A solution to a sex ratio puzzle in Melittobia wasps

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

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

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

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

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

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

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

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

The evolution of red color vision is linked to coordinated rhodopsin tuning in lycaenid butterflies

Color vision has evolved multiple times in both vertebrates and invertebrates and is largely determined by the number and variation in spectral sensitivities of distinct opsin subclasses. However, because of the difficulty of expressing long-wavelength (LW) invertebrate opsins in vitro, our understanding of the molecular basis of functional shifts in opsin spectral sensitivities has been biased toward research primarily in vertebrates. This has restricted our ability to address whether invertebrate G_q protein-coupled opsins function in a novel or convergent way compared to vertebrate G_t opsins. Here we develop a robust heterologous expression system to purify invertebrate rhodopsins, identify specific amino acid changes responsible for adaptive spectral tuning, and pinpoint how molecular variation in invertebrate opsins underlie wavelength sensitivity shifts that enhance visual perception. By combining functional and optophysiological approaches, we disentangle the relative contributions of lateral filtering pigments from red-shifted LW and blue short-wavelength opsins expressed in distinct photoreceptor cells of individual ommatidia. We use in situ hybridization to visualize six ommatidial classes in the compound eye of a lycaenid butterfly with a four-opsin visual system. We show experimentally that certain key tuning residues underlying green spectral shifts in blue opsin paralogs have evolved repeatedly among short-wavelength opsin lineages. Taken together, our results demonstrate the interplay between regulatory and adaptive evolution at multiple G_q opsin loci, as well as how coordinated spectral shifts in LW and blue opsins can act together to enhance insect spectral sensitivity at blue and red wavelengths for visual performance adaptation.

Opsins belong to a diverse multigene family of G protein-coupled receptors that bind to a small nonprotein retinal moiety to form photosensitive rhodopsins and enable vision across animals (1–4). The tight relationship between opsin genotypes and spectral sensitivity phenotypes offers an ideal framework to analyze how specific molecular changes give rise to adaptations in visual behaviors (5). Notably, independent opsin gene gains and losses (6–13), genetic variation across opsins (14–16), spectral tuning mutations within opsins (17–21), and alterations in visual regulatory networks (22, 23) have contributed to opsin adaptation. Yet, the molecular and structural changes underlying the remarkable diversification of spectral sensitivity phenotypes identified in some invertebrates, including crustaceans and insects (24–27), are far less understood than those in vertebrate lineages (28–32).The diversity of opsin-based photoreceptors observed across animal visual systems is produced by distinct ciliary vertebrate c-opsin and invertebrate rhabdomeric based r-opsin subfamilies that mediate separate phototransduction cascades (31, 33–35). Vertebrate c-opsins function through the G protein transducing (G_t) signaling pathway, which activates cyclic nucleotide phosphodiesterase, ultimately resulting in a hyperpolarization response in photoreceptor cells through the opening of selective K⁺ channels (31, 36). By contrast, insect opsins transmit light stimuli through a G_q-type G protein (33, 37) with phosphoinositol (PLCβ) acting as an effector enzyme to achieve TRP channel depolarization in the invertebrate photoreceptor cell (34, 38).All vertebrate visual cone opsins derive from four gene families: short-wavelength-sensitive opsins SWS1 (or ultraviolet [UV]) with λ_max 344 to 445 nm and SWS2 with λ_max 400 to 470 nm, and longer-wavelength-sensitive opsins that specify the green MWS (or Rh2) pigments with λ_max 480 to 530 nm and red-sensitive LWS pigments with λ_max 500 to 570 nm (5, 30). Most birds and fish have retained the four ancestral opsin genes (39), with notable opsin expansions in cichlid fish opsins (23, 40), whereas SWS1 is extinct in monotremes, and SWS2 and M opsins are lost in marsupials and eutherian mammals (41). In primates, trichromatic vision is conferred through SWS1 (λ_max = 414 nm) and recent duplicate MWS (λ_max = 530 nm) and LWS opsins (λ_max = 560 nm) (42–44). In vertebrates, molecular evolutionary approaches and well-established in vitro opsin purification have identified the complex interplay between opsin duplications, regulatory and protein-coding mutations controlling opsin gene tuning, and spectral phenotypes notably in birds, fish, and mammals (45–47).Insect opsins are phylogenetically distinct but functionally analogous to those of vertebrates, and the ancestral opsin repertoire consists of three types of light-absorbing rhabdomeric G_q-type opsin specifying UV (350 nm), short-wavelength (blue, 440 nm) and long-wavelength pigments (LW, 530 nm) (48). Given the importance of color-guided behaviors and the remarkable photoreceptor spectral diversity observed in insects (26, 27), the dynamic opsin gene diversification found across lineages (Fig. 1) highlights their potentially central role in adaptation (27, 49, 50), yet the molecular basis of opsin functionality of rhabdomeric invertebrate G_q opsins remains understudied.Open in a separate windowFig. 1.Visual opsin gene evolution and spectral tuning mechanisms in insects. Visual opsin genes of the Atala hairstreak (E. atala, Lepidoptera, Lycaenidae) in comparison with those encoded in the genomes of diverse insects. The opsin types are highlighted in gray for UV, in blue for short wavelength (SW), and in green for long wavelength (LW). Numbers indicate multiple opsins, whereas no dot indicates gene loss. Colored circles indicate instances of shifted spectral sensitivities in at least one of the encoded opsins. The direction of shift is inferred from the opsin lambda max that departs from the typical range of absorbance in the opsin subfamily using wavelength boundaries for the various colors: UV <380 nm, violet 380 to 435 nm, blue 435 to 492 nm, green 492 to 530 nm, and red shifted >530 nm. Coleopteran lineages, and some hemipterans, lost the blue opsin locus and compensated for the loss of blue sensitivity via UV and/or LW gene duplications across lineages (11, 12). In butterflies, extended photosensitivity at short wavelengths is observed in Heliconius erato with two UV opsins at λ_max = 355 nm and 398 nm (10) and in P. rapae with two blue opsins with λ_max = 420 and 450 nm (17). A blue opsin duplication occurred independently in lycaenid butterflies (61). LW opsin duplications occurred independently in most major insect lineages (6, 16, 55) and confer a variable range of LW sensitivities with or without additional contributions from lateral filtering. In order to extend spectral sensitivity at longer wavelengths while sharpening blue acuity, some lycaenid butterflies have evolved a new color vision mechanism combining spectral shifts at a duplicate blue opsin and at the LW opsin. Images credit: Christopher Adams (illustrator).The recurrent evolution of red receptors in insects in particular suggests that perception of longer wavelengths can play an important role in the context of foraging, oviposition, and/or conspecific recognition (6, 27, 51–54). In butterflies, several mechanisms are likely to have provided extended spectral sensitivity to longer wavelengths. LW opsin duplications along with the evolution of lateral filtering between ommatidia has been demonstrated in two papilionids, Papilio xuthus (27) and Graphium sarpedon (55), as well as in a riodinid (Apodemia mormo) (6, 54). Lateral filtering pigments are relatively widespread across butterfly lineages, e.g., Heliconius (56), Pieris (57), Colias erate (58), and some moths [Adoxophyes orana (59) and Paysandisia archon (60)]. These pigments absorb short wavelengths and aid in shifting the sensitivity peak of green LW photoreceptors to longer wavelengths (27, 51, 56, 57, 61, 62). Despite creating distinct spectral types that can contribute to color vision, as identified in nymphalid (56), pierid (57), and lycaenid (62) species, all of which lack duplicated LW opsins (61, 63), lateral filtering alone cannot extend photoreceptor sensitivity toward the far red (700 to 750 nm) beyond the exponentially decaying long-wavelength rhodopsin absorbance spectrum (51). Thus, molecular variation of ancestral LW opsin genes is likely to have contributed an as yet underexplored mechanism to the diversification of long-wavelength photoreceptor spectral sensitivity. However, disentangling the relative contributions of lateral filtering and pure LW opsin properties has remained technically challenging using classical electrophysiological approaches (14, 64, although see, e.g., refs. 65, 66, 67) and has been limited by the lack of in vitro expression systems suitable for LW opsins.While opsin duplicates have been identified in numerous organisms, the spectral tuning mechanisms and interplay between new opsin photoreceptors in invertebrate visual system evolution are less well understood. Here we combine physiological, molecular, and heterologous approaches to start closing this gap in our knowledge of invertebrate G_q opsin evolution by investigating the functions, spectral tuning, and implications of evolving new combinations of short- and long-wavelength opsin types in lycaenid species. This butterfly group, comprising the famous blues, coppers, and hairstreaks, is the second largest family with about 5,200 (28%) of the some 18,770 described butterfly species (68). In light of their remarkable behavioral, ecological, and morphological diversity (69, 70), as well as pioneer studies in the Lycaena and Polyommatus genera supporting the rapid evolution of color vision in certain lineages (56, 61, 62), lycaenids provide an ideal candidate system for investigating opsin evolution and visual adaptations. Using the Atala hairstreak, Eumaeus atala, as a molecular and ecological model, we find coordinated spectral shifts at short- and long-wavelength G_q opsin loci and demonstrate that the combination of six ommatidial classes of photoreceptors in the compound eye uniquely extend spectral sensitivity at long wavelengths toward the far-red while concurrently sharpening acuity of multiple blue wavelengths. Together, these findings link the evolution of four-opsin visual systems to adaptation in the context of finely tuned color perception critical to the behavior of these butterflies.     

Modulation of immune cell reactivity with cis-binding Siglec agonists

Inflammatory pathologies caused by phagocytes lead to numerous debilitating conditions, including chronic pain and blindness due to age-related macular degeneration. Many members of the sialic acid-binding immunoglobulin-like lectin (Siglec) family are immunoinhibitory receptors whose agonism is an attractive approach for antiinflammatory therapy. Here, we show that synthetic lipid-conjugated glycopolypeptides can insert into cell membranes and engage Siglec receptors in cis, leading to inhibitory signaling. Specifically, we construct a cis-binding agonist of Siglec-9 and show that it modulates mitogen-activated protein kinase (MAPK) signaling in reporter cell lines, immortalized macrophage and microglial cell lines, and primary human macrophages. Thus, these cis-binding agonists of Siglecs present a method for therapeutic suppression of immune cell reactivity.

Sialic acid-binding immunoglobulin (IgG)-like lectins (Siglecs) are a family of immune checkpoint receptors that are on all classes of immune cells (1–5). Siglecs bind various sialoglycan ligands and deliver signals to the immune cells that report on whether the target is healthy or damaged, “self” or “nonself.” Of the 14 human Siglecs, 9 contain cytosolic inhibitory signaling domains. Accordingly, engagement of these inhibitory Siglecs by sialoglycans suppresses the activity of the immune cell, leading to an antiinflammatory effect. In this regard, inhibitory Siglecs have functional parallels with the T cell checkpoint receptors CTLA-4 and PD-1 (6–9). As with these clinically established targets for cancer immune therapy, there has been a recent surge of interest in antagonizing Siglecs to potentiate immune cell reactivity toward cancer (10). Conversely, engagement of Siglecs with agonist antibodies can suppress immune cell reactivity in the context of antiinflammatory therapy. This approach has been explored to achieve B cell suppression in lupus patients by agonism of CD22 (Siglec-2) (11, 12), and to deplete eosinophils for treatment of eosinophilic gastroenteritis by agonism of Siglec-8 (13). Similarly, a CD24 fusion protein has been investigated clinically as a Siglec-10 agonist for both graft-versus-host disease and viral infection (14, 15).Traditionally, Siglec ligands have been studied as functioning in trans, that is, on an adjacent cell (16–18), or as soluble clustering agents (9, 19). In contrast to these mechanisms of action, a growing body of work suggests that cis ligands for Siglecs (i.e., sialoglycans that reside on the same cell membrane) cluster these receptors and maintain a basal level of inhibitory signaling that increases the threshold for immune cell activation. Both Bassik and coworkers (20) and Wyss-Coray and coworkers (21) have linked the depletion of cis Siglec ligands with increased activity of macrophages and microglia, and other studies have shown that a metabolic blockade of sialic acid renders phagocytes more prone to activation (22).Synthetic ligands are a promising class of Siglec agonists (17, 23, 24). Many examples rely on clustering architectures (e.g., sialopolymers, nanoparticles, liposomes) to induce their effect (19, 23–26). Indeed, we have previously used glycopolymers to study the effects of Siglec engagement in trans on natural killer (NK) cell activity (16). We and other researchers have employed glycopolymers (16, 23), glycan-remodeling enzymes (27, 28), chemical inhibitors of glycan biosynthesis (22), and mucin overexpression constructs (29, 30) to modulate the cell-surface levels of Siglec ligands. However, current approaches lack specificity for a given Siglec.We hypothesized that Siglec-specific cis-binding sialoglycans displayed on immune cell surfaces could dampen immune cell activity with potential therapeutic applications. Here we test this notion with the synthesis of membrane-tethered cis-binding agonists of Siglec-9 (Fig. 1). Macrophages and microglia widely express Siglec-9 and are responsible for numerous pathologies including age-related inflammation (31), macular degeneration (32), neural inflammation (33), and chronic obstructive pulmonary disease (34). We designed and developed a lipid-linked glycopolypeptide scaffold bearing glycans that are selective Siglec-9 ligands (pS9L-lipid). We show that pS9L-lipid inserts into macrophage membranes, binds Siglec-9 specifically and in cis, and induces Siglec-9 signaling to suppress macrophage activity. By contrast, a lipid-free soluble analog (pS9L-sol) binds Siglec-9 but does not agonize Siglec-9 or modulate macrophage activity. Membrane-tethered glycopolypeptides are thus a potential therapeutic modality for inhibiting phagocyte activity.Open in a separate windowFig. 1.Lipid-tethered glycopolypeptides cluster and agonize Siglecs in cis on effector cells. (A) Immune cells express activating receptors that stimulate inflammatory signaling. (B) Clustering of Siglec-9 by cis-binding agonists stimulates inhibitory signaling that quenches activation.     

Capillary equilibrium of bubbles in porous media

In geologic, biologic, and engineering porous media, bubbles (or droplets, ganglia) emerge in the aftermath of flow, phase change, or chemical reactions, where capillary equilibrium of bubbles significantly impacts the hydraulic, transport, and reactive processes. There has previously been great progress in general understanding of capillarity in porous media, but specific investigation into bubbles is lacking. Here, we propose a conceptual model of a bubble’s capillary equilibrium associated with free energy inside a porous medium. We quantify the multistability and hysteretic behaviors of a bubble induced by multiple state variables and study the impacts of pore geometry and wettability. Surprisingly, our model provides a compact explanation of counterintuitive observations that bubble populations within porous media can be thermodynamically stable despite their large specific area by analyzing the relationship between free energy and bubble volume. This work provides a perspective for understanding dispersed fluids in porous media that is relevant to CO₂ sequestration, petroleum recovery, and fuel cells, among other applications.

Bubbles are generated, trapped, and mobilized within porous media as a consequence of incomplete fluid–fluid displacements (1, 2), phase changes (3, 4), chemical and biochemical reactions (5, 6), or injection of emulsified fluids and foams (7, 8). Compared to continuously connected phases, the behavior of dispersed bubbles, or ganglia, are far less understood. In particular, the thermodynamic stability of bubbles, despite their large specific surface area, remains a puzzle. The difficulty comes from the fact that each bubble can attain a volume (V), topology, and capillary pressure (P_c) that is distinct from other bubbles in the medium (9). The variability poses challenges to understanding the transport and trapping mechanisms of bubbles in geologic CO₂ sequestration (10, 11), hydrocarbon recovery (12, 13), fuel cell water management (14, 15), and vadose zone oxygen supply (16, 17).The dominant factor controlling a bubble’s behavior in a porous medium is capillarity, which is typically much larger than either viscous, gravitational, or inertial forces (18, 19). Capillary pressure, P_c, allows a closure relationship for two-phase Darcy Eqs. (20–22) and influences thermodynamic properties like phase partition (23). Capillary pressure is derived from the Young–Laplace equation P_c = γκ, where γ is the interfacial tension and κ is the surface curvature. In an open space without obstacles, a bubble spontaneously evolves into a sphere to minimize its total interfacial energy. Thus, P_c is a continuous and monotonically decreasing function of V (Fig. 1A). However, in a porous medium, bubble’s P_c–V relation is more complicated due to the geometric confinement imposed by the porous structure and topological evolution (24). A bubble can no longer remain spherical as it grows in size but must conform to the geometry of the pore(s) it occupies. Therefore, a bubble’s P_c is a function of not only its volume and interfacial tension but also its topology as dictated by the confining porous medium, as confirmed by recent laboratory experiments and numerical simulations (25–29). The mere presence of confinement therefore engenders a host of phenomena that would otherwise be absent, such as capillary trapping (30, 31), anticoarsening of bubble populations (32, 33), and complex ganglion dynamics (11, 18). Furthermore, theoretical studies in mathematical topology (28, 34, 35) prove that immiscible fluids can be fully characterized by d+1 Minkowski functionals, where d is the problem dimension. Such characterizations remove the path-dependent (or hysteretic) behavior common to these systems (34, 35).Open in a separate windowFig. 1.(A) Spherical bubbles inside a bulk fluid. (B) Micromodel observations show that bubbles are nonspherical in porous media and may occupy multiple pores. This image is from SI Appendix, Movie S1. (C) A 2D porous medium comprised of an ordered array of identical circular grains. A bubble occupying multiple pores including a zoom-in to a portion of it. (D) Illustration of the full state. (E) Illustration of the critical state. (F) Decomposition of a bubble into four distinct parts: minor arc menisci shown by dark blue cap-shaped regions, throats shown by light blue diamond-shaped regions, inner bulk bodies shown by red star-shaped regions, and major arc menisci shown by dark green cap-shaped regions.Recent developments in microfluidics and micro computed tomography imaging allow detailed pore-scale visualizations of fluids inside porous media, including the morphology of bubbles and ganglia (25, 36–39). Garing et al. (25) experimentally measured the equilibrium capillary pressure of trapped air bubbles inside sandstone and bead-pack samples. They found that, unlike bubbles within a bulk fluid, the P_c of trapped bubbles shows no clear dependence on V and seems to fall within a bounded interval, except for vanishingly small V. Xu et al. (40) proposed an empirical correlation for the P_c trapped bubbles based on microfluidic observations. In this correlation, as V increases, P_c decreases until a minimum is reached and then increases linearly. In the first stage, the bubble is unconfined, whereas in the second, it is reshaped by the surrounding solid walls. The proposed correlation, however, is only valid for bubbles in a single pore and not bubbles that span multiple pores. The latter seems to be rather common in nature as evidenced by recent direct observations (Fig. 1B) (2, 25).Here, we propose a simple conceptual model to describe the equilibrium states of a bubble with arbitrary size trapped inside a porous medium. The model accounts for the bubble’s morphology, the geometry of the solid matrix, and the wettability between the two. We derive all metastable configurations of the bubble analytically and highlight the thermodynamic states the bubble assumes when it is static, growing, or shrinking. We also show that the relationship between surface free energy (F) and volume (V) of large bubbles is approximately linear, which explains the previously counterintuitive observation that such bubbles are thermodynamically stable despite having large surface areas. Our work provides a step toward understanding the capillary state, stability, and evolution of dispersed immiscible fluids in porous media.     

Superelastic oxide micropillars enabled by surface tension–modulated 90° domain switching with excellent fatigue resistance

Superelastic materials capable of recovering large nonlinear strains are ideal for a variety of applications in morphing structures, reconfigurable systems, and robots. However, making oxide materials superelastic has been a long-standing challenge due to their intrinsic brittleness. Here, we fabricate ferroelectric BaTiO₃ (BTO) micropillars that not only are superelastic but also possess excellent fatigue resistance, lasting over 1 million cycles without accumulating residual strains or noticeable variation in stress–strain curves. Phase field simulations reveal that the large recoverable strains of BTO micropillars arise from surface tension–modulated 90° domain switching and thus are size dependent, while the small energy barrier and ultralow energy dissipation are responsible for their unprecedented cyclic stability among superelastic materials. This work demonstrates a general strategy to realize superelastic and fatigue-resistant domain switching in ferroelectric oxides for many potential applications.

Superelastic materials are capable of recovering large amount of nonlinear “plastic” strains, way beyond their linear elastic regimes (1–4). They are ideal for a variety of applications from morphing structures, reconfigurable systems, to robots (5–8). The effects have traditionally been associated with macroscopically compliant/ductile rubbers (2) or microscopically phase-transforming shape memory alloys (SMAs) (7–11). The only macroscopically brittle oxide recently discovered to be superelastic is ZrO₂-based micropillars or particles (12–20), which is realized via austenite-martensite phase transformation similar to SMAs. Although ultimate strengths approaching the theoretical limit have been demonstrated in nanoscale samples (21, 22), long fatigue life is elusive, which is arguably more important for most applications. As a matter of fact, poor fatigue life has been a long-standing challenge for oxide ceramics in general (23, 24). Even for ductile SMAs that enjoy excellent fatigue life, irrecoverable residual strains gradually accumulate over cycling, leading to substantial variations in stress–strain curves at different cycles (9, 10, 25). We overcome these difficulties by reporting superelastic barium titanate (BaTiO₃ [BTO]) micropillars enabled by surface tension–modulated 90° domain switching, which exhibit excellent fatigue resistance, while bulk BTO crystals or ceramics are rather brittle. The demonstration of over one million cycles of loading and unloading without accumulating residual strains or noticeable variation in stress–strain curves is unprecedented among superelastic materials.BTO is a ferroelectric oxide exhibiting modest piezoelectric strains around 0.1 to 0.2% (26) and fracture toughness of ∼1 MPa ⋅ m^1/2, and thus it is quite brittle (27). Considerable research efforts have been devoted to enhancing its electric field–induced strain via 90° ferroelectric domain switching (28–30). However, the process is often irreversible, and external mechanisms such as restoring force (28, 29) and internal mechanisms such as defect pinning (30) have to be invoked to make the electrostrain recoverable. Nevertheless, it hints at the possibility of BTO being made superelastic by taking advantage of the stress-induced 90° domain switching (6). Earlier works suggest that surface tension induces an in-plane compressive stress that favors the axial polarization in one-dimensional ferroelectrics at small size (31, 32), which may provide the necessary restoring mechanism for the stress-switched domains. Thus, if a compressive axial force is applied, reversible domain switching may occur during unloading, leading to superelasticity. To verify this hypothesis, we fabricated single-crystalline BTO micropillars from [001]-oriented bulk crystals (SI Appendix, Fig. S1A) via focused ion beam (FIB), as detailed in Materials and Methods and SI Appendix, Fig. S1B. The diameters (Φ) of the micropillars range from 0.5 μm to 5 μm, with their height to diameter ratio fixed at 3. No visible defects can be seen from the scanning electron microscopy (SEM) images of these micropillars shown in Fig. 1 A–D, and their surfaces appear to be quite smooth, suggesting that no apparent damages are induced by FIB.Open in a separate windowFig. 1.Superelastic BTO micropillars below a critical size. (A–D) SEM images of the micropillars with Φ = 5, 3, 2, and 0.5 μm. (E–G) The first and second cycles of stress–strain curves for BTO micropillars with Φ = 5, 2, and 0.5 μm. (H) S_r/S_max and ΔW/W_max during the first cycle for BTO micropillars of different diameters. Here, S_r and S_max denote the residual strain and the maximum strain (SI Appendix, Fig. S6A), while ΔW and W_max are energy dissipated and stored in the first cycle, respectively (SI Appendix, Fig. S6F).     

“Soft” oxidative coupling of methane to ethylene: Mechanistic insights from combined experiment and theory

The oxidative coupling of methane to ethylene using gaseous disulfur (2CH₄ + S₂ → C₂H₄ + 2H₂S) as an oxidant (SOCM) proceeds with promising selectivity. Here, we report detailed experimental and theoretical studies that examine the mechanism for the conversion of CH₄ to C₂H₄ over an Fe₃O₄-derived FeS₂ catalyst achieving a promising ethylene selectivity of 33%. We compare and contrast these results with those for the highly exothermic oxidative coupling of methane (OCM) using O₂ (2CH₄ + O₂ → C₂H₄ + 2H₂O). SOCM kinetic/mechanistic analysis, along with density functional theory results, indicate that ethylene is produced as a primary product of methane activation, proceeding predominantly via CH₂ coupling over dimeric S–S moieties that bridge Fe surface sites, and to a lesser degree, on heavily sulfided mononuclear sites. In contrast to and unlike OCM, the overoxidized CS₂ by-product forms predominantly via CH₄ oxidation, rather than from C₂ products, through a series of C–H activation and S-addition steps at adsorbed sulfur sites on the FeS₂ surface. The experimental rates for methane conversion are first order in both CH₄ and S₂, consistent with the involvement of two S sites in the rate-determining methane C–H activation step, with a CD₄/CH₄ kinetic isotope effect of 1.78. The experimental apparent activation energy for methane conversion is 66 ± 8 kJ/mol, significantly lower than for CH₄ oxidative coupling with O₂. The computed methane activation barrier, rate orders, and kinetic isotope values are consistent with experiment. All evidence indicates that SOCM proceeds via a very different pathway than that of OCM.

The oxidative coupling of methane (OCM) with O₂ would seem to be a concise, direct route to convert methane, one of the most Earth-abundant carbon sources (1), to ethylene (2CH₄ + O₂ → C₂H₄ + 2H₂O), a key chemical intermediate (2, 3), and this process has been extensively studied (1, 4–19) since 1982 (20). Nevertheless, the widespread use of OCM is challenged by methane overoxidation to CO₂ and other oxygenates. Furthermore, the severe reaction conditions of nonoxidative pathways (2, 21–28) typically risk carbon deposition and catalyst deactivation (2, 21–26). In preliminary studies, we reported a 2CH₄ + S₂ → C₂H₄ + 2H₂S coupling process that moderates the methane overoxidation driving force using gaseous disulfur (S₂) as a “soft” oxidant (SOCM; Fig. 1A) (29). S₂ is isoelectronic with O₂, the major sulfur vapor species at 700 to 925 °C (30–32), and is a less aggressive oxidant than O₂ (33). In this scenario, elemental sulfur is recovered from the H₂S coproduct via the known Claus process (Fig. 1B) (30), in a cycle where sulfur mediates/moderates the high nonselective O₂ reactivity. SOCM achieved promising ethylene selectivity, raising intriguing mechanistic questions and the possibility of higher selectivity. Methane + S_2(g) ethylene selectivities near ∼20% are achieved over a PdS/ZrO₂ catalyst (29), and oxide precatalysts give selectivities near 33% (34).Open in a separate windowFig. 1.Energetic comparison between the oxidative coupling of methane with O₂ (OCM) and with S₂ (SOCM) and the pathway to recover elemental sulfur from H₂S. (A) Gibbs free energy of desired and overoxidation processes in OCM and SOCM at 800 and 1,050 °C. (B) Industrialized catalytic Claus process used to recover elemental sulfur from H₂S.Nevertheless, in contrast to extensive OCM (17, 35–39) and nonoxidative CH₄ coupling studies (40), far less is known about the SOCM reaction pathway. Post-SOCM X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and elemental analysis (29, 34) indicate that the oxide precatalysts are predominantly sulfided. Density functional theory (DFT) analyses of molybdenum sulfide catalysts suggest that methane is activated at M–S or S–S sites to form surface-bound CH₃* species which dehydrogenate to form CH₂* (methylidene) species, which then couple to produce C₂H₄. It was proposed that CH₃* species can also desorb as methyl radicals which couple to form ethane (29). The overoxidation product, CS₂, was suggested to form via sulfur addition to methylidene surface intermediates (29).Kinetic, mechanistic, and theoretical analyses are needed to better understand the CH₄ conversion pathways to C₂H₄ and other products. In principle, there are two plausible pathways following methane activation: 1) H abstraction from adsorbed methyl species forms methylidene (CH₂*) and methylidyne (CH*) species then couple to C₂ products or undergo oxidation to CS₂ or 2) coupling of surface or gas phase methyl species form ethane, which then dehydrogenates to form ethylene or oxidizes to CS₂. For further SOCM optimization it is important to determine which pathways are operative, their relative rates, and the C₂ and CS₂ formation sites.Here we investigate SOCM pathways over a sulfided Fe₃O₄ precatalyst which affords C₂H₄ selectivities near 33%, complete oxide to sulfide conversion, minimal carbon deposition (coking), and 48-h SOCM stability at 950 °C (34). We first summarize SOCM phenomenology, followed by analysis of the Fe phases during sulfurization and SOCM. Next, kinetic/mechanistic studies focus on the methane and S₂ reaction orders, activation energetics, and isotope effects and probe the pathways governing C₂ vs. CS₂ formation. Complementary DFT calculations focus on reaction mechanisms, the active sites, and their role in product formation. The results are used in a microkinetic model to simulate reaction rates, apparent activation barriers, and reaction rate orders and to compare with experiment. Finally, SOCM and OCM are compared, revealing that they follow distinctly different pathways.     

Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance

In [NiFe]-hydrogenases, the active-site Ni is coordinated by four cysteine-S ligands (Cys; C), two of which are bridging to the Fe(CO)(CN)₂ fragment. Substitution of a single Cys residue by selenocysteine (Sec; U) occurs occasionally in nature. Using a recent method for site-specific Sec incorporation into proteins, each of the four Ni-coordinating cysteine residues in the oxygen-tolerant Escherichia coli [NiFe]-hydrogenase-1 (Hyd-1) has been replaced by U to identify its importance for enzyme function. Steady-state solution activity of each Sec-substituted enzyme (on a per-milligram basis) is lowered, although this may reflect the unquantified presence of recalcitrant inactive/immature/misfolded forms. Protein film electrochemistry, however, reveals detailed kinetic data that are independent of absolute activities. Like native Hyd-1, the variants have low apparent K_MH₂ values, do not produce H₂ at pH 6, and display the same onset overpotential for H₂ oxidation. Mechanistically important differences were identified for the C576U variant bearing the equivalent replacement found in native [NiFeSe]-hydrogenases, its extreme O₂ tolerance (apparent K_MH₂ and V_max [solution] values relative to native Hyd-1 of 0.13 and 0.04, respectively) implying the importance of a selenium atom in the position cis to the site where exogenous ligands (H⁻, H₂, O₂) bind. Observation of the same unusual electrocatalytic signature seen earlier for the proton transfer-defective E28Q variant highlights the direct role of the chalcogen atom (S/Se) at position 576 close to E28, with the caveat that Se is less effective than S in facilitating proton transfer away from the Ni during H₂ oxidation by this enzyme.

Hydrogenases catalyze highly efficient H₂ activation, providing a paradigm for renewable hydrogen technologies (1). In a small subgroup of [NiFe]-hydrogenases from sulfate-reducing bacteria and methanogens, natural substitution of cysteine (Cys; C) for selenocysteine (Sec; U) occurs in the active site (Fig. 1) (2). The [NiFeSe]-hydrogenases (group 1a) are reported to have higher activity than their [NiFe] counterparts—a feature seen in other enzymes where C and U are swapped (2–8). Escherichia coli produces [NiFe]-hydrogenase-1 (Hyd-1) (group 1d, O₂-tolerant) and Hyd-2 (group 1c, O₂-sensitive) membrane-bound [NiFe]-hydrogenases (3, 9–12). At neutral pH in vitro Hyd-1 performs H₂ oxidation only, whereas Hyd-2 can also produce H₂ (reduce H⁺) (13). Hydrogen oxidation activity in vivo is linked to reduction of different terminal electron acceptors depending on the bacterial species, availability of different oxidants, and their redox potential, for example fumarate or, in the case of the Knallgas bacterium Ralstonia eutropha, O₂. The production of Hyd-1 and Hyd-2 is maximal using fumarate as the terminal electron acceptor under anaerobic conditions (13). E. coli does not produce a [NiFeSe]-hydrogenase; Hyd-3 (group 4a) is U-containing in the formate dehydrogenase (FdhF) subunit only (3, 14).Open in a separate windowFig. 1.(A) Amino acid alignment of selected hydrogenases (Hyd-1 numbering) highlighting key residues (Cys/Sec, red; E28, green; D118, pink; R509, yellow) and differences (cyan). See also SI Appendix, Fig. S1 and Table S1. (B and D) The extended active site of Hyd-1 (Protein Data Bank [PDB] ID code 5A4M) (B) and Desulfomicrobium baculatum NiFeSe (PDB ID code 1CC1) (D) hydrogenases. (C) Representation of the active site, where “X” denotes the atom in a bridging position between the Ni and Fe atoms, the identity of which depends on the (in)active state of the enzyme (SI Appendix, Fig. S3).Minimally, Hyd-1 has two membrane-extrinsic subunits: HyaB containing the NiFe active site, and HyaA housing three FeS clusters to mediate long-range electron transfer (SI Appendix, Figs. S1 and S2) (15). The resulting complex, a (HyaA)₂(HyaB)₂ dimer, transfers electrons to a b-type cytochrome in a membrane-intrinsic HyaC subunit (10). For periplasmic [NiFeSe]-hydrogenases from sulfate-reducing bacteria, the normal redox partner is a soluble cytochrome c₃ (16). In protein film electrochemistry (PFE; see below), the FeS clusters connect the active site to an electrode, enabling catalysis to be controlled and recorded (1).The active-site metal atoms are coordinated by four conserved Cys residues (Fig. 1), two of which are terminal to the Ni and two of which are bridging (μ) between the Ni and Fe atoms. In [NiFeSe] homologs it is usually a terminal Cys (C576; Fig. 1B) that is replaced by Sec (Fig. 1D), although purported examples exist in which a bridging Cys residue C579 is substituted (2). Additionally, a nearby aspartate is substituted by serine in the active-site “canopy” of [NiFeSe]-hydrogenases (17). Other important residues include a strictly conserved arginine (R509) essential for fast and efficient H₂ oxidation in Hyd-1 (4, 17), and a glutamate (E28) adjacent to C576 which appears to be a universal proton gate (18, 19).The FeS relays are disparate (SI Appendix, Fig. S2): [NiFeSe]-hydrogenases coordinate three [4Fe-4S] clusters at all positions proximal, medial, and distal to the active site, whereas the medial site in [NiFe]-hydrogenases is a [3Fe-4S] cluster having a more positive reduction potential (20–23). In group 1d O₂-tolerant hydrogenases, such as Hyd-1, all the FeS clusters have more positive reduction potentials (24, 25); importantly, the proximal cluster is a unique [4Fe-3S]_6-Cys center that is essential for O₂ tolerance (9), a property requiring the invading O₂ molecule to be reduced to harmless water (1, 26). The [4Fe-3S]_6-Cys proximal cluster can perform two one-electron transfers back to the active site upon O₂ exposure during H₂ oxidation, a process requiring substantial conformational change to form the “superoxidized state” (11, 24, 25, 27, 28). A third electron is available from the high-potential medial [3Fe-4S] cluster and a fourth stems from oxidation of the Ni (SI Appendix, Fig. S3). A truly O₂-tolerant [NiFe]-hydrogenase is thus also an oxidase (26). Although [NiFeSe]-hydrogenases are considered “O₂-tolerant” (29), this property, requiring reductive destruction of O₂, may be limited to H₂ evolution (30).Selenocysteine, the versatile 21st amino acid, appears in proteins from all domains of life (31). Sec is structurally similar to Cys, except the thiol is replaced by a selenol (Fig. 2A). Selenium and sulfur are chalcogens; thus U and C share certain chemical properties, but the electronic structures of S and Se differ sufficiently to give selenoproteins distinctive catalytic efficacies (6). The much lower pKa of selenol compared with thiol renders it fully ionized at physiological pH (32), selenoproteins are more resistant to irreversible oxidation than their C-containing homologs (5), and diselenide bonds are more stable to reduction than disulfide bonds (7, 33). Most natural selenoenzymes are oxidoreductases having an essential (for efficient catalysis) U active-site residue, and many have Cys homologs from which they evolved (34).Open in a separate windowFig. 2.(A) Chemical structures of Cys and Sec show the selenol moiety (red) to be the only difference. (B) EF-Tu–driven site-specific incorporation of Sec at a UAG codon. mRNA, messenger RNA. (C) Coomassie blue-stained denaturing sodium dodecyl sulfate-polyacrylamide gel of Hyd-1 variants (C76U, C79U, C576U, and C579U) shows high purity in each case, comprising HyaA (37 kDa) and HyaB (66 kDa) only. (D) Tandem mass spectra of C76U and C576U show Sec incorporation at the desired position in the designated peptide. Red lines correlate with the cleavage products depicted in the peptide sequence with an accuracy of 5 ppm. See also SI Appendix, Figs. S4–S8.Advances in genetic code expansion have provided tools for effective, site-specific UAG-programmed Sec insertion into recombinant proteins in E. coli. These in vivo methods (35–37) rely on elongation factor EF-Tu, thus bypassing the natural complex U-specific selenoprotein synthesis machinery programmed by UGA (Fig. 2B). Recently, one of these methods was used to replace active-site Cys residues with Sec in ribonucleotide reductase (38). This encouraged us to produce Cys-to-Sec variants of Hyd-1 in E. coli by the same strategy (35, 37).A recent paper described the consequences of replacing the Sec residue of a natural [NiFeSe]-hydrogenase with Cys, thereby retroengineering it to resemble a [NiFe]-hydrogenase (39). Here we report the opposite and complementary study, substituting each active-site Cys residue for Sec at all four coordination positions in Hyd-1. The resulting data highlight why one particular position has special significance.     

Atomic-scale evidence for highly selective electrocatalytic N−N coupling on metallic MoS2

Molybdenum sulfide (MoS₂) is the most widely studied transition-metal dichalcogenide (TMDs) and phase engineering can markedly improve its electrocatalytic activity. However, the selectivity toward desired products remains poorly explored, limiting its application in complex chemical reactions. Here we report how phase engineering of MoS₂ significantly improves the selectivity for nitrite reduction to nitrous oxide, a critical process in biological denitrification, using continuous-wave and pulsed electron paramagnetic resonance spectroscopy. We reveal that metallic 1T-MoS₂ has a protonation site with a pK_a of ∼5.5, where the proton is located ∼3.26 Å from redox-active Mo site. This protonation site is unique to 1T-MoS₂ and induces sequential proton−electron transfer which inhibits ammonium formation while promoting nitrous oxide production, as confirmed by the pH-dependent selectivity and deuterium kinetic isotope effect. This is atomic-scale evidence of phase-dependent selectivity on MoS₂, expanding the application of TMDs to selective electrocatalysis.

Transition-metal dichalcogenides (TMDs) have gained considerable attention in recent years due to their variable crystal phases, which allow for precise tuning of their electronic, optical, magnetic, and catalytic properties (1, 2). For example, molybdenum sulfide (MoS₂), which is one of the most extensively studied TMDs, exists as different polymorphs depending on the orientation of sulfur atoms around the molybdenum center. In octahedral coordination (1T phase), MoS₂ exhibits metallic behavior, whereas the material acts as a semiconductor in trigonal prismatic coordination (2H phase) (3–6). In addition to higher conductivity, 1T-MoS₂ has enlarged layer spacing and more electrochemical active sites (7, 8), making it a promising next-generation material for batteries (9, 10), memristors (11, 12), capacitors (13, 14), and numerous other energy-related applications (15–17).In the field of electrocatalysis, phase engineering has mainly been used to enhance catalytic activity. For instance, exchanging 2H-MoS₂ for 1T-MoS₂ results in a marked increase toward the hydrogen evolution reaction (18, 19). Considering the advantage of TMDs being able to control the atomic-scale structure, phase engineering may also open possibilities to control the selectivity of multielectron/proton reactions with multiple possible products, such as CO₂ reduction (20–23), denitrification (NO₃⁻/NO₂⁻ reduction) (24–26), and the electrosynthesis of functional molecules (27–30). Selectivity is a critical requirement for cascade catalysis, one-pot reaction systems, and multistep catalytic processes, and strategies to guide the complex chemical reaction network toward the desired end product are necessary (31, 32). However, to the best of our knowledge, no studies have attempted to exploit the advantages of phase-engineered materials for selective electrocatalysis.One effective approach to explore phase-engineered MoS₂ for selectivity control is to utilize the newly proposed concept of sequential proton−electron transfer (SPET) (off-diagonal pathways, Fig. 1A) (33, 34). In contrast to the extensively studied concerted proton−electron transfer (CPET) pathway, the energy landscape of sequential (decoupled) proton−electron transfer (SPET) pathways is pH-dependent (Fig. 1B). This leads to pH-dependent reaction rates (Fig. 1C), where the maximum reaction rate can be obtained at a pH close to the pK_a of the reaction intermediate (33, 34). This was recently observed experimentally for nitrite reduction to dinitrogen – an artificial analog of biological denitrification – on partially oxygenated molybdenum sulfide (oxo-MoS_x), and the record high selectivity toward dinitrogen was achieved by simple pH optimization (35). In contrast, this pH dependence was absent in the case of crystalline 2H-MoS₂, demonstrating that the SPET pathway is a unique property of oxo-MoS_x and is therefore probably phase-dependent. However, the origin of the SPET behavior on this material remains unclear. Therefore, elucidating the mechanism at the atomic level would help rationalize the relationship between selectivity and crystal phases, thus providing significant insight into the newly proposed SPET mechanism (33, 34) to enhance the selectivity of multistep electrochemical processes.Open in a separate windowFig. 1.Selectivity control of MoS₂ based on SPET theory. (A) Diagram showing the possible pathways for proton−electron transfer on MoS₂. In the blue pathway (CPET), protons and electrons are transferred in a single elementary step. In contrast, stepwise pathways (SPET) generate an intermediate whose charge depends on whether the electron or proton transfers first (red and black pathways, respectively). (B) Diagram showing the energetic landscape of SPET. The landscape depends on the relationship between the pK_a of the reaction intermediate and the solution pH. (C) Influence of pH on reaction selectivity. The rates of SPET reactions (red lines) show a pH dependence with a maximum corresponding to the pK_a of the intermediate. Therefore, the relative rate of one reaction over another can be tuned by changing the pH. In contrast, the rate of CPET reactions are pH-independent, and therefore, their relative rates are also constant with respect to pH.Here, we identified the atomic-scale origin of SPET-driven selectivity on MoS₂ using continuous-wave electron paramagnetic resonance (CW-EPR), Raman, and pulsed ¹H/²H electron−nuclear double-resonance (ENDOR) spectroscopy. Specifically, a proton located at the first coordination sphere (∼3.26 Å) of a redox-active Mo center was found to have a pK_a value matching that involved in the pH-dependent electrocatalytic selectivity and H/D kinetic isotope effect (KIE). The observed pH-dependent behavior is specific to 1T-MoS₂, as oxo-MoS_x was assigned to the 1T phase using high-resolution transmission electron microscopy (HRTEM), Raman- and X-ray photoelectron spectroscopy (XPS). These results not only provide atomic-scale evidence of SPET in heterogeneous catalysis, but also demonstrate how the phase engineering of TMDs can be used to enhance their electrocatalytic selectivity.     

Photosynthesis tunes quantum-mechanical mixing of electronic and vibrational states to steer exciton energy transfer

Photosynthetic species evolved to protect their light-harvesting apparatus from photoxidative damage driven by intracellular redox conditions or environmental conditions. The Fenna–Matthews–Olson (FMO) pigment–protein complex from green sulfur bacteria exhibits redox-dependent quenching behavior partially due to two internal cysteine residues. Here, we show evidence that a photosynthetic complex exploits the quantum mechanics of vibronic mixing to activate an oxidative photoprotective mechanism. We use two-dimensional electronic spectroscopy (2DES) to capture energy transfer dynamics in wild-type and cysteine-deficient FMO mutant proteins under both reducing and oxidizing conditions. Under reducing conditions, we find equal energy transfer through the exciton 4–1 and 4–2-1 pathways because the exciton 4–1 energy gap is vibronically coupled with a bacteriochlorophyll-a vibrational mode. Under oxidizing conditions, however, the resonance of the exciton 4–1 energy gap is detuned from the vibrational mode, causing excitons to preferentially steer through the indirect 4–2-1 pathway to increase the likelihood of exciton quenching. We use a Redfield model to show that the complex achieves this effect by tuning the site III energy via the redox state of its internal cysteine residues. This result shows how pigment–protein complexes exploit the quantum mechanics of vibronic coupling to steer energy transfer.

Photosynthetic organisms convert solar photons into chemical energy by taking advantage of the quantum mechanical nature of their molecular systems and the chemistry of their environment (1–4). Antenna complexes, composed of one or more pigment–protein complexes, facilitate the first steps in the photosynthesis process: They absorb photons and determine which proportion of excitations to move to reaction centers, where charge separation occurs (4). In oxic environments, excitations can generate highly reactive singlet oxygen species. These pigment–protein complexes can quench excess excitations in these environments with molecular moieties such as quinones and cysteine residues (1, 5–7).The Fenna–Matthews–Olson (FMO) complex, a trimer of pigment–protein complexes found in the green sulfur bacterium Chlorobaculum tepidum (8), has emerged as a model system to study the photophysical properties of photosynthetic antenna complexes (9–19). Each subunit in the FMO complex contains eight bacteriochlorophyll-a site molecules (Protein Data Bank, ID code: 3ENI) that are coupled to form a basis of eight partially delocalized excited states called excitons (Fig. 1) (20–23). Previous experiments on FMO have observed the presence of long-lived coherences in nonlinear spectroscopic signals at both cryogenic and physiological temperatures (11, 13). The coherent signals are thought to arise from some combination of electronic (24–26), vibrational (16–18), and vibronic (27) coherences in the system (28–30). One previous study reported that the coherent signals in FMO remain unchanged upon mutagenesis of the protein, suggesting that the signals are ground state vibrational coherences (17). Others discuss the role of vibronic coupling, where electronic and nuclear degrees of freedom become coupled (29). Other dimeric model systems have demonstrated the regimes in which these vibronically coupled states produce coherent or incoherent transport and vibronic coherences (31–33). Recent spectroscopic data has suggested that vibronic coupling plays a role in driving efficient energy transfer through photosynthetic complexes (27, 31, 33, 34), but to date there is no direct experimental evidence suggesting that biological systems use vibronic coupling as part of their biological function.Open in a separate windowFig. 1.(Left) Numbered sites and sidechains of cysteines C353 and C49 in the FMO pigment–protein complex (PDB ID code: 3ENI) (20). (Right) Site densities for excitons 4, 2, and 1 in reducing conditions with the energy transfer branching ratios for the WT oxidized and reduced protein. The saturation of pigments in each exciton denotes the relative contribution number to the exciton. The C353 residue is located near excitons 4 and 2, which have most electron density along one side of the complex, and other redox-active residues such as the Trp/Tyr chain. C353 and C49 surround site III, which contains the majority of exciton 1 density. Excitons 2 and 4 are generally delocalized over sites IV, V, and VII.It has been shown that redox conditions affect excited state properties in pigment-protein complexes, yet little is known about the underlying microscopic mechanisms for these effects (1, 9). Many commonly studied light-harvesting complexes—including the FMO complex (20), light-harvesting complex 2 (LH2) (35), the PC645 phycobiliprotein (36), and the cyanobacterial antenna complex isiA (37)—contain redox-active cysteine residues in close proximity to their chromophores. As the natural low light environment of C. tepidum does not necessitate photoprotective responses to light quantity and quality, its primary photoprotective mechanism concerns its response to oxidative stress. C. tepidum is an obligate anaerobe, but the presence of many active anoxygenic genes such as sodB for superoxide dismutase and roo for rubredoxin oxygen oxidoreductase (38) suggests that it is frequently exposed to molecular oxygen (7, 39). Using time-resolved fluorescence measurements, Orf et al. demonstrated that two cysteine residues in the FMO complex, C49 and C353, quench excitons under oxidizing conditions (1), which could protect the excitation from generating reactive oxygen species (7, 40–42). In two-dimensional electronic spectroscopy (2DES) experiments, Allodi et al. showed that redox conditions in both the wild-type and C49A/C353A double-mutant proteins affect the ultrafast dynamics through the FMO complex (9, 43). The recent discovery that many proteins across the evolutionary landscape possess chains of tryptophan and tyrosine residues provides evidence that these redox-active residues may link the internal protein behavior with the chemistry of the surrounding environment (41, 43).In this paper, we present data showing that pigment–protein complexes tune the vibronic coupling of their chromophores and that the absence of this vibronic coupling activates an oxidative photoprotective mechanism. We use 2DES to show that a pair of cysteine residues in FMO, C49 and C353, can steer excitations toward quenching sites in oxic environments. The measured reaction rate constants demonstrate unusual nonmonotonic behavior. We then use a Redfield model to determine how the exciton energy transfer (EET) time constants arise from changing chlorophyll site energies and their system-bath couplings (44, 45). The analysis reveals that the cysteine residues tune the resonance between exciton 4–1 energy gap and an intramolecular chlorophyll vibration in reducing conditions to induce vibronic coupling and detune the resonance in oxidizing conditions. This redox-dependent modulation of the vibronic coupling steers excitations through different pathways in the complex to change the likelihood that they interact with exciton quenchers.     

Atomistic investigation of surface characteristics and electronic features at high-purity FeSi(110) presenting interfacial metallicity

Iron silicide (FeSi) is a fascinating material that has attracted extensive research efforts for decades, notably revealing unusual temperature-dependent electronic and magnetic characteristics, as well as a close resemblance to the Kondo insulators whereby a coherent picture of intrinsic properties and underlying physics remains to be fully developed. For a better understanding of this narrow-gap semiconductor, we prepared and examined FeSi(110) single-crystal surfaces of high quality. Combined insights from low-temperature scanning tunneling microscopy and density functional theory calculations (DFT) indicate an unreconstructed surface termination presenting rows of Fe–Si pairs. Using high-resolution tunneling spectroscopy (STS), we identify a distinct asymmetric electronic gap in the sub-10 K regime on defect-free terraces. Moreover, the STS data reveal a residual density of states in the gap regime whereby two in-gap states are recognized. The principal origin of these features is rationalized with the help of the DFT-calculated band structure. The computational modeling of a (110)-oriented slab notably evidences the existence of interfacial intragap bands accounting for a markedly increased density of states around the Fermi level. These findings support and provide further insight into the emergence of surface metallicity in the low-temperature regime.

Iron silicide (FeSi; cf. Fig. 1A) is an archetypical B20 compound (1, 2) featuring a cubic unit cell without an inversion center and a remarkable sevenfold coordination of the constituents. The ε-FeSi B20 phase presents unusual temperature-dependent physical properties explored by a multitude of seminal experimental (3–13) and groundbreaking theoretical investigations guided by different conceptual approaches (14–21). There is a general consensus to classify FeSi as a prototypical d-electron–based narrow-gap semiconductor (gap width Δ < 100 meV) whose intriguing material characteristics are strongly affected by electronic correlations and provide prospects for technological applications (21–23).Open in a separate windowFig. 1.Surface structure of the FeSi(110) single crystal. (A) FeSi B20 unit cell containing eight atoms. (B) Photo of the FeSi(110) single crystal (top view). (C) LEED pattern of FeSi(110) prepared in UHV (E_electron = 82 eV); reciprocal lattice vectors are indicated. (D) Large-scale STM image of the FeSi(110) surface (V_b = −100 mV, I_t = 500 pA). (Scale bar: 20 nm.) (E) High-resolution STM image of a flat terrace where atomic-like features are resolved (V_b = 1 V, I_t = 100 pA). (Scale bar: 45 Å.) (F) 2D-FFT of E featuring a rectangular reciprocal lattice in good agreement with the LEED observation. (Scale bar: 0.015 Å⁻¹.) (G) Zoomed-in STM image of the surface lattice (V_b = 300 mV, I_t = 500 pA). (H) Representative STM image showing adatoms and vacancies on the surface (V_b = −200 mV, I_t = 1 nA). (Scale bars in G and H: 10 Å.)The behavior of FeSi bulk specimens in the low-temperature (LT) regime proved particularly interesting and remains an enigmatic subject. The resistivity saturates or even slightly decreases at low temperatures and exhibits a metallization at elevated temperatures, well below those nominally to be expected from the gap size (7, 24–27). Based on estimates of impurity concentrations in the samples, defect or impurity states in the gap accounting for residual conductivity were hypothesized, though also arguments in favor of intrinsic behavior exist (21). Further evidence pointing toward the existence of in-gap states has been associated with spin-polaronic phenomena (28) or localized excitonic states (29). Quite recently, two groups independently reported the detection of an electron-dominated high-mobility surface conduction channel for T < ∼20 K via careful electrical transport measurements on high-purity samples of systematically varied geometric shape and Fe concentration, respectively (26, 27). Finding surface-related conduction channels would allow to reconcile the mixed metallic and semiconducting nature of FeSi at low temperatures in a simpler picture, that is, without violating the Ioffe-Regel criterion (30, 31).The physical properties of FeSi feature striking similarities with so-called heavy-fermion semiconductors or Kondo insulators (KI), as assessed notably for Ce₃Bi₄Pt₃ (21, 22) or SmB₆ (32). Accordingly, FeSi was repeatedly considered as a special member of this class, although the key feature of canonical f-electron KIs is the hybridization between the f band and a conduction band. The latter entails the formation of a hybridization gap between bands of mixed character. A long-standing puzzle in exemplary KIs is an LT resistivity saturation, which was ascribed to surface conductivity (33–37). Inspired by the discovery of topological insulators, it was also proposed that KIs can host topologically protected surface states (32, 38–40). The topological KI (TKI) concept provides a compelling explanation for robust metallic conduction channels that has been invoked in turn for FeSi (26), although earlier studies questioned the classification of FeSi as KI (12). In SmB₆, inversion of heavy quasiparticle bands at the Fermi level generates linearly dispersive heavy Dirac fermions residing in the narrow energy gap (32, 40), which were lately deduced for T < ∼5 K at the SmB₆(100)−(2 × 1) reconstructed surface, inducing characteristic changes in the local density of states within the gap region (41). Related findings exist for unreconstructed SmB₆(100) (42); however, there are notorious difficulties in the preparation of homogenous surfaces with this material, and certain aspects underlying the data interpretation are an unsettled matter of debate (43–45). Finally, a refined orbital-selective KI scenario was recently proposed for FeSi based on a dynamic mean-field theory analysis disentangling the eminent role of different Fe 3d components in the gap formation (21).This motivated us to get an exemplary high-quality FeSi single crystal surface under control for direct scrutiny. Despite numerous investigations of FeSi samples, an atomic-level characterization of the pertaining surface characteristics and related local electronic features is missing. Cleavage methods for B20 materials provide samples of limited quality. Accordingly, angle-resolved photoemission spectroscopy (ARPES) investigations emphasized the need of well-defined surfaces and appropriate preparation protocols (8, 12), albeit the atomistic nature of the investigated systems could hitherto not be probed. First electron tunneling spectroscopic measurements were carried out on cleaved samples without recognizing the surface structure, influence of impurities, surface defects, and morphology (46).Herein, we report on well-defined single crystalline FeSi surfaces with extended atomically flat terraces that were reproducibly prepared from polished high-quality (110)-oriented bulk samples via sputtering and annealing treatments. Employing LT-scanning tunneling microscopy (STM) as well as extensive computational modeling, we determined the surface atomic arrangement and termination with element-specific registry. Furthermore, by high-resolution scanning tunneling spectroscopy (STS) measurements, the narrow energy gap with distinct temperature-dependent characteristics is clearly resolved. For T < ∼10 K, it exhibits a markedly asymmetric shape with subtle features of in-gap states located on both sides of the Fermi level, where nonzero density of states (DOS) is present. Furthermore, the band structure obtained via density functional theory (DFT) calculations of a slab with (110) surface termination reproduces key features of STS spectra in a qualitative way, showing surface-related bands crossing the bulk energy gap. Our findings confirm that samples fabricated with the highest purity standards and examined with atomistic precision are a prerequisite for both developing a basic understanding of complex materials and making further progress toward harnessing their application potential. Specifically, we provide unambiguous evidence that the appearance of surface conductivity channels for the FeSi system in the LT regime can be attributed to interfacial symmetry breaking. Moreover, our findings reveal that the electronic properties of FeSi not only resemble that of the archetypical KI SmB₆ regarding bulk characteristics but also in the appearance of surface metallicity at low temperatures.     

Type III secretion system effector proteins are mechanically labile

Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins—such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)—impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy (ΔGunfold0) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published ΔGunfold0 for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force (F_unfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δx^‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust (F_unfold > 80 pN) and brittle (Δx^‡ < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

Type III secretion systems (T3SS) are large nanomachines utilized by both pathogenic and symbiotic bacteria to inject effector proteins directly into the cytoplasm of host cells (1–3). Once delivered, effector proteins facilitate host cell colonization through a variety of mechanisms (4–7), including down-regulation of the host immune response (8) and rearrangement of the cytoskeleton (9, 10). The T3SS apparatus, known as the injectisome, is a syringe-like structure with a hollow needle that spans the inner and outer bacterial membranes, the extracellular space, and the host membrane, enabling proteins to pass directly from bacteria to host cells (Fig. 1A) (2). Specialized bacterial chaperones often bind the N-terminal 50 to 100 amino acids (aa) of the effector proteins, known as the chaperone binding domain, and help maintain the effector N-terminal domain in an extended conformation. C-terminal to the chaperone binding domain, effector proteins contain one or more globular domains, which adopt their folded conformations even when in complex with their cognate chaperone (4, 11, 12). The effector proteins, or their chaperone complexes, are recognized by the base of the injectisome prior to secretion (13). At its narrowest point, the injectisome needle’s inner diameter is less than 2 nm (14–16). As a result, effector proteins must be mostly unfolded to be secreted (17–20). Secretion is thus thought to proceed by a “threading-the-needle mechanism,” where the N-terminal extended domain is released from the chaperone and fed to the injectisome, followed by unfolding of the C-terminal effector domain (21).Open in a separate windowFig. 1.Thermodynamic stability of T3SS effector proteins SptP_CD and SopE2_CD. (A) Schematic depiction of protein transport through the T3SS showing effector proteins, which are at least partially folded in the bacterial cytoplasm. Such effector proteins interact with an associated unfoldase to passage through the T3SS, which has an inner channel with a diameter <2 nm. Once inside the host cytoplasm, effector proteins refold to carry out their function. (B) Crystal structures of SptP_CD (Protein Data Bank [PDB] ID code 1G4U) and SopE2_CD (PDB ID code 1R9K). (C) Ellipticity from CD at λ = 222 nm plotted as a function of urea concentrations for SptP_CD (orange) and SopE2_CD (green). A fit of the data with Eq. 1 yielded the free energy of unfolding ΔGunfold0 for SptP_CD (6.9 ± 0.2 kcal/mol [mean ± fit error]) and SopE2_CD (6.0 ± 0.2 kcal/mol [mean ± fit error]). Data points are the result of at least three independent measurements. Error bars represent SD.Before proteins are secreted through the T3SS, they interact with a hexameric ATPase at the base of the T3SS that is capable of mediating chaperone release from effector proteins and effector-protein unfolding (15, 22). Indeed, most in vivo unfolding is catalyzed by unfoldases that work from one end of the substrate protein in stark contrast to the global effects of temperature, pH, or chemical denaturants. The most common examples of targeted protein unfolding are catalyzed by ATPases of the AAA(+) family that mechanically unfold their substrates (23, 24). For example, the AAA(+) ATPase ClpX forms a ring-shaped hexamer that mechanically pulls its substrates through its narrow central pore to unfold them (25). These are powerful unfoldases that can unfold even tightly packed proteins such as GFP, ubiquitin, and dihydrofolate reductase (DHFR) (23, 24, 26, 27). However, the T3SS ATPase does not belong to the AAA(+) family of ATPases. Instead, it is structurally similar to the catalytic β-subunit of the F₁F₀ ATP synthase, a rotary motor that normally couples proton gradient dissipation to ATP synthesis but can also run in reverse and hydrolyze ATP to do work (15, 28–30). The T3SS ATPase is not as powerful an unfoldase as the AAA(+) family, as fusions of effector proteins with GFP, ubiquitin, or DHFR stall in the injectisome and are poorly secreted (20, 22, 31, 32). These observations have led to the current model that T3SS effector proteins have low thermodynamic stability to facilitate their secretion (22, 31–33).While thermodynamic stability is the most common metric of protein stability, mechanical stability is a distinct metric that quantifies how easily a protein unfolds under force (F_unfold). Mechanical stability is typically measured by pulling across the N and C termini of single molecules via force spectroscopy using optical tweezers (34, 35) or an atomic force microscope (AFM) (36). Early force spectroscopy studies showed that thermodynamic stability does not correlate with mechanical stability (37–41). For example, titin’s I28 domain requires ∼20% more force to unfold than titin’s I27 domain [I85 and I91, respectively, in the new nomenclature (42)], despite I27 having approximately twofold higher thermodynamic stability (43). Importantly, AFM studies have shown that GFP (44), ubiquitin (45), and DHFR (46) are mechanically robust, requiring high forces to unfold despite their typical thermodynamic stabilities. These three proteins each stall the T3SS; thus, mechanical stability may be the physical determinant to proteins being secreted by the T3SS, rather than thermodynamic stability.Here, we determine the thermodynamic and mechanical stabilities of SptP and SopE2, two effector proteins from Salmonella enterica. These effectors are ideal candidates for this study as they have known crystal structures (10, 47), have characterized in vivo secretion kinetics (48), and represent effector proteins of different size and structure (Fig. 1B). We show that the catalytic domains of SptP and SopE2 have unremarkable thermodynamic stabilities, similar to many other previously characterized proteins, including GFP, ubiquitin, and DHFR. Conversely, our AFM-based force spectroscopy measurements demonstrate that SptP and SopE2 are among the most mechanically labile proteins studied to date by AFM. These two T3SS effector proteins are therefore mechanically labile while being thermodynamically stable, supporting the hypothesis that it is mechanical stability, not thermodynamic stability, that predicts efficient protein secretion by the T3SS.     

A new sea-level record for the Neogene/Quaternary boundary reveals transition to a more stable East Antarctic Ice Sheet

Sea-level rise resulting from the instability of polar continental ice sheets represents a major socioeconomic hazard arising from anthropogenic warming, but the response of the largest component of Earth’s cryosphere, the East Antarctic Ice Sheet (EAIS), to global warming is poorly understood. Here we present a detailed record of North Atlantic deep-ocean temperature, global sea-level, and ice-volume change for ∼2.75 to 2.4 Ma ago, when atmospheric partial pressure of carbon dioxide (pCO₂) ranged from present-day (>400 parts per million volume, ppmv) to preindustrial (<280 ppmv) values. Our data reveal clear glacial–interglacial cycles in global ice volume and sea level largely driven by the growth and decay of ice sheets in the Northern Hemisphere. Yet, sea-level values during Marine Isotope Stage (MIS) 101 (∼2.55 Ma) also signal substantial melting of the EAIS, and peak sea levels during MIS G7 (∼2.75 Ma) and, perhaps, MIS G1 (∼2.63 Ma) are also suggestive of EAIS instability. During the succeeding glacial–interglacial cycles (MIS 100 to 95), sea levels were distinctly lower than before, strongly suggesting a link between greater stability of the EAIS and increased land-ice volumes in the Northern Hemisphere. We propose that lower sea levels driven by ice-sheet growth in the Northern Hemisphere decreased EAIS susceptibility to ocean melting. Our findings have implications for future EAIS vulnerability to a rapidly warming world.

The instability of polar continental ice sheets in a warmer future is an issue of major societal concern (1–5). Based on linear extrapolation of recent sea-level rise (2), mean global sea level could increase by 65 ± 12 cm by 2100 relative to the 2005 baseline, consistent with Intergovernmental Panel on Climate Change projections (1) of a ∼30- to 100-cm increase by 2100. Further, satellite observations (4) document substantial mass loss of both the Greenland Ice Sheet (GIS) and the West Antarctic Ice Sheet (WAIS) over the past decade—the two ice sheets that are most susceptible to global warming because of rapidly rising Arctic air temperatures (1) (GIS) and vulnerability to ocean-atmospheric warming (5, 6) (WAIS). The mass balance of the much larger EAIS and its contribution to ongoing sea-level change, however, remain poorly constrained (1).The role of atmospheric partial pressure of carbon dioxide (pCO₂) as a driver of long-term changes in ice volume and sea level over the Cenozoic Era (past ∼66 My) is widely documented (7–9) and there is compelling evidence (6, 10–12) of East Antarctic Ice Sheet (EAIS) retreat during warm intervals of the Pliocene epoch between ∼5.3 and 3.3 Ma when pCO₂ levels (13, 14) last reached values close to the present day (∼400 parts per million volume [ppmv]; Fig. 1 A and B and see SI Appendix, section S1). However, there is disagreement over EAIS behavior under pCO₂ levels (13) similar to those of preindustrial Quaternary times (<280 ppmv). A compilation of marine geochemical paleo-sea-level and pCO₂ records suggests that the EAIS was stable under these conditions (7). In contrast, while the amplitudes of change are controversial (15) (SI Appendix, section S2), sea-level reconstructions from paleoshorelines (16) and benthic geochemical data (9, 17, 18) (Fig. 2) imply EAIS melting during the Quaternary “super-interglacials” of Marine Isotope Stage (MIS) 11 (∼400 ka) and 31 (∼1.07 Ma) under relatively low pCO₂ conditions. Supporting evidence for EAIS retreat during the most recent “super-interglacial” MIS 11 comes from isotope measurements in mineral deposits recording past changes in subglacial East Antarctic waters (19), as well as records of ice-rafted debris (IRD) and detrital sediment neodymium isotopes from offshore the Wilkes Subglacial Basin (20). The latter records (20) also indicate EAIS retreat during the last interglacial MIS 5e (∼120 ka). Melting of the EAIS as inferred in the late Quaternary was likely driven by ocean–atmosphere warming around Antarctica and grounding-line retreat in response to ice–ocean interactions (19, 20).Open in a separate windowFig. 1.Neogene to Quaternary climate and sea-level evolution. (A) LR04 stack (21) for the past 5 My; arrow indicates the iNHG (∼3.6 to 2.4 Ma) and its culmination (thick-arrowed interval) (22); green line indicates the benthic δ¹⁸O level associated with MIS 101. (B) Atmospheric pCO₂ estimates of refs. 13 (blue) and 23 (purple) for the past 5 My; the late Quaternary glacial–interglacial pCO₂ range (1) is indicated as preindustrial pCO₂ band. Yellow shading in A and B highlights the study interval (∼2.75 to 2.4 Ma). (C and D) Site U1313 benthic δ¹⁸O and Mg/Ca raw data, respectively. (E) Site U1313 deep-sea temperature. (F) Site U1313 δ¹⁸O_sw-based sea level relative to present (black line); blue shading: 95% probability interval from Monte Carlo simulations (2σ); red line: threshold (11.6 msle) above which a smaller-than-present EAIS is signaled (24–26); m = marine part of EAIS, t = terrestrial part of EAIS. Glacials are highlighted in gray.Open in a separate windowFig. 2.Implication of different sea-level-δ¹⁸O_sw conversions for estimates of interglacial ice-volume loss. y axis shows lower-than-modern δ¹⁸O_sw values (∆δ¹⁸O_sw) and the x axis (log-scale) the corresponding sea-level increase for commonly used conversion factors (27–29) (0.011 [black], 0.010 [purple], and 0.008 ‰⋅m⁻¹ [red]) and those for Antarctica only (11) (0.014 ‰⋅m⁻¹) ignoring (yellow) and incorporating (brown) the impact of its marine-based ice sheets. Stars mark ∆δ¹⁸O_sw for interglacials of this study and corresponding sea-level equivalents in dependence of the conversion applied. Orange, blue, and purple diamonds show the same for MIS 31, 11 (18), and 5e (17), respectively. Vertical lines indicate the sea-level increase resulting from complete melting of the GIS (+7.3 m), WAIS (+4.3 m), and EAIS (+53.3 m) (24–26).To further investigate past EAIS response to climate forcing we studied the Neogene/Quaternary transition when mean pCO₂ (13, 23) fell from levels similar to the anthropogenically perturbed values of today into the Quaternary range, leading to progressive high-latitude cooling and the intensification of Northern Hemisphere Glaciation (21, 30–33) (iNHG; Fig. 1 A and B). Our approach is based on a simple approximation that, once estimated past global sea level exceeds 11.6 m sea-level equivalent (msle) above modern, which corresponds to the complete melting of the present-day GIS [7.3 msle (24, 25)] and the marine- and land-based WAIS [3.4 and 0.9 msle (25, 26), respectively], EAIS instability (i.e., a retreat from its present-day size) can be inferred (see SI Appendix, section S4.1 for details). We quantified sea-level and ice-volume changes for the interval ∼2.75 to 2.4 Ma (MIS G7 to 95) by measuring the oxygen-isotope composition (δ¹⁸O) and Mg/Ca ratio in well-preserved benthic foraminiferal calcite (Oridorsalis umbonatus) from Integrated Ocean Drilling Program (IODP) Site U1313 [41°0′N, 32°57′W; 3,426-m water depth (34)] in the North Atlantic Ocean (Fig. 1 C and D). Using this approach we reconstructed changes in seawater δ¹⁸O (δ¹⁸O_sw), a proxy for global sea level and continental ice volume (35). This was done by 1) calculating bottom-water temperatures (BWT) derived from Mg/Ca (36) (Fig. 1E), 2) combining Mg/Ca-derived BWTs with δ¹⁸O to determine δ¹⁸O_sw (37) (Fig. 1F), and 3) converting δ¹⁸O_sw to sea level using a relationship between changes in sea level and δ¹⁸O_sw of 0.011 ‰⋅m⁻¹ (27) (Materials and Methods and Fig. 1F). Ninety-five percent probability intervals calculated through Monte Carlo simulations for individual sea-level data points yield an average uncertainty for our sea-level estimates of ± 28 m (∼2σ [SD]) (Materials and Methods and Fig. 1F), roughly equivalent to the decay/growth of ice four times greater than the GIS. Our approach was validated by reconstructing δ¹⁸O_sw for the recent (∼0 to 7 ka) at IODP Site U1313 and for late Holocene core-top (multicorer) samples from a neighboring site (MSM58) which are indistinguishable from the observed modern-day values (see Materials and Methods and SI Appendix, section S4.2.8 for details).     

A switch to feeding on cycads generates parallel accelerated evolution of toxin tolerance in two clades of Eumaeus caterpillars (Lepidoptera: Lycaenidae)

We assembled a complete reference genome of Eumaeus atala, an aposematic cycad-eating hairstreak butterfly that suffered near extinction in the United States in the last century. Based on an analysis of genomic sequences of Eumaeus and 19 representative genera, the closest relatives of Eumaeus are Theorema and Mithras. We report natural history information for Eumaeus, Theorema, and Mithras. Using genomic sequences for each species of Eumaeus, Theorema, and Mithras (and three outgroups), we trace the evolution of cycad feeding, coloration, gregarious behavior, and other traits. The switch to feeding on cycads and to conspicuous coloration was accompanied by little genomic change. Soon after its origin, Eumaeus split into two fast evolving lineages, instead of forming a clump of close relatives in the phylogenetic tree. Significant overlap of the fast evolving proteins in both clades indicates parallel evolution. The functions of the fast evolving proteins suggest that the caterpillars developed tolerance to cycad toxins with a range of mechanisms including autophagy of damaged cells, removal of cell debris by macrophages, and more active cell proliferation.

The genus Eumaeus Hübner (Lycaenidae, Theclinae) arguably contains the most aposematically colored caterpillars and butterflies among the ∼4,000 Lycaenidae in the world (1–6). The brilliant red and gold gregarious caterpillars (Fig. 1) sequester cycasin from the leaves of their cycad food plants (Zamiaceae), which deters predators (3–9). Other secondary metabolites in cycads (e.g., 10‒11) may also deter predators. Eumaeus adults have a bright orange-red abdomen and an orange-red hindwing spot (except for one species) (Fig. 2). Blue and green iridescent markings are especially conspicuous on a black ground color. Eumaeus adults are among the largest lycaenids and have more rounded wings and a slower, more gliding flight than most Theclinae (1). Cycads are among the most primitive extant seed-plants (9), and the “plethora of aposematic attributes suggests a very ancient association between Eumaeus and the cycad host plants” (3).Open in a separate windowFig. 1.Caterpillars and pupae of Theorema eumenia (Top) and Eumaeus godartii (Bottom) in Costa Rica. Clockwise from Upper Left, second or third instar (length, ∼13 mm), fourth (final) instar (∼20 mm), pupa (∼18 mm), pupa (∼24 mm), fourth (final) instar (∼27 mm), second or third instar (∼20 mm). (Images from authors W.H. and D.H.J.).Open in a separate windowFig. 2.Adult wing uppersides and undersides. Eumaeus childrenae (two Upper Left images), E. atala (two Upper Right images), Theorema eumenia (two Lower Left images), and Mithras nautes (two Lower Right images). Scale bar, 1 cm.Eumaeus has been classified as a separate family (12–14), a genus in the Riodinidae (15‒16), or a monotypic subfamily or tribe of the Lycaenidae (17–20). Alternatively, others called it a typical member of the Neotropical Lycaenidae (21‒22). The evolutionary question behind this discordant taxonomic history is whether Eumaeus is a phylogenetically isolated lineage long associated with cycads (3) or an embedded clade in which a recent food plant shift to cycads resulted in the rapid evolution of aposematism. Recent molecular evidence for a limited number of taxa suggested the latter (23). To answer this question definitively, we analyzed genomic sequences of Eumaeus and its relatives.To trace the evolution of cycad feeding, we report the caterpillar food plants of the genera most closely related to Eumaeus and illustrate their immature stages (Fig. 1 and SI Appendix). This natural history information combined with analyses of genome sequences is the foundation for investigating the subsequent evolutionary impact on the Eumaeus genome of the switch to eating cycads.     

Stable metal anodes enabled by a labile organic molecule bonded to a reduced graphene oxide aerogel

Metallic anodes (lithium, sodium, and zinc) are attractive for rechargeable battery technologies but are plagued by an unfavorable metal–electrolyte interface that leads to nonuniform metal deposition and an unstable solid–electrolyte interphase (SEI). Here we report the use of electrochemically labile molecules to regulate the electrochemical interface and guide even lithium deposition and a stable SEI. The molecule, benzenesulfonyl fluoride, was bonded to the surface of a reduced graphene oxide aerogel. During metal deposition, this labile molecule not only generates a metal-coordinating benzenesulfonate anion that guides homogeneous metal deposition but also contributes lithium fluoride to the SEI to improve Li surface passivation. Consequently, high-efficiency lithium deposition with a low nucleation overpotential was achieved at a high current density of 6.0 mA cm⁻². A Li|LiCoO₂ cell had a capacity retention of 85.3% after 400 cycles, and the cell also tolerated low-temperature (−10 °C) operation without additional capacity fading. This strategy was applied to sodium and zinc anodes as well.

Rechargeable batteries based on metal anodes including lithium (Li), sodium (Na), and zinc (Zn) show great promise in achieving high energy density (1–3). Unfortunately, the electrochemical interface of the metal anodes is not favorable for metal deposition. Metal nucleation is inhomogeneous at the surface, leading to the growth of metal dendrites (4–7) and the formation of an unstable solid–electrolyte interphase (SEI) that is incapable of protecting metals from the side reactions with the electrolyte (8–12).Substantial efforts have been devoted to stabilizing the interface of metal anodes, especially for Li metal. These include the design of artificial protective layers (13–17), alternative electrolytes (18–24), and sacrificial additives (25–30) to stabilize the metal–electrolyte interface, the development of mechanically robust coatings (31–34) to block Li dendrite growth, and the use of structured scaffolds to host dendrite-free Li deposition by reducing local current densities (35–43). However, the performance of metal anodes remains poor under high-current or low-temperature conditions. This is because the inhomogeneous Li nucleation and unstable SEI problems have not been well addressed, and these problems at the interface are even exacerbated under critical operating conditions, especially high-current densities and low temperatures (5, 6, 44).Toward this end, we report a simple molecular approach for regulating the electrochemical interface of metal anodes, which enables even Li deposition and stable SEI formation in a conventional electrolyte. This was realized by bonding a labile organic molecule, benzenesulfonyl fluoride (BSF), to a reduced graphene oxide (rGO) aerogel surface as the Li anode host (Fig. 1A). During Li deposition, BSF molecules electrochemically decompose at the interface and generate benzenesulfonate anions bonded to the rGO aerogel (Fig. 1B). The conjugated anions have a strong binding affinity for Li, serving as lithiophilic sites on the rGO surface to synergistically induce homogeneous Li nucleation of Li on the rGO surface. At the same time, BSF molecules contribute LiF to the SEI layer, which facilitates Li surface passivation (Fig. 1C). As a result, high-efficiency (99.2%) Li deposition was achieved at a Li deposition amount of 6.0 mAh cm⁻² and a current density of 6.0 mA cm⁻²; the barrier to Li nucleation was markedly reduced, as evidenced by the low nucleation overpotentials at high-current density (6.0 mA cm⁻²) or at a low temperature (−10 °C). A 400-cycle life with a capacity retention of 83.6% was achieved for a Li|LiCoO₂ (LCO) cell in a conventional carbonate electrolyte. Moreover, with the organic molecule-tuned interface, the Li|LCO cell can be stably cycled at a low operating temperature (−10 °C). This approach was applied to Na and Zn metal anodes as well.Open in a separate windowFig. 1.Illustration of a stable interface for Li deposition using a labile organic molecule, benzenesulfonyl fluoride (BSF). (A) Covalently bonded BSF on the rGO aerogel surface. (B) In situ generation of a lithiophilic conjugated anion (benzenesulfonate) and LiF on the surface during Li deposition. (C) Li nucleation preferentially occurs at the conjugated anion sites owing to the strong Li binding affinity, which leads to uniform Li deposition. In addition, the LiF that is formed is in the SEI layer and passivates the Li surface.