Natural Systems Research Articles

A brain microbiome in salmonids at homeostasis.

Ectotherms have peculiar relationships with microorganisms. For instance, bacteria are recovered from the blood and internal organs of healthy teleosts. However, the presence of microbial communities in the healthy teleost brain has not been proposed. Here, we report a living bacterial community in the brain of healthy salmonids with bacterial loads comparable to those of the spleen and 1000-fold lower than in the gut. Brain bacterial communities share >50% of their diversity with gut and blood bacterial communities. Using culturomics, we obtained 54 bacterial isolates from the brains of healthy trout. Comparative genomics suggests that brain bacteria may have adaptations for niche colonization and polyamine biosynthesis. In a natural system, Chinook salmon brain microbiomes shift from juveniles to reproductively mature adults. Our study redefines the physiological relationships between the brain and bacteria in teleosts. This symbiosis may endow salmonids with a direct mechanism to sense and respond to environmental microbes.

Flash behaviors protect prey from avian predators

Abstract It has been proposed that flash behaviors, defined as the transient display of conspicuous color patches during escape, serve as an anti-predator defense in camouflaged prey. While recent studies in humans have demonstrated the effectiveness of flash behaviors in improving the survival of artificial prey, it remains unclear whether these benefits also arise in more natural systems involving non-human predators. This study investigated the adaptive significance of flash behaviors using chicks (Gallus gallus domesticus) as predators and virtual prey. Our experiments revealed that prey employing flash behaviors consistently enhanced their survival rate compared to non-flashing prey. Additionally, prey with non-conspicuous escape colors, though distinct from their resting colors, also showed a higher survival rate than non-flashing prey, challenging the conventional belief that conspicuous colors are required for search interference. An interplay between prey size and flash behaviors in enhancing survival was evident, with larger prey benefiting more than smaller prey. We found no evidence to suggest that being hidden before initiating an escape confers a survival advantage to flashing prey compared to being visible before escape. This study sheds light on the adaptive benefits of flash behaviors against natural predators and contributes to our understanding of the widespread occurrence of hidden color signals in camouflaged prey. We encourage further studies to explore the effectiveness of remaining hidden in flashing prey when confronted with natural predators, as well as how predators might learn to overcome the deception of flash behaviors.

Integration of Green and Gray Infrastructures – Conceptualization from the Perspective of Gray Engineers

Green infrastructure preserves, enhances, or restores elements of a natural system to provide higher-quality, more resilient, and lower-cost infrastructure services. Ecosystem disaster prevention and mitigation is also one of the features of green infrastructure. It is thus expected to integrate gray and green infrastructure to achieve greater resilience and sustainability. While several concepts of integrated green and gray infrastructure (IGGI), or hybrid infrastructure, have been proposed at the landscape scale, hybrid possibilities could exist at the single structural element and material scales. This paper proposes three types of IGGI concepts that could be beneficial for gray engineers to implement green infrastructure in terms of the gray engineering perspectives (safety, serviceability, and durability), primarily focusing mainly on the scale of single- or multiple-structure scales smaller than landscape scale. By categorizing the past and feasible IGGI cases into three concepts, the performance requirements for each infrastructure (green or gray) will become apparent, accelerating the discussion to develop a performance curve model as an IGGI. Also, the work needed to realize IGGI is discussed, emphasizing the urgent need for gray engineers to understand the benefits of integrating green infrastructure into gray infrastructure and thus promote the implementation of IGGI.

Anthropic-Induced Variability of Greenhouse Gasses and Aerosols at the WMO/GAW Coastal Site of Lamezia Terme (Calabria, Southern Italy): Towards a New Method to Assess the Weekly Distribution of Gathered Data

The key to a sustainable future is the reduction in humankind’s impact on natural systems via the development of new technologies and the improvement in source apportionment. Although days, years and seasons are arbitrarily set, their mechanisms are based on natural cycles driven by Earth’s orbital periods. This is not the case for weeks, which are a pure anthropic category and are known from the literature to influence emission cycles and atmospheric chemistry. For the first time since it started data gathering operations, CO (carbon monoxide), CO2 (carbon dioxide), CH4 (methane) and eBC (equivalent black carbon) values detected by the Lamezia Terme WMO/GAW station in Calabria, Southern Italy, have been evaluated via a two-pronged approach accounting for weekly variations in absolute concentrations, as well as the number of hourly averages exceeding select thresholds. The analyses were performed on seven continuous years of measurements from 2016 to 2022. The results demonstrate that the analyzed GHGs (greenhouse gasses) and aerosols respond differently to weekly cycles throughout the seasons, and these findings provide completely new insights into source apportionment characterization. Moreover, the results have been combined into a new parameter: the hereby defined WDWO (Weighed Distribution of Weekly Outbreaks) normalizes weekly trends in CO, CO2, CH4 and eBC on an absolute scale, with the scope of providing regulators and researchers alike with a new tool meant to better evaluate anthropogenic pollution and mitigate its effects on the environment and human health.

Sustainable treatment of combined industrial wastewater: synergistic phytoremediation with Eichhornia crassipes, Pistia stratiotes, and Arundo donax in biofilm wetlands

This study investigates the treatment of combined wastewater from Hattar Industrial Estate using Biofilm Wetlands (BW) planted with monoculture species: Eichhornia crassipes (EAC), Pistia stratiotes (WL), and Arundo donax (GR). Each species showed distinct capabilities in organic degradation, metal uptake, and pH stabilization. BW2, planted with EAC, achieved the highest total solids (TS) and total suspended solids (TSS) removal efficiencies of 66% and 65%, respectively. GR effectively reduced initial COD concentrations from 232 mg/L to 58.67 mg/L, while EAC and WL reached reductions to 72.78 mg/L and 70.67 mg/L, respectively. Overall, the plant efficiency ranking was EAC > GR > WL. These findings underscore the potential of these plant species in synergistic BW systems, highlighting their role as natural solutions for remediating complex industrial effluents. This research contributes to advancing eco-friendly wastewater treatment approaches, suggesting promising applications for sustainable practices in industrial contexts. RESEARCH HIGHLIGHTS This research assessed the effectiveness of phytoremediation using Eichhornia crassipes, Pistia stratiotes, and Arundo donax for removing pollutants i.e. heavy metals (Cd, Pb, Ni, K, Ca, Mg, Na, Fe, Hg) nitrates, phosphates and sulfates from combined industrial wastewater of Hattar Industrial Estate Pakistan. It highlighted the potential of selected plant species’ as natural treatment systems, providing crucial insights into their efficiency. Findings contribute to understanding nature-based solutions for complex industrial effluents.

MAPPING THE NEED FOR A CIRCULAR ECONOMY FOR FUTURE ECONOMIC BENEFITS: POTENTIALS AND PROJECTIONS FOR SMALL AND MEDIUM-SIZED ENTERPRISES IN NORTH MACEDONIA

The circular economy, a concept based on the principles of designing waste and pollution, keeping products and materials in use for a longer time, and regenerating natural systems is still in its infancy in the Western Balkans, especially in many analyses related to the development and future projections of small and medium enterprises. Over the years, there has been a steady increase in per capita waste generation in the region, with the municipal waste recycling rate among economies being very low, while resource intensity as a measure of resource efficiency has remained relatively high. In addition, enterprises in the region are rarely aware of the opportunities of a circular economy, such as reduced environmental impact, more sustainable use, and conservation of natural resources, lower production costs, increased innovation, increased competitiveness, greater resistance to external shocks through reduced dependence on resources or the creation of new markets and jobs. Of particular importance is the identification of key economic sectors that should be directly involved in the process of circular economic transformation. In addition, it is important in the European framework, to map of the good practices that could be transferred to the economy in the Republic of North Macedonia, when it comes to the circular economy. In the framework of this paper, through the application of a methodological framework of content analysis, comparison, synthesis, and generalization, a diagnosis of the current situation regarding a circular economy in North Macedonia will be presented, as well as the identification of priority sectors/areas as the main key for the future perspectives of the circular economy within these society frameworks. It requires the involvement of all key stakeholders, including government institutions, the private sector, civil society, and academia, to apply a comprehensive approach in setting a future roadmap for a circular economy and encouraging a circular transition for the greater benefit of society of the Republic of North Macedonia, with special reference to improving the future of small and medium enterprises.

Life-Cycle Analysis of Natural Treatment Systems for Wastewater (NTSW) Applied to Municipal Effluents

The objective of the described activity is to develop technologies or proposals that can be implemented within the cycle to enhance the relationship between climate change, water, energy, and food. The focus is on analyzing natural treatment systems for wastewater (NTSW) within the context of Macaronesia, considering factors such as life-cycle assessment (LCA), carbon footprint, impacts, and mitigation capacity. The analysis of real case data from the Canary Islands and Cape Verde will inform the development of appropriate technologies tailored to different areas and scales within Macaronesia. This work includes a comprehensive life-cycle analysis of the Santa Catarina (Cape Verde) NTSW. This analysis encompasses: (a) Inventory analysis of the construction phase: This involves the assessment of inputs and outputs associated with the construction of the NTSW, including materials, energy consumption, transportation, and waste generation. The maintenance and operation phases are then evaluated, with a focus on the ongoing maintenance and operation activities required for the NTSW, including energy consumption, water usage, chemical inputs (if any), labor, and equipment maintenance. (b) Finally, the impacts of the NTSW are evaluated. The environmental, social, and economic impacts generated by the NTSW are assessed. This includes an analysis of factors such as carbon emissions, water usage, land use, ecosystem impacts, human health effects, and economic costs. By conducting a comprehensive analysis of the Santa Catarina NTSW, the document aims to provide insights into the environmental performance and sustainability of the system. This information can then be used as a tool and experience of educational innovation for final-year undergraduate students to identify areas for improvement, develop mitigation strategies in the water sector, and inform decision-making processes regarding wastewater treatment technologies in Macaronesia. Furthermore, lessons learned from real case studies in the Canary Islands and Cape Verde can be applied to similar regions within the Macaronesia archipelago (IDIWATER project).

Discrete Laplacian thermostat for flocks and swarms: the fully conservedInertial Spin Model

Abstract Experiments on bird flocks and midge swarms reveal that these natural systems are well described by an active theory in which conservation laws play a crucial role. By building a symplectic structure that couples the particles’ velocities to the generator of their internal rotations (spin), the Inertial Spin Model (ISM) reinstates a second-order temporal dynamics that captures many phenomenological traits of flocks and swarms. The reversible structure of the ISM predicts that the total spin is a constant of motion, the central conservation law responsible for all the novel dynamical features of the model. However, fluctuations and dissipation introduced in the original model to make it relax, violate the spin conservation law, so that the ISM aligns with the biophysical phenomenology only within finite-size regimes, beyond which the overdamped dynamics characteristic of the Vicsek model takes over. Here, we introduce a novel version of the ISM, in which the irreversible terms needed to relax the dynamics strictly respect the conservation of the spin. We perform a numerical investigation of the fully conservative model, exploring both the fixed-network case, which belongs to the equilibrium class of Model G, and the active case, characterized by self-propulsion of the agents and an out-of-equilibrium reshuffling of the underlying interaction network. Our simulations not only capture the correct spin wave phenomenology of the ordered phase, but they also yield dynamical critical exponents in the near-ordering phase that agree very well with the theoretical predictions.

Repurposing weather modification for cloud research showcased by ice crystal growth

Abstract The representation of cloud processes in models is one of the largest sources of uncertainty in weather forecast and climate projections. While laboratory settings offer controlled conditions for studying cloud processes, they cannot reproduce the full range of conditions and interactions present in natural cloud systems. To bridge this gap, here we leverage weather modification, specifically glaciogenic cloud seeding, to investigate ice growth rates within natural clouds. Seeding experiments were conducted in supercooled stratus clouds (at −8 to −5 °C) using an uncrewed aerial vehicle, and the created ice crystals were measured 4-10 min downwind by in situ and ground-based remote sensing instrumentation. We observed substantial variability in ice crystal growth rates within natural clouds, attributed to variations in ice crystal number concentrations and in the supersaturation, which is difficult to reproduce in the laboratory and which implies faster precipitation initiation than previously thought. We found that for the experiments conducted at −5.2 °C, the ice crystal populations grew nearly linearly during the time interval from 6 to 10 minutes. Our results demonstrate that the targeted use of weather modification techniques can be employed for fundamental cloud research (e.g., ice growth processes, aerosol-cloud interactions), helping to advance cloud microphysics parameterizations and to improve weather forecasts and climate projections.

Manipulating conductivity and noise for transitioning between stochastic and inverse stochastic resonances in liquid–crystal electroconvection

Noise can play a constructive role in nature and various engineering systems. Over the past four decades, noise-induced stochastic resonances (SRs) have been extensively documented, showing enhancement in system performance. Additionally, inverse SR has been observed in various systems. Typically, these resonances were studied independently. A transition between these resonances was recently observed in an alternating current-driven liquid–crystal electroconvection (EC) system using combined amplitude and phase noises. This study uses internal (material) and external (noise) parameters to demonstrate the control of this transition. Specifically, the nonmonotonic threshold voltage behavior of the EC system, indicative of the resonances, was numerically examined using additional parameters. Experimental tests were conducted to confirm the effects of these parameters. The findings reveal that the transition between these resonances can be systematically controlled to meet specific needs, whether desirable or undesirable system performances. Notably, this study illustrates how to modify the behavior of both resonances in colored noise by adjusting its cutoff frequency and steepness and phase noise, which is often overlooked. Moreover, this study provides valuable insights for various noise-related applications.

Study on the Evolutionary Process and Balancing Mechanism of Net Load in Renewable Energy Power Systems

With the rapid development of renewable energy sources such as wind and solar, the net load characteristics of power systems have undergone fundamental changes. This paper defines quantitative analysis indicators for net load characteristics and examines how these characteristics evolve as the proportion of wind and solar energy increases. By identifying inflection points in the system’s adjustment capabilities, we categorize power systems into low, medium, and high renewable energy penetration. We then establish adjustment models that incorporate traditional coal power, hydropower, natural gas generation, adjustable loads, system interconnections, pumped-storage hydroelectricity, and new energy storage technologies. A genetic algorithm is employed to optimize and balance the net load curves under varying renewable energy proportions, analyzing the mechanism behind net load balance. A case study, based on real operational data from 2023 for a provincial power grid in western China, which is rich in renewable resources, conducts a quantitative analysis of the system’s adjustment capability inflection point and net load balancing strategies. The results demonstrate that the proposed method effectively captures the evolution of the system’s net load and reveals the mechanisms of net load balancing under different renewable energy penetration levels.

Polymers as Efficient Non-Viral Gene Delivery Vectors: The Role of the Chemical and Physical Architecture of Macromolecules

Gene therapy is the technique of inserting foreign genetic elements into host cells to achieve a therapeutic effect. Although gene therapy was initially formulated as a potential remedy for specific genetic problems, it currently offers solutions for many diseases with varying inheritance patterns and acquired diseases. There are two major groups of vectors for gene therapy: viral vector gene therapy and non-viral vector gene therapy. This review examines the role of a macromolecule’s chemical and physical architecture in non-viral gene delivery, including their design and synthesis. Polymers can boost circulation, improve delivery, and control cargo release through various methods. The prominent examples discussed include poly-L-lysine, polyethyleneimine, comb polymers, brush polymers, and star polymers, as well as hydrogels and natural polymers and their modifications. While significant progress has been made, challenges still exist in gene stabilization, targeting specificity, and cellular uptake. Overcoming cytotoxicity, improving delivery efficiency, and utilizing natural polymers and hybrid systems are vital factors for prospects. This comprehensive review provides an illuminating overview of the field, guiding the way toward innovative non-viral-based gene delivery solutions.

Token-modification adversarial attacks for natural language processing: A survey

Many adversarial attacks target natural language processing systems, most of which succeed through modifying the individual tokens of a document. Despite the apparent uniqueness of each of these attacks, fundamentally they are simply a distinct configuration of four components: a goal function, allowable transformations, a search method, and constraints. In this survey, we systematically present the different components used throughout the literature, using an attack-independent framework which allows for easy comparison and categorisation of components. Our work aims to serve as a comprehensive guide for newcomers to the field and to spark targeted research into refining the individual attack components.

“Letting the ancestors speak”: collaborative archaeology at the Hastings Natural History Reservation

Since 2021, collaborative research at the University of California (UC), Berkeley's Hastings Natural History Reservation between the Esselen Tribe of Monterey County, UC-Berkeley, and the University of Oregon has demonstrated the mutual benefits of a co-created and Indigenous-centered archaeological research program within North America. While initial archaeological studies on the Hastings Reservation occurred over five decades ago, our project represents one of the first Tribal-centered archaeological field programs within the UC Natural Reserve System and, therefore, a model of collaborative research in the post-NAGPRA and CalNAGPRA era. The broader purpose of our research is to work with the Esselen Tribe to collect historical ecological information about the legacies of Native people and their relationship with the environment to help inform contemporary stewardship goals in their Tribal territory. Our archaeological research has confirmed the Indigenous occupation at the Hastings Reserve before, during, and after the Spanish Colonial period (1769–1821) in central California. By weaving archaeology, ethnobotany, ethnohistory, historical documents, ethnography, and Indigenous knowledge, this project documents the critical role of maintaining a connection to cultural landscapes despite three successive waves of European and Euro-American colonialism in the region. These findings provide an alternative narrative of cultural persistence, survivance, and resilience contrary to the narratives of culture loss and erasure espoused by 20th-century California anthropology regarding the Indigenous societies that occupied the central California coast.

Mathematical Graph Based Urban Simulations as a Tool for Biomimicry Urbanism?

Biomimicry studies natural systems and attempts to use the gained knowledge and understanding to solve human problems. Can biomimicry, if applied in urban planning, help to make our cities more sustainable or, precisely, more friendly for walkable and 15-minute city models? Various researchers identify the following features of natural systems as form fits function, catalysis of cooperation, local contextuality, continuity of development, diversity, integrity, redundancy, decentralization, multifunctionality, and less energy consumption (e.g. TOD if the energy needed for transportation is considered), hierarchy, fractality, etc. The presented research has two objectives: analyze the possibility of describing and evaluating the degree of expression of the features of natural systems in urban spatial structures to use them for comparison, benchmarking, or creation of a biomimicry index; evaluate if cities with more strongly expressed biomimicry features could be considered more walkable and close to the 15-minute city concept. To give answers to the above-presented questions Riga, Vilnius, and Tallinn were investigated. They represent both certain similarities and differences in terms of history, urban planning, political context, size, etc. A simulative mathematical graph model was used as the main tool to describe and analyze complex urban structures based on spatial configurations. Various graph centralities were employed to describe and compare the expression of natural systems’ features in the selected cities. OSM data was used to overlap graph models with such information as the density of inhabitants, concentrations of various points of interest in order to evaluate walkability and 15-minute city friendliness. The research results could be seen as an important step for better understanding of informal principles of biomimicry that can help transform urban areas.

Potential nutrient, carbon and fisheries impacts of large-scale seaweed and shellfish aquaculture in Europe evaluated using operational oceanographic model outputs

Large-scale seaweed and shellfish aquaculture are increasingly being considered by policymakers as a source of food, animal feed and bioproducts for Europe. These aquacultures are generally thought to be low impact or even beneficial for marine ecosystems as they are ‘extractive’ – i.e., growing passively on foodstuff already available in seawater, and with potential habitat provision, fisheries, climate mitigation and eutrophication mitigation benefits. At some scale however, over-extraction of nutrients or chlorophyll could potentially have a negative effect on natural systems. Understanding the likely impacts of aquaculture production at scale is important to identify when safe limits are being approached. Taking seaweed aquaculture as the primary focus, this work uses operational oceanographic model outputs to drive prognostic growth models to predict the likely optimal distribution of seaweed farms across European waters to meet different production scenarios. A novel nutrient transport scheme is then used to model the interacting ‘footprints’ of nutrient drawdown from aquaculture facilities to demonstrate the likely spatial impact of large-scale aquaculture. Evaluation of both seaweed and shellfish contributions to CO2 balance under large scale production, and the potential impact on fisheries are also considered. The study finds that the impact of intensive seaweed aquaculture on nutrient availability could be significant where many farms are placed close together; but at the regional/basin scale even the highest level of production considered does not significantly impact total nutrient budgets. Seaweed aquaculture has the potential to extract large amounts of carbon dioxide, but the impact on carbon budgets depends on the end-use of the extracted seaweed. Shellfish aquaculture is a net source of CO2 due to the impact of calcification of shells on the carbonate system (i.e., alkalinity removal). However, gram-for-gram the CO2 impact of shellfish production is likely to be less than the impact of land-based meat production. Whilst operational oceanographic models are useful for taking a ‘broad brush’ approach to likely placement and impacts of aquaculture, reliable yield predictions for individual locations across European waters would require models integrating more physical and biogeochemical factors (wave environment, local currents, riverine inputs) at a finer scale than currently achievable.

Artificial Photosynthetic Cell with Molecular Biomimetic Thylakoid.

The construction of solar-to-chemical conversion system by mimicking the photosynthetic network of the chloroplast holds great promise on efficient solar energy utilization. We developed an artificial photosynthetic cell (APC) based on molecular biomimetic thylakoid (CoTPP-FePy) to split water into hydrogen and oxygen (H2 and O2) at low driving voltage (1.1 V) and neutral condition (pH≈7). The CoTPP-FePy can emulate the light reaction in thylakoids to produce O2 by coupling light harvesting, photocatalysis, and electron/energy storage (FeIII/FeII-Py). Subsequently, a membrane electrode assembly (MEA) were employed to simulate the dark reaction, wherein the proton, electron and energy generated by the light reaction can drive the H2 producing process. By a temporally and spatially coupling of the light and dark reactions, the resulting APC achieved a solar conversion efficiency of 3.1%, exceeding that of natural photosynthetic systems and demonstrating the potential of artificial photosynthesis.

Artificial Photosynthetic Cell with Molecular Biomimetic Thylakoid

The construction of solar‐to‐chemical conversion system by mimicking the photosynthetic network of the chloroplast holds great promise on efficient solar energy utilization. We developed an artificial photosynthetic cell (APC) based on molecular biomimetic thylakoid (CoTPP‐FePy) to split water into hydrogen and oxygen (H2 and O2) at low driving voltage (1.1 V) and neutral condition (pH≈7). The CoTPP‐FePy can emulate the light reaction in thylakoids to produce O2 by coupling light harvesting, photocatalysis, and electron/energy storage (FeIII/FeII‐Py). Subsequently, a membrane electrode assembly (MEA) were employed to simulate the dark reaction, wherein the proton, electron and energy generated by the light reaction can drive the H2 producing process. By a temporally and spatially coupling of the light and dark reactions, the resulting APC achieved a solar conversion efficiency of 3.1%, exceeding that of natural photosynthetic systems and demonstrating the potential of artificial photosynthesis.

Of heroes and villains – How coalitions shape their narratives and what the public conservation debate is actually about?

Policy narrative analyses provide important insight to understand mechanisms and dynamic of policy change but also to explore how narratives shape or bind coalitions in a policy subsystem. The overall aim of our analysis was to check what are the dominant narratives about nature conservation manifested in public debate and who generates them. We applied a Narrative Policy Framework (NPF) that allowed for a systematic analysis of narratives generated in public debate on conservation in Poland. We collected and analyzed over 1200 sources of policy narratives published in the most popular nationwide newspapers in Poland between 2004 and 2020.Our results illustrate that the public discussion on conservation is focused on policy actors and their actions and beliefs rather than on scientific evidence. While wild nature itself is barely discussed, it is often positioned as an important, active actor in generated narratives. We identified two main coalitions that present different beliefs on nature and human role in natural systems maintenance. Both mutualists coalition and the coalition of traditionalists describe themselves as heroes, knowing the right solutions for conservation challenges, and their opponents as villains - impeding the effective conservation actions.The study illustrates the applicability of the NPF to explore beliefs and values expressed in policy narratives. The results highlight a polarization of conservation debate in a country in transition.

Benchmarking sample pooling for epigenomics of natural populations.

DNA methylation (DNAm) is a mechanism for rapid acclimation to environmental conditions. In natural systems, small effect sizes relative to noise necessitates large sampling efforts to detect differences. Large numbers of individually sequenced libraries are costly. Pooling DNA prior to library preparation may be an efficient way to reduce costs and increase sample size, yet there are to date no recommendations in ecological epigenetics research. We test whether pooled and individual libraries yield comparable DNAm signals in a natural system exposed to different pollution levels by generating whole-epigenome data from two invasive molluscs (Corbicula fluminea, Dreissena polymorpha) collected from polluted and unpolluted localities (Italy). DNA of the same individuals were used for pooled and individual epigenomic libraries and sequenced with equivalent resources per individual. We found that pooling effectively captures similar genome-wide and global methylation signals as individual libraries, highlighting that pooled libraries are representative of the global population signal. However, pooled libraries yielded orders of magnitude more data than individual libraries, which was a consequence of higher coverage. We would therefore recommend aiming for a high initial coverage of individual libraries (15×) in future studies. Consequently, we detected many more differentially methylated regions (DMRs) with the pooled libraries and a significantly lower statistical power for regions from individual libraries. Computationally pooled data from the individual libraries produced fewer DMRs and the overlap with wet-lab pooled DMRs was relatively low. We discuss possible causes for discrepancies, list benefits and drawbacks of pooling, and provide recommendations for future epigenomic studies.