Isotopic signals of summer denitrification in a northern hardwood forested catchment

Despite decades of measurements, the nitrogen balance of temperate forest catchments remains poorly understood. Atmospheric nitrogen deposition often greatly exceeds streamwater nitrogen losses; the fate of the remaining nitrogen is highly uncertain. Gaseous losses of nitrogen to denitrification are especially poorly documented and are often ignored. Here, we provide isotopic evidence (δ¹⁵N_NO3 and δ¹⁸O_NO3) from shallow groundwater at the Hubbard Brook Experimental Forest indicating extensive denitrification during midsummer, when transient, perched patches of saturation developed in hillslopes, with poor hydrological connectivity to the stream, while streamwater showed no isotopic evidence of denitrification. During small rain events, precipitation directly contributed up to 34% of streamwater nitrate, which was otherwise produced by nitrification. Together, these measurements reveal the importance of denitrification in hydrologically disconnected patches of shallow groundwater during midsummer as largely overlooked control points for nitrogen loss from temperate forest catchments.Many forested catchments export far less nitrogen (N) in streamwater than they receive in atmospheric deposition (1, 2). The rest of the deposited N may accumulate in vegetation or soil organic matter, or be lost in gaseous form. Losses of N to denitrification, the microbial reduction of aqueous nitrate (NO₃⁻) to nitrous oxide (N₂O, a greenhouse gas) and N₂ gas, are extremely difficult to measure due to the difficulty in directly measuring N₂ fluxes and due to the high degree of spatiotemporal variability in redox conditions and substrate sources (3). Many past studies using a range of measurements (streamwater nitrate isotopic composition, the acetylene block technique, N₂O emissions, and mass balance calculations) have concluded that denitrification in temperate forests is highly uncertain or generally unimportant (e.g., refs. 4–8).Nitrogen budgets are particularly perplexing in the northern hardwood forests at the Hubbard Brook Experimental Forest (HBEF) in the White Mountains of New Hampshire, USA, where atmospheric deposition has supplied 6–8 kg N ha⁻¹⋅yr⁻¹ for half a century, a rate ∼5–10 times preindustrial levels (7–10). Accumulation of N in plant biomass ceased in the early 1990s (10, 11), while streamwater inorganic N export from catchments across the HBEF and nearby streams decreased to <1 kg N ha⁻¹⋅yr⁻¹, for reasons that remain elusive (9, 10, 12). These N flux measurements imply increasingly important roles for N gas loss or storage in soil organic matter. However, both processes are so difficult to quantify that the fate, drivers, and consequences of the “missing” N remain unknown, at the HBEF and elsewhere (8–10, 12).Measurement of the dual isotopic composition of NO₃⁻ (δ¹⁵N_NO3 and δ¹⁸O_NO3) provides a powerful tool to identify NO₃⁻ sources and to infer its loss to denitrification (13–16). Values of δ¹⁸O_NO3 differ greatly between NO₃⁻ in precipitation and NO₃⁻ produced by nitrification (refs. 13–16, Table S1), which is the microbial oxidation of NH₄⁺ to NO₃⁻. Measurements of δ¹⁸O_NO3 have enabled detection of direct contributions of precipitation NO₃⁻ to streamwater (Table S1), especially during snowmelt, when catchments often release large quantities of NO₃⁻ (7, 8). However, past δ¹⁸O_NO3 measurements show that nitrification—not precipitation—supplies the vast majority of NO₃⁻ in streamwater at the HBEF (10, 17, 18) and other forested catchments (refs. 14 and 19, Table S1).Nitrate isotopic composition reflects not only NO₃⁻ sources but also fractionation from a range of processes (14, 20). During denitrification, heterotrophic microbes consume organic carbon using NO₃⁻ as an electron acceptor under low-oxygen conditions, in a 5:4 molar ratio of carbon:NO₃⁻. If NO₃⁻ is not replenished or consumed by other processes, denitrification progressively enriches both ¹⁸O and ¹⁵N in the residual NO₃⁻, with an O:N fractionation ratio of 0.4–0.7 in the field (14, 20, 21) and up to 1.0 in laboratory studies (22). The fractionation ratio is the slope of the relationship between δ¹⁸O_NO3 and δ¹⁵N_NO3. Dual isotopic enrichment and these enrichment ratios provide evidence of denitrification. Dual isotope analysis of NO₃⁻ has provided evidence for denitrification in large aquifers (e.g., ref. 20) and in drainage waters receiving heavy agricultural N loads (e.g., refs. 21 and 23), whereas recent catchment studies in a subtropical forest (15) and a warm Mediterranean grassland (24) have reported isotopic evidence of denitrification in soil or groundwater. However, dozens of past studies of stream δ¹⁸O_NO3 and δ¹⁵N_NO3 in naturally vegetated temperate and boreal catchments (refs. 14 and 19, Table S1), including the HBEF (10, 17), have revealed little if any isotopic evidence of denitrification.To investigate the role of denitrification at the HBEF, we measured NO₃⁻ isotopic composition throughout watershed 3 (WS3), a hydrologic reference catchment drained by Paradise Brook within the HBEF (Fig. 1), during the first two weeks of July 2011, close to the warmest part of the year (Fig. S1). Sampling encompassed nine shallow groundwater wells, a seep, and 19 stream sites along Paradise Brook and its tributaries. The shallow groundwater wells accessed water from saturated soil within the solum above the C horizon at depths between 30 and 115 cm. Three of the nine wells were close to (<2 m) or within the perennial stream channel; the other six were more distal (≥4 m) and upgradient from the perennial channel. Four rain events occurred during the sampling period (0.8–12.3 mm, 26.8 mm total); all contained NO₃⁻ and NH₄⁺ concentrations that exceeded those in streamwater and groundwater by an order of magnitude (Fig. 2 A and B). Nitrogen export over the study period amounted to 0.006 ± 0.003 kg N ha⁻¹, consisting of 24% NO₃⁻, 13% NH₄⁺, and 63% dissolved organic nitrogen (DON). Streamflow over the sampling period (5.3 mm) exported less than 2% of rainfall N input (0.335 ± 0.087 kg N ha⁻¹). The remaining 98% was retained within the catchment or lost via denitrification. If this 98% were denitrified in soil or shallow groundwater, it gives a maximum denitrification rate of 5.0 (3.6–6.4) kg N ha⁻¹ if extrapolated over the growing season. Although this figure represents the maximum loss of N to denitrification, recent extrapolations of N₂ and N₂O flux measurements from soil cores from the HBEF found denitrification rates during the growing season higher than previous estimates and equal to or higher than atmospheric deposition while follow-up measurements found rates ranging from 4–10 kg N ha⁻¹⋅y⁻¹ at HBEF (25).Open in a separate windowFig. 1.Location of HBEF in the northeast United States (Top Right), showing HBEF watersheds 1–9 (Bottom Right) and watershed 3 (WS3; Left). WS3 shows drainage network comprising Paradise Brook and tributary channels, with sampling locations from the weir (triangle), streams (filled circles), wells (empty circles; including wells ≥4 m from surface streamflow in July 2011 (JD05, JD17, JD18, JD19, JD29, JD30) and those <2 m (R12, east bank; R13 in-stream; R14, west bank) and a seep (dash). Contour interval is 3 m; elevation range is 537–732 m.

Table 1.

Concentrations of NO₃⁻, NH₄⁺, DON, and DOC (μM), and δ¹⁵N_NO3 and δ¹⁸O_NO3 of water samples from watershed 3, Hubbard Brook Experimental Forest, New Hampshire July 2011

Pliocene reversal of late Neogene aridification

The Pliocene epoch (5.3–2.6 Ma) represents the most recent geological interval in which global temperatures were several degrees warmer than today and is therefore considered our best analog for a future anthropogenic greenhouse world. However, our understanding of Pliocene climates is limited by poor age control on existing terrestrial climate archives, especially in the Southern Hemisphere, and by persistent disagreement between paleo-data and models concerning the magnitude of regional warming and/or wetting that occurred in response to increased greenhouse forcing. To address these problems, here we document the evolution of Southern Hemisphere hydroclimate from the latest Miocene to the middle Pliocene using radiometrically-dated fossil pollen records preserved in speleothems from semiarid southern Australia. These data reveal an abrupt onset of warm and wet climates early within the Pliocene, driving complete biome turnover. Pliocene warmth thus clearly represents a discrete interval which reversed a long-term trend of late Neogene cooling and aridification, rather than being simply the most recent period of greater-than-modern warmth within a continuously cooling trajectory. These findings demonstrate the importance of high-resolution chronologies to accompany paleoclimate data and also highlight the question of what initiated the sustained interval of Pliocene warmth.Our knowledge of Pliocene climates is based predominantly on the rich marine sediment record (1), but understanding of Pliocene climates on land remains limited because the few existing terrestrial archives tend to have poor age control and are of short duration. This deficiency is no more evident than in paleovegetation records, which are integral to modeling Pliocene climates because vegetation is both a critical indicator of regional precipitation and also makes a large contribution to planetary albedo (2, 3). Several syntheses of Pliocene vegetation have been compiled (3, 4), but their value is hampered by substantial uncertainty surrounding the synchroneity of the records. For example, vegetation syntheses have focused on the Late Pliocene (the Piacenzian Age, 3.6–2.6 Ma) because this period is considered likely to be a closer biological and geological analog for future warming than the Early Pliocene (1, 5). However, only 6 of 32 Southern Hemisphere records interpreted by Salzmann et al. (4) as documenting the nature of Late Pliocene vegetation are both based on plant fossils and can confidently be assigned to the Late Pliocene; the remaining records have such poor chronologies that their possible ages range from Late Miocene to Early Pleistocene, or the records only infer the nature of vegetation indirectly from geomorphology or vertebrate fossils (SI Appendix, Table S1). As a result, current understanding of the response of Southern Hemisphere vegetation to Late Pliocene climates (2–4) may conflate the climate and vegetation history of ≥5 My of the late Cenozoic (Fig. 1), an interval that is characterized by global cooling (6) and, in subtropical- to midlatitudes, increasing aridity (7–10). This uncertainty is problematic for two reasons. First, it has become clear that the peak of Pliocene warmth globally was not generally within the Late Pliocene but occurred earlier, within the Early Pliocene (11). Second, largely because of a lack of continuous and well-dated records, the relationship between the longer-term cooling/aridification trend and Pliocene warmth remains unclear. Thus, was Pliocene warmth a discrete interruption of late Cenozoic cooling/aridification trends (6), or was the Pliocene merely an interval immediately preceding the abrupt steepening of these trends associated with the onset of extensive Northern Hemisphere glaciation (12, 13)?Open in a separate windowFig. 1.Conservative estimates of the age ranges of Southern Hemisphere vegetation records accepted by Salzmann et al. (3) as indicative of Late Pliocene (Piacenzian, 2.6–3.6 Ma) terrestrial vegetation. Sample code numbers are those used by Salzmann et al. Colors represent record type (pollen, wood, vertebrate, sediment). Bold colors indicate records clearly falling within the Late Pliocene, and faint colors indicate records either falling outside of the Late Pliocene or with broader age ranges. Of the 32 records, only 6 based on plant fossil data can be confidently assigned a Late Pliocene age. For Makapan, two age estimates are provided, reflecting uncertainty whether the record can be attributed to the Pliocene as a whole or to the Late Pliocene.To place the temporal evolution of Southern Hemisphere Pliocene vegetation and climate on a firmer chronological footing, we generated fossil pollen records from radiometrically dated speleothems (secondary cave carbonate deposits such as stalagmites and flowstones) from southern Australia. Pollen assemblages were extracted from samples collected in five caves on the Nullarbor Plain (Fig. 2 and SI Appendix, Fig. S1), a large (200,000 km²) karst province uplifted above sea level during the late Miocene (14). Consistent with its position in Southern Hemisphere subtropical latitudes, the Nullarbor Plain is currently semiarid, receiving mean annual precipitation (MAP) of ca. 180–270 mm (SI Appendix, Fig. S1), with a weak winter-maximum. Mean annual temperature is ∼18 °C. The vegetation is largely treeless chenopod shrubland, grading coastward into sparse, low open woodlands dominated by Acacia (Mimosaceae) or Eucalyptus (Myrtaceae). Nullarbor speleothems grew during the late Neogene (15) but negligible calcite speleothem growth occurs today. Recent development and refinement of the uranium-lead (U-Pb) chronometer has allowed high-precision geochronology of such ancient speleothem samples (15–18). Our fossil pollen record is composed of 13 polleniferous samples (out of 81 explored for pollen), the oldest dated at 5.59 ± 0.15 Ma, in the latest Miocene, and the youngest 0.41 ± 0.07 Ma, in the Middle Pleistocene (Open in a separate windowFig. 2.Locality map showing Nullarbor caves in southern Australia and sites mentioned in the text. The map was produced using Ocean Data View (odv.awi.de).

Table 1.

Age and pollen yield of Nullarbor speleothems

Multitarget,quantitative nanoplasmonic electrical field-enhanced resonating device (NE2RD) for diagnostics

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients’ homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE²RD), which addresses all these impediments on a single platform. The NE²RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE²RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE²RD’s broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients’ homes.Biosensing platforms have enabled various applications in different fields of clinical medicine such as biomarker/drug discovery and initiation and monitoring of therapy (1–3). However, material cost, accessibility, ease of operation, lack of portability, and complexity in readout remain major challenges for developing robust diagnostic assays (SI Appendix, Table S1). Recent advances in nanotechnology and biosensing have created new avenues to address these issues (4–9). Technically, they have provided integration of high-throughput sampling with readout systems for quantitative detection of disease-specific biotargets. Therefore, they have demonstrated great potential to revolutionize medical diagnostics. However, from a clinical and technological perspective, existing platforms still face several challenges. First, lengthy assay time hinders physicians from making early clinical decisions. Second, examining clinical samples with diverse pH range, ionic content, and ionic strength requires extensive sample preprocessing steps to avoid signal fluctuations and inaccuracies, and this requirement increases the complexity of current systems. Third, temperature is an extrinsic factor that needs to be tightly controlled for reliable measurements, thus resulting in additional cost for developing diagnostic technologies. Fourth, a wide dynamic linear range is needed to distinguish concentrations of biotargets reliably. Fifth, sensitivity is hindered by the complex composition of biological specimens, thus requiring labeling (10, 11), which further increases assay cost significantly. Sixth, since a panel of parameters is monitored for complex diseases, detection of multiple biotargets is critically needed. Seventh, portability is another important parameter to deploy a biosensing platform at point-of-care (POC) settings. These practical barriers seriously reduce the broad clinical applicability of current platforms for diagnostic implementations.The development of various sensitive detection modalities has addressed some of these requirements. The mass-sensitive piezoelectric and microcantilever-based systems that use surface oscillations or changes in surface stress for molecular interactions and mass input (12, 13) have simplified sample-preparation steps. Likewise, electrical detection systems such as electrochemical sensors and interdigitated electrodes sense changes in molecular charges (14–17) and can provide affordable, simple, and high-throughput measurements. Further, plasmonic-based platforms that use an electrical field around metal surfaces/nanoparticles coupled with light (5, 18–22) have minimized readout complexity and demonstrated quantitative and sensitive measurements (23–26). In particular, surface plasmons on 3D-oriented surfaces generate highly sensitive spots that provide a sevenfold stronger electrical field than 2D-oriented plasmonic sensors (27–29). However, these advances have only reduced the effort required for sample preparation and the readout complexity, and some critical challenges remain unaddressed as summarized in

Fast–slow continuum and reproductive strategies structure plant life-history variation worldwide

The identification of patterns in life-history strategies across the tree of life is essential to our prediction of population persistence, extinction, and diversification. Plants exhibit a wide range of patterns of longevity, growth, and reproduction, but the general determinants of this enormous variation in life history are poorly understood. We use demographic data from 418 plant species in the wild, from annual herbs to supercentennial trees, to examine how growth form, habitat, and phylogenetic relationships structure plant life histories and to develop a framework to predict population performance. We show that 55% of the variation in plant life-history strategies is adequately characterized using two independent axes: the fast–slow continuum, including fast-growing, short-lived plant species at one end and slow-growing, long-lived species at the other, and a reproductive strategy axis, with highly reproductive, iteroparous species at one extreme and poorly reproductive, semelparous plants with frequent shrinkage at the other. Our findings remain consistent across major habitats and are minimally affected by plant growth form and phylogenetic ancestry, suggesting that the relative independence of the fast–slow and reproduction strategy axes is general in the plant kingdom. Our findings have similarities with how life-history strategies are structured in mammals, birds, and reptiles. The position of plant species populations in the 2D space produced by both axes predicts their rate of recovery from disturbances and population growth rate. This life-history framework may complement trait-based frameworks on leaf and wood economics; together these frameworks may allow prediction of responses of plants to anthropogenic disturbances and changing environments.Demographic schedules of survival, growth, and reproduction, which comprise life-history strategies, are fundamental to our understanding of a range of ecological and evolutionary processes, such as invasions and local extinctions (1–3), community structure (4, 5), and species diversification (6, 7). Consequently, the development and careful testing of theory on how organisms allocate resources to survival, growth, and reproduction are important goals for evolutionary biology, ecology, and conservation biology (8). Indeed, calls for the development of a “periodic table” to classify species based on their life-history strategies and to predict population dynamics and community composition go back to the early development of evolutionary biology as a discipline (9).A main axiom of life-history theory is that trade-offs (i.e., budgetary compromises) between different aspects of an organism’s demographic schedules, such as survival, growth, and/or reproduction, constrain and optimize the range of possible life-history strategies that can evolve across the tree of life (10, 11). However, the plant kingdom encompasses a vast amount of life-history variation; plant longevity, for instance, ranges from weeks to millennia (12). Many plant species’ life cycles include cryptic life stages such as seedbanks (13) or dormant adults (similar to animal hibernation) (14). Reproduction also can be highly variable among plants, with seed mass and per-capita seed production ranging across six orders of magnitude (15). Previous classifications of plant life-history strategies have been limited in geographic (16, 17), taxonomic, and phylogenetic scales (17) and in the ability to differentiate life-history trade-offs (17–19).Here we propose an approach analogous to that developed decades ago for vertebrates (20) to study the drivers behind plant life-history variation. We combine demographic, phylogenetic, and ecological data from natural populations of 418 plant species worldwide (Fig. 1) to address the following questions: (i) What are the main axes of variation of plant life-history strategies? (ii) To what extent do phylogenetic ancestry, habitat, growth form, and size constrain plant life-history variation? We then test whether the position of a species on these axes predicts two important metrics of population performance: population growth rate and speed of recovery from disturbances. If clear patterns emerge, they may form the basis for a satisfactory classification and predictive framework of plant responses to the changing environment and for cross-taxonomic comparisons.Open in a separate windowFig. 1.The 418 plant species examined include all major terrestrial habitats (A) and growth forms (B). Growth form is categorized according to the position of the plant’s shoot apical meristems in relation to ground level. Tissue shown in gray is typically renewed every year; tissue shown in black is perennial.We use the COMPADRE Plant Matrix Database (21) to address these questions, drawing from the demographic, biogeographic, anatomic, and phylogenetic information of the 418 plant species covering 105 families (Dataset S1). Together, the selected species represent 825 natural populations worldwide across all major terrestrial habitats and vascular plant growth forms (Fig. 1) for which at least 4 years of high-resolution demographic field data exist. For each species, we use their population matrix models (22) to calculate a set of representative life-history traits that inform on schedules of survival, growth, and reproduction (11), and we then evaluate the variation in these traits along major axes using phylogenetically corrected principal component analyses (PCA) (23).

Table 1.

Loading of the life-history traits grouped by their relation to turnover and strategies for longevity, growth, and reproduction onto the first two PCA axes

Spin-dependent electron transport in protein-like single-helical molecules

We report on a theoretical study of spin-dependent electron transport through single-helical molecules connected by two nonmagnetic electrodes, and explain the experiment of significant spin-selective phenomenon observed in α-helical protein and the contradictory results between the protein and single-stranded DNA. Our results reveal that the α-helical protein is an efficient spin filter and the spin polarization is robust against the disorder. These results are in excellent agreement with recent experiments [Mishra D, et al. (2013) Proc Natl Acad Sci USA 110(37):14872–14876; Göhler B, et al. (2011) Science 331(6019):894–897] and may facilitate engineering of chiral-based spintronic devices.Spintronics is a multidisciplinary field that manipulates the electron spin transport in solid-state systems and has been receiving much attention among the physics, chemistry, and biology communities (1–4). Recent experiments have made significant progress in this research field, finding that double-stranded DNA (dsDNA) molecules are highly efficient spin filters (5–7). This chiral-induced spin selectivity (CISS) is surprising because the DNA molecules are nonmagnetic and their spin-orbit couplings (SOCs) are small. Additionally, the CISS effect opens new opportunities for using chiral molecules in spintronic applications and could provide a deeper understanding of the spin effects in biological processes. For the above reasons, there has been considerable interest in the spin transport along various chiral systems including dsDNA (8–11), single-stranded DNA (ssDNA) (12–15), and carbon nanotubes (16). However, no spin selectivity was measured in the ssDNA above the experimental noise (5).Very recently, spin-dependent electron transmission and electrochemical experiments were performed on bacteriorhodopsin—an α-helical protein of which the structure is single helical—embedded in purple membrane which was physisorbed on a variety of substrates (17). It was reported by means of two distinct techniques that the electrons transmitted through the membrane are spin polarized, independent of the experimental environments, implying that this α-helical protein can exhibit the ability of spin filtering. Meanwhile, a chiral-based magnetic memory device was fabricated by using self-assembled monolayer of another α-helical protein called polyalanine (18). All of these results seem to be inconsistent with previous experiments’ conclusions that the single-stranded helical molecules, such as ssDNA, may not polarize the electrons (5). We note that the electron transport/transfer has been widely investigated in many proteins (19–26). However, to our knowledge, the underlying physics is still unclear for spin-selective phenomenon observed in the α-helical protein and for the contradictory behaviors between the protein and the ssDNA.In this paper, we propose a model Hamiltonian to explore the spin transport through single-helical molecules connected by two nonmagnetic electrodes, and provide an unambiguous physical mechanism for efficient spin selectivity observed in the protein and for the contrary experimental results between the protein and the ssDNA. Our results reveal that the α-helical protein is an efficient spin filter, whereas the ssDNA presents extremely small spin filtration efficiency with the order of magnitude being 10⁻⁵, although both molecules possess single-helical structure as illustrated in Fig. 1, where the circles represent the amino acids (nucleobases) for the protein (ssDNA). The underlying physics is attributed to the intrinsic structural difference between the two molecules that the distance l_j of the jth neighboring sites (e.g., sites n and n + j) increases much slower with increasing j for the protein than for the ssDNA, because the stacking distance Δh between the nearest neighbor (NN) sites is shorter in the protein. This can be seen from Open in a separate windowFig. 1.Single-helical molecule with radius R and pitch h, where the circles (sites) denote the amino acids for protein and the nucleobases for DNA. Because of the electron wave function overlap, the electrons can hop between two neighboring sites with the Euclidean distance l_j. Here, lj=[2R⁡sin(jΔφ/2)]2+(jΔh)2 with Δφ and Δh being the twist angle and the stacking distance between the nearest neighbor sites, respectively. The space angle between the solid line and the x−y plane is defined as θ_j = arccos[2R sin (jΔφ/2)/l_j].

Table 1.

Structural parameters of the α-helical protein and the regular B-form DNA

From the Cover: American mastodon extirpation in the Arctic and Subarctic predates human colonization and terminal Pleistocene climate change

Existing radiocarbon (¹⁴C) dates on American mastodon (Mammut americanum) fossils from eastern Beringia (Alaska and Yukon) have been interpreted as evidence they inhabited the Arctic and Subarctic during Pleistocene full-glacial times (∼18,000 ¹⁴C years B.P.). However, this chronology is inconsistent with inferred habitat preferences of mastodons and correlative paleoecological evidence. To establish a last appearance date (LAD) for M. americanum regionally, we obtained 53 new ¹⁴C dates on 36 fossils, including specimens with previously published dates. Using collagen ultrafiltration and single amino acid (hydroxyproline) methods, these specimens consistently date to beyond or near the ∼50,000 y B.P. limit of ¹⁴C dating. Some erroneously “young” ¹⁴C dates are due to contamination by exogenous carbon from natural sources and conservation treatments used in museums. We suggest mastodons inhabited the high latitudes only during warm intervals, particularly the Last Interglacial [Marine Isotope Stage (MIS) 5] when boreal forests existed regionally. Our ¹⁴C dataset suggests that mastodons were extirpated from eastern Beringia during the MIS 4 glacial interval (∼75,000 y ago), following the ecological shift from boreal forest to steppe tundra. Mastodons thereafter became restricted to areas south of the continental ice sheets, where they suffered complete extinction ∼10,000 ¹⁴C years B.P. Mastodons were already absent from eastern Beringia several tens of millennia before the first humans crossed the Bering Isthmus or the onset of climate changes during the terminal Pleistocene. Local extirpations of mastodons and other megafaunal populations in eastern Beringia were asynchrononous and independent of their final extinction south of the continental ice sheets.Last appearance dates (LADs) are crucial for evaluating hypotheses regarding the timing and causes of species disappearance in the fossil record (1). In principle, species extinction and local extirpation chronologies can be rigorously established by determining LADs using a variety of radiometric dating methods. In practice, however, problematic and incomplete chronological data inevitably affect the precision and accuracy of LADs. This may in turn affect how potential extinction mechanisms are evaluated. A case in point is the radiocarbon (¹⁴C) record of Pleistocene American mastodon (Mammut americanum) fossils from the unglaciated regions of Alaska and Yukon in northwest North America, collectively known as eastern Beringia (Fig. 1).Open in a separate windowFig. 1.Known fossil localities of M. americanum across North America [Table S1, with late Pleistocene glacial limits (white) and glacial/pluvial lakes (light blue) following ref. 22]. CIS, Cordilleran Ice Sheet; IIS, Innuitian Ice Sheet; LIS, Laurentide Ice Sheet. Dashed line and question mark denote uncertainty over northwest LIS limits (23). Localities with fossils analyzed in this study in Alaska and Yukon (northwest North America) are designated by red circles. Locality data for American mastodons across the continent, designated as green circles, are from refs. 5–8, 11–17, 19, 20, and 24 (see Table S1) in addition to collections data from the United States National Park Service, Royal Ontario Museum, and Royal British Columbia Museum.The American mastodon was one of roughly 70 species of mammals in North America that died out during the late Quaternary extinctions (2, 3). American mastodon appears in the North American fossil record roughly 3.5 million years ago and was the terminal member of a lineage that arose from its presumed ancestor Miomastodon merriami, which had crossed the Bering Isthmus from Eurasia during the middle Miocene (4). Over the course of the late Pleistocene (∼125,000–10,000 y ago), M. americanum became widespread, occupying many parts of continental North America, as well as peripheral areas as mutually remote as the tropics of Honduras and the Arctic coast of Alaska (5−8) (Fig. 1). Despite their Old World roots, and unlike American populations of their distant relative the woolly mammoth (Mammuthus primigenius) (9), there is no evidence that American mastodons managed to cross the Bering Isthmus westward into Eurasia (4).The rich and well-dated record of American mastodons living in the midlatitudes, particularly near the Great Lakes and Atlantic coast regions, demonstrates they were among the last members of the megafauna to disappear in North America near the end of the Pleistocene (4, 10–13). The current LAD for mastodons south of the former Laurentide and Cordilleran ice sheets is ∼10,000 ¹⁴C years B.P. (B.P. = years before A.D. 1950), based on enamel and ultrafiltered bone collagen from the Overmyer specimen from northern Indiana (11). Paleoenvironmental data from this region are consistent with the view that American mastodons preferentially inhabited coniferous or mixed forests or lowland swampy habitats in what can plausibly be regarded as the most persistent portion of their late Pleistocene geographic range (14–16) (Fig. 1 and Table S1). Mastodon remains at archeological sites south of the former continental ice sheets underscore the prominent role this mammal species has played in ongoing discussions of the late Quaternary extinctions (17).American mastodon and woolly mammoth differed in both habitat and dietary preference. As large grazers that relied on grasses and forbs, woolly mammoths were well adapted to semiarid, generally treeless steppe-tundra habitats that were widespread in eastern Beringia during Pleistocene glacial intervals (18). Conversely, mastodons were browsing specialists, relying on woody plants and preferentially inhabiting coniferous or mixed woodlands with lowland swamps (4). The large, bunodont teeth of mastodons were effective at stripping and crushing twigs, leaves, and stems from shrubs and trees (4). Plant remains from purported coprolites and stomach contents found in association with several mastodon skeletons found south of the former continental ice sheets include masticated or partially digested stick fragments, twigs, deciduous leaves, conifer needles, and conifer cones (4, 19). In places where American mastodons and woolly mammoths coexisted during the late Pleistocene, stable isotope and other paleoecological data establish these two proboscideans occupied and exploited distinct environmental niches and did not compete for the same resources (16, 20).In light of their preferred diet and habitat, the Arctic and Subarctic during the late Pleistocene would seem to be unlikely places for mastodon populations to live. Indeed, their fossils are quite rare; in the course of more than a century of collecting, American mastodon accounts for <5% of all proboscidean fossils recovered in Alaska and Yukon (7). Nevertheless, American mastodons certainly lived at high latitudes, either in small numbers or, more probably, for limited intervals. The likeliest proposed scenario is that American mastodons occupied the Arctic and Subarctic region only intermittently, during warm, Pleistocene interglacial periods, when widespread boreal forests and muskeg wetlands were established (21). During the Wisconsinan glaciation, when much of high-latitude North America was ice covered (22, 23), mastodons were probably absent in the cold, dry, unglaciated refugium of eastern Beringia. Indeed, this hypothesis was put forth decades before the advent of radiocarbon dating (24).It is thus surprising that the meager published ¹⁴C record has complicated, rather than corroborated, the long-standing hypothesis (24) that mastodons were Pleistocene interglacial residents of the Arctic and Subarctic and were absent during glacial periods (7, 25) (Table S2). In contrast, published dates on fossils from the Ikpikpuk River, Alaska (26), and Herschel Island, Yukon (27), are nonfinite (i.e., greater than ∼50,000 y B.P., the effective limit of ¹⁴C dating methods) and were interpreted to mean that mastodons lived along the Arctic coast only during the Last Interglacial (MIS 5: ∼125,000–75,000 y ago). If American mastodons did survive in eastern Beringia during the MIS 2 full-glacial period, and possibly even as late as the terminal Pleistocene as suggested by the two finite ages on Yukon molars (7, 25) (2, 17), abrupt climate change (28, 29), or extraterrestrial impact (30, 31).

Table 1.

Radiocarbon (¹⁴C) dates on American mastodon fossils from Alaska and Yukon

Mechanical resilience and cementitious processes in Imperial Roman architectural mortar

The pyroclastic aggregate concrete of Trajan’s Markets (110 CE), now Museo Fori Imperiali in Rome, has absorbed energy from seismic ground shaking and long-term foundation settlement for nearly two millenia while remaining largely intact at the structural scale. The scientific basis of this exceptional service record is explored through computed tomography of fracture surfaces and synchroton X-ray microdiffraction analyses of a reproduction of the standardized hydrated lime–volcanic ash mortar that binds decimeter-sized tuff and brick aggregate in the conglomeratic concrete. The mortar reproduction gains fracture toughness over 180 d through progressive coalescence of calcium–aluminum-silicate–hydrate (C-A-S-H) cementing binder with Ca/(Si+Al) ≈ 0.8–0.9 and crystallization of strätlingite and siliceous hydrogarnet (katoite) at ≥90 d, after pozzolanic consumption of hydrated lime was complete. Platey strätlingite crystals toughen interfacial zones along scoria perimeters and impede macroscale propagation of crack segments. In the 1,900-y-old mortar, C-A-S-H has low Ca/(Si+Al) ≈ 0.45–0.75. Dense clusters of 2- to 30-µm strätlingite plates further reinforce interfacial zones, the weakest link of modern cement-based concrete, and the cementitious matrix. These crystals formed during long-term autogeneous reaction of dissolved calcite from lime and the alkali-rich scoriae groundmass, clay mineral (halloysite), and zeolite (phillipsite and chabazite) surface textures from the Pozzolane Rosse pyroclastic flow, erupted from the nearby Alban Hills volcano. The clast-supported conglomeratic fabric of the concrete presents further resistance to fracture propagation at the structural scale.The builders of the monuments of Imperial Rome (from 27 BCE, when Octavian became Emperor Augustus, through the fourth century CE) used pyroclastic volcanic rock to create unreinforced concrete structures with dramatic vaulted spans, as at the Markets of Trajan (110 CE) (1, 2) (Fig. 1A). The concrete foundations, walls, and vaulted ceilings are composed of decimeter-sized volcanic tuff and brick coarse aggregate (caementa) bound by volcanic ash–lime mortar (Fig. 1B). The conglomeratic fabric of the concretes is analogous to sedimentary rocks made of coarse rock fragments and a matrix of finer grained material. The concretes have resisted structural scale failure during moderate-magnitude earthquakes (<8 on the Mercalli–Cancani–Sieberg intensity scale) associated with slip on Appennine fault systems 80–130 km to the northeast, as well as chemical decay associated with repeated inundations of foundations and walls by Tiber River floods (3–5). To date, at least six episodes of moment magnitude 6.7–7 ground shaking and damage to monuments have been recorded since 508 CE (4). The concrete structures contain common macroscale fractures, with rough surfaces that link by complex segment overlap and bridging, and either follow or traverse caementa interfacial zones (Fig. 1C). Many monuments remain in active use as residences, offices, museums, and churches. In addition to the Markets of Trajan, these include the Theater of Marcellus (44–13 BCE), Mausoleum of Hadrian (123–39 CE), Pantheon (ca. 126 CE), and Baths of Diocletian (298–306 CE). The monuments that did undergo sectional failure, for example at the Colosseum (70–90 CE), Baths of Caracalla (ca. 215 CE), and Basilica of Maxentius (ca. 313 CE), mainly did so in Late Antiguity or the Middle Ages, when they were several centuries old and had become vulnerable through subsurface instabilities; problematic structural design; removal of marble and travertine dimension stone, columns, and cladding; and lack of regular maintenance (4, 6, 7).Open in a separate windowFig. 1.Markets of Trajan concretes. (A) Great Hall, vaulted ceiling and brick-faced concrete walls; reprinted with permission from Archives, Museo Fori Imperiali. (B) Drill core with Pozzolane Rosse volcanic ash (harena fossicia) mortar and conglomeratic aggregate (caementa). (C) Fractures in vaulted ceiling, Grande Emiciclo: 1, crack follows caementa perimeter; 2, crack traverses caementa. Wall concrete contains ∼88 vol % pyroclastic rock: 45–55% tuff (and brick) as caementa, ∼38% volcanic ash pozzolan, and ∼12% lime paste, with 3:1 ash:lime volumetric ratio (de Architectura 2.5.1) in the mortar (18).The pozzolanic mortar perfected by Roman builders during first century BCE (8) is key to the durability of concrete components in structurally sound monuments well maintained over two millennia of use. [Pozzolans, named after pumiceous ash from Puteoli (now, Pozzuoli) in the Campi Flegrei volcanic district, react with lime in the presence of moisture to form binding cementitious hydrates (9)]. By the Augustan era (27 BCE–14 CE), after experimenting with ash mixtures for >100 y, Romans had a standardized mortar formulation using scoriaceous ash of the mid-Pleistocene Pozzolane Rosse pyroclastic flow (Fig. S1) that substantially improved the margin of safety associated with increasingly daring structural designs (10, 11). They used this mortar formulation in the principal Imperial monuments constructed in Rome through early fourth century CE (8). Pozzolane Rosse erupted at 456 ± 3 ka from nearby Alban Hills volcano (12), filling valleys and covering topographic plateaus across the Roman region; the ash has a highly potassic tephritic composition (13). Romans made the architectural mortars by calcining limestone at ∼900 °C to produce quicklime [CaO], hydrating the quicklime to form portlandite [Ca(OH)₂], a trigonal calcium hydroxide] putty, and laboriously incorporating granular Pozzolane Rosse ash. This is the red and black excavated sand (harena fossicia) described by the Roman architect Vitruvius in first century BCE (de Architectura 2.4.1–2.4.3; 2.5.1–2.5.3) (14). The strongly alkaline portlandite solution attacks the surfaces of the scoriaceous pozzolan; volcanic glass and silicate mineral textures dissociate; their alkali ions dissolve in the liquid phase; and calcium is adsorbed on the scoria surfaces, forming cementitious hydrates (9). These phases are regarded as central to the chemical durability that is an essential component of the impressive record of survival of many monuments, but their role in resisting mechanical degradation through obstructing microcrack propagation has never been examined.Fracture-mechanical properties offer important insight into a cementitious material’s long-term survivability (i.e., its ability to absorb energy from applied loads without failing catastrophically). Two common properties are uniaxial tensile strength [f_t (megapascals)], which refers to the stress at which a macrocrack initiates, and fracture energy [G_F (joules per square meter)], the amount of mechanical work required to propagate a macrocrack to create one square unit of new surface area (15). Experimental characterization of the fracture behavior of the Imperial-age mortar through tests of ancient material is difficult, because it occurs in narrow, irregular zones that are bonded to caementa (Fig. 1B), and the heterogeneous fabric of the concrete requires large test dimensions (16). We therefore duplicated the Imperial-age mortar using the volcanic ash–quicklime proportions described by Vitruvius (de Architectura 2.4–2.5) (14) and petrographic and mineralogical characterization of mortar samples from the Great Hall of Trajan’s Markets (17) to formulate a mix design that closely mimics the Trajanic formulation. Fracture-mechanical properties, as well as Young’s modulus, were previously determined at 28, 90, and 180 d hydration via an innovative arc-shaped three-point bending test (18) that reproduced half-slices of hollow 20-cm-diameter drill cores from the Great Hall (Fig. 1B), so that the behavior of the mortar reproduction can be compared with that of Trajanic concrete in a future experimental testing program. All measured properties increase with age, with the 180-d mortar producing values for Young’s modulus and uniaxial tensile strength around 1/10 of modern structural concrete, whereas fracture energy is close to one-half (Fig. S2) of the crack arrays in the reproduction at 28, 90, and 180 d hydration provide visualization of millimeter-scale fracture processes to which macroscale toughness associated with previously published G_F values are attributed (18). X-ray microdiffraction experiments with synchroton radiation are a critical analytic component, because they provide very fine-scale identifications of the cementitious mineral assemblage that evolved over 1,900 y. The Markets of Trajan concrete provides a proven prototype for innovations in monolithic concretes (19) that are reinforced by a clast-supported conglomeratic fabric at the macroscale and an enduring crystalline fabric at the microscale. New concrete materials formulated with pyroclastic aggregate based on the Imperial Roman prototype could reduce carbon emissions, produce crystalline cementitious reinforcements over long periods of time, enhance durability in seismically active regions, and extend the service life of environmentally sustainable buildings.

Table 1.

Mechanical properties of mortar reproductions

Late Pleistocene horse and camel hunting at the southern margin of the ice-free corridor: Reassessing the age of Wally’s Beach,Canada

The only certain evidence for prehistoric human hunting of horse and camel in North America occurs at the Wally’s Beach site, Canada. Here, the butchered remains of seven horses and one camel are associated with 29 nondiagnostic lithic artifacts. Twenty-seven new radiocarbon ages on the bones of these animals revise the age of these kill and butchering localities to 13,300 calibrated y B.P. The tight chronological clustering of the eight kill localities at Wally’s Beach indicates these animals were killed over a short period. Human hunting of horse and camel in Canada, coupled with mammoth, mastodon, sloth, and gomphothere hunting documented at other sites from 14,800–12,700 calibrated y B.P., show that 6 of the 36 genera of megafauna that went extinct by approximately 12,700 calibrated y B.P. were hunted by humans. This study shows the importance of accurate geochronology, without which significant discoveries will go unrecognized and the empirical data used to build models explaining the peopling of the Americas and Pleistocene extinctions will be in error.The only known late Pleistocene horse and camel kill and butchering localities occur at the southern margin of the ice-free corridor in the rolling Prairie of southwest Alberta, Canada, at the Wally’s Beach site (DhPg-8), about 180 km south of Calgary (Fig. 1). Here, seven butchered horses (Equus conversidens) (1) and one butchered camel (Camelops hesternus) (2) were found associated with nondiagnostic lithic artifacts. These sites were originally assigned to the Clovis complex based on five inaccurate radiocarbon ages and horse protein residue extracted from two unassociated fluted projectile points. Here we present 27 new radiocarbon ages that revise the age of these kill localities. These new ages show that accurate radiocarbon geochronology requires rigorous sample pretreatment and isolation of chemically pure fractions, especially to accurately date bone (SI Text and Figs. S1–S4). Without such care, significant archaeological discoveries will go unrecognized and be misinterpreted. The new ages for Wally’s Beach also allow us to explore the role of hunting in the extinction of megafauna at the end of the Pleistocene.Open in a separate windowFig. 1.(A) Map showing the location of Wally’s Beach (j) and other sites: a, Colby, WY; b, Murray Springs, AZ; c, Blackwater Draw, NM; d, Lehner, AZ; e, Domebo, OK; f, Dent, CO; g, Lange-Ferguson, SD; h, Lubbock Lake, TX; i, El Fin del Mundo, Mexico; k, Firelands, OH; l, Manis, WA; m, Lindsay, MT; n, Schaefer, WI; o, Page-Ladson, FL; p, Hebior, WI. (B) Butchered horse carcass with large stone and artifacts (Horse B). (C) Butchered camel carcass with artifacts. (D) Biface fragment associated with Horse 1. (E) Edge-modified flake tool associated with Horse C. (F) Edge-modified flake tool associated with Horse B. (G) Large biface associated with Horse 2. (H) Large unifacial chopper associated with Horse 2. (I) Core tool associated with camel.The eight kill and butchering localities at Wally’s Beach lay 30 m above the St. Mary River. Individual carcasses and associated artifacts were buried to a depth of 1.5–2.0 m in aeolian loess and sand that overlies Wisconsinan glacio-fluvial sediments. Each carcass was isolated and horizontally separated from other carcasses by 25–100 m over a distance of 500 m (1, 2). At each locality, some bones of each carcass were still articulated, although most were scattered and with some elements missing (Fig. 1). Multiple cutmarks made by stone tools occur on the hyoid bone of Horse B and on a cervical vertebra of the camel. The ribs of the camel display spiral fractures where they were broken away from the vertebral column. These taphonomic patterns are consistent with human disarticulation; the absence of carnivore gnaw marks and other damage to the bones suggests rapid burial of the horse and camel localities (1, 2). Twenty-nine lithic artifacts and one rounded cobble were found at the horse localities (1), with at least one lithic artifact associated with each horse. Three lithic artifacts were associated with the camel (2). These are all nondiagnostic artifacts and are mostly flakes, used flakes, and core tools (Fig. 1). Most are made of quartzite that is locally available in the glacial till, but some are made of chert.Previously, four radiocarbon ages ranging from 10,980 ± 80 ¹⁴C y B.P. (TO-7691) to 11,350 ± 80 ¹⁴C y B.P. (TO-8972) were obtained from bison, horse, muskox, and caribou bones from the aeolian sediments at Wally’s Beach (1). A single date of 11,070 ± 80 ¹⁴C y B.P. (TO-13513) was obtained on the rib of the butchered camel (2). All of these dates were on gelatin and are considered inaccurate because of the chemical fraction dated (SI Text). None of the seven horses with butchering evidence and associated with artifacts was directly dated in the original study.

Table 1.

Previous radiocarbon ages from Wally’s Beach site, Canada

Transoceanic drift and the domestication of African bottle gourds in the Americas

Bottle gourd (Lagenaria siceraria) was one of the first domesticated plants, and the only one with a global distribution during pre-Columbian times. Although native to Africa, bottle gourd was in use by humans in east Asia, possibly as early as 11,000 y ago (BP) and in the Americas by 10,000 BP. Despite its utilitarian importance to diverse human populations, it remains unresolved how the bottle gourd came to be so widely distributed, and in particular how and when it arrived in the New World. A previous study using ancient DNA concluded that Paleoindians transported already domesticated gourds to the Americas from Asia when colonizing the New World [Erickson et al. (2005) Proc Natl Acad Sci USA 102(51):18315–18320]. However, this scenario requires the propagation of tropical-adapted bottle gourds across the Arctic. Here, we isolate 86,000 base pairs of plastid DNA from a geographically broad sample of archaeological and living bottle gourds. In contrast to the earlier results, we find that all pre-Columbian bottle gourds are most closely related to African gourds, not Asian gourds. Ocean-current drift modeling shows that wild African gourds could have simply floated across the Atlantic during the Late Pleistocene. Once they arrived in the New World, naturalized gourd populations likely became established in the Neotropics via dispersal by megafaunal mammals. These wild populations were domesticated in several distinct New World locales, most likely near established centers of food crop domestication.In independent centers of plant domestication worldwide, distinct suites of food crops tend to emerge from native flora under human selection. An exception to this is the bottle gourd (Lagenaria siceraria, Cucurbitaceae), which is native to Africa, but was used by diverse human cultures not only in Africa, but also across Eurasia, the Pacific Islands, and the New World during pre-Columbian times (1–4). Although bottle gourd fruits are edible, they are used by humans mostly for other purposes, including as lightweight, durable containers, fishnet floats, and musical instruments (5). This variety of utilitarian applications likely explains why bottle gourds are so globally pervasive.In the New World, bottle gourds appear in archaeological contexts as early as 10,000 BP (6) (9). Bottle gourds were long proposed to have arrived in the Americas via long-range dispersal on ocean currents (10–13). However, an analysis of DNA from living and archaeological gourds suggested that the bottle gourd may have been transported into the New World by the first colonizing humans (6). In this scenario, the bottle gourd, like the dog (14), crossed the Bering Land Bridge with colonizing humans already in its domestic form, making the bottle gourd one of the earliest domesticated species (1, 6).

Table 1.

Archaeological samples and associated AMS radiocarbon dates for gourd rind fragments used in this study

Early Lapita skeletons from Vanuatu show Polynesian craniofacial shape: Implications for Remote Oceanic settlement and Lapita origins

With a cultural and linguistic origin in Island Southeast Asia the Lapita expansion is thought to have led ultimately to the Polynesian settlement of the east Polynesian region after a time of mixing/integration in north Melanesia and a nearly 2,000-y pause in West Polynesia. One of the major achievements of recent Lapita research in Vanuatu has been the discovery of the oldest cemetery found so far in the Pacific at Teouma on the south coast of Efate Island, opening up new prospects for the biological definition of the early settlers of the archipelago and of Remote Oceania in general. Using craniometric evidence from the skeletons in conjunction with archaeological data, we discuss here four debated issues: the Lapita–Asian connection, the degree of admixture, the Lapita–Polynesian connection, and the question of secondary population movement into Remote Oceania.The first human settlement of Vanuatu is indicated by the Lapita culture, whose earliest signature appears in the northwestern Melanesian islands toward the end of the interval 3,470–3,250 y B.P. or slightly later (1). The Lapita culture is defined by a set of artifacts including highly decorated pottery displaying a distinctive design system, long-distance exchanges of raw material and finished items, translocations of plants and animals, and the initial incursion of humans into the pristine island environments of Remote Oceania to the east of the main Solomon chain between 3,000 and 2,800 y B.P. (1, 2). In Vanuatu, as in the rest of Remote Oceania, Lapita quickly evolved, within 200–300 y, into distinctive local cultures in conjunction with increased population size and sedentism by the end of the Lapita period (3).The question of the biological nature of the Lapita populations is routinely approached with data collected from protohistoric/historic or extant populations used as proxies. Analysis of skull morphology and morphometrics of protohistoric/historic populations from Oceania shows a geographical pattern of variation, separating northern and southern Melanesia from western and eastern Polynesia (4–6). More generally, the results indicate two contrasting divisions, an Australo-Melanesian pole comprising groups from the western part of Remote Oceania (Island Melanesia) and an Asian pole including groups from the (far) eastern part of Remote Oceania (Polynesia). This pattern suggests separate origins for the indigenous inhabitants of these two regions. Evidence from inherited genetic markers indicates that the populations living today in Vanuatu and generally in the region first settled by Lapita groups share a common origin in an area that encompasses Island South East Asia, the north coast of New Guinea, and the Bismarck Archipelago (7–13). These populations display haplogroups attributed both to the Pleistocene settlement of the northern Melanesian/Near Oceanic region and to the Lapita diaspora, with chronological estimates based on genetic data. Geographical variations in haplotype frequencies distinguish the western part of the initial Lapita region from the eastern part, with a smaller diversity in the eastern populations in what is today Western Polynesia.Studies on Lapita skeletal morphology (14–20). In a recent biodistance study of mandibles from Watom (New Britain), Pietrusewsky et al. (16) conclude that “expectation that skeletons associated with the Lapita Cultural Complex, Early or Late Lapita, biologically resemble the modern-day inhabitants of Remote Oceania is not supported” and challenge “the prevailing orthodox view that the origin of Polynesians is associated with Lapita culture.” However, whether the few analyzed individuals represent initial “Lapita people” is open to question. Because they postdate the initial appearance of the Lapita culture in the region (20), they may actually reflect subsequent gene flow and migratory events within the Melanesian region, saying more “about the contemporary indigenous inhabitants of eastern Melanesia than … about the ancestors of the Polynesians,” as noted by Pietrusewsky et al. (18). Alternatively, the possibility that these late Lapita and (immediately) post-Lapita individuals derive directly from the initial “Lapita population” is not excluded, because heterogeneity among the early populations of the region and among the Lapita groups themselves might be expected (21–23).

Table S1.

List of Lapita specimens known so far

NO binding kinetics in myoglobin investigated by picosecond Fe K-edge absorption spectroscopy

Diatomic ligands in hemoproteins and the way they bind to the active center are central to the protein’s function. Using picosecond Fe K-edge X-ray absorption spectroscopy, we probe the NO-heme recombination kinetics with direct sensitivity to the Fe-NO binding after 532-nm photoexcitation of nitrosylmyoglobin (MbNO) in physiological solutions. The transients at 70 and 300 ps are identical, but they deviate from the difference between the static spectra of deoxymyoglobin and MbNO, showing the formation of an intermediate species. We propose the latter to be a six-coordinated domed species that is populated on a timescale of ∼200 ps by recombination with NO ligands. This work shows the feasibility of ultrafast pump–probe X-ray spectroscopic studies of proteins in physiological media, delivering insight into the electronic and geometric structure of the active center.Diatomic molecules, such as CO, NO, and O₂, are the receptors that bind to and activate heme proteins. Among them, NO has been highlighted as a key biological messenger (1) and its level controls various physiological responses, such as NO synthases, message transduction (soluble guanylyl cyclases) (2, 3), NO transport and oxidation [hemoglobin, myoglobin (Mb), and nitrophorin] (4–6), and regulation of the NO/O₂ balance (neuroglobin) (7, 8). In all of these cases, the heme group that binds the NO ligand is chemically identical, and therefore, variations in the reactivity and function are thought to be closely related to the spin, electronic configuration, and geometric structure on binding (9) and/or suggestive of different steric and electronic interactions of the bound NO with neighboring protein residues (10). Consequently, there is great interest in understanding the nature of NO binding to heme proteins and its biochemical role.The binding kinetics of NO in Mb have been studied by a variety of time-resolved spectroscopic techniques. Ligand dissociation from the heme iron was triggered by excitation into either the Soret or the Q bands, whereas the ensuing dynamics were probed using transient absorption (TA) in the UV visible (UV-Vis) (11–20), the near IR (21–23), and the mid-IR (10, 23, 24) or by resonance Raman spectroscopy (20). For UV-Vis and near-IR TA spectroscopy, the signals are dominated by the π-orbitals of the porphyrin, whereas mid-IR TA of the NO stretch mode is sensitive to the orientation of the NO dipole. Resonance Raman spectroscopy maps several vibrational modes of the porphyrin, but most studies have focused on the important Fe-N stretch vibration (at 220 cm⁻¹) with the proximal histidine (20, 25–27), which is sensitive to the position of the iron atom out of the heme plane and to the strain that the protein exerts on the heme through movements of the helices (28, 29).All of the TA studies report multiexponential recombination kinetics with time constants spanning from subpicoseconds to several hundreds of picoseconds or even longer. 10, 12, 13, 16–18, 20, 21, 24). Based on temperature-dependent UV-Vis TA studies, Champion and coworkers (17) argued that the transition state associated with the fast recombination kinetics (8–15 ps) has no barrier and is caused by rebinding of NO from the center of the distal pocket very close to the iron. A barrierless recombination is thought to occur, because the unpaired NO electron forms a transition state with the antibonding d_z² orbitals without structural distortions of the protein matrix (17). The slow component in their work (170–200 ps) was assigned to recombination of NO from the more distant Xe4 pocket (30). The rebinding occurs on a similar timescale as the structural fluctuations of the protein architecture (13, 23, 28, 29), and therefore, relaxation of the active site after dissociation gives rise to a small time-dependent barrier (∼3 kJ mol⁻¹) (17). From their mid-IR TA studies, Lim and coworkers (23, 24) similarly concluded that the slow recombination component (133 ps in their case) is caused by the protein environment surrounding the distal side of the heme, and conformational relaxation of the protein after photolysis may raise the barrier to NO rebinding. In both cases (long and short components), it was hypothesized that NO binds to an out-of-plane iron (domed porphyrin), in agreement with theoretical calculations (31–33). Finally, τ₄ in ProbeExcitation wavelength (nm)τ₁ (ps)τ₂ (ps)τ₃ (ps)τ₄ (ns)SourceUV-Vis TA^*57427.6 (52%)279.3 (48%)12UV-Vis TA^*Times change with mutation3–518.9 (41%)126.4 (49%)∞ (10%)11UV-Vis TA^†5709.1 (40%)200 (50%)∞ (10%)14UV-Vis TA^*5642–413 (40%)148 (50%)∞ (10%)Resonance Raman^†560–57030 ± 1020Near-IR TA^‡560–5702–427.5 ± 5 (42%)293 ± 30 (33%)∞ (25%)21Visible TA^‡5642–412 ± 3 (40%)205 ± 30 (37%)∞ (23%)21Mid-IR TA—1 and 4 (35%)42 (29%)238 (36%)10Mid-IR TA5805.3 (54%)133 (46%)24UV-Vis TA^§4001.8 (42%)13.8 (24%)200 (34%)∞ (<5%)16UV-Vis TA^§5801.1 (50%)8 (30%)170 (20%)16UV-Vis TA40013.8 (41%)200 (59%)17UV-Vis TA5808 (60%)170 (40%)17X-ray absorption532192 ± 44 (75%)>1.3 (25%)This workOpen in a separate window^*Single-wavelength detection at 480 nm (Soret band).^†Single-wavelength detection at 435 nm (Soret band).^‡Single-wavelength detection at 615 nm (Q bands).^§White light continuum detection.More recent studies by Negrerie and coworkers (20, 21) combined UV-Vis, near-IR (in the region of band III) TA, and resonance Raman measurements. The resonance Raman mainly probed the Fe-N_histidine mode that is sensitive to the heme iron out-of-plane position, just like the near-IR band III (27). Although the UV-Vis TA results in the Soret and Q-band regions agree with the results in refs. 16 and 18, the resonance Raman results and the TA studies of band III reveal an additional ∼30-ps component [also reported in one of the mid-IR studies (10) and one UV-Vis TA study (12)]. Negrerie and coworkers (21) also concluded that GR leads to the formation of a transient six-coordinated domed heme, with a rise time of ∼10 ps and a decay of 30 ± 10 ps, corresponding to the return to the planar form. Thus, the transition from domed to planar is not a prompt one [contrary to the reverse process (34)], and Negrerie and coworkers (21) attributed its timescale to the constraints that the protein exerts on the porphyrin. Indeed, using picosecond UV resonance Raman studies of MbCO, Mizutani and coworkers (29) found that, on photodissociation of CO, the signal of the Tryptophan situated on the A helix showed a prompt decrease followed by a recovery in ∼50 ps that was attributed to changes in the protein tertiary structure that exerts strain on the heme–protein link. Negrerie and coworkers (20) studied other heme proteins and found that the timescale of the primary domed to planar heme transition was ∼15 ps for hemoglobin, ∼7 ps for dehaloperoxidase, and ∼6 ps for Cytochrome c (Cytc), and they attributed these timescales to the constraints exerted by the protein structure on the heme cofactor (20). Negrerie and coworkers (20) noted, however, that dehaloperoxidase and Cytc have similar time constants, despite their different structure and heme linking, revealing that several factors, not only the protein strain, can influence the heme response kinetics. Similar time constants were reported using IR TA by Lim and coworkers (22) for Cytc and a model heme, microperoxidase-8, but they attributed the faster ligand rebinding in Cytc compared with Mb to the fact that the former does not have a primary docking site-like structure that slows down NO rebinding.On the theory side, Franzen (31) calculated potential curves along the Fe-NO distance for the different spin states of a compound consisting of an imidazole (Im) ligand bound to iron-porphine (FeP) trans to the diatomic NO ligand (Im-FeP-NO): the electronic ground state (S = 1/2) and the excited quartet (S = 3/2) and sextet (S = 5/2) states. For the planar geometry, Franzen (31) found, as expected for the ground state, that the doublet is the most stable configuration. The quartet is somewhat less binding, whereas the sextet state is even less binding with an Fe-NO equilibrium distance above 2 Å. Franzen (31) attributed the ∼10-ps component to the S = 5/2→S = 3/2 relaxation and the >100-ps timescale to a sequential relaxation S = 5/2→S = 3/2→S = 1/2 process. More refined calculations by Strickland and Harvey (32) found that the quartet state is the most likely state to be populated on recombination. Contrary to the works in refs. 16, 17, 23, and 24, these calculations exclusively invoke intramolecular electronic relaxation, neglect the role of the environment, and do not include the strain on the porphyrin.Because the measured time constants somewhat vary with the observable (35–37). However, solid samples are far from the physiological conditions under which proteins operate, and it is desirable to investigate the ligand dynamics of heme proteins in physiological solutions. In addition, the latter can be flowed continuously to ensure the renewal of the sample and to decrease the X-ray dose on it. Adopting such an approach, X-ray scattering studies of MbCO in solution with a 100-ps resolution were recently reported (38–40), but the spatial resolution is such that tertiary and global structural changes can be probed but not atomic-scale changes. In addition, this approach does not deliver information about the electronic structure of the active site, which plays a central role in the biochemistry and reactivity of heme proteins (41, 42). The valence 3d electrons of the iron atom are significantly delocalized over the porphyrin ligand π*-orbitals, and the ability of the heme to redistribute charge and spin density plays an important role in the formation and stabilization of a variety of intermediates important for biological function (42–44). Time-resolved X-ray absorption spectroscopy (XAS) (45) offers the advantage of interrogating the electronic and geometric structure of the biochemically active center of the system with elemental selectivity (i.e., the Fe atom). It was used to investigate the recombination of CO to Mb after its photodissociation from MbCO (46–48). In this case, ligand recombination occurs on times up to milliseconds (49), and the transient XAS was recorded using alternating intervals of data acquisition for the laser-excited sample at a 100-ps time delay and the unexcited sample, contrary to the pulse-to-pulse data acquisition where the unexcited and excited XASs are recorded sequentially (50). The resulting transient reflects the formation of the deoxy-Mb species (46), indicating the absence of GR of CO at this time delay. Very recently, Levantino et al. (51) reported a femtosecond XAS study of MbCO at the Fe K edge using the Stanford Free Electron Laser LCLS. Levantino et al. (51) presented kinetic traces exhibiting two time components (∼70 and ∼400 fs), which they tentatively attributed to a structural rearrangement induced by photolysis involving essentially only the heme chromophore and a residual Fe motion out of the heme plane that is coupled to the displacement of Mb F helix, respectively. However, without transient spectra, these conclusions are highly speculative.The high-repetition rate scheme for picosecond XAS studies (46) was originally developed to investigate photoinduced processes in highly dilute media (52), such as proteins in physiological solutions, which have concentrations (1–4 mM) that are one to two orders of magnitude lower than those of the metal complexes that we investigated (50). Here, we show for the first time, to our knowledge, its implementation to address the nature of the recombination of NO to the porphyrin Fe atom with 70-ps resolution. Nitrosyl-Mb (MbNO) is excited at 532 nm into the Q bands, and the system is probed at the Fe K edge near 7.12 keV. We carried out two series of experiments under somewhat different conditions, which are both described in SI Experimental Details, Figs. S1–S3, and 16 and 17, forming the domed hexacoordinated species, which relaxes to the planar configuration in ∼30 ps (20, 21). The experimental setups and sample preparation are described in SI Experimental Details. We also performed simulations of the transient X-ray absorption near-edge structure (XANES) spectra using multiple scattering theory (MST) (details in ref. 42 and SI Simulations of Transient XANES Spectra).Open in a separate windowFig. S1.Comparison of the time-resolved XAS signal at 70-ps time of the different series of measurements: series I (red) and series II (black). The static difference spectrum is in blue. Norm. Trans. Abs., normalized transient absorption.Open in a separate windowFig. S3.ei Sets of data points making up a full transient spectrum: e1 (Green), e2 (red), e3 (blue), and e4 (yellow). Note that not all datasets have been measured a similar number of times (details in SeriesSample type (window thickness, optical path; μm)Laser focus (μm²)Laser fluence (mJ/cm²)X-ray focus (μm²)Incident X-ray photons per pulseDetected X-ray (photons per point)Sample stabilityIFlat diamond flow cell (50, 250)85 × 656540 × 402 × 10⁴5 × 10⁶12 hII^*Cylindrical quartz capillary (10–20, 300)70 × 702430 × 302 × 10⁴5 × 10⁶24 h^†Open in a separate window^*Similar values for 300 ps.^†The sample was rereduced every 4 h, and each time, the UV-Vis spectrum immediately showed characteristic Soret and Q bands of MbNO. After 24 h, a new sample was used.

Table S4.

Statistics of the two series of measurements

Rationale and mechanism for the low photoinactivation rate of bacteria in plasma

The rate of bacterial photoinactivation in plasma by methylene blue (MB), especially for Gram-negative bacteria, has been reported to be lower, by about an order of magnitude, than the rate of inactivation in PBS and water solutions. This low inactivation rate we attribute to the bleaching of the 660-nm absorption band of MB in plasma that results in low yields of MB triplet states and consequently low singlet oxygen generation. We have recorded the change of the MB 660-nm-band optical density in plasma, albumin, and cysteine solutions, as a function of time, after 661-nm excitation. The transient triplet spectra were recorded and the singlet oxygen generated in these solutions was determined by the rate of decrease in the intensity of the 399-nm absorption band of 9, 10-anthracene dipropionic acid. We attribute the bleaching of MB, low singlet oxygen yield, and consequently the low inactivation rate of bacteria in plasma to the attachment of a hydrogen atom, from the S–H group of cysteine, to the central nitrogen atom of MB and formation of cysteine dimer.Even though infections from bacterially contaminated plasma are thought to be very few (1) and the frequency of patient sepsis caused by transfusion of blood contaminated by bacteria is as low or less than the transfusion-associated hepatitis C virus infection (2), the possibility of such infection cannot be entirely discounted in view of the fact that new or even more resistant strains of known bacteria are being encountered. To a large extent, pooled human plasma is sterilized by the solvent–detergent method (3–5), whereas single-donor human plasma is often decontaminated by methylene blue (MB) photoexcitation, which generates reactive oxygen species that safely inactivate many viruses (6, 7). However, several Gram-positive and especially Gram-negative bacteria are found to be very resistant to photoinactivating agents such as porphyrins and thiazine dyes, possibly because the outer walls of Gram-negative bacteria, such as Serratia marcescens (SM), are composed of negatively charged lipopolysaccharides, which are resistant to photoinactivation by these dyes (8).Gram-positive bacteria and several Gram-negative bacteria are relatively easily inactivated, especially in high-pH MB–PBS solutions, after irradiation with 661-nm light for a rather short period, i.e., 10 min (9). Gram-negative bacteria usually require longer exposure times for inactivation. 10). This inactivation deficiency, observed in plasma, cannot be attributed to the decrease in light intensity due to absorption or scattering, because the fresh human plasma used is clear and neither absorbs in the 661-nm excitation wavelength region nor scatters the LED light. Singlet oxygen is the dominant active species responsible for the photoinactivation of bacteria (11–21), with MB being the primary photosensitizing agent used for the generation of singlet oxygen and the sterilization of plasma (22–27). To that effect we investigated possible reactions between plasma and MB that may be responsible for decrease in singlet oxygen generation that would explain the low bacteria inactivation rate in plasma.

Table 1.

Inactivation of Gram-positive and Gram-negative bacteria in MB–PBS and MB–plasma solutions as a function of irradiation time with 6.8-mW, 661-nm LED light

Structure of subcomplex Iβ of mammalian respiratory complex I leads to new supernumerary subunit assignments

Mitochondrial complex I (proton-pumping NADH:ubiquinone oxidoreductase) is an essential respiratory enzyme. Mammalian complex I contains 45 subunits: 14 conserved “core” subunits and 31 “supernumerary” subunits. The structure of Bos taurus complex I, determined to 5-Å resolution by electron cryomicroscopy, described the structure of the mammalian core enzyme and allowed the assignment of 14 supernumerary subunits. Here, we describe the 6.8-Å resolution X-ray crystallography structure of subcomplex Iβ, a large portion of the membrane domain of B. taurus complex I that contains two core subunits and a cohort of supernumerary subunits. By comparing the structures and composition of subcomplex Iβ and complex I, supported by comparisons with Yarrowia lipolytica complex I, we propose assignments for eight further supernumerary subunits in the structure. Our new assignments include two CHCH-domain containing subunits that contain disulfide bridges between CX₉C motifs; they are processed by the Mia40 oxidative-folding pathway in the intermembrane space and probably stabilize the membrane domain. We also assign subunit B22, an LYR protein, to the matrix face of the membrane domain. We reveal that subunit B22 anchors an acyl carrier protein (ACP) to the complex, replicating the LYR protein–ACP structural module that was identified previously in the hydrophilic domain. Thus, we significantly extend knowledge of how the mammalian supernumerary subunits are arranged around the core enzyme, and provide insights into their roles in biogenesis and regulation.In mammalian mitochondria, respiratory complex I (NADH:ubiquinone oxidoreductase) (1, 2) contains 45 subunits with a combined mass of 1 MDa (3–6). Fourteen core subunits, conserved from bacteria to humans, constitute the minimal complex I and catalyze the energy transducing reaction: NADH oxidation and ubiquinone reduction coupled to proton translocation across the inner membrane. Complex I is thus critical for mammalian metabolism: it oxidizes the NADH generated by the tricarboxylic acid cycle and β-oxidation, produces ubiquinol for the rest of the respiratory chain, and contributes to the proton-motive force that supports ATP synthesis and transport processes. Seven of the core subunits are hydrophilic proteins that are encoded in the nucleus and imported to mitochondria, and the other seven are hydrophobic, membrane-bound proteins (known as the ND subunits) that are encoded by the mitochondrial genome. These two sets of seven subunits form the two major domains of complex I, in the matrix and inner membrane, which confer its characteristic L shape.The protein composition of complex I from Bos taurus heart mitochondria has been characterized extensively, and is the blueprint for the human enzyme (3–6). In addition to the 14 core subunits it contains 31 supernumerary subunits (including two copies of the acyl-carrier protein, SDAP; ref. 2). Several supernumerary subunits are homologous to proteins of known function and some are required for the assembly/stability of the complex, but the roles of most of them are unknown: they may be involved in regulation, homeostasis, mitochondrial biogenesis, or protection against oxidative damage (2, 5, 7, 8).Work to determine the structure of bacterial complex I culminated in the 3.3-Å resolution structure of intact Thermus thermophilus complex I (9–11). The bacterial structures revealed many catalytically important elements, including the flavin mononucleotide that oxidizes NADH, the iron–sulfur (FeS) clusters that transfer electrons from the flavin to ubiquinone, the ubiquinone-binding site, and elements of the proton transfer apparatus: four antiporter-like structures in the membrane, and a long transverse helix that appears to strap the membrane domain together. These features are closely conserved in the medium resolution structures of the complexes from B. taurus (2) (4UQ8.pdb) and the yeast Yarrowia lipolytica (12) (4WZ7.pdb). Furthermore, in the 5-Å resolution electron cryomicroscopy (cryo-EM) structure of the B. taurus enzyme, 18 supernumerary transmembrane helices (TMHs) were observed, and it was possible to propose assignments and partial models for 14 supernumerary subunits (2). One of these assignments (for the 13-kDa subunit) was confirmed recently in Y. lipolytica complex I using the anomalous diffraction from a bound zinc ion (13). Currently, 17 supernumerary subunits remain unassigned, and 11 supernumerary TMHs in the cryo-EM model have no subunits assigned to them.Fractionation of B. taurus complex I into subcomplexes Iα, Iλ, and Iβ has provided a wealth of information on its protein composition, and enabled subunits to be assigned to its major domains (3, 5, 14). Subcomplex Iλ contains all of the redox cofactors and represents the hydrophilic domain; subcomplex Iα contains subcomplex Iλ plus additional subunits from the interdomain region; and subcomplex Iβ constitutes the distal section of the membrane domain. Subcomplex Iβ represents the domain of complex I that is the least-well-characterized structurally because it contains many unassigned supernumerary subunits (2) (5, 8)

Oil platforms off California are among the most productive marine fish habitats globally

Secondary (i.e., heterotrophic or animal) production is a main pathway of energy flow through an ecosystem as it makes energy available to consumers, including humans. Its estimation can play a valuable role in the examination of linkages between ecosystem functions and services. We found that oil and gas platforms off the coast of California have the highest secondary fish production per unit area of seafloor of any marine habitat that has been studied, about an order of magnitude higher than fish communities from other marine ecosystems. Most previous estimates have come from estuarine environments, generally regarded as one of the most productive ecosystems globally. High rates of fish production on these platforms ultimately result from high levels of recruitment and the subsequent growth of primarily rockfish (genus Sebastes) larvae and pelagic juveniles to the substantial amount of complex hardscape habitat created by the platform structure distributed throughout the water column. The platforms have a high ratio of structural surface area to seafloor surface area, resulting in large amounts of habitat for juvenile and adult demersal fishes over a relatively small footprint of seafloor. Understanding the biological implications of these structures will inform policy related to the decommissioning of existing (e.g., oil and gas platforms) and implementation of emerging (e.g., wind, marine hydrokinetic) energy technologies.Secondary production is the sum of new biomass from growth for all individuals in a given area during a unit of time. Some of the original motivations for understanding biological productivity stem from the need to estimate the annual production of fishes that can be taken from a body of water (1, 2). By integrating multiple metrics that can individually reflect aspects of fitness (e.g., density, biomass, growth, fecundity, survivorship, body size, life span), secondary production can be thought of as a general criterion of success for a population (3, 4). Recent studies have extended this idea, using secondary fish production to provide a measure of the productive capacity and economic value of specific habitats within an ecosystem (5, 6) and, in a few instances, to evaluate the efficacy of creating artificial reefs and other forms of habitat restoration (7–9). In ecological studies, static properties such as density or biomass are typical structural response variables, whereas the use of secondary production, a functional measure, has been mostly limited to freshwater and marine benthic invertebrate studies (4). Meanwhile, marine ecologists and fisheries scientists continue to advocate for incorporating more ecosystem-based approaches to managing marine resources (10–12). This includes calls to add more elements of community and trophic ecology to the concept of essential fish habitat (12) and will likely involve the development of functional measures or indicators that incorporate several processes from within an ecosystem (13, 14).The decommissioning of the >7,500 oil and gas platforms around the world (15, 16) is an unavoidable issue. Understanding the potential effects of the different decommissioning options on the biology of fishes living in such habitats will be important information to consider in the process. These options include “rigs-to-reefs” approaches where some portion of the platform is left in the water to continue functioning as an artificial reef. A main unresolved issue is the degree to which these types of structures enhance ecosystem function, and in particular secondary fish production, compared with nearby natural reefs (16–20). Additionally, with the current global emphasis on developing sources of renewable energy, deployment of new structures in the marine environment associated with offshore wind and wave energy extraction is increasing (21–23). These deployments may create opportunities to incorporate design elements that may enhance the conservation value and fisheries production associated with these structures.Here, we compare the annual secondary production of fish communities on oil and gas platforms to those on natural reefs off the coast of southern California (Fig. 1) and to secondary production estimates of fish communities from other marine ecosystems. To calculate the annual secondary production for a fish community, referred to here as “Total Production,” we develop a model based on fisheries-independent density and size structure data of fishes from visual surveys performed from a manned submersible once per year for between 5 and 15 y at each site. We define Total Production of the fish community as the sum of two components: “Somatic Production,” which is the difference between the observed biomass during surveys and the biomass predicted 1 y later using species-specific morphometric, growth, and mortality functions, and “Recruitment Production,” which estimates production from the growth of postlarval and pelagic juvenile fishes that settled or immigrated and survived during a 1-y time interval. Metrics for a “complete platform” were scaled to per square meter of seafloor, i.e., overall values were calculated for an entire platform, and then divided by the surface area of seafloor beneath the footprint of the platform. This permits a more direct comparison among platforms and natural reefs in the present study, and among estimates of secondary production of fishes in other ecosystems from the literature, which are also typically scaled to per square meter of seafloor (Open in a separate windowFig. 1.Platform diagram and map of the study area. The platform midwater habitat encompasses the hard substrate of the platform structure from the water surface to 2 m above the seafloor, whereas the platform base habitat is the bottom 2 m of the platform structure. The platform structure consists of outer vertical pilings and horizontal crossbeams (i.e., the platform jacket) and the vertical oil and gas conductors in the center. Note this is a general display diagram and the designs of these structures vary from platform to platform. The 16 platforms (filled circles; names in all capital letters) and seven natural reefs (open circles) used in the study were surveyed for at least 5 (up to 15) y between 1995 and 2011.

Table 1.

Estimates of secondary production of fishes from various marine ecosystems

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

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

Table S1.

USR fish vertebrae samples subjected to aDNA analysis

The evolution of social parasitism in Formica ants revealed by a global phylogeny

Studying the behavioral and life history transitions from a cooperative, eusocial life history to exploitative social parasitism allows for deciphering the conditions under which changes in behavior and social organization lead to diversification. The Holarctic ant genus Formica is ideally suited for studying the evolution of social parasitism because half of its 172 species are confirmed or suspected social parasites, which includes all three major classes of social parasitism known in ants. However, the life history transitions associated with the evolution of social parasitism in this genus are largely unexplored. To test competing hypotheses regarding the origins and evolution of social parasitism, we reconstructed a global phylogeny of Formica ants. The genus originated in the Old World ∼30 Ma ago and dispersed multiple times to the New World and back. Within Formica, obligate dependent colony-founding behavior arose once from a facultatively polygynous common ancestor practicing independent and facultative dependent colony foundation. Temporary social parasitism likely preceded or arose concurrently with obligate dependent colony founding, and dulotic social parasitism evolved once within the obligate dependent colony-founding clade. Permanent social parasitism evolved twice from temporary social parasitic ancestors that rarely practiced colony budding, demonstrating that obligate social parasitism can originate from a facultative parasitic background in socially polymorphic organisms. In contrast to permanently socially parasitic ants in other genera, the high parasite diversity in Formica likely originated via allopatric speciation, highlighting the diversity of convergent evolutionary trajectories resulting in nearly identical parasitic life history syndromes.

The complex societies of eusocial insects are vulnerable to exploitation by social parasites that depend on their host colonies for survival and reproduction without contributing to colony maintenance and brood care (1–4). Social parasitism is common among eusocial Hymenoptera and evolved independently in distantly related lineages, including bees, wasps, and ants (3–8). Many studies on social parasitism have focused on the evolution of cooperation and conflict in colonies of eusocial insects and on coevolutionary arms race dynamics between hosts and parasites (9–12). However, the evolutionary origins of social parasitism and the coevolutionary factors causing speciation and thereby contributing to the high diversity of social parasite species in eusocial insects are not well understood (13, 14). Comparative evolutionary studies of social parasites are promising, because they are expected to provide insights into the conditions associated with a behavioral change from cooperative eusociality to exploitative social parasitism as well as into the consequences of the life history transitions on speciation and biological diversification.Social parasitism is a life history strategy that evolved at least 60 times in ants, and more than 400 socially parasitic species are known from six distantly related subfamilies (4). Despite the high diversity, three main life history strategies can be recognized across social parasites: 1) temporary, 2) dulotic, and 3) permanent social parasitism (1, 3, 15–21). The queens of temporary socially parasitic ant species invade the host nest and kill the resident queen(s), and the host workers raise the parasite’s offspring (16). In the absence of an egg-laying host queen, the host workforce is gradually replaced until the colony is composed solely of the temporary social parasite species. The queens of dulotic social parasites start their colony life cycle as temporary social parasites, and once sufficient parasitic workers have been reared, they conduct well-organized raids of nearby host nests to capture their brood (22). Some brood is eaten, but most workers eclose in the parasite’s nest and contribute to the workforce of the colony. By contrast, most permanent social parasite (i.e., inquiline) species are tolerant of the host queen, allowing her to continuously produce host workers, whereas the inquiline queens focus their reproductive effort on sexual offspring (1, 13). Inquilines obligately depend on their hosts and most inquiline species lost their worker caste entirely (1, 18, 19, 23).The evolutionary origins of social parasitism have been debated since Darwin’s On the Origin of Species by Means of Natural Selection (24). Entomologists have long noticed that ant social parasites and their hosts are close relatives (15, 16, 25–29), an observation subsequently referred to as “Emery’s rule” (30). Strictly interpreted, Emery’s rule postulates a sister group relationship between host and parasite, whereas a less restrictive or “loose” interpretation signifies for example a congeneric, but not necessarily a sister taxon relationship (13, 31–33). Consequently, two competing hypotheses were developed for explaining the speciation mechanisms of social parasites: 1) The interspecific hypothesis proposes that host and social parasite evolved reproductive isolation in allopatry, whereas 2) the intraspecific hypothesis postulates that the social parasite evolved directly from its host in sympatry (3, 13, 18, 20, 21, 31–37). Empirical studies of temporary, dulotic, and host queen-intolerant workerless ant social parasites generally provide support for the interspecific hypothesis (14, 38–48), whereas recent phylogenetic studies lend support to the intraspecific hypothesis for queen-tolerant inquilines (33, 36, 49–51). In some cases, host shifts, secondary speciation events of hosts and/or parasites, and extinctions obscure the original evolutionary conditions under which social parasitism originated (52–54).To explore the origin and evolution of diverse socially parasitic life histories in eusocial insects, we reconstructed the evolutionary history of the Holarctic ant genus Formica. Formica ants are ideally suited for comparative studies of social parasitism because the genus has the highest number of social parasite species in any ant genus (84 of 172; Fig. 1 and ​and2).2). In addition, colonies of Formica species vary significantly in colony-founding behavior as well as in nest and colony structures, providing an opportunity to explore the interplay between colony organization and life history at the origin of social parasitism. Some Formica species use independent colony foundation (ICF), when new colonies are started by a single queen (i.e., haplometrosis) or a group of cooperating queens (i.e., pleometrosis). Queens of other species rely on dependent colony founding (DCF), cooperating with groups of conspecific workers to found a new colony (i.e., budding) or invading an existing heterospecific colony as a temporary social parasite (TSP) or a permanent social parasite (PSP) (1, 56–62). In contrast to other studies (63), we regard TSP as a form of DCF because the socially parasitic queen relies on the social environment of the host for colony founding and rearing of the first brood. Furthermore, Formica colonies can have a single or multiple functional queens (monogyny vs. polygyny) and comprise one (monodomous) or multiple (polydomous) to thousands of interconnected physical nests covering a large area (supercolonial) (64).Table 1.Diversity of social parasites in the genus Formica compared to all other ants