Ecology

The structure and microstructure of the fibresThe surfaces of the natural fibres are presented from Figs. 6, 7, 8, 9, 10 and of the synthetic fibres are presented in Figs. 11 and 12.Figure 6SEM of jute fibre [Fot.M.Kurpińska].Full size imageFigure 7SEM of bamboo fibre [Fot.M.Kurpińska].Full size imageFigure 8SEM of sisal fibre [Fot.M.Kurpińska].Full size imageFigure 9SEM of cotton fibre [Fot.M.Kurpińska].Full size imageFigure 10SEM of ramie fibre [Fot.M.Kurpińska].Full size imageFigure 11SEM of polymer fibre [Fot.M.Kurpińska].Full size imageFigure 12SEM of polypropylene (PP) fibre [Fot.M.Kurpińska].Full size imageThe basic components of natural fibres influencing their properties are cellulose, hemicellulose, lignin, waxes, oils, and pectin. Cellulose is mainly composed of three elements such as carbon, hydrogen, and oxygen, and it is the material basis that forms the cell wall natural fibre. Typically, cellulose remains in the form of micro-fibrils within the cell wall of a plant. Cellulose is the main factor affecting the tensile strength along natural fibre and the cellulose content is closely related to the plant’s age and content decreases with the increasing age of the plant6.Hemicellulose is an amorphous substance offering a low degree of polymerization and it exists between fibres. Hemicellulose is a complex polysaccharide with xylan as the predominant chain, and the branches mainly include 4-O-methyl-D-glucuronic acid, L-arabinose, and D-xylose. Lignin is a kind of polymer with complex structures and of many types. The basic units of lignin include: guaiacyl, syringyl monomers, and p-hydroxyphenyl monomers. The structural units in lignin are mainly connected by ether bonds and carbon–carbon single bonds. Usually, lignin is not evenly distributed in the plant fibre wall9.In addition to three main components, lignin often contains various sugars, fats, protein substances, and a small amount of ash elements. These chemical compositions affect not only the properties of natural fibres, but also the possibility of a specific application of fibre. The composition of individual natural fibres and their properties are presented in Table 1. Figure 6a–c shows longitudinal and cross-sectional views of the untreated jute fibre. Externally, the fibre is smooth and shiny. The presence of hemicellulose influences the high hygroscopicity of jute fibres. The structure of the jute fibre shows that the fibre swells when it absorbs water. Possible swelling of the fibre in the cross-section by approx. 30%. The microscope scans of indicate the succinylated regions. This is due to the chemical bonding of the succinic anhydride molecule with the hydroxyl group of the cellulose present in the fibre. The encircled region in the top side shows an unsuccinylated region with naturally waxy impurities16.Figure 7a shows the scanning electron micrograph (SEM) of the bamboo fibre. According to the SEM analysis, the microstructure of bamboo is anisotropic. At the Fig. 7b–c it can be recognized that the orientation of cellulose fibrils was placed almost along the fibre axis which may affect to maximize the modulus of elasticity. Factors affect the mechanical properties of bamboo fibres are the chemical composition and structure of bamboo fibres, moisture content, age of bamboo, etc. In addition, the age of the plant affects the chemical composition and structure of fibre. These factors and the natural humidity influence their change of mechanical properties. The hemicellulose content directly influences the tensile strength. This parameter increases with the decrease in the hemicellulose content in the bamboo fibre18.The cell structure of bamboo fibres is complex, and the middle layer of the cell wall has a multi-layer structure. The lignification of the thin and thick layers in the multilayer structure varies. The multi-layered cell wall structure leads to better fracture resistance and promotes internal sliding between the cell wall layers during tension. The angle of the microfiber alignment is also an important factor influencing the mechanical properties of the fibre. Typically, the tensile strength and modulus of elasticity of a fibre increase as the angle between the interposition of the microfibers decreases. Hence, the smaller microfibril angle is an important factor that contributes to the good mechanical properties of bamboo fibre. Large voids between bamboo fibre molecules can be seen, which impact good hygroscopicity19. The moisture content is an important factor affecting the mechanical properties of bamboo fibres. Figure 8a–c shows the morphology of the sisal fibre. The surface of the sisal fibre has higher roughness, and it increases the bonding area between the fibre and cement paste. This leads to increase the mechanical properties of the composites38.Figure 9a–c shows images of the cotton fibres. At the microscope image, a cotton fibre looks like a twisted ribbon or a collapsed and twisted tube. These twists are called convolutions: there are about 60 convolutions per centimetre. The weaves give the cotton an uneven surface of the fibres, which increases the friction between the fibres, but at the same time they can prevent fibres from evenly dispersing in the cement matrix. The outer layer, the cuticle is a thin film of mostly fats and waxes. Figure 9b shows the waxy layer surface with some smooth grooves. The waxy layer forms a thin sheet over the primary wall that forms grooves on the cotton surface19. The cotton fibre surface comprises non-cellulosic materials and amorphous cellulose in which the fibrils are arranged in a criss-cross pattern. Owing to the non-structured orientation of cellulose and non-cellulosic materials, the wall surface is unorganized and open. This gives flexibility to the fibre. The basic ingredients, responsible for the complicated interconnections in the primary wall, are cellulose, hemicelluloses, pectin, proteins, and ions. In the core of fibre, only the crystalline cellulose is present, what is highly ordered and has a compact structure with the cellulose fibrils lying parallel to one another18.SEM micrograph of the surface and cross section of the ramie fibre are shown at Fig. 10a–c. The surface of the ramie fibres is dense but porous. There are many micropores and continuous bubbles in the porous structure of a single bundle of a ramie fibre Fig. 10c. This structure has some effect for low absorption of water, moreover, it is also related to the fibre distribution in the cement composites. In case of the short ramie fibre, due to its random distribution in composites, the strength of the composite may be affected. Cellulose, lignin, and hemicellulose weight materials can form a dense layer on the surface of the ramie fibres, so the water absorptivity is low. This special structure of the fibre with a dense matrix, and at the same time, with a characteristic pore arrangement has an influence on the adhesion of the cement matrix and the strength of the cement composite18.The surface and cross section of multifilament macrofibre is demonstrated at Fig. 11a–c. From the chemical point of view, this type of fibres belongs to the polymers from the group of polyolefins, composed of units of the formula: –[CH2CH (CH3)]–. They are obtained by low-pressure polymerization of propylene. They are made of 100% pure co-polymer twisted bundles of multifilament fibres Fig. 11c. Polypropylene is one of two most commonly used plastics, in addition to polyethylene. Polypropylene is a hydrocarbon thermoplastic polymer2.Figure 12a–c shows the structure of a bundle of polypropylene (PP) fibres in the form of a 3D mesh. They are made of isotactic polypropylene, called propylene, CH2=CHCH3 obtained from crude oil. They are one of the finest polypropylene fibres. The surface of the fibres is smooth Fig. 12b 2.The consistency—fluidityThe results of fluidity are shown at Fig. 13. The fluidity of the composite not modified with fibres is 145 mm and is a reference to other test results. The use of bamboo fibres increased the composite fluidity and composite flow by 8.6% (157.5 mm). The use of polymer fibers and jute increased the consistency by about 7%, while the use of sisal fibres by 3%. The use of PP fibres (122.5 mm) had the greatest impact on the loss of consistency by 15.5%. The use of cotton and frame fibres resulted in a reduction of workability and consistency by 13.8% and 3.5%, respectively.Figure 13Results of fluidity test.Full size imageBased on the research results, it was found that in the case of using bamboo fibres characterizing a high absorption of 120–145%, the consistency of composite increased by 8.2% compared to the consistency of composite without fibres. In the case of a change in consistency, the chemical composition of natural fibres, their surface, and the total length in the volume of composite are significant, too. There is a noticeable regularity related to the cellulose content in natural fibres. If the higher cellulose content, it reduces the consistency of the composite. For example, the cellulose content in bamboo fibres is the lowest and amounts to 40–45%, while the cellulose content in cotton fibres is the highest, ranging from 80 to 94%. It can also be recognized that consistency and workability will be influenced by the hemicellulose content.The higher the hemicellulose content, it impacts the higher consistency of the composite. It is similar referring to the content of lignin. It was noticed that the higher the lignin content, the higher the composite consistency was found. Regarding the total length of the fibres, a regularity is apparent that the greater the total length of fibres, e.g., in the case of cotton fibres, the greater decrease in consistency is visible. In the case of polymer and polypropylene (PP) fibres, the consistency is influenced by the surface of the fibre, the number of fibres, and their total length in the volume of the composite. Increasing the total length of PP fibres by approx. 15% resulted in a reduction of the consistency of approx. 20%.Flexural and compressive strengthAssigning mechanical properties of fibre reinforced composite, particular emphasis was placed on the determination of the flexural strength of the composite. This parameter was appointed by the 3-point test. Figure 14. shows the flexural strength of plain composite and 7 groups of different fibre reinforced composites on the 2nd, 7th, 28th, and 56th days.Figure 14Flexural strength test results.Full size imageIt can be seen that the bending strength of composites with the addition of natural fibres, ramie, bamboo, jute, and sisal are similar. The bending strength of composites with PP and polymer fibres is lower. It should be noted that the strength of the cotton fibre-reinforced composite is much lower than that of all the others tested. The reason may be the low tensile strength of the cotton fibres used. When mixing the composites, a tendency to create conglomerates of cotton fibres was also noticed, which may affect the strength of the composites.The test results clearly show that the effectiveness of the added natural fibres depends on the chemical composition and mechanical properties, and above all, on their adhesion to the cement matrix. The adhesion of the natural fibre to the cement matrix has a significant influence on the mechanical properties of the cement composite, in particular on compression and bending strength. The highest bending strength was achieved by cement composites modified with ramie fibres. Ramie fibres are characterized by the highest tensile strength among the tested synthetic and natural fibres, ranging from 400 to 1000 MPa. The results of the compressive strength are shown in Fig. 15.Figure 15Compressive strength test results.Full size imageThe analysis of the test results shows that the use of dispersed fibres reduced the early compressive strength after 2 days from 8.5 to 33%. The exception is the ramie fibres, the use of which increased the early strength by 6.6%. Within 28 days, as in the case of early strength, the use of all types of synthetic and natural fibres resulted in a decrease in strength from 4.6 to 26.5%. The exception is the use of ramie fibres, which increased the compressive strength by 7.2% after 28 days. After 56 days, a decrease in strength was noticed in the case of using PP and polymer synthetic fibres as well as natural cotton and bamboo from 5.5 to 11.9%.On the other hand, the increase in compressive strength after 56 days from 5.8 to 16.4% was visible in the case of using fibres such as sisal, jute and ramie. The highest compressive strength was achieved by the composite with a ramie fibre. The fibre of the ramie is characterized by the highest modulus of elasticity ranging from 24.5 to 128 GPa and is over 100% higher than the Young’s modulus of the other fibres.Shrinkage testFigure 16A shows that the samples after demolding showed expansion for about 2 days, and from the third day after demolding, the length of the samples was shortened. The lowest degree of expansion in the first days was shown by samples without fibres and samples containing cotton fibres. In this case, the expansion did not exceed 0.02 mm/m. However, the same samples finally showed the highest shrinkage after 180 days, which was 0.06 mm/m.Figure 16Testing the change in length of samples.Full size imageThe highest expansion within 48 h after deformation was shown by samples containing sisal fibres, while these samples finally after 180 days showed the lowest deformation of the length of the samples, which was 0.001 mm/m. The samples containing the synthetic fibres showed an expansion of about 0.02–0.03 mm/m in 48 h and the final shrinkage after 180 days was 0.03 mm/m for both the polymer and PP fibre samples. The bamboo and ramie fibres initially showed an expansion of 0.04–0.06 mm/m while their final shrinkage was 0.02 mm/m. The samples with jute fibres showed an expansion of 0.04 mm/m and the final shrinkage of the samples was 0.04 mm/m. Figure 16a,b shows the results of testing the change in length of samples over time.After 180 days, the total deformation of the samples was determined. Samples containing sisal fibers showed a slight expansion of about 0.001 mm/m, while the highest deformation (shrinkage) was shown for composite samples without fibers and with cotton fibres, which was 0.06 mm/m. Samples with bamboo, jute, PP, polymer and ramie fibres showed a shrinkage from 0.02 to 0.04 mm/m. Only the samples containing the sisal fibre showed a slight expansion of 0.001 mm/m.Ultimately, the samples containing sisal fibres were characterized by the lowest deformability. This phenomenon is related to the fibre structure and the total length of the fibres in a sample with dimensions of 40 × 40 × 160mm. For example, in a sample containing sisal fibres, their total length is 5856.7 m. Otherwise, a sample containing jute fibres, their total length in the sample is only 7.4 m. Therefore it was found that the fibre structure, its diameter, the cellulose content and the total length of the fibres in the element are important factors of deformation as a result of shrinkage or expansion of the fibre reinforced composite.Water absorption of composite testHigher water absorption (8.5%) compared to the composite without fibres was noticed in the case of using both synthetic fibres and with the exception of the use of ramie fibres, which caused a slight reduction in water absorption to 8.2%. It can be recognized that the water absorption rate of the 8 groups of samples is slightly different, the highest is the polymer fibre-reinforced composite (9.2%); the lowest water absorption rate refers to ramie fibre-reinforced composite (8.2%). The difference in water absorption rates is presented at Fig. 17.Figure 17Water absorption of composite (%).Full size imageExcept for cotton fibre-reinforced composite, the water absorption rate of another plant fibre-reinforced composite is lower than that of synthetic fibre-reinforced composite. Probably because of the fact that ramie, sisal, and jute fibres all have good moisture absorption and release properties. It is commonly known that plant fibre-reinforced cement-based materials have reduced strength and initial properties due to their performance degradation in a humid environment, so their long-term durability could become problematic. Sisal fibres (with noticed absorption of 95–100%) have absorbed more cement slurry on their surface than jute fibres (absorption of fibre 7–12%). This phenomenon could be explained by the fact that the slurry became the impregnation of the fibre. The absorbability of the composite was tested after the composite had completely hardened. Probably a fibre that is characterized by high absorption—sisal is very well “embedded” in the matrix, therefore the bending strength results for composites with sisal fibre were higher by 8–10%. More

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Simulation experimental set upLaboratory monitoring of leakage migration process can provide an important basis for field tests. The designed and improved ERT device can better describe the migration range of leakage in soil41. In this experiment, a parallel potential monitoring device was used to improve the monitoring of leakage fluid migration. The simulation experiment in the laboratory is carried out in a (100 cm*100 cm*50 cm) plexiglass tank. Sand and clay shall be screened with a 2.36 mm square sieve, watered and compacted with a board to ensure that the soil layer is in close contact with the measuring electrode.Electrode arrangementThe ground wire of high-density electrical method instrument is connected to the electrodes arranged around the bottom of the tank as the power electrode C2, as shown in Fig. 2a. The host is connected to the electrode system. The electrode system consists of 47 electrode grids with a spacing of 0.08 m. The measuring electrode P1 is connected to the mainframe through a wire 0.05 m below the grid center. The geomembrane is located 0.03 m above the measuring electrode P1. The collection device is used as a monitoring system for various leachate. The arrangement of electrodes is shown in Fig. 2b. The power supply electrode C1 is placed at a certain depth in the middle of the saturated sand to provide a constant current. The location of electrode C1 and leakage point is shown in Fig. 2c. The layers from the bottom of the tank are silty clay, geomembrane, silty clay and saturated sand, as shown in Fig. 2d.Figure 2Set-up of leachate migration simulation experiment: (a) Schematic diagram of electrode C2 layout; (b) Schematic diagram of electrical system laying; (c) Position of electrode C1 and leakage point; (d) Schematic diagram of simulated experimental soil layer.Full size imageComposition of monitoring systemThe electrode system is used to monitor the background electric field and artificial electric field of the landfill site. In the experiment, the electrode system is laid in the clay layer under the geomembrane. It is composed of detection electrodes distributed in a grid at a certain distance.The electrical signal conversion system adjusts the measurement mode, sampling accuracy, acquisition frequency and other parameters of the electrode in the field according to the instructions of the mainframe, and transmits the collected electrical signal to the mainframe.The mainframe can control the operation of the monitoring system. The possible leachate points and their pollution range are determined by collecting data. The system mainly includes mainframe and its software system, power supply, etc., as shown in Fig. 3.Figure 3Se2432 parallel electric method instrument.Full size imageLeachate devicePlace 4 leakage bottles above the tank. No.1 and No.4 bottled water are used to simulate the leakage liquid formed by the direct infiltration of rainwater in slag through geomembrane and as a reference. Because Cl-1 is a typical pollutant in the landfill. No. 2 bottle containing 20 g/L NaCl solution is used to simulate inorganic salt leakage in urban life. No. 3 bottle containing 20 ml/L ethanol solution is used to simulate the leakage liquid containing a large amount of organic matter in municipal solid waste. The characteristics of leachate have been summarized in Table1.Table 1 The characteristics of leachate.Full size tableBefore the experiment, configure four solutions, close the injection, use an electric meter to check the conductivity of each measuring point. After each measuring point has no open circuit, supply power to the soil layer through the mainframe to measure the background electric field of the soil. Then open the injection, adjust the flow rate, release the solution at a fixed flow rate, record the soil electric field in the process of leakage every half an hour, collect the potential values of each measuring point, process the data through the potentiometry and potential difference method, and form the relevant potential horizontal profile and longitudinal section of the soil.Principle of potentiometric detection technologyWhen there are leakage points in the landfill, power is supplied to the landfill, and the current forms a current loop through the geomembrane. If there are n (n = 1,2,3…) leakage points in the geomembrane, the power supply current is I, and the artificial electric field will form a leakage electric field at the leakage point, which can be used as a point power supply.$$I = int dI = int j cdot dS$$
(1)
where I is the current intensity, j is the current density vector, and S is the area passing through the current.When there are n leakage points, I will be shunted. If a leakage point is regarded as a finite surface, the current intensity I as:$$I = {I_1} + {I_2} + cdot cdot cdot + {I_{text{n}}} = sumlimits_{i = 1}^n {int_{S_i} {jdS} }$$
(2)
Generally, the power supply current field of landfill site will be affected by the formation medium structure. It is assumed that the formation medium structure is composed of three layers, each layer has uniform properties and stable conductivity, and the layers from top to bottom are: landfill layer, with resistivity of ρ1. The saturated leakage liquid layer above the geomembrane has a resistivity of ρ2. The clay layer under the geomembrane has a resistivity of ρ3. The electrode C1 is arranged in the garbage layer for power supply, and the electrode C2 is arranged at the lower part of the geomembrane away from the electrode system area. The electrode C2 can be regarded as a far pole.Because of the ρ1  > ρ2, the conductivity of the saturated leakage liquid layer at the upper part of the geomembrane is better than that of the landfill layer, so that there is almost no reflected current between the ρ1 layer and the ρ2 layer, that is, the current generated by the power supply electrode C1 is all transmitted to the ρ2 layer. Because of the ρ3  > ρ2, it can be considered that the interface between ρ2 layer and ρ3 layer has both a reflection current, and a transmission current through the leakage point. The potential generated at the detection electrode P1 under the geomembrane is formed by the action of transmission current. The total potential of point P1 is obtained by the superposition of the potential of point power supply passing through n leakage points at P1.$${U_{P1}} = sumlimits_{i = 1}^n {frac{{{I_i}{rho_3}}}{{2pi {{text{r}}_{iP1}}}}}$$
(3)
Parallel potential difference methodThe test adopts pole–pole arrangement, and the calculation formula of apparent resistivity is as follows:$$rho = 2pi {text{aR}}$$
(4)
where ρ is apparent resistivity; a is the distance between electrodes C1 and P1; R is measuring resistivity.When there are loopholes in the geomembrane of the landfill, the leakage liquid will gradually penetrate into the soil layer under the geomembrane through the loopholes, resulting in the change of the conductivity of the soil layer under the geomembrane. The pole-pole acquisition mode of Se2432 parallel electrical instrument is used to obtain the original data (potential difference) of each measuring point on the grid. After current normalization, the apparent resistivity of the soil layer is obtained. The electrical properties of different depths of the soil layer can be obtained by inversion of the apparent resistivity data of the soil layer, so as to determine the occurrence point and distribution range of leakage.The monitoring grid is 5 × 5. The spacing between measuring points is 0.08 m. The measurement method adopted by Se2432 parallel electric method instrument is cross diagonal measurement method. Figure 4 shows that it only needs to measure the potential values on the measuring points on the horizontal, vertical and 45° diagonal lines.Figure 4Schematic diagram of cross-diagonal measurement method.Full size imageTheoretical calculation of test modelTheoretical results of 10 × 10 grid monitoringAccording to the experimental model and statistical data, the resistivity of the clay layer under the geomembrane is assumed ρ = 10 Ω· m, the resistivity ratio of tap water, NaCl solution and ethanol solution after penetrating into the soil layer ρNo.1:ρNo.2:ρNo.3 = 5:3:10. If the four leakage points set by the model are regarded as four conductive resistors, the ratio of the current passing through the four leakage points is INo. 1:INo. 2:INo. 3:INo. 4 = 6:10:3:6.The calculation model is 10 × 10 grid, and the spacing of measuring points is 0.05 m. The potential value on each measuring point is calculated according to Eq. 3, and the obtained data is processed with surfer software to obtain the potential contour map, as shown in Fig. 5. Among them, points 1, 2, 3 and 4 are the leakage positions of water, NaCl solution, ethanol solution and water respectively, and the spacing between leakage points is 0.15 m.Figure 510 × 10 Grid theory detection potential contour map.Full size imageFigure 5 shows that the leakage fields formed by the four kinds of leaking liquids interfere with each other from the theoretical calculation results. The leachate current at point 2 is larger, the high potential closed loop is obvious, and its center corresponds to the leakage center. The reason for this is that the NaCl solution contains conductive particles that increase the conductivity of the leak point. Point 1 and 4 are the same as water, and the leakage electric field is almost the same. Its closed loop is obvious, and the high potential center also corresponds to their leakage position. There is almost no closed loop effect at point 3 under the influence of 1, 2 and 4. The results show that the leakage field formed by high resistance leakage liquid is not easy to be detected by potentiometric detection, and low resistance leakage is suitable to be detected by potentiometric detection.Theoretical results of 12 × 12 grid monitoringThe resistivity of the clay layer under the geomembrane is assumed ρ = 10Ω·m. In consideration of the mutual influence between the leachate and appropriately reduce its influence effect, the resistivity ratio of water, NaCl solution, and ethanol solution after penetrating into the soil layer is set as ρNo.1:ρNo.2:ρNo.3 = 20:15:24, the ratio of the current passing through the four leakage points is INo.1:INo.2:INo.3:INo.4 = 6:8:5:6. And adjust the distance between the two points to 0.28 m. 12 × 12 grid was used for detection, and the spacing of detection points is 0.04 m. Calculate the potential value of each detection point according to Eq. 3, and use Surfer to obtain the detection contour map of four kinds of leakage, as shown in Fig. 6.Figure 612 × 12 Grid theory detection potential contour map.Full size imageTheoretical calculation results show that when the distance between the leakage points is large and the distance between the detection points is small, the potentiometric method can detect the leakage position of various leachates well. At the same time, the diffusion range of different leachates in the same plane is roughly the same, and they all gradually diffuse outward from the center of the leakage point, and the potential value gradually decrease. Point 2 has the largest potential closed loop range, which also has a certain impact on the leakage points of adjacent points 1 and 3. Point 1 and point 4 are water leakage. Affected by different leakage liquids, the leakage electric field of the two same leakage liquids is obviously different. The potential closed loop range of point 1 is larger than that of point 4. Point 3 is the leakage of ethanol solution. Because its resistance is the largest, the range of potential closed loop is the smallest.Figure 7 shows that the leakage fields around the leachates are funnel-shaped, and its size is related to the type of leachate. Therefore, different network density should be designed for different types of leakage liquid, so as to use the most economical scheme to detect the leakage point.Figure 712 × 12 Grid theory detects potential 3d view.Full size imageInterpretation and discussion of resultsLaboratory simulation experiment researchFigure 8a shows the background electric field potential of soil layer. The four injection pipes are opened at the same time and adjusted to the same flow rate. Under the condition of continuous leakage, monitor the leakage field potential at an interval of 1 h. Figure 8b shows the leakage electric field potential value for 1 h. Reduce the injection pipes flow rate to 1/2 of the initial value. Figure 8c shows the monitoring results of 2 h soil layer leakage field potential. Figure 8d shows the soil leakage field potential monitored after 30 min of sealing the injection pipes.Figure 8Leakage field potential diagram of soil layer: (a) Background electric field of soil layer; (b) Potential distribution of soil layer after 1 h of leakage; (c) Potential distribution of soil layer after 2 h of leakage; (d) Potential distribution of soil layer after closing the injection tube for 30 min.Full size imageFigure 8a shows that the background potential contour of the experimental soil layer is at a lower value. Few current lines pass through the monitoring area. A dense closed potential circle of high potential value is formed at point 2. The current flow at point 2 is greater than the other points 1, 3 and 4. The analysis result may be that in the process of watering and compaction, the clay layer under the geomembrane is not uniform, and the compaction degree of the soil layer is different, resulting in different potential values ​​obtained by monitoring. The permeability at point 2 is better than other points, so when the flow rate of the leakage liquid is large, the leakage liquid under the geomembrane gathers near point 2 and spreads out around. After the clay is watered and compacted, the soil compaction is small and the pore water content is large, resulting in a high potential abnormal area in the lower left corner of point 3.Point 2 forms a closed loop of anomaly potential contour much higher than the background electric field, while the value of potential contour coil at leakage point 3 is lower than the surrounding value. It can be analyzed that positions 2 and 3 are leakage points. The leachate at point 2 is a high concentration NaCl solution containing more conductive particles, which will reduce the resistivity of the soil layer under the geomembrane at point 2. Thus, the passing current is increased to form a high potential closed loop. The leachate at point 3 is ethanol solution, which will increase the resistivity of the soil layer under the geomembrane at point 3. So as to reduce the passing current and form a low potential closed loop. Figure 8b shows that the potential contour is consistent with the influence of NaCl solution and ethanol solution on the soil layer under the geomembrane. It can be concluded that point 2 and point 3 are leakage points. The electric field formed after water leakage at point 1 and point 4 cannot clearly distinguish the leakage points.During the monitoring process, the leachate was continuously released from the injection pipe, and the results reflected the dynamic characteristics. Figure 8b shows the phenomenon that the leachate from point 1 and point 4 aggregates around point 2, which is consistent with the inference of better permeability at point 2. Figure 8b,c show that when the flow rate of the leachate is changed and the flow rate of the injection pipe is reduced, the high-potential region of the entire electric field is reduced. Under the influence of gravity, the leachate will migrate longitudinally, and the closed-loop abnormally high-potential regions and abnormally low-potential regions at points 2 and 3 also decrease.Compared with the surrounding potential contours, the difference is more obvious. Figure 8d shows that when the injection pipe stops leaking for a period of time, the leachate migrates longitudinally along the leakage point. At this time, the electric field of the soil layer is similar to the original background electric field, but the potential value is higher than the background electric field, indicating that the leachate is stagnant in the pores of the soil layer, it is the result of changing the electrical properties of the soil layer. The parallel potential method can collect the potential value of each point in the field at one time, which provides a basis for real-time monitoring of landfill leachate.Figure 9 shows the inversion results of the horizontal section of the experimental model. The blue area corresponds to the distribution range of the low resistance anomaly. There are no jump or distortion points in the profile. The resistivity in the longitudinal direction basically shows a change from low to high. The upper layer seepage liquid migrates, and the bottom soil layer is characterized by low humidity and high resistivity. The low-resistance areas formed by the leakage of NaCl solution are widely distributed in the horizontal section. The distribution range is 0–0.28 m, and the migration scale of the leakage liquid can be clearly seen. The morphological characteristics of water leakage in different parts are basically the same. The distribution range is 0–0.18 m. The leakage of ethanol solution is only reflected at 0–0.06 m, and the distribution range is the smallest at the same depth. The ethanol solution also had the slowest migration rate.Figure 9Inversion map of plane section at different depths.Full size imageFigure 10 shows the inversion results of the X–Z longitudinal section of the test model. The two apparent resistivity profiles at Y = 0.24 m and Y = 0.32 m show that there is no low-resistance area in the shallow layer on the soil layer, indicating that the geomembrane in this area is not damaged. The low resistance zone in the middle is caused by the lateral migration of leakage fluid. The low-resistance anomaly area at the top of the profile can be judged as a leak point or formed by the migration of nearby leachate. Combined with the horizontal section, the leakage depth is similar, and the lateral migration speed of leachate is faster than the longitudinal migration speed. Four leak points can be distinguished, delineating the general location of the leak.Figure 10X–Z longitudinal section on different Y axes.Full size imagePhysical model experimentThe potential value of each electrode was monitored after 2 h of leakage, and the resistivity profiles at different positions were obtained by the potential difference method.It can be seen from Fig. 11 that the potential difference method can monitor the leakage of leachate in different directions. The morphological features of the plume formed by the downward migration of the leak point are approximately funnel-shaped in longitudinal section. The affected area of ​​the soil layer can be obtained in time. Figure 11b shows that the potential difference at the monitoring point is very different on both sides. After 2 h of leakage, a large amount of leakage liquid exists in the soil layer. When the water content in the soil layer increases, the diffusion rate of the ethanol solution increases, showing high resistance characteristics. At the same time, due to the action of gravity, there is a lot of vertical migration, and the potential value changes greatly. The profile clearly shows that the distribution area of ​​high potential difference is large, and the distribution of low potential is small. Figure 11c shows that since the migration rate of leachate in the horizontal direction is greater than that in the vertical direction, the potential difference of the monitoring point in the middle region is smaller, and a closed region of a high-potential circle is formed in the middle. The difference between the two results in a smaller potential difference area. Figure 11d shows that almost all the low-potential areas on the monitoring point are on the left side, because the leakage rate of NaCl solution in the horizontal direction is similar to that in the vertical direction under the condition of good soil compaction. At this time, a large number of conductive particles are contained, resulting in a large high-potential region. The difference between the two forms a large area of ​​low potential difference on the left. This is in good agreement with the lower resistance characteristics of the NaCl solution. Figure 11e shows that the two low-resistance regions correspond to the two leakage centers. The low potential difference region is formed by migration around the leak point. The migration speed in the horizontal direction is similar to that in the vertical direction, and the water migration speed on the left is slower than that of the sodium chloride solution on the right. Figure 11e,f show that the monitoring results are the same, but the resulting potential difference is also increased. This is affected by the distance between the monitoring point and the leak point. When the monitored point and the leakage point are located on the same section, the soil layer is the most severely affected area by leakage. Through the change of the potential difference, the leakage range and the location of the leakage point can be better judged.Figure 11Electrical resistivity tomograms of profile: (a) Resistivity of the slitting profile Y = 0; (b) Resistivity of the slitting profile Y = 0.08; (c) Resistivity of the slitting profile Y = 0.16; (d) Resistivity of the slitting profile Y = 0.24; (e) Resistivity of the slitting profile Y = 0.32; (f) Resistivity of the slitting profile Y = 0.4.Full size image More

Phakamani M’Afrika Xaba speaks at a botanical workshop.Credit: Nong Nooch/Tropical Botanical Garden

For Black communities in today’s South Africa, the legacies of colonialism and apartheid still prevail, shaping social structure and limiting access to opportunities. Colonialism displaced Black South Africans from the mid-seventeenth century, eroding cultural and social systems.From the 1950s, apartheid legalized systematic racial discrimination against disenfranchised, mainly Black, people. It limited their economic opportunities and social standing, prescribing an inferior education system to deliberately shape a poor quality of life. The policy fuelled systemic sexism, sexual-orientation discrimination, ageism, and the use of ethnicity as a divide-and-conquer strategy.Seventy years later, even after more than 25 years of democracy following the end of apartheid in 1994, schools and suburbs are still predominantly segregated, with government funding unevenly allocated in terms of facilities and quality of education.Former South African president Nelson Mandela once said, “In Africa there is a concept known as ubuntu — the profound sense that we are human only through the humanity of others; that if we are to accomplish anything in this world, it will in equal measure be due to the work and achievement of others.”As three past and present employees of the South African National Biodiversity Institute (SANBI), a conservation organization founded in 2004 to manage the country’s biodiversity resources, we have been advocating for a culture of treating others in the way we want to be treated: by applying universal shared human values, redefining institutional culture and systems to be inclusive, and opening safe spaces for a diversity of ideas. We have proposed a ground-up approach that aims to focus on holistic transformation at different levels in our organization.Our approach was to initiate a platform to identify inclusivity challenges, foster awareness and collaboration among staff and collectively develop innovative ideas and solutions. These would be aligned to existing organizational values, such as ubuntu, growth, respect and tolerance, excellence, accountability and togetherness. We strive to bring about institutional cultural change through facilitated, constructive conversations, by strengthening connections and cohesion among staff and through creative and proactive problem-solving across our institution.Mentorship that thrivesInstitutional culture needs to enable successful mentoring by creating a safe space. For example, SANBI’s mentoring programme for interns, students and early-career scientists involves quarterly meetings between them and dedicated human-resources staff — check-ins that provide a space to engage with programme coordinators without early-career researchers’ supervisors being present. In addition to sharing feedback on institutional policies and procedures, early-career scientists have the opportunity to discuss challenges they might face because of their supervisor or work placement. When issues are identified early, transfers or exchanges between work programmes can be arranged.Every year, we each sign up to mentor junior researchers to provide a supportive environment for guidance, counselling and the transfer of skills. We develop structured workplans with specific goals and outputs, and we discuss expectations with our protégés. In addition, we offer shared workspaces for interns and encourage peer learning, so that interns can form a peer support network. In these relationships, trust is crucial and can be a gateway to broader professional and personal networks.

Early-career researchers doing fieldwork training at the Stellenbosch University Experimental Farms in South Africa.Credit: Tlou Masehela

Institutions should recruit outside of their walls, if necessary, to ensure that appropriately skilled mentors are paired with early-career researchers. For mentorship to thrive, institutions must also create an enabling environment. In non-supportive environments, staff — particularly those from under-represented groups — who remain inadequately skilled and work without guidance become frustrated. Some can even feel they don’t belong because they see themselves as lagging behind their peers.Institutions often focus too strongly on outputs — such as publications, products or technologies — at the expense of reflecting on the values that uphold the institution. These values might be outdated and out of touch with those of staff, or with those held by partners, stakeholders or society at large. If staff cannot relate to the institutional culture and systems, job satisfaction and retention rates can suffer.Until a few years ago, for example, venues at our organization were named after former staff, as a way of acknowledging their contributions. Inevitably, these were mostly white, male, senior staff, such as Harold Pearson, the first director of Kirstenbosch National Botanical Garden, and Brian Rycroft, who served as director in the 1950s. But the contributions of staff who were employed in junior positions for 20–30 years also needed to be acknowledged. After an outcry around 2014, then-chief-executive Tanya Abrahamse, the first Black woman to hold the post, decided to acknowledge contributions of staff no matter their position. As a result, we now have Richard Crowie Hall, an exhibition space named after one of SANBI’s longest-serving staff members.The protracted legacy of apartheid in South Africa means that if institutional implicit biases are left unaddressed, they can create a fertile ground for racial, ethnic, tribal, financial and gender tensions. We urge more institutional recognition of the contributions of all.Fostering safe spacesThrough our engagements with each other, we have discovered a set of shared values, aligned with those of our institution, and have set out to establish a space to build our vision of a supportive, safe environment based on these values. Safe spaces are required for expressing controversial or uncomfortable views and to do the hard work of finding solutions to inequities. Confidentiality and trust cultivate such safe spaces, which can be created initially in small groups, then expanded to constructive formal or informal spaces. The conversations and suggestions of informal discussion groups about staff development and transformation can be elevated to management for implementation.
Decolonizing science toolkit
Safe spaces are a necessity for institutions that wish to truly address their legacies of racism and colonialism. Policies alone will not create these spaces — they require dedicated staff, too. Such spaces should ensure that those who speak up can do so without fear of being labelled as troublemakers or victimized.As a first step in pursuing this vision, we met with the senior teams at our organization to share ideas around the need for and benefits of an inclusive culture. We highlighted that inclusivity improves work–life balance, productivity and mental well-being for all employees.Any change, transformative or otherwise, is a process that takes perseverance, patience and determination. For any individual scientist to grow and flourish, they need a supportive environment, rich mentorship, a safe space and an enabling culture. It’s time for those factors to apply to all scientists equitably, no matter their gender, race, ethnicity or tribe. By fostering this mindset, we aim to reframe the narrative of our history and, in doing so, give all South African scientists their chance to thrive. More

Variation in environmental parameters across space and time in Bohai BayThe environmental parameters of samples collected near the Tianjin coastal area from different stations and seasons exhibited high temporal and spatial heterogeneity. The seawater temperature was 28.09 ± 0.53 °C in Aug, 17.48 ± 2.36 °C in May, and 19.55 ± 1.26 °C in Oct (Table 1). The seasonal variation in seawater temperature corresponded to the meteorological characteristics in Bohai Bay, with warm seawater in summer and relatively cool seawater in spring. The salinity was 29.69 ± 2.71‰ in Aug, 33.19 ± 0.33‰ in May, and 30.15 ± 1.63‰ in Oct. Seasonal variations in salinity may be mainly related to freshwater loading. According to the precipitation observed data of Bohai Bay in previous years, the rainfall amount and days in summer are the most19, which may lead to the increase in runoff and the relatively low salinity in summer. Chlorophyll a (Chl a) was highest in May, with lower levels in Aug and Oct. The dissolved inorganic nitrogen (DIN) was significantly higher in May and Aug than in Oct. The higher level of DIN in May and Aug may be related to terrestrial input and supply for the demand of phytoplankton growth. In October, the temperature and DIN content were both not suitable for phytoplankton growth, and Chl_a showed the lowest value. Spatially, the DIN distribution across the three seasons was rather similar, with high values observed in nearshore waters and low values in offshore waters (Dataset S1 & Fig. S1), which suggested that terrestrial input was an important source of DIN. The pH, soluble reactive phosphate (SRP) and chemical oxygen demand (COD) showed relatively higher values in October than in August and May, which may be caused by the dead phytoplankton release and terrestrial loadings through coasts and rivers. The dissolved oxygen (DO), conductivity and depth did not show significant variation among sampling times (Table 1), while the conductivity and depth had relatively higher values at offshore stations (Dataset S1) since the more remote the sampling water was, the greater the depth was in Bohai Bay and the closer it was to the open sea with higher salinity and conductivity. The ordination plot showed distinct partitioning of samples from nearshore and offshore sites along principal component axis 1 (PC1) (Fig. 1). The ordination plot could explain 73.49% of the total variation in the geo-physical–chemical parameters and revealed a linear positive correlation between different parameters (Fig. 1). AN, DIN, nitrate and Chl_a were most crucial in the partitioning of samples from May and the other 2 months; salinity, longitude, depth and conductivity were crucial for the partitioning of samples from offshore and nearshore stations; pH, COD, SRP, nitrite and temperature were crucial for the partitioning of samples from nearshore stations in August and October and samples from offshore stations. Overall, the principal component analysis (PCA) plot clearly showed both the temporal and spatial variation of the measured environmental parameters, indicating that complex biogeochemical processes and hydrodynamic conditions lead to the variation among sites and seasons.Table 1 The independent-samples t test of environmental variables and α-diversity among different months.Full size tableFigure 1Biplot of the principal component analysis (PCA) for environmental parameters in the seawater samples of the Bohai Bay coastal area across different seasons and sites. The two principal components (PC1 and PC2) explained 73.49% of the total variation in the environmental data and showed clear partitioning of offshore samples (in blue font) from other nearshore samples along PC1 and partitioning of May samples from August and October along PC2. The variables transparency and latitude were strongly correlated with PC1, and the variables ammonia nitrogen (AN), COD, pH, soluble reactive phosphate (SRP), and nitrite were strongly correlated with PC2. Chlorophyll a (Chl_a), dissolved inorganic nitrogen (DIN), nitrate and DO were mainly positively correlated with samples from May, while salinity, longitude, depth and conductivity were mainly positively correlated with offshore samples. Blue arrows represent environmental parameters, and circles in color represent sampling points.Full size imageProkaryotic α/β-diversity variationMeasures of α-diversity showed significant differences in shannon, evenness, faith_pd and OTU richness between samples from May/Aug and Oct (Fig. 2, Table 1). Principal coordinates analysis (PCoAs) based on weighted UniFrac (WUF) distance and unweighted UniFrac (UUF) distance showed that the PCC from different sampling months separated across the first and second principal coordinates (Fig. 3A-B). Both the analysis of similarity (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA/ADONIS) results indicated that the prokaryotic communities varied significantly across different sampling months when using a WUF distance metric (ANOSIM, r = 0.709, P = 0.001; ADONIS, R2 = 40.0%, P = 0.001) and UUF distance metric (ANOSIM, r = 0.934, P = 0.001; ADONIS, R2 = 38.7%, P = 0.001). At the same time, the prokaryotic α– and β-diversity both showed high within-month variability in Aug (Figs. 2, 3C–D), which indicated that the community varied greatly among different sites in Aug.Figure 2Alpha diversity of shannon, eveness, faith_pd (phylogenetic diversity) and OTU richness value of the prokaryotic community of all the samples from different stations at different sampling times.Full size imageFigure 3Principal coordinate analysis (PCoA) based on unweighted (A) and weighted (B) UniFrac distances for prokaryotic communities in the surface waters; box plots showing the unweighted (C) and weighted (D) UniFrac distances among each station at different sampling times.Full size imageCorrelation between prokaryotic α/β-diversity and physical, chemical and geographic factorsThe α-diversity measurements exhibited significant positive correlations with temperature, pH, SRP, AN and un_ionN (Dataset S2). The correlation between α-diversity indexes and geo factors (longitude and latitude) was not strong or significant both in samples across the three sampling times or from each sampling time (Dataset S2).The environmental variation significantly correlated with β-diversity among the three seasons (r_weighted = 0.4558, r_unweighted = 0.4631, P = 0.001, Table 2), with pH, AN, temperature, un_ionN, COD, nitrite, SRP, salinity, DO and DIN as the main individual determinants. However, it did not show significant correlations with β-diversity at any sampling time except in Oct (Table S1).Table 2 Spearman’s rank correlation between environmental/spatial variability (Euclidean distance) and prokaryotic β-diversity (weighted/unweighted UniFrac distance) among all samples from different season.Full size tableThe geographic distance was not correlated with prokaryotic β-diversity (variation in community composition; r  0.05; Table 2) among the three sampling times. However, samples from Aug and Oct exhibited a significant correlation between β-diversity and geographic distance (Table S1).Factors driving the PCC variationPERMANOVA using the UUF/WUF distance indicated that temperature variation explained the largest part of community variation among the investigated factors (34.90%/19.83%, P = 0.001, Dataset S3), with AN (31.84%/13.56%, P = 0.001) and salinity (12.91%/6.21%, P = 0.001) as the second and third most significant sources of variation.The variance partitioning analysis (VPA) conducted on both UUF/WUF distances showed that almost 100% percent of the variation in PCC among all three sampling times was explained by the detected environmental factors. In May, no environmental or spatial factors could be selected as significantly explain the PCC variation; in Aug, the joint effects of environmental and spatial factors could explain 49.5% of the variation; in Oct, based on WUF distance, the spatial factors could purely explain 10.5%, environmental factors could purely explain 38.8%, their joint effects could explain 28.2%, and based on UUF distance, the joint effects of environmental factors and trend could explain 13.7% of the PCC variation. These results indicated dramatic shifts in the spatial or environmental factor effects on the PCC variation at different sampling times in Bohai Bay (Table 3).Table 3 Variance partitioning analysis of prokaryotic community in Bohai Bay according to seawater environmental factors and geospatial factors. The spatial factors including linear trend and PCNM variables. Forward selection procedures were used to select the best subset of environmental, trend, and PCNM variables explaining community variation, respectively. The community variation was calculated on the weighted and unweighted UniFrac distance matrix, respectively. Monte Carlo permutation test was performed on each set without the effect of the other by permuting samples freely (999 permutations).Full size tableDistinct seasonal features at the phylum and OTU levelsThere were notable differences in the proportions of various phyla among different seasons (sampling month). In May, there was a greater proportion of Alphaproteobacteria (41.41%), Planctomycetes (6.42%), Actinobacteria (3.86%), Firmicutes (1.48%), Acidobacteria (0.45%), TM7 (0.16%), Tenericutes (0.16%), OD1 (0.13%), and WPS-2 (0.09%) than in Aug and Oct, whereas Gammaproteobacteria (44.23%), GN02 (0.08%) and SAR406 (0.04%) were depleted in May and Aug but enriched in Oct. In Aug, Bacteroidetes (13.98%), Deltaproteobacteria (6.93%), Verrucomicrobia (4.5%), Chloroflexi (0.36%), Lentisphaerae (0.97%), TM6 (0.25%), Nitrospirae (0.08%), Chlamydiae (0.07%), Chlorobi (0.07%), Spirochaetes (0.04%) and OP8 (0.03%) were significantly enriched than in the other two sampling times (Duncan test; Table S2).At the OTU level, OTUs with relative abundance  > 0.01% (1040 OTUs) were used to perform the difference analysis, and 175 OTUs in May, 281 OTUs in Aug, and 210 OTUs in Oct were identified as seasonal specific OTUs (ssOTUs). The cooccurrence network showed that the ssOTUs were clustered separately (Fig. 4A). Furthermore, the separation of the three modules contained most of the ssOTUs specific to different seasons (Fig. 4A-B). All the ssOTUs of different seasons comprised a taxonomically broad set of prokaryotes at the phylum (phylum Proteobacteria is grouped at the class level) level (Fig. 4C) belonging to various phyla with different proportions. Betaproteobacteria, Verrucomicrobia, Gemmatimonadetes, Epsilonproteobacteria, PAUC34f., and Euryarchaeota did not show significant differences among the three sampling times at the phylum level, but features belonging to these phyla showed differences at the OTU level (Fig. 4C, Dataset S4). In addition, the phylum ssOTUs belonging to, such as Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, Actinobacteria, and Deltaproteobacteria, were not only enriched at one sampling time (Dataset S4) but also enriched at the other two sampling times (Fig. 4C, Dataset S4). These results revealed that different seasons do not strictly select specific microbial lineages at the phylum level, but a finer level analysis could more strictly reflect the seasonal variation.Figure 4Co-occurrence patterns of seasonal sensitive OTUs (A). Co-occurrence network visualizing significant correlations (ρ  > 0.7, P  0.01%. Different colors represent ssOTUs in May (green), Aug (red) and Oct (blue). Cumulative relative abundance (as counts per million, CPM; y-axis in × 1000) of all the sensitive modules in the networks (B). The phylum attribution of ssOTUs in each season (C). The y-axis is the percentage of the number of OTUs that belong to a particular phylum that accounts for the total number of all the OTUs.Full size imageRegression analysis between the relative abundance of modules to which the ssOTUs belonged and the environmental factors was also conducted, and module 1 abundance, to which the Aug-ssOTUs belonged, showed a significant positive correlation with temperature (R2 = 0.77, P = 6.609e−62), AN (R2 = 0.43, P = 7.416e−25), and un_ionN (R2 = 0.75, P = 1.366e−58) and a negative correlation with SRP (R2 = 0.81, P = 6.762e-17). This may be caused by the functional role of the microbes in Aug. In the Aug-ssOTUs, Deltaproteobacteria showed a higher ratio than in the other 2 months (Fig. 4c), and in the following functional analysis, Deltaproteobacteria contributed to the genes related to nitrogen fixation, which may help to explain why there was a positive correlation of Aug-ssOTUs to AN and un_ionN. The module 2 abundance to which the May-ssOTUs belonged showed a significant negative correlation with pH (R2 = 0.65, P = 4.026e−44), temperature (R2 = 0.19, P = 2.325e−10), un_ionN (R2 = 0.025, P = 0.01779), and SRP (R2 = 0.12, P = 4.104e−07) and a positive correlation with AN (R2 = 0.26, P = 5.174e−14). In the May-ssOTUs, the ratio of Alphaproteobacteria was the highest, and Alphaproteobacteria were reported to be pH-sensitive groups in marine environments20, which prefer neutral pH environments21. In this study, the pH in May was 8.04 ± 0.07, in Aug was 8.39 ± 0.09, in Oct was 8.38 ± 0.07, and the pH in May was the closest to neutral, and the ratio decreased with increasing pH in Oct and Aug. The abundance of module 3, to which the Oct-ssOTUs belonged, showed a significant positive correlation with SRP (R2 = 0.81, P = 0.16e-10) and pH (R2 = 0.054, P = 0.00075) and a negative correlation with temperature (R2 = 0.44, P = 2.276e−25), AN (R2 = 0.75, P = 4.51e−58), and un_ionN (R2 = 0.6, P = 3.995e-39) (Fig. S2). Phosphate has been identified to limit primary productivity22, which is of great importance in the structure of dominant bacterial taxa in marine environments23. In the Oct-ssOTUs, the ratio of Gammaproteobacteria was the highest, as reported. Gammaproteobacteria was significantly explained by SRP during the seasonal variation in the Western English Channel, with Rho equal to 0.7523, which suggested the sensitivity of it to SRP, and in that study, it also showed a negative correlation between temperature and Gammaproteobacteria and a positive correlation between SRP and Gammaproteobacteria. Although the correlation was not significant, the variation trend was consistent, which indicates that the phenomenon observed in this study was not unexpected. In addition, most ammonia-oxidizing bacteria belong to the Betaproteobacteria and Gammaproteobacteria classes are chemolithoautotrophs that oxidize ammonia to nitrite24. Gammaproteobacteria and Betaproteobacteria both had higher ratios in Oct-ssOTUs, and the functional prediction results also showed that pmoA/amoA and pmoB/amoB, which encode ammonia monooxygenase, were mainly contributed by OTUs from Gammaproteobacteria and Betaproteobacteria (Dataset S10). The utilization of ammonia may explain the negative correlation between the Oct-ssOTUs and AN.Community assembly processes across different sampling months and sitesBased on the analysis of phylogenetic turnover, unweighted βNTI mostly ranged from -2 to 2 across different sites at a single sampling time in May, Aug and Oct, revealing that PCC variations across different sampling sites at a single time were mostly affected by stochastic processes. The unweighted βNTI was greater than 2 across May–Aug, May–Oct and Aug-Oct (Fig. 5A), which revealed that the variations in PCC across different sampling times were mostly affected by deterministic processes. The RCbray values across any two sampling times were equal to 1, and in each sampling time, the RCbray values ranged from − 1 to 1 (Fig. 5B). Combining the βNTI and RCbray values, the community assembly processes were quantified at each sampling time and at any two sampling times. As shown in Fig. 5C, turning over of the community during different sampling times was mainly governed by selection; among the different sites in May and Oct, it was mainly governed by “undominated” processes; community turn over in Aug was mainly governed by the influence of “Dispersal Limitation”. These results indicated that the shifts in the assembly of prokaryotic communities during different sampling times were caused by strong “heterogeneous selection” (βNTI  > 2), and the community variation at each sampling time was mainly caused by stochastic processes.Figure 5Patterns of distribution of unweighted βNTI (A) and RCbray (B) values across different sampling times. Quantification of the features that impose community assembly processes in and among different sampling times. (C) Pie charts give the percent of turnover in community composition governed primarily by Selection acting alone (white fill), Dispersal Limitation (green line fill), Homogenizing Dispersal (blue line fill) and undominated process (cyan fill).Full size imagePrediction of the metabolic potential at different sampling timesThe NSTI scores of each sample ranged from 0.033 to 0.096, with a mean of 0.058 (Dataset S5). Microbial functions were detected in all the samples from the three sampling times, and it was found that the relative abundances of 242 pathways were significantly changed between samples from May and samples from Aug (Dataset S6). The relative abundances of 321 pathways were significantly changed between samples from May and Oct (Dataset S7), and the relative abundances of 370 pathways were significantly changed between samples from Aug and Oct (Dataset S8).Genes related to energy metabolism were given more attention. For nitrogen metabolism genes relevant with nitrogen fixation (nifD, nifK) were detected only enriched in Aug, while genes relevant with nitrate reduction and denitrification (narG, narZ, nxrA, narH, narY, nxrB, narI, narV, nirD, nasA, nasB) were detected enriched in May, genes related with ammonia oxidation were both detected enriched in Oct and Aug. For sulfur metabolism, genes relevant with thiosulfate oxidation (soxA, soxB, soxC, soxX, soxY and soxZ) were only enriched in Aug, while genes relevant with sulfate and sulfite reduction (cysNC, aprA, aprB, cysJ, cysI, cysK, dsrA) were detected enriched in May and Oct (Fig. 6).Figure 6The LEfSe analysis indicated significantly differential abundances of PICRUSt predicted genes relevant to energy metabolism in different months of samples.Full size imageProkaryotic taxa contributed to the significantly varied functional genes related to nitrogen and sulfur metabolism at different sampling times. At the species level, the taxa contributing to nifK and nifD mainly belonged to Deltaproteobacteria and Firmicutes, and the taxa contributing to the sox-series genes mainly belonged to Alphaproteobacteria and Gammaproteobacteria (Fig. S3). The denitrification-related functional genes that were enriched in May were mainly contributed by members from Alphaproteobacteria and Gammaproteobacteria. The taxa contributing to dsrA, aprA and aprB were mainly from Deltaproteobacteria, including members of Desulfarculaceae, Desulfobacteraceae, Desulfobulbaceae, Desulfovibrionaceae and Syntrophobacteraceae (Fig. S4). More

Study sites and coloniesAll the experiments were carried out during the apple and pear blooming seasons of 2007, 2008, 2011, 2013 and 2014 in different locations of the province of Rio Negro, Argentina, while some laboratory experiments performed in the city of Buenos Aires. We used individual foragers of Apis mellifera L. and their colonies containing a mated queen, brood, and food reserves in ten-frame Langstroth hives. All beehives used had similar sizes and the same management history from the beekeeper. The honey bees studied belonged to commercial Langstroth-type hives rented to pollinate these plots. Each hive had a fertilized queen, 3 or 4 capped brood frames, reserves and approximately 15,000 individuals56.Testing generalization of memories from pear mimic odours to pear and apple natural floral scentsThe absolute conditioning assays were performed in the laboratories of the School of Exacts and Natural Sciences of the University of Buenos Aires (34° 32′ S, 58° 26′ W), Buenos Aires, Argentina. We used honey bee foragers collected at the entrance of the hives settle in the experimental field of the School of Exacts and Natural Sciences. The apple (‘Granny Smith’ and ‘Red Delicious’ varieties) and pear (‘Packham’ and ‘D’anjou’ varieties) bud samples that we used as conditioned stimuli (CS) during the conditioning were collected at the end of the blossom of 2011 in Ingeniero Huergo (39° 03′ 27.5″ S; 67° 13′ 53.5″ W), province of Río Negro, Argentina, and taken to the laboratory in the city of Buenos Aires, Argentina, to be used within the following 2 days.We first developed the three different synthetic mixtures (PM, PMI and PMII) that could be generalized to the fragrance of the pear flower by foraging bees. The pear synthetic mixtures were formulated considering the previously reported volatile profile of pear blossoms57. Then, we chose the synthetic mixture most perceptually similar to the pear flower fragrance and measured its generalisation response to the apple flower fragrance to test the compounds’ specificity. The chemical compounds used to prepare the different synthetic mixtures for the behavioural assays were obtained from Sigma-Aldrich, Steinheim, Germany. The compounds used for the three pear mixtures (PM, PMI and PMII) were composed by alpha-pinene, 2-ethyl-hexanol, (R)-(+)-limonene, and (±)-linalool. For details of the PM and mixture proportions see Patent PCT/IB2018/05555058.To test generalization, we took advantages of the fact that honey bees reflexively extend their proboscises when sugar solution is applied to their antennae59. The proboscis extension reflex (PER) can be used to condition bees to an odour if a neutral olfactory stimulus (CS) is paired with a sucrose reward as unconditioned stimulus, US60. Conditioned honey bees extend their proboscises towards the odour alone, a response that indicates that this stimulus has been learned and predicts the oncoming food reward. Conditioned bees can generalize such a learned response to a novel odour if it is perceived like the conditioned one (CS). Then we performed three absolute PER conditionings where we paired each of the three PMs with a sucrose-water solution (30%) reward along three learning trials (exp. 4.2a). Afterwards, pear floral scent was presented as novel odour to test generalization. Based on the generalization level to the pear odour, we chose the synthetic mixture that showed the highest generalisation towards pear flower fragrance, and we used it in all the experiments that follow. In an additional 3-trial PER conditioning with the chosen mixture, we quantified generalisation towards both the pear and apple fragrances as novel stimuli (exp. 4.2b).The experimental bees were all foragers, captured from colonies that had no access to any pear and/or apple tree, hence completely naïve for the CSs. Immediately after capture, bees were anaesthetized at 4 °C and harnessed in metal tubes so that they could only move their mouthparts and antennae60. They were fed 30% weight/weight unscented sucrose solution for about three seconds and kept in a dark incubator (30 °C, 55% relative humidity) for about two hours. Only those bees that showed the unconditioned response (the reflexive extension of the proboscis after applying a 30% w/w sucrose solution to the antennae) and did not respond to the mechanical air flow stimulus were used. Trials lasted 46 s and presented three steps: 20 s of clean air, 6 s of odour presentation (CS) and the last 20 s of clean air. During rewarded trials (CS), the reward (US, a drop of 30% w/w sucrose solution) was delivered upon the last 3 s of CS presentation. The synthetic mixtures (PM) were delivered in a constant air flow (15 ml/s) that passed through a 1 ml syringe containing 4 µl of the synthetic mixture on a small strip of filter paper. On the other hand, pear and apple floral volatiles were swept from a 100 g of fresh pear buds (var. ‘D’Anjou’ and ‘Packham’) or apple buds (var. ‘Granny Smith’, ‘Gala’ and ‘Red Delicious’) inside a kitasato by means of an air flow (54 ml/s).Testing discrimination between mimics and natural floral scentsThe differential conditioning assays were performed in a field laboratory in Ingeniero Huergo, province of Río Negro, Argentina. Conditioning trials with AM as CS were carried out in September 2007 and 2008, prior to the beginning of flowering of the fruit trees. Conditioning trials with PM as CS were carried out in September 2011 in the same area (Ingeniero Huergo, province of Río Negro, Argentina). Apple and pear bud samples used as CS were collected in plots that start blooming located around Ingeniero Huergo, but distant (more than 1 km) from the plot where we collected the bees. The bud samples presented the following varieties: M. domesticus sp., ‘Granny Smith’, ‘Gala’, and ‘Red Delicious’; P. communis sp., ‘Packham’ and ‘D’Anjou’.With the aim to develop a synthetic mixture that presents difficult to discriminate with the fragrance of the apple flower by foraging bees, an apple synthetic mixture (AM) was formulated considering the previously reported volatile profile of apple blossoms61. The chemical compounds used to prepare the apple synthetic mixtures for the behavioural assays were obtained from Sigma-Aldrich, Steinheim, Germany. Apple mimic (AM) was composed by benzaldehyde, limonene and citral. For details of the AM proportions see Patent AR2011010244162. Jasmine mimic (JM) was a commercial extract obtained from Firmenich S.A.I.C. y F, Argentina.If the synthetic mixture chosen were perceptually similar to the apple flower fragrance, experimental bees should have difficult to discriminate to the apple flower fragrance to test the compounds’ specificity. Thus, we performed differential PER conditioning between synthetic mixtures (AM and Jasmine mimic, JM) or between synthetic mixtures (AM or JM) and the apple natural fragrance. We followed a differential PER conditioning34 to assess to what extent the bees were able to discriminate the synthetic mimics from their natural flower scents. PER differential conditioning consisted of four pairs of trials, four rewarded trials (CS+) and four non-rewarded trials (CS−) that were presented in a pseudo-randomized manner. Conditionings were performed using the synthetic mixtures PM and AM and the natural floral scents, pear and apple, either as CS+ and CS−. We followed the same procedure that in 3.3 to capture the bees and to present the stimuli during trials.Feeding protocolWe used the offering of scented sucrose solution in the hive as a standardized procedure to establish long-term olfactory memory in honey bees23,24,24,26,63. Scented sucrose solution was obtained by diluting 50 µl of PM or AM per litre of sucrose solution (50% weight/weight, henceforth: w/w). For the ‘apple’ series, colonies were fed 1500 ml of sugar solution offered in an internal plastic feeder for 2 days, about 3 days before the apple trees began to bloom. For the ‘pear’ series, hives were fed 500 ml of sugar solution that we spread over the top of the central frames. Both feeding procedures have been found to be functional for establishing olfactory in-hive memories26. Depending on the pear varieties, the scented sucrose solution was offered when the pear trees were 10–40% in bloom.Colony activityThe effects of the AM-treatment on colony nest entrance activity were studied in 18 colonies located in an agricultural setting of apple and pear trees in Ingeniero Huergo, on an 8-ha plot, half of which was planted with apple trees (varieties: ‘Granny Smith’, ‘Gala’ and ‘Red Delicious’) and the other 4 ha with pear trees (varieties: ‘Packham’ and ‘D’anjou’). The effect of the PM-treatment on colony activity was studied in 14 colonies located in three adjoining pear plots (total surface: 8 ha) in Otto Krause (39° 06′ 22″ S 66° 59′ 46″ O, Supplementary Fig. S5), province of Río Negro, Argentina. The varieties of these plots corresponded to ‘Packham’ and ‘Williams’. Pollen collection (exp. 4.5.2) was also studied in colonies located in these plots.We focused on the nest entrance activity since once the first successful foragers return to the hive and display dances and/or unload the food collected, it promotes the activation or reactivation of inactive foragers and, in a minor proportion, those hive mates ready to initiate foraging tasks39,65,66,67,67. Then, we choose number of incoming bees as an indicator of colony foraging activity, since most of these bees are expected to return from foraging sites33. Thus, we compared the activity level at the nest entrance between 7 SS + PM-treated colonies and 7 SS-treated colonies. We also compared the nest entrance activity level between 5 colonies treated with SS + AM and 5 colonies fed with SS. This activity value was estimated by the number of incoming foragers at the entrance of the hive for one minute, every morning at the same time (10:30 a.m.) during the entire experiment (9 consecutive days). A first measurement was done one day before feeding the colonies (used as covariate) and 7 measurements afterwards.We measured the amount of pollen loads collected by two colonies: one fed with SS + PM and one fed with SS. Pollen loads were collected using conventional pollen traps (frontal-entrance trap), consisting of a wooden structure with a removable metal mesh inside. Pollen samples were collected for 3 days, two hours per day during the late morning, 3, 7 and 8 days after the offering of SS + PM or SS. Pollen pellets identified based on pollen colour as coming from the pear flower or from other species were separated and counted. In addition, we estimated the weight of pear pollen loads during a 5 days period, from 6 to 10 days after the offering of scented or unscented sucrose solution. To reduce measurement error, pollen loads were weighed in groups of 10.Crop yieldPear crop yield was studied in pear plots in General Roca (39° 02′ 00″ S; 67° 35′ 00″ O, Supplementary Fig. S4, Supplementary Table S3), province of Río Negro, Argentina. In an area of 15.2 ha (4 plots of 3.8 ha each), 45 beehives were equidistantly located in groups. We measured the number of fruits per tree set of 30 trees in the surrounding areas of the PM-treated colonies (2 groups of 8 hives) and control colonies (2 groups of 8 hives). A third group category contained 13 untreated colonies. The varieties of the pear trees were ‘D’Anjou’ and ‘Packham’.Apple crop yield estimated by means of number of fruits per plant was studied in General Roca (Supplementary Fig. S2, Supplementary Table S1), province of Río Negro, Argentina. We measured fruit set in the two plots that covered a surface of 3.8 ha and contained a total of 74 colonies distributed in groups (the control plot, 39 SS-treated-colonies treated with SS; and the treated plot, 35 SS + AM-treated-colonies treated with SS + AM). The varieties of the apple trees were ‘Red Delicious’ (clone 1), ‘Royal Gala’ and ‘Granny Smith’.A second studied on apple fruit yield by means of kg of fruits per hectare was performed in Coronel Belisle (39° 11′ 00″ S 65° 59′ 00″ O, Supplementary Fig. S3, Supplementary Table S2), province of Río Negro, Argentina. Four apple plots with ‘Granny Smith’, ‘Hi Early’ and ‘Red Delicious’, clone 1 varieties of 15.4 ha each were randomly assigned to different treatments (treated plot 1, 40 SS + AM-treated-hives treated with SS + AM; treated plot 2, 40 SS + AM-treated-hives treated with SS + AM; control plot 1, 40 SS-treated-hives treated with SS; control plot 2, 40 SS-treated-hives treated with SS).During the fruit harvest, the fruit yield was estimated in the surroundings (150 m around) of two groups of 8 colonies each. We fed one group SS + PM and the other unscented sucrose solution (SS). Yield was estimated as the number of fruits per trees in 30 randomly selected trees within each area, alternating the counts between the North and South faces of the plots. Following the same procedure, we also estimated the number of fruits per trees in the surroundings of two groups of 14 colonies each that pollinated apple crops. Again, we fed one group SS + AM and the other SS. Additionally, a total of 218 colonies in General Roca and 180 colonies in Coronel Belisle have been separated in the two experimental groups, in which yield had been provided by the producer and expressed in kg of fruits per ha. It is worth remarking that in some plots the distance between treated and control beehive groups was around 300 m, suggesting that might have been overlapping flying areas between treated and control hives. Additionally, the apple fields studied in the surrounding of Coronel Belisle, presented many trees without flowers. It was considered that the absence of flowers in numerous trees would bias the counts performed in those fields. Then, to quantify this situation, which might be associated with the masting phenomenon68, samples with the proportions of trees without flowers for every 20 trees in each plot was done. Trees that had between 80 and 100% of their surface devoid of flowers were considered “without flowers” trees, and “trees with available flowers” those that had more than 20% of their surface covered with flowers. An average of 30% of the trees within these plots were devoid of flowers. Thus, a correction factor was considered to evaluate the yield data provided by the grower per plot analysed (Supplementary Table S4).StatisticsAll statistical analyses were performed with R Core Team 201969. For Experiment 4.2 and 4.3, we analysed PER proportion by means of a binomial multiplicative generalized linear mixed model using the “glmer” function of the ‘lme4’ package70.For experiment 4.2a we considered the pear mimics (three-level factor corresponding to PM, PMI and PMII) and the event (two-level factor corresponding to 3rd trial and test) as fixed factors and each “bee” as a random factor.For experiment 4.2b we considered the tested odours (three-level factor corresponding to Apple, Pear and PM) as fixed factors.For experiment 4.3 we considered the tested odours (two-level factor corresponding to CS+ and CS−) as fixed factors. Post hoc contrasts were conducted on models to assess effects and significance between fixed factors using the “emmeans” function of the ‘emmeans’ package version 1.7.071 with a significance level of 0.05.For experiment 4.5.1 we analysed “rate of incoming bees” using a generalized linear mixed model. As Poisson model for incoming bees was overdispersed72, we used a negative binomial distribution using the ‘glmmTMB’ package (function ‘glmmTMB’73. We considered “treatment” [two-level factor corresponding to SS + AM (or SS + PM) and SS], “days” (7-level factor corresponding to the date after treatment), the rate of incoming bees before the offering of food (to control for pre-existing colony differences) as covariate (a quantitative fixed effects variable), and “colony” as a random factor.For experiment 4.6, we analysed fruits per trees by means of a negative binomial multiplicative generalized linear mixed model using the “log” function of the ‘ml’ package70. Post hoc contrasts were conducted on models to assess effects and significance between fixed factors using the “emmeans” function of the ‘emmeans’ package version 1.8.071 with a significance level of 0.05. For experiment 4.6b we analysed “yield” (as weight of fruits per unit area) using a general linear mixed model. We checked homoscedasticity and normality assumptions (Levene and Shapiro–Wilk tests, respectively). We considered “treatment” (two-level factor corresponding to SS + AM and SS) and “apple varieties” (3-level factor corresponding to Hi Early, Granny Smith and Chañar 28) as fixed factors and “location” (2-level factor corresponding to General Roca and Coronel Belisle) as random factors. More

Population sampling and sequencingWe generated whole genome sequence data from 302 wild-caught individual An. minimus female mosquitoes collected from five different field sites in Cambodia using the Illumina HiSeq 2000 platform with 150 bp paired-end reads with a target coverage of 30X for each. Mosquito collections in Thmar Da, in Eastern Cambodia, were done in 2010. Longitudinal monthly collections were performed from February 2014 to January 2015 in two sites in each of the Preah Vihear, and Ratanakiri provinces. Quarterly collections were also done in 2016 in one site in Preah Vihear province, Cambodia.Variant discoveryThe methods for sequencing and variant calling closely follow those of the Anopheles gambiae 1000 Genomes project phase 2 (Ag1000G)27. Sequence reads were aligned to the An. minimus reference genome AminM128. We restricted our analysis to the largest 40 contigs, which cover 96.6% of the AminM1 reference genome, as many smaller-sized contigs can confound diversity and divergence calculations. We found that 138,161,075 (75.4%) of sites within these 40 largest contigs pass our site filters and thus were accessible to SNP calling. Of these, we discovered 38,000,285 segregating single nucleotide polymorphisms (SNPs) that passed all of our quality control filters of 55,307,039 total segregating SNPs. 13.4% of these SNPs were multiallelic, with 32,906,471 biallelic SNPs. There were 4,807,355 triallelic and 286,459 quadriallelic SNPs. A total of 100,160,790 sites were invariant. The median genome-wide coverage was 35X.Population structureA principal component analysis (PCA) over biallelic SNPs distributed over the genome of 302 individual field-collected mosquitoes showed that there is clear population structure of An. minimus in Cambodia. Samples collected from five sites in three provinces split into three distinct clusters; here, we report on 283 individuals that could be clearly assigned to these clusters (Fig. 1), excluding 9 anomalous and 10 outlying individuals. One cluster includes all samples from the western collection site Thmar Da and the northern collection sites in Preah Vihear province, with two further clusters with samples from Ratanakiri province in the northeast. These clusters split primarily along the first and second principal components. This was a surprising finding because this population structure did not correlate to the geographic sampling of these mosquitoes. Individuals collected from the western and northern sites cluster tightly together despite being hundreds of kilometers apart.Fig. 1: Population structure of An. minimus in Cambodia.The map indicates the five Cambodian collection sites. Principal component analysis (PCA) of whole genome sequences of 283 individual An. minimus s.s. collected in five villages in Cambodia shows that there is a distinct population structure and three populations. When performing the same PCA on a large X-chromosomal contig (KB664054), these individuals break into four populations: TD from the West, PV from the northern province in Preah Vihear, and RK1 and RK2, both collected in two sites in Ratanakiri province in the Northeast.Full size imageTo further explore this population structure, we performed the same PCA over individual contigs from different regions of the genome. Performing PCA over the largest X-chromosomal contig KB664054 resulted in a splitting of the western and northern samples, indicating four distinct populations of An. minimus in Cambodia (Fig. 1). PCA from this contig on a quickly evolving sex chromosome revealed more population structure compared to autosomal contigs. The populations defined by these PCA clusters are designated in this study as TD from Thmar Da, in Western Cambodia (n = 41), on the Thai-Cambodian border, PV from the Northern province Preah Vihear (n = 156), and the two distinct populations collected in Ratanakiri province in the Northeast, each including individuals collected at both collection sites, these are designated as populations RK1 (n = 58) and RK2 (n = 28).To confirm our results from PCA, we also performed an admixture analysis. We ran admixture on each of the largest 10 contigs for values of K between 2 and 6 (Supplemental Fig. 1). At K = 2, the samples from Northeastern Cambodia split from Northern and Western Cambodia samples. At K = 3, the two different groups in Ratanakiri were separated, consistent with the PCA results. At K = 4, there was some evidence for geographical population structure between the Western TD and Northern PV populations, but the admixture results did not perfectly correspond with geographic sampling, with some evidence of mixed ancestry in the PV samples. Again, this is consistent with the PCA groupings, with the generally weaker evidence of geographic population structure between TD and PV. A cross-validation analysis showed the lowest cross-validation error for K = 2 and K = 3, consistent with the strongest evidence for population structure between the two RK groups and other populations. Cross-validation error was higher at K = 4, consistent with the weaker differentiation between TD and PV. At no point was their an indication of admixture between RK1 and RK2.To examine population differentiation, we computed differences in allele frequencies between each population using Pairwise Fst. Pairwise Fst between all 4 populations over the largest contig, KB663610, representing 16% of the An. minimus genome, (Fig. 2) shows that differentiation was relatively low between populations of TD and PV with an average pairwise Fst of 0.003, while the difference between RK2 and the other three populations is tenfold higher, around 0.03. Pairwise Fst estimates comparing these populations over other large An. minimus contigs indicate a similar level of differentiation, with average pairwise Fst values over 0.03 (Supplementary Data 3). The two sympatric populations from the Ratanakiri collection sites are as differentiated from each other as they are from the northern and western clusters.Fig. 2: Population diversity and divergence.Nucleotide diversity (π), Watterson’s Theta (θW), and Tajima’s D statistics were calculated over fourfold degenerate sites on autosomal contigs. The error bars indicate 95% confidence intervals calculated over 100 bootstrap replicates over samples. An average pairwise Fst in the table here was calculated in 20 kb windows over the largest contig KB663610.Full size imageThis level of differentiation of RK2, even from the RK1 population, might indicate an emerging cryptic species within An. minimus A or a newly diverging clade. RK1 and RK2 are sympatric populations, both being collected in the same two sites in Northeastern Cambodia. The differences seen here between RK1 and RK2 populations are consistent with cryptic taxa in other anopheline groups. For example, in the An. gambiae complex, the level of differentiation between recently diverged sibling species An. coluzzii and An. gambiae in West Africa is approximately 0.0319.Population diversity and variationTo characterize population diversity among these populations, nucleotide diversity (π), Watterson’s Theta (θW), and Tajima’s D statistics were calculated over 4-fold degenerate sites on autosomal contigs larger than 2 megabases with 100 bootstrap replicates over samples. These 17 contigs represent 80% of the Anopheles minimus genome (Fig. 2). The populations were downsampled for these calculations to have sizes equal to that of the smallest population RK2 (n = 28).There are small but significant differences in the magnitude of the genetic diversity summary statistics between these four different populations. In particular, there were notable differences between the putatively cryptic taxa RK1 and RK2, two populations that were collected in the same sites in Northeastern Cambodia. RK1 had higher levels of nucleotide diversity and lower levels of Tajima’s D than RK2. These differences are consistent with different population size histories between these sympatric groups. Lower values of Tajima’s D suggest stronger population growth in RK1. Comparing all four populations, higher levels of genetic diversity indicate larger effective population sizes of TD and PV compared to RK1 and RK2.RK2 has a significantly reduced nucleotide diversity and Watterson’s Theta compared to the other three populations. This may indicate a smaller population size and a recent bottleneck of the RK2 population in Cambodia. All four An. minimus populations have a negative Tajima’s D, indicating an excess of rare variants, particularly in RK1. This suggests recent population expansions in all populations.Signals of evolutionary selectionWe used Fst to scan across the Anopheles minimus genome to look for regions of the genome with increased differentiation. When we scanned the genome using pairwise Fst, there were no apparent long signals of differentiation that might indicate a large inversion or other structural variants, known to be major drivers of adaptive evolution in other Anopheles groups. To investigate increased differentiation across large regions of the genome, we performed scans of nucleotide diversity (π), Watterson’s Theta (θW), and Tajima’s D over the largest 14 contigs (representing 80% of the An. minimus genome). As with the Fst scans, there were no large regions of higher differentiation in any of the populations that might indicate major structural variants or inversions (Supplementary Figs. 2–4).Whole-genome sequencing allowed us to identify pointed signals occurring across the entire genome using scans of average pairwise Fst. Isolated points of high differentiation were compared over single contigs with average pairwise Fst calculated over windows of 1000 SNPs each and plotted over whole contigs. The strongest signals, indicated by the highest Fst value at the peak of a strong signal of differentiation, were ranked and compared. The five top signals in each of the six comparisons between the four populations are listed in Table 1. These isolated points of high differentiation are one indication of a signal of evolutionary selection. The most differentiated regions by Fst occurred when comparing the RK2 population to the other three populations, with the highest selection peaks with pairwise Fst over 0.4. RK2 also had more distinct signals of selection when compared to the other populations than RK1. Since these signals of differentiation were highly localized, we could look to known gene annotations and gene predictions across the AminM1 reference genome to see which genes were within 100 kbp of the peaks of these signals. We have noted candidate genes of interest that were near the strongest Fst signal peaks and also had known or predicted gene functions (Table 1, Supplementary Fig. 6, Supplementary Fig. 8).Table 1 The top five Fst signals of high differentiation within each of six population comparisons are reported here.Full size tableThere is almost no indication of selection when comparing the Thmar Da population with Preah Vihear, with all but one signal with Fst values below 0.05. The one strong signal between TD and PV (Fst = 0.125) is near a Carbohydrate sulfotransferase, which is involved in detoxification processes. Comparing TD to RK1 and RK2 reveals multiple strong signals of selection, some which are present in both Northeastern populations, as well as many unique RK2-specific signals (Fig. 3, Supplementary Fig. 5).Fig. 3: Signals of selection over a single autosomal contig.Pairwise Fst was calculated in 1000 SNP windows over autosomal contig KB664266, comparing the Thmar Da population to the three other populations, Ratanakiri 2, Ratanakiri 1, and Preah Vihear. There is almost no indication of selection when comparing Thmar Da with Preah Vihear. There is a strongly supported signal of differentiation in both Ratanakiri 1 and Ratanakiri 2 populations at 7.5 Mbp, which is in the same location as a cluster of GSTe genes, including GSTe2, which are known to be involved in metabolic resistance to DDT and pyrethroids. The signal with the highest Fst peak here in RK2, at 6 Mbp is close to an Ecdysteroid UDP-glucosyltransferase gene, shown to confer pyrethroid insecticide resistance in other anophelines. These are a few of many selection signals identified in this study that may be associated with insecticide pressure on these An. minimus populations.Full size imageMany of the strongest signals identified in this study may be associated with insecticide pressure on these An. minimus populations. The strongest selection signals in every population comparison were close to genes that are involved in detoxification, signal transduction, and adaptations to oxidative stress, or have been functionally validated to have mutations that confer resistance to insecticides (Table 1). Some signals of interest include a strongly supported signal of selection in both RK1 and RK2 populations at 7.5 Mbp on the contig KB664266, which is in the same location as a cluster of glutathione-S-transferases, including GSTe2, which has been shown to be involved in the metabolism of DDT and pyrethroids, mutations in which mediate metabolic insecticide resistance29. The signal with the highest pairwise Fst peak on the same contig KB664266, at 6 Mbp is an RK2-specific signal and close to an Ecdysteroid UDP-glucosyltransferase gene, which has been shown to confer pyrethroid insecticide resistance in An. stephensi30.Another notable signal is between the RK1 and RK2 populations on the contig KB663610, a Peptidase S1 domain-containing protein AMIN002286, which has been shown to be involved in response to parasite pathogens in insects31. The signals of selection observed in this study are mostly distinct from the main selection signals seen in An. gambiae complex mosquitoes19, the primary vectors of Plasmodium falciparum in Africa.Insecticide resistanceWe report here variants at known insecticide resistance-associated alleles for each of the four An. minimus populations. Variants occurring at a frequency of more than 2% in at least one of the four populations are reported in the known insecticide-resistance-associated genes Ace1, Rdl, KDR, and GSTe2 (Supplementary Data 2). GSTe2 mutants are present in multiple populations, at a low rate, and there are a few individuals in Thmar Da and Preah Vihear with the Rdl resistance mutation, which is known to confer resistance to cyclodiene insecticides, despite evidence from other studies that species in this region lack this resistance mutation32. We did not investigate copy number variation, which is one mechanism by which GSTe2 confers insecticide resistance. These SNP variants indicate variation throughout these insecticide-resistance-associated genes, and though most of these populations do not currently have high rates of validated insecticide resistance-associated mutations, this underlying variation provides the potential for structural and transcriptional events resulting in greater levels of insecticide resistance in An. minimus populations. More