spp.), which account for tens of thousands of construction beams, have been absent from Chaco Canyon for at least 12,000 y and could have only been logged from distant, higher-elevation sites (2,500–3,450 m above sea level) (4). An inadequate supply of timbers in Chaco Canyon and its immediate surroundings during Puebloan occupation strongly suggests long-distance procurement from surrounding mountain ranges, where all three conifers now grow in abundance. This inference was corroborated by strontium isotope (⁸⁷Sr/⁸⁶Sr)-based sourcing. Through a comparison of ⁸⁷Sr/⁸⁶Sr values from great-house timbers to ₈₇Sr/₈₆Sr values from conifer stands growing today in mountains surrounding the San Juan Basin, two studies concluded that the Chuska Mountains (75 km west) and Mount Taylor (85 km southeast) were the most likely sources for spruce, fir, and ponderosa pine trees (6, 7). Recently, the explanation of long-distance timber transport and the related interpretations of ⁸⁷Sr/⁸⁶Sr evidence have been challenged and an alternative has been proposed that most great-house timbers (particularly ponderosa pine) were just as likely to have originated from nearby and low-elevation sites within, east, and south of Chaco Canyon (8, 9).We assessed probable timber origins independently from previous efforts by applying tree-ring width-based sourcing techniques to a set of 170 beams from our archives at the University of Arizona. These beams comprise six tree species from seven great-house structures (17) (Fig. S1). Each site chronology, as the average of 40–100 trees, represents tree-ring growth patterns peculiar to an individual landscape. This method of identifying the probable origin of timbers has been applied widely in Europe in the study of archaeological and nautical timbers and artifacts, musical instruments, and paintings on oak panels (17–20). These techniques are underused in North America, but recent efforts in the northeastern United States have revealed distant, inland sources for 18th- and 19th-century nautical timbers (21, 22).Table S1.Number of sourced beams by species and great-house structureFig. S1.An example of sourcing a great-house beam via tree-ring-width methods. (A) An individual beam (black line), the ponderosa pine JPB-88 from Pueblo del Arroyo, and the Chuskas chronology (red line). (B) Bivariate plot comparing the ring-width indices of ...Tree-ring sourcing can only be applied where tree growth patterns are distinguishable between the potential locations of origin. In the southwestern United States tree growth primarily responds to regionally coherent winter precipitation (23, 24), and as a consequence trees across the region tend to share roughly half of their interannual variability (25). Differences between site chronologies are predominantly attributed to variations in topography and subregional-scale climate conditions (26).We compared great-house beams to the site chronologies of eight potential harvesting areas surrounding the San Juan Basin. Chaco Canyon was not included as one of our sites because it lacked enough remnant wood from the Chaco era to build a local site chronology. To assess the efficacy and accuracy of the tree-ring sourcing method within the San Juan Basin, we tested whether tree-ring growth patterns could be distinguished between the various mountain ranges surrounding Chaco Canyon by applying sourcing methods to living trees of known origin (SI Text and Fig. S2).Table S2.Tree-ring sites used to evaluate tree-ring-based sourcing in the San Juan BasinFig. S2.Evaluation of dendroprovenance in the San Juan Basin. (A–F) Each tile provides a different test for a set of modern trees (green triangles). Circle sizes are proportional to the number of trees (labeled in the circle) sourcing to a given location. ...     

Long-term reliability of the Athabasca River (Alberta,Canada) as the water source for oil sands mining

Exploitation of the Alberta oil sands, the world’s third-largest crude oil reserve, requires fresh water from the Athabasca River, an allocation of 4.4% of the mean annual flow. This allocation takes into account seasonal fluctuations but not long-term climatic variability and change. This paper examines the decadal-scale variability in river discharge in the Athabasca River Basin (ARB) with (i) a generalized least-squares (GLS) regression analysis of the trend and variability in gauged flow and (ii) a 900-y tree-ring reconstruction of the water-year flow of the Athabasca River at Athabasca, Alberta. The GLS analysis removes confounding transient trends related to the Pacific Decadal Oscillation (PDO) and Pacific North American mode (PNA). It shows long-term declining flows throughout the ARB. The tree-ring record reveals a larger range of flows and severity of hydrologic deficits than those captured by the instrumental records that are the basis for surface water allocation. It includes periods of sustained low flow of multiple decades in duration, suggesting the influence of the PDO and PNA teleconnections. These results together demonstrate that low-frequency variability must be considered in ARB water allocation, which has not been the case. We show that the current and projected surface water allocations from the Athabasca River for the exploitation of the Alberta oil sands are based on an untenable assumption of the representativeness of the short instrumental record.Over the past several decades, the province of Alberta has had Canada’s fastest growing economy, driven largely by the production of fossil fuels. Climatic change, periodic drought, and expanding human activities impact the province’s water resources, creating the potential for an impending water crisis (1). The Athabasca River (Fig. 1) is the only major river in Alberta with completely unregulated flows. It is the source of surface water for the exploitation of the Alberta oil sands, the world’s third-largest proven crude oil reserve at roughly 168 billion barrels. The oil and gas industry accounted for 74.5% of total surface water allocations in the Athabasca River Basin (ARB) in 2010 (Fig. 2) (2). An almost doubling of ARB water allocations since 2000, or 13 times the provincial average, is attributable to expanding oil sands production, which began in 1967 (Fig. 2).Open in a separate windowFig. 1.Alberta’s seven major drainage basins. The numbers refer to hydrometric gauges identified in 6). Reproduced with permission from the Government of Alberta.Open in a separate windowFig. 2.Licensed water allocations by sector and decades to 2000 and selected subsequent years in the Athabasca River Basin, 1900–2010. Reproduced with permission from the Government of Alberta.According to the Canadian Association of Petroleum Producers, in 2012, surface mining of the oil sands and in situ extraction (drilling) required 3.1 and 0.4 barrels of fresh water, respectively, to produce a barrel of crude oil (3). This amounted to 187 million cubic meters of fresh water use in 2012, or the equivalent of the residential water use of 1.7 million Canadians (4). Within the next decade, cumulative water use for oil sands production is projected to peak at about 505 million cubic meters per year or a rate of 16 m³⋅s⁻¹ (5). The current (2010 data) total water allocation represents only 4.4% of the mean annual Athabasca River flow (2) (Fig. 3); however, allocation and use as a proportion of average water levels does not account for the large variability in flow between seasons and years (interannual coefficient of variation = 22%). During 1952–2013, the Athabasca River gauge at Athabasca recorded mean seasonal flows of 100 m³⋅s⁻¹ in winter (December−February) and 911 m³⋅s⁻¹ in summer (June−August), with extreme monthly flows of 48 m³⋅s⁻¹ in December 2000 and 2,280 m³⋅s⁻¹ in June 1954. The mean, maximum, and minimum annual flows were 422 m³⋅s⁻¹, 702 m³⋅s⁻¹, and 245 m³⋅s⁻¹, respectively (Water Survey of Canada, wateroffice.ec.gc.ca/search/search_e.html?sType=h2oArc).Open in a separate windowFig. 3.Mean annual flow of the Athabasca River at Athabasca (gauge 07BE001, Water Survey of Canada) for the water year (October 1 to September 30, 1913–2013).Assessing the sustainability of ARB surface water availability is difficult because most regional streamflow gauges have been operational for a few years to a few decades, with long intervals of missing values, including during the 1930s and 1940s when drought was prevalent throughout the North American western interior. Various critics (e.g., ref. 4) emphasize the intraannual variability and express concern about water withdrawals in the low-flow winter season. Less attention has been given to flow variability at interannual to decadal scales associated with climate oscillations such as the Pacific Decadal Oscillation (PDO) and Pacific North American mode (PNA), which are known to have significant impacts on runoff from the Rocky Mountains, including in the ARB (6–9), and the potential consequences of a long period of predominantly low flow. We investigate the response of ARB flows to the low-frequency components of the PDO and PNA. This paper is the first, to our knowledge, to explicitly model the variability linked to the PDO and PNA, when testing for trends in discharge, thereby removing the tendencies associated with these climatic oscillations, which can confound trend detection in the relatively short instrumental records (6, 7). However, the available instrumental hydrologic records provide a limited sample of these low-frequency fluctuations; exploring longer records of this variability is critical. Therefore, we also reconstructed the Athabasca River annual flow from the growth rings in moisture-sensitive conifers at a network of sites in the upper reaches of the ARB and in an adjacent watershed (Fig. S1). We compare 900 y of inferred proxy flows to the streamflow variability recorded in recent decades, identifying extended periods of paleo low flow that exceeded the worst-case scenario in the instrumental records. We consider (i) the extent to which gauged flows (e.g., Fig. 3), which are the basis for surface water allocation, fail to capture the full range of hydroclimatic variability and extremes evident in a longer proxy record and (ii) the temporal evolution of low-frequency Athabasca River variability, related to the PDO and PNA, with its consequences for ARB surface water availability.Open in a separate windowFig. S1.The tree-ring sampling sites within and near the Athabasca River Basin in north-central Alberta. The colored triangles represent four coniferous tree species.     

One-step catalytic asymmetric synthesis of all-syn deoxypropionate motif from propylene: Total synthesis of (2R,4R,6R,8R)-2,4,6,8-tetramethyldecanoic acid

In nature, many complex structures are assembled from simple molecules by a series of tailored enzyme-catalyzed reactions. One representative example is the deoxypropionate motif, an alternately methylated alkyl chain containing multiple stereogenic centers, which is biosynthesized by a series of enzymatic reactions from simple building blocks. In organic synthesis, however, the majority of the reported routes require the syntheses of complex building blocks. Furthermore, multistep reactions with individual purifications are required at each elongation. Here we show the construction of the deoxypropionate structure from propylene in a single step to achieve a three-step synthesis of (2R,4R,6R,8R)-2,4,6,8-tetramethyldecanoic acid, a major acid component of a preen-gland wax of the graylag goose. To realize this strategy, we focused on the coordinative chain transfer polymerization and optimized the reaction condition to afford a stereo-controlled oligomer, which is contrastive to the other synthetic strategies developed to date that require 3–6 steps per unit, with unavoidable byproduct generation. Furthermore, multiple oligomers with different number of deoxypropionate units were isolated from one batch, showing application to the construction of library. Our strategy opens the door for facile synthetic routes toward other natural products that share the deoxypropionate motif.The deoxypropionate motif, an alternately methylated alkyl chain containing multiple stereogenic centers, is a common substructure found in natural products synthesized by bacteria, fungi, and plants (Fig. 1) (1). Because of the range of biological activities and abundance of this motif in natural products, its synthesis has received a great amount of attention (2, 3).Open in a separate windowFig. 1.Selected examples of natural products containing the deoxypropionate motif.In nature, the deoxypropionate motif is synthesized by using propionyl-CoA (or methylmalonyl-CoA) as a C3 building block (Fig. 2A). The deoxypropionate chain propagates by Claisen condensation of propionyl-CoA and acyl-CoA moiety at the chain end. After consecutive reduction of the β-ketone, dehydration, and asymmetric reduction of the carbon–carbon double bond, the deoxypropionate motif is elongated. We predicted that if the preparation of the deoxypropionate motif were possible by the asymmetric oligomerization of propylene, which is one of the simplest C3 building blocks, we could construct the analog of biosynthetic pathway in an even simplified manner (Fig. 2B).Open in a separate windowFig. 2.Synthesis of the deoxypropionate motif. (A) Biosynthetic scheme. (B) Synthesis by asymmetric oligomerization of propylene (current study). (C) Synthesis by iterative asymmetric carboalumination (7). (D) Synthesis by iterative asymmetric conjugate addition (11).To demonstrate our strategy, we chose (2R,4R,6R,8R)-2,4,6,8-tetramethyldecanoic acid 1 as a synthetic target. This carboxylic acid is a natural product containing the deoxypropionate motif, and is a major acid component of preen-gland wax of the graylag goose (4). Its total synthesis has been reported by two groups, both involving the oxidation of (2R,4R,6R,8R)-2,4,6,8-tetramethyldecan-1-ol 2. By using our strategy, this intermediate 2 can be constructed in a single step, significantly shortening the overall synthetic route.Conventionally, the deoxypropionate motif has been synthesized mainly using iterative reactions of complex building blocks or stoichiometric amount of organometallic reagents with unavoidable byproduct generation (e.g., inorganic salts) at each step. Previously reported synthetic routes include enolate alkylation (5, 6), carboalumination (7), organocuprate displacement (8), homologation of boronic esters (9), and conjugate addition (10). In addition, due to their iterative nature, long reaction sequences of 3–6 steps per unit were required to yield the desired products. As for the synthesis of 2, Liang et al. used an asymmetric carboalumination (Fig. 2C) (7), whereas ter Horst et al. used an asymmetric conjugate addition of methylcopper species (Fig. 2D) (11). Due to the iterative nature of methyl-branched chiral center formation, these syntheses required a total of 8–17 steps. Recently, convergent strategies for the synthesis of the deoxypropionate motif have been reported to shorten the synthetic route but generation of byproducts remains unavoidable (12, 13).Notably, propylene polymerization catalysts have rarely been used in the stereoselective oligomerization for synthesis of short oligomers, despite the great effort that has been devoted to the development of both homogeneous and heterogeneous catalysts for the highly isoselective propylene polymerization (14, 15). In 1987, Pino et al. reported an asymmetric propylene oligomerization catalyzed by enantiomerically pure chiral zirconocene in the presence of dihydrogen to afford saturated isotactic oligopropylenes (16). Kaminsky et al. later reported the preparation of moderately stereoregular oligomers with unsaturated chain end, which can be converted to other functional groups (17). To achieve natural product synthesis by the asymmetric oligomerization of propylene, a combination of stereoselectivity, functionalizability, and control over initiating groups is required. We herein report the one-step diastereoselective and enantioselective construction of the deoxypropionate motif by coordination chain transfer polymerization using an alkylmetal species as a chain-transfer agent (CTA) (18, 19). Our objective is to achieve highly stereoselective propylene oligomerization using this method. In addition, it is expected that the resulting oligomers will be end-capped with metals, thus enabling further functionalization.