Early Holocene greening of the Sahara requires Mediterranean winter rainfall

The greening of the Sahara, associated with the African Humid Period (AHP) between ca. 14,500 and 5,000 y ago, is arguably the largest climate-induced environmental change in the Holocene; it is usually explained by the strengthening and northward expansion of the African monsoon in response to orbital forcing. However, the strengthened monsoon in Early to Middle Holocene climate model simulations cannot sustain vegetation in the Sahara or account for the increased humidity in the Mediterranean region. Here, we present an 18,500-y pollen and leaf-wax δD record from Lake Tislit (32° N) in Morocco, which provides quantitative reconstruction of winter and summer precipitation in northern Africa. The record from Lake Tislit shows that the northern Sahara and the Mediterranean region were wetter in the AHP because of increased winter precipitation and were not influenced by the monsoon. The increased seasonal contrast of insolation led to an intensification and southward shift of the Mediterranean winter precipitation system in addition to the intensified summer monsoon. Therefore, a winter rainfall zone must have met and possibly overlapped the monsoonal zone in the Sahara. Using a mechanistic vegetation model in Early Holocene conditions, we show that this seasonal distribution of rainfall is more efficient than the increased monsoon alone in generating a green Sahara vegetation cover, in agreement with observed vegetation. This conceptual framework should be taken into consideration in Earth system paleoclimate simulations used to explore the mechanisms of African climatic and environmental sensitivity.

Moisture availability in northern Africa, from the Sahel to the Mediterranean coast, is a critical issue for both ecosystems and human societies yet represents one of the largest uncertainties in future climate simulations (1, 2). The humid time span in the African Sahel and Sahara, known as the African humid period (AHP) (3–13), occurred in northern Africa after the last glacial period (4, 10, 11, 14–16) and lasted from ca. 14.5 to 5 ky ago (ka), with an optimum between 11 and 6 ka (11, 16). This prominent climatic event allowed semiarid, subtropical, and tropical plant species to spread outside their modern ranges (14) into the Sahara and human populations to inhabit what is known as the green Sahara (5, 17).The green Sahara is an example of extreme environmental change, which highlights the region’s extraordinary sensitivity and the need to better understand its hydroclimatic variability. Current explanations for the greening of the Sahara point to the Earth’s orbital changes during the Early Holocene, leading to increased boreal summer (JJA) insolation, which drove the intensification and northward expansion of the JJA monsoon over northern Africa (15, 18), aided by strong positive feedbacks from the land surface (19–22). Reproducing the green Sahara has posed a lasting challenge for climate modelers. The influence of the African monsoon extends only to ∼24° N (with or without interactive vegetation) in most Middle Holocene simulations, which is insufficient to sustain a vegetated Sahara. Models that integrate vegetation, dust, and soil feedbacks push the monsoon influence further north but still have discrepancies with proxy data (18, 23, 24).When all surface feedbacks are prescribed, simulated precipitation in the northern Sahara is still too low compared to paleoclimatic evidence for substantially increased moisture at 31° N (11, 13) or too high in the 15 to 20° N range (20), creating incompatibility with prescribed vegetation (22). Additional sources of moisture (25, 26) may have contributed to an AHP that extended toward the Mediterranean borderlands through different mechanisms. However, identifying the moisture sources over North Africa during the AHP requires paleoclimate records of both winter (DJF) and JJA precipitation.In the High Atlas Mountains, we collected an 8.5-m sediment core from Lake Tislit (ca. 32° N). The lake traps pollen grains from the surrounding landscape and, as a closed lake, is highly sensitive to hydroclimate fluctuations. It is ideally located for capturing the climatic variability of the Mediterranean and northwestern Sahara (Fig. 1). The Tislit sequence yielded unique hydrological data from leaf-wax stable isotopes and ostracod stable oxygen isotopes (δ¹⁸O), as well as a quantified time series of seasonal rainfall from the fossil pollen assemblages. Based on the findings from the Tislit record, we propose a precipitation regime for the AHP, including both Mediterranean DJF precipitation and monsoon JJA precipitation increases. Using a dynamic vegetation model for a conceptual experiment with 9 ka boundary conditions, we evaluate how a change in the seasonal distribution of precipitation over the Sahara can affect its revegetation.Open in a separate windowFig. 1.Maps showing the location of Lake Tislit, core GC27 (11), and Lake Yoa (6), along with the schematic position of the Inter Tropical Convergence Zone, with modern mean JJA (A) and DJF (B) rainfall (56). Map C shows the correlation coefficients (r) between Tislit and northern Morocco for DJF precipitation variability over the 1901 to 2010 time period (using the 20th century reanalysis of National Oceanic and Atmospheric Administration; https://psl.noaa.gov/data/20thC_Rean/). The limit of statistical significance (0.05 level) is shown by the dashed black line. Gray contours indicate annual precipitation isohyets (millimeter/year).     

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.     

From the Cover: Neurophysiological coordination of duet singing

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

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

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

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

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

Early,intensive marine resource exploitation by Middle Stone Age humans at Ysterfontein 1 rockshelter,South Africa

Modern human behavioral innovations from the Middle Stone Age (MSA) include the earliest indicators of full coastal adaptation evidenced by shell middens, yet many MSA middens remain poorly dated. We apply ²³⁰Th/U burial dating to ostrich eggshells (OES) from Ysterfontein 1 (YFT1, Western Cape, South Africa), a stratified MSA shell midden. ²³⁰Th/U burial ages of YFT1 OES are relatively precise (median ± 2.7%), consistent with other age constraints, and preserve stratigraphic principles. Bayesian age–depth modeling indicates YFT1 was deposited between 119.9 to 113.1 thousand years ago (ka) (95% CI of model ages), and the entire 3.8 m thick midden may have accumulated within ∼2,300 y. Stable carbon, nitrogen, and oxygen isotopes of OES indicate that during occupation the local environment was dominated by C₃ vegetation and was initially significantly wetter than at present but became drier and cooler with time. Integrating archaeological evidence with OES ²³⁰Th/U ages and stable isotopes shows the following: 1) YFT1 is the oldest shell midden known, providing minimum constraints on full coastal adaptation by ∼120 ka; 2) despite rapid sea-level drop and other climatic changes during occupation, relative shellfish proportions and sizes remain similar, suggesting adaptive foraging along a changing coastline; 3) the YFT1 lithic technocomplex is similar to other west coast assemblages but distinct from potentially synchronous industries along the southern African coast, suggesting human populations were fragmented between seasonal rainfall zones; and 4) accumulation rates (up to 1.8 m/ka) are much higher than previously observed for dated, stratified MSA middens, implying more intense site occupation akin to Later Stone Age middens.

The Middle Stone Age (MSA) defines an interval in which many “modern” human behaviors emerged, including the first indications of artistic and symbolic expression, the use of personal adornments, advancements in tool making, effective hunting of large mammal game, and the exploitation of harsher environments (e.g., refs. 1–3). The earliest records of humans intensely and systematically exploiting marine resources also derive from MSA sites (4), and archaeological sequences rich in marine shellfish remains provide insight into several key aspects of human behavioral and cultural evolution during the MSA. For example, marine foods such as shellfish and marine mammals (scavenged or hunted) have been proposed as uniquely rich sources of key nutrients promoting brain development and perhaps leading to enhanced cognitive development and higher reproductive fitness (5–8). Some workers have suggested that adaptation to systematic coastal foraging with its rich, geographically stable and predictable resources may have promoted behaviors considered to be unique and potent adaptations of Homo sapiens such as territoriality, intergroup competition, and high levels of nonkin cooperation (9). Differences in marine shell sizes and accumulation rates between MSA and Later Stone Age (LSA) coastal sequences have been interpreted to provide insight into, respectively, relative population size and intensity of site occupation through time (10, 11). Compared to LSA people representing comparably larger populations, smaller populations of MSA people are thought to have exploited coastal resources less intensively, more selectively, or both (4). However, without robust chronological frameworks at MSA sites, lithic technologies and evidence of behavioral innovations cannot be confidently compared between sites. We show that precise geochronology and refined paleoenvironmental information can shed light on human behavioral development, adaptations, and population sizes in the MSA.MSA shell middens occur on the South African coastline (Fig. 1) (10, 12–21), and their ages are constrained largely by diverse techniques with precision typically of the order ∼10% or more at 2σ relative uncertainty (22). Here, we apply a recently developed approach to uranium-series (hereafter U-series, or ²³⁰Th/U) dating of ostrich eggshells (OES) at Ysterfontein 1 (YFT1), a well-stratified, carefully excavated MSA shell midden on the west coast of the Western Cape Province, South Africa, hosting a record of early shellfishing (10, 12, 13).Open in a separate windowFig. 1.YFT1 site location, locations of other sites with marine shell-rich layers, and YFT1 site stratigraphy. (A) Distribution of shell middens along the coasts of South Africa, with modern winter seasonal rainfall isopleths; (B, Inset) detailed map (A, pink outline) of shell middens on the western coast in the winter rainfall zone. Maps in A and B are modified from ref. 83. (C) YFT1 composite stratigraphy, numbered LG, and placement of OES dated by ²³⁰Th/U burial dating and/or with stable isotope data, after ref. 12; Inset are the OES sampled from LG12 for stable isotopes and ²³⁰Th/U burial dating. Site acronyms for maps A and B are as follows: BBC, Blombos Cave; BNK, Byneskranskop Cave; DK1, Die Kelders 1; DKS, Diepkloof Shelter; EBC, Elands Bay Cave; HDP, Hoedjiespunt; KRM, Klasies River Mouth; NBC, Nelson Bay Cave; PP, Pinnacle Point; SH, Sea Harvest; and YFT1, Ysterfontein 1.Unlike other U-series dating of OES, “²³⁰Th/U burial dating” accounts for the secondary uptake of U in OES from soil pore water upon burial and uses geochemical criteria inherent to the ²³⁰Th/U data to help identify reliable ages (23–27). The method was first tested against radiocarbon (¹⁴C) dating in LSA contexts (23), and this paper describes results from an extension of the method into archaeological contexts beyond the range of ¹⁴C dating (∼50 thousand years ago; hereafter, ka), which has not been previously attempted. Light stable isotope–paleoenvironmental proxies from OES also provide paleoenvironmental context directly related to the archaeological site (28–31), which we apply to OES recovered from YFT1. OES fragments at YFT1 are interspersed throughout the 3.8 m stratified sequence (SI Appendix), providing abundant sample material for dating. The chronological framework and paleoenvironmental context for YFT1 presented here provides key implications for the spatiotemporal coherence of South African lithic technologies and early evidence of intensive shellfishing, a coastal adaptation thus far considered unique to modern humans (9).     

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: Summer warmth of the past six interglacials on Greenland

The relative warmth of mid-to-late Pleistocene interglacials on Greenland has remained unknown, leading to debates about the regional climate forcing that caused past retreat of the Greenland Ice Sheet (GrIS). We analyze the hydrogen isotopic composition of terrestrial biomarkers in Labrador Sea sediments through interglacials of the past 600,000 y to infer millennial-scale summer warmth on southern Greenland. Here, we reconstruct exceptionally warm summers in Marine Isotope Stage (MIS) 5e, concurrent with strong Northern Hemisphere summer insolation. In contrast, “superinterglacial” MIS11 demonstrated only moderate warmth, sustained throughout a prolonged interval of elevated atmospheric carbon dioxide. Strong inferred GrIS retreat during MIS11 relative to MIS5e suggests an indirect relationship between maximum summer temperature and cumulative interglacial mass loss, indicating strong GrIS sensitivity to duration of regional warmth and elevated atmospheric carbon dioxide.

The Greenland Ice Sheet (GrIS) is projected to contribute between +5 and +33 cm to global sea level by 2100 CE under continued strong anthropogenic forcing (1). Significant uncertainty in projections results, in part, from a lack of constraints on the regional terrestrial climate changes causing past large-scale ice sheet mass loss (2, 3). Extensive retreat of the GrIS likely occurred most recently during Marine Isotope Stage (MIS) 11 (ca. 425 to 375 thousand years before present [ka]), indicated by evidence of coniferous forest cover in southern Greenland coincident with a cessation in the delivery of glacially eroded silts to the Labrador Sea (4, 5) (Figs. 1 and ​and2).2). Curiously, Northern Hemisphere summer insolation and atmospheric carbon dioxide (CO₂) forcing were lower during MIS11 than other Pleistocene interglacials through which continental-scale ice persisted on Greenland. For example, the Last Interglacial (MIS5e) (ca. 130 to 115 ka) was associated with stronger Northern Hemisphere summer insolation and briefly higher atmospheric CO₂ concentrations (6, 7). Yet basal sections of seven ice cores contain ice deposited during MIS5e (8), suggesting ice was present on much of the island within this stage.Open in a separate windowFig. 1.Map of study region. Location of Eirik Drift core sites (black point), including Ocean Drilling Program Site 646 used in this study. Dotted lines separate silt provenances as in Fig. 2H (5, 12). White is the modern glacier extent. Solid lines are the modern schematic surface ocean currents: the West Greenland Current (WGC), Baffin Island Current (BIC), and Irminger Current (IC). The dashed line is the Deep Western Boundary Undercurrent (WBUC). Points are the Greenland ice cores (27), with ice dated to peak (dark red) or late (light red) MIS5e (28–31) and Holocene δ²H_C28 records (yellow) (17, 24). The inset map is of Lake El’Gygytgyn (Lake E) (9), Arctic Ocean core HLY-06 (10), and the Faroe Islands (FI) (25).Open in a separate windowFig. 2.Interglacial records from MIS13 to MIS1. Datasets are plotted on their own age scales and not synchronized, except those from the same sites. Formally defined MIS and peak substage (22) (as in Fig. 4) are shaded. (A) June 21st insolation at 65°N (6). (B) Atmospheric CO₂ concentration (7). (C) Site 646 δ²H_C28 (this study). Analytical error is smaller than point size (SI Appendix). (D) Site 646 δ²H_C28 as an anomaly relative to the last millennium. (E) Global benthic (blue) and Site 646 planktic foraminifera δ¹⁸O (black) (4, 22). (F) Stable carbon isotopes (δ¹³C, ‰ VPDB [Vienna Pee Dee Belemnite]) of Cibicidoides wuellerstorfi from U1305 (34). (G) SSTs from U1305 (summer: black and red error envelope; winter: black and blue error envelope) (11) and Site 646 (summer: red; winter: blue) (4). (H) Provenance of MD99-2227 silts as in Fig. 1 (5, 12). (I) Mean temperature of the warmest month (MTWM) from Lake El’Gygytgyn (9). (J) Site 646 pollen concentrations (4).Sparse paleoclimate evidence suggests that Arctic climate responded nonlinearly to global-scale forcings during past interglacials. For example, MIS11 was one of a few Pleistocene “superinterglacials” identified in the eastern Arctic, with inferred summer air temperatures 4 to 5 °C higher than the current interglacial (MIS1, the Holocene, 11.7 to 0 ka) (9) (Fig. 2I). Outstanding Arctic warmth during MIS11 is supported by ostracod assemblages in the Arctic Ocean, indicating summer sea surface temperatures (SSTs) 8 to 10 °C higher than modern (10). Yet regional Arctic temperatures likely differed; summer Labrador SSTs were cooler during MIS11 than MIS1 or MIS5e (4, 11) (Fig. 2G). Terrestrial climate on Greenland, where summer air temperature directly influences ice sheet mass balance, remains unconstrained by geologic evidence throughout most Pleistocene interglacials older than MIS5e, including MIS11.     

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 absolute chronology of Boker Tachtit (Israel) and implications for the Middle to Upper Paleolithic transition in the Levant

The Initial Upper Paleolithic (IUP) is a crucial lithic assemblage type in the archaeology of southwest Asia because it marks a dramatic shift in hominin populations accompanied by technological changes in material culture. This phase is conventionally divided into two chronocultural phases based on the Boker Tachtit site, central Negev, Israel. While lithic technologies at Boker Tachtit are well defined, showing continuity from one phase to another, the absolute chronology is poorly resolved because the radiocarbon method used had a large uncertainty. Nevertheless, Boker Tachtit is considered to be the origin of the succeeding Early Upper Paleolithic Ahmarian tradition that dates in the Negev to ∼42,000 y ago (42 ka). Here, we provide ¹⁴C and optically stimulated luminescence dates obtained from a recent excavation of Boker Tachtit. The new dates show that the early phase at Boker Tachtit, the Emirian, dates to 50 through 49 ka, while the late phase dates to 47.3 ka and ends by 44.3 ka. These results show that the IUP started in the Levant during the final stages of the Late Middle Paleolithic some 50,000 y ago. The later IUP phase in the Negev chronologically overlaps with the Early Upper Paleolithic Ahmarian of the Mediterranean woodland region between 47 and 44 ka. We conclude that Boker Tachtit is the earliest manifestation of the IUP in Eurasia. The study shows that distinguishing the chronology of the IUP from the Late Middle Paleolithic, as well as from the Early Upper Paleolithic, is much more complex than previously thought.

The spread of modern humans from Africa into Eurasia is certainly one of the most important events in human history (1–3). The appearance of Homo sapiens at the transition between the Middle Paleolithic (MP) and the Upper Paleolithic (UP) periods corresponds with the demise of Neanderthals in Europe and west Asia (4). This demographic process, known in the literature as the “Recent African Origin” (5), has undergone refinements since it was first introduced (6). Today, this dispersal event is thought to be a multifaceted process that involved several events and genetic admixture between H. sapiens and Neanderthals (7–12).Recognizing demographic changes in the archaeological record is not always straightforward, mainly because of a lack of human fossils. Still, transformations in material culture are often conceived as a reliable indicator for demographic change (13, 14). In the Levant, as in Europe, such changes occurred during the transition from the MP to the UP, namely, the replacement of Levallois technology by blade technologies and the introduction of systematically produced tools on bone and antler (15–18). The nature and timing of the MP to UP transition has been investigated for almost a century (18–24). While the characteristics of the material culture changes are more or less defined, the absolute chronology and the origins of the transitional industries are under debate (25–30).One of the major reasons for this uncertainty is the fact that many of the key sites with supposedly “transitional” lithic industries in the south Levant, such as Emireh and el-Wad caves, were excavated in the beginning of the 20th century and their stratigraphies are challenging (24). An important exception is the site of Boker Tachtit in the Negev Highlands, Israel, that comprises a series of intact stratigraphic layers with refitted lithic assemblages, which are separated by sterile sediments (31, 32). Here, we report the results of an excavation at Boker Tachtit and in particular the chronology based on radiocarbon and optically stimulated luminescence (OSL) dates.Boker Tachtit is located in the Wadi Zin basin in the central Negev region, Israel (Fig. 1). The site was discovered and excavated by A. Marks in the framework of the Central Negev Project (31). The excavation revealed well-preserved archaeological horizons (Levels 1 through 4 from the bottom up) composed of flint artifacts, a few hammer stones, and charcoal pieces, including the presence of a hearth feature in Level 1. Comprehensive lithic studies enabled technological reconstructions of the lithic industries at the site as well as spatial aspects of these occupations (32–34). The refitting study demonstrated a technological continuity from the lowermost Level 1 to the uppermost Level 4 and indicated on-site flint knapping. This later point is additionally supported by a study of the microflints from Marks’ excavation section D (35). Marks conceived Boker Tachtit as an MP to UP transitional site bearing two consecutive cultural phases: the Emirian (Levels 1 through 3), which he associated with the MP, and the Initial UP (IUP) (Level 4), which was predominantly UP (28). Later studies by Kuhn that included sites in the north Levant redefined the IUP and incorporated the Emirian into this phase (16). In this paper, we follow Kuhn''s definition for the IUP.Open in a separate windowFig. 1.Location of Boker Tachtit and other sites mentioned in the text.The original chronology of Boker Tachtit was based on five radiocarbon dates using the ¹⁴C decay counting method (31). Four samples (GY-3642, SMU-184, SMU-580, and SMU-259) were from Level 1 and one sample (SMU-579) from Level 4. Two samples were of infinite ages (GY-3642, >33,000 BC; SMU-184, >43,620 BC), and one (SMU-579, 33,105 ± 4,100 BC) appeared to be an outlier. Another (SMU-580, 44,330 ± 9,050 BC) had an extremely wide uncertainty range of 9,000 y. The only supposedly reliable date (SMU-259, 44,980 ± 2,420 BC) was used to set the chronology of the site to ∼47 ka BP. The latter date was perceived by Marks and many others to reflect the age of the MP to UP transition in the Levant (1, 13). The few samples analyzed and the large uncertainties are related to the methodology used and probably also to the quality of the charred material analyzed.More recent MP to UP chronological studies based on radiocarbon dating of charred material and marine shells from new and old excavations at other sites have initiated a debate about the chronology of the transition, and the age of Boker Tachtit was suggested to be too old compared with northern Levantine sites (25, 27, 29, 30, 36). The problems lie in the disparities in the documented timing of the transition. While these differences may reflect a time lag in the transition between Boker Tachtit and the northern Levantine sites, problems with the quality of dated material or their context should not be overlooked (i.e., stratigraphic provenience and/or diagenesis) (e.g., refs. 25, 29, 30).     

A rate threshold mechanism regulates MAPK stress signaling and survival

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

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

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.     

Heading perception depends on time-varying evolution of optic flow

There is considerable support for the hypothesis that perception of heading in the presence of rotation is mediated by instantaneous optic flow. This hypothesis, however, has never been tested. We introduce a method, termed “nonvarying phase motion,” for generating a stimulus that conveys a single instantaneous optic flow field, even though the stimulus is presented for an extended period of time. In this experiment, observers viewed stimulus videos and performed a forced-choice heading discrimination task. For nonvarying phase motion, observers made large errors in heading judgments. This suggests that instantaneous optic flow is insufficient for heading perception in the presence of rotation. These errors were mostly eliminated when the velocity of phase motion was varied over time to convey the evolving sequence of optic flow fields corresponding to a particular heading. This demonstrates that heading perception in the presence of rotation relies on the time-varying evolution of optic flow. We hypothesize that the visual system accurately computes heading, despite rotation, based on optic acceleration, the temporal derivative of optic flow.

James Gibson first remarked that the instantaneous motion of points on the retina (Fig. 1A) can be formally described as a two-dimensional (2D) field of velocity vectors called the “optic flow field” (or “optic flow”) (1). Such optic flow, caused by an observer’s movement relative to the environment, conveys information about self-motion and the structure of the visual scene (1–15). When an observer translates in a given direction along a straight path, the optic flow field radiates from a point in the image with zero velocity, or singularity, called the focus of expansion (Fig. 1B). It is well known that under such conditions, one can accurately estimate one’s “heading” (i.e., instantaneous direction of translation in retinocentric coordinates) by simply locating the focus of expansion (SI Appendix). However, if there is angular rotation in addition to translation (by moving along a curved path or by a head or eye movement), the singularity in the optic flow field will be displaced such that it no longer corresponds to the true heading (Fig. 1 C and D). In this case, if one estimates heading by locating the singularity, the estimate will be biased away from the true heading. This is known as the rotation problem (14).Open in a separate windowFig. 1.Projective geometry, the rotation problem, time-varying optic flow, and the optic acceleration hypothesis. (A) Viewer-centered coordinate frame and perspective projection. Because of motion between the viewpoint and the scene, a 3D surface point traverses a path in 3D space. Under perspective projection, the 3D path of this point projects onto a 2D path in the image plane (retina), the temporal derivative of which is called image velocity. The 2D velocities associated with all visible points define a dense 2D vector field called the optic flow field. (B–D) Illustration of the rotation problem. (B) Optic flow for pure translation (1.5-m/s translation speed, 0° heading, i.e., heading in the direction of gaze). Optic flow singularity (red circle) corresponds to heading (purple circle). (C) Pure rotation, for illustrative purposes only and not corresponding to any experimental condition (2°/s rightward rotation). (D) Translation + rotation (1.5 m/s translation speed, 0° heading, 2°/s rightward rotation). Optic flow singularity (red circle) is displaced away from heading (purple circle). (E) Three frames from a video depicting movement along a circular path with the line-of-sight initially perpendicular to a single fronto-parallel plane composed of black dots. (F) Time-varying evolution of optic flow. The first optic flow field reflects image motion between the first and second frames of the video. The second optic flow field reflects image motion between the second and third frames of the video. For this special case (circular path), the optic flow field evolves (and the optic flow singularity drifts) only due to the changing depth of the environment relative to the viewpoint. (G) Illustration of the optic acceleration hypothesis. Optic acceleration is the derivative of optic flow over time (here, approximated as the difference between the second and first optic flow fields). The singularity of the optic acceleration field corresponds to the heading direction. Acceleration vectors autoscaled for visibility.Computer vision researchers and vision scientists have developed a variety of algorithms that accurately and precisely extract observer translation and rotation from optic flow, thereby solving the rotation problem. Nearly all of these rely on instantaneous optic flow (i.e., a single optic flow field) (4, 9, 16–25) with few exceptions (26–29). However, it is unknown whether these algorithms are commensurate with the neural computations underlying heading perception.The consensus of opinion in the experimental literature is that human observers can estimate heading (30, 31) from instantaneous optic flow, in the absence of additional information (5, 10, 15, 32–34). Even so, there are reports of systematic biases in heading perception (11); the visual consequences of rotation (eye, head, and body) can bias heading judgments (10, 15, 35–37), with the amount of bias typically proportional to the magnitude of rotation. Other visual factors, such as stereo cues (38, 39), depth structure (8, 10, 40–43), and field of view (FOV) (33, 42–44) can modulate the strength of these biases. Errors in heading judgments have been reported to be greater when eye (35–37, 45, 46) or head movements (37) are simulated versus when they are real, which has been taken to mean that observers require extraretinal information, although there is also evidence to the contrary (10, 15, 33, 40, 41, 44, 47–50). Regardless, to date no one has tested whether heading perception (even with these biases) is based on instantaneous optic flow or on the information available in how the optic flow field evolves over time. Some have suggested that heading estimates rely on information accumulated over time (32, 44, 51), but no one has investigated the role of time-varying optic flow without confounding it with stimulus duration (i.e., the duration of evidence accumulation).In this study, we employed an application of an image processing technique that ensured that only a single optic flow field was available to observers, even though the stimulus was presented for an extended period of time. We called this condition “nonvarying phase motion” or “nonvarying”: The phases of two component gratings comprising each stationary stimulus patch shifted over time at a constant rate, causing a percept of motion in the absence of veridical movement (52). Phase motion also eliminated other cues that may otherwise have been used for heading judgments, including image point trajectories (15, 32) and their spatial compositions (i.e., looming) (53, 54). For nonvarying phase motion, observers exhibited large biases in heading judgments in the presence of rotation. A second condition, “time-varying phase motion,” or “time-varying,” included acceleration by varying the velocity of phase motion over time to match the evolution of a sequence of optic flow fields. Doing so allowed observers to compensate for the confounding effect of rotation on optic flow, making heading perception nearly veridical. This demonstrates that heading perception in the presence of rotation relies on the time-varying evolution of optic flow.