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In order to study the impact of biochar application into the soil for the emanation coefficient and radon exhalation rate, biochar was applied to the soil in various doses. Then, after the soil stabilization period, samples were taken for laboratory tests and field measurements of exhalation arte were carried out. Based on the obtained results, a discussion was conducted focused on the perspective of environmental changes in the context of radon emission from soil depending on the dose of biochar. A detailed description of the materials and measurement methods used to conduct the research is presented in the following subsections.BiocharThe biochar incorporated in the field experiment was produced from sunflower husk in the pyrolysis process in the temperature range of 450–550 °C and consist on grains with diameters from 50 μm up to 10 mm. The biochar was characterized by specific surface area of 2.0 m2 g−1 in occupancy of Cu ions and 5.1 m2 g−1 for Ag ions23.Field experimentThe field experiment was conducted in ten plots, each with a dimension of 1.1 m × 1.1 m, located in Lublin/Felin at the Institute of Agrophysics of Polish Academy of Science. In addition one plot was left without biochar as a reference. The biochar was applied into the soil in April 2018. The soil presented on the fields were classified as Haplic Luvisol with 66% of sand, 23% of silt 11% of clay and 0.91% of organic matter (data for 0–15 cm layer)32. The following doses of biochar were applied for the following fields: 1, 5, 10, 20, 30, 40, 50, 60, 80, and 100 Mg ha−1 (which corresponded to the percentage of biochar per unit mass of soil: 0.05, 0.24, 0.48, 0.95, 1.43, 1.90, 2.28, 2.86, 3.81, and 4.76%, respectively). The fields were kept without vegetation by application of herbicide (Roundup 360 PLUS at 2.5 L ha−1). For the purpose of presented work six fields were investigated: with 0 (control), 20, 40, 60, 80 and 100 Mg ha−1 biochar doses.Soil sample collection and preparationThe soil samples were collected from five experimental plots, where the biochar was applied into the soil at the doses of 20, 40, 60, 80, and 100 Mg ha−1, and from a control field, where no biochar was applied (denoted as 0 Mg ha−1). Soil samples were collected from each field at five statistically chosen points to get about 2 kg of soil. After collection, the soil was mixed and dried at room temperature for two weeks. One sample for laboratory examinations was prepared from each part of the soil. The sample was a 4.7 cm high and 5.2 cm diameter steel cylinder, as presented in Fig. 1. The volume of each sample was 100 cm3. The bulk density of each sample was evaluated. The soil net weight of each sample was measured by measurements of each sample and subtraction of the weight of the steel cylinder. Next, the cylinders were closed on the bottom with a rubber cap to reduce the radon emanation surface to the size of 19.62 cm2 at the top of the sample.Figure 1The example of sample collected from the experimental field and prepared for measurements within the small accumulation chamber in the process of radon emanation assessment.Full size imageTotal porosity measurementsThe total porosity was measured with the weight method after water saturation. First, the samples were dried at 105 °C for 1 h and weighed to measure the total mass of the soil-biochar samples. To assess the total pore volume, the samples were placed in a tray filled with water for 24 h to reach saturation and the weight measurements were repeated. The total porosity η was calculated according to the equation:$$eta =frac{{V}_{p}}{{V}_{s}}=frac{{m}_{w}}{{rho }_{w}cdot {V}_{s}}=frac{left({m}_{s}-{m}_{d}right)}{{rho }_{w}cdot {V}_{s}}$$where ms is the mass of a saturated sample, md is the mass of a dried sample, Vs and Vp represent the sample volume (100 cm3) and pore volume, respectively, and ρw is water density. The weight measurements were realized by electronic laboratory balance with the accuracy of 0.1 mg. The total uncertainty of the method was assessed for 5%.The uncertainty for radon in air concentration measurements were assessed for 18% basing on the data provided by the instrument.Emanation coefficient assessmentThe radon emission from samples in the laboratory environment were measured using an AlphaGUARD instrument equipped with a measuring chamber made of stainless steel, as presented on the left in Fig. 2. The chamber was 11 cm in diameter and 12 cm in height, giving 0.00114 m3 of volume. The measurement of the radon concentration in air was made setting a 1 dm3 min−1 flow rate and 10 min reading cycles. Data were next averaged for 1 h, resulting to one data point represents the average values from six originally measured data points. Measurement for one sample took 20 h. Values of radon concentration in air was registered with the uncertainty for each data point assessed directly by the instrument.Figure 2Set up for radon accumulation measurements for assessment of the emanation coefficient in the laboratory environment. Set include the small accumulation chamber with the total volume of 0.00114 m3 (present on the left side) operating with the AlphaGuard instrument (presented on the right).Full size imageThe emanation coefficient ε was determined based on the Ra-226 activity concentration and radon potential Ω according to the methodology proposed by33 and making an additional assumption that the samples were dried to zero humidity, which implied that no pores filled with water were present within the samples during measurements. The assumption reduces the equation for calculating the time bound exhalation constant.The emanation coefficient expressed in % was evaluated according to the equation:$$varepsilon =frac{{Omega }}{{Ac}_{Ra}}cdot 100{%}$$
(2)
where AcRa represents the Ra-226 activity concentration in a soil sample expressed in Bq kg−1.The AcRa was assessed using gamma spectrometry and a high purity germanium detector according to the methodology described by25. For properly Ra-226 activity concentration in soil assessment the impact of 185.7 keV gammas from U-235 was subtracted after its evaluation basing on the 63.3 keV peak of Th-234.The main advantage of the proposed method is the ability to assess the emanation coefficient based on short-time measurements (below 24 h) with a cumulative chamber method. Originally, in the methodology developed by33, the assessment requires evaluation of Ω according to the formula:$$Omega = frac{{a + lambda _{{eff}} cdot C_{{Rn}}^{0} }}{{lambda _{{Rn}} }}~frac{{V_{e} }}{m}$$
(3)
where a (Bq m−3 s−1) represents the slope of the linear fit of radon exhalation rate data series measured in the cumulative chamber, λeff is an effective time constant (s−1), CRn0 is the initial Rn-222 concentration in the accumulation chamber (Bq m−3), λRn denotes the Rn-222 decay constant, Ve describes the effective accumulation volume in the experimental setup (m3), and m (kg) is the mass of the sample.The effective time constant describes the effective time of the presence of radon exhaled within the experimental set up and is the sum of the Rn-222 decay constant λRn (s−1), the bound exhalation constant λb (s−1) characterizing the sample, and the leakage constant characterizing the accumulation chamber equipment λl (s−1):$${lambda }_{eff}={lambda }_{Rn}+{lambda }_{b}+{lambda }_{l}$$
(4)
The λb coefficient is dependent on the soil sample porosity according to the simplified equation:$${lambda }_{b}={lambda }_{Rn}eta frac{{V}_{0}}{{V}_{e}}$$
(5)
where η represents the total porosity of the sample, as the assumption of zero humidity of samples during measurements was made, and V0 represents the volume of the sample.λl was evaluated experimentally by measuring the Rn-222 decay in the empty accumulation chamber system with a natural radon concentration at the starting point. The most significant compound of total uncertainty for the emanation coefficient was associated with estimation for slope a, CRn0 and with assessment of the total porosity for the samples η. The uncertainty was evaluated using differentiation method.Radon exhalation rate assessmentThe radon exhalation rate (ERn) was assessed according to the methodology presented in25. The field radon exhalation rate was assessed indirectly by measurement of radon concentration in air using an AlphaGUARD instrument equipped with accumulation open-wall chamber placed on the ground as presented on the Fig. 3. The chamber volume was 0.024 m3. The setup measuring parameters were the same as in the case of the laboratory measurements with the small closed chamber but the measuring time was 70 min giving seven data points for each field. Values of radon concentration in air was registered with the uncertainty for each data point assessed directly by the instrument.Figure 3Setup for assessment of the radon exhalation rate in the field measurements. The set consist of the accumulation chamber with 0.024 m3 in volume and the AlphaGuard instrument.Full size imageThe increase of radon concentration in air (CRn) in accumulation box were registered and the linear function was interpolated basing on seven measuring points registered for each experimental field. Basing on the measurement of radon concentration in air the radon exhalation rate could be assessed according to the equation:$${E}_{Rn}=frac{V}{A}frac{partial {C}_{Rn}}{partial t}$$
(6)
where V/A are the ratio of accumulation chamber volume to area covered by the chamber and is a constant value of 0.2, (frac{partial {C}_{Rn}}{partial t}) is a change of radon concentration in air registered in accumulation chamber in time t and was represented as a linear fit into the experimental data as:$${C}_{Rn}=acdot t+b$$
(7)
After differentiation we can compute the radon exhalation rate as slope, a of linear fit scaled by 0.2:$${E}_{Rn}=0.2a$$
(8)
The uncertainty for radon exhalation rate assessment was associated mainly with the assessment of the slope of linear fit ad was calculated as a standard deviation for data point used for linear fits. More

Nature-based solutions (NBS) are increasingly recognised as an effective response to a number of major urban challenges. These include heatwaves1,2,3, flooding4,5,6, water quality7,8 and public health and wellbeing9,10,11. While the concept of NBS emerged as recently as 201512, the idea of using urban nature to address these issues also features prominently in the more established fields of ecosystem services (which emerged in 2005)13 and green infrastructure (2002)14.Despite mounting evidence of their benefits, strategies built around NBS are seldom practically realised15; implementation in cities has been slow, inconsistent and often limited to demonstration sites16,17,18,19,20,21. For example, 6 years after Copenhagen embraced green infrastructure as a response to its acute flooding problems in 2011, the implementation of green infrastructure was only just ‘taking off’ in 2017, and remained highly contested22. Even retaining existing urban NBS remains a challenge; tree canopy cover, central to mitigation of urban heat island effects, is declining in many cities23,24. For example, metropolitan Melbourne experienced a loss of 2000 hectares between 2014 and 201825. In the US, an average of 36 million trees were lost each year from urban areas between 2009 and 201426.Barriers within the organisations responsible for implementing NBS are frequently identified as a primary reason for limited NBS delivery16,17,23,27,28,29. Delivery organisations have significant path dependencies; existing regimes are self-enforcing, and change is difficult30,31.The reasons for non-delivery have been characterised in detail in a broad range of literature. Barriers are highlighted in studies focused on urban forestry32,33, urban water management18,29,34, nature-based solutions35,36 and climate adaptation37,38. The issue has been investigated through the lenses of mainstreaming39,40, governance19,21,33,34,41,42, transitions31,36,43,44,45 and general analyses of barriers17,23,46,47. Papers describing the barriers to NBS have drawn on interviews with experts17,23,33,34,36,48, and direct project experiences49,50. At the time of writing, we are aware of nine review papers that present typologies of barriers to NBS delivery, based on systematic reviews of the considerable literature16,18,21,29,32,37,41,46,47.These studies have identified a largely consistent set of eight essential (and frequently lacking) traits for successful NBS implementation in local government. Leadership support is critical, both at the political and executive level18,20,21,29,34,41,51,52. A project team with the right capacity and timeframes to implement projects is also important32,37,51,53,54, as is a framework of internal mechanisms that facilitate the delivery of NBS, including clear approval processes, supportive policies and laws, and well-established standards for NBS design and maintenance16,18,20,35,47,55,56,57. A positive, supportive organisational culture for delivering new projects is also necessary, recognising that new NBS projects often have inherent (and novel) risks and trade-offs17,19,20,32,33,47,52,58. Finally, access to teams within the organisation that are both suitably skilled and supportive is vital16,18,23,46,54,59,60. Beyond the organisation itself, it is common for other levels of government to play an important role in approving aspects of NBS projects; an absence of support or clear process from higher regulatory authorities can pose a significant barrier32,33,38,44,51. Effective community engagement is also noted as important, recognising that many NBS need public support and/or private property owner consent to be successful23,37,38,46,49,61,62,63,64.While the barriers to NBS delivery have been the subject of significant attention, the implementation gap persists, with recent publications continuing to note the difficulty of NBS delivery33,36,52,57.A range of theoretical frameworks offer insight into how the implementation gap might be addressed. In the field of governance, The Policy Arrangement Model65 has been used to conceptualise governance in urban forestry32,33 and urban stormwater management21,34 as the temporary balance of a set of actors, discourses, rules and resources; changes to these variables may lead to changes in governance.In the Policy Arrangement Model, each of these four elements is significant, as is their interplay33. The actors included (or excluded) in a policy arrangement are crucial, given the range of agendas in typical stakeholders (e.g. politicians, community groups, chambers of commerce, financiers etc.), as are the relations between these actors (some may operate as coalitions, or as antagonists). Discourses include tacit and explicit conceptualisations of what the policy problem is, how it should be solved, and what values matter most. These are important in lending legitimacy to rules, which define interactions and roles between actors. These may be as formal as laws and design standards, or as informal as a set of undocumented organisational processes and norms (e.g. “talk to Anne in our compliance branch, she usually decides what is safe”). These elements are all vital in determining who deploys resources such as staff time, skills, budgets or equipment, and how they are deployed. Collectively, the dynamics between these four elements constitute a policy arrangement; changes in one element have the potential to affect others, and in turn spark shifts in governance65. However, these systems can be strongly entrenched66. Governance shifts are theorised to be typically driven by at least four factors: policy entrepreneurs (or ‘champions’), shock events, socio-political changes and ‘adjacent arrangements’ (developments in policy domains in related sectors or institutions)67.Policy entrepreneurs are also a focus of mainstreaming research, which highlights how these individuals advance NBS uptake by working within organisations to involve key stakeholders, engage citizens and contract technical expertise while incrementally introducing NBS considerations into planning practice35. This work is conceptualised as ‘horizontal’ mainstreaming, as officers champion NBS across their organisations, but it is argued that this must be supported by ‘vertical’ actions by top-down actors (such as executives and elected leaders) with the power to determine resource allocations and organisational structures39,40.To support NBS development and planning, the European Union’s Horizon 2020 programme initiated a series of large international demonstration projects, each involving collaborations between a number of cities, consultancies and universities. These include the UnaLab, ProGIreg, Connecting Nature, GrowGreen, Urban GreenUP and EdiCitNet projects68. These projects generally fund dedicated staff, as well as on-ground delivery of NBS, and have potential to address some or all of the barriers to NBS delivery that cities face. When considered in terms of the Policy Arrangement Model, the new actors, discourses and resources introduced by these projects all challenge the ‘temporary balance’ theorised to constitute the organisational status quo65. These projects also may encourage governance shifts67, by both facilitating the hiring of NBS policy entrepreneurs, and increasing a city’s exposure to influential adjacent arrangements in other centres of NBS expertise, such as university research units or exemplar municipalities. With the involvement of organisational champions/policy entrepreneurs, mainstreaming activities such as stakeholder outreach, citizen engagement and intra-organisational collaboration become increasingly possible35.This paper investigates the Horizon 2020 NBS project, Urban GreenUP. Urban GreenUP focuses on supporting partner cities to prepare NBS plans, as well as funding a multi-million Euro programme of investment in NBS interventions including floating vegetated islands, green walls on private structures, and streambank renaturalisation. The seven cities participating actively as project partners are Liverpool (UK), Ludwigsburg (Germany), Mantova (Italy), Valladolid (Spain), Izmir (Turkey), Quy Nhon (Vietnam) and Medellín (Colombia). This group of cities represents a wide range of governance arrangements and urban contexts in which NBS delivery occurs; Liverpool is a significant post-industrial centre emerging from sustained economic challenges compounded by government austerity, while Mantova has large areas of UNESCO world heritage and a legacy of industrial pollution. Quy Nhon is a coastal holiday town fairly new to NBS, while Ludwigsburg has extensive environmental legislation and has already successfully carried out major streambank restoration works on their local river. The former is largely governed by provincial government, with more operational management at a local level, whereas the latter has individual portfolio mayors, including one for the city’s environment. Valladolid has a population of 300,000 and a fairly compact urban form, whereas Medellin numbers over two million residents. We were able to work with each of these cities, effectively capturing the full range of capabilities and experiences in the Urban GreenUP project, and a significant variety of landscapes and organisational contexts in which NBS may be implemented.While the ‘generalisability’ of case studies is often limited, the constraints can be at least partially addressed through strategic sampling of cases69. Our sample is diverse, and while limited to seven cities, it does represent the full cohort of cities participating in this major EU programme designed to promote NBS innovation. A smaller sample size allows for a close, qualitative study of each case. Urban GreenUP presents a valuable opportunity to investigate the persistence of NBS barriers within local governments with ambitions for NBS implementation, with implications both for future innovation-oriented programmes such as Horizon 2020, and potentially the broader practice of NBS delivery in cities.Many cities beyond the Urban GreenUP group are preparing new NBS plans and programmes, and could benefit from insights arising from this study. Each GreenUP city is embarking on NBS planning and delivery and, at the time of our research, each had an individual or team employed with a specific NBS delivery role (with potential to serve the policy entrepreneur role highlighted in the literature). Local government often plays a key role in the implementation of urban NBS39,47,70, and Urban GreenUP places these organisations at its centre. Citizen engagement is an explicit focus of the project, as is the making of plans; these are both emphasised as opportunities for mainstreaming new practices35,71.We analyse the NBS implementation capacity of the cities within this study using an approach generally consistent with the practice of theory-based evaluation72,73. This ‘theory-based’ method breaks an implementation programme into its component elements, and assesses each element against available theory regarding what is required for success. This poses significant advantages over other evaluative methods because it focuses on the causative elements that lead to policy success or failure, rather than just the final outcomes achieved74, and is therefore more conducive to reforms of the institutional barriers discussed above.Theory-based evaluation typically takes place at the end of projects, but ours is ex ante; an approach noted by Weiss in her seminal outline of theory-based evaluation as having the potential to improve programme planning72. Evaluative practices have been noted as a particular weakness in local government NBS programmes, both at the political and officer level, due to a fear that acknowledging problems would lead to criticism of failures36. We sought to mitigate this issue both through use of an ex ante approach, and by designing a tool that creates distance between evaluators and individual practitioners.This paper investigates the enduring difficulties faced by cities seeking to deploy NBS, in the context of a major NBS project spanning seven cities. We had two key research questions. First, do case study cities have the capabilities required for successful NBS delivery, or are there barriers that continue to make this difficult? Second, does identifying and measuring a city’s NBS delivery capabilities facilitate improvements in these capabilities?We elicited organisational barriers from NBS practitioners in participating cities using a purpose-built diagnostic tool. We used this approach because tools that enable organisations to learn about their success factors have the potential to address implementation gaps44,71.The tool was developed to bring lessons from the literature into an operational context, by enabling practitioners to assess and rate their organisation’s capability levels across eight key areas, such as political support, alignment between teams, and technical knowledge (refer to Table 1). The tool posed a series of questions pertaining to each of these eight capability areas; users answered by selecting from a set of pre-defined answers. The tool associated each answer with a level of capability, which was reported as a final assessment of an organisation’s capabilities. This was provided to the practitioner directly on completion of the tool’s questions, enabling an immediate estimate of their organisation’s NBS delivery capacity, and a diagnosis of any key barriers they would be likely to face in future NBS projects. These results formed the basis for our reflections on NBS delivery capacity within Urban GreenUP, as well as discussions with practitioners to understand how results of the tool were received.Table 1 Eight success factors for urban NBS in local government.Full size tableOur research proceeded in three steps. First, we drew on the literature to define a set of eight key capabilities—which we call ‘success factors’—for NBS; these formed the basis of the tool. Second, practitioners within NBS teams in participating cities used the tool to identify their capability levels. Finally, we interviewed users to evaluate the impact of the tool. More

Evaluation of the lithological, geochemical, and fossil diatom evidenceWe obtained a short, 270-cm sediment core ( 250 m40. Lake Chalco lies in the central region of the Trans-Mexican Volcanic Belt (Fig. 1). It is bounded to the north by the Sierra de Santa Catarina, to the east by the Sierra Nevada (Volcanoes Popocatepetl, Iztaccíhuatl, Tláloc and Telapón), and to the west, by Volcano Teuhtli, which is the closest volcano to the waterbody (~ 6.5 km)41. Lake sediments are composed mainly of clays that have been described as impermeable. The Lake Chalco Basin, however, is a highly complex system and seismically active. Therefore, the presence of active fractures during the Holocene is possible. Active fractures may result in inflow from the deep aquifer. Historical data indicate the existence of freshwater springs in upper parts of the aquifer, mainly at the eastern piedmont42. For instance, mineral and thermal waters at Peñón del los Baños, near the Mexico City airport (33 km from Lake Chalco), have been reported since Aztec times around AD 1,325. This spring is associated with a system of seismically active fractures. No thermal waters have been reported in modern Lake Chalco, however phreatomagmatic activity ( > 100,000 years BP) from the Xico Volcano has been documented43. Recent studies showed that the water table position changes from the upper parts of the watershed to central Texcoco, 45 km from Lake Chalco. In this study four components of the flow system were identified, including waters of recent infiltration and local circulation, evidencing intermediate chemical evolution, and waters more chemically evolved of large flow trajectories and of deep circulation44.The lithology of the studied sediment sequence is as follows: (1) Sediments from 270 to 250 cm are characterized by lapilli, ash and black to light brown silty ashes (massive to stratified); (2) Sediments in the interval 250–235 cm are composed of diatomite (yellow); (3) From 235 to 200 cm, sediments are characterized by massive black to brown sandy silts (brown); (4) From 200 to 70 cm, sandy brown to reddish, banded laminated silts, with scattered or banded pumice fragments (red) are present; (5) Sediments from 70 to 60 cm are black silty sands with organic material; (6) From 60 to 50 cm sediments are sandy brown to reddish, laminated silt, with scattered or banded pumice fragments (red); (7) Sediments from 50 to 40 cm are characterized by lapilli, ash and black to light brown silty ashes, massive to stratified, and (8) uppermost sediments from 40 to 0 cm are black silty sands with organic material (Fig. 3)36.Figure 3Taxonomic diversity revealed by metagenomic and fossil diatom analysis, and geochemical variables from the Lake Chalco Holocene sediment sequence. Each horizontal bar represents a collected sample, with the exception of the upper row, which shows the average of surface samples S1 (0 cm, i.e., modern) and S2 (0 cm, i.e., modern) (Supplementary Table S1). The lithology of the 270-cm sediment sequence is shown in the first column. The Upper Toluca Pumice (UTP) is represented as tephra underneath the first column (from left to right). Taxonomic diversity is depicted as the relative abundance of phyla Bacteria (green), Archaea (pink), and Eukarya (blue) (columns 2–4). Percent values correspond to the diversity of peptide sequences corresponding to each domain. Geochemical variables related to biological processes and past conditions are shown in columns 5–7. Results of fossil diatom analysis are shown in column 8. Dark horizontal lines show the boundaries for each delimited paleoenvironmental zone: (1) freshwater, (2) hyposaline and (3) subsaline. Edited in CorelDRAW 2020 version 22.0.Full size imageElemental geochemistry and fossil diatoms in sediment cores can be used as indicators of past wet and dry climate intervals. We measured geochemical indicators including element concentrations and ratios, and Total Organic Carbon (TOC) throughout the core (Supplementary Table S1). Total organic carbon is an important component of sediments and soils and can be used to assess the environmental status of terrestrial and aquatic ecosystems45. Maximum TOC values characterize the period of hyposaline conditions. The Mn/Fe ratio, often used to track past O2 content in bottom waters and changing redox conditions, was used as a proxy for water-column oxygen concentration46. The Mn/Fe ratios display highest values at depths of 200, 150 and 60 cm, suggesting periods of permanent anoxia during warmer conditions and excessive nutrient inputs47. The Fe/Ti ratio provides information about fluvial sediment sources. Changes in the abundance of iron oxides can be used to infer fluctuations in inputs of land‐derived detrital material48. We observed increasing Fe/Ti ratio values in superficial layers, and highest values at 100 cm. We performed cluster analysis based on Euclidean distance which revealed three groups of samples (Supplementary Fig. S1).We identified three zones in the Lake Chalco Holocene sediment sequence, based on geochemical analysis and diatom assemblages, which reflect different paleoenvironmental conditions: (1) a cool, freshwater lake (235–210 cm), (2) a warm, hyposaline lake (185–60 cm), and (3) a temperate, subsaline lake (50–0 cm) (Figs. 3, 5, Supplementary Fig. S1, Table S1).Fossil diatom assemblages provide information about past environmental changes and water quality7,49. Fossil diatom analysis, along with knowledge of species ecological preferences, enables inference of past limnological variables such as temperature, salinity, pH, electrical conductivity, and phosphorus concentration7. Inferences from our fossil diatom record concur with an earlier diatom-based paleoclimate study from Lake Chalco. Our studies revealed that during the last deglacial (~ 19,500–11,500 cal years BP), conditions were colder and much wetter than present. Assemblages are dominated by small araphid diatoms Gomphonema affine and Cocconeis placentula (Fig. 3). From 11,500 to 4,500 cal years BP, Lake Chalco was characterized by hyposaline conditions, with higher evaporation rates until ~ 6,500 cal years BP. Typical diatoms taxa include Anomoeoneis costata, Halamphora veneta and small araphids. After ~ 6,500 cal years BP, salinity in Lake Chalco declined, mean annual precipitation increased slightly, and summer insolation, seasonality, and evaporation decreased7. Assemblages are composed of H. veneta, Nitzschia frustulum, Cyclotella meneghiniana and small araphid diatoms.Meta-taxonomic analysis of Prokaryote and Eukaryote diversityOur metagenomic analysis identified 36,722 OTUs (genera) in the sediments of Lake Chalco. Among those genera, 81% correspond to bacteria (29,818 ± 106 identified to genera [ig]), 15% to Archaea (5,710 ± 118 ig), 3% to Eukarya (1,147 ± 6 ig), and 76. Iron is considered a potentially harmful element (PHE), which may be indicative of human-mediated contamination. For instance, high Fe concentrations in surficial sediments could be related to inputs of clastic sediments, and often reflect agricultural activities77. We determined 13 plant genera, five belonging to the family Poaceae (58%), including the genus Zea (corn), known to have been cultivated and consumed by early settlers78, and Oryza, a fast-growing weed that is indicative of human-mediated habitat disturbance. Twelve microscopic fungal genera were observed, including taxa that are pathogenic on wheat and rice (Gibberella and Cladochytrium), plants, keratin, and flies (Supplementary Fig. S10)79, and a protozoan that is pathogenic in humans (Giardia). We found high abundances of the family Culicidae (18%) (Supplementary Fig. S10) during the period of human occupation. The subsaline zone displays 290 unique pfams and the highest number of representatives of potassium metabolism throughout the entire Holocene (Supplementary Fig. S4B). The abundance of genes related to Cyanobacteria in this zone is much lower (9%), in contrast with findings from the Blastp against MetaProt database of the freshwater (30%) and hyposaline zones (60%) (Supplementary Fig. S10, Table S3).Our findings suggest that the biota in and around Lake Chalco during the Holocene responded mainly to changes in temperature, salinity, and trophic state, reflecting climate and human impacts over the last 6,000 years. This implies landscape modifications, agricultural activities and accelerated lake eutrophication. Furthermore, prokaryotic assemblages revealed gradual deposition of microbial communities capable of anaerobic fermentation of organic material and methanogenesis, as well as evidence for volcanic activity, inferred from the metabolic potential for sulfur cycling in the deeper zones (hyposaline and freshwater). This study generated information on Neotropical Prokaryote and Eukaryote diversity and microbial metabolic pathways during the Holocene. Nevertheless, we recommend that future studies focus on detailed characterization of microbial substrates and constrain post-depositional processes. Such studies should also consider selection processes that result from gradual depletion of substrates during burial52,59,63. Finally, we highlight the importance of including additional geochemical measures, such as porewater chemistry80. and sedimentology81 in future studies and measuring biomass by qPCR assay. More

Study site and soil samplingThe soil was collected at 5 − 20 cm (Ap horizon) from an agricultural field in Southern Germany (Freising, Bavaria, 48°23’53.8“N, 11°38’39.7“E) in December 2017. The sampling area is situated within the lower Bavarian upland, and characterized by a mean annual temperature of 7.8 °C and mean annual precipitation of 786 mm. The soil type is a Cambisol (silty clay loam; 32% clay, 53% silt, and 14% sand) with a considerable amount of loess mixed with underlying Neogene sandy sediments. The soil was selected to represent a widely distributed soil type and land use. The collected soil was oven-dried (2 days, 40 °C), sieved ( More