Formation of these solid solutions is demonstrated to cause significant changes to the thermal stability and ammonia reactivity of the samples, highlighting the potential use of compositional variation to control the properties of the material in gas storage and catalytic applications.Li–N–H materials, particularly lithium amide and lithium imide, have been explored for use in a variety of energy storage applications in recent years. Compositional variation within the parent lithium imide, anti-fluorite crystal structure has been related to both its facile storage of hydrogen and impressive catalytic performance for the decomposition of ammonia. Here, we explore the controlled solid-state synthesis of Li–N–H solid-solution anti-fluorite structures ranging from amide-dominated (Li A wide solid solution based on the lithium imide anti-fluorite structure is demonstrated and related to its energy storage functions. Compositional variation, and the ability to exert synthetic control over such variation, lies at the heart of much of functional materials chemistry research, impacting a wide range of materials properties from metal alloy strength to superconductivity. The ability of solid structures to accommodate variable quantities of metal ions is central to lithium-ion battery chemistry, which has transformed the field of portable devices and is currently changing the landscape of passenger transportation.1,2 The hydrogenation of lithium nitride (Li3N) produces lithium imide (Li2NH) and then lithium amide (LiNH2), with stoichiometric equivalents of lithium hydride (LiH) x(NH)x1− or Lix2−NHx1+, 0 < x < 1) were observed during hydrogen storage and release reactions, and facilitate a smooth structural transformation between hydrogenated and dehydrogenated states.8,9 By analogy with interstitial metal hydrides, such as PdH0.8, this provides an explanation for the facile hydrogen storage observed in the system.Variable anion content is at the heart of applications of metal–nitrogen–hydrogen solid state materials. The Li–N–H materials system consists of lithium salts of anions formed from nitrogen and/or hydrogen, and is most well-known for its favourable hydrogen storage properties.de (LiH) . These r10,11 Partial or complete conversion of ammonia to hydrogen allows for flexible end-use of the stored energy in combustion engines, gas turbines and fuel cells.12–14 Lithium imide based catalysts have shown comparable or superior performance to state-of-the-art ruthenium catalysts.15–18 In this context, in situ neutron powder diffraction experiments indicated that the lithium imide becomes non-stoichiometric under operating conditions, with a mixed amide–imide composition dependent on the temperature and conversion.15 Isotope exchange experiments have confirmed that this variable composition allows for bulk nitrogen and hydrogen exchange between the catalyst and ammonia.19More recently, lithium imide has also sparked interest as a highly active catalyst for the decomposition of ammonia to produce hydrogen (and nitrogen). Ammonia decomposition is likely to become increasingly important as a component of renewable energy export markets, where hydrogen produced by water electrolysis is converted to ammonia and shipped to areas with energy demand.20–22 The formation of solid solutions of electride material dicalcium nitride (Ca2N) and calcium nitride hydride (Ca2NH) under ammonia synthesis conditions has been hypothesised to promote high activity through (i) the formation of a defect structure which lowers the material work function, increasing electron donation to the ruthenium particles and (ii) the ability to act as a reservoir for hydrogen, avoiding the inhibition of catalytic activity by hydrogen which is often observed for ruthenium catalysts.22The broader significance of compositional variation in M–N–H materials is illustrated by recent reports of their use as strong promoters for ammonia synthesis in ruthenium catalysts.It is clear that the impressive performance of the M–N–H materials in these applications relates to compositional flexibility, which derives from the close structural relationships between the phases involved. Lithium amide and lithium imide both have crystal structures based on a cubic close packed arrangement of anions with occupancy of the tetrahedral holes by cations: the anti-fluorite structure. Lithium amide has a layered arrangement of lithium ions occupying half the holes, while in lithium imide these holes are fully occupied .4NH, a × a × 2a supercell of the anti-fluorite structure; the charge imbalance between nitride (3−) and hydride (1−) displaces the lithium ions from the tetrahedral site towards a trigonal coordination. As such, lithium nitride–hydride could also be described as an anti-scheelite structure, which is an ordered variant of the anti-fluorite structure. This close structural relationship between anti-fluorite and anti-scheelite suggests that mixed lithium imide–nitride–hydride phases (Lix2+(NH)x1−NxHx or Lix2+NH, 0 < x < 2) can be formed. This possibility was first reported by Marx, but without accompanying experimental data.23 However, evidence for the existence of such phases has been published in the context of hydrogen storage, where a cubic phase termed “quasi imide” was reported in neutron powder diffraction studies of the low-pressure hydrogenation of lithium nitride.24–26 The presence of imide–nitride–hydride was also proposed as an explanation for the ammonia decomposition activity of lithium imide, given the formation and subsequent ammoniation of lithium nitride–hydride within the imide structure is a possible ammonia decomposition cycle:19Structural similarities are also clear between lithium imide and lithium nitride–hydride Li4NH, . Here, n2 < 0.1 ppm, H2O < 0.1 ppm) because of the water sensitivity of the samples.All sample handling was performed in an argon-filled glove box  were synthesised by the reaction of lithium amide  with lithium nitride  or lithium hydride  according to the following reactions:13 samples of LiFd3̄m) lithium imide phase for the x = 0 sample . Tungsten carbide milling balls (3 mm diameter) were added to achieve a 40 : 1 mass ratio with the powder. The jar was then sealed and transferred into a planetary ball mill (Retsch PM100) and milled for 1 hour at 150 rpm, with the direction of rotation reversed at 10 minutes intervals.−1, and then dwelled at that temperature for 12 hours.0.5 g of the resultant mixture was transferred into a cylindrical alumina crucible and placed in the bottom of a glass boiling tube fitted with a Young's tap T-piece to prevent air ingress. The sample was then heated in a tube furnace (Lenton LTF12/38/500) under a continuous flow of argon to 300 °C at a heating rate of 2 °C minx2+NH  were synthesised by the solid state reaction of lithium imide with lithium nitride–hydride according to the following reaction:13 samples of Lix1+NHx2− samples (x = 1.000). Lithium nitride–hydride was synthesised by the reaction of lithium hydride  and lithium nitride :−1 and dwelled for 12 hours.Lithium imide for the reaction was synthesised as for the Lix2+NH samples from lithium imide and lithium nitride–hydride was the same as for the other samples, except that the milling procedure used a rotation rate of 400 rpm, and the furnace temperature set point was 540 °C.Synthesis of the mixed Liλ = 0.826021 Å) at the Diamond Light Source.27 Powder samples were loaded into 0.7 mm borosilicate glass capillaries, which were flame sealed. The samples were mounted onto the diffractometer using a robotic arm, and rotated while diffraction data (8–20 minutes scans) were collected using the five Multi-Analyser Crystal (MAC) detectors.Structural analysis of the sample was performed using synchrotron X-ray diffraction; data were collected on the I11 high-resolution powder diffraction beamline  using the TOPAS Academic package (v6).Spectroscopic analysis of the samples was performed using a Raman microscope (Bruker Senterra). Each sample was loaded into a 0.7 mm wide borosilicate glass capillary to a height of approximately 10 mm and sealed with grease. Each capillary was then individually loaded onto the adjustable table, moved into position and brought into focus using the built-in microscope with ×10 lens. Raman data were collected using a 20 mW, 532 nm laser, with an aperture of 50 × 1000 μm to ensure sampling across multiple powder grains. Peak areas and positions were analysed using the TOPAS Academic package.−1, then immediately cooled back to 25 °C at a rate of 10 °C min−1. Each imide–nitride–hydride sample was heated to 575 °C at a rate of 10 °C min−1, dwelled for 10 minutes and cooled back down to 100 °C. A flowing argon atmosphere (50 cm3 min−1) was used throughout each experiment.The thermal behaviour of the samples was analysed by differential scanning calorimetry (DSC), with experiments run on a Mettler Toledo DSC 1 instrument. 10 mg of powdered sample was loaded into a 20 μL aluminium pan and sealed with an aluminium lid in the glove box. After loading onto a carousel, a robotic arm individually loaded each sample into an inert atmosphere, pierced the lid and started the heating cycle. For amide–imide samples, each sample was heated to 450 °C from 25 °C at a rate of 5 °C min−1, dwelled at temperature for 5 hours and cooled back down to room temperature. The instrument was then purged with inert gas to remove the sample safely.The reactivity of the lithium imide–nitride–hydride samples when heated under ammonia was studied in a simultaneous thermal analysis (STA) apparatus (Netzsch F1 Jupiter). Each sample was loaded into a 20 μL aluminium pan, sealed with an aluminium lid and pierced before loading to allow gas exchange. The samples were heated to 350 °C under ammonia at a rate of 5 °C minFd3̄m structure which is a 2 × 2 × 2 supercell of the antifluorite structure, formed as a result of ordered N–H bond orientations facilitated by the formation of a Frenkel defect by 1/8 of the lithium ions. This structure is most obviously evidenced by presence of the (111) peak at around 8°. It can be seen that this ordered structure is specific to samples very close to Li2NH stoichiometry; the peak is not observed for any of the imide–nitride–hydride samples, and in the Li1.917NH1.083 amide–imide sample, it is present as a weak and broad feature, indicative of a coherence length of around 300 Å.Synchrotron powder X-ray diffraction data provides a basis for assessment of the structural variation across the Li–N–H samples and the successful formation of solid solutions. These data are shown in Fm3̄m peaks, indicating a disordered (solid solution) average structure. The amide–imide samples show a single well-defined phase up to Li1.333NH1.667 (x = 0.667), two-thirds substitution of amide within the structure. A similar trend was observed for amide–imide samples synthesised via the lithium amide lithium hydride reaction. This reflects an improved synthesis protocol over previous attempts to synthesise amide–imide phases, where a single phase was only observed29 up to x = 0.4. For imide–nitride–hydride samples, an Fm3̄m structure is observed up to Li3.167NH (x = 1.167). These samples displayed much more complex microstructure than the amide–imide samples, with asymmetric peak shapes that are likely to be indicative of regions of variable composition. This likely reflects the higher enthalpic barriers to ion migration in the nitride–hydride–imide array compared with the amide–imide, where a lower-energy proton exchange and lithium ion migration is required. Grinding and re-heating these samples did not improve the peak symmetry. While the use of higher temperatures to synthesise those samples risks thermal degradation , further optimisation of milling routines may yet yield more uniform solid solutions. The data in the imide–nitride–hydride samples were modelled with a series of solid solution Fm3̄m phases with a constrained lattice parameter – composition relationship in the Rietveld analysis  peak of P4̄ phase is most likely related to a change in the lithium occupancy in the layered structure. The I4̄ structure has an alternating 0.25/0.75 occupancy of the tetrahedral sites in layers along the c-axis. Refinement of the crystallographically unique lithium sites in the P4̄ structure indicates a disruption of that ordering NH2.04(6)) is not significantly different from lithium amide. It is likely that this phase represents a kinetically stabilised phase, perhaps as a result of the synthesis procedure; further examination of its thermal stability may confirm this.The loss of body-centring implied by the ordering . The preFm3̄m phase in the imide–nitride–hydride samples is a weighted average of the three lattice parameters of the fitted phases.In order to gain insight into the nature of the compositional variation across the samples, lattice parameter data for the different phases are shown in x ≤ 1, but show a drop in c/a for the x = 0.750 sample, which may reflect some compositional variation within the tetragonal structures. Conversely, the lithium nitride–hydride structure shows a steady drop in c/a as the composition moves away from stoichiometric Li4NH, which is likely to be associated with partial site disordering with imide incorporation. Indeed, initial reports of the structure of lithium nitride–hydride via powder diffraction measurements refined ∼5% anion site disorder,23 while single crystal diffraction data indicated a perfectly ordered structure.30The lithium amide lattice parameters  are stabFm3̄m phase in the amide–imide samples displays an apparent minimum at x = 0.667 (Li1.333NH1.667), consistent with a maximum level of amide incorporation in the solid solution phase; further amide remains in the tetragonal structure. Between x = 0.583 and x = 0.333 the lattice parameter change is approximately linear before tailing off to a maximum at x = 0.167 and decreasing for Li1.917NH1.083 and Li2NH. This would appear to reflect the imide proportion at which onset of the ordered imide structure occurs, which relies on the ability to form lithium vacancies that are tetrahedrally coordinated by four imide groups.31,32The lattice parameter of the 2NH/Li4NH than Li2NH/LiNH2. The lattice parameter of Li3NH appears to break the monotonous trend in the lattice parameter; this may reflect local ordering of the anions given the equal quantities of NH2− and (N3− + H−) at this composition, though no evidence of long-range ordering was observed. Additionally, the lattice parameter does not simply level off after x = 1.167, whereupon further increasing the proportion of nitride–hydride results in tetragonal lithium nitride–hydride formation. Instead, it continues to decrease, implying a continuing change in composition, though possibly at a less rapid rate, which would imply a reduced nitride–hydride component compared to the overall stoichiometry of the sample. It is also clear from an examination of the diffraction data that the microstructure of the solid solution phase and lithium nitride–hydride in these samples differs from the other imide–nitride–hydride mixtures. The Bragg peaks of both phases are significantly strain-broadened. One possible interpretation of these data is that a solid solution is possible across the whole compositional range, but that in these samples, the solid solution is only stable at elevated temperatures, and that rapid separation upon cooling leads to two highly strained, inhomogeneous phases. This possibility will be discussed further in relation to the thermal analysis of the samples.The lattice parameter trend for the imide–nitride–hydride samples is more complex. A small proportion of nitride–hydride incorporation produces a more substantial response in the solid solution lattice parameter given the larger lattice mismatch for Lix groups in the samples, with the spectra shown in Raman spectroscopy was used in order to provide an insight into the local environments of the NH2 – symmetric NH2 stretch at 3262 cm−1, asymmetric NH2 stretches at 3314 and 3324 cm−1.LiNH2NH – broad NH stretch at 3180 cm−1.Li4NH – sharp phonon modes at 452, 584 and 775 cm−1.Li29 the tetragonal lithium amide gives sharp NH2 stretch signals which remain at constant wavenumber, reflecting the compositional stability of the phase. A second set of broadened amide stretch peaks can be observed which represent amide ions in solid solution; these are the only amide peaks present from Li1.333NH1.667 onwards, reflecting the observations from the XRD data. These peaks, and the imide peak, can be observed to move to lower wavenumber as the solid solution becomes more imide-like and the average coordination number of lithium ions around each NHx group increases . All the remaining amide–imide solid solutions display no significant thermal events on heating or cooling, aside from the Fd3̄m to Fm3̄m phase transition in Li1.913NH1.083 and Li2NH at 60 °C, highlighted in the small inset box in Fm3̄m phases were observed (Li2NH to Li3.167NH aside from Li3NH).The melting of lithium amide is seen as a sharp endotherm at 370 °C, just prior to the onset of decomposition to lithium imide , though has a large hysteresis, with the corresponding exotherm on cooling observed at 465 °C. The Li3NH and Li3.5NH samples also display exotherms in this region, perhaps indicating incomplete mixing of imide and nitride–hydride. For the other samples in this compositional range, a broad exotherm is observed in the 315–340 °C range. It is probable that this feature corresponds to phase segregation of the high temperature solid solution into the Fm3̄m and tetragonal Li4NH phases observed in the diffraction data. This is consistent with the observed broadened Bragg peaks for these phases as discussed earlier.For the samples which had both solid solution and tetragonal Li1.333NH1.667 to Li3.167NH, and indications of wider stability for nitride–hydride rich samples above 500 °C. A summary of the stable composition range and some of the local structural features which characterise different regions of the solid solution are given in The Li–N–H solid solution demonstrates remarkable tolerance for compositional variation, with samples successfully synthesised for the composition range Li+ and H+ diffusion through the structure.8,34 However, the temperature for reversible hydrogen storage is still significantly higher than could be achieved using waste heat from a PEM fuel cell, reducing overall efficiency. Given the tetragonal amide structure is known to be a very poor Li+ conductor (∼10−10 S cm−1 at room temperature35), it might be expected that the hydrogen storage properties would be improved by keeping the system within the more conductive lithium imide structure . Having revealed an apparent upper limit to amide content of the solid solution at Li1.333NH1.667, it may be possible to achieve a more favourable operating temperature by restricting the hydrogenation capacity to two thirds of the theoretical maximum.In the field of hydrogen storage, the reversibility of the hydrogen storage process has been attributed to solid solution formation. This process is facilitated by Li37 It would be preferable to use a packed bed reactor in order to maximise the exposure of ammonia to the catalyst surface. While the precise stoichiometry has been shown to vary under the reaction conditions chosen, it is clear that maintaining a more imide-like stoichiometry than Li1.33NH1.667 will prevent melting of the sample.Loss of the tetragonal structure is also important to the deployment of lithium amide–imide catalysts for ammonia decomposition, where loss of catalyst material through melting will degrade activity over time and also thwart attempts to create high surface area formulations to maximise turnover frequency. In our recent demonstration of a lithium imide ammonia cracking unit coupled to a PEM fuel cell, a Dreschel bottle style reactor was used to avoid blocking of the reactor in case of catalyst melting.+, H−, N3−, NH2− and NH2− within the structure suggests that these phases could play an analogous role to that ascribed to the Ca–N–H supports for ruthenium based ammonia synthesis catalysts, acting as a reservoir of reactive species which might otherwise occupy surface sites on the metal catalyst. Similarly, an analogy may be drawn to the metal nitride catalysts such as Co3Mo3N and Li–Mn–N phases, where the proposed Mars–van Krevelen mechanism involves reactivity of the lattice nitrogen in ammonia synthesis.38–40 The bulk transport of ions within the solid may also help explain the effectiveness of the materials as catalysts/supports despite generally low surface areas. Previous work has demonstrated that bulk nitrogen/hydrogen exchange is possible with ammonia, but these solid solutions also react with hydrogen, and lithium nitride–hydride is also able to fix nitrogen.23,33 Thus these phases show promise not only as catalysts in their own right, but also as components of composite catalyst formulations, where multi-site approaches are increasingly seen as the most promising way to escape the bottleneck of energy scaling relations on single-site catalyst surfaces.41–43The demonstrated ability of the anti-fluorite Li–N–H structure to accommodate a wide variety of ions also has implications for understanding its role in catalysis. Certainly, the existence of a wide range of stable imide–nitride–hydride solid solutions supports the possibility that nitride–hydride formation could play a role in the catalytic decomposition of ammonia by lithium imide. Additionally, the accommodation of H2NH) and three (for Li4NH) equivalents of ammonia. Indeed, mass uptake is observed. However, it is clear that even very minor incorporation of nitride–hydride into the solid solution leads to a dramatic change in the absorption profile, which more strongly resembles that of lithium nitride–hydride. The onset of rapid ammonia reaction was shifted by more than 100 °C and a generally slower kinetic profile can be seen, evidenced by the significantly reduced extent of reaction of the nitride–hydride containing samples over the duration of the experiment. Imide to amide conversion is likely to be facilitated by Grotthuss-style proton transfer through the solid, while conversion of nitride–hydride appears to be more kinetically hindered. These results are not inconsistent with the hypothesised stabilisation of nitride–hydride regions in lithium imide under ammonia decomposition conditions.19 A more comprehensive assessment of reactivity–composition relationship, including under catalytic conditions, is now possible with this library of compounds. As illustrated in It is therefore of significant interest the extent to which these compositional modifications perturb the electronic structure and reactivity of the materials. An initial demonstration of such variation is shown in Through the synthesis of mixtures of lithium amide, lithium imide and lithium nitride–hydride, a solid solution with wide-ranging anion composition has been revealed. A highly disordered, anti-fluorite structure was observed for samples from majority lithium amide through to pure lithium imide and majority lithium nitride–hydride, with evidence for greater nitride–hydride incorporation at high temperatures. In contrast, the tetragonal lithium amide and nitride–hydride structures were found to show only limited evidence of compositional variation.x environment. Further work focused on other structural and spectroscopic probes of the local structure, along with computational investigations, will be able to provide further insight as to the relationship between local structural features in solid solutions and the parent phases.While imide–nitride–hydride solid solution formation is somewhat kinetically hindered, Raman spectroscopy of amide–imide samples was found to show monotonic variation in the local NHThe formation of solid solutions has been related to a number of key functional properties of the materials, from a composition range for maintaining the more highly-conductive cubic structure for optimising hydrogen storage characteristics, to broadening the thermal stability of the material, and demonstrating significant changes in the reactivity of lithium imide with ammonia upon small amounts of nitride–hydride incorporation. With the ability to select the desired anion mixture and its related vacancy and defect phenomena, the Li–N–H system now represents a rich library of candidates for materials applications in catalysis and energy storage, and a basis for expanded investigations into composition control in M–N–H materials.Conceptualisation: JWM, WIFD and TJW. Investigation: JMB, ASM, JWM, TJW and CAM. Formal analysis: JMB, ASM, JWM, TJW and WIFD. Software: TJW. Writing – original draft: JWM, JMB, ASM. Writing – review and editing: all authors.There are no conflicts of interest to declare.CP-023-D1CP02440J-s001


East African ecosystems have been shaped by long-term socio-ecological–environmental interactions. Although much previous work on human–environment interrelationships have emphasised the negative impacts of human interventions, a growing body of work shows that there have also often been strong beneficial connections between people and ecosystems, especially in savanna environments. However, limited information and understanding of past interactions between humans and ecosystems of periods longer than a century hampers effective management of contemporary environments. Here, we present a late Holocene study of pollen, fern spore, fungal spore, and charcoal analyses from radiocarbon-dated sediment sequences and assess this record against archaeological and historical data to describe socio-ecological changes on the Laikipia Plateau in Rift Valley Province, Kenya. The results suggest a landscape characterised by closed forests between 2268 years before present  and 1615 cal year BP when there was a significant change to a more open woodland/grassland mosaic that continues to prevail across the study area. Increased amounts of charcoal in the sediment are observed for this same period, becoming particularly common from around 900 cal year BP associated with fungal spores commonly linked to the presence of herbivores. It is likely these trends reflect changes in land use management as pastoral populations improved and extended pasture, using fire to eradicate disease-prone habitats. Implications for contemporary land use management are discussed in the light of these findings. Understanding the detail of longer-term climate–human–ecosystem interaction is essential for the sound and sustainable management of cultural landscapes. Past research in eastern Africa has provided insights into the relationships between human activities, rainfall, soil fertility, wildlife, livestock grazing, and deforestation over both decadal and centennial scales, particularly with respect to pastoral communities and their links to conservation of rangelands and wildlife across Africa Acacia anmon Fig. .Cyperus with Acacia drepanolobium and Euclea divinorum. Marura swamp is surrounded by settlements and irrigated cultivation dominated by Brassicaceae, Casuarina, Eucalyptus, Grevillea robusta, and Zea mays. Cabbage, spinach, and other vegetables are grown during the dry season through irrigation and form an important source of local income. The wetland is important for the bordering communities and their animals after the Ol’ Pejeta ranch was fenced to prevent encroachment. The swamp provides habitat for harvestable plants and is a vital source of drinking water for stock, especially when short rains between March and April fail and during longer droughts. Between the 1920s and the 1970s, the dominant land use was large-scale ranching and nomadic pastoralism with pockets of smallholder agriculture especially after land redistribution following independence in 1963  radiocarbon dating of five bulk sediment subsamples provided a geochronology for the Marura sediment core. Samples were submitted to either Beta Direct AMS or Beta Analytic, USA, and were alkali-acid–alkali pre-treated, combusted to COSediments were prepared for microfossil pollen and fungal spores based on modified standard procedures published in Faegri and Iversen  and Van Microscopic charcoal was counted on slides prepared for pollen analysis. Charcoal is regarded as black, uniformly opaque, angular particles with a long axis greater than 2.9 µm. Charcoal occurrence at 80 depths were counted using the point counting method adapted from Clark  to estim14C year BP) were calibrated using the SHCal20 calibration curve , Olea (1–30%), Juniperus (0–5%), Cyathea (5–20%), Polyscias (0–5%), Schefflera (5–15%), and Rapanea (5%). The woodland forest category accounts for less than 20% of the pollen sum. Among the herbaceous taxa, Amaranthaceae contribute up to 60%, while Asteraceae and Justicia are rare in this zone recording less than 5%. Fungal (cercophora) spores dominate towards the upper boundary of this zone (0–98%) with Cyperaceae contributing up to 50% of the local pollen and cryptogam spores. The fungal spores are dominated by Acospore (20–80%), Reniform (10–40%) and Valsaria type (5–20%). The local pollen, fern spore, and fungal spore concentrations are generally low at the base of the zone, rising towards the upper pollen zone boundary  range from 0 to 5% and are evenly distributed. Afromontane taxa such as Juniperus (1–20%), Olea (0–40%) and Podocarpus decreases, except for the uppermost sample, to the end of the zone (20–80%), with low occurrence of Ericaceae (0–10%), Ilex (0–10%), Polyscias (5–20%), Rapanea (0–15%), and Syzygium (0–15%). Among the herbaceous taxa, Poaceae are the most abundant taxa in the zone. Aloe (5–20%), Asteraceae (10–40%), Artemisia (< 5%), Amaranthaceae (1–10%), and Sansevieria (10%) are all present. Local fungal and fern spores are dominated by Acospore type (90%-40%), Reniform (5–60%), Valsaria type (0–20%), Glomus (0–40%), Trichoglossum (20%), Spirogyra (3%), Cyathea, Lycopodium, and Helicoon (0–15%). Local pollen was dominated by Typha (10–99%) with Cyperaceae having a high percentage (80%) at 52 cm , Olea (20–1%), Juniperus (5–0%), and Polyscias (10–0%) all decrease towards the core top. Schefflera reached a peak at 40%, before being absent from the record. Alchornea, Myrica, Nyctaginaceae, Ricinus, Rubiaceae, and Terminalia are present but low in percentages with the latter attaining 10% in the upper part of the core. Canthium, Capparidaceae, Cleome, Commicarpus, Croton, Euclea, and Merremia all attain less than 10%, with Euphorbia rising to 20%. Asteraceae are abundant ranging from 10 to 60% where other taxa are present in lower amounts: Aloe (0–10%), Artemisia (0–3%), Amaranthaceae (10–20%), Cucurbitaceae (0–5%), Orchidaceae (2%), Rumex (4%), and Sansevieria (1–5%). Cereal-like Poaceae grains measuring < 60 µm are increasingly common (10–80%) and a constant part of the pollen signature over the past 500 years following a large peak around 800–900 cal year BP. The exotic tree species of Pinus and Eucalyptus are consistently present in the uppermost five samples that provide a chronostratigraphic marker for the arrival of colonial forestry plantations. Other herbaceous taxa such as Commelina (10–20%), Labiatae (1–20%), Justicia (10–25%), Phyllanthus (1–45%), and Solanum (2–5%) are common. Cyperaceae dominates (40–80%) the local pollen and spores. Reniform spores and Ascospore type fluctuate widely (1–90%); indeed, fungal spores are well represented in this zone compared to other zones, ranging from 50 to 80%. Pteridophytes (5%), Lycopodium (3%), and Helicoon spores (5–20%) are all present  towards the core top, although it remains the most significant size-class Fig. . CharcoaSedimentary data from Marura Swamp provide a record of ecosystem history of the Laikipia Plateau dating from ca. 2268 cal year BP. By combining a range of proxies and bringing in context from archaeological sites, we can disentangle the roles that climate variability, taphonomic processes , local riverine moist evergreen forests persisted as documented by Cyathea, ferns such as Pteris and fungal spores (Sordaria-type) indicative of edaphically wet conditions close to the coring site. The record of swamp forest growth is a good indication that Marura Swamp was functioning well ecologically, able to retain sediments and nutrients. Today, swamps like Marura have crucially important functions in the extensive savanna landscape as they provide a locus of dry season grazing for wildlife and domestic stock , and a steady expansion of pastoralism numbers from c. 3000 cal year BP and especially after ca. 1200 cal year BP, as reflected by the increase in the number of archaeological sites  in the uppermost samples. Eucalyptus-type pollen may be derived from the exotic timber tree Eucalyptus; however, this pollen type could also be derived from Eugenia or Syzygium which produce similar morphotypes  in the adjacent Rift Valley lowlands at least by c. 450 cal year BP  and ruderal taxa reflecting settlement increased after c. 800 cal year BP, consistent with archaeological data suggesting increased human activity on the Laikipia Plateau associated with the Pastoral Iron Age. The archaeological record provides evidence of shifts in settlement and mobility, while other patterns of human distribution indicate that these pre-industrial populations were susceptible to climatic changes prompting a variety of adaptive responses. Later colonial administration expanded to these areas introducing exotics to replace the cleared Afromontane forests around 1915, shortly after forced removal of most of the remaining pastoralist populations.The initiation of a regular cycle of pastoralist settlement formation, occupation, and abandonment would have led to the generation of distinctive nutrient hotspots across these savanna grasslands (Boles and Lane In summary, this combination of different environmental proxies from a sedimentary archive on the Laikipia Plateau, interpreted against the context of a growing body of archaeological data, provides a rich insight into the vegetation history of the area in a manner that can help contextualise current debates around conservation, rangeland management, and ecosystem response to climate change and human interaction.


Solution blow spinning (SBS) has recently emerged as a novel method that can produce nano- and microfiber structures suitable for tissue engineering. Gelatin is an excellent precursor for SBS as it is derived mainly from collagens that are abundant in natural extracellular matrices. Here we report, for the first time the successful generation of 3D thermally crosslinked preforms by using SBS from porcine gelatin. These SBS mats were shown to have three-dimensional fibrous porous structure similar to that of mammalian tissue extracellular matrix. In pharma industry, there is an urgent need for adequate 3D liver tissue models that could be used in high throughput setting for drug screening and to assess drug induced liver injury. We used SBS mats as culturing substrates for human hepatocytes to create an array of 3D human liver tissue equivalents in 96-well format. The SBS mats were highly cytocompatible, facilitated the induction of hepatocyte specific CYP gene expression in response to common medications, and supported the maintenance of hepatocyte differentiation and polarization status in long term cultures for more than 3 weeks. Together, our results show that SBS-generated gelatin scaffolds are a simple and efficient platform for use in vitro for drug testing applications. In pharma industry, there is an urgent need for an adequate evaluation of the potential of the drug to induce drug induced liver injury (DILI) as DILI represents an important cause of morbidity and mortality2. To pursue drug metabolism studies an easily scalable model to study libraries of slowly metabolizing drug candidates is needed. Current practices to assess DILI include animal models that are expensive, non-human and ethically questionable as well as two-dimensional (2D) cultures of human hepatocytes and hepatocyte co-cultures with nonparenchymal cells that involve a complicated experiment setup2. 2D cultures are suitable for initial assessment of drug metabolism, however, their disadvantage lies in their short-term nature as hepatocytes dedifferentiate into nonfunctional cells within a few days in the standard culture conditions3. In addition, 2D cultured cells lack cell-extracellular matrix contact, intercellular communication and spatial heterogeneity that are essential for maintaining long-term liver structure and functions4. Three-dimensional (3D) culture models facilitate the maintenance of a similar tissue architecture to that occurring in in vivo situation, which supports correct cell polarization and differentiation status. In recent years several 3D hepatocyte culture and co-culture methods have been described5, which include both non-scaffold-based methods utilizing 3D liver spheroids6  and scaffold-based methods . More recent techniques in hepatocyte culture include 3D liver microtissue biochips coupled to a microfluidic device15 and bioprinting17. Despite of these advances there is a continuous need for a cost-efficient, simple and reliable long-term 3D culture for primary human hepatocytes to assess DILI. Currently the most commonly used long term hepatocyte model system is a pseudo 3D sandwich culture where the hepatocytes are cultured in 2D conditions between two layers of collagen18 or collagen and Matrigel19. However, this model is time consuming and costly to set up, and the results may vary due to the batch-to-batch variation of the culture reagents.Liver is the central drug-metabolizing organ and hepatotoxicity is a major cause for drug withdrawal5. Whereas a wide range of methods have been applied to design fibrous gelatin-based preforms for tissue engineering, electrospinning stands out as the most widely used approach. In an earlier work we have characterized a thermally cross-linked glucose-containing electrospun gelatin material that was suitable for growing primary human fibroblasts20. However, we20 and other research groups have shown that electrospinning typically produces nonwovens of in-plane oriented fibers that limit cell migration into deeper layers resulting in culture constructs that are essentially 2D and as such not suitable for modelling of parenchymal organs such as liver23. In recent years, solution blow spinning (SBS) has emerged as alternative method that can produce three-dimensional nano- and microfiber structures at higher throughput using a significantly simpler and less specific setup25. SBS has been successfully applied for creating 3D structures from fish gelatin; however, the single published attempt to utilize mammalian (porcine) gelatin as the input was unsuccessful26.It is known that artificially generated natural protein-based matrices may mimic several aspects of the native environment. Gelatin is a physiological non-synthetic material that consists of a mix of collagens—the main constituents of the extracellular matrix (ECM) of animal tissues and is prepared from natural sources with minimal processing. Furthermore, gelatin forms an excellent base for creating semi-artificial materials that mimic natural ECM as this has similar tensile strength to natural ECM when cross-linked in a suitable manner and it is possible to enrich the gelatin-derived structures with other relevant proteins to enhance the homing and differentiation status of the contained cellsIn the present work we, to our best knowledge, for the first time successfully applied the SBS method and glucose-enhanced thermal crosslinking to produce 3D scaffolds from mammalian (porcine) gelatin that faithfully mimic main aspects of natural extracellular collagen matrix. Furthermore, we used these 3D scaffolds as culturing substrates for human primary hepatocytes to create 3D human liver tissue equivalents that maintained the characteristic differentiation status and main physiological properties of these cells in long term cultures. We conclude that combining the SBS-generated mammalian gelatin scaffolds with human primary hepatocytes can be used for creation of a simple and efficient platform for in vitro research and drug testing applications related to human liver.2 in the middle. The deposited material was removed from the collector fabric and heated at in an oven at 175 °C for 1 h This time is sufficient for cross linking to occur and does not cause gelatine melting or decomposition. 7 mm round punch was used to cut round pieces from the mat to fit the wells in the 96-well tissue culture plate  used in cell culture studies. The fiber samples were fixed to the bottom of the wells by placing simple polypropylene ribbon as spring adaptors on their edges.Gelatin , d-( +)-glucose and glacial acetic acid were purchased from Sigma-Aldrich. Gelatin was dissolved in 10 M aqueous acetic acid solution at about 40 °C by vigorous stirring to obtain solutions containing 20% gelatin. Glucose was then added to the mixture in 1:6 ratio to gelatin. Obtained solution was then transferred to a commercial Tamiya airbrush . Compressed air from a central line in the building was adjusted to 2 bar pressure using a generic valve and fed into the airbrush with the gas inlet and nozzle in fully open positions. The airbrush was held in hand and the fibers were collected on a nylon mesh fabric on a 40 × 40 cm frame and placed at 70 distance under a fume hood. Systematic trial and error experiments were conducted to reach the described setup that produced fairly uniform fiber material with about 300 µm thickness over several cmPrior cell culture the mats were enriched for 3 h at 4 °C with either Matrigel Matrix , laminin-111  or vitronectin  in hepatocyte culture medium. Control mats were incubated in culture medium alone. After the incubation the mats were briefly washed with phosphate buffered saline (PBS) pH 7.4 before seeding the cells.To simulate the behaviour of the SBS generated mats in an aqueous environment, a separate sample was prepared by soaking in water for 24 h and subsequent drying by critical point drying (Leica EM CPD300). Scanning electron microscope (Tescan Vega) was used to visualize the microstructural features of samples that were prior coated with 5 nm gold layer by Polaron SC7640 sputter coater. The images were obtained with Vega TC software. Fiber thickness was estimated from the captured images using ImageJ/FIJI software .5 cells were seeded into each well of 96-well plate containing a piece of mat in 100 µl Iscove's Modified Dulbecco's Medium containing 10% fetal bovine serum (both Corning) and 1% penicillin/streptomycin . Cells were cultured for 3 days with daily media changes. Next, the mats were processed for immunofluorescence microscopy as described below.Hep G2 cells were a kind gift from Dr Reet Kurg . Approximately 1 × 10® 3D Cell Viability Assay (Promega) according to the manufacturer’s instructions.Cell viability and cytocompatibility assessment were carried out using CellTiter-Glol-glutamine (Sigma-Aldrich) and 1% penicillin/streptomycin . For long-term experiments the cells were cultured for up to 24 days with media changes every second day. Approximately 2 × 105 cells were seeded into each well of 96-well plate containing a mat or on collagen I coated 24-well plates (Corning) which were overlaid with Matrigel (MG) Matrix (Corning) according to manufacturer’s instructions. The percentage of live, matrix-attached cells in culture was estimated by counting the number of detached cells at every medium renewal and at final time-point. Additionally, CellTiter-Glo 3D Cell Viability Assay (Promega) was performed to evaluate the adequacy of detached cell counting for assessing live cell numbers.Human cryopreserved hepatocytes  were cultured in serum-free hepatocyte defined growth medium (Corning), containing epidermal growth factor (10 ng/ml), 2 mM 27. Briefly, 20 days after seeding the hepatocytes 50 µM omeprazole, 20 µM rifampicin or similar volume of dimethyl sulfoxide solvent  were added to hepatocyte culture medium for 4 days with daily media replacement.CYP inducibility in cultured hepatocytes was assessed as described previouslyCYP1A2, CYP2B6 and CYP3A4 activity was measured by using P450-Glo™ CYP1A2 (#V8421), CYP2B6 (#V8321) and CYP3A4 (#V9001) Assay systems (Promega) according to manufacturer’s protocols. Results were calculated against the number of viable cells in the culture which was determined by using CellTiter-Glo 3D Cell Viability Assay (Promega) according to manufacturer’s instructions.g for 10 min at 16 °C. Following centrifugation, a colourless aqueous upper phase was transferred into a gDNA Eliminator spin column from RNeasy Plus Mini Kit (Qiagen) and RNA was further isolated with the same RNA purification kit according to the manufacturer’s protocol.For RNA extraction hepatocytes were lysed in 500 µl Trizol (Gibco) by repetitive pipetting. 100 µl chloroform was added to the lysates and the mixture was incubated 2–3 min at room temperature and centrifuged at 12,000Total RNA was reverse transcribed with High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to manufacturer’s instructions.CYP1A2, CYP2B6 and CYP3A4 qPCR was performed according to manufacturer’s protocols by using TaqMan Universal PCR Master Mix (Applied Biosystems) and FAM-MGB Taqman probes directed against CYP1A2 (Thermo Fisher #Hs00167927), CYP2B6 (Thermo Fisher #Hs03044634), CYP3A4 (Thermo Fisher #Hs00604506) and HPRT1 (Thermo Fisher #Hs02800695) as a loading control. HNF4a, Vimentin and RPLP0 qPCR was performed by using Maxima SYBR Green/ROX qPCR Master Mix reagents (Thermo Fisher Scientific) with the following primers: HNF4a forw TGGTGGACAAAGACAAGAGGAAC, rev GAGCGCATTGATGGAGGGCA, Vimentin forw GACCAGCTAACCAACGACAAAG, rev GGTGTTTTCGGCTTCCTCTCT and RPLP0 forw CTGGAGGGTGTCCGCAATGT, rev AGCAGCCACAAAGGCAGATGGAT. All qPCR reactions were carried out with a LightCycler® 480 Instrument II (Roche) and the data acquired were analysed with LightCycler® 480 Software (Roche). The results were calculated by using the ΔΔCT method against HPRT1 or RPLP0 as a loading control. The qPCR reactions were performed at least 3 times for each sample.Mats were fixed in 4% paraformaldehyde for 10 min and permeabilized in 0.2% Triton X-100 in PBS solution for 15 min. After blocking with 5% normal donkey serum (Sigma-Aldrich) for 60 min, the slides were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature. Where indicated, the actin filaments were stained with phalloidin-Alexa568 . Nuclei were counterstained with DAPI . Used antibodies are listed in Supplementary Table Human primary hepatocytes were cultured in serum-free hepatocyte defined growth medium as described in section “P < 0.05 was considered significant.Statistical significance was determined by one-way ANOVA followed by Turkey’s post-test (multiple comparisons) or Student’s t-test (two groups). 24. SEM analysis of dry mats demonstrated the placement of collagen fibers in wavy bundles that were orientated in all three spatial dimensions confirming the presence of an ECM-like 3D structure  would modulate cell growth. We cultured Hep G2 cells on either uncoated SBS mats or mats preincubated with MG, laminin-111 or vitronectin solution in tissue culture media —the marker of apoptosisres Fig. A. While res Fig. E.To study the cell attachment and maintenance dynamics we evaluated the number of cells that remained unattached and were floating in the tissue culture media 3 h after plating and assumed that the rest of the cells were attached. Although the SBS mats had a lower cell attachment percentage  the proportion of live matrix-attached cells remained essentially consistent from 4 to 24 days (Supplementary Fig. We examined the presence of two polarization markers, the junctional protein ZO1 and the transcytotic marker CD13 in human primary hepatocytes grown on uncoated or MG, laminin-111 or vitronectin–coated SBS mats; as a comparison we used MG bilayer culture. Immunofluorescence analysis showed that ZO1 is expressed at cell membranes at cell–cell contacts Fig. . ZO1 expNext, we tested whether the human primary hepatocytes grown on SBS mats were able to upregulate the expression of CYP1A2, CYP2B6 and CYP3A4 mRNA in response to known inducers of CYP transcription (Fig. To further substantiate our findings, we performed functional analysis of CYP enzyme activity of hepatocytes grown for 24 days in SBS mats or in MG bilayer Fig. . We founIn this work we demonstrate that the SBS method can be successfully utilized to produce 3D glucose-gelatin preforms that are excellent culturing substrates for human primary hepatocytes and enable the creation of 3D human liver tissue equivalents that recapitulate the main aspects of human functional liver tissue. This novel 3D hepatocyte culture model was shown to be superior to the most commonly used alternative to hepatocyte culture—MG-collagen bilayer—particularly in long term culture experiments where the SBS mats supported the maintenance of hepatocyte differentiation in serum-free culture medium for more than 3 weeks.26. Here we report, for the first time, the creation of 3D ECM-like mats from porcine gelatin by utilizing SBS method. The advantages of using porcine gelatin over the fish equivalent are its more similar composition to human collagen matrix, better availability and lower cost. An advantage of our SBS mats over natural matrices such as decellularized tissue is that the mats consist of thicker threads with increased porosity. As this artificial matrix is less dense than decellularized liver, it facilitates effective cell penetration of cells into the artificial ECM in culture conditions. Nevertheless, the microstructure of SBS mats was shown to be very similar to a decellularized liver matrix in 3D fiber orientation and microstructure. Compared to most commonly used pseudo-3D hepatocyte culture model where the cells obtain a relatively flat morphology between collagen-MG layers, the fibrous structure of SBS mats creates a more natural extracellular environment and cells can spread in 3D.Although SBS is a very simple method for producing fine fibers from various materials, finding suitable parameters for producing continuous and uniform fibers is challenging. We used relatively long nozzle-to-collector distance for fiber production, which leaves sufficient time for the acetic acid and water to evaporate before deposition of the formed fibers. Based on our prior testing with various gelatins, we found that lower bloom gelatins are more suitable for electrospinning by SBS. Previous studies applied SBS to generate 3D nonwovens from fish gelatin while the generation of similar materials from porcine gelatin was unsuccessful30. Secreted ECM component vitronectin can be used as a substrate to culture many types of primary cells including embryonic stem cells31 and we have previously shown that it accumulates in damaged human and mouse livers32. However, the differences between the ability to support the metabolic activity and differentiation status of primary liver cells were generally marginal as also non-enriched SBS mats without any additional components were proven to be an excellent substrate for the maintenance and growth of both immortalized and primary hepatocytes.Collagens are the main components of fibrous matrices in natural ECMs; however, biological matrices contain several proteins that can have a substantial impact on cell growth and differentiation. Laminins have been studied for decades, mainly because of their role in basal membrane assembly, embryogenesis and potential utilization as a substrate for tissue engineering33. Previous studies with in vitro cultured hepatocytes have shown that 2D cultured hepatocytes lose or show significantly altered expression of cell polarization markers33. Here we show that SBS mats support the expression of hepatocyte polarization markers tight junction component ZO1 and the transcytotic marker CD13. This was particularly evident in long term cultures where the 3D substrate was more efficient in supporting hepatocyte polarization and differentiation when compared to the most often utilized primary hepatocyte cultivating system—the MG bilayer.Hepatocyte polarity is essential for liver structure and function. In vitro studies with murine hepatocytes have indicated that cell polarity is directly correlated with the maintenance of cell differentiation34. Although very relevant for studying hepatotoxicity such systems lack the mechanical supportive mesh that has been shown to play a major role in regulating hepatocyte behaviour as substrate stiffness has been demonstrated to be an important factor in tissue development, differentiation and disease37. The extent to which the lack of supportive structures affects the reactivity of primary hepatocytes to external stimuli is yet to be determined in future head-head comparisons.Recently, 3D spheroid-based cytotoxic assays have emerged as a promising approach for DILI evaluation. A recent multicenter study showed that such systems are superior to conventional MG bilayer cultures in respect of proteome stability and displayed increased sensitivity in long-term repeated exposure experimentsIn conclusion, our data suggest that the gelatin mats generated via SBS are well-suited and easy to use alternatives for long term hepatocyte drug screening studies, as the 3D substrate allows maintenance of hepatocyte-specific functions stable over long time periods. Furthermore, the mats can be easily inserted into the wells of 96-well plates, the cells in the mats are easily accessible to various chemicals including cell lysis buffers and there is no need for elimination of the MG layer prior cell lysis for further analysis. The 3D liver cultivation method is potentially more easily maintained by robotic systems and is anticipated to be well compatible with high-throughput screening methods Fig. .Figure 8Supplementary Information.


Mylodon darwinii was an opportunistic omnivore. This direct evidence of omnivory in an ancient sloth requires reevaluation of the ecological structure of South American Cenozoic mammalian communities, as sloths represented a major component of these ecosystems across the past 34 Myr. Furthermore, by analyzing modern mammals with known diets, we provide a basis for reliable interpretation of nitrogen isotopes of amino acids of fossils. We argue that a widely used equation to determine trophic position is unnecessary, and that the relative isotopic values of the amino acids glutamate and phenylalanine alone permit reliable reconstructions of trophic positions of extant and extinct mammals.Fossil sloths are regarded as obligate herbivores for reasons including peculiarities of their craniodental morphology and that all living sloths feed exclusively on plants. We challenge this view based on isotopic analyses of nitrogen of specific amino acids, which show that Darwin’s ground sloth  Little is known, however, about the ecological structure of mammalian communities before the megafaunal extinctions of the last Pleistocene ice age, when more than 80% of mammals above 40 kg became extinct2. Characterizing the paleoecology of South American (SA) fossil mammals is particularly challenging, because those communities bear little resemblance to modern or fossil guilds from other continents due to marked taxonomic and phylogenetic disparities and unusually large numbers of entirely extinct clades3. Another aspect of this perceived uniqueness is the depauperate abundance and diversity of mammalian carnivores throughout the Cenozoic. Lack of placental mammalian carnivores is particularly puzzling for the Pleistocene because all large marsupials  and non-mammalian predators such as terrestrial crocodiles  and large flightless “terror birds” , which likely maintained terrestrial food web energy transfer balances in the Tertiary, were already extinct by this epoch. Allometric functions relating population densities to body mass indicate that Pleistocene SA mammalian communities were ecologically imbalanced because the herbivore biomass markedly surpassed the energetic requirements of carnivores4. It has been further suggested that Pleistocene ecosystems could not have supported the plant biomass demands of all the presumed large herbivores, which implies that secondary consumers disguised as herbivores must exist in the fossil record but have not been recognized as such4. Because of their high taxonomic diversity and simple yet potentially functionally versatile dentitions, fossil sloths  have been identified as the most likely candidates to have occupied scavenger niches5. All modern sloth species, however, are obligate tropical arboreal herbivores, with extreme feeding specializations involving the exploitation of food of low nutritional quality, with the extra physiological challenges entailed by the poor digestibility of plant allelochemicals6. The traditional consensus that fossil sloths were also obligate herbivores like their modern relatives is based mostly on craniodental morphology analyses and a presumption of shared, phylogenetically conserved dietary adaptation across the group. Dental structures and jaw biomechanics of sloths, however, do not preclude the ingestion of foods that require little or no preparation, as in certain kinds of scavenging . Furthermore, their extremely high diversity and incredibly broad range of body sizes, geographic distributions, and inferred habitats, including even a marine sloth7, suggest that some of the hundreds of known species of fossil sloths may have been much more versatile ecologically than traditionally thought. Sloths  comprised a substantial proportion of South American mammalian diversity until as recently as 10,000 years ago8. Reliably determining the trophic relationships of xenarthrans is thus crucial for understanding the evolution of Neotropical mammalian biodiversity, as well as the interplay between resource availability, ecological functions among xenarthrans, and competition within and across mammalian clades in South America over the Cenozoic.Home to more than half of Earth’s land biota, South America contains the highest diversity of extant terrestrial mammals of any continent9. Stable isotope analyses therefore offer a rare route to directly document the consumed diet itself, rather than relying on indirect inferences based on morphological proxies, representing a particularly useful and potentially more reliable method to be applied to fossils. Traditionally, nitrogen stable isotope analyses (δ15N) of bulk tissue are used to evaluate trophic levels due to an empirically documented increase of 3–5‰ in δ15N values per trophic level change11. However, substantial variation in δ15N values has also been observed within trophic guilds12, indicating that the use of δ15N values alone can be a misleading proxy to identify trophic levels. Indeed, interpretation of bulk tissue δ15N values often fails to acknowledge variations in baseline δ15N values within an ecosystem13, which can greatly differ depending on the isotopically different types of inorganic nitrogen sources available for the primary producers , the way nitrogen was obtained (direct uptake of nitrogen from soil or through symbiotic microbes), and where nitrogen was assimilated by the plant .Isotopic analyses are now widely applied in ecological and paleoecological studies. These techniques rely on two underlying premises. First, that isotopic values of any given chemical element differ across diverse reservoirs in the biosphere, yielding isotopically varied foods and waters. Second, because an organism’s body tissues are synthesized from the food and water they ingest, they are isotopically linked to those sources in a predictable way15N values of specific amino acids, overcomes the issue of unknown or variable baseline δ15N values because it records both baseline  and trophic information in the same organism18. Indeed, studies have shown that certain amino acids (AAs) experience little change in their δ15N values from ingested diet to consumer body tissues, whereas others differ significantly15. The explanation behind this observation relies on whether or not the metabolic pathways of amino acids are engaged in transamination or deamination reactions (involving the breakdown of C-N bonds) and the extent to which amino acids exchange their amino-nitrogen with the overall metabolic pool of nitrogen19. Amino acids that readily exchange their amino-nitrogen, thereby experiencing large isotopic fractionations, have been termed “trophic” amino acids, whereas those implicated in metabolic processes that involve little or no breakdown or formation of C-N bonds, and thus are similar to baseline δ15N values, are known as “source” amino acids19. The higher the trophic level of an organism, the more metabolic cycles their amino-nitrogen will be involved in, and therefore the difference between the δ15N values of trophic versus source amino acids will progressively increase. Among “trophic” AAs, glutamic acid  usually experiences a large increase in its δ15N values with each trophic level transfer20, as its central role in nitrogen metabolism of heterotrophs involves transamination reactions21. In contrast, among source amino acids, phenylalanine (Phe) has been observed to experience minimal increase in its δ15N values per each trophic level, as its main initial metabolic step, the formation of tyrosine, does not involve breaking C–N bonds22. Constancy of these distinct patterns between Glx and Phe across organisms makes them the two canonical “trophic” and “source” amino acids23. Indeed, the most popular equation 20 to calculate trophic positions (hereafter referred to as “TP Eq”) uses the δ15N values of those two amino acids, although a few studies have instead proposed a combination of multiple trophic and source amino acids to calculate trophic levels25.In contrast, amino acid compound-specific isotope analysis (AACSIA), a more recent technique that measures and evaluates the δ26), application of the AACSIA TP Eq is not without caveats, as this equation relies on constants  that have not been thoroughly evaluated in terrestrial ecosystems. Indeed, the equation relies on the assumption that the β value  and the TDF  are both constant across all organisms within an ecosystem. For terrestrial ecosystems, β values of − 8.4‰ and − 0.4‰ are considered representative of C3 and C4 plant-dominated ecosystems, respectively27; however, a recent meta-analysis of β values, demonstrated considerably more variation than currently acknowledged28. On the other hand, a standard TDF value of 7.6‰ has been used to calculate trophic positions of all consumers, whether terrestrial or aquatic, modern or extinct, vertebrate or invertebrate, yet its assumption of constancy has only been tested on marine invertebrates, fishes, birds, insects, and heterotrophic bacteria, where it is also known to vary31. The only prior controlled experimental study we are aware of on a terrestrial mammal, Bos taurus32, showed different β (− 4.5‰) and TDF (4‰) values than those used in the TP Eq, suggesting that these “universal” values cannot be applied uniformly across food webs and ecosystems.Although widely applied in archaeological, anthropological, and paleontological research . We argue instead that this equation is not necessary in any case, and that the δ15N values of glutamate (δ15NGlx) and phenylalanine (δ15NPhe) alone permit reliable determinations of trophic positions of mammals. To document this, we first tested the predictive power of this relationship in modern mammalian species under different, known feeding regimes or diets (in both controlled and wild environments), to provide a basis for reliable interpretation of δ15N AACSIA of fossil samples. We then used this proxy to test the hypothesis of carnivory and consumption of proteins of animal origin in the targeted fossil sloths, Mylodon darwinii and Nothrotheriops shastensis.For these reasons, we posit that the TP Eq cannot be used to confidently calculate the trophic position of mammals  and Nothrotheriops shastensis (δ15NPhe = 17.7‰). The greatest intraspecific δ15NPhe variation (8.4‰ span) is observed in Choloepus , with hair keratin δ15NPhe values ranging from 3.2 to 11.6‰. Intraspecific variation in δ15NGlx values is smaller than that for δ15NPhe, with Choloepus and Mylodon showing the greatest variation among individuals (2.5‰). Inter-specific variation in δ15NGlx spans 14‰, with the extreme values also bracketed by the fossil sloths Mylodon (median δ15NGlx = 5.6‰) and Nothrotheriops (δ15NGlx = 19.6‰).The seven xenarthran species included in this study (sloths [extant and extinct] and anteaters) display substantial intra- and inter-specific variation in their δ15N values of bulk dietary samples for zoo specimens are bracketed by the results for the two living sloth species, the mixed plant/animal diet of Choloepus  and the rubber plant Ficus elastica, Bradypus’s monospecific diet  for Ficus elastica is − 7.6‰ , ranging from − 1‰ for the diet of the pygmy anteater Cyclopes to 0.8‰ for that of Choloepus. The dietary Δδ15NGlx − δ15NPhe result for Myrmecophaga and Tamandua (both species fed the same diet at the zoo) was − 0.4‰  than that of omnivores  = − 10.2, p < 0.001) and marine consumers  (Table 15NGlx − δ15NPhe (2.7‰) is bracketed by two artiodactyls, the buffalo Syncerus caffer (− 2.3‰) and sheep Ovis aries , bracketed by humans with high plant content in their diets  and the common genet Genetta genetta (10.7‰). The range of variation in Δδ15NGlx − δ15NPhe in marine consumers also is large (11.6‰), ranging from 12.1‰ in a population of Homo sapiens from South Africa to 23.7‰ in the fish-eating bat Myotis vivesi. The ∆δ15NGlx − δ15NPhe values in marine consumers are significantly higher  than for any terrestrial feeding guild . Data for terrestrial carnivores is limited to three species with ∆δ15NGlx − δ15NPhe values lower than for all marine consumers: the hyena Crocuta crocuta (∆δ15NGlx − δ15NPhe = 11.2‰), the fringe-lipped bat Trachops cirrhosus (∆δ15NGlx − δ15NPhe = 10.2‰), and the spectral bat Vampyrum spectrum (∆δ15NGlx − δ15NPhe = 11.5‰). Fossil sloths were not placed in any dietary category a priori. The Δδ15NGlx − δ15NPhe values for Mylodon overlap those of taxa within the omnivore category (median = 2.7‰) while Nothrotheriops best fit values in the herbivore category  Table , S5. Amo15NGlx and δ15NPhe for 59 mammalian species yielded strong correlations between these two AAs for herbivores , marine consumers , and wild omnivores  . Our results show that there is a significant effect of diet on δ15NPhe, but no significant interaction  = 0.36, p = 0.78; Table 15NGlx, observed as differences in intercepts among the regression lines  = 116.5, p < 0.01; Table Ordinary least square (OLS) analyses of δdf) Fig. . The ∆δ1nge Fig. . Values nge Fig. , when ex15NGlx and δ15NPhe remained strong (p < 0.001), but R2 decreased from 0.96 to 0.86. When only Nothrotheriops is considered as an herbivore, however, the correlation between δ15NGlx and δ15NPhe for herbivores is as strong  as when only modern taxa are analyzed . However, results from robust regression and diagnostic analyses of fossil and extant taxa together identified Nothrotheriops  as influential outliers for the omnivorous correlation (Cook’s D > 4/n). The exclusion of these two outliers, and inclusion of Mylodon as an omnivore, greatly improved the strength of the omnivore correlation  analyses and robust regressions were carried out classifying fossil sloths either as herbivores or as omnivores, to evaluate the best dietary category fit for these species relative to known dietary correlations in extant taxa Fig. . Fossil zed Fig. . When bo15NGlx and δ15NPhe values alone , which are therefore preferable to relying on an equation with poorly constrained constants introducing uncertainties. Indeed, the TP Eq often failed to place captive and wild mammalian omnivores in their correct trophic positions , which introduces uncertainties that are not accounted for when using single constant β and TDF factors . Low ∆δ15NGlx − δ15NPhe values for the captive anteaters (− 2.2 and − 0.3 for Tamandua and Cyclopes respectively) are likely due to the fact that these individuals are fed diets containing protein supplements with higher δ15NPhe values than expected for secondary consumers, consequently blurring the ∆δ15NGlx − δ15NPhe values naturally observed in other species in that trophic level. The weaker correlation between δ15NGlx and δ15NPhe for wild omnivores compared to herbivores or marine consumers is likely due to the fact that this feeding category includes mammals incorporating widely different proportions of distinct source foods  in their diets.Our results document that we can accurately reconstruct the trophic position of mammal species from δ15NGlx − δ15NPhe values  match those of modern omnivores, such as the American marten. In contrast, although the ∆δ15NGlx − δ15NPhe value obtained for the fossil sloth Nothrotheriops (1.9) fit that of some low-meat consuming omnivores , it was found as an outlier in the omnivore regression and thus is conservatively interpreted as an herbivore  is likely due to the arid conditions in which this animal lived, as plant δ15N values increase with decreasing water availability38 and the taxonomic composition of the plants identified in its diet (from dung) consistently correspond to desert taxa still present in the area40.Based on the OLS analysis and their ∆δues Fig. , Mylodonre Table . There i15NGlx and δ15NPhe values  is significantly different in plants performing C3 (~ − 8.4‰) versus C4 (~ 0.4‰) photosynthesis20, although substantial variation also exists among C3 plants  depending on the degree of vascularization28 and amount of lignin produced by the plant species27. Mammalian herbivores, whether feeding on C3 or C4 resources, however, do not differ significantly in ∆δ15NGlx − δ15NPhe values . Thus, the almost 1:1 correlation between these two amino acids in terrestrial herbivores is independent of the type of plant consumed  and data on wild mammals feeding on C3 or C4 plants (SI Dataset) both corroborate this observation.The offset between δ15NGlx − δ15NPhe and linear relationships between δ15NGlx and δ15NPhe in extant mammals) suggests that while the Shasta ground sloth Nothrotheriops shastensis was likely an obligate herbivore, Darwin’s sloth Mylodon darwinii was not, instead occupying a higher trophic position. AA δ15N values indicates that the feeding ecology of the fossil sloth Mylodon darwinii best fits within the mixed-feeder/omnivore dietary category, with a degree of meat-eating omnivory comparable to that of the extant American marten. Further, the strength of the correlation between δ15NGlx − δ15NPhe for modern herbivores, regardless of its ecosystem  or the type of vegetation consumed  makes it extremely unlikely for Mylodon to have been an obligate herbivore.Assessing the results of all analyses together (∆δNothrotheriops shastensis coprolites (from Rampart and Gypsum Caves in the US41), revealed a variety of plant species but no evidence for a non-herbivorous diet. Mylodon darwinii dung has been subject to ancient DNA studies aimed to explore evolutionary relationships of this species43, but only one study attempted to characterize its diet from dung44. This macro- and micromorphological study of dung content found Mylodon’s diet to be composed mostly of grasses and sedges, concluding that it most likely was a grazer44, in contrast to morphological interpretations45. The absence of hardly digestible material  in any Mylodon’s coprolites shows that this species could not have been a strict carnivore (or a hyena-type scavenger) but does not exclude an opportunistic omnivore behavior because other animal protein-rich dietary items  are rapidly assimilated after ingestion. In this case, fecal components will be biased towards plant dietary items because cellulose is difficult to digest. Indeed, macro- and micromorphological analyses of feces do not allow precise identification of an animal’s full dietary composition, as has been exhaustively demonstrated in archaeological studies of human coprolites46.Macroscopic analysis and DNA sequences extracted from 47. Hindgut fermenting grazers, such as horses, have greater relative OSAs than do cows (ruminant foregut fermenters); by being more efficient at orally reducing the particle size of the ingested food, horses compensate for shorter digesta retention times than ruminants48. Compared to modern herbivores, fossil sloths (with the sole exception of Megatherium americanum) had smaller occlusal areas than expected for their body masses49, indicating that they had a lower capacity for food processing than do extant herbivores of similar sizes.Anatomical studies on modern herbivores, on the other hand, showed a strong correlation of dental occlusal surface area (OSA) with efficiency and capacity of food processing, digestive physiology, and food qualityMylodon darwinii) had the lowest relative OSA values, reflecting poor oral food processing efficiency. These results were interpreted as likely due to lower energetic requirements of fossil sloths, suggesting in turn that fossil sloths might have required lower food intake than other mammals of similar sizes49. Although low basal metabolic rates might partially account for these seemingly incongruent digestive physiology inferences, there is no compelling evidence for unusually low metabolic rates in fossil sloth species. McNab50 proposed that Mylodon and Nothrotheriops would have had basal metabolic rates of 50% of that expected from their body mass. Those estimations are not convincing, however, because they were based on the length of hair strands and fur thickness, while energy transfer along hairs also depends on other traits such as hair diameter and number and arrangement of hair in the fur51. Even assuming those estimations were roughly correct, Mylodon is thought to have faced ambient temperatures well below its thermoneutrality50, and therefore it would have needed to increase its metabolism to maintain a constant body temperature in cooler conditions. Furthermore, a fact considered crucial in explaining the low metabolic rates in modern xenarthrans is their food preferences, including plant items with extremely low energy content and allelochemical defenses against herbivory50.Of all fossil sloth species analyzed, mylodontids . Foregut fermenters tend to have smaller large intestines  than hindgut fermenters, which results in reduced colonic water reabsorption52. The foregut of Mylodon would have represented ~ 20%− 24% of its body weight  which is comparable to that observed in modern hippos. However, high water loss resulting from a reduced caecum and colon in the hippos is hypothetically overcome by their amphibious lifestyles52. An amphibious lifestyle for Mylodon, however, has never been proposed, nor would one be consistent with habitats in which they are known to have lived in. For other mylodontids, such as the giant ground sloth Lestodon (estimated weight > 4 tons), the issue of colonic water reabsorption is even more extreme because their foregut would have represented almost 40% of their total weight .That fossil sloths were foregut fermenters is highly likely because extant sloths, phylogenetically bracketing almost the entire diversity of fossil sloths, are foregut fermentersMylodon in particular, would be ingestion of a higher quality of food. Indeed, if Mylodon was an opportunistic scavenger and incorporated, even if only sporadically, meat or other animal dietary elements, then its relatively small oral food processing capacity would be more congruent with the apparent physiological constraints imposed by its body mass and foregut fermentation strategy. The energetic requirements of Mylodon, with its need for higher metabolic rates due to air temperatures below its thermoneutrality, also are more consistent with omnivory than obligate herbivory. Our δ15N AACSIA results support this interpretation, as they clearly indicate that Mylodon was not an obligate herbivore, but instead was an omnivore showing consumption of animal proteins in proportions comparable to the American marten and other mixed feeders.An alternative explanation for these results on fossil sloths, and 15NGlx and δ15NPhe values in modern mammals with known diets, and from distinct ecosystems, digestive physiologies, and phylogenetic relationships. These relationships indicate that analyses of δ15NGlx and δ15NPhe alone are preferable to inferences of trophic position using an equation (TP Eq) with inaccurate or poorly documented assumptions, and permit reliable reconstruction of diets in fossil taxa from terrestrial ecosystems. More δ15NGlx and δ15NPhe data on modern secondary consumers may permit differentiation of insect-eating from meat-eating omnivores, as well as from terrestrial hypercarnivores, by teasing apart patterns of interspecific variation in δ15NGlx and δ15NPhe relative to particular food sources for omnivores.Increased sampling and a new analytical framework for AACSIA data document the high predictive power of relationships between δ15NGlx and δ15NPhe, enable us to conclude that while Nothrotheriops was likely an obligate herbivore, Mylodon was not. Our data, from direct isotopic evidence of the food consumed, clearly indicate that Mylodon was not an exclusive herbivore, as commonly presumed, but rather that its feeding behavior better fits that of an omnivore, consuming plant material but sometimes also incorporating items of animal origin in its diet. Although the craniodental and skeletal morphology of sloths does not support them being active predators, an opportunistic scavenging behavior for Mylodon would be in agreement with our results obtained via AACSIA. Our conclusions, from direct isotopic evidence of the food consumed, are also supported by studies on masticatory muscle structure reconstructions, biomechanics, and morphogeometry for Mylodon, indicating that it would have been inefficient at processing food and at a disadvantage (in terms of resource competition) relative to other mammalian herbivores49.The newly established relationships between dietary category and δ53. Furthermore, as large herbivores greatly impact the vegetation structure, soil moisture, and the carbon cycle of an ecosystem54, removal of some fossil ground sloth taxa from the continental herbivore guild would change previous estimations of the net primary production required to sustain a given number of megaherbivores, as well as the type of vegetation dominating the biome floor . This would be the case in particular if, in addition to Mylodon, other fossil sloth species also had more versatile feeding behaviors than traditionally thought and would help explain the perplexing and long-assumed paucity of mammalian secondary consumers during the Cenozoic in South America.These results, providing the first direct evidence of omnivory in an ancient sloth species, demands reevaluation of the entire ecological structure of mammalian communities in Cenozoic South America, as sloths represented a major component of these ecosystems across the past 34 million years. For instance, in the well-studied Pleistocene Lujanian fauna in Argentina, xenarthrans would have represented almost half of the mammalian herbivore guild, with sloths alone representing almost 20%p = 0.005), likely because the omnivore and marine consumers categories both include a broad range of diets (see main text), but differences between groups also were significant under an unequal variance t-test  of the isotopic signal recorded in the modern sloth with a monospecific diet  and in the fossil species (Mylodon and Nothrotheriops) with unknown diets, hair strands for these three species were sectioned in 0.5 or 1 cm and analyzed for bulk δ15N prior to AACSIA , the proximal 1 cm  were homogenized and analyzed for δ15N (bulk and for AACSIA). For Cyclopes, possessing very thin and short hair, entire hair strands were homogenized for both bulk and AACSIA δ15N. Dietary items for the species with mixed diets were isolated and analyzed for bulk δ15N. Mixed diets were homogenized in the proportion of each individual dietary item given to the individual animals at the zoo and then analyzed for δ15N AACSIA. Variability of the isotopic values for the two modern sloths’ diets in the zoo was accounted for by analyzing samples on a weekly, monthly, and yearly basis  δSIA Fig. . If signtio Fig. . For alls Tables –S10.15N value of the proximal first centimeter was higher than the remaining length of the hair for both species, so this first centimeter was conservatively excluded for AACSIA  were found between Mylodon darwinii’s hair segments 3–4 plus 7–10 (mean = 2‰ ± 0.1), and segments 2 plus 5–6 (mean = 3.5‰ ± 0.6). Therefore, we homogenized segments 3–4 plus 7–10, and 2 plus 5–6, and analyzed those for AACSIA δ15N , the δ15N values of the remaining hair segments of Nothrotheriops are extremely consistent and do not differ significantly from each other , glycine (Gly), isoleucine (Iso), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr), and valine . Additionally, the terminal amide groups in glutamine (Gln) and aspartamine (Asn) are cleaved during the chemical isolation of amino acids, the result being the conversion of these amino acids to glutamic acid (Glu) and aspartic acid (Asp), respectively. Thus, we measured the isotope values of combined Glu + Gln (termed Glx) and Asn + Asp (termed Asx). Internal reference compounds, L-2-Aminoadipic acid (AAA) and L-(+)-Norleucine (Nor) of known nitrogen isotopic composition, were co-injected with samples and used to determine accuracy and precision. Between triplicate runs of each sample, a suite of these 14 amino acids of known isotopic composition were analyzed; AAA and Nor were also co-injected with these amino acids. For isotopic correction of unknown amino acids, a linear correction is derived from the amino acid suites run immediately before and after the triplicate sample analysis and applied to measured isotope ratios. All amino acids were analyzed in triplicate and isotopic values are reported in δ-notation relative to atmospheric N2.Amino acids were chemically isolated and purified from sample material prior to derivatization and esterification in preparation for GC-IRMS analysis, as described inThe protocols used to collect hair samples from museum/zoo specimens conform to the legal requirements and institutional guidelines of the Peruvian and US Governments . All applicable international, national, and/or institutional sampling methods were carried out in accordance with relevant guidelines and regulations. When appropriate, this research adheres to the ARRIVE guidelines.Supplementary Information 1.Supplementary Information 2.


Few-layer ZrTe5 is anticipatedto exhibit the quantum spin Hall effect due to topological statesinside the band gap, but sample degradation inflicted by ambient conditionsand processing has so far hampered the fabrication of high qualitydevices. The quantum Hall effect (QHE), serving as the litmus testfor 2D systems to be considered of high quality, has not been observedso far. Only a 3D variant on bulk was reported. Here, we succeededin preserving the intrinsic properties of thin films lifting the carriermobility to ∼3500 cm2 V–1 s–1, sufficient to observe the integer QHE and a bulkband gap related zero-energy state. The magneto-transport resultsoffer evidence for the gapless topological states within this gap.Topological matterplays a central role in today’s condensedmatter research. Zirconium pentatelluride (ZrTe It is an orthorhombic layered materialbelonging to the Cmcm space group. In the monolayerform, this should result in a quantum spin Hall insulator (QSHI)5 with a bulk band gap up to 0.1 eV.12 Because this system is located at the boundary of various topologicalphases,3 even moderate disturbances inthe lattice constant or carrier density and possibly also disorderdetails seem to cause significantly different experimental observations.The latter have instigated an intense debate on the nontrivial bandtopology of bulk ZrTe5 and whether it should be regardedas a Dirac semimetal,14 a strong,15 or a weak topological insulator.17 When moving from bulk to thin films, additional complexity is addedbecause of the poor stability of ZrTe5 when exposed toambient conditions and the increased sensitivity to the surface anddisorder. Complex transport behavior as well as low mobility havebeen observed in thin films, whereas the intrinsic physics remainedelusive.21 Here, significant advances in observing the intrinsic magneto-transportproperties of thin ZrTe5 are reported by fabricating andmeasuring devices without any exposure to ambient air. Clean singleband transport, incipient quantum Hall effect behavior, and evidencefor a bulk energy gap in this promising quantum spin Hall insulatormaterial are the outcome of these experiments.ZrTe5 synthesizedusing either the tellurium flux method or chemicalvapor transport.22 Crystals are needlelike and deviceswere fabricated by transferring mechanically exfoliated thin filmson top of buried contacts inside atomically flat hBN to achieve thebest sample quality ). The entire fabrication and measurement procedure avoidedexposure to air, solvents, as well as elevated temperatures, all ofwhich cause quality degradation . A sample schematic is illustrated in 5 layer is shown in panel SI.Our studies wereperformed on high-quality single crystals of ZrTexx, and Hall resistivity, ρxy, between 20 and 240 K. A pronounced peak is observed in ρxx below 200 K. It is reminiscent of the chargeneutrality peak in graphene24 and is attributed tothe transition from majority hole transport in the valence band tomajority electron transport in the conduction band as confirmed inρxy. However, above ∼220K the Hall resistivity remains positive. Transport is apparently holedominated at all back-gate voltages and the chemical potential residesdeep inside the valence band. This behavior heralds the temperature-inducedLifshitz transition previously reported in the literature18 . xx in the -plane and confirms this interpretation.Two distinct regions can be identified separated by a fairly sharpboundary in the low-temperature regime that marks the transition fromhole to electron transport. The transition is less well-defined athigh temperatures where thermal excitation of charge carriers playsa role. Three criteria were adopted to determine this transition fromhole to electron transport with less uncertainty. The Hall resistivitycrossing through zero at a given T and Vg signals the charge inversion. Errors due to offset voltageswere minimized by symmetrizing ρxy. Exemplary raw data are shown in Figure S4b of the SI. The crossing points are included in xx data recorded at fixed temperature canbe used as criterion . Finally, the previously reported anomalous resistivity peak inthe temperature dependence of ρxx is also a manifestation of charge carrier inversion that accompaniesthe temperature-induced Lifshitz transition of the Fermi surface16 . At low temperature, all three criteria yield nearly the same transitionpoint. However, data points drift apart for T >∼200K and Vg > ∼20 V. Below 100K,the gate voltage where the transition occurs remains nearly fixed,yet it changes rapidly at higher temperature.13 Forthe sake of completeness, we note that the carrier density itselfis not a good criterion as it changes rapidly with the temperature13 and gate voltage . The electron density can be tuned up to 1012 cm–2, which is 1 order of magnitude larger than the sheetdensity in bulk samples which varies between 1010 and 1011 cm–2.13 This exceptionaldensity tunability allows us to map this Lifshitz transition diagramacross a much wider range. It also offers the opportunity to studytransport behavior across this entire density range within a singlesystem, which is not possible for bulk systems.Figure S5b, Supporting Information), using the expression25xx is the sheet conductivity, Vg isthegate voltage, and e is theelementary charge. For our back-gate displacedfrom the ZrTe5 by a 300 nm thick SiO2 layerand a hBN flake, a fit to gate-dependent carrier density data fromHall measurements yields Cg = 0.970 F/cm2 . The mobilitiesare shown in 2 V–1 s–1 is achieved at low temperature, whereas the electronmobility is much smaller (∼1000 cm2 V–1 s–1). This suggests an asymmetry in the band structure.16The field effectmobility for holes and electrons is obtained byreplotting the data of 5 (∼105 cm2V–1s–1),13 QHE features at integerLandau level filling ν can be observed ) and Hall conductivity (σxy =−ρxy/(ρxxρyy + ρxy2)) instead (20 (α= ρyy/ρxx) should be taken into consideration when calculating the conductivitytensor elements for ZrTe5. Even if so, such an anisotropycorrection has no influence on the main conclusion . xy reaches ∼1.068 e2/h, close to the fully quantizedvalue of e2/h. The plateauis also accompanied by a deep minimum in σxx (1.67 × 10–7 S ∼ 0.043 e2/h). At filling ν =6, σxx also exhibits a strong minimumand at σxy a plateau starts to develop.Weaker QHE features are available at ν = 2 and 4. This QHE isdistinct from the previously reported 3D QHE on bulk ZrTe5 as the latter Hall effect requires a gap opening in the third directionbelieved to be mediated by a transition to a charge density wave state.4 Because the effect occurs in the bulk, it is far less vulnerablecompared to the reduced dimensionality needed here.Although these mobilities are far away from those obtainedon bulkZrTeobserved 2. In ear instead 2b. Note xy centered aroundzero filling, while at the sametime ρxx has increased significantly.These are the signatures of a true insulating ground state. A colormap of ρxx across the -plane is displayed in B-dependence of ρxx and ρxy at a fixed back gate voltage of Vg = −35 V are illustrated in 16 Lower temperature is required to resolve the field-induced Zeemansplitting due to substantial disorder broadening . We note that the QHE in 21 They may be the result of additionalscattering. For thicker samples where surface inhomogeneities playa lesser role, Shubnikov–de Haas (SdH) oscillations can alsobe observed for electrons .Particularly interestingis the plateau in σB-periodic oscillations can bedescribed by the Lifshitz–Kosevich formula13RT, RD, and RS are dampingfactors accounting for phase smearing due to temperature, disorderscattering, and spin splitting, respectively. The maximal Fermi surfacesection perpendicular to the B-field is denoted as BF and γ is the Berry phase.24Rxx ata fixed gate voltage of Vg = −35V as a function of 1/B for different temperatures.A smooth background was subtracted from the raw magneto-resistancedata to obtain ΔRxx and improve the visibility of the oscillations. Arrows mark integerfillings where minima are expected. Minima due to the Zeeman splittingare resolved only in traces recorded at the lowest temperatures. Afast Fourier transform of T) equals kB isthe Boltzmann constant, ℏ is the reduced Planckconstant, and the cyclotron mass m* is a geometricalaverage of the effective masses in the plane. Some fits of the T-dependence of B-fieldsare illustrated in Vg = −35 V. Theyyield a hole cyclotron mass m* of about 0.085 ±0.01 me, where me is the free electron mass. This cyclotron mass is significantlylarger than the value of ∼0.02 me reported for bulk.13m* extracted fromthe T-dependent analysis of the Shubnikov–deHaas oscillations for different densities revealing a strong dependence.The effective mass is highest for large hole density and graduallydrops with decreasing density. Scattering is also responsible fora B-dependent damping of the Shubnikov–deHaas oscillations. It is taken care of in D is equal to RxxB sinh λ(T)] as a functionof 1/B the quantum lifetime τq canbe determined . The large Zeeman splitting further complicates the picture asit can distort the phase of the oscillations. In bulk systems,13 low magnetic field data  were chosen to avoidthe Zeeman splitting related phase shift in the oscillations. It ispossible to estimate the g-factor through the thirddamping factor 13 , a g-factor of 11.2 ±2.8 is obtained. Forbulk ZrTe5, this g-factor can even beas large as 15.8 or 21.3.14 An estimate of theratio of the cyclotron energy . These topological surface states, responsible forthe quantum spin Hall state, are however easily destroyed when timereversal symmetry gets broken through the application of a large enough B-field. The sample is then expected to turn into an insulator.If instead we are dealing with a 3D Dirac semimetal, then there isno bulk gap and we would not expect to observe the v = 0 ground state.To determine the gap size, the temperature-dependenceof ρot28 of  4c. The g5.3 ARPES and STM measurements performed on the surface ofZrTe5 bulk material confirmed a bulk gap with a value of80–100 meV.11 However, subsequent theoreticalwork as well as high-resolution spectroscopy studies suggested thatthe energy gap is strongly temperature dependent with a maximum ofabout 30 meV.32 Recent optical spectroscopy measurementseven obtained a gap value as small as 6 meV.33 These discrepancies between different reports raise controversyabout the actual gap value. Part of the problem is no doubt that experimentalstudies performed on actual monolayers or ultrathin films are lacking.Despite these discrepancies, our work confirms the existence of aband gap in magneto-transport. Taken altogether our experimental observationsseem to exclude that the ZrTe5 sample is a 3D Dirac semimetal,while it can be either a weak or strong topological insulator. Furtherprogress in sample quality will likely turn thin ZrTe5-basedfield effect devices into a useful platform to investigate topologicalphase transitions and search for the quantum spin Hall state in thishighly tunable system.The gap value we report here is much smallerthan previously reportedvalues. The first theoretical work predicted a quantum spin Hall gapas large as 100 meV in monolayer ZrTe