Continental erosion and the Cenozoic rise of marine diatoms

Marine diatoms are silica-precipitating microalgae that account for over half of organic carbon burial in marine sediments and thus they play a key role in the global carbon cycle. Their evolutionary expansion during the Cenozoic era (66 Ma to present) has been associated with a superior competitive ability for silicic acid relative to other siliceous plankton such as radiolarians, which evolved by reducing the weight of their silica test. Here we use a mathematical model in which diatoms and radiolarians compete for silicic acid to show that the observed reduction in the weight of radiolarian tests is insufficient to explain the rise of diatoms. Using the lithium isotope record of seawater as a proxy of silicate rock weathering and erosion, we calculate changes in the input flux of silicic acid to the oceans. Our results indicate that the long-term massive erosion of continental silicates was critical to the subsequent success of diatoms in marine ecosystems over the last 40 My and suggest an increase in the strength and efficiency of the oceanic biological pump over this period.Unlike the majority of other phytoplankton, diatoms (unicellular photosynthetic microalgae) depend on the availability of silicic acid (in the form of orthosilicic acid—H₄SiO₄) to construct their cell walls (1, 2). Once the supply of H₄SiO₄ is exhausted, diatom blooms collapse and a large fraction sinks rapidly out of the surface layer to the ocean interior (Fig. 1). Over geological time, this phenomenon is thought to have decreased the concentration of H₄SiO₄ in the surface waters of the oceans to unprecedented levels in the history of Earth systems (3, 4), with important consequences for the biogeographic distribution and morphometric evolution of marine silicifiers (5–7).Open in a separate windowFig. 1.Global map showing the surface concentration of silicic acid in the modern oceans. Although biogenic silica tends to dissolve throughout the water column and in the sediments, enough silica is buried to keep the surface ocean undersaturated (contour lines, % biogenic silica in sediments) (23). In the modern oceans, diatoms thrive along continental margins, the Equatorial Pacific, and the Southern Ocean where increased inputs of H₄SiO₄ stimulate their growth. The locations of the Deep Sea Drilling Project and Ocean Drilling Program sites used to delineate the evolutionary trajectory of diatoms are also plotted (dots).Like diatoms, radiolarians (amoeboid protozoa, some of which harbor photosynthetic symbiotic algae) build intricate tests of amorphous silica by precipitating H₄SiO₄ from seawater. Fossil evidence strongly suggests that the rise of marine diatom diversity over the latter half of the Cenozoic era is mirrored by a simultaneous decrease in the weight of radiolarian tests (7–9), suggesting that an increasingly larger proportion of the ocean silica reservoir has been appropriated by diatoms. The most pronounced decrease in radiolarian test weight is coeval with a pulse of diatom diversity across the Eocene−Oligocene (E/O) transition (∼38–32 Ma), supporting the hypothesis that competition for H₄SiO₄ played a role in the rise of diatoms (7, 8). This evolutionary model assumes a constant (steady-state) input of H₄SiO₄ to the ocean. However, changes in the rates of continental weathering and erosion could also increase the concentration of H₄SiO₄ in the surface waters of the oceans over geological time, and thus represent a plausible alternative hypothesis for the rise of diatoms to ecological prominence (10, 11). Here we combine the analysis of data from sedimentary records with numerical simulations to quantify the extent to which the erosion of continental silicates facilitated the ecological expansion of marine diatoms during the Cenozoic era.     

Four hundred million years of silica biomineralization in land plants

Biomineralization plays a fundamental role in the global silicon cycle. Grasses are known to mobilize significant quantities of Si in the form of silica biominerals and dominate the terrestrial realm today, but they have relatively recent origins and only rose to taxonomic and ecological prominence within the Cenozoic Era. This raises questions regarding when and how the biological silica cycle evolved. To address these questions, we examined silica abundances of extant members of early-diverging land plant clades, which show that silica biomineralization is widespread across terrestrial plant linages. Particularly high silica abundances are observed in lycophytes and early-diverging ferns. However, silica biomineralization is rare within later-evolving gymnosperms, implying a complex evolutionary history within the seed plants. Electron microscopy and X-ray spectroscopy show that the most common silica-mineralized tissues include the vascular system, epidermal cells, and stomata, which is consistent with the hypothesis that biomineralization in plants is frequently coupled to transpiration. Furthermore, sequence, phylogenetic, and structural analysis of nodulin 26-like intrinsic proteins from diverse plant genomes points to a plastic and ancient capacity for silica accumulation within terrestrial plants. The integration of these two comparative biology approaches demonstrates that silica biomineralization has been an important process for land plants over the course of their >400 My evolutionary history.In modern ecosystems, land plants play a major role in the silica cycle through the accumulation and synthesis of amorphous biominerals composed of SiO₂, known as phytoliths or silica bodies. It is widely appreciated that actively accumulating plants such as grasses are important components of the terrestrial biological pump of silica (1–3). Plant silica also plays a key role in connecting the terrestrial and marine carbon cycles, because silica is an important nutrient for marine silica-biomineralizing primary producers (i.e., diatoms) (1, 2, 4–7). However, both grasses and diatoms evolved in the latter part of the Mesozoic Era (8–10) and rose to ecological dominance within the Cenozoic Era (6, 9, 11–14). Determining precisely when and how the terrestrial–marine silica teleconnections evolved remains an obstacle to reconstructing the history of the silica cycle.Direct analysis of silica bodies in the fossil record provides limited insight into this problem. When fossiliferous material is macerated, it is often challenging to identify whether residual silica bodies are the result of primary biomineralization or secondary diagenetic processes, and if a living plant origin is suspected, it is often difficult to assign taxonomic identity to the phytolith producer. In addition, with rare exceptions (e.g., ref. 15), lagerstätten that preserve exceptional anatomical detail in fossils, and might therefore be expected to preserve silica bodies, tend to be oversaturated with respect to silica (e.g., ref. 16) or extremely undersaturated with respect to silica (e.g., refs. 17, 18). To account for this, efforts to understand the history of silica biomineralization in terrestrial plants have taken a comparative biology approach (1, 5).Silica is widely used within plants for structural support and pathogen defense (19–21), but it remains a poorly understood aspect of plant biology. Recent work on the angiosperm Oryza sativa demonstrated that silica accumulation is facilitated by transmembrane proteins expressed in root cells (21–24). Phylogenetic analysis revealed that these silicon transport proteins were derived from a diverse family of modified aquaporins that include arsenite and glycerol transporters (19, 21, 25, 26). A different member of this aquaporin family was recently identified that enables silica uptake in the horsetail Equisetum, an early-diverging fern known to accumulate substantial amounts of silica (27). However, despite a growing number of fully sequenced genomes, angiosperm-type silicon transporters are not found within the gymnosperms or in spore-bearing plants, including plant lineages that are known to contain many weight-percent silica (25, 28) (Fig. 1). A more complete understanding of the distribution and mechanisms of silica accumulation within these early-diverging lineages is a necessary precondition for assessing the evolutionary history of silica biomineralization in terrestrial plants.Open in a separate windowFig. 1.Stratigraphic ranges (42) and evolutionary relationships (59–61) between major terrestrial plant lineages. Although the angiosperm macrofossil record only extends to the Early Cretaceous Period (62), a strict interpretation of their position as sister group to all other seed plant clades implies an earlier origin, shown here with a dashed line (60, 63). For the purposes of this article, we define Araucarian-type conifers as Araucariaceae and extinct relatives, and Podocarpus-type conifers as Podocarpaceae and extinct relatives. Filled stars mark clades that accumulate >1 wt % silica (dry matter), color coded for identified and unidentified silicon transport proteins.     

Microstructure provides insights into evolutionary design and resilience of Coscinodiscus sp. frustule

We conducted in situ three-point bending experiments on beams with roughly square cross-sections, which we fabricated from the frustule of Coscinodiscus sp. We observe failure by brittle fracture at an average stress of 1.1 GPa. Analysis of crack propagation and shell morphology reveals a differentiation in the function of the frustule layers with the basal layer pores, which deflect crack propagation. We calculated the relative density of the frustule to be ∼30% and show that at this density the frustule has the highest strength-to-density ratio of 1,702 kN⋅m/kg, a significant departure from all reported biologic materials. We also performed nanoindentation on both the single basal layer of the frustule as well as the girdle band and show that these components display similar mechanical properties that also agree well with bending tests. Transmission electron microscopy analysis reveals that the frustule is made almost entirely of amorphous silica with a nanocrystalline proximal layer. No flaws are observed within the frustule material down to 2 nm. Finite element simulations of the three-point bending experiments show that the basal layer carries most of the applied load whereas stresses within the cribrum and areolae layer are an order of magnitude lower. These results demonstrate the natural development of architecture in live organisms to simultaneously achieve light weight, strength, and exceptional structural integrity and may provide insight into evolutionary design.Diatoms are single-cell algae that form a hard cell wall made of a silica/organic composite. The ability to produce a functional biosilica shell presents several natural precedents that fascinate and inspire scientists and engineers. One fascinating aspect of such silica glass shells is their intricate, varied, and detailed architecture. Diatoms are generally classified based on the symmetry of their shells: Centric diatoms display radial symmetry whereas pennate diatoms have bilateral symmetry. Fig. 1A shows a schematic of a typical centric diatom and reveals that the shells are composed of two halves, called frustules, that fit together in a Petri-dish configuration. The frustules are attached to each other around the perimeter of the shell by one or several girdle bands. The frustules are usually porous with pore size and density varying between species. The frustule shell can also be composed of multiple layers with a cellular structure within the shell.Open in a separate windowFig. 1.(A) Schematic of the diatom frustule shell. (B) Cross-section of shell demonstrating the honeycomb sandwich plate configure of the silica shell. (C) Cribrum, the outer layer of the frustule shell, displays hexagonal arrangements of pores. (D) The basal plate, the inner layer of the frustule shell, is punctuated by reinforced pores called foramen.The proposed evolutionary functions for these intricate shell designs include nutrient acquisition, control of diatom sinking rate, control of turbulent flow around the cell, and protection from grazing and viral attack (1). Evidence in favor of a protective function is that the degree of shell silification depends on the environment, with greater amounts of silica found in shells grown in a predatory environment (2). As a deterrent to predation, the frustule makes use of an inherently brittle glass as a structurally protective material while balancing other evolutionary pressures. A denser shell may provide greater protection but will cause the diatom to sink beyond depths suitable for photosynthesis. A solid shell might also prevent exchange of resources and waste between the diatom cell and its environment. This requires adaption through control of the frustule micromorphology or modification of the constituent silica/organic composite material (3). The protective aspects of the frustule shell are clear; what remains an open question is how much the intricate pore structure and cellular design contribute to the amplified structural resilience vs. biological function.The size of most diatom species ranges from 2 to 200 µm (4, 5), which renders most of the traditional mechanical testing methods inadequate to characterize such complex materials; a few mechanical studies on diatoms have been reported (6–11). The majority of studies perform atomic force microscopy (AFM) indentation (6–9) on a full frustule of centric or pennate diatoms. Reported values of hardness ranged from 0.06–12 GPa and values of elastic modulus from 0.35–22.4 GPa. Differences in local pore structure and the nonplanar geometry of the frustule were often cited for the variance in mechanical properties. Three-point bending tests on beams that were extracted from the diatom frustule reported failure strengths of 336 ± 73 MPa but were complicated by local penetration of the indenter tip and tilting of the frustule during testing (10, 11).This overview demonstrates a wide range in the reported hardnesses and elastic moduli for biosilica shells. Most of these experiments were performed on full diatom shells, which in some instances contained organic cellular material; it is unclear whether the measured mechanical data represent the deformation of the constituent biosilica or the overall deformation of the shell through bending, local twisting, pivoting, and so on. Indentation using AFM can introduce inaccuracies such as tip sliding, and the resulting uncertainty in compliance within a single set of experiments, as well as among the data obtained with different instruments, makes it challenging to compare mechanical properties of the diatoms across the reported experiments. Within a single species, these mechanical data may provide qualitative trends in the structural response of the diatom shells; it is difficult to make any conclusions on the mechanical properties of the constituent biosilica. The mechanisms of silica biogenesis likely varies among the species (12), but it is unclear to what extent these differences reflect the variation in elastic modulus and hardness between species and within an individual frustule.To investigate the mechanical properties of the diatom frustule and constituent biosilica as decoupled from the full-shell structural response, we conducted in situ three-point bending experiments on beams with roughly 3.5-µm-square cross-sections fabricated from the frustule of Coscinodiscus sp. performed in a scanning electron microscope (SEM) equipped with a nanoindenter, as well as ex situ nanoindentation on an individual basal plate that had been isolated from a frustule and the girdle band. We determined the elastic modulus to be 36.4 ± 8.3 GPa and the failure strength to be 1.1 ± 0.3 GPa. We discuss these results, as well as deformation and failure mode of the diatoms, in the context of their atomic-level microstructure obtained from transmission electron microscopy (TEM) and finite element method (FEM) simulations of the three-point bending tests.     

Teamwork in the viscous oceanic microscale

Nutrient acquisition is crucial for oceanic microbes, and competitive solutions to solve this challenge have evolved among a range of unicellular protists. However, solitary solutions are not the only approach found in natural populations. A diverse array of oceanic protists form temporary or even long-lasting attachments to other protists and marine aggregates. Do these planktonic consortia provide benefits to their members? Here, we use empirical and modeling approaches to evaluate whether the relationship between a large centric diatom, Coscinodiscus wailesii, and a ciliate epibiont, Pseudovorticella coscinodisci, provides nutrient flux benefits to the host diatom. We find that fluid flows generated by ciliary beating can increase nutrient flux to a diatom cell surface four to 10 times that of a still cell without ciliate epibionts. This cosmopolitan species of diatom does not form consortia in all environments but frequently joins such consortia in nutrient-depleted waters. Our results demonstrate that symbiotic consortia provide a cooperative alternative of comparable or greater magnitude to sinking for enhancement of nutrient acquisition in challenging environments.

Global models of oxygen and carbon dioxide alterations depend upon transfer rates between small phytoplankton cells and surrounding surface waters of the world’s oceans (1–3). Although these cells are important on large scales, their individual interactions occur at microscopic dimensions that are dominated by viscosity. In this viscous environment, critical cellular processes, such as the exchange of nutrients, metabolites, and wastes, rely upon diffusion (4). While diffusion is an effective means of nutrient transport for the smallest microbes (5), it also creates a depleted region around the cell surface, referred to as the diffusion boundary layer (DBL), that limits nutrient consumption and cell growth (6, 7). The DBL for a cell at rest extends nine cell radii from the cell surface before the nutrient concentration reaches 90% of ambient levels (8), creating formidable disadvantages for nutrient acquisition by large cells requiring nutrient diffusion across large distances relative to their cell size (9). Phytoplankton have evolved mechanisms to mitigate the limitations of diffusive transfer rates by swimming or sinking (10) to generate relative motion between the cell and surrounding fluid. Diatoms—barrel-shaped, nonmotile eukaryotes—are considered to be one of the most ecologically important groups of phytoplankton (11–14) that absorb nutrients across their whole cell surface (15) but often increase sinking rates when experiencing nutrient limitation (16). Sinking thins the DBL surrounding the diatom cell and reduces the distance over which diffusion limits nutrient transport (8, 17). One potential disadvantage of this mechanism for DBL reduction is the high probability for a cell to sink out of sunlit regions, and sinking diatoms are major contributors to organic mass flux from surface to deep oceanic waters (1). A widespread but unevaluated alternative for such large cells involves teamwork with other smaller, motile cells that combine to form multicell consortia. Consortia are typically comprised of larger nonmotile host cells with smaller, surface-adhering motile cells termed epibionts. Although infrequently studied, epibionts are ubiquitous in the micrometer-scale world of planktonic organisms (18), and flagellated or ciliated epibionts often attach to larger objects (19) or marine snow (20). The selective forces favoring epibiont attachment remain in question. A range of fluid dynamic effects on prey encounter as well as biological factors such as reduced predator encounter risk or elevated prey availability surrounding host attachment sites have been proposed to explain the widespread nature of epibiont attachment (21, 22). However, the movement of motile epibiont cilia or flagella alters flows and, hence, creates an altered fluid dynamic environment surrounding the consortia. What are the consequences for the consortium host—does a larger, nonmotile cell benefit by membership in consortia? While advantages to the epibiont have been examined, the impacts on the host cells within a consortium have remained largely unconsidered. One reason for this is that physical associations between members of consortia are often temporary, and the short time scales of these relationships have hampered direct evaluation of the fluid mechanical interactions between consortium members.Here, we describe experimental work in combination with mathematical models that quantify the effect of the epibiont’s advective currents on nutrient availability to host cells within consortia formed by a large diatom, Coscinodiscus wailesii, and its peritrich ciliate epibiont, Pseudovorticella coscinodisci (Fig. 1A). Consortia composed of C. wailesii and P. coscinodisci are common along the Atlantic coast of South America (23, 24) and have provided a valuable opportunity for measuring fluid interactions characterizing a planktonic host–epibiont association.Open in a separate windowFig. 1.Diatom–ciliate association: (A) diatom C. wailesii and its epibiont P. coscinodisci. Scale bars represent 100 microns in length. (B) Flow around a single ciliate directed toward the diatom cell surface. (C) Velocity field for the same diatom–ciliate pair with a blue line indicating the transect line used for measurement of flow field velocities. The magenta segment represents the microcurrent component that directly intercepts the ciliary crown.     

From the Cover: Thick-shelled,grazer-protected diatoms decouple ocean carbon and silicon cycles in the iron-limited Antarctic Circumpolar Current

Diatoms of the iron-replete continental margins and North Atlantic are key exporters of organic carbon. In contrast, diatoms of the iron-limited Antarctic Circumpolar Current sequester silicon, but comparatively little carbon, in the underlying deep ocean and sediments. Because the Southern Ocean is the major hub of oceanic nutrient distribution, selective silicon sequestration there limits diatom blooms elsewhere and consequently the biotic carbon sequestration potential of the entire ocean. We investigated this paradox in an in situ iron fertilization experiment by comparing accumulation and sinking of diatom populations inside and outside the iron-fertilized patch over 5 wk. A bloom comprising various thin- and thick-shelled diatom species developed inside the patch despite the presence of large grazer populations. After the third week, most of the thinner-shelled diatom species underwent mass mortality, formed large, mucous aggregates, and sank out en masse (carbon sinkers). In contrast, thicker-shelled species, in particular Fragilariopsis kerguelensis, persisted in the surface layers, sank mainly empty shells continuously, and reduced silicate concentrations to similar levels both inside and outside the patch (silica sinkers). These patterns imply that thick-shelled, hence grazer-protected, diatom species evolved in response to heavy copepod grazing pressure in the presence of an abundant silicate supply. The ecology of these silica-sinking species decouples silicon and carbon cycles in the iron-limited Southern Ocean, whereas carbon-sinking species, when stimulated by iron fertilization, export more carbon per silicon. Our results suggest that large-scale iron fertilization of the silicate-rich Southern Ocean will not change silicon sequestration but will add carbon to the sinking silica flux.Diatoms—silica-shelled unicellular phytoplankton—are major exporters of organic carbon from the surface to the deep ocean and sediments and, hence, influence ocean nutrient cycles and atmospheric CO₂ levels (1, 2). However, silicate concentrations, for which diatoms have an obligate demand, vary widely over the nutrient-rich regions of the oceans (3). This is largely due to processes decoupling silicon cycling from that of other nutrients and carbon in surface waters of the Antarctic Zone (AZ), the southernmost belt of the Antarctic Circumpolar Current (ACC) (4). Thus, Si concentrations decline across the AZ from >70 mmol Si⋅m⁻³ in upwelling waters along its southern boundary (the Antarctic Divergence) (5) to <5 mmol Si⋅m⁻³ along the Antarctic Polar Front (APF) (6). The corresponding decline in nitrate is much smaller, from 30 to 23 mmol N⋅m⁻³. The resulting Si/N export ratio of 9/1 is much higher than the average diatom Si/N ratio of ∼1/1 (7, 8). The paradox (9) can partly be explained by increasing Si/N ratios with iron deficiency recorded in many species (10–13) in addition to the exceptionally thick frustules of some ACC diatom species (14), which can reach Si/N ratios of >4:1 in Fragilariopsis kerguelensis (15).A portion of the silica shells (frustules) sinking out of the northward-propagating surface Ekman layer dissolves in the southward-propagating deep water and is returned as Si to the surface in upwelling water along the Antarctic Divergence (1). This vertical recycling loop between surface and deep water supports growth of thick-shelled diatoms in the surface and functions as a global ocean silicon trap in the deep-water column. Another portion, mainly comprising robust frustules of comparatively few species, of which Fragilariopsis kerguelensis and Thalassiothrix antarctica are particularly common (16–18), is buried as diatom ooze in sediments underlying the iron-limited ACC, which functions as a major global silicon sink (19), accounting for 42–48% of the total marine silica removal (20). In contrast, the sediments underlying productive regions in the ACC, where phytoplankton blooms fertilized by iron input from land masses (21, 22), shelf sediments or dust occur regularly (23), have 10-fold higher carbon contents (>2% C of dry matter) (24), and are dominated by spores of the ubiquitous diatom genus Chaetoceros (25, 26).The massive removal of silicon relative to nitrogen from the surface layer by the diatoms of the low-productive, iron-limited AZ ecosystem implies that, in addition to the heavy silicification of ACC diatoms (14), a significant proportion of their nitrogen demand will have to be provided by a highly efficient recycling system in the surface layer (27). In contrast to phytoplankton, copepod-dominated zooplankton stocks of high-nutrient, low-chlorophyll (HNLC) regions of the oceans are comparatively large (28, 29). In fact, their grazing pressure was considered to control phytoplankton biomass in HNLC regions before iron limitation was firmly established (30). It has since been hypothesized that copepod feeding and defecation are part of the recycling system (31) and that the phytoplankton species that accumulate biomass in the face of heavy grazing pressure will have evolved some form of defense (32), most likely the heavily silicified frustules characteristic of ACC diatoms (14).As the Si-depleted northern ACC surface layer is the major source of nutrients upwelling in low latitudes (4), Si retention in the ACC constrains diatoms from forming blooms over large, nutrient-rich areas of the ocean (3) with far-reaching repercussions on food webs and ocean carbon sequestration. A better understanding of the deep water silicon trap and sedimentary sink is necessary to explain functioning of the glacial Southern Ocean (33) and its impact on CO₂ drawdown, but also to predict the response of Southern Ocean biota to large-scale and long-term artificial iron fertilization (34). Ocean iron fertilization experiments provide the necessary conditions for the quantitative investigation of these mechanisms because they simulate the effect of natural iron input on pelagic ecosystems with their full complement of grazers and pathogens (34).     

Chlorination of arenes via the degradation of toxic chlorophenols

Aryl chlorides are among the most versatile synthetic precursors, and yet inexpensive and benign chlorination techniques to produce them are underdeveloped. We propose a process to generate aryl chlorides by chloro-group transfer from chlorophenol pollutants to arenes during their mineralization, catalyzed by Cu(NO₃)₂/NaNO₃ under aerobic conditions. A wide range of arene substrates have been chlorinated using this process. Mechanistic studies show that the Cu catalyst acts in cooperation with NO_x species generated from the decomposition of NaNO₃ to regulate the formation of chlorine radicals that mediate the chlorination of arenes together with the mineralization of chlorophenol. The selective formation of aryl chlorides with the concomitant degradation of toxic chlorophenol pollutants represents a new approach in environmental pollutant detoxication. A reduction in the use of traditional chlorination reagents provides another (indirect) benefit of this procedure.

Chlorophenols are widely encountered moieties present in herbicides, drugs, and pesticides (1). Owing to the high dissociation energies of carbon‒chloride bonds, chlorophenols biodegrade very slowly, and their prolonged exposure leads to severe ecological and environmental problems (Fig. 1A) (2–4). Several well-established technologies have been developed for the treating of chlorophenols, including catalytic oxidation (5–11), biodegradation (12–15), solvent extraction (16, 17), and adsorption (18–20) Among these methods, adsorption is the most versatile and widely used method due to its high removal efficiency and simple operation, but the resulting products are of no value, and consequently, these processes are not viable.Open in a separate windowFig. 1.Background and reaction design. (A) Examples of chlorophenol pollutants. (B) Examples of aryl chlorides. (C) The chlorination process reported herein was based on chloro-group transfer from chlorophenol pollutants.With the extensive application of substitution reactions (21, 22), transfunctionalizations (23, 24), and cross-coupling reactions (25, 26), aryl chlorides are regarded as one of the most important building blocks widely used in the manufacture of polymers, pharmaceuticals, and other types of chemicals and materials (Fig. 1B) (27–31). Chlorination of arenes is usually carried out with toxic and corrosive reagents (32–34). Less toxic and more selective chlorination reagents tend to be expensive [e.g., chloroamides (35, 36)] and are not atom economic (37–39). Consequently, from the perspective of sustainability, the ability to transfer a chloro group from unwanted chlorophenols to other substrates would be advantageous.Catalytic isofunctional reactions, including transfer hydrogenation and alkene metathesis, have been widely exploited in organic synthesis. We hypothesized that chlorination of arenes also could be achieved by chloro-group transfer, and since stockpiles of chlorophenols tend to be destroyed by mineralization and chlorophenol pollutants may be concentrated by adsorption (18–20), they could be valorized as chlorination reagents via transfer of the chloro group to arene substrates during their mineralization, thereby adding value to the destruction process (Fig. 1C). Although chlorophenol pollutants are not benign, their application as chlorination reagents, with their concomitant destruction to harmless compounds, may be considered as not only meeting the criteria of green chemistry but also potentially surpassing it. Herein, we describe a robust strategy to realize chloro-group transfer from chlorophenol pollutants to arenes and afford a wide range of value-added aryl chlorides.     

Electrochemical borylation of carboxylic acids

A simple electrochemically mediated method for the conversion of alkyl carboxylic acids to their borylated congeners is presented. This protocol features an undivided cell setup with inexpensive carbon-based electrodes and exhibits a broad substrate scope and scalability in both flow and batch reactors. The use of this method in challenging contexts is exemplified with a modular formal synthesis of jawsamycin, a natural product harboring five cyclopropane rings.

Boronic acids are among the most malleable functional groups in organic chemistry as they can be converted into almost any other functionality (1–3). Aside from these versatile interconversions, their use in the pharmaceutical industry is gaining traction, resulting in approved drugs such as Velcade, Ninlaro, and Vabomere (4). It has been shown that boronic acids can be rapidly installed from simple alkyl halides (5–19) or alkyl carboxylic acids through the intermediacy of redox-active esters (RAEs) (Fig. 1A) (20–24). Our laboratory has shown that both Ni (20) and Cu (21) can facilitate this reaction. Conversely, Aggarwal and coworkers (22) and Li and coworkers (23) demonstrated photochemical variations of the same transformation. While these state-of-the-art approaches provide complementary access to alkyl boronic acids, each one poses certain challenges. For example, the requirement of excess boron source and pyrophoric MeLi under Ni catalysis is not ideal. Although more cost-effective and operationally simple, Cu-catalyzed borylation conditions can be challenging on scale due to the heterogeneity resulting from the large excess of LiOH•H₂O required. In addition to its limited scope, Li and coworkers’ protocol requires 4 equivalence of B₂pin₂ and an expensive Ir photocatalyst. The simplicity of Aggarwal and coworkers’ approach is appealing in this regard and represents an important precedent for the current study.Open in a separate windowFig. 1.(A) Prior approaches to access alkyl boronic esters from activated acids. (B) Inspiration for initiating SET events electrochemically to achieve borylation. (C) Summary of this work.At the heart of each method described above, the underlying mechanism relies on a single electron transfer (SET) event to promote decarboxylation and form an alkyl radical species. In parallel, the related borylation of aryl halides via a highly reactive aryl radical can also be promoted by SET. While numerous methods have demonstrated that light can trigger this mechanism (Fig. 1B) (16, 25–31), simple electrochemical cathodic reduction can elicit the same outcome (32–35). It was postulated that similar electrochemically driven reactivity could be translated to alkyl RAEs. The development of such a transformation would be highly enabling, as synthetic organic electrochemistry allows the direct addition or removal of electrons to a reaction, representing an incredibly efficient way to forge new bonds (36–40). This disclosure reports a mild, scalable, and operationally simple electrochemical decarboxylative borylation (Fig. 1C) not reliant on transition metals or stoichiometric reductants. In addition to mechanistic studies of this interesting transformation, applications to a variety of alkyl RAEs, comparison to known decarboxylative borylation methods, and a formal synthesis of the polycyclopropane natural product jawsamycin [(–)-FR-900848] are presented.     

Pulsed rise and growth of the Tibetan Plateau to its northern margin since ca. 30 Ma

The onset of mountain building along margins of the Tibetan Plateau provides a key constraint on the processes by which the high topography in Eurasia formed. Although progressive expansion of thickened crust underpins most models, several studies suggest that the northern extent of the plateau was established early, soon after the collision between India and Eurasia at ca. 50 Ma. This inference relies heavily on the age and provenance of Cenozoic sediments preserved in the Qaidam basin. Here, we present evidence in the northern plateau for a considerably younger inception and evolution of the Qaidam basin, based on magnetostratigraphies combined with detrital apatite fission-track ages that date the basin fills to be from ca. 30 to 4.8 Ma. Detrital zircon-provenance analyses coupled with paleocurrents reveal that two-stage growth of the Qilian Shan in the northeastern margin of the Tibetan Plateau began at ca. 30 and at 10 Ma, respectively. Evidence for ca. 30 and 10 to 15 Ma widespread synchronous deformation throughout the Tibetan Plateau and its margins suggests that these two stages of outward growth may have resulted from the removal of mantle lithosphere beneath different portions of the Tibetan Plateau.

Geodynamic processes in the interior of broad, high-elevation orogenic plateaus have a pronounced effect on the outward growth of those plateaus over time (1–3). The Cenozoic collision of India and Eurasia has built the high-elevation Tibetan Plateau (>4.5 km) that extends almost 2,000 km from the Himalaya into central Asia. Resolving the geodynamic processes that controlled development of high topography in Tibet underpins understanding the nature of intracontinental deformation (3–5), mechanisms of surface uplift of orogenic plateaus (2, 6, 7), and their impact on regional and global climate change (8, 9). In the northeastern Tibetan Plateau, numerous Cenozoic basins are separated by narrow mountain ranges (Fig. 1A); deformation within the basins and growth of the intervening mountain ranges (10) have led some to propose geodynamic mechanisms for the outward and upward growth of the Tibetan Plateau and its northeastern margin including: 1) progressive, south-to-north propagation in stepwise fashion along lithospheric strike-slip faults (3, 11); 2) lateral flow of middle-lower crust from the main body of the Tibetan Plateau into its margins (2, 6, 12); and 3) the removal of mantle lithosphere beneath all or part of the Tibetan Plateau (1, 4, 13). All of these models rely heavily on the ages of Cenozoic sediments preserved in Tibetan basins.Open in a separate windowFig. 1.(A) Regional shaded relief map of the Tibetan Plateau showing major faults, terranes, volcanic rock ages, and paleoelevation study sites. The black rectangles outline extent of B and the swath of Fig. 5 B and C. (B) Generalized tectonic and topographic map of the East Kunlun Shan, Qilian Shan, and the Qaidam basin with magnetostratigraphic section locations (solid squares) in the north margin of the basin. ATF, Altyn Tagh fault; EKLF, East Kunlun fault; NQTB, North Qaidam thrust belt; and HYF, Haiyuan fault. (C) Geological map of the Hongshan region [modified after Qinghai Bureau of Geology and Mineral Resources (17)]. Shown are the distributions of Cenozoic stratigraphic units (Lulehe, Xia Ganchaigou, Shang Ganchaigou, Xia Youshashan, Shang Youshashan, Shizigou, and Qigequan formations) in the study area and Hongshan West section (WS) as well as Hongshan East section (ES).As the largest Cenozoic basin with >10-km–thick deposits in the northeastern Tibetan Plateau (14), the Qaidam basin’s inception, development, and extinction provides critical insights into the timing, processes, and mechanisms of Tibetan Plateau growth. Here, we combine detrital zircon provenance with magnetostratigraphies and detrital apatite fission-track (DAFT) from the Hongshan East and West sections in the northern basin margin (Fig. 1B) to decipher the inception of Cenozoic sedimentation in the Qaidam basin and the emergence of ranges around it. Constrained by youngest detrital apatite fission-track peak ages, our paleomagnetic investigation provides a temporal framework for the Qaidam basin depositions spanning from ca. 30 to 4.8 Ma, within which detrital zircon provenance analysis reveals the ca. 30 Ma initial growth of the South Qilian Shan, followed by a second accelerated growth of the entire Qilian Shan since ca. 10 Ma. We compare this growth history with major deformational events within the central and northern Tibetan Plateau to demonstrate that pulses of outward growth on the northern margin of Tibetan Plateau occurred during pulses of rapid uplift in the plateau’s interior. Such pulsed uplift is consistent with multistage removal of dense lower lithosphere as an important process governing the upward and outward growth of orogenic plateaus in collisional tectonic settings.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

Volatile-consuming reactions fracture rocks and self-accelerate fluid flow in the lithosphere

Hydration and carbonation reactions within the Earth cause an increase in solid volume by up to several tens of vol%, which can induce stress and rock fracture. Observations of naturally hydrated and carbonated peridotite suggest that permeability and fluid flow are enhanced by reaction-induced fracturing. However, permeability enhancement during solid-volume–increasing reactions has not been achieved in the laboratory, and the mechanisms of reaction-accelerated fluid flow remain largely unknown. Here, we present experimental evidence of significant permeability enhancement by volume-increasing reactions under confining pressure. The hydromechanical behavior of hydration of sintered periclase [MgO + H₂O → Mg(OH)₂] depends mainly on the initial pore-fluid connectivity. Permeability increased by three orders of magnitude for low-connectivity samples, whereas it decreased by two orders of magnitude for high-connectivity samples. Permeability enhancement was caused by hierarchical fracturing of the reacting materials, whereas a decrease was associated with homogeneous pore clogging by the reaction products. These behaviors suggest that the fluid flow rate, relative to reaction rate, is the main control on hydromechanical evolution during volume-increasing reactions. We suggest that an extremely high reaction rate and low pore-fluid connectivity lead to local stress perturbations and are essential for reaction-induced fracturing and accelerated fluid flow during hydration/carbonation.

Hydration and carbonation reactions in the crust and mantle transport H₂O and CO₂ from Earth’s surface to the interior and control volatile budgets within the Earth (1–6). These reactions are characterized by solid-volume increase, by up to several tens of vol%, which induces stress that may lead to fracturing (7–10). The driving force of such stress generation is the thermodynamic free energy released when metastable anhydrous/noncarbonate minerals react with fluids (7). The stress generated by the reaction has the potential to cause rock fracture and fragmentation (7, 11–13), thereby increasing the reactive surface area and fluid flow and further accelerating the reactions (7, 8, 14). Such chemical breaking of rocks, or reaction-induced fracturing, appears to be important in driving hydration and carbonation reactions to completion (8, 15, 16) in an otherwise self-limiting process where reaction products can clog pores and suppress fluid flow, thereby hindering the reaction (15, 17).Observations of naturally serpentinized and fractured ultramafic rocks indicate a volume increase of 20 to 60% during hydration reactions (13, 18–20), providing evidence of an accelerated supply of fluids during hydration (Fig. 1 A and B). Natural carbonation of ultramafic rocks is also associated with extensive fracture networks, and reaction-induced fracturing is considered a key process in mineral carbonation (Fig. 1C) (7, 8, 21). Numerical simulations indicate a positive feedback between volume-increasing reaction, fracturing, and fluid flow (10, 22–32). Laboratory experiments partially reproduce fracturing during peridotite carbonation, serpentinization, and periclase hydration (29, 33–36); however, hydrothermal flow-through experiments of peridotite serpentinization and carbonation show a decrease in permeability and deceleration of fluid flow and reaction rate (37–42). Observations of the natural carbonation of serpentinized peridotite indicate the decrease in permeability and reduced fluid flow and reaction rate are a consequence of pore clogging related to carbonation (43). Until now, no experimental studies have shown a clear increase in permeability during expansive fluid–rock reactions under confining pressure. As such, despite their geological and environmental importance, the evolution of expansive fluid–rock reactions remains difficult to predict, owing to the complex hydraulic–chemical–mechanical feedbacks underlying these reactions (15, 16, 44). The processes controlling the self-acceleration or deceleration of these reactions remain largely unknown.Open in a separate windowFig. 1.Reaction-induced fractures related to natural hydration/carbonation. (A) Polygonal block of serpentinite cut by planar lizardite veins, extracted from a serpentinite body, San Andreas Lake, California. (B) Photomicrograph of mesh structure in partly serpentinized peridotite, Redwood City serpentinite, California [crossed-polarized light (61)]. (C) Quartz veins in silica–carbonate rocks (i.e., listvenite, a carbonated ultramafic rock) that occur along the boundaries of serpentinite bodies, San Jose, California. ol, olivine; serp, serpentine (lizardite ± antigorite mixture); br, brucite.Here, we use the hydration of periclase to brucite [MgO + H₂O → Mg(OH)₂] as an analog for solid-volume–increasing reactions in the Earth. This reaction produces an extreme solid-volume increase of 119%, with a high reaction rate at 100 to 600 °C (45). Previous experimental studies on periclase hydration have revealed that extensive fracturing occurs under certain conditions (29, 33, 35), yet the links between fracturing experiments (periclase hydration), nonfracturing experiments (peridotite hydration/carbonation), and natural observations are unknown. On the basis of in situ observations of fluid flow during the reactions, we clearly show that fluid flow and associated permeability are strongly enhanced by solid-volume–increasing reactions under confining pressure (i.e., at simulated depth). Based on the experimental results and nondimensional parameterization, we propose that the ratio of the initial fluid flow rate to the reaction rate has a primary control on the self-acceleration and deceleration of fluid flow and reactions during hydration and carbonation within the Earth.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Chirality-matched catalyst-controlled macrocyclization reactions

Macrocycles, formally defined as compounds that contain a ring with 12 or more atoms, continue to attract great interest due to their important applications in physical, pharmacological, and environmental sciences. In syntheses of macrocyclic compounds, promoting intramolecular over intermolecular reactions in the ring-closing step is often a key challenge. Furthermore, syntheses of macrocycles with stereogenic elements confer an additional challenge, while access to such macrocycles are of great interest. Herein, we report the remarkable effect peptide-based catalysts can have in promoting efficient macrocyclization reactions. We show that the chirality of the catalyst is essential for promoting favorable, matched transition-state relationships that favor macrocyclization of substrates with preexisting stereogenic elements; curiously, the chirality of the catalyst is essential for successful reactions, even though no new static (i.e., not “dynamic”) stereogenic elements are created. Control experiments involving either achiral variants of the catalyst or the enantiomeric form of the catalyst fail to deliver the macrocycles in significant quantity in head-to-head comparisons. The generality of the phenomenon, demonstrated here with a number of substrates, stimulates analogies to enzymatic catalysts that produce naturally occurring macrocycles, presumably through related, catalyst-defined peripheral interactions with their acyclic substrates.

Macrocyclic compounds are known to perform a myriad of functions in the physical and biological sciences. From cyclodextrins that mediate analyte separations (1) to porphyrin cofactors that sit in enzyme active sites (2, 3) and to potent biologically active, macrocyclic natural products (4) and synthetic variants (5–7), these structures underpin a wide variety of molecular functions (Fig. 1A). In drug development, such compounds are highly coveted, as their conformationally restricted structures can lead to higher affinity for the desired target and often confer additional metabolic stability (8–13). Accordingly, there exists an entire synthetic chemistry enterprise focused on efficient formation and functionalization of macrocycles (14–18).Open in a separate windowFig. 1.(A) Examples of macrocyclic compounds with important applications. HCV, hepatitis C virus. (B) Use of chiral ligands in metal-catalyzed or mediated stereoselective macrocyclization reactions. (C) Remote desymmetrization using guanidinylated ligands via Ullmann coupling. (D) This work: use of copper/peptidyl complexes for macrocyclization and the exploration of matched and mismatched effect.In syntheses of macrocyclic compounds, the ring-closing step is often considered the most challenging step, as competing di- and oligomerization pathways must be overcome to favor the intramolecular reaction (14). High-dilution conditions are commonly employed to favor macrocyclization of linear precursors (19). Substrate preorganization can also play a key role in overcoming otherwise high entropic barriers associated with multiple conformational states that are not suited for ring formation. Such preorganization is most often achieved in synthetic chemistry through substrate design (14, 20–22). Catalyst or reagent controls that impose conformational benefits that favor ring formation are less well known. Yet, critical precedents include templating through metal-substrate complexation (23, 24), catalysis by foldamers (25) or enzymes (26–29), or, in rare instances, by small molecules (discussed below). Characterization of biosynthetic macrocyclization also points to related mechanistic issues and attributes for efficient macrocyclizations (30–34). Coupling macrocyclization reactions to the creation of stereogenic elements is also rare (35). Metal-mediated reactions have been applied toward stereoselective macrocyclizations wherein chiral ligands transmit stereochemical information to the products (Fig. 1B). For example, atroposelective ring closure via Heck coupling has been applied in the asymmetric total synthesis of isoplagiochin D by Speicher and coworkers (36–40). Similarly, atroposelective syntheses of (+)-galeon and other diarylether heptanoid natural products were achieved via Ullman coupling using N-methyl proline by Salih and Beaudry (41). Finally, Reddy and Corey reported the enantioselective syntheses of cyclic terpenes by In-catalyzed allylation utilizing a chiral prolinol-based ligand (42). While these examples collectively illustrate the utility of chiral ligands in stereoselective macrocyclizations, such examples remain limited.We envisioned a different role for chiral catalysts when addressing intrinsically disfavored macrocyclization reactions. When unfavorable macrocyclization reactions are confronted, we hypothesized that a catalyst–substrate interaction might provide transient conformational restriction that could promote macrocyclization. To address this question, we chose to explore whether or not a chiral catalyst-controlled macrocyclization might be possible with peptidyl copper complexes. In the context of the medicinally ubiquitous diarylmethane scaffold, we had previously demonstrated the capacity for remote asymmetric induction in a series of bimolecular desymmetrizations using bifunctional, tetramethylguanidinylated peptide ligands. For example, we showed that peptidyl copper complexes were able to differentiate between the two aryl bromides during C–C, C–O, and C–N cross-coupling reactions (Fig. 1C) (43–45). Moreover, in these intermolecular desymmetrizations, a correlation between enantioselectivity and conversion was observed, revealing the catalyst’s ability to perform not only enantiotopic group discrimination but also kinetic resolution on the monocoupled product as the reaction proceeds (44). This latter observation stimulated our speculation that if an internal nucleophile were present to undergo intramolecular cross-coupling to form a macrocycle, stereochemically sensitive interactions (so-called matched and mismatched effects) (46) could be observed (Fig. 1D). Ideally, we anticipated that transition state–stabilizing interactions might even prove decisive in matched cases, and the absence of catalyst–substrate stabilizing interactions might account for the absence of macrocyclization for these otherwise intrinsically unfavorable reactions. Herein, we disclose the explicit observation of these effects in chiral catalyst-controlled macrocyclization reactions.     

Volcanically driven lacustrine ecosystem changes during the Carnian Pluvial Episode (Late Triassic)

The Late Triassic Carnian Pluvial Episode (CPE) saw a dramatic increase in global humidity and temperature that has been linked to the large-scale volcanism of the Wrangellia large igneous province. The climatic changes coincide with a major biological turnover on land that included the ascent of the dinosaurs and the origin of modern conifers. However, linking the disparate cause and effects of the CPE has yet to be achieved because of the lack of a detailed terrestrial record of these events. Here, we present a multidisciplinary record of volcanism and environmental change from an expanded Carnian lake succession of the Jiyuan Basin, North China. New U–Pb zircon dating, high-resolution chemostratigraphy, and palynological and sedimentological data reveal that terrestrial conditions in the region were in remarkable lockstep with the large-scale volcanism. Using the sedimentary mercury record as a proxy for eruptions reveals four discrete episodes during the CPE interval (ca. 234.0 to 232.4 Ma). Each eruptive phase correlated with large, negative C isotope excursions and major climatic changes to more humid conditions (marked by increased importance of hygrophytic plants), lake expansion, and eutrophication. Our results show that large igneous province eruptions can occur in multiple, discrete pulses, rather than showing a simple acme-and-decline history, and demonstrate their powerful ability to alter the global C cycle, cause climate change, and drive macroevolution, at least in the Triassic.

The Carnian Pluvial Episode (CPE; ca. 234 to ∼232 Ma; Late Triassic) was an interval of significant changes in global climate and biotas (1, 2). It was characterized by warming (3, 4) and enhancement of the hydrological cycle (5–7), linked to repeated C isotope fluctuations (8–11) and accompanied by increased rainfall (1), intensified continental weathering (9, 12), shutdown of carbonate platforms (13), widespread marine anoxia (4), and substantial biological turnover (1, 2, 10). Available stratigraphic data indicate that the Carnian climatic changes broadly coincide with, and could have been driven by, the emplacement of the Wrangellia large igneous province (LIP) (2, 4, 7, 8, 10, 14, 15) (Fig. 1A). It is postulated that the voluminous emission of volcanic CO₂, with consequent global warming and enhancement of a mega-monsoonal climate, was responsible for the CPE (9, 16), although the link is imprecise (2, 17) because the interval of Wrangellian eruptions have not yet been traced in the sedimentary records encompassing the CPE.Open in a separate windowFig. 1.Location and geological context for the study area. (A) Paleogeographic reconstruction for the Carnian (∼237 to 227 Ma) Stage (Late Triassic), showing locations of the study area and volcanic centers (revised after ref. 4, with volcanic data from refs. 4, 7, 49, and 50). (B) Tectono-paleogeographic map of the NCP during the Late Triassic (modified from ref. 21), showing the location of the study area. (C) Stratigraphic framework of the Upper Chunshuyao Formation (CSY) to the Lower Yangshuzhuang (YSZ) Formation from the Jiyuan Basin (modified from ref. 20). Abbreviations: LIP, Large Igneous Province; QDOB, Qingling-Dabie Orogenic Belt; S-NCP, southern NCP; SCP, South China Plate; Fm., Formation; m & s, coal, mudstone, and silty mudstone; s., sandstone; c, conglomerate; Dep. env., Depositional environment; and C.-P., Coniopteris-Phoenicopsis.The CPE was originally identified because of changes in terrestrial sedimentation, but most subsequent studies have been on marine strata (2, 4, 7–10). By contrast, much less is known about the effects of this climatic episode on terrestrial environments (2), although there were major extinctions and radiations among animals (including dinosaurs, crocodiles, turtles, and the first mammals and insects) and modern conifer families (2). Some of the new organisms may have flourished because of the spread of humid environments, such as the turtles and metoposaurids (18, 19).In this study, we have investigated terrestrial sediments from the Zuanjing-1 (ZJ-1) borehole in the Jiyuan Basin of the southern North China Plate (NCP) and use zircon U–Pb ages from two tuffaceous claystone horizons, fossil plant biostratigraphy, and organic C isotope (δ¹³C_org) and Hg chemostratigraphy to identify the CPE and volcanic activity.     

Number neurons in the nidopallium of young domestic chicks

Numerical cognition is ubiquitous in the animal kingdom. Domestic chicks are a widely used developmental model for studying numerical cognition. Soon after hatching, chicks can perform sophisticated numerical tasks. Nevertheless, the neural basis of their numerical abilities has remained unknown. Here, we describe number neurons in the caudal nidopallium (functionally equivalent to the mammalian prefrontal cortex) of young domestic chicks. Number neurons that we found in young chicks showed remarkable similarities to those in the prefrontal cortex and caudal nidopallium of adult animals. Thus, our results suggest that numerosity perception based on number neurons might be an inborn feature of the vertebrate brain.

Be it a number of conspecifics in a group (1), a number of food items (2), or a number of motifs in a song (3), correct estimation of quantities is of vital importance for animals. Several behavioral studies have confirmed that numerical competence is not a prerogative of human beings but is a widespread phenomenon in the animal kingdom (reviewed by refs. 4 and 5). Mammals (6–8), birds (3, 9, 10), reptilians (11), amphibians (12), fishes (13), and invertebrates (14), although evolutionarily distant, all can spontaneously assess quantities using an approximate number system (15).For the approximate number system, which is based on Weber’s law (16), the perception of cardinal numbers resembles the perception of continuous physical stimuli, and the just noticeable difference is proportionate to the quantity being estimated. As a consequence, discrimination of quantities is imprecise and depends on the numerical distance between stimuli. In other words, it is easier to tell apart 5 and 10 than 9 and 10. Moreover, discrimination of quantities becomes increasingly difficult with the numerical size. For a given numerical distance (e.g., one), it is easier to discriminate between numbers with low magnitudes (1 vs. 2) than with high magnitudes (9 vs. 10).Recent research has uncovered that the approximate number system relies on the activity of a specific neuronal population. Neurons that respond to abstract numerosity irrespective of objects’ physical appearance (shape, color, size) have been found in the forebrain of human and nonhuman primates (17, 18) and in crows (19). In mammals, numerical responses were recorded in the parietal and the prefrontal cortices (PFCs) (17). In birds, similar neurons have been described in the caudolateral nidopallium (NCL) (19). The NCL is believed to be an analog of the PFC in the avian brain (20) and is involved in a variety of cognitive processes, including memory formation (21, 22), abstract rule learning (23), and action planning (24).Both monkeys and crows are among the most evolutionarily advanced species of their phylogenetic groups. They independently developed sophisticated intellectual capacities (25), and both possess enlarged forebrains (26). The neural representations of numerosities described in these species also share remarkable similarities (19, 27–29). In both species, the number neurons show the strongest response to a preferred numerosity, which gradually decreases along with the numerical distance (numerical distance effect, but see ref. 30). Their tuning curves are skewed toward larger numerosities and become progressively broader (less selective) with increasing numerosities (numerical size effect). However, it is unclear whether the presence of similar number neurons in these two species emerges as a consequence of their elaborate cognitive skills and enlarged forebrains. To understand the evolution of the number sense, we need to explore its neural correlates in distant bird species with more ancestral traits.Moreover, until now, number neurons have been described only in adult animals (e.g., refs. 19, 27–29, and 31). At the same time, behavioral data from human infants (32) and young domestic chicks (10, 33) indicate that some core numerical abilities might be an inborn or spontaneously emerging (34, 35) property of the vertebrate brain. Testing the presence of number neurons in young and untrained organisms is crucial to verify this hypothesis.In our study, we aimed to describe the neural correlates of the number sense in domestic chicks (Gallus gallus), which belong to a sister group of modern Neoaves (36). The domestic chick is a well-established developmental model for studying numerical cognition. Soon after hatching, these birds are already capable of discriminating quantities (33, 37) and even performing basic arithmetic operations (10). It has also been shown that young chicks represent numbers across the mental number line (38), a cognitive ability that had been previously attributed only to humans.We hypothesized that neural processing of numerical information in young untrained chicks might be similar to that in crows, despite them having evolved independently over the last ∼70 million years (36). In a domestic chicken, the NCL is morphologically different from that of corvids (39), but it is unclear whether this reflects any functional difference. Therefore, we decided to search for neural responses to numerical stimuli in the NCL of domestic chicks. For this purpose, we habituated young chicks to a computer monitor, where numerical stimuli were presented (Fig. 1A). We explored neural responses to numerosities from one to five. To control for nonnumerical parameters, we presented three different categories of stimuli: “radius-fixed,” “area-fixed,” and “perimeter-fixed” (Fig. 1B).Open in a separate windowFig. 1.Experimental design. (A) Schematic drawing of the experimental setup. Young chicks were placed in a small wooden box in front of the screen, where numerical stimuli appeared. They were trained to pay attention to the stimuli without any further discrimination between different numerosities. (B) Examples of different types of numerosity stimuli that we presented in every neural recording: “radius-fixed,” “area-fixed,” and “perimeter-fixed.”     

A 5,000-year vegetation and fire history for tierra firme forests in the Medio Putumayo-Algodón watersheds,northeastern Peru

This paper addresses an important debate in Amazonian studies; namely, the scale, intensity, and nature of human modification of the forests in prehistory. Phytolith and charcoal analysis of terrestrial soils underneath mature tierra firme (nonflooded, nonriverine) forests in the remote Medio Putumayo-Algodón watersheds, northeastern Peru, provide a vegetation and fire history spanning at least the past 5,000 y. A tree inventory carried out in the region enables calibration of ancient phytolith records with standing vegetation and estimates of palm species densities on the landscape through time. Phytolith records show no evidence for forest clearing or agriculture with major annual seed and root crops. Frequencies of important economic palms such as Oenocarpus, Euterpe, Bactris, and Astrocaryum spp., some of which contain hyperdominant species in the modern flora, do not increase through prehistoric time. This indicates pre-Columbian occupations, if documented in the region with future research, did not significantly increase the abundance of those species through management or cultivation. Phytoliths from other arboreal and woody species similarly reflect a stable forest structure and diversity throughout the records. Charcoal ¹⁴C dates evidence local forest burning between ca. 2,800 and 1,400 y ago. Our data support previous research indicating that considerable areas of some Amazonian tierra firme forests were not significantly impacted by human activities during the prehistoric era. Rather, it appears that over the last 5,000 y, indigenous populations in this region coexisted with, and helped maintain, large expanses of relatively unmodified forest, as they continue to do today.

More than 50 y ago, prominent scholars argued that due to severe environmental constraints (e.g., poor natural resources), prehistoric cultures in the Amazon Basin were mainly small and mobile with little cultural complexity, and exerted low environmental impacts (1, 2). Contentious debates ensued and have been ongoing ever since. Empirical data accumulated during the past 10 to 20 y have made it clear that during the late Holocene beginning about 3,000 y ago dense, permanent settlements with considerable cultural complexity had developed along major watercourses and some of their tributaries, in seasonal savannas/areas of poor drainage, and in seasonally dry forest. These populations exerted significant, sometimes profound, regional-scale impacts on landscapes, including with raised agricultural fields, fish weirs, mound settlements, roads, geometric earthworks called geoglyphs, and the presence of highly modified anthropic soils, called terra pretas or “Amazonian Dark Earths” (Fig. 1) (e.g., refs. 3–15).Open in a separate windowFig. 1.Location of study region (MP-A) and other Amazonian sites discussed in the text. River names are in blue. The black numbers represent major pre-Columbian archaeological sites with extensive human alterations (1, Marajó Island; 2, Santarém; 3, Upper Xingu; 4, Central Amazon Project; 5, Bolivian sites) (3, 5–10, 14, 15). ADE, terra preta locations (e.g., refs. 19 and 20); triangles are geoglyph sites (6, 8). The white circles are terrestrial soil locations previously studied by Piperno and McMichael (29, 31–33, 54) (Ac, Acre; Am, Amacayacu; Ay, Lake Ayauchi; B, Barcelos; GP, lakes Gentry-Parker; Iq, Iquitos to Nauta; LA, Los Amigos; PVM, Porto Velho to Manaus; T, Tefe).An important, current debate that frames this paper centers not on whether some regions of the pre-Columbian Amazon supported large and complex human societies, but rather on the spatial scales, degrees, and types of cultural impacts across this continental-size landscape. Some investigators drawing largely on available archaeological data and studies of modern floristic composition of selected forests, argue that heavily modified “domesticated” landscapes were widespread across Amazonia at the end of prehistory, and these impacts significantly structure the vegetation today, even promoting higher diversity than before (e.g., refs. 14–21). It is believed that widespread forms of agroforestry with planted, orchard-like formations or other forest management strategies involving the care and possible enrichment of several dozens of economically important native species have resulted in long-term legacies left on forest composition (e.g., refs. 14–22). Some (20) propose that human influences played strong roles in the enrichment of “hyperdominant” trees, which are disproportionately common elements in the modern flora (sensu ref. 23). Some even argue that prehistoric fires and forest clearance were so spatially extensive that post-Columbian reforestation upon the tragic consequences of European contact was a principal contributor to decreasing atmospheric CO₂ levels and the onset of the “Little Ice Age” (24, 25).However, modern floristic studies are often located in the vicinity of known archaeological sites and/or near watercourses (26). Many edible trees in these studies are early successional and would not be expected to remain as significant forest elements for hundreds of years after abandonment. Historic-period impacts well-known in some regions to have been profound have been paid little attention and may be mistaken for prehistoric legacies (26–28). Moreover, existing phytolith and charcoal data from terrestrial soils underneath standing tierra firme forest in some areas of the central and western Amazon with no known archaeological occupations nearby exhibit little to no evidence for long-term human occupation, anthropic soils, agriculture, forest clearing or other significant vegetation change, or recurrent/extensive fires during the past several thousand years (Fig. 1) (29–33). Even such analyses of terrestrial soils of lake watersheds in western Amazonia known to have been occupied and farmed in prehistory revealed no spatially extensive deforestation of the watersheds, as significant human impacts most often occurred in areas closest to the lakes (Fig. 1) (34). Furthermore, vast areas have yet to be studied by archaeologists and paleoecologists, particularly the tierra firme forests that account for 95% of the land area of Amazonia.To further inform these issues, we report here a vegetation and fire history spanning 5,000 y derived from phytolith and charcoal studies of terrestrial soils underneath mature tierra firme forest in northeastern Peru. Phytoliths, the silica bodies produced by many Neotropical plants, are well preserved in terrestrial soils unlike pollen, and are deposited locally. They can be used to identify different tropical vegetational formations, such as old-growth forest, early successional vegetation typical of human disturbances including forest clearings, a number of annual seed and root crops, and trees thought to have been cultivated or managed in prehistory (e.g., refs. 29–33 and 35).     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA’s physiological roles in vivo. We set out to design antagonists that block receptor–PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT–PYL10 complex (1.86 Å) reveals that ANT’s peptidotriazole headgroup is positioned to sterically block receptor–PP2C interactions in the 4′ tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA–ANT. Equilibrium dissociation constants for TAMRA–ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA–receptor function for dissecting and manipulating ABA signaling.

The phytohormone abscisic acid (ABA) controls numerous physiological processes in plants ranging from seed development, germination, and dormancy to responses for countering biotic and abiotic stresses (1). ABA binds to the PYR/PYL/RCAR (Pyrabactin Resistance 1/PYR1-like/Regulatory Component of ABA Receptor) soluble receptor proteins (2, 3) and triggers a conformational change in a flexible “gate” loop flanking the ligand-binding pocket such that the ABA–receptor complex can then bind to and inhibit clade A type II C protein phosphatases (PP2Cs), which normally dephosphorylate and inactivate SNF1-related protein kinase 2 (SnRK2). This, in turn, leads to SnRK2 activation, phosphorylation of downstream targets, and multiple cellular outputs (4, 5).Chemical modulators of ABA perception have been sought as both research tools for dissecting ABA’s role in plant physiology and for their potential agricultural utility (6, 7). Dozens of ABA receptor agonists, which reduce transpiration and water use by inducing guard cell closure, have been developed and are being explored as chemical tools for mitigating the effects of drought on crop yields (7–23), most of them either being analogs of ABA or sulfonamides similar to quinabactin (24). ABA receptor antagonists could conceivably be useful in cases where water is not limiting, for example, to increase transpiration and gas exchange under elevated CO₂ in glasshouse agriculture, as germination stimulators, and for studying the ABA dependence of physiological processes, among other applications (25–31). Thus, both ABA receptor agonists and antagonists have potential uses as research tools and for plant biotechnology.In principle, there are at least two mechanisms for blocking ABA receptor activation: by preventing gate closure, which is necessary for PP2C binding, or by sterically disrupting the activated, closed-gate receptor conformer from binding to PP2Cs. Prior efforts to design antagonists have focused on the latter strategy and include multiple ABA-derived ligands such as AS6 (25), PanMe (26), 3′-alkyl ABA (30–32), 3′-(phenyl alkynyl) ABA (33), or ligands derived from tetralone ABA (34) with varying degrees of conformational restriction (27, 28, 35). With the exception of PanMe, these antagonists have linkers attached to the 3′ carbon of ABA or 11′ carbon of tetralone ABA, which is positioned to disrupt receptor–PP2C interactions by protruding through the 3′ tunnel. PanMe was created by modifying ABA’s C4′ (Fig. 1) with a toluylpropynyl ether substituent designed to occupy the 4′ tunnel, a site of close receptor–PP2C contact (26). Structural studies showed that this 4′ moiety adopts two conformations, one that resides in the 4′ tunnel and another that occupies the adjacent 3′ tunnel (26). Collectively, these elegant studies have demonstrated that antagonists of receptor–PP2C interactions can be designed by modifying agonists at sites situated proximal to the 3′ or 4′ tunnels. Despite these advances, current antagonists have limitations. For example, PanMe, which has low nanomolar affinity for the subfamily II receptor PYL5, is limited by relatively low activity on subfamily I and III ABA receptors, and as we show here, the ABA antagonist AA1 (36) (Fig. 1) lacks detectable antagonist activity in vitro and is, therefore, unlikely to be a true ABA receptor antagonist. Together, these data suggest that higher-affinity pan-antagonists and/or molecules with increased bioavailability will be necessary to more efficiently block endogenous ABA signaling. We set out to address these limitations by modifying the scaffold of the synthetic ABA agonist opabactin (OP), which has an approximately sevenfold increase in both affinity and bioactivity relative to ABA (21). We describe an OP derivative called antabactin (ANT) and show that it is a high-affinity binder and antagonist of ABA receptors that disrupts ABA-mediated signaling in vivo.Open in a separate windowFig. 1.Structures of ABA, PanMe, and AA1.     

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

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

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

Electrophilic aromatic substitution reactions of compounds with Craig-Möbius aromaticity

Electrophilic aromatic substitution (EAS) reactions are widely regarded as characteristic reactions of aromatic species, but no comparable reaction has been reported for molecules with Craig-Möbius aromaticity. Here, we demonstrate successful EAS reactions of Craig-Möbius aromatics, osmapentalenes, and fused osmapentalenes. The highly reactive nature of osmapentalene makes it susceptible to electrophilic attack by halogens, thus osmapentalene, osmafuran-fused osmapentalene, and osmabenzene-fused osmapentalene can undergo typical EAS reactions. In addition, the selective formation of a series of halogen substituted metalla-aromatics via EAS reactions has revealed an unprecedented approach to otherwise elusive compounds such as the unsaturated cyclic chlorirenium ions. Density functional theory calculations were conducted to study the electronic effect on the regioselectivity of the EAS reactions.

Aromaticity, a core concept in chemistry, was initially introduced to account for the bonding, stability, reactivity, and other properties of many unsaturated organic compounds. There have been many elaborations and extensions of the concept of aromaticity (1, 2). The concepts of Hückel aromaticity and Möbius aromaticity are widely accepted (Fig. 1A). A π-aromatic molecule of the Hückel type is planar and has 4n + 2 conjugated π-electrons (n = 0 or an integer), whereas a Möbius aromatic molecule has one twist of the π-system, similar to that in a Möbius strip, and 4n π-electrons (3, 4). Since the discovery of naphthalene in 1821, aromatic chemistry has developed into a rich field and with a variety of subdisciplines over the course of its 200-y history, and the concept of aromaticity has been extended to other nontraditional structures with “cyclic delocalization of mobile electrons” (5). For example, benzene-like metallacycles—predicted by Hoffmann et al. as metallabenzenes—in which a metal replaces a C–H group in the benzene ring (6), have garnered extensive research interest from both experimentalists and theoreticians (7–12). As paradigms of the metalla-aromatic family, most complexes involving metallabenzene exhibit thermodynamic stability, kinetic persistence, and chemical reactivity associated with the classical aromaticity concept (13–15). Typically, like benzene, metallabenzene can undergo characteristic reactions of aromatics such as electrophilic aromatic substitution (EAS) reactions (16–18) (Fig. 1B, I) and nucleophilic aromatic substitution reactions (19–21).Open in a separate windowFig. 1.Schematic representations of aromaticity classification (A) and EAS reactions (B) of benzene, metallabenzene, and polycyclic metallacycles with Craig-Möbius aromaticity.The incorporation of transition metals has also led to an increase in the variety of the aromatic families (22–25). We have reported that stable and highly unusual bicyclic systems, metallapentalenes (osmapentalenes), benefit from Craig-Möbius aromaticity (26–30). In contrast to other reported Möbius aromatic compounds with twisted topologies, which are known as Heilbronner-Möbius aromatics (31–34), the involvement of transition metal d orbitals in π-conjugation switches the Hückel anti-aromaticity of pentalene into the planar Craig-Möbius aromaticity of metallapentalene (35–38) (Fig. 1A, III). Both the twisted topology and the planar Craig-Möbius aromaticity are well established and have been accepted as reasonable extensions of aromaticity (39–43). There has been no experimental evidence, however, as to whether these Möbius aromatic molecules can undergo classical aromatic substitution reactions, such as EAS reactions, instead of addition reactions. Given the key role of EAS in aromatic chemistry to obtain various derivatives, we sought to extend the understanding of the reactivity paradigm in the metalla-aromatic family.Our recent synthetic efforts associated with the metallapentalene system prompted us to investigate whether typical EAS reactions could proceed in these Craig-Möbius aromatics. If so, how could substitution be achieved in the same way that it is with traditional Hückel aromatics such as benzenes? In this paper, we present EAS reactions, mainly the halogenation of osmapentalene, osmafuran-fused osmapentalene, and osmabenzene-fused osmapentalene, which follow the classic EAS mechanistic scheme (Fig. 1B). With the aid of density functional theory (DFT) calculations, we characterized the effects on EAS reactivity and regioselectivity.