Hot spring oases in the periglacial desert as the Last Glacial Maximum refugia for temperate trees in Central Europe.

Northern glacial refugia are a hotly debated concept. The idea that many temperate organisms survived the Last Glacial Maximum (LGM; ~26.5 to 19 thousand years) in several sites across central and northern Europe stems from phylogeographic analyses, yet direct fossil evidence has thus far been missing. Here, we present the first unequivocal proof that thermophilous trees such as oak (Quercus), linden (Tilia), and common ash (Fraxinus excelsior) survived the LGM in Central Europe. The persistence of the refugium was promoted by a steady influx of hydrothermal waters that locally maintained a humid and warm microclimate. We reconstructed the geological and palaeohydrological factors responsible for the emergence of hot springs during the LGM and argue that refugia of this type, allowing the long-term survival and rapid post-LGM dispersal of temperate elements, were not exceptional in the European periglacial zone.

Pengzhenrongella phosphoraccumulans sp. nov., isolated from high Arctic glacial till, and emended description of the genus Pengzhenrongella.

A yellow pigmented, Gram-stain-positive, motile, facultatively anaerobic and irregular rod-shaped bacteria (strain M0-14T) was isolated from a till sample collected from the foreland of a high Arctic glacier near the settlement of Ny-Ålesund in the Svalbard Archipelago, Norway. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that M0-14T formed a lineage within the family Cellulomonadaceae, suborder Micrococcineae. M0-14T represented a novel member of the genus Pengzhenrongella and had highest 16S rRNA gene sequence similarity to Pengzhenrongella sicca LRZ-2T (97.3 %). Growth occurred at 4-25 °C (optimum 4-18 °C), at pH 6.0-9.0 (optimum pH 7.0), and in the presence of 0-5 % (w/v) NaCl. The predominant menaquinone was MK-9(H4) and the major fatty acids were anteiso-C15 : 0, C16 : 0 and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). The major polar lipids were phosphatidylglycerol, phosphatidylinositol mannosides, phosphatidylinositol, one undefined phospholipid and five undefined phosphoglycolipids. The cell-wall diamino acid was l-ornithine whereas rhamnose and mannose were the cell-wall sugars. Polyphosphate particles were found inside the cells of M0-14T. Polyphosphate kinase and polyphosphate-dependent glucokinase genes were detected during genomic sequencing of M0-14. In addition, the complete pstSCAB gene cluster and phnCDE synthesis genes, which are important for the uptake and transport of phosphorus in cells, were annotated in the genomic data. According to the genomic data, M0-14T has a metabolic pathway related to phosphorus accumulation. The DNA G+C content of the genomic DNA was 70.8 %. On the basis of its phylogenetic relationship, phenotypic properties and chemotaxonomic distinctiveness, strain M0-14T represents a novel species of the genus Pengzhenrongella, for which the name Pengzhenrongella phosphoraccumulans sp. nov. is proposed. The type strain is M0-14T (= CCTCC AB 2012967T = NRRL B-59105T).

Tibetan terrestrial and aquatic ecosystems collapsed with cryosphere loss inferred from sedimentary ancient metagenomics.

Glacier and permafrost shrinkage and land-use intensification threaten mountain wildlife and affect nature conservation strategies. Here, we present paleometagenomic records of terrestrial and aquatic taxa from the southeastern Tibetan Plateau covering the last 18,000 years to help understand the complex alpine ecosystem dynamics. We infer that steppe-meadow became woodland at 14 ka (cal BP) controlled by cryosphere loss, further driving a herbivore change from wild yak to deer. These findings weaken the hypothesis of top-down control by large herbivores in the terrestrial ecosystem. We find a turnover in the aquatic communities at 14 ka, transitioning from glacier-related (blue-green) algae to abundant nonglacier-preferring picocyanobacteria, macrophytes, fish, and otters. There is no evidence for substantial effects of livestock herding in either ecosystem. Using network analysis, we assess the stress-gradient hypothesis and reveal that root hemiparasitic and cushion plants are keystone taxa. With ongoing cryosphere loss, the protection of their habitats is likely to be of conservation benefit on the Tibetan Plateau.

One-third of Southern Ocean productivity is supported by dust deposition.

Natural iron fertilization of the Southern Ocean by windblown dust has been suggested to enhance biological productivity and modulate the climate1-3. Yet, this process has never been quantified across the Southern Ocean and at annual timescales4,5. Here we combined 11 years of nitrate observations from autonomous biogeochemical ocean profiling floats with a Southern Hemisphere dust simulation to empirically derive the relationship between dust-iron deposition and annual net community production (ANCP) in the iron-limited Southern Ocean. Using this relationship, we determined the biological response to dust-iron in the pelagic perennially ice-free Southern Ocean at present and during the last glacial maximum (LGM). We estimate that dust-iron now supports 33% ± 15% of Southern Ocean ANCP. During the LGM, when dust deposition was 5-40-fold higher than today, the contribution of dust to Southern Ocean ANCP was much greater, estimated at 64% ± 13%. We provide quantitative evidence of basin-wide dust-iron fertilization of the Southern Ocean and the potential magnitude of its impact on glacial-interglacial timescales, supporting the idea of the important role of dust in the global carbon cycle and climate6-8.

Trajectory of increased iceberg kill-off in West Antarctica's shallows.

Compared with low latitude coasts, many polar latitudes are still little impacted by intense and direct anthropogenic stressors. Climate forcing is now bringing rapid physical change to nearshore polar realms. In the shallow coastal waters adjacent to the United Kingdom's Rothera Research Station in the West Antarctic Peninsula (WAP), 225 seabed markers at 5-25 m depth have been surveyed and replaced every year from 2002-2023 (75 markers at each of 5, 10 and 25 m). This is one of the longest continuously running marine disturbance experiments in the world, in one of Earth's fastest changing environments. Different categories of sea ice are recorded (including when the sea surface freezes into fast ice) at Rothera since the 1980s, and losses of marine ice in both polar regions are one of the striking responses to a warming planet1. Five to ten years of seabed marker hit rate data (marker broken or moved) showed that reduced sea ice cover is correlated with disturbance and mortality on the seabed2,3.

Individual diet variability shapes the architecture of Antarctic benthic food webs.

Antarctic biodiversity is affected by seasonal sea-ice dynamics driving basal resource availability. To (1) determine the role of intraspecific dietary variability in structuring benthic food webs sustaining Antarctic biodiversity, and (2) understand how food webs and the position of topologically central species vary with sea-ice cover, single benthic individuals' diets were studied by isotopic analysis before sea-ice breakup and afterwards. Isotopic trophospecies (or Isotopic Trophic Units) were investigated and food webs reconstructed using Bayesian Mixing Models. As nodes, these webs used either ITUs regardless of their taxonomic membership (ITU-webs) or ITUs assigned to species (population-webs). Both were compared to taxonomic-webs based on taxa and their mean isotopic values. Higher resource availability after sea-ice breakup led to simpler community structure, with lower connectance and linkage density. Intra-population diet variability and compartmentalisation were crucial in determining community structure, showing population-webs to be more complex, stable and robust to biodiversity loss than taxonomic-webs. The core web, representing the minimal community 'skeleton' that expands opportunistically while maintaining web stability with changing resource availability, was also identified. Central nodes included the sea-urchin Sterechinus neumayeri and the bivalve Adamussium colbecki, whose diet is described in unprecedented detail. The core web, compartmentalisation and topologically central nodes represent crucial factors underlying Antarctica's rich benthic food web persistence.

Holed up, but thriving: Impact of multitrophic cryoconite communities on glacier elemental cycles.

Cryoconite holes (water and sediment-filled depressions), found on glacier surfaces worldwide, serve as reservoirs of microbes, carbon, trace elements, and nutrients, transferring these components downstream via glacier hydrological networks. Through targeted amplicon sequencing of carbon and nitrogen cycling genes, coupled with functional inference-based methods, we explore the functional diversity of these mini-ecosystems within Antarctica and the Himalayas. These regions showcase distinct environmental gradients and experience varying rates of environmental change influenced by global climatic shifts. Analysis revealed a diverse array of photosynthetic microorganisms, including Stramenopiles, Cyanobacteria, Rhizobiales, Burkholderiales, and photosynthetic purple sulfur Proteobacteria. Functional inference highlighted the high potential for carbohydrate, amino acid, and lipid metabolism in the Himalayan region, where organic carbon concentrations surpassed those in Antarctica by up to 2 orders of magnitude. Nitrogen cycling processes, including fixation, nitrification, and denitrification, are evident, with Antarctic cryoconite exhibiting a pronounced capacity for nitrogen fixation, potentially compensating for the limited nitrate concentrations in this region. Processes associated with the respiration of elemental sulfur and inorganic sulfur compounds such as sulfate, sulfite, thiosulfate, and sulfide suggest the presence of a complete sulfur cycle. The Himalayan region exhibits a higher potential for sulfur cycling, likely due to the abundant sulfate ions and sulfur-bearing minerals in this region. The capability for complete iron cycling through iron oxidation and reduction reactions was also predicted. Methanogenic archaea that produce methane during organic matter decomposition and methanotrophic bacteria that utilize methane as carbon and energy sources co-exist in the cryoconite, suggesting that these niches support the complete cycling of methane. Additionally, the presence of various microfauna suggests the existence of a complex food web. Collectively, these results indicate that cryoconite holes are self-sustaining ecosystems that drive elemental cycles on glaciers and potentially control carbon, nitrogen, sulfur, and iron exports downstream.

Giant viral signatures on the Greenland ice sheet.

BACKGROUND: Dark pigmented snow and glacier ice algae on glaciers and ice sheets contribute to accelerating melt. The biological controls on these algae, particularly the role of viruses, remain poorly understood. Giant viruses, classified under the nucleocytoplasmic large DNA viruses (NCLDV) supergroup (phylum Nucleocytoviricota), are diverse and globally distributed. NCLDVs are known to infect eukaryotic cells in marine and freshwater environments, providing a biological control on the algal population in these ecosystems. However, there is very limited information on the diversity and ecosystem function of NCLDVs in terrestrial icy habitats. RESULTS: In this study, we investigate for the first time giant viruses and their host connections on ice and snow habitats, such as cryoconite, dark ice, ice core, red and green snow, and genomic assemblies of five cultivated Chlorophyta snow algae. Giant virus marker genes were present in almost all samples; the highest abundances were recovered from red snow and the snow algae genomic assemblies, followed by green snow and dark ice. The variety of active algae and protists in these GrIS habitats containing NCLDV marker genes suggests that infection can occur on a range of eukaryotic hosts. Metagenomic data from red and green snow contained evidence of giant virus metagenome-assembled genomes from the orders Imitervirales, Asfuvirales, and Algavirales. CONCLUSION: Our study highlights NCLDV family signatures in snow and ice samples from the Greenland ice sheet. Giant virus metagenome-assembled genomes (GVMAGs) were found in red snow samples, and related NCLDV marker genes were identified for the first time in snow algal culture genomic assemblies; implying a relationship between the NCLDVs and snow algae. Metatranscriptomic viral genes also aligned with metagenomic sequences, suggesting that NCLDVs are an active component of the microbial community and are potential "top-down" controls of the eukaryotic algal and protistan members. This study reveals the unprecedented presence of a diverse community of NCLDVs in a variety of glacial habitats dominated by algae.

Interaction between phytoplankton and heterotrophic bacteria in Arctic fjords during the glacial melting season as revealed by eDNA metabarcoding.

The hydrographic variability in the fjords of Svalbard significantly influences water mass properties, causing distinct patterns of microbial diversity and community composition between surface and subsurface layers. However, surveys on the phytoplankton-associated bacterial communities, pivotal to ecosystem functioning in Arctic fjords, are limited. This study investigated the interactions between phytoplankton and heterotrophic bacterial communities in Svalbard fjord waters through comprehensive eDNA metabarcoding with 16S and 18S rRNA genes. The 16S rRNA sequencing results revealed a homogenous community composition including a few dominant heterotrophic bacteria across fjord waters, whereas 18S rRNA results suggested a spatially diverse eukaryotic plankton distribution. The relative abundances of heterotrophic bacteria showed a depth-wise distribution. By contrast, the dominant phytoplankton populations exhibited variable distributions in surface waters. In the network model, the linkage of phytoplankton (Prasinophytae and Dinophyceae) to heterotrophic bacteria, particularly Actinobacteria, suggested the direct or indirect influence of bacterial contributions on the fate of phytoplankton-derived organic matter. Our prediction of the metabolic pathways for bacterial activity related to phytoplankton-derived organic matter suggested competitive advantages and symbiotic relationships between phytoplankton and heterotrophic bacteria. Our findings provide valuable insights into the response of phytoplankton-bacterial interactions to environmental changes in Arctic fjords.

The distinctive weathering crust habitat of a High Arctic glacier comprises discrete microbial micro-habitats.

Sunlight penetrates the ice surfaces of glaciers and ice sheets, forming a water-bearing porous ice matrix known as the weathering crust. This crust is home to a significant microbial community. Despite the potential implications of microbial processes in the weathering crust for glacial melting, biogeochemical cycles, and downstream ecosystems, there have been few explorations of its microbial communities. In our study, we used 16S rRNA gene sequencing and shotgun metagenomics of a Svalbard glacier surface catchment to characterise the microbial communities within the weathering crust, their origins and destinies, and the functional potential of the weathering crust metagenome. Our findings reveal that the bacterial community in the weathering crust is distinct from those in upstream and downstream habitats. However, it comprises two separate micro-habitats, each with different taxa and functional categories. The interstitial porewater is dominated by Polaromonas, influenced by the transfer of snowmelt, and exported via meltwater channels. In contrast, the ice matrix is dominated by Hymenobacter, and its metagenome exhibits a diverse range of functional adaptations. Given that the global weathering crust area and the subsequent release of microbes from it are strongly responsive to climate projections for the rest of the century, our results underscore the pressing need to integrate the microbiome of the weathering crust with other communities and processes in glacial ecosystems.

Survival in nunatak and peripheral glacial refugia of three alpine plant species is partly predicted by altitudinal segregation.

Mountain biota survived the Quaternary cold stages most probably in peripheral refugia and/or ice-free peaks within ice-sheets (nunataks). While survival in peripheral refugia has been broadly demonstrated, evidence for nunatak refugia is still scarce. We generated RADseq data from three mountain plant species occurring at different elevations in the southeastern European Alps to investigate the role of different glacial refugia during the Last Glacial Maximum (LGM). We tested the following hypotheses. (i) The deep Piave Valley forms the deepest genetic split in the species distributed across it, delimiting two peripheral refugia. (ii) The montane to alpine species Campanula morettiana and Primula tyrolensis survived the LGM in peripheral refugia, while high-alpine to subnival Saxifraga facchinii likely survived in several nunatak refugia. (iii) The lower elevation species suffered a strong population decline during the LGM. By contrast, the higher elevation species shows long-term stability of population sizes due to survival on permanently ice-free peaks and small population sizes at present. We found peripheral refugia on both sides of the Piave Valley, which acted as a major genetic barrier. Demographic modelling confirmed nunatak survival not only for S. facchinii but also for montane to alpine C. morettiana. Altitudinal segregation influenced the species' demographic fluctuations, with the lower elevation species showing a significant population increase at the end of the LGM, and the higher elevation species either showing decrease towards the present or stable population sizes with a short bottleneck. Our results highlight the role of nunatak survival and species ecology in the demographic history of mountain species.

Alzheimer's Amyloid Hypothesis and Antibody Therapy: Melting Glaciers?

The amyloid cascade hypothesis for Alzheimer's disease is still alive, although heavily challenged. Effective anti-amyloid immunotherapy would confirm the hypothesis' claim that the protein amyloid-beta is the cause of the disease. Two antibodies, aducanumab and lecanemab, have been approved by the U.S. Food and Drug Administration, while a third, donanemab, is under review. The main argument for the FDA approvals is a presumed therapy-induced removal of cerebral amyloid deposits. Lecanemab and donanemab are also thought to cause some statistical delay in the determination of cognitive decline. However, clinical efficacy that is less than with conventional treatment, selection of amyloid-positive trial patients with non-specific amyloid-PET imaging, and uncertain therapy-induced removal of cerebral amyloids in clinical trials cast doubt on this anti-Alzheimer's antibody therapy and hence on the amyloid hypothesis, calling for a more thorough investigation of the negative impact of this type of therapy on the brain.

Dynamics and drivers of mycorrhizal fungi after glacier retreat.

The development of terrestrial ecosystems depends greatly on plant mutualists such as mycorrhizal fungi. The global retreat of glaciers exposes nutrient-poor substrates in extreme environments and provides a unique opportunity to study early successions of mycorrhizal fungi by assessing their dynamics and drivers. We combined environmental DNA metabarcoding and measurements of local conditions to assess the succession of mycorrhizal communities during soil development in 46 glacier forelands around the globe, testing whether dynamics and drivers differ between mycorrhizal types. Mycorrhizal fungi colonized deglaciated areas very quickly (< 10 yr), with arbuscular mycorrhizal fungi tending to become more diverse through time compared to ectomycorrhizal fungi. Both alpha- and beta-diversity of arbuscular mycorrhizal fungi were significantly related to time since glacier retreat and plant communities, while microclimate and primary productivity were more important for ectomycorrhizal fungi. The richness and composition of mycorrhizal communities were also significantly explained by soil chemistry, highlighting the importance of microhabitat for community dynamics. The acceleration of ice melt and the modifications of microclimate forecasted by climate change scenarios are expected to impact the diversity of mycorrhizal partners. These changes could alter the interactions underlying biotic colonization and belowground-aboveground linkages, with multifaceted impacts on soil development and associated ecological processes.

Plant water source effects on plant-soil feedback for primary succession of terrestrial ecosystems in a glacier region in China.

Despite the extensive research conducted on plant-soil-water interactions, the understanding of the role of plant water sources in different plant successional stages remains limited. In this study, we employed a combination of water isotopes (δ2H and δ18O) and leaf δ13C to investigate water use patterns and leaf water use efficiency (WUE) during the growing season (May to September 2021) in Hailuogou glacier forefronts in China. Our findings revealed that surface soil water and soil nutrient gradually increased during primary succession. Dominant plant species exhibited a preference for upper soil water uptake during the peak leaf out period (June to August), while they relied more on lower soil water sources during the post-leaf out period (May) or senescence (September to October). Furthermore, plants in late successional stages showed higher rates of water uptake from uppermost soil layers. Notably, there was a significant positive correlation between the percentage of water uptake by plants and available soil water content in middle and late stages. Additionally, our results indicated a gradual decrease in WUE with progression through succession, with shallow soil moisture utilization negatively impacting overall WUE across all succession stages. Path analysis further highlighted that surface soil moisture (0- 20 cm) and middle layer nutrient availability (20- 50 cm) played crucial roles in determining WUE. Overall, this research emphasizes the critical influence of water source selection on plant succession dynamics while elucidating underlying mechanisms linking succession with plant water consumption.

Unprecedented insights into extents of biological responses to physical forcing in an Arctic sub-mesoscale filament by combining high-resolution measurement approaches.

In Fram Strait, we combined underway-sampling using the remote-controlled Automated Filtration System for Marine Microbes (AUTOFIM) with CTD-sampling for eDNA analyses, and with high-resolution optical measurements in an unprecedented approach to determine variability in plankton composition in response to physical forcing in a sub-mesoscale filament. We determined plankton composition and biomass near the surface with a horizontal resolution of ~ 2 km, and addressed vertical variability at five selected sites. Inside and near the filament, plankton composition was tightly linked to the hydrological dynamics related to the presence of sea ice. The comprehensive data set indicates that sea-ice melt related stratification near the surface inside the sub-mesoscale filament resulted in increased sequence abundances of sea ice-associated diatoms and zooplankton near the surface. In analogy to the physical data set, the underway eDNA data, complemented with highly sampled phytoplankton pigment data suggest a corridor of 7 km along the filament with enhanced photosynthetic biomass and sequence abundances of sea-ice associated plankton. Thus, based on our data we extrapolated an area of 350 km2 in Fram Strait with enhanced plankton abundances, possibly leading to enhanced POC export in an area that is around a magnitude larger than the visible streak of sea-ice.

Arctic plasmidome analysis reveals distinct relationships among associated antimicrobial resistance genes and virulence genes along anthropogenic gradients.

Polar regions are relatively isolated from human activity and thus could offer insight into anthropogenic and ecological drivers of the spread of antibiotic resistance. Plasmids are of particular interest in this context given the central role that they are thought to play in the dissemination of antibiotic resistance genes (ARGs). However, plasmidomes are challenging to profile in environmental samples. The objective of this study was to compare various aspects of the plasmidome associated with glacial ice and adjacent aquatic environments across the high Arctic archipelago of Svalbard, representing a gradient of anthropogenic inputs and specific treated and untreated wastewater outflows to the sea. We accessed plasmidomes by applying enrichment cultures, plasmid isolation and shotgun Illumina sequencing of environmental samples. We examined the abundance and diversity of ARGs and other stress-response genes that might be co/cross-selected or co-transported in these environments, including biocide resistance genes (BRGs), metal resistance genes (MRGs), virulence genes (VGs) and integrons. We found striking differences between glacial ice and aquatic environments in terms of the ARGs carried by plasmids. We found a strong correlation between MRGs and ARGs in plasmids in the wastewaters and fjords. Alternatively, in glacial ice, VGs and BRGs genes were dominant, suggesting that glacial ice may be a repository of pathogenic strains. Moreover, ARGs were not found within the cassettes of integrons carried by the plasmids, which is suggestive of unique adaptive features of the microbial communities to their extreme environment. This study provides insight into the role of plasmids in facilitating bacterial adaptation to Arctic ecosystems as well as in shaping corresponding resistomes. Increasing human activity, warming of Arctic regions and associated increases in the meltwater run-off from glaciers could contribute to the release and spread of plasmid-related genes from Svalbard to the broader pool of ARGs in the Arctic Ocean.

Extended ozone depletion and reduced snow and ice cover-Consequences for Antarctic biota.

Stratospheric ozone, which has been depleted in recent decades by the release of anthropogenic gases, is critical for shielding the biosphere against ultraviolet-B (UV-B) radiation. Although the ozone layer is expected to recover before the end of the 21st century, a hole over Antarctica continues to appear each year. Ozone depletion usually peaks between September and October, when fortunately, most Antarctic terrestrial vegetation and soil biota is frozen, dormant and protected under snow cover. Similarly, much marine life is protected by sea ice cover. The ozone hole used to close before the onset of Antarctic summer, meaning that most biota were not exposed to severe springtime UV-B fluxes. However, in recent years, ozone depletion has persisted into December, which marks the beginning of austral summer. Early summertime ozone depletion is concerning: high incident UV-B radiation coincident with snowmelt and emergence of vegetation will mean biota is more exposed. The start of summer is also peak breeding season for many animals, thus extreme UV-B exposure (UV index up to 14) may come at a vulnerable time in their life cycle. Climate change, including changing wind patterns and strength, and particularly declining sea ice, are likely to compound UV-B exposure of Antarctic organisms, through earlier ice and snowmelt, heatwaves and droughts. Antarctic field research conducted decades ago tended to study UV impacts in isolation and more research that considers multiple climate impacts, and the true magnitude and timing of current UV increases is needed.

Modeling study on oil spill transport in the Great Lakes: The unignorable impact of ice cover.

The rise in oil trade and transportation has led to a continuous increase in the risk of oil spills, posing a serious worldwide concern. However, there is a lack of numerical models for predicting oil spill transport in freshwater, especially under icy conditions. To tackle this challenge, we developed a prediction system for oil with ice modeling by coupling the General NOAA Operational Modeling Environment (GNOME) model with the Great Lakes Operational Forecast System (GLOFS) model. Taking Lake Erie as a pilot study, we used observed drifter data to evaluate the performance of the coupled model. Additionally, we developed six hypothetical oil spill cases in Lake Erie, considering both with and without ice conditions during the freezing, stable, and melting seasons spanning from 2018 to 2022, to investigate the impacts of ice cover on oil spill processes. The results showed the effective performance of the coupled model system in capturing the movements of a deployed drifter. Through ensemble simulations, it was observed that the stable season with high-concentration ice had the most significant impact on limiting oil transport compared to the freezing and melting seasons, resulting in an oil-affected open water area of 49 km2 on day 5 with ice cover, while without ice cover it reached 183 km2. The stable season with high-concentration ice showed a notable reduction in the probability of oil presence in the risk map, whereas this reduction effect was less prominent during the freezing and melting seasons. Moreover, negative correlations between initial ice concentration and oil-affected open water area were consistent, especially on day 1 with a linear regression R-squared value of 0.94, potentially enabling rapid prediction. Overall, the coupled model system serves as a useful tool for simulating oil spills in the world's largest freshwater system, particularly under icy conditions, thus enhancing the formulation of effective emergency response strategies.