Smog chamber experimental systems, which have been widely used in laboratory simulation for studying atmospheric processes, are comprehensively reviewed in this paper. The components, development history, main research topics and main achievements of smog chambers are introduced. Typical smog chambers in the world, including their volumes, wall materials, light sources and features, are summarized and compared. Key factors of smog chambers and their influences on the simulation of the atmospheric environment are discussed, including wall loss, wall emission and background pollutants. The features of next-generation smog chambers and their application prospect in future studies of the atmospheric environment are also outlined in this paper. This review summarizes the key features of smog chambers and their application in atmospheric process studies, bringing new insights into development directions for next-generation smog chambers. Air pollution and climate change are two of the most important ecological environmental problems that humanity must confront. Serious air pollution events have taken place in developed countries, such as the ‘London smog’ and ‘Los Angeles smog’, and are taking place in many developing countries, for example the ‘haze’ in China and India. Polluted air poses a major threat to health and ecosystems. Understanding the mechanism and evaluating the environmental impact of air pollution are crucial for policy making with regard to air pollution control and related scientific research.2.5, particulate matter with aerodynamic diameter less than 2.5 μm), SO2, NOx, NH3 and volatile organic compounds (VOCs). The cocktail of high concentrations of these pollutants, or so-called highly complex air pollution, has given rise to frequent haze pollution episodes. Despite the high intensity of primary emissions, secondary inorganic and organic species dominated PM2.5 during haze formation in terms of both mass and number concentrations [In recent years, the rapid urbanization and economic growth in developing countries, such as China and India, have led to high emissions of various pollutants from coal combustion, motor vehicle exhaust and various industrial emissions, and resulted in high concentrations of fine particles , +1% for NH3 and −27% for CO. The different change rates of pollutants also resulted in changes in atmospheric processes and particle compositions. Taking Beijing as an example, a much larger decline in sulfate than nitrate and ammonium led to a rapid transition from sulfate-driven to nitrate-driven aerosol pollution during the wintertime [This serious air pollution prompted strict air pollution controls, and remarkable achievements have been made in some developing countries. In China, a new air quality standard was set in 2012, in which the PMes, etc. , with coes, etc.  and emises, etc. . Despite8 period ,18,19, s[2 [3 [3 . Emissiontertime . These pField observation, laboratory study and numerical simulation are the three main approaches to investigating the physical and chemical processes in the atmosphere and their impact on our environment. Each of them has both advantages and disadvantages in studying the atmospheric environment. For example, field observation studies the real atmosphere directly but without reproducibility, and studies the contribution of different factors together but cannot isolate one factor and study it independently; laboratory study usually investigates atmospheric processes under well-controlled conditions but there is always the problem of whether it represents the real atmosphere; numerical simulation is helpful for integrating complex processes together, but its input and verification rely on laboratory studies and field observations. No single method can be omitted in the study of the atmospheric environment. For laboratory study, the smog chamber is well known for its ability to set experimental conditions close to those in the real environment and can also be carefully controlled. In this review, we summarize the application of smog chambers in studying the atmospheric environment in China and all over the world, discuss the factors influencing chamber simulations, and propose perspectives on future development of smog chambers.In general, a smog chamber is a reactor that simulates atmospheric processes under well-controlled conditions. A smog chamber can either be an outdoor chamber located on top of a building or a bracing structure, or an indoor chamber located in a building. The sizes of outdoor smog chambers are usually larger than indoor ones due to there being fewer space limitations. However, controlling the temperature and repeating an experiment are more difficult in outdoor smog chambers due to the significant greenhouse effects and the changing weather conditions. Typical indoor and outdoor smog chambers around the world are summarized in Tables S1 and S2 in the online Supplementary Data, respectively. They usually contain an enclosed space where the concentrations of pollutants and reaction conditions such as the temperature, relative humidity (RH) and irradiation can be controlled. Taking the indoor chamber in the Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (RCEES-CAS) as an example , a schemA smog chamber experimental system usually contains the following parts: an enclosed reactor, inlet system, light sources, temperature control system, monitoring system and auxiliary system.The reactor of a smog chamber can be made of Teflon film, stainless steel, Pyrex (borosilicate glass), quartz and so on. Teflon is the most widely used material for constructing the reactor of smog chambers, due to its good chemical stability and transmittance. A Teflon reactor also allows the operation of the smog chamber in static-state mode, i.e. simulating the reaction without adding continuous flow into the reactor, due to the fact that a Teflon reactor can collapse, to some extent, to adjust its volume with continuous sampling. Stainless steel has the advantage of reducing particle loss due to its good conductivity. The pressure in a reactor made of stainless steel can be adjusted to simulate atmospheric processes at different altitudes. Quartz could be a good material for constructing a reactor, but experience in building large reactors with quartz is still lacking. The pros and cons of different materials can be found in Finlayson-Pitts and Pitts .3 to hundreds of m3. To better simulate the real atmosphere, overly strong wall effects should be avoided. To minimize the wall effect, one can enlarge the volume of the reactor and choose inert wall materials. A large reactor volume can reduce the surface to volume ratio (S/V) and can also allow a bigger sampling volume for the monitoring instruments. However, the reactor volume is usually limited by many factors, such as the laboratory space, budget and so on. Besides, a larger volume will also increase the difficulty of maintaining homogeneous conditions in the reactor, which is usually crucial for accurately evaluating chemical mechanisms. To overcome this shortcoming in achieving homogeneous conditions, a mixing fan or mixing fans are usually placed in reactors with large volume. These fans will also help to increase the cleaning efficiency when flushing the reactor, and they can be either on or off during the reaction. In addition, mixing the reactor with fans will also increase wall deposition. In some smog chambers, the fans can mix the reactor at different speeds, aiming to strike a balance between homogeneity and wall deposition.The reactor volume of smog chambers varies from <1 mTo simulate the real atmosphere, different gases and aerosols need to be introduced into the reactor. The background clean air is usually the main component in the reactor. It can either be generated from purified air, which is usually called zero air, with concentrations of aerosol and gas pollutants as low as possible, or be made up of a mixture of nitrogen and oxygen. The air cleanliness is critical in studying chemical mechanisms, especially for experiments carried out with atmospherically relevant concentrations of pollutants. After cleaning and filling the reactor with background clean air, gas pollutants are usually introduced into the chamber from standard gas bottles at known concentrations or generated from liquid samples with a known volume through temperature-controlled tubes. Aerosols can be introduced into the smog chamber by different methods according to their physicochemical properties. The atomizer is widely used to generate aerosols, with a diffusion drier to remove water or organic solvent ,26. Besi2) and a good UV cutoff at ∼300 nm. UV lights cannot represent the visible light in solar irradiation, however, and these visible wavelengths may be important for photolysis [2 photolysis rate (JNO2), which has been reported to be from 0.1 min–1 to higher than 1 min–1 in different smog chambers (Table S1). Constant and diurnally varying light conditions also have an impact on O3 formation [To simulate photochemical processes, light sources are needed in the smog chamber. For an outdoor chamber, direct solar irradiation has always been utilized, which has the advantage of consistency with real atmospheric conditions. Indoor chambers must use artificial irradiation to simulate photochemical processes. As summarized in previous publications ,31, diffotolysis  and someotolysis . To bettotolysis . The irrormation . TherefoTemperature is one crucial parameter that influences most reaction processes. Due to the introduction of irradiation, the temperature in the reactor will increase to unrealistic values without a cooling system. Besides, reactions occurring at lower temperatures also need to be simulated, such as those in the upper troposphere. In order to simulate atmospheric reactions at precisely controlled temperatures, different temperature control systems have been designed. For indoor smog chambers, the reactor is usually placed in a temperature-controlled room or an enclosure with an air conditioner. Meanwhile, the enclosure is usually lined with clear sheeting to maximize and homogenize the interior light intensity. For outdoor smog chambers, cooling the floor of the reactor, allowing the solar light to pass through the reactor, and using two layers of film can help to reduce the temperature increase due to absorption of solar irradiation.The monitoring system is the most important part of a smog chamber system, since it determines how much we will know about the simulated atmospheric process. The monitoring system can be divided into three categories, i.e. meteorological conditions, gas phase and aerosol phase. The main instruments or technologies used in smog chamber experimental systems can be found in Table S3.3 and organic peroxides was the photochemical reactions of VOCs and NOx [In the 1940s, the Los Angeles smog began to attract the attention of researchers. Haagensmit successfully simulated the formation of this pollution with the smog chamber technique, and revealed that the main source of secondary air pollutants such as O and NOx . Smog ch and NOx . These i and NOx . The conet al. built a 6 m3 indoor smog chamber with an aluminum alloy coated with a fluorinated ethylene propylene (FEP) film [et al. [3 outdoor smog chambers with FEP film and studied photochemical reactions with solar irradiation. The transmittance of 280–460 nm light through the FEP film is >80%, which avoids the big difference in irradiation between the smog chamber and the real atmosphere. The functional testing and characterization of these indoor and outdoor smog chambers helped us understand the use of smog chambers for studying the atmospheric environment. Interference by the reactor wall was recognized.In the 1970s, researchers began to build smog chambers aimed at revealing the chemical processes in the atmospheric environment. Pitts EP) film . This ch [et al.  built tw3 and SO2. Meanwhile, due to the diversity of the ambient air used, the reproducibility of smog chamber experiments was not good. In order to minimize the interference of ambient air, purified air began to be used to clean the smog chambers in the 1980s. Adding specified pollutants into a smog chamber with a clean background allowed the study of specific atmospheric chemical reactions, such as O3 formation from the oxidation of VOCs. With the development of measurement technology of submicron particle numbers and sizes, smog chambers began to be used to investigate SOA formation, which became a research hotspot in post-1980s works [Lots of experiments on the formation of photochemical smog and secondary inorganic aerosol, initially focused on sulfate, were carried out in these chambers with ambient air. Limited by the simple monitoring instruments available, the concentrations of pollutants were relatively high, and only some normal gas pollutants were measured, such as O0s works .3 Aerosol Interaction and Dynamics in the Atmosphere (AIDA) chamber built in 1990 [3 European Photoreactor (EUPHORE) outdoor chambers built in 1995 [3 chamber built in Jülich in 1996 [3 indoor chambers built in Riverside in 2000 [3 from a single VOC, and further, to foster the development of air quality models. For example, the 256 m3 chamber built in Jülich was designed with a top that can be cooled and a bottom that can be heated. The temperature gradient then resulted in vertical mixing like that in the troposphere. This chamber was mainly used to study the NOy chemical mechanisms at night-time. Based on the results obtained with these smog chambers, several atmospheric chemical mechanisms were developed, including the Carbon Bond Mechanism (CBM), Statewide Air Pollution Research Center Mechanism (SAPRC), Regional Acid Deposition Mechanism (RADM), Regional Atmospheric Chemical Mechanism (RACM), Master Chemical Mechanism (MCM) and Common Representative Intermediates (CRI). These mechanisms are still widely used in modern air quality models and play important roles in supporting the control of photochemical pollution.In the 1990s, more advanced smog chambers were built, and in greater numbers, including aerosol chambers and indoor and outdoor photochemical chambers, such as the 84 m in 1990 ,41, the  in 1995 ,43, the  in 1996  and the  in 2000 . These cx on the reactor wall was recognized as an important OH radical source in the smog chamber. Wall losses were found to be underestimated in small smog chambers, and therefore resulted in significant underestimation of the yields of peroxides, O3 and secondary aerosols. Increasing the volume of chambers to reduce the wall effects became one obvious trend in chamber construction.In this period, wall effects, including absorption, deposition and heterogenous reactions of the reaction precursors, reactive intermediates and products on the wall, became further understood. The photolysis of HONO generated in the hydrolysis of NOAfter 2000, newly built chambers aimed at achieving lower background effects and utilizing comprehensive monitoring technologies. The low background pollutant concentrations allowed simulation of the real atmosphere even in a very clean environment, meaning these more advanced chambers obtained more accurate information on atmospheric transformation. Meanwhile, the comprehensive monitoring systems had the ability to measure precursors, some key intermediates and radicals, and complex oxidation products in both the gas phase and aerosol phase on-line. Benefiting from these advanced monitoring technologies, researchers were able to simulate more complicated reaction systems, such as the photo-oxidation of emissions from mobile vehicles, biomass burning and plants rather than the photo-oxidation of a single VOC. More attention was also given to the effects of pollutants and their atmospheric oxidation products on health, climate and ecosystems. The two most representative chambers are SAPHIR and CLOUD, which were built in 2000 and 2006, respectively, and represented the current technical level. The details of some notable chambers and main research concerns are introduced here.3 [5 Pa) and RH (0%–100%) [This chamber was made from aluminum (AlMg3), with a volume of 84 m3 . It is a0%–100%) . It was 3 each [EUPHORE had a wide involvement of institutes and advanced facility design, and was one of the most famous smog chambers in the world. EUPHORE has two FEP Teflon hemispherical reactors with a volume of 200 m3 each ,43. The 3 each .3 cuboid reactors made from FEP film [The UCR indoor chamber comprises two 90 mFEP film . These tFEP film . This ch3 [x and RO2 radicals. SAPHIR is used to investigate the key precursors and factors that determine the concentrations of radicals, the driving factors of photo-oxidant generation and degradation, the key parameters influencing the decay of trace pollutants and the physicochemical properties of aerosols. SAPHIR made serious improvements in the oxidation mechanism of semi-volatile organics and the formation of SOA, contributed to model development for ozonolysis and SOA formation chemistry, and did lots of work on evaluation of hygroscopic/optical properties and the health effects of SOA. Besides, with the help of SAPHIR, multiple monitoring technologies and instruments were developed, including those for the direct measurement of radicals . Recently, a new plant chamber facility, PLant chamber Unit for Simulation (PLUS), was coupled to SAPHIR to investigate biogenic emissions and related atmospheric chemistry [SAPHIR, which is made from two layers of FEP, is the largest outdoor smog chamber, with a volume of 280–370 m3 . The airhemistry .3 stainless steel chamber built in the European Organization for Nuclear Research (CERN). The chamber was designed to achieve high-level cleanliness. The chamber is cleaned with dry high-pressure ultrapure air at room temperature, wet ultrapure air at high temperature (373 K) and ultrapure air with a ppm level of O3. The nucleating agents in CLOUD are controlled at realistic concentration levels (ppq level) [–2), which can adjust the H2SO4 production rate in the chamber. The most unique feature of the chamber is that the ion concentrations in the chamber can be adjusted. The chamber can be exposed to either galactic cosmic rays or a 3.5 GeV c–1 secondary pion beam (π beam) from the CERN Proton Synchrotron. The beam intensity can be adjusted from 200 to 600 ion-pair cm–3, corresponding to the natural ion-pair concentration range from ground level to stratospheric values [–1) can be generated inside the chamber, which can reduce the ion-pair concentration to a few ion-pair cm–3 within a second. State-of-the-art instruments are available for use with the chamber during the experiments, including various chemical-ionization mass spectrometer (CIMS) and particle sizers to measure ions, particles and trace gases.The CLOUD chamber is a 26 mq level) ,49. Orgaq level) . The RH q level) . The UV c values . A high-et al. [3 have been developed around China to study secondary aerosol and O3 formation under various conditions, such as the 30 m3 indoor smog chambers at the Guangzhou Institute of Geochemistry, Chinese Academy of Sciences (GIG-CAS) [3 outdoor smog chamber in the Chinese Research Academy of Environmental Sciences (CRAES) [2.5 and O3 pollution in China, the development of smog chambers has become imperative to deeply understand the complex air pollution, especially in China's megacities. A total of 35 research institutes and groups in China have built smog chamber systems, of which indoor smog chambers are the main ones (http://www.bjkwnt.com/ditu/index.html). Compared with smog chambers in developed countries, the volume of chambers in China is still relatively small. Meanwhile, since one chamber is usually operated by a single small research group, the equipped instrumentation is relatively lacking, especially for the detection of radicals and intermediate products. Therefore, more high-quality smog chambers with larger volumes, equipped with state-of-the-art instruments, are urgently needed in China.Smog chamber research in China started relatively late. In the 1980s, Tang et al.  in PekinGIG-CAS)  and RCEEGIG-CAS) , and the (CRAES) . In ordeWith the development of smog chamber technology, atmospheric chemistry saw great progress. Since many review articles and books have summarized these achievements ,56–60, h3 generation has been confirmed in photochemical smog. The NO-NO2-O3 redox cycle  and SOA). The coupled NOx-HOx-ROx cycle was revealed as the key reaction scheme in atmospheric photochemical reactions. The massive coexistence of NOx and VOCs was revealed as the main cause of O3 pollution in Los Angeles smog, and these pollutants were mainly derived from vehicular emissions and industrial fumes. These scientific findings supported the control of NOx and VOCs emissions from transportation and industry in order to reduce photochemical pollution in the city.Ever since the famous flask experiments , O3 gene3 pollution is still a problem in many cities in both developing countries and developed countries. The broad array of sources of the precursors and the nonlinear response of O3 to its precursors provide a significant challenge to O3 pollution control. As VOCs and NOx were recognized as the two most important precursors for O3 formation, their relative abundance was further known to be important for determining O3 concentrations [3 formation to VOCs or NOx concentrations was calculated using the empirical kinetic modeling approach (EKMA) chemical model, which then provided the basic scientific support for the control of O3 pollution. As there are hundreds of VOCs in the atmosphere, it is crucial to know which VOCs were the most important precursors. The O3 formation potential of single VOCs has been widely investigated in smog chambers to evaluate the contribution of VOC emission to O3 formation. Alternatively, the incremental reactivity to yield O3 formation can also be tested by adding a VOC to the mixture and recording the change in O3 production. Knowledge of the contribution of speciated VOCs or emission sources based on smog chamber experiments was the precondition of an accurate control of O3 pollution.To this day, Otrations . The sen3, NO3, etc. have been developed over the past 70 years  [x, and polymerization reactions, were recognized as the main transformation paths of VOCs in atmospheric oxidation. With the help of smog chamber experiments, oxidation mechanisms of VOCs, with consideration of the key factors influencing oxidation, such as NOx level [3 and SOA formation.Large quantities of VOCs  are emitted into the troposphere from anthropogenic and biogenic sources. Understanding their chemical behaviors is crucial to predicting the atmospheric environment. Gas-phase oxidation mechanisms of VOCs initiated by OH, Otes (CI) ,63, as wtes (CI) –66, reacOx level , were reOx level ,69, are et al. [et al. [NPF is an important source of cloud condensation nuclei and has been subjected to intensive study. The picture of NPF and growth is not fuzzy anymore with the aid of chamber simulation, despite the fact that nucleation mechanisms in various atmospheric environments are still to be elucidated, which hampers the understanding of the aerosol effect on climate. Studies based on CLOUD chamber have reported the nucleation mechanism from sulfuric acid-water-ammonium, sulfuric acid-water-ammine, sulfuric acid-organic vapor-water, pure organics, and iodic acid ,70–72. Wet al.  reveal t [et al.  pointed  [et al. ,71. Thes2SO4 and its clusters leads to the enhanced growth rate of newly formed particles [et al. [After nucleation, the growth of these newly formed particles is governed by the condensation of low volatility vapors, which is also called gas-particle partitioning ,76. The articles ,78. In aarticles . Wang et [et al.  showed t2 in photochemical reactions will form sulfuric acid and sulfate, while the low-volatility organic products produced from the oxidation of VOCs will partition into the aerosol phase and result in SOA formation. For the nitrate formation, the oxidation of NO2 by OH during the day and the hydrolysis of N2O5 at night are the dominant reaction pathways. Sulfate, nitrate and SOA are important compositions of atmospheric fine particles and are still research hotspots today. It was found that hydrocarbons and oxygenates with a carbon number of >2 would have the potential to contribute to SOA formation [2 [A large amount of secondary aerosol mass was also found to be generated, accompanied by the oxidation of gas precursors, which was verified in laboratory experiments . The oxiormation . Smog chormation , SOA yieormation  and volaormation . FAC is ormation . To charormation . Besidesormation ,87. Thesation [2 . Chamberation [2  and in r 000 reaction pathways and thousands of trace species have been constructed since the 1950s [et al. [et al. [y and a series of primary emitted VOCs. Meanwhile, the smog chamber dataset was used to update and evaluate the MCM mechanisms to constantly improve the gap between model and measurement [2 in the presence of NO2 and NH3 helped to improve sulfate simulation in heavy haze [et al. [One major contribution of smog chamber data is to verify and evaluate atmosphere-related mechanisms. Detailed gas-phase mechanisms involving more than 20he 1950s . Jenkin  [et al.  and Saun [et al.  developesurement . Other csurement , RADM [9surement  and the surement  were alssurement . The devavy haze . Some fi [et al.  construcinsitu surface analysis technologies to suspended aerosols in the chamber. The interferences from the chamber wall, which usually lead to uncertainty in smog chamber results, always exist and should be carefully considered. These interferences, and the attempt to better represent real atmospheric environmental conditions, also result in a relatively complex reaction system in the chamber, and therefore it is usually not easy to quantify the contribution of a specific reaction pathway. Besides, due to wall losses, it is difficult for smog chambers to simulate atmospheric aging processes over long residence times (such as multiple days). Numerical simulation and smog chamber simulation have to work together to explore the complex processes in the atmospheric environment.There are also a few limitations when applying smog chamber results to air quality models. A smog chamber has the advantage of simulating gas-phase reactions, while it is usually difficult to explore microscopic heterogeneous chemical processes due to the difficulty of applying x and VOCs initiated a cooperative research entity with industrial or motor vehicle representatives and led to the creation of the Air Pollution Foundation in 1954 [x to reduce O3 pollution in California. These studies on the Los Angeles smog caused by the photoreaction of NOx and VOCs gave birth to the famous ‘Clean Air Act’ in the United States, administered by the US Environmental Protection Agency (EPA). This is one of the first and most influential modern environmental laws in the US, as well as one of the most comprehensive air quality laws in the world. This law promoted the development of vehicle exhaust pollution control technology. This also in turn promoted the development of major smog chamber facilities around the world to ultimately deal with the air quality. Meanwhile, the Community Multiscale Air Quality (CMAQ) model developed by the US EPA during this period has also become a universal and mainstream air quality model worldwide.The knowledge of atmosphere-related mechanisms and the further development of air quality models provided crucial support for the proposal of regulatory measures, and contributed scientific support for air pollution control. For example, smog chamber simulation of the photochemical reactions of NO in 1954 , and guiIn order to obtain a high-fidelity simulation of accurate atmospheric chemistry and kinetics, smog chambers are expected to have sufficiently low background pollutant levels, low wall reactivity and wall loss, good simulation of solar irradiation, well-controlled temperature and RH, and so on. Some of these factors and their influence on the simulation of the atmospheric environment are discussed here.6F6), sulfur hexafluoride (SF6) or acetonitrile (CH3CN), can be introduced for leak detection. FEP Teflon, which is the most widely used material for smog chambers to date, is known to allow the permeation of water molecules, and can cause leaks when the welding of FEP Teflon pieces is not tight enough. Different thicknesses of FEP Teflon (50–250 μm) have been used for different smog chambers (Tables S1 and S2). The thickness of the FEP Teflon will influence the permeation, weld and subsequent leaks in the chamber, while these differences have not been comprehensively studied as far as we know. Before a smog chamber experiment, a continuous flow of clean air is usually introduced into the chamber for cleaning purposes. Besides flushing, heating the reactor, introducing high O3 concentrations and UV irradiation were also used to remove the residual organics in the chamber. Further photochemical cleaning was also performed by using H2O2 and NO in some chambers [The background pollutant level can be minimized by purifying the background air and taking steps to reduce the introduction of ambient pollutants due to leaks or permeation. Non-reactive tracer compound, such as Hexafluorobenzene . For example, the loss of OH radicals to the wall or aerosols will be enhanced by elevated RH [3 and SOA formation. Due to the extremely high occurrence of molecular collision, it can be speculated that the loss of radicals is also greatly affected by the volume of the smog chamber due to their extremely high activity. Chambers with larger volume would have relatively lower wall loss of radicals. Organic vapor and particle loss on Teflon surfaces will cause an underestimation of particle yield [x and O3. Considering that real-time detection instruments for low-volatility or semi-volatile organics are not widely available, it is difficult to quantify or even identify their wall losses experimentally, but they can be accounted for empirically. Wall loss of SVOCs and LVOCs depends on the equivalent wall OA concentration (Cw) and their vapor saturation concentrations (Ci*) [kw) is dependent on the S/V ratio of the chamber, the degree of turbulent mixing in the chamber, the molecular diffusivity of the vapor and the mass-accommodation coefficient [kw and the S/V ratio can be expressed as follows:w is the mass-accommodation coefficient of vapors onto the chamber walls, ke is the coefficient of eddy diffusion and Dgas is the gas-phase diffusivity, which is assumed to vary with molecular weight (MW) and is equal to –5 m2 s–1.The wall loss of radicals  will greatly affect the formation of secondary pollutants  ,104. Thefficient ,105,106.–1 K–1), T is the experimental temperature, and MW is the molecular weight.For a given vapor molecule, the mean thermal speed The deposition rate of aerosol is size-dependent, and a series of methods have been developed to correct aerosol wall loss according to the available instruments. According to the test results in some experiments, aerosols with a diameter of ∼300 nm usually deposit slowest, while smaller aerosols and larger particles deposit faster due to diffusion and gravity deposition, respectively . Some prFor a better application of smog chambers, a series of comprehensive computational models were also developed to understand the complex physicochemical processes associated with the deposition of vapors on the wall and on suspended aerosols, deposition of aerosols on the wall, and coagulation and condensation of suspended aerosols –108.3 from deposited ammonium sulfate was reported to be the main reason for background NH3 [2 on the wall has been recognized as a HONO source for a long time. The photolysis of HONO will then provide OH radical and initiate the oxidation of VOCs and many other pollutants. The uptake of NO2 and HONO formation rates showed orders of magnitude differences in different chambers. For example, under irradiation conditions, the uptake constant of NO2 was reported to be as high as 10–2 s–1 with a HONO yield of 10% in a 4.2 m3 stainless steel chamber [–5 s–1 in large Teflon chambers (24 and 27 m3) [2 [5–107 molecules cm–3 s–1 [2 and water was postulated to be responsible for the formation of HONO [2, the photolysis of nitrate adsorbed on the surface was also postulated as a HONO source [Besides acting as deposition sinks for pollutants, walls can also act as emission sources. The pollutants deposited on the wall can partition back to the gas phase when the gas wall equilibrium changes. For example, the emission of NHound NH3 . More im chamber , while id 27 m3) ,110. Thed 27 m3) , and wasd 27 m3) . Smog chd 27 m3) . The pho7 m3) [2 . With ircm–3 s–1 ,115. In cm–3 s–1 , and the of HONO . The pho of HONO . BesidesO source , while tSmog chambers have been built worldwide and will continuously make significant contributions to the study of the atmospheric environment and the improvement of air quality. With more than 70 years’ development, current smog chambers are already successful in some specific investigation directions, although the technologies can always be further improved. Despite the progress in atmospheric chemistry related to smog chambers, there are also emerging problems in our atmospheric environment that the developed mechanisms could not fully explain, such as the temperature increase in outdoor chambers due to the greenhouse effect, underestimation of wall effects, limited knowledge about the chemical composition of the generated aerosols, and the difficulty in detecting intermediate compounds, such as radicals and clusters. No heavily invested large-volume chambers have been built since 2010. For next-generation smog chambers, more diversified simulation would be expected with more advanced and more comprehensive simulation technologies, and the following development directions may need to be considered.To simulate atmospheric chemical processes under conditions as close as possible to those in the real atmosphere, a reactor with a large volume is needed to minimize wall effects, including both deposition and heterogeneous reaction on the wall. The large volume will also help in longer-duration simulation, for example, longer than one day, which is important for studying the atmospheric aging process. A well-designed mixing system should be introduced to ensure homogeneity in the chamber and to overcome the potential accumulation of air stratification, as summarized by Hidy .The background pollutant concentrations should be controlled at minimal levels. The influence of the background pollutants on the investigated reaction system should be well characterized.Experimental conditions should be controlled as much as possible to conform to those in the real atmosphere. The pollutant reactants as well as secondary reaction products should be constrained within concentration ranges in the real atmosphere. One should try to use ambient irradiation or at least well-characterized artificial irradiation sources, or both. The temperature, RH, and air pressure in the chamber should be well-documented.The ability to accurately detect relevant species is crucial for revealing atmospheric processes. An advanced monitoring system, including detection of gases, radicals, ions and particles, should be developed according to the properties of the chamber facility and the experimental purposes. The chamber should be designed to be compatible with the fast development of instruments.As pointed out by Hidy , smog chAs mentioned earlier, field observation, laboratory study and numerical simulation are the three main approaches to investigating the physical and chemical processes in the atmosphere and their impacts on our environment, with both advantages and disadvantages for each. Chamber study should work closely with field observation and numerical simulation. The experimental conditions should always be comparable to those observed in the field, and the experimental results should be validated as much as possible with field observations. Applying smog chamber results in numerical simulation is the most common method to evaluate the importance of a laboratory finding. Therefore, with input from observations, simulation in smog chambers serves the development of air quality models, which in turn will be validated by observations, forming a closed chain for studying the atmospheric environment. In addition, it is highly recommended that a ‘numerical chamber’ associated with the smog chamber is developed, with knowledge of the wall effects, background impact and possible dilution and leakage considered. Comparisons between the mechanistic calculations and smog chamber measurements will provide detailed evaluation of chemical mechanisms and should be quite helpful in identifying accurate mechanisms and kinetics from smog chamber simulations.The next-generation smog chamber, with more advanced simulation technology, more comprehensive functions and closer cooperation with observation and numerical simulation, will certainly prove its mettle in supporting the continuous improvement of air quality in the world.nwab103_Supplemental_FileClick here for additional data file.


Lensless holographic microscope (LHM) is an emerging very promising technology that provides high-quality imaging and analysis of biological samples without utilizing any lens for imaging. Due to its small size and reduced price, LHM can be a very useful tool for the point-of-care diagnosis of diseases, sperm assessment, or microfluidics, among others, not only employed in advanced laboratories but also in poor and/or remote areas. Recently, several LHMs have been reported in the literature. However, complete characterization of their optical parameters remains not much presented yet. Hence, we present a complete analysis of the performance of a compact, reduced cost, and high-resolution LHM. In particular, optical parameters such as lateral and axial resolutions, lateral magnification, and field of view are discussed into detail, comparing the experimental results with the expected theoretical values for different layout configurations. We use high-resolution amplitude and phase test targets and several microbeads to characterize the proposed microscope. This characterization is used to define a balanced and matched setup showing a good compromise between the involved parameters. Finally, such a microscope is utilized for visualization of static, as well as dynamic biosamples. Light microscopy is one of the most employed techniques for analysis in biomedicine, mainly due to its capability to provide images of microscopic specimens using a real-time and non-invasive operation principle. In that technique, high-quality imaging is typically provided by optical compound microscopes. However, such microscopes are relatively bulky, and often include complicated lens systems which can significantly increase their prices. Those aspects can negatively restrict their use only to advanced laboratories and/or make them neither field-portable nor affordable in practice for developing countries . By contLensless holographic microscopy (LHM) arises from the digital implementation of Gabor holography ,24. In LFOV), and resolution when using objectives of medium numerical apertures (NAs) (~0.4–0.5 NA) [FOV is practically equal to the size of the sensitive area of the digital sensor. By contrast, the resolution is usually modest incoming from relatively low NA values (~0.2 NA) and limited by the pixel geometrical constraints, and not by diffraction unlike DIHM. Nonetheless, several pixel superresolution approaches were also implemented in order to decrease the effective pixel size [LHM is usually implemented using two opposite arrangements with particular significance, depending on the position of the sample between the illumination point source and the digital sensor. When the sample is located in close proximity to the illumination source but far from the digital sensor plane, the microscope implements a digital in-line holographic microscopy (DIHM) configuration ,25,27. I–0.5 NA) , althoug–0.5 NA) ,36,37. O–0.5 NA) ,9. On-chxel size ,38,39. Fxel size ,40. FOV, and extended depth of field (DOF) are interrelated and discussed into detail, comparing the experimental results with the expected theoretical predictions for different axial positions of the sample. Moreover, we utilize the microscope to characterize static as well as dynamic biosamples. Hence, Due to its strong practical potential and applicability, LHMs have been mainly proposed under an application-oriented perspective, that is, validation of their capabilities to monitor/measure/analyze/quantify/etc. different items  but aimed at specific applications. However, theoretical–practical analysis and characterization of the performance in a LHM is not as common in the literature where only a few references partially cover these issues ,21. For NA focusing lens. The light emerging from such a lens is focused on a point located less than 1 mm away from the lens, creating a quasi-point light source. In order to define a DIHM configuration, the sample is placed in close proximity to such a spherical divergent illumination source at a distance z1. The interference pattern generated by the coherent overlapping between the light scattered and not scattered by the object is recorded by a monochrome digital sensor , thus creating an in-line digital hologram at a distance Z away from the divergent illumination. Our lensless microscope is presented in In the picture included in x,y) the spatial coordinates at the recording plane, then the intensity distribution in such a plane is given by,The mathematical principles of our lensless holographic microscope are based on DIHM. In DIHM, the object is illuminated with a spherical divergent beam. Part of this light is scattered by the object, considered as object beam, whereas the other part is not, forming the reference beam. Then, both beams interfere at the recording plane, creating an in-line digital hologram. Assuming that the complex amplitude of the object and reference beams are The complex amplitude distribution at the object plane can be reconstructed by digital propagation of the hologram using scalar diffraction theory . Howeverod (ASM)  to propa2 is the distance between the recording and object planes (see k is the wavenumber z2 limitation [In Equation (2), anes see a, kx andmitation .FOV, NA, and lateral and axial spatial resolutions. In the following, we introduce the mathematical expressions that define such parameters for a lensless microscope. All optical microscopes are partly characterized by a set of different optical parameters that give an idea of the aspect and quality of the images provided by them. Perhaps the most significant parameters when describing a microscope are lateral magnification, M comes from the geometric projection of the sample from the point source onto the recording plane. Hence, M only depends on the ratio between the axial distances z1 and Z according to the expression [In DIHM, the lateral magnification pression ,(3)M= ZFOV is directly related to M and the sensor area of the digital camera, and can be calculated asN being the number of pixels and p being the pixel size of the camera.By contrast, the ρ in DIHM, it can be limited by several factors, such as diffraction, spatial aliasing, limited spatial and temporal coherence of the source, or reduced signal-to-noise ratio (SNR) [λ of the illumination source and the NA of the imaging system in the way,k is a constant parameter that takes different values for incoherent (k = 0.61) and coherent (k = 0.82) light, and for near-field imaging (k = 1), all of them given for circular apertures and following the Rayleigh criterion [a being the radius of the circular aperture, and z being the distance from such an aperture. For our specific case, where z~15 mm, λ = 405 nm, and a~6 mm, the Fresnel number is F~6000 >> 1. Hence, we will consider that k = 1 in our case, despite being our imaging system limited by the sensor area (square aperture). Additionally, NA is directly related to half of the size of the sensor area (Np/2) and its distance z2 with the object plane as [Regarding the lateral resolution io (SNR) . Here, writerion . DIHM utplane as ,(6)NA=sOn the other hand, the geometrical parameters of the digital sensor can also reduce the resolution achieved by the lensless microscope, according to the Nyquist-Shannon sampling theorem . Thus, aNA and is given by the following expressionIn addition, the axial resolution is inversely proportional to the squared Finally, the proposed lensless holographic microscope also allows phase imaging. Numerical propagation of a digital hologram provides a complex amplitude distribution, containing not only intensity but also phase information of the light field in a given plane. Thus, if we propagate towards the image plane of a phase object, we can achieve the phase delays introduced by the different elements of that object, which is directly related to the thickness and the refractive index (RI) of such elements, according to the expression,Three different samples, namely, synthetic microspheres, fixed prostate cancer cells, and live sperm cells, are prepared following different procedures. On one hand, the polystyrene microbeads with 3 μm in diameter are first homogenized, and second placed in an Eppendorf tube, where they are highly diluted into water (1:100). After that, we pipette one drop of the solution on a microscope slide and another one on a cover glass. Once the water is evaporated and the samples are completely dry, we build a lab-made chamber where both surfaces containing microspheres are facing each other and separated by 1 mm.2 in a humidified incubator. Once the cells reach a confluent stage, they are released from the culture support and centrifuged. The supernatant fluid is discarded by centrifugation, and the cells are resuspended in a cytopreservative solution and mounted in a microscopy slide.On the other hand, two different types of prostate cancer cells (PC-3 and LnCaP cell lines) are prepared following a similar procedure. The cells are cultured in a RPMI 1640 medium with 10% fetal bovine serum, 100 U/mL Penicillin, and 0.1 μg/mL streptomycine at standard cell culture conditions (37 °C) in 5% COFinally, a human commercial insemination dose from Proiser R+D S.L. is received after conventional refrigerated transport (less than 24 h). After homogenization of the dose, 1 mL is placed in an Eppendorf tube, maintained for 20 min at 37 °C. Next, the semen sample is inserted by pipetting into a commercially available counting chamber (100 μm thickness). FOV ). The target area is a quartz substrate with opaque lines of chrome with 100 nm in thickness. In this process, we obtain the calibration curves concerning lateral magnification , FOV . Indeed, both the experimental results and the theoretical values are directly derived from it, whose experimental errors are calculated using the error propagation formula, Mδ the experimental error of the magnification M. The dependence of the FOV with the distance z1 is presented in FOV increases quadratically with the distance z1, approaching to 0 mm2 when z1 → 0 mm. This behavior can be also seen when combining Equations (3) and (4). Furthermore, the experimental values perfectly match with the theoretical ones.The quantification of the ρ of the microscope as a function of z1, we perform the following procedure: (1) we visualize which elements of the test target are close to the resolution limit at best focused image; (2) we perform an intensity profile along the perpendicular direction of such elements; (3) we quantify the intensity differences between the chromed elements, their immediate background, and the external background; and (4) when the central peaks (immediate background) are found to rise about or more than 27% [For the analysis of the lateral resolution than 27%  above thz1 = 0.7 ± 0.1 mm as well as the calibration curve. Here, the best focused image is shown in z1 is a value between 1.74 μm (G9-E2 resolved) and 1.55 μm (G9-E3 not resolved). The final resolution limit considered here is the intermediate resolution value (ρexp = 1.65 ± 0.09), whereas the uncertainty in the measurement is the difference between such values.z1, we then represent in a single plot  to 3.7 μm (z1 = 5 mm). By contrast, the experimental resolution limit is progressively increasing from 1.65 ± 0.09 μm to 3.7 ± 0.2 μm when changing from z1 = 0.7 ± 0.1 mm to z1 = 4.7 ± 0.1 mm. Moreover, those experimental values are above these theoretical ones and sometimes differ significantly from them. A reason for such a discrepancy could be because the test target utilized for the resolution analysis does not fully satisfy the Gabor’s condition. Since the USAF test target presents different chromed elements of different sizes, it could be that some of them block a considerable part of the reference light necessary to have interferences of high frequency, thus reducing the number of interference fringes employed for reconstruction and eventually the resolution. In addition, the presence of the twin image may also hinder the resolution analysis. In any case, and leaving aside the discrepancy between theoretical and experimental values, the experimental trend has a reasonable behavior since it approaches the geometrical resolution limit as z1 increases. Once the procedure is performed for all gle plot c the latz1. For that purpose, we first find the best focused image for each z1, setting this axial position as Δz1 = 0 ± 2 μm, where 2 μm is the axial digital step between propagated planes considered here. Next, we digitally propagate such focused image backward and forward until the test target element defining the lateral resultion limit will be lost. Here, we use the same resolution criterion as in z1 is taken as the maximum numerical propagation distance that still allows to resolve the test element defining the resolution limit at the distance z1. z1 = 3.2 z1. Hence, the axial resolution for this z1 is ρaxial = 20 z1, we can obtain the full set of experimental ρaxial values, and the results are depicted in The axial resolution of our lensless microscope is also experimentally analyzed with respect to the distance 1 mm see a,b. FiguNA considered in this equation is an effective NA, which is computed from Equation (5) considering the lateral resolution values achieved in z1.These values are then compared with the estimated values when considering Equation (8). In this case, the A major advantage of holographic microscopy in comparison to conventional microscopy is the obtention of an extended DOF. In a compound microscope, the DOF decreases quickly when improving resolution limit, requiring mechanical refocusing to visualize/analyze a large volume. On the contrary, in holographic microscopy, once a digital hologram has been recorded, one can visualize/analyze different objects located at different axial planes by digitally refocusing the recorded optical field, so that three-dimensional (3D) information can be extracted from a two-dimensional (2D) recording/digital hologram .z1 = 0.7 ± 0.1 mm and 1.7 ± 0.1 mm, respectively, for the region enclosed in the yellow rectangle included in In order to validate this capability for our lensless microscope, we have prepared a sample consisting of two layers of 3 μm diameter beads that are axially separated at two different planes see . Under tz1 distance to be retrieved as long as the weakly diffractive assumption and the requirements of magnification and resolution are fulfilled. Another aspect clearly visible in those reconstructed images is the variation of the lateral magnification. The microspheres located at z1 = 0.7 mm are magnified with a 21.5X, whereas a magnification of 8.8X is considered for the ones placed at z1 = 1.7 mm, according to the calibration curve provided in z1. Looking at z1 = 2.2 ± 0.1 mm, and has a nominal thickness of 50 nm according to the manufacturer. Here, the recorded in-line hologram is presented in The demonstration of phase imaging with our microscope is performed involving a phase test target. Such a phase target is made by Benchmark Technologies and includes several structures with different thickness raised on a Corning Eagle XG glass substrate, having a RI ~ 1.52 for the wavelength of the microscope. Moreover, the thickness of the different elements is perfectly established by the manufacturer, so it is perfect for phase imaging validation. Regarding the quantitative phase values measured for such structures, these values will be directly affected by the phase disturbances induced by the twin image presence, modifying their values accordingly. However, we can do a rough comparison between the experimental values obtained in NA microscope objective for comparison. Thus, intensity and phase images retrieved by our lensless microscope are included in Next, the lensless microscope is utilized for imaging several fixed and undyed biosamples. More concretely, the biosamples are different types of prostate cancer cells (PC-3 and LnCaP cell lines). The cells are prepared as described in NA microscope lens, but with the advantage of having not only intensity but also phase information, which is crucial in biosamples for label-free high-contrast imaging.Looking at the images provided by the lensless microscope, we can visualize the different cellular types. Moreover, magnified areas (yellow squares) including only a few cells serve to identify internal structures, such as nucleus inside the cells. On the other hand, images recorded by the optical compound microscope reveal that such internal structures are indeed present in the cells, and they are not artifacts caused by the twin image or other noise sources. That means our holographic microscope can also image some inner components of the cells, such as the compound microscope using a medium Finally, the proposed lensless microscope is employed to visualize and track a live human sperm sample. Such a biosample is prepared as described in ρtheo~1 μm, ρexp~1.65 μm) in real time and without the use of any lens for imaging. Furthermore, the lateral magnification and FOV can be modified by simply changing the axial position of the sample. Moreover, two major advantages of the lensless holographic microscope in comparison to conventional compound microscopes are the extended DOF and phase imaging capability. On one hand, the DOF is usually small (~μm) in a compound microscope and decreases sharply with improving resolution, so that the visualization of a large volume requires of refocusing to different planes. By contrast, in DIHM, a single 2D hologram can be numerically propagated to different planes in order to produce a 3D image of the whole sample volume [The presented lensless holographic microscope includes all optical, mechanical, and electronic components in a small quasi-cubic structure (20 × 16 × 13 cm), being easily transportable in a backpack or a bag. In addition, such a microscope is able to obtain images with a lateral resolution up to 1.65 μm  since they work with a magnification equal to 1. By contrast, although our microscope is compact, portable, and cost-effective, there exist other technologies that are smaller [3, 14.9 × 11.2 × 12.2 cm3 and 7.0 × 6.5 × 6.5 cm3, respectively, or proposed by Ozcan’s group in [3. On the other hand, although our microscope is very light (around 500 g), there exist lighter LHMs, having a weight of less than 280 g [When comparing our microscope with the existing LHM technologies, it compares favorably in several aspects. For instance, due to its optimized DIHM configuration and optical components, our system experimentally demonstrates higher lateral resolution than most of the reported LHMs ,20,21,22on value ,18,21,22on value ,8,12 pre smaller ,21,22, l smaller ,8,12,21, smaller ,21,22, wFOV, lateral resolution, etc., without exploiting the versality of lensless configurations to easily provide either higher magnification and resolutions or bigger FOVs. In addition, some of those contributions were mainly focused on the biological applicability of such LHMs, rather than to provide an in-depth characterization of the optical parameters as a function of the axial position of the sample. Some of these applications were the analysis of cells [FOV as a function of the distance, but they did not provide neither experimental validation of them nor comparison between theoretical and experimental values. On the other hand, in [FOVs achievable by their LHM, and experimentally demonstrated those parameters for only one  or three axial positions (FOV). By contrast, in our contribution, we have conducted a much more detailed and rigorous analysis, not only of those parameters, but also of magnification and axial resolution, providing theoretical and experimental values and comparing them for several distances, covering a broad range of axial positions.Another crucial aspect to consider here is the thorough analysis performed of the principal optical parameters of our microscope. It is worth noting that most of the previous contributions reporting on LHMs ,18,20,22of cells ,18,20, tof cells , just toof cells ,21 partiof cells , the autFOV, and lateral and axial resolutions have been analyzed into detail for different axial positions of the sample by comparing experimental results with the theoretical predictions. Such a lensless microscope has been finally applied for bioimaging involving different types of not only static, but also dynamic, biosamples. The whole analysis performed here may serve as a guide for future developments of other lensless holographic microscopes based on DIHM principle. Due to its reduced size and price, this microscope appears as a great tool to be used in laboratories with limited sources and/or in remote areas, which has applications in sperm assessment, microfluidics, or point-of-care diagnosis, among others.Hence, in this contribution we have presented a complete analysis of a robust, cost-effective, and high-resolution DIHM for biomedical applications. Several major features such as lateral magnification,


Captive breeding programmes represent the most intensive type of ex situ population management for threatened species. One example is the Cuvier’s gazelle programme that started in 1975 with only four founding individuals, and after more than four decades of management in captivity, a reintroduction effort was undertaken in Tunisia in 2016, to establish a population in an area historically included within its range. Here, we aim to determine the genetic consequences of this reintroduction event by assessing the genetic diversity of the founder stock as well as of their descendants. We present the first whole‐genome sequencing dataset of 30 Cuvier’s gazelles including captive‐bred animals, animals born in Tunisia after a reintroduction and individuals from a genetically unrelated Moroccan population. Our analyses revealed no difference between the founder and the offspring cohorts in genome‐wide heterozygosity and inbreeding levels, and in the amount and length of runs of homozygosity. The captive but unmanaged Moroccan gazelles have the lowest genetic diversity of all genomes analysed. Our findings demonstrate that the Cuvier’s gazelle captive breeding programme can serve as source populations for future reintroductions of this species. We believe that this study can serve as a starting point for global applications of genomics to the conservation plan of this species. RoH represent tracts of the genome that are homozygous due to the inheritance of equal haplotypes from a common ancestor  is a medium‐sized antelope species endemic to the mountain and hill ranges of the Maghreb (northwest Africa), with neighbouring ranges in Morocco, Algeria and Tunisia, where it inhabited the Tunisian Dorsal. Its historical range once spanned from the Mediterranean and Atlantic coast to the northern border of the Sahara . The remaining males were housed in individual enclosures for mating in the following breeding seasons , the population increased more than expected as indicated by the number of females giving birth, the number of offspring born and those surviving to 30 days from the first (2017) to the third (2019) breeding season  from four founders: 1 male and 3 females  evaluate the impact of a Cuvier's gazelle reintroduction event on the genomic diversity of their descendants, (ii) estimate and compare inbreeding levels using both genomic and pedigree data and (iii) determine the genetic differences between gazelles from two populations: one managed and one unmanaged. To accomplish these objectives, we have sequenced the genomes of the first and second cohort individuals born after the reintroduction in Tunisia and compared these to the genomes of the reintroduction founders as well as to an unrelated captive but unmanaged population.22.1We included samples from two different Cuvier’s gazelle populations. One population is located in Tunisia  as part of a reintroduction project initiated in October 2016 from captive‐bred founders  originated from two captive populations in Spain . To facilitate rewilding and to minimize risk of death , calves were not tagged  of Cuvier's gazelle were born in Tunisia (Jardin Zoologique de Rabat (Morocco).In contrast, the MOR population had no human intervention in mating decisions and although it is kept in a fenced area, we consider it to be an unmanaged but captive population. These Moroccan individuals were unrelated to those reintroduced and consequently to those animals born in Tunisia. This population was founded in 1997 with seven founders  from the We obtained whole blood samples and sequenced a total of 30 individuals from these two populations Table . Specifi2.2We extracted genomic DNA from whole blood samples using either the Qiagen DNeasy Blood and Tissue Kit (Qiagen), the MagAttract HMW Kit (Qiagen) or the GEN‐IAL All Tissue DNA kit (GEN‐IAL) following manufacturer's protocols Table . Then, w2.3Nanger dama) reference assembly (NCBI GenBank accession GCA_019969365.1) using BWA‐MEM (version 0.7.12)   to the corresponding libraries as well as to remove duplicates (MarkDuplicates), we used the Picard software suite (version 1.95)  software package  segmentation method. A 3‐state HMM with normally distributed emissions was fitted for each sample, where the state with the lowest heterozygous position density corresponded to the RoH segments and the two remaining states captured any other heterozygosity fluctuations occurring in the genome. For that purpose, the HMM emissions and transitions were fitted as follows: (a) prior emission probabilities were defined by partitioning any heterozygosity windows containing <60,000 noncallable bps into three clusters using the skit‐learn v0.21.2 implementation of the unidimensional k‐means algorithm in python 3.5.2 . For these analyses, we only indicated the significant differences based on the p‐adjusted value of each test.For the comparison of genome‐wide heterozygosity between groups, we used a Tukey Honest Significant Difference test (tukey_hsd from the rstatix R package). To test the significance of the difference in the proportion of the genome in RoH between groups, we used a Wilcoxon test  from the ggpubr R package   . For this, we generated three random subsets of gazelles for each of these two groups excluding the SPA‐CAN gazelle from any of the Founder subsets.The site frequency spectrum (SFS) was estimated using ANGSD  was calculated from the studbook pedigree including all the gazelles in the breeding programme   and the BCFTools/RoH estimates (FROH2). FROH is the ratio between the length of the genome inside RoH and the total length of the autosomes. Spearman's rho coefficients for all possible pairs (rcorr (type = ‘spearman’) from the Hmisc R package; https://hbiostat.org/R/Hmisc/) were calculated to test the correlation between the different inbreeding estimates. Since we only have FPED estimates for 15 gazelles, its correlation with the other inbreeding estimates only considers these 15 individuals. On the other hand, for the remaining genomic inbreeding estimates the correlation was estimated using all the study gazelles (N = 30). We used the Wilcoxon test  from the ggpubr R package) to study whether there were significant differences between groups in each inbreeding coefficient and we only retained and report the ones that were significant.Within the EEP population, the inbreeding coefficient (F2.7N = 13) and two gazelles from the Offspring   Figure .33.1We evaluated the genomic diversity and population differentiation through different cohorts of a reintroduced Cuvier's gazelle population originated from an initial ex situ captive breeding programme. We generated whole‐genome sequences for a total of 30 individuals to an average depth of coverage of ~7× (4.77×–14.60×) Table , which h(SPA‐ALM vs. SPA‐CAN) = 0.02 and Weighted Fst(Founders vs. Moroccan) = 0.258), even though we only have one individual from SPA‐CAN. To remove any potential bias introduced by having related individuals in the PCA, we performed this analysis including a single gazelle of each group . This PCA shows a similar clustering of the gazelles as the one including all individuals   Figure . The secs Figure . The str) Figure .3.2Moroccan = 2.1 × 10−4) compared to the individuals born in Tunisia . The median genome‐wide heterozygosity in the offspring of the reintroduced gazelles is similar to that of the Founder gazelles . When separating the Offspring gazelles into F1 and F2, we observe the highest genome‐wide heterozygosity levels in the first cohort , which significantly differ from all the other sampled individuals. We observe that heterozygosity is variable among different scaffolds; Hi‐C_scaffold_10 has the highest heterozygosity levels, while scaffolds 6 and 18 have the lowest , 521.875 ± 37.64 (F1), 580 ± 20.73 (F2) and 620 ± 21.54 (Moroccan) events per gazelle group. Moroccan gazelles have a significantly higher proportion of RoH compared to the other gazelles, while there is no statistical difference between the Founder and the Offspring groups Figure . BreakinWe also analysed RoH with the publicly available software BCFTools/RoH, a different HMM‐based method. The RoH estimated by this method are highly fragmented and therefore shorter, which results in about half the genomic coverage of RoH as our custom HMM method. Nonetheless, both methods have a significant positive correlation Figure .Runs of Homozygosity are differentially distributed along the Hi‐C scaffolds among individuals and groups of gazelles  Figure A. After 3.3PED) and any other genomic inbreeding estimates   Figure . In contem Table . All genem Table .Kinship analysis confirms that Moroccan gazelles are not related to any of the other gazelles but they show high levels of relatedness amongst themselves Figure . Overall4Genomic approaches are becoming increasingly used by captive breeding programmes to analyse the levels of genetic diversity and inbreeding of captive‐bred animals. They can be applied to study the progress of reintroduction programmes on the recovery of an endangered species. To assess the genetic diversity of the cohort born after the reintroduction of 43 Cuvier's gazelles back into the wild, we have sequenced 30 complete genomes, representing the first genomic dataset available from this species. Our study includes a subset of captive‐bred gazelles that were reintroduced into Tunisia as founders, as well as some of their offspring. These gazelles are compared to one another and to an unmanaged Cuvier's gazelle population in Morocco.Nanger dama). Recent studies have shown the usefulness of employing assemblies from other species for conservation management  where both founder populations are clearly separated. Overall, our results suggest that mixing the SPA‐ALM and SPA‐CAN gazelles, which had no gene flow for two generations, has avoided the loss of genetic diversity and even resulted in a slight increase in genome‐wide heterozygosity in the F1 Offspring cohort. Our findings are in line with previous publications stating that genetic diversity in several endangered species can be increased, by including individuals from different populations  or even reduced (FHOM).Levels of inbreeding play a fundamental part in species survival and the success of reintroductions. Keeping a complete and accurate pedigree of a species in captivity (Ito et al., We performed a kinship analysis to further examine the underlying relationship between gazelles and confirmed that the Moroccan gazelles are unrelated to the other gazelles studied. For the offspring group, we hypothesize that G15 and G19 are dizygotic twins from inbred parents because they exhibit the most extreme levels of shared RoH in our dataset, and the coefficient of kinship for this pair is higher than for any other pair of gazelles, even parent‐offspring relationships. From the field records of the reintroduction programme (Moreno et al., In this study, we showed how genomic tools are useful to assess the genomic composition of individuals that are part of an ex situ conservation project and their offspring. In this particular case, we have assessed the consequences of a reintroduction event after it was carried out. The characterization of the genomic status of the founder gazelles and the first offspring born after the reintroduction sets the grounds for future projects of genetic monitoring of the descendants of this initial release. When possible, future conservation plans could go one step further and use genomics not only to evaluate the success after the reintroduction has already been carried out but also to provide further information for the selection of the best‐fitted animals as founder stock, together with other indicators that may be used to select individual animals for reintroduction. It would have been very interesting to compare the diversity and inbreeding of the founders of the captive population in La Hoya, founded in 1975, to the diversity of the gazelles used as Founders in Tunisia. Sadly, the only remaining samples from the gazelles from the former population were skins chemically treated for preservation in a museum, which made DNA retrieval impossible. Sampling of specimens is advisable for endangered species whenever possible. Blood and skin samples from animals in the La Hoya Field Station are stored in a Genetic Rescue Bank that was started in 2003.Considerable uncertainty exists regarding the ability to maintain genetic diversity in captive‐bred populations in long‐term management programs and the suitability of these animals for the re‐establishment of populations into the native historical range of a species. Altogether, we have demonstrated that captive‐reared individuals of EEP Cuvier's gazelle were suitable as founders for a reintroduction in Tunisia during 2016, as proven by both animal survival and other fitness traits (Moreno et al., The authors declare no conflict of interest.Fig S1‐S12Click here for additional data file.Table S1‐S3Click here for additional data file.


Environmental variation related to ecological habitat is the main driver of plant adaptive divergence. Longitude plays an important role in the formation of plant population structure, indicating that environmental differentiation can significantly shape population structure.C. dactylon (L.) Pers. (common bermudagrass) individuals were sampled from 13 geographic sites along the longitude (105°57′34″–119°27′06″E).Genetic diversity and population genetic structure were estimated using 105 expressed sequence tag-derived simple sequence repeat (EST-SSR) loci. A total of 249 C. dactylon along longitude gradients. Significantly positive Mantel correlation  was found between genetic distance and geographical interval while no significant partial Mantel correlation after controlling the effect of mean annual precipitation, which indicated geographic distance correlated with mean annual precipitation affect genetic distance. The genetic diversity of C. dactylon with higher ploidy level was higher than that with lower ploidy level and groups of individuals with higher ploidy level were separated further away by genetic distance from the lower ploidy levels. Understanding the different genetic bases of local adaptation comparatively between latitude and longitude is one of the core findings in the adaptive evolution of plants.There was no obvious linear trend of intra-population genetic diversity along longitude and the intra-population genetic diversity was not related to climate in this study. Low gene flow (Nm = 0.7701) meant a rich genetic differentiation among populations of  Plants will undergo adaptive genomic and phenotypic changes in response to environmental heterogeneity on a spatial scale . IdentifCynodon dactylon (L.) Pers.) is a genus of the tribe Chlorideae (Poaceae), which inhabits most countries and islands, usually in between about 45°N and 45°S and penetrates to approximately latitude 53°N in Europe , which is a crucial step of studying genetic evolution  quantify population genetic diversity of 249 individuals from 13 populations ranging from different longitudes using EST-SSRs; (2) estimate the spatial population genetic structure and gene flow along longitude; (3) test the relationship between ploidy levels and genetic diversity; (4) compare genetic pattern between longitude and latitude. Studying genetic variation of the wild C. dactylon along longitude gradients can promote broadening the genetic resources, understanding the genetic process of adaptation to environmental variation along with longitude and providing some valuable information for the better utilization of wild germplasms of different ploidy levels in breeding.The lack of genomic information makes genotyping difficult, but molecular markers enhance the study of plant genetic diversity . SSRs arvolution . Althougus study . SamplinC. dactylon individuals were sampled at 13 different geographic origins locating 105°57′34″–119°27′06″E, China , 0.5 µL each of forward and reverse primers, 0.2 µL of Taq DNA polymerase, and 17.8 µL of nuclease-free water.The 20 EST-SSR primer pairs from previous research  producedThe amplifications were performed with the following PCR protocol used in the previous research : first 1Genetic diversity for all populations and ploidy levels was calculated using POPGENE V. 1.32 Software . These iC. dactylon at different spatial scales was also investigated using NTSYS pc version 2.0  between observed and expected mismatch distributions  and g is the generation time in years (1 year was used as an approximation for g according to age of first reproduction of C. dactylon). Fu’s FS , per year ), k is t Fu’s FS  and Taji Fu’s FS  neutraliF = 0.027, P = 0.974). However, populations 4, 5, 6 and 8 at mid-longitude and populations 1, 12, 13 at lowest and highest longitude had higher genetic diversity than other populations according to I and He (P > 0.05) between intra-population genetic diversity and climate , suggesting that temperature and precipitation showed little influence on intra-population genetic diversity along longitudes.The genetic diversity parameters including the PIC value, He and I were calculated for EST-SSR markers in 13 populations . The PICI and He . In gener = 0.276, P = 0.022). Environmental measures along longitude gradients ; mean annual precipitation ) were correlated with genetic distance, while mean annual temperature was not . However, a partial Mantel test accounting for mean annual precipitation was used to estimate that genetic distance was not independently correlated with geographic distance (P = 0.146). Gene flow (Nm) among these C. dactylon populations along longitude gradients was 0.7701 (<1), which mainly reflected low communication frequency of gene among populations.AMOVA results indicated that 38.23% of the genetic variation was attributed to difference among populations. About 61.77% of the genetic variation showed within population, which accounted for a greater part of the whole genetic variation . Mantel’C. dactylon located at different regions along longitude gradient. Populations 7, 9, 10 clustered together and population 3 was close to population 5. The third mixture cluster contained plants coming exclusively from interval locations along the longitude, such as Shanxian (4), Zhengzhou (6), Sanmenxia (8), and Baoji (12). In addition, each of four populations in the mixture cluster had higher intra-population genetic diversity. The Principal coordinate analysis (PCoA) (A UPGMA dendrogram dependent on Nei’s genetic distance showed that 13 different populations were grouped into three distinct clusters . The firs (PCoA)  revealedC. dactylon was used to conduct separate calculation from K = 2 to K = 20. The results clearly demonstrated that the appropriate K values were obtained when the 13 populations were subdivided into 4 or 8 groups  and the cluster with green consisted of populations 4, 6, 8, 12. Populations 5, 7, 10, 13 were differentiated from populations 3, 9, 11 compared to K = 3. And when K = 8, one group included populations 3, 5, 7, 9, 10, and another group consisted of the populations 8 and 12. Each of the other six groups consisted of only one population respectively, which indicated a rich genetic differentiation along longitude gradients. Admixture among populations of medium longitude  could be observed from the structure of C. dactylon populations (K = 2 to 13), the clustering of populations at low and high longitude were relatively stable except for the populations 1, 12, 13.Bayesian analysis of the population structure of 8 groups . The resdactylon ; Fig. S2C. dactylon  and hexaploid  . Diploid and triploid respectively had significantly lower genetic diversity than other polyploidy. All the individuals of C. dactylon at different ploidy levels fell into three major clusters based on UPGMA. The first and second clusters contained diploid and triploid individuals, respectively. Tetraploid, pentaploid, and hexaploid individuals formed the third cluster  results of 0 for I) . The gen cluster . The genC. dactylon along longitude gradients had a remarkable genetic variation within population based on the study. Genetic diversity can be improved by sexual propagation, and clonal propagation by rhizome and stolon can promote stable inheritance not being easily affected by genetic drift  than other populations at low- and high-longitudes, because outcrossing and self-incompatibility of C. dactylon could produce low communication among different populations of the east–west transitional locations. Genetic differences among distant geographical sites with unique alleles can increase genetic diversity  also exhibits a certain broad-scale population genetic structure in east-to-west gradients  . The rese hybrid . Higher g season .C. dactylon along a latitudinal gradient, low genetic differentiation and notable admixture structure existed among populations along latitudes  means a rich genetic differentiation along longitude gradients (Nm < 1) . In our atitudes . Low genatitudes  than thaern ones . The easern ones . East Asern ones . A probaC. dactylon has been conducted to evaluate genetic variation and population structure along a longitude gradient. Landscape heterogeneity and geographical isolation along several mountain ranges can lead to formation of locally adapted populations with reduced gene flow between habitats. Geographic distance correlated with precipitation may influenced genetic diversity among populations along longitude gradients. A higher level of genetic differentiation among populations of C. dactylon along longitude gradients than those along latitudinal gradients indicated a stronger gene flow from the north-south than east–west direction. Decreased genetic diversity is estimated for individuals with lower ploidy level compared to higher ploidy level, and relatively rich genetic differentiation exists among different ploidy levels. Identifying genetic diversity along longitude gradients in contrast with latitudinal gradients provides important genetic information and improves the ability to protect biodiversity.A large-scale population genetic survey of genetic diversity of 10.7717/peerj.11953/supp-1Supplemental Information 1C. dactylon (L.) Pers. (common bermudagrass) individuals sampled from 13 geographic sites along the longitude from 105° 57′34″E to 119° 27′06″E.Raw data of 105 expressed sequence tag-derived simple sequence repeat (EST-SSR) loci of 249 Click here for additional data file.10.7717/peerj.11953/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11953/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.11953/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.11953/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.11953/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.11953/supp-7Supplemental Information 7Click here for additional data file.


Rhagoletis pomonella species group (RPSG) hypothesized to have diverged in sympatry via adaptation to different host fruits. We use a large-scale population genetic survey of 1568 flies across 33 populations to: (1) detect on-going hybridization, (2) determine whether the RPSG is derived from the same proximate ancestor, and (3) examine patterns of clustering and differentiation among sympatric populations. We find that divergence of each in-group RPSG taxon is occurring under current gene flow, that the derived members are nested within the large pool of genetic variation present in hawthorn-infesting populations of R. pomonella, and that sympatric population pairs differ markedly in their degree of genotypic clustering and differentiation across loci. We conclude that the RPSG provides a particularly robust opportunity to make direct comparisons to test hypotheses about how ecological speciation proceeds despite on-going gene flow. New species form through the evolution of genetic barriers to gene flow between previously interbreeding populations. The understanding of how speciation proceeds is hampered by our inability to follow cases of incipient speciation through time. Comparative approaches examining different diverging taxa may offer limited inferences, unless they fulfill criteria that make the comparisons relevant. Here, we test for those criteria in a recent adaptive radiation of the  Speciation is the process responsible for generating new twigs on the tree of life. New species form as genetically based barriers to gene flow evolve between formerly interbreeding populations ,2,3. OneHowever, drawing inferences from patterns of population genetic differentiation is complicated by several factors. One important consideration is the need to confirm that on-going gene flow is actually occurring between taxa. The possibility of incomplete lineage sorting means that shared allelic variation is insufficient to demonstrate that speciation is indeed proceeding in the face of gene flow. Heterogeneous topologies of divergence can still occur between sibling taxa, even in the complete absence of gene flow, driven by differences in post-divergence selection histories or demographics in the diverging taxa ,11. AnotRhagoletis pomonella species group (RPSG), to test some underlying assumptions of comparative approaches to studying the speciation continuum and examine patterns of population genetic differentiation at different stages of speciation in this system. The most well-studied component of this species group is the apple-infesting host race of R. pomonella, which arose as the result of a shift from ancestral hawthorn (Crataegus spp.) to introduced apple  in recent historical times, less than 170 years ago ,82EST foted loci B. The dip > 0.05 for each). Observed allele frequency differences between the mayhaw and green hawthorn population under an assumed gross migration rate of 5% can be maintained by an average selection coefficient of 0.114 (0.014–0.29) against green hawthorn alleles in mayhaw flies and 0.095 (0.014–0.2) in the reverse direction. For apple and downy hawthorn flies at 4.5% gene flow, the distribution of estimated selection coefficients were highly similar . Selection coefficients for blueberry hawthorn and mayhaw at 4% gene flow both averaged 0.094 with ranges of 0.021–0.199 and 0.018–0.244, respectively. Distributions were also similar for R. mendax and downy hawthorn, assuming 2% gene flow, averaging 0.115 (0.028–0.258) and 0.096 (0.024–0.226), respectively. However, the comparisons for sympatric hawthorn with flowering dogwood and R. zephyria both showed markedly stronger selection coefficients and considerable asymmetry across sympatric taxa. While the means selection coefficients did not differ, the variance was markedly higher in the derived compared to the ancestral taxa in both comparisons . At a conservatively estimated 3% gene flow, selection against flowering dogwood alleles in downy hawthorn flies had a mean coefficient of 0.139 (0.059–0.339), while selection against downy hawthorn alleles in flowering dogwood flies averaged 0.219 (0.44–1.0). The value of 1 for section against hawthorn alleles in flowering dogwood for a single locus (P23) indicates that the observed (pooled) allele frequency could not be maintained in the face of 3% migration at equilibrium. Selection coefficients necessary to maintain the observed allele frequency differences given estimated rates of gene flow between sympatric taxa showed similar distributions across most pairs of taxa . None ofR. zephyria comparison assumed a migration rate of 1%, consistent with observed hybridization rates in the Pacific Northwest [R. zephyria alleles in black hawthorn flies averaged 0.131 (0.019–0.623), with 0.259 (0.019–1.0) for the reverse. In this case, two loci (P70 and P16) had allele frequency differences that were too high to be maintained by selection at 1% gene flow at equilibrium. Given the observed presence of hybrids between hawthorn and both the dogwood fly and R. zephyria, it is unlikely that the lack of fit of these extreme estimated coefficients to our model is the result of unrealistic migration rates. Instead, these results are likely due to the simplifications of symmetrical migration rates or lack of dominance effects. Nevertheless, these results indicate a role for very strong post-zygotic selection in maintaining genomic differentiation along the speciation continuum in the RPSG. The black hawthorn–orthwest . SelectiSpecies complexes involving on-going divergence in the face of gene flow among populations and taxa that differ in the magnitude of reproductive isolation and population genetic differentiation may offer unique windows into the evolutionary processes and genomic conditions underlying the formation of new biodiversity. While such systems do not represent a true chronoseries of events in a single speciation trajectory , comparaR. pomonella and each of the named in-group taxa of the RPSG , the relationship of R. mendax and R. zephyria to that ancestral latitudinal variation was not well resolved in the network  in the nsu Funk , throughThe taxa in the earlier host race portion of the divergence continuum in the RPSG show strong phenotypic differentiation in traits directly related to both host plant adaptation and reproductive isolation ,35,36,37R. pomonella  than the dogwood fly  the primary axes of divergence and reproductive isolation for each case involve the same facets of host-plant adaptation ,34,36,37ulations , (3) theulations , and (4)ulations . The RPSulations A. This sulations B. These The microsatellite data presented here are too coarse-grained to adequately represent the dynamics of diverging genomes. The obvious extension of this work in the RPSG will be to compare the genomic topology of divergence among these taxa using high-coverage SNP data. These data will almost certainly bring additional insights into how ecology, genetic variation, and genome structure interact in the origin of species and allow for more rigorous tests of theoretical predictions of how genomic differentiation proceeds in the face of gene flow. One important consideration for future studies in the RPSG will be to combine population genomic analyses with functional genomics of the traits under divergent selection. Our results here also highlight how the most powerful systems for understanding speciation may be those for which it is still an on-going process. This requires taking an explicitly population-based approach  in systems that are best described by population genetic distance rather than fixed molecular genetic distances and post-speciation lineage sorting.