Ecology

We applied a modified version of a planning framework for CWR conservation25,26 which has been used by numerous countries of Europee.g.29,63,64, Americae.g.65, Africa30 and Asia66,67. We addressed the following main steps of the toolkit (see Spanish version49): (i) CWR checklist, i.e., creating a list of CWR taxa distributed in an area (Supplementary Data 1), (ii) CWR inventory, i.e., taxa selection and collation of ancillary data, including taxonomic data (Supplementary Data 2), (iii) taxa extinction risk assessment (Table 1, Supplementary Data 3), and (iv) a systematic conservation planning assessment, i.e., spatial analyses to assess conservation areas (Fig. 1). We only provide a brief description of steps i-iii, as these are thoroughly described in Goettsch et al.2. Here, we focus on the systematic conservation planning assessment, introducing an approach in order to identify conservation areas for CWR that account for genetic differentiation in a spatially explicit way, through the use of proxies of genetic differentiation (Fig. 1).During the process -framed under the project “Safeguarding Mesoamerican crop wild relatives” (https://www.darwininitiative.org.uk/project/23007/)- more than 100 experts from academic, governmental, and non-governmental organizations from El Salvador, Guatemala, Honduras, Mexico, the UK, and IUCN participated in six workshops, shared data, and provided fundamental knowledge and feedback at each project stage to ensure accurate, reliable and robust information for next steps. The checklist, inventory and risk assessment were collaboratively developed between partners of El Salvador, Guatemala, and Mexico (hereafter, Mesoamerica; Goettsch et al.2). The spatial analysis to identify areas for in situ and ex situ conservation of CWR was done independently by each country.To assess conservation areas of CWR in Mexico, we developed proxies of genetic differentiation that account for evolutionary processes by including historical and environmental drivers of genetic diversity (see the Methods section ‘Proxies of genetic differentiation’). In addition, we used criteria such as information on taxon-specific tolerance to human-modified habitats and IUCN extinction risk category. We applied a systematic conservation planning approach and performed spatial analysis using the software Zonation50. We compared different scenarios to represent genetic diversity of CWR based on potential species distribution models (SDM) and proxies of genetic differentiation.Study areaMesoamerica is a cultural region encompassing the territories of Belize, Guatemala, El Salvador, the southern part of Mexico and parts of Honduras, Nicaragua and Costa Ricasee 2. In this study, we also included the dry areas of northern Mexico that are part of Aridamerica68 and the Nearctic biogeographic realm69 to account for the full extent of the geographic range of many taxa included in the extinction risk assessment2.For the assessment of conservation areas, we focused on Mexico, which is one of the most biodiverse countries in the world70. The Mexican territory covers 80% of the landscapes of the region called Mesoamerica. Its high biological diversity is attributed to its geographic, topographic, climatic, geological and cultural characteristics, which, among other factors, shaped the distribution of an extraordinary variety of ecosystems and species with high levels of endemism and species turnover among different regions32,71,72,73. In particular, the high genetic variation within populations of landraces and CWR is the result of past and ongoing sociocultural processes occurring in a wide range of distinct environmental conditions74,75.(i) CWR checklist and (ii) CWR inventoryThe compiled CWR checklist included ~3000 species and subspecies of 92 genera and 45 families of plants that belong to the same genus of a crop cultivated in Mesoamerica, or wild plant collected for food or other uses in the region (Supplementary Data 1).The first set of criteria were established in preparation for the first stakeholder workshop. The following criteria were applied at the genus level to compile the CWR inventory: (1) occurrence of wild relatives of cultivated plants or crops that were domesticated in Mesoamerica; (2) existence of research groups working on taxa that could support the extinction risk assessment; and (3) relation to a crop of economic and nutritional importance at local, national and regional levels, or cultivars known to require genetic improvement.To narrow the list for the inventory and extinction risk assessment, similar criteria were agreed upon in the same workshop and applied at the species level: (1) native distribution in Mesoamerica, incl. Aridamerica; (2) related to a crop of economic or social importance based on production and nutritional value; (3) related to a taxon for which Mesoamerica is the center of origin or domestication; (4) constitutes part of the primary or secondary gene pool, and in some cases the tertiary gene pool76. The primary gene pool consists of wild plants of the same species as the crop and thus their mating produces strong fertile progeny. The secondary gene pool is composed of wild relatives distinct from cultivated species but closely related as to produce some fertile offspring (same taxonomic series or section in the absence of crossing and genetic diversity information, see the ‘taxon group’ concept proposed by Maxted and collaborators77, Supplementary Note 5). The tertiary gene pool (same subgenus in the taxon group concept) corresponds to CWR that are more distant relatives to the taxa of the primary gene pool, but can have important adaptive traits which can be used with specific breeding techniques. This provided a preliminary list of 514 CWR taxa related to avocado, cotton, amaranth, cocoa, squash, sweet potato, chayote, chili pepper, cempasuchil, bean, sunflower, maize, papaya, potato, vanilla, and yuca (Supplementary Data 2).The list had to be further reduced due to time and funding restrictions to include those genera which when added together would include no more than 250 taxa, and that the taxonomic groups could be comprehensively assessed and their taxa evaluated throughout their entire range. Thus, not all species in the group necessarily met the criteria previously mentioned. See the final Mesoamerican CWR inventory in Supplementary Data 3; see summary in Table 1.(iii) Taxa extinction risk assessmentFull methodological details and results of this section are described in Goettsch et al.2. Summarizing, during the process 224 taxa were evaluated according to the International Union for Conservation of Nature, IUCN, Red List Categories and Criteria78. The IUCN Red List is a critical indicator to identify species most vulnerable to extinction considering a set of criteria, i.e., species’ population trends, size, structure, and geographic ranges. A Red List workshop with the participation of 25 experts from different project partner institutions and IUCN specialists was organized to assess the extinction risk of taxa. The threat analysis included not only species, but subspecies and subpopulations (i.e. races) for some groups (Supplementary Data 3, see summary in Table 1).(iv) Systematic conservation planning assessmentTo undertake the following spatial analyses we focused on the dataset of 224 CWR described above, which is representative of the CWR of the main crops of Mesoamerica (10 genera, Table 1).Species distribution modelingTo compile occurrence records, hundreds of data sources were consulted, including published and personal databases of the project participantse.g.79,80,81,82, the Agrobiodiversity Atlas of Guatemala (https://www.ars.usda.gov/northeast-area/beltsville-md-barc/beltsville-agricultural-research-center/national-germplasm-resources-laboratory/docs/atlas-of-guatemalan-crop-wild-relatives), the Global Biodiversity Information Facility (GBIF, https://www.gbif.org/), and Mexico’s Biodiversity Information System (SNIB, http://snib.mx/).To generate potential species distribution models (SDM), we used more than 13,000 occurrence records (Supplementary Data 4), that were standardized and curated by experts to generate the range maps of taxa as part of the extinction risk assessment, which were published in IUCN Red List (https://www.iucn.org/news/species/202109/threats-crop-wild-relatives-compromising-food-security-and-livelihoods). Spatial resolution of the SDM was 1 km2. SDM were obtained for taxa with more than 20 unique occurrence data in a 1 km2 grid covering the study extent to reduce uncertainty when using smaller sample sizes83. We used 19 bioclimatic variables and other climatic variables, such as annual potential evapotranspiration, aridity index, annual radiation, slope, and altitude84,85,86. Climate data represents annual and seasonal patterns of climate between 1950 and 2000. Also, we used a variable that described the percentage of bare soil and cultivated areas87. Collinearity between variables was assessed with the ‘corselect’ function of the package fuzzySim version 1.088, using a value of 0.8 and the variance inflation factors as criteria to exclude highly correlated variables.We used MaxEnt version 3.3.1, a machine-learning algorithm that uses the maximum entropy principle to identify a target probability distribution, subject to a set of constraints related to the occurrence records and environmental data89,90. Model calibration area for each taxon included those ecoregions where the taxon has been recorded; we used the terrestrial ecoregions dataset69. We did this based on the calibration area or ‘M element’ of the BAM diagram that refers to areas that have been accessible to the taxon via dispersal over relevant periods of time91,92. We randomly sampled 10,000 background localities from the selected areas.To reduce model complexity without compromising model performance, we built several models by varying the feature classes (FC) and regularization multipliers (RM) (see refs. 93,94,95) using R 3.6.096 and ‘ENMeval’ version 0.3.0 package97. FC determines the flexibility of the modeled response to the predictor variables, while the RM penalizes model complexity93. Occurrence records were randomly divided into 70% for model selection, and 30% of data was withheld for model validation. ENMeval carries out an internal partition of localities to test each combination of settings. Therefore, we selected the random k-fold method to divide localities into four bins. We build models with six FC combinations and varied RM values ranging from 0.5 to 4.0 in 0.5 increments. Optimal models were selected using Akaike’s Information Criterion corrected for small sample sizes (⍙AICc = 0). This method penalizes overly complex models and helps to choose those with an optimal number of parameters. However, it has been shown that the number of model parameters may not correctly estimate degrees of freedom98, and that model selection should not be selected solely with one measure99. Thus, we used 30% of the withheld data to test the area under the curve (AUC) of the receiver operating characteristic, and the omission error under a 10 percentile training threshold.We used the ten percentile or minimum training presence threshold to obtain binary maps of the presence and absence of suitable areas for species distribution. We asked experts of each taxonomic group who were also involved in the extinction risk assessment to select one of these two options and to indicate possible overestimated areas, which were then eliminated case by case using the information of Mexican ecoregions100 and watersheds101. Eight models were binarized with the minimum training presence threshold; for the other models we used the 10 percentile threshold. See MaxEnt performance and significance of SDM at Supplementary Data 5. AUC values ranged from 0 to 1; 0.5 indicated a model performance not better than random, while values closer to 1 indicated a better model performance; here we used SDM showing AUC values higher than 0.7. For Phaseolus and Zea, we used SDM that were previously generated by Delgado-Salinas et al.102, and Sánchez González et al.103, respectively. SDM for 116 taxa were validated by experts of each taxonomic group. See references and download links at Supplementary Data 6.For the conservation planning analysis of Mexico, we clipped the models to the Mexican territory, and trimmed the continuous SDM using the binary SDM to keep pixel values of areas with elevated probability of taxa presence. For taxa without SDM, we included the occurrence records of these taxa in the spatial analysis by using the information on observation location, i.e., coordinates (see Supplementary Data 3). This is done by enabling the function ‘species of special interest’ (SSI). See further details in the method section ‘Final conservation analysis’.Proxies of genetic differentiationTo identify proxies of genetic differentiation in an explicit, efficient, and repeatable way, we included environmental and historical drivers of genetic diversity. For this, we first divided Mexico into 27 Holdridge life zones (Supplementary Fig. 2, Supplementary Data 8), which we then subdivided according to phylogeographic studies that have found genetic differentiation among populations of several taxa (see division of each life zone into proxies in Supplementary Fig. 4; Supplementary Fig. 3 provides a general geographical overview of Mexico and main geographic references mentioned in Supplementary Fig. 4). The literature review was done searching for the words “phylogeography” and one of the following: (i) name of the Mexican biogeographic zones, (ii) “Mexico” + an ecosystem name (e.g. “Mexico” “rainforest”) or (iii) “Mexico” + lowlands/highlands. See list of references used in this study in Supplementary Data 9.In addition, we manually reviewed the citations to the most cited papers of the previous search. Reviews and meta-analyses were also included, although we excluded studies performed in CWR to show that our approach can be used without prior information on this group. As more studies on such taxa become available, they can be used to fine-tune the proxies of genetic differentiation. We focused on terrestrial species including plants, animals, and fungi (Supplementary Data 10) except to subdivide a life zone covering the coasts of the California Peninsula, where we could not find studies on terrestrial taxa so we included studies on fish species (see Supplementary Fig. 4).Since most of the life zones cover large territories, and complete phylogeographic congruence among different taxa is uncommon, we targeted to represent general trends that would likely occur across diverse species, instead of trying to represent fine idiosyncratic patterns of genetic differentiation. For instance, although distribution ranges of highland taxa shifted during the Pleistocene climate fluctuations, in general populations persisted (glacial-interglacial periods) within the main mountain ranges, while lowland populations were ephemeral (only glacial periods). So, gene flow among mountain ranges was more limited than within them. As a result, genetic differentiation among mountain ranges of different biogeographic provinces has been widely documented32, so we used this general pattern to subdivide the life zones that occur in highlands. These types of patterns are particularly relevant for a country like Mexico, due to its complex topography, tropical latitude, and geographic features of different ages, which promote population differentiation among the Mexican main geographic features. To translate the phylogeographic information into a spatial context, we used biogeographic regions, basins, topographic or edaphic data to split the life zones into different subzones using the best fitting cartography to represent the phylogeographic patterns (Supplementary Fig. 4).We obtained 102 proxies of genetic differentiation for Mexico (Supplementary Fig. 5). We validated our findings by using available genomic data of an empirical study of a wild relative of maize, the teosinte Zea mays subsp. parviglumis, which was not included in the literature review in order to test the usefulness of our approach regarding the lack of genetic data. The dataset includes ca. 1800 occurrence records and ca. 30,000 SNPs48. Sampling localities were not used for distribution modeling. Admixture groups per population were estimated for K1 to 60. According to the population analysis, Z. mays subsp. parviglumis is structured in 13 genetic clusters along a longitudinal gradient (Fig. 3a–c). We used the K = 13 for plotting based on the Cross-Validation error. The proportion of each genetic cluster was estimated by sampling locality and plotted using pie charts over the map (Supplementary Fig. 6). Then, using the data layer of the SDM subdivided by proxies of genetic differentiation, we extracted which was the proxy most frequent in a 5 km buffer for each sampling locality. The Admixture plot was ordered by all genetic clusters and subdivided by the proxy of genetic differentiation most frequent for each locality. In addition, we calculated a principal component analysis (PCA) and projected into a score plot the first three components. Individual samples were colored by the proxies where they fell in the 5 km buffer (Fig. 3c). To compare how genetic variation was represented by the different scenarios we plotted the proportion of the area of each proxy as given by the potential SDM according to two different scenarios (only considering SDM; combining SDM*PGD) considering 20% of Mexico’s terrestrial area (Fig. 3d). Analyses were run in R version 3.5.196 using the R packages pcadapt version 4.3.3104, ggplot2 version 2_3.3.3105, readr version 1.4.0106, gridExtra version 2.3107, ggnewscale version 0.4.5108, scatterpie version 0.1.5109, pophelper version 2.3.1110, raster version 3.4-5111, rgdal version 1.4-8112, rgl version 0.107.10113, and sp version 1.4-4114,115.Habitat preferenceWe considered habitat preference to refine the presence of CWR in the planning process; thus minimizing commission errors and highlighting areas that more probably contain taxa116. For each taxon, experts assessed its habitat preference (1: high preference; 0.5: low preference; 0.1: no preference) according to the following categories: (i) well-conserved vegetation (i.e. primary vegetation), (ii) human-impacted vegetation (i.e. secondary vegetation), (iii) less intensive rainfed and moisture agriculture, (iv) intensive rainfed and moisture agriculture, (v) irrigated agriculture, (vi) induced and cultivated grasslands and forests, and vii) urban areas (Supplementary Data 11). To spatially delimit these classes, we used the land use cover and vegetation map for Mexico117, and assessed seven main categories of land cover by grouping the map legend (Supplementary Fig. 9). To differentiate between less intensive and intensive cultivated areas, we followed Bellon et al.56, who associated the presence of native maize varieties of Mexico to occur in municipalities with average yields of less than or equal to 3 t ha-1 using agricultural production data from 2010 from the Information System of Agrifood and Fisheries (SIAP), and selected the municipalities with the established average maize yield. We combined the municipality layer with the land cover map to differentiate areas of high and low agricultural intensity. To generate taxon-specific habitat layers, we associated the habitat preference classes established by experts to the land cover map aggregated into seven major land cover categories, using R 3.6.096 and the following packages: raster version 3.4-5111 and rgdal version 1.4-8112. We obtained habitat maps for 116 taxa with SDM.Preliminary analysisWe generated five preliminary scenarios to explore different approaches to include conservation features for maximizing the representation of intraspecific diversity as given by taxa and proxies of genetic differentiation, i.e., representation of proxies within a taxa range (Supplementary Fig. 7): (i) “SDM” scenario, included 116 SDM, which we used as base scenario to examine the representation of taxa and proxies of genetic variability (n = 116); (ii) “SDM + LZ” scenario, included 116 SDM and 27 layers representing Holdridge life zones to consider environmental variation (n = 143); (iii) “SDM + PGD” scenario, included 116 SDM and 102 layers representing each proxy of genetic differentiation individually (n = 218); (iv) “SDM*PGD” scenario, included 5004 input layers representing the intersection of SDM and PGD (n = 5004; combining 116 SDM with 102 proxies resulted in 11,832 layers, but as some of the intersections produced empty outputs given the extension of SDM that do not cover all Mexico, for further analysis we used 5004 input layers with value data. To subdivide the layers, we used ArcGIS version 10.2.2118; to filter the layers, we used R 3.5.196.); (v) “SDM and PGD as ADMU” scenario, included 116 SDM as the main conservation features, while integrating one single layer of proxies of genetic differentiation to consider each of them as planning units by using the ‘Administrative units’ function. Analysis was done in Zonation50,119.We compared the results by assessing 20% of Mexico’s terrestrial area (Fig. 5b) to perform statistical analysis in R 3.5.196 using the following packages: purrr version 0.3.4120, ‘dplyr’ version 1.0.2121, ‘ggplot2’ version 2_3.3.3105, ‘raster’ version 3.4-5111, ‘scales’ version 1.2.0122, ‘sp’ version 1.4-4114,115, ‘tidyr’ version 1.0.2123, and ‘vegan’ version 2.6-2124. The area threshold was established based on Aichi target 11 and on comparisons of performance curves to efficiently represent taxa ranges delimited by SDM and proxies of genetic differentiation (Fig. 6). As using SDM combined with proxies of genetic differentiation showed the highest representation of genetic diversity (“SDM*PGD” scenario), we used this approach for the final analyses.Final conservation analysisWe identified areas of high conservation value for CWR in Mexico by using the software Zonation version 4.050,119, a systematic conservation planning tool that allows optimizing representation of species, taxa, or other conservation features, e.g., proxies of genetic differentiation, in a given study area. The program hierarchically ranks areas by removing cells of low conservation value, as given, for example, by a reduced number of taxa or occurrence of low weighted features, while considering multiple criteria such as the weighting of taxa and habitat preference of taxa. We applied the core-area zonation removal rule (CAZ) to maximize the representation of all conservation features in a minimal possible area51. Zonation generates two main outputs: (a) a hierarchical landscape priority rank map, that allows decision makers establishing different area thresholds to highlight areas of conservation interest; and (b) a representation curve showing species or conservation features range distribution in a given area. The curve also allows identifying how much area is needed to cover a certain taxon range or the distribution of a feature of conservation interest.For the conservation scenarios, we integrated the following inputs in the Zonation software: (1) 5,004 layers, i.e., SDM intersected with proxies of genetic differentiation (as described by “SDM*PGD” scenario, Fig. 4), (2) occurrence records of 98 taxa; only for those taxa without SDM, see Supplementary Data 3), (3) taxa specific habitat layers (according to Supplementary Data 11 and Supplementary Fig. 9), and (4) IUCN threat category (Supplementary Data 3) as an additional parameter to weight taxa differently to consider their vulnerability to extinction, see details below. See Zonation configuration at Supplementary Note 6.Data from different sources can be mixed in the same analysis, which is useful to not lose or omit information of any taxa of interest in the assessment. Here, we included information of a total of 214 taxa (see Supplementary Data 3). Distribution data of 116 taxa were represented by 5004 layers that resulted from combining 116 SDM and 102 PGD. This approach showed the highest proportion of area of taxa ranges (on average 41%) and highest representation of PGD within the area of each taxon (on average 76%; Fig. 4; see description in the main text). For some taxa, e.g. Cucurbita pepo, Physalis cinerascens, and Zea mays information on its distribution was assessed at subspecies level rather than at species level, explaining the difference in numbers of CWR taxa.In addition, we included occurrence data of 98 taxa without SDM to prevent missing important areas of taxa known distribution that are important to conserve (see Supplementary Data 3). We enabled the function ‘species of special interest’ (SSI) of Zonation, and included a SSI feature list file, listing the taxon names, as well as taxon-specific coordinate file for each of the 98 taxa that have been reviewed by the experts of each group. The spatial reference system was World Mercator projection. Occurrence data and SDM are treated similarly in the Zonation analysis, i.e., cells where taxa occur will be retained in the solution as long as possible to maximize its representation in the solution.We assigned weights to the 116 taxa with SDM by using IUCN threat categories (according to Supplementary Data 3), giving highest values to taxa with highest risk of extinction that urgently need management actions to further avoid genetic erosion. By including conservation feature weights, Zonation estimates the conservation value of a cell not only based on the presences of a taxa and their distribution range, but also on the weight. A high weight indicates a high conservation value of cells where these taxa are distributed. As there is no rule for weight setting, we assigned values between 1 and 0 regardless of taxa distribution ranges, which is automatically considered in the Zonation algorithm to guarantee the representation of locations where limited-range distributed taxa occur within the most valuable conservation area. Thus, weights were assigned as follows: Critically endangered, CR: 1; Endangered, EN: 1; Vulnerable, VU: 0.8; Near threatened, NT: 0.5; Data deficient, DD: 0.3; Least concern, LC: 0.2 Not evaluated, NE: 0.1. SSI taxa were all weighted similarly with 1 in order to represent the 98 SSI taxa and their occurrences in the top fraction of the most valuable conservation area, as these areas could be considered as ‘irreplaceable’ in terms of conservation. The conservation of these taxa that are only known in a few locations is crucial to maintain their populations. Information on weights for taxa with and without SDM is included in the file that lists the 5004 conservation features and the SSI file, respectively.To include the information on habitat, we included 116 habitat maps which guide the selection of cells to areas where its presence is more probable (see the Methods section: “Habitat preference”). This option can only be used for taxa represented by a raster layer, and is not available for SSI taxa included via occurrence records. By enabling the “landscape condition” option of Zonation, each habitat map is linked to a specific conservation feature layer. Areas with unfavorable habitats will quickly be masked out during the selection of cells in order to obtain a solution that favors conservation areas within areas of preferred habitat.We generated three final scenarios to identify conservation areas for (a) all taxa, (b) taxa exclusively distributing in natural vegetation, and (c) taxa associated with a wider range of habitats such as natural vegetation, agricultural and urban areas. The Zonation configuration remained similar among the three scenarios. When taxa were not included in a given scenario, we assigned a value weight of 0. This excluded the feature to be considered for the hierarchical prioritization of the landscape, but still allowed to evaluate the taxa during post-processing.To evaluate the spatial results (Supplementary Fig. 11), we analyzed performance curves to represent proxies of genetic differentiation within each taxon range (Supplementary Fig. 12). Also, we considered the most valuable 20% area of Mexico to calculate the coincidence of the three scenarios (Supplementary Fig. 13), and the overlap with federal protected areas125 and indigenous regions126,127 (Supplementary Fig. 14), and land cover data used in the analyses (Supplementary Figs. 9, 15).We discussed the proposed methodological framework, input layer and criteria during a fourth workshop in Mexico. It is worth mentioning that we ran several analyses including additional layers, such as areas where indigenous communities live that promote the presence of CWR in the landscape6. However, as the output indicated no evident difference by including this information, final analyses did not consider these data. We neither included protected areas nor tried to expand on the current 12% protected area system, because most management plans do not specifically address CWR management (but see the management program of the Protected Area of ‘Sierra de Manantlán’128), and thus generally do not adequately plan for wild and native genetic resources129. We also discussed different approaches to consider connectivity for taxa, habitats and proxies of genetic differentiation in the Zonation processing. Still, we finally decided to run the analysis without particularly accounting for connectivity as we had no taxa-specific information on dispersal abilities or possible effects of fragmentation, and we did not want to lose efficiency of the solution to represent taxa by or include lower-quality habitats by forcing the solution to an aggregation of pixels.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. More

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Thermogravimetric/differential thermogravimetry analyses of corn stoverThermogravimetric/Differential Thermogravimetry (TG/DTG) curves are shown in Fig. 2. The pyrolysis process of corn stover could be divided into three stages. The first stage was the dehydration stage, which occurred at approximately 55–125 °C, and the weight loss was mainly accounted for by water19. The second stage was the pyrolysis stage, which occurred at approximately 200–400 °C and mainly involved the decomposition of cellulose, hemicellulose and a small amount of lignin. This process involved the generation of CO and CO2 and the breaking of carbonaceous polymer bonds20. In addition, a shoulder peak in the range of 265 to 300 °C in the DTG diagram could be caused by side chain decomposition and glycosidic bond cleavage of xylan during the pyrolysis of corn stover21. The third stage was the carbonization stage, which occurred above 400 °C; this stage mainly involved the decomposition of lignin22,23. The carbonization process was relatively slow after 600 °C; this process was called the passive pyrolysis stage24. In general, the TG loss in the pyrolysis process of corn stover was mainly from the moisture in the biomass sample in the first stage. Hemicellulose and cellulose decomposition occurred in the second stage, and lignin decomposition occurred in the third stage25. In this experiment, the minimum pyrolysis temperature for the preparation of biochar was 400 °C. Therefore, the pyrolysis of biochar was relatively complete.Figure 2TG/DTG curves of corn stover.Full size imageCharacterization of biocharYield and specific surface area analysesThe yield and SBET are presented in Table 2. BC, BC-H and BC-OH represent the origin, acid-modified, and base-modified biochar, respectively. The yield of corn stover biochar exhibited a negative correlation with the temperature and decreased from 39.65 to 28.26% when the pyrolysis temperature increased from 400 to 700 °C. This phenomenon could have occurred due to the loss of more volatile substances and the thermal degradation of lignocellulose with increasing temperature, thus reducing the yield of biochar26,27. The SBET of the original biochar showed little difference below 700 °C but increased significantly at 700 °C. Combined with the SEM analysis (Fig. 3), at low temperatures, more ashes on the surface of biochar could block its pores so that the change in SBET was not obvious. At 700 °C, because the ash content significantly reduced and the pyrolysis was more sufficient, the pores of the biochar were more developed, and the SEBT significantly increased. The SBET of the acid/base-modified biochar increased with increasing temperature. The SBET of biochar was larger than that of the original biochar after acid and base modification at 400–600 °C. This phenomenon occurred because the porous structure of biochar was enhanced by acid and base modification28. Moreover, pickling removed most of the inorganic substances in biochar and reduced ash content, while alkali washing removed the tar on the surface of biochar to a certain extent29. However, at 700 °C, the SBET of biochar after acid/base modification was lower than that of the original biochar. Combined with the SEM (Fig. 4), the acid/base modification caused the nanopores of biochar to collapse into mesopores or macropores30. Therefore, the well-developed pore structure of the biochar prepared at 700 °C was destroyed by acid/base modification, resulting in a significant decrease in SBET.Table 2 Yield and SBET of different biochars.Full size tableFigure 3SEM (ZEISS) images of biochar at different pyrolysis temperatures: (a) C1, (b) C8, (c) C12, and (d) C16.Full size imageFigure 4SEM (OPTON) images of C16 biochar and its acid/base modification: (a) C16, (b) C16-H, and (c) C–OH.Full size imageScanning electron microscopy analysisThe C1, C8, C12 and C16 biochars had the highest Cd2+ removal rates at 400, 500, 600 and 700 °C, respectively. Therefore, these BCs were selected for SEM analysis. Figure 3 clearly showed that as the pyrolysis temperature increased from 400 to 700 °C, the pore structure of biochar became more developed, with a smaller pore size and more pores. Although there were numerous pores at 500 °C, the pores were not fully developed and were blocked inside. At 700 °C, the skeleton structure appeared, and the particle size of ash blocked in the pores decreased.By taking C16 biochar with the highest removal rate of Cd2+ as the research object, the changes in the biochar surface before and after modification were compared. C16-H and C16-OH represent acid-modified and base-modified biochar, respectively. After acid/base modification, the ash content on the surface of the biochar decreased, and the pore size increased (Fig. 4). Therefore, some skeleton structures could collapse after corrosion, which was consistent with the previous SBET results. Sun et al. discovered that citric acid-modified biochar would lead to micropore wall collapse and micropore loss, resulting in a reduction in SBET31. This finding was in agreement with the results of our study.Fourier transform infrared spectroscopy analysisThe FTIR spectra of biochar at different pyrolysis temperatures are presented in Fig. 5a.Figure 5FTIR spectra of corn stover biochar: (a) different pyrolysis temperatures and (b) different modification treatments.Full size imageAs the pyrolysis temperature increased from 300 to 700 °C, the absorption peak intensity showed a downwards trend. There was a remarkable decrease in features associated with stretch O–H (3400 cm−1)32. The vibration peaks of C–H (2924 cm−1) and C=O (1610 cm−1) decreased with increasing temperature, which could be due to the reduction in –CH2 and –CH3 groups of small molecules and the pyrolysis of C=O into gas or liquid byproducts at high temperatures33. In addition, the peak at 1435 cm−1 was identified as the vibration of C=C bonds belonging to the aromatic skeleton of biochar. A decrease in the absorbance peaks was found at 1115 cm−1, which corresponded to C–O–C bonds. The ratio of intensities for C=C/C=O (1550–1650 cm−1) and C–O–C (1115 cm−1) to the shoulder (1100–1200 cm−1) gradually decreased, and the loss of –OH at 3444 cm−1 indicated that the oxygen content in biochar reduced. The cellulose and wood components were dehydrated, and the degree of biochar condensation increased at higher temperatures. The bending vibration peaks of Ar–H at 856 and 877 cm−1 changed little at different temperatures, which showed that the aromatic rings were relatively stable below 700 °C34. Combined with the above analysis the condensation degree of biochar increased gradually above 400 °C35,36. In summary, as the pyrolysis temperature increased, the degree of aromatization of biochar improved, and the numbers of oxygen-containing functional groups decreased continuously.Figure 5b showed that after acid/base modification, the absorbance peaks at 3444 cm−1, 1610 cm−1 and 1115 cm−1 increased, indicating that the number of oxygen-containing functional groups increased. However, the stretching vibration peak of aromatic ring skeleton C=C (1435 cm−1) and the bending vibration peaks of Ar–H (856–877 cm−1) changed little. The number of functional groups of acid-modified biochar increased more than that of alkali-modified biochar. Mahdi et al. found that acid modification increased the number of functional groups in a study of biochar modification37. After acid/base modification, the number of oxygen-containing functional groups, such as hydroxyl and carboxyl groups, increased.Optimization of biocharFigure 6 illustrates that the removal rates of Cd2+ by corn stover biochar (original, acid-modified, and base-modified biochars) consistently increased with increasing pyrolysis temperature. The highest removal rate reached 95.79% at 700 °C. The removal rate decreased after modification, especially after pickling. The results showed that C16 biochar had the best removal effect on Cd2+.Figure 6Cd2+ removal rate of different biochars (BC: original biochar, BC-OH: alkali-modified biochar, and BC-H: acid-modified biochar).Full size imageIntuitive and variance analyses were employed to explore the influences of biochar preparation conditions on the removal rate of Cd2+.

1.

Intuitive analysis
The intuitive analysis of the orthogonal experiment is shown in Table 3 and Fig. 7. The pyrolysis temperature had the most significant influence on the removal of Cd2+, followed by the retention time and finally the heating rate. Therefore, the optimal conditions for biochar preparation were a pyrolysis temperature of 700 °C, a retention time of 2.5 h, and a heating rate of 5 °C/min.

2.

Variance analysis
Variance analysis showed that the effect of pyrolysis temperature on the removal rate of Cd2+ was very significant (Table 4). The effects of retention time and heating rate were not significant. This phenomenon was consistent with the conclusions obtained in the intuitive analysis.

Table 3 Intuitive analyses of influencing factors of biochar preparation.Full size tableFigure 7Intuitive analysis diagram of influencing factors for biochar preparation.Full size imageTable 4 Variance analysis.Full size tableAnalysis of adsorption mechanismThe SBET of the unmodified biochar did not change significantly with temperature, which indicated that SBET could potentially not be a critical factor for Cd2+ adsorption. Qi et al. obtained a similar conclusion when studying the adsorption of Cd2+ in water by chicken litter biochar38. In addition to SBET, the four primary mechanisms involved in the removal of heavy metal ions by biochar were as follows: (1) Ion exchange: the alkali or alkaline earth metals in biochar (K+, Ca2 +, Na+, and Mg2+) were the dominant cations in ion exchange39. (2) The complexation of oxygen-containing functional groups mainly included hydroxyl and carboxyl groups40. (3) Mineral precipitation: Cd2+ was precipitated by minerals on the surface of biochar to form Cd3(PO4)2 and CdCO341. Soluble cadmium precipitated with some anions released by biochar, such as CO32−, PO43− and OH−42,43. (4) π electron interaction: Cd2+ coordinated with the π electrons of C=C or C=O at low pyrolysis temperatures43,44. Biochar contains more aromatic structures at high pyrolysis temperatures, which could provide more π electrons. Therefore, the π electron interaction in adsorption of Cd2+ was effectively enhanced45.C1, C8, C12 and C16 were selected to study the adsorption mechanism. Related physicochemical properties are given in Table 5.Table 5 Physicochemical properties of biochar at different pyrolysis temperatures.Full size tableThe CEC of biochar gradually increased as the pyrolysis temperature increased, reaching a maximum at 600 °C and slightly decreasing at 700 °C. This phenomenon could have occurred because the crystalline minerals under high pyrolysis temperatures inhibited the exchange of cations on the surface of biochar with Cd2+ in aqueous solution46. Nevertheless, CEC did not change significantly with temperature; thus, CEC was not the main adsorption mechanism. With increasing pyrolysis temperature, the number of acidic functional groups decreased gradually, while the number of alkaline functional groups increased. The main functional groups used to remove Cd2+ were generally considered acidic oxygen-containing functional groups. However, the number of these functional groups decreased with increasing pyrolysis temperature, which weakened the complexation on the surface of the biochar. However, this result was contradictory to the results of Cd2+ adsorption. Therefore, the functional groups were not the main adsorption mechanism.To further explore the adsorption mechanism of Cd2+, the biochar before and after the adsorption of Cd2+ was characterized by XRD. As shown in Fig. 7a, C16-100Cd and C16-200Cd represented the biochar after Cd2+ adsorption when the concentrations of cadmium solution were 100 mg/l and 200 mg/l, respectively. The results showed that new peaks appeared at 30.275° and 36.546° after adsorption, corresponding to CdCO3. The spike at 29.454° was due to Cd(OH)2. Additionally, the intensity of the CdCO3 peak increased significantly from C16-100Cd to C16-200Cd, indicating that mineral precipitation occurred in adsorption. Liu et al. found similar results in a study on removing Cd2+ from water by blue algae biochar12. However, as the concentration of Cd2+ increased from 0 to 200 mg/L, the diffraction peak at 2θ = 29.454° first increased and then decreased. This because the peak position of CaCO3 at 2θ = 29.369° was very close to Cd(OH)2 at 2θ = 29.454°. At low concentrations, the production of Cd(OH)2 was greater than that of CdCO3. When the initial concentration of Cd2+ increased, more CO32− released by CaCO3 combined with Cd2+ to form CdCO3, resulting in a reduction in the diffraction peak.As presented in Fig. 8b, the peak intensities of CdCO3 and Cd(OH)2 gradually increase with increasing pyrolysis temperature. On the one hand, this phenomenon could be ascribed to the increase in the mineral content of biochar with increasing pyrolysis temperature. On the other hand, the pH value of biochar increased with increasing pyrolysis temperature. In this way, more OH− was released, thus forming more Cd(OH)2. Wang et al. obtained similar results42. Moreover, the peak intensity of KCl at 2θ = 28.347° decreased after adsorption, as shown in Fig. 8a, which indicated that ion exchange took part in adsorption.Figure 8XRD images: (a) before and after adsorption of Cd2+ on C16 biochar and (b) Cd2+ adsorption by biochar at different pyrolysis temperatures.Full size imageIn addition, the FTIR spectra showed that the number of functional groups, such as C=C and C=O, in biochar decreased with increasing pyrolysis temperature, leading to the weakening of cation–π interactions between Cd2+ and C=C and C=O. In contrast, due to the enhanced aromatization of functional groups on the surface of biochar, many lone pair electrons existed in the electron-rich domains of the graphene-like structure, which in turn enhanced the cation–π interactions. Harvey et al., based on the study of Cd2+ adsorption by plant biochar, concluded that the electron-rich domain bonding mechanism between Cd2+ and the graphene-like structure on the surface of biochar played a more significant role in biochar with a high degree of carbonization45. Therefore, π-electron interactions could play a dominant role in Cd2+ adsorption on high-temperature pyrolysis biochar. Moreover, the results showed that the number of alkaline functional groups increased while acidic functional groups decreased with the increase in pyrolysis temperature. It is generally believed that acidic functional groups could withdraw electrons, and basic functional groups could donate electrons47,48. The biochar with higher pyrolysis temperature contained more alkaline functional groups, which improved the electron donating ability of biochar and enhanced the cation–π electron effect.In summary, mineral precipitation and π electron coordination were the main mechanisms of removing Cd2+ from water by corn stover biochar. This phenomenon explained why the Cd2+ removal rate of acid/base–modified biochar decreased. After modification, the functional groups on the surface of biochar increased, but the inorganic minerals were removed. Pickling resulted in the loss of soluble minerals and alkaline functional groups on the surface of biochar, which was not conducive to adsorption49. After alkaline washing, more PO43−, CO32− and HCO3− were released, thereby reducing the mineral precipitation50,51. Since NaOH had a weaker destructive effect than HCl and introduced some OH−, alkaline washing had little effect on the removal rate of Cd2+.Adsorption isotherm and adsorption kineticsAdsorption isothermThe adsorption isotherms were fitted with Langmuir (Eq. 3) and Freundlich (Eq. 4) models, as shown in Fig. 9, and the fitting parameters are listed in Table 6.Figure 9Adsorption isotherm.Full size imageTable 6 Fitting parameters of the adsorption isotherm model.Full size tableThe Langmuir model (R2  > 0.963) was more suitable than the Freundlich model (R2  > 0.919), indicating that the adsorption sites of biochar were evenly distributed, and adsorption was mainly monolayer. Parameter KL reflected the difficulty of adsorption and was generally divided into four types: unfavourable (KL  > 1), favourable (0  More

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