Ecology

Monitoring sites and speciesThe monitoring survey was conducted at five sites in Fukushima Prefecture (sites 1–4 and A1) and at one site in Ibaraki Prefecture (site 5), Japan (Fig. 1). Sites 1, 2, and A1 are located in Kawauchi Village, site 3 in Otama Village, site 4 in Tadami Town, and site 5 in Ishioka City. Monitoring at sites 1–5 was started in 2011 or 2012, and site A1 was additionally monitored since 2017. The tree species, age, mean diameter at breast height, initial deposition density of 137Cs, and sampling year of each sample at each site are listed in Table 1. The dominant tree species in the contaminated area, namely, Japanese cedar (Cryptomeria japonica [L.f.] D.Don), Japanese cypress (Chamaecyparis obtusa [Siebold et Zucc.] Endl.), konara oak (Quercus serrata Murray), and Japanese red pine (Pinus densiflora Siebold et Zucc.) were selected for monitoring. Japanese chestnut (Castanea crenata Siebold et Zucc.) was supplementally added in 2017. The cedar, cypress, and pine are evergreen coniferous species, and the oak and chestnut are deciduous broad-leaved species. Sites 1 and 3 each have three plots, and each plot contains a different monitoring species. Site A1 has one plot containing two different monitoring species, and the remaining sites each have one plot with one monitoring species, giving ten plots in total.Figure 1Locations of the monitoring sites and initial deposition densities of 137Cs (decay-corrected to July 2, 2011) following the Fukushima nuclear accident in Fukushima and Ibaraki Prefectures. Open circles indicate the monitoring sites and the cross mark indicates the Fukushima Dai-ichi Nuclear Power Plant. Data on the deposition density were provided by MEXT19,20 and refined by Kato et al.21. The map was created using R (version 4.1.0)22 with ggplot2 (version 3.3.5)23 and sf (version 1.0–0)24 packages.Full size imageTable 1 Description of the sampled trees and monitoring sites.Full size tableSample collection and preparationBulk sampling of bark and wood disks was conducted by felling three trees per year at all sites during 2011–20168,25 and at sites 3–5 and A1 during 2017–2020. Partial sampling from six trees per year was conducted at sites 1 and 2 during 2017–2020 (from seven trees at site 2 in 2017) to sustain the monitoring trees. All the samples were obtained from the stems around breast height. During the partial sampling, bark pieces sized approximately 3 cm × 3 cm (axial length × tangential length) were collected from four directions of the tree stem using a chisel, and 12-mm-diameter wood cores were collected from two directions of the tree stem using an automatic increment borer (Smartborer, Seiwa Works, Tsukuba, Japan) equipped with a borer bit (10–101-1046, Haglöf Sweden, Långsele, Sweden). Such partial sampling increases the observational errors in the bark and wood 137Cs concentrations in individual trees26. To mitigate this error and maintain an accurate mean value of the 137Cs concentration, we increased the number of sampled trees from three to six. The sampling was conducted mainly in July–September of each year; the exceptions were site-5 samples in 2011 and 2012, which were collected irregularly during January–February of the following year. The collected bark pieces were separated into outer and inner barks, and the wood disks and cores were split into sapwood and heartwood. The outer and inner bark samples during 2012–2016 were obtained by partial sampling of barks sized approximately 10 cm × 10 cm from 2–3 directions on 2–3 trees per year.The bulk samples of bark, sapwood, and heartwood were air-dried and then chipped into flakes using a cutting mill with a 6-mm mesh sieve (UPC-140, HORAI, Higashiosaka, Japan). The pieces of the outer and inner bark were chipped into approximately 5 mm × 5 mm pieces using pruning shears, and the cores of the sapwood and heartwood were chipped into semicircles of thickness 1–2 mm. Each sample was packed into a container for radioactivity measurements and its mass was measured after oven-drying at 75 °C for at least 48 h. Multiplying this mass by the conversion factor (0.98 for bark and 0.99 for wood)8 yielded the dry mass at 105 °C.Radioactivity measurementsThe radioactivity of 137Cs in the samples was determined by γ-ray spectrometry with a high-purity Ge semiconductor detector (GEM20, GEM40, or GWL-120, ORTEC, Oak Ridge, TN). For measurements, the bulk and partial samples were placed into Marinelli containers (2.0 L or 0.7 L) and cylindrical containers (100 mL or 5 mL), respectively. The peak efficiencies of the Marinelli containers, the 100-mL container, and the 5-mL container were calibrated using standard sources of MX033MR, MX033U8PP (Japan Radioisotope Association, Tokyo, Japan), and EG-ML (Eckert & Ziegler Isotope Products, Valencia, CA), respectively. For the measurement of the 5-mL container, a well-type Ge detector (GWL-120) was used under the empirical assumption that the difference in γ-ray self-absorption between the standard source and the samples is negligible27. The measurement was continued until the counting error became less than 5% (higher counting errors were allowed for small or weakly radioactive samples). The activity concentration of 137Cs in the bark (whole) collected by partial sampling was calculated as the mass-weighted mean of the concentrations in the outer and inner barks; meanwhile, the concentration in the wood (whole) was calculated as the cross-sectional-area-weighted mean of sapwood and heartwood concentrations. The activity concentrations were decay-corrected to September 1, 2020, to exclude the decrease due to the radioactive decay.Trend analysesThe yearly representative values (true states) of 137Cs activity concentration in each stem part in each plot were estimated using a DLM, a state-space model in which the noise follows a normal distribution and the relationship between variables is linear. One basic DLM is the local linear trend model defined by the following equations:$$Y_{t} = mu _{t} + varepsilon _{t} ,quad quad quad varepsilon _{t} sim Normal left( {0,sigma _{varepsilon }^{2} } right)$$
(1)
$$mu_{t} = mu_{t – 1} + beta_{t – 1} + eta_{t} ,quad quad quad eta_{t} sim Normal left( {0,sigma_{eta }^{2} } right)$$
(2)
$$beta_{t} = beta_{t – 1} + zeta_{t} ,quad quad quad zeta_{t} sim Normal left( {0,sigma_{zeta }^{2} } right)$$
(3)
where Yt, μt, and βt are the observation values, level (true state), and slope, respectively, and εt, ηt, and ζt denote their corresponding noises. The subscript t is the time index. The noises εt, ηt, and ζt follow normal distributions with a mean of 0 and variances of ({sigma }_{varepsilon }^{2}), ({sigma }_{eta }^{2}), and ({sigma }_{zeta }^{2}), respectively. To detect relatively long-term trends, we employed the smooth local linear trend model28 (also called the smooth trend model, integrated random walk model, or second-order trend model), which is obtained by considering that μt and βt are driven by the same noise. The trend changes are assumed to be smoother in this model than in the local linear trend model28,29. Combining Eqs. (2) and (3), μt in the smooth local linear trend model is finally obtained as$$mu_{t} = 2mu_{t – 1} – mu_{t – 2} + eta_{t} ,quad quad quad eta_{t} sim Normal left( {0,sigma_{eta }^{2} } right)$$
(4)
The parameters μt, ({sigma }_{eta }^{2}), and ({sigma }_{varepsilon }^{2}) of each stem part in each plot were determined by Bayesian estimation with a Markov chain Monte Carlo (MCMC) method. The Bayesian estimation was performed in R (version 4.1.0)22 with the rstan package (version 2.21.2)30. Uninformative prior distributions were used for μ1, μ2, ({sigma }_{eta }^{2}), and ({sigma }_{varepsilon }^{2}). The log-transformed values of the 137Cs activity concentration (decay-corrected to September 1, 2020) were given as Yt (the observed values of multiple individuals in each year were passed via the segment function of Stan). MCMC sampling was conducted for four chains of 50,000 iterations (the first 25,000 were discarded as warmup), obtaining 100,000 MCMC samples for each parameter. The MCMC was judged to have converged when the maximum value of Rhat was less than 1.05 and the divergent transitions after warmup were fewer than 1,000 (i.e., less than 1% of the MCMC sample size). On the datasets of the outer and inner barks from site-3 oaks and all stem parts from site-A1 pines and chestnuts, the MCMC converged poorly owing to the small number of monitoring years. Thus, the temporal trends in these datasets were not analyzed (the observational data at site A1 are shown in Supplementary Fig. S1 and Table S1).To detect decadal trends rather than yearly variations, we determined the temporal trends in the true state (μ) by setting 2–4 delimiting years and examining whether μ varied significantly from one delimiting year to the next. As the delimiting years, we selected the initial and final years of monitoring and the years in which the median µ was highest (µ-max year) and lowest (µ-min year). When the µ-max year and/or the µ-min year coincided with the initial year and/or final year of monitoring, the number of delimiting years reduced from four to two or three. The trend in µ between two delimiting years was determined to be increasing and decreasing when the 95% credible interval of µ2nd delimiting year − µ1st delimiting year (obtained from the MCMC samples) was higher and lower than zero, respectively. A flat trend (no significant variation) was detected when the 95% credible interval included zero. If the 3rd and 4th delimiting years existed, the trends between the 2nd and 3rd delimiting years and between the 3rd and 4th delimiting years were determined in the same manner.The 137Cs CRs of outer bark/inner bark, heartwood/sapwood, and inner bark/sapwood were also subjected to the above trend analyses. On datasets with less than five years of monitoring, the MCMC did not converge so the trend analysis was not attempted. More

Thamdrup, B. New Pathways and processes in the global nitrogen cycle. Annu. Rev. Ecol. Evol. Syst. 43, 407–428 (2012).
Google Scholar 
Lam, P. et al. Revising the nitrogen cycle in the Peruvian oxygen minimum zone. Proc. Natl. Acad. Sci. USA 106, 4752–4757 (2009).CAS 

Google Scholar 
Behrendt, A., de Beer, D. & Stief, P. Vertical activity distribution of dissimilatory nitrate reduction in coastal marine sediments. Biogeosciences 10, 7509–7523 (2013).
Google Scholar 
Fossing, H. et al. Concentration and transport of nitrate by the mat-forming sulphur bacterium. Thioploca. Nature 374, 713–715 (1995).CAS 

Google Scholar 
McHatton, S. C., Barry, J. P., Jannasch, H. W. & Nelson, D. C. High nitrate concentrations in vacuolate, autotrophic marine Beggiatoa spp. Appl. Environ. Microbiol. 62, 954–958 (1996).CAS 

Google Scholar 
Kamp, A., Høgslund, S., Risgaard-Petersen, N. & Stief, P. Nitrate storage and dissimilatory nitrate reduction by eukaryotic microbes. Front. Microbiol. 6, 1492 (2015).
Google Scholar 
Eppley, R. W. & Rogers, J. N. Inorganic nitrogen assimilation of Ditylum brightwellii, a marine plankton diatom. J. Phycol. 6, 344–351 (1970).CAS 

Google Scholar 
Lomas, M. & Glibert, P. Comparisons of nitrate uptake, storage, and reduction in marine diatoms and flagellates. J. Phycol. 36, 903–913 (2000).CAS 

Google Scholar 
Jørgensen, B. B. & Gallardo, A. Thioploca spp.: filamentous sulfur bacteria with nitrate vacuoles. FEMS Microbiol. Ecol. 28, 301–313 (1999).
Google Scholar 
Schulz, H. N. et al. Dense populations of a giant sulfur bacterium in Namibian shelf sediments. Science 284, 493–495 (1999).CAS 

Google Scholar 
Risgaard-Petersen, N. et al. Evidence for complete denitrification in a benthic foraminifer. Nature 443, 93–96 (2006).CAS 

Google Scholar 
Kamp, A., de Beer, D., Nitsch, J. L., Lavik, G. & Stief, P. Diatoms respire nitrate to survive dark and anoxic conditions. Proc. Natl. Acad. Sci. USA 108, 5649–5654 (2011).CAS 

Google Scholar 
Stief, P. et al. Dissimilatory nitrate reduction by Aspergillus terreus isolated from the seasonal oxygen minimum zone in the Arabian Sea. BMC Microbiol. 14, 35 (2014).
Google Scholar 
Høgslund, S., Cedhagen, T., Bowser, S. S. & Risgaard-Petersen, N. Sinks and sources of intracellular nitrate in gromiids. Front. Microbiol. 8, 617 (2017).
Google Scholar 
Harold, F. M. The Vital Force: A Study of Bioenergetics (WH Freeman & Co., 1986).Katz, M. E., Finkel, Z. V., Grzebyk, D., Knoll, A. H. & Falkowski, P. G. Evolutionary trajectories and biogeochemical impacts of marine eukaryotic phytoplankton. Annu. Rev. Ecol. Evol. Syst. 35, 523–556 (2004).
Google Scholar 
Villareal, T. A., Altabet, M. A. & Culverrymsza, K. Nitrogen transport by vertically migrating diatom mats in the North Pacific Ocean. Nature 363, 709–712 (1993).CAS 

Google Scholar 
Kamp, A., Stief, P. & Schulz, H. N. Anaerobic sulfide oxidation with nitrate by a freshwater Beggiatoa enrichment culture. Appl. Environ. Microbiol. 72, 4755–4760 (2006).CAS 

Google Scholar 
Merz, E. et al. Nitrate respiration and diel migration patterns of diatoms are linked in sediments underneath a microbial mat. Environ. Microbiol. 23, 1422–1435 (2021).CAS 

Google Scholar 
Leblanc, K. et al. A global diatom database–abundance, biovolume and biomass in the world ocean. Earth Syst. Sci. Data 4, 149–165 (2012).
Google Scholar 
Benoiston, A. S. et al. The evolution of diatoms and their biogeochemical functions. Phil. Trans. R. Soc. B 372, 20160397 (2017).
Google Scholar 
Nelson, D. M., Tréguer, P., Brzezinski, M. A., Leynaert, A. & Queguiner, B. Production and dissolution of biogenic silica in the ocean-revised global estimates, comparison with regional data and relationship to biogenic sedimentation. Global Biogeochem. Cycl. 9, 359–372 (1995).CAS 

Google Scholar 
Sarthou, G., Timmermans, K. R., Blain, S. & Tréguer, P. Growth physiology and fate of diatoms in the ocean: a review. J. Sea Res. 53, 25–42 (2005).CAS 

Google Scholar 
Dortch, Q., Clayton, J. R., Thoresen, S. S. & Ahmed, S. I. Species differences in accumulation of nitrogen pools in phytoplankton. Mar. Biol. 81, 237–250 (1984).CAS 

Google Scholar 
Kamp, A., Stief, P., Knappe, J. & de Beer, D. Response of the ubiquitous pelagic diatom Thalassiosira weissflogii to darkness and anoxia. PLoS ONE 8, e82605 (2013).
Google Scholar 
Kamp, A., Stief, P., Bristow, L. A., Thamdrup, B. & Glud, R. N. Intracellular nitrate of marine diatoms as a driver of anaerobic nitrogen cycling in sinking aggregates. Front. Microbiol. 7, 1669 (2016).
Google Scholar 
Needoba, J. A. & Harrison, P. J. Influence of low light and a light:dark cycle on NO3− uptake, intracellular NO3−, and nitrogen isotope fractionation by marine phytoplankton. J. Phycol. 40, 505–516 (2004).CAS 

Google Scholar 
Lomas, M. W. & Glibert, P. M. Temperature regulation of nitrate uptake: A novel hypothesis about nitrate uptake and reduction in cool-water diatoms. Limnol. Oceanogr. 44, 556–572 (1999).CAS 

Google Scholar 
Lomas, M. W., Rumbley, C. J. & Glibert, P. M. Ammonium release by nitrogen sufficient diatoms in response to rapid increases in irradiance. J. Plankton Res. 22, 2351–2366 (2000).CAS 

Google Scholar 
Van Tol, H. M. & Armbrust, E. V. Genome-scale metabolic model of the diatom Thalassiosira pseudonana highlights the importance of nitrogen and sulfur metabolism in redox balance. PLoS ONE 16, e0241960 (2021).
Google Scholar 
Piña-Ochoa, E. et al. Widespread occurrence of nitrate storage and denitrification among Foraminifera and Gromiida. Proc. Natl. Acad. Sci. USA 107, 1148–1153 (2010).
Google Scholar 
García-Robledo, E., Corzo, A., Papaspyrou, S., Jimenez-Arias, J. L. & Villahermosa, D. Freeze-lysable inorganic nutrients in intertidal sediments: dependence on microphytobenthos abundance. Mar. Ecol. Prog. Ser. 403, 155–163 (2010).
Google Scholar 
Marchant, H. K., Lavik, G., Holtappels, M. & Kuypers, M. M. M. The fate of nitrate in intertidal permeable sediments. PLoS ONE 9, e104517 (2014).
Google Scholar 
Villareal, T. A. & Lipschultz, F. Internal nitrate concentrations in single cells of large phytoplankton from the Sargasso Sea. J. Phycol. 31, 689–696 (1995).CAS 

Google Scholar 
Smith, G. J., Zimmerman, R. C. & Alberte, R. S. Molecular and physiological responses of diatoms to variable levels of irradiance and nitrogen availability: Growth of Skeletonema costatum in simulated upwelling conditions. Limnol. Oceanogr. 37, 989–1007 (1992).CAS 

Google Scholar 
Montagnes, D. J. S. & Franklin, D. J. Effect of temperature on diatom volume, growth rate, and carbon and nitrogen content: Reconsidering some paradigms. Limnol. Oceanogr. 46, 2008–2018 (2001).CAS 

Google Scholar 
Smith, S. R. et al. Evolution and regulation of nitrogen flux through compartmentalized metabolic networks in a marine diatom. Nat. Commun. 10, 4552 (2019).
Google Scholar 
Behrenfeld, M. J. et al. Thoughts on the evolution and ecological niche of diatoms. Ecol. Monogr. 91, e01457 (2021).
Google Scholar 
Bourke, M. F. et al. Metabolism in anoxic permeable sediments is dominated by eukaryotic dark fermentation. Nat. Geosci. 10, 30–35 (2017).CAS 

Google Scholar 
Härnström, K., Ellegaard, M., Andersen, T. J. & Godhe, A. Hundred years of genetic structure in a sediment revived diatom population. Proc. Natl. Acad. Sci. USA 108, 4252–4257 (2011).
Google Scholar 
Pelusi, A., Santelia, M. E., Benvenuto, G., Godhe, A. & Montresor, M. The diatom Chaetoceros socialis: spore formation and preservation. Europ. J. Phycol. 55, 1–10 (2020).CAS 

Google Scholar 
Petterson, K. & Sahlsten, E. Diel patterns of combined nitrogen uptake and intracellular storage of nitrate by phytoplankton in the open Skagerrak. J. Exp. Mar. Biol. Ecol. 138, 167–182 (1990).
Google Scholar 
Petterson, K. Seasonal uptake of carbon and nitrogen and intracellular storage of nitrate in planktonic organisms in the Skagerrak. J. Exp. Mar. Biol. Ecol. 151, 121–1137 (1991).
Google Scholar 
Bode, A., Botas, J. A. & Fernandez, E. Nitrate storage by phytoplankton in a coastal upwelling environment. Mar. Biol. 129, 399–406 (1997).CAS 

Google Scholar 
Stief, P., Kamp, A., Thamdrup, B. & Glud, R. N. Anaerobic nitrogen turnover by sinking diatom aggregates at varying ambient oxygen levels. Front. Microbiol. 7, 98 (2016).
Google Scholar 
Jensen, M. M. et al. Intensive nitrogen loss over the Omani Shelf due to anammox coupled with dissimilatory nitrite reduction to ammonium. ISME J. 5, 1660–1670 (2011).CAS 

Google Scholar 
Magalhaes, C. M., Wiebe, W. J., Joye, S. B. & Bordalo, A. A. Inorganic nitrogen dynamics in intertidal rocky biofilms and sediments of the Douro River estuary (Portugal). Estuaries 28, 592–607 (2005).CAS 

Google Scholar 
Burgin, A. J. & Hamilton, S. K. Have we overemphasized the role of denitrification in aquatic ecosystems? A review of nitrate removal pathways. Front. Ecol. Environ. 5, 89–96 (2007).
Google Scholar 
Kühl, M., Glud, R. N., Ploug, H. & Ramsing, N. B. Microenvironmental control of photosynthesis and photosynthesis-coupled respiration in an epilithic cyanobacterial biofilm. J. Phycol. 32, 799–812 (1996).
Google Scholar 
Heisterkamp, I. M. et al. Shell biofilm-associated nitrous oxide production in marine molluscs: processes, precursors and relative importance. Environ. Microbiol. 15, 1943–1955 (2013).CAS 

Google Scholar 
Fernandez-Mendez, M. et al. Composition, buoyancy regulation and fate of ice algal aggregates in the Central Arctic Ocean. PLoS ONE 9, e107452 (2014).
Google Scholar 
Boetius, A. et al. Export of algal biomass from the melting Arctic sea ice. Science 339, 1430–1432 (2013).CAS 

Google Scholar 
Abed, R. M. M. & Garcia-Pichel, F. Long-term compositional changes after transplant in a microbial mat cyanobacterial community revealed using a polyphasic approach. Environ. Microbiol. 3, 53–62 (2001).CAS 

Google Scholar 
Al-Najjar, M. A. A., de Beer, D., Kühl, M. & Polerecky, L. Light utilization efficiency in photosynthetic microbial mats. Environ. Microbiol. 14, 982–992 (2012).CAS 

Google Scholar 
Heisterkamp, I. M., Kamp, A., Schramm, A. T., de Beer, D. & Stief, P. Indirect control of the intracellular nitrate pool of intertidal sediment by the polychaete Hediste diversicolor. Mar. Ecol. Prog. Ser. 445, 181–192 (2012).
Google Scholar 
García-Robledo, E., Corzo, A. & Papaspyrou, S. A fast and direct spectrophotometric method for the sequential determination of nitrate and nitrite at low concentrations in small volumes. Mar. Chem. 162, 30–36 (2014).
Google Scholar 
Grasshoff, K. In Methods of Seawater Analysis (eds Grasshoff, K., Ehrhardt, M., Kremling, K.) 143–150 (Verlag Chemie Weinheim, 1983).Braman, R. S. & Hendrix, S. A. Nanogram nitrite and nitrate determination in environmental and biological materials by vanadium(III) reduction with chemiluminescence detection. Anal. Chem. 61, 2715–2718 (1989).CAS 

Google Scholar 
Meier, D. V. et al. Limitation of microbial processes at saturation-level salinities in a microbial mat covering a coastal salt flat. Appl. Environ. Microbiol. 87, e00698–21 (2021).CAS 

Google Scholar 
Sode, K., Horikoshi, K., Takeyama, H., Nakamura, N. & Matsunaga, T. Online monitoring or marine cyanobacterial cultivation based on phycocyanin fluorescence. J. Biotechnol. 21, 209–217 (1991).CAS 

Google Scholar 
Berns, D. S., Scott, E. & Oreilly, K. T. C-phycocyanin-minimum molecular weight. Science 145, 1054–1055 (1964).CAS 

Google Scholar 
Hillebrand, H., Durselen, C. D., Kirschtel, D., Pollingher, U. & Zohary, T. Biovolume calculation for pelagic and benthic microalgae. J. Phycol. 35, 403–424 (1999).
Google Scholar 
Zimmermann, J., Jahn, R. & Gemeinholzer, B. Barcoding diatoms: evaluation of the V4 subregion on the 18S rRNA gene, including new primers and protocols. Org. Divers. Evol. 11, 173–192 (2011).
Google Scholar 
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).
Google Scholar 
Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583 (2016).CAS 

Google Scholar 
Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucl. Acids Res. 41, D590–D596 (2013).CAS 

Google Scholar 
Round, F. E., Crawford, R. M. & Mann, D. G. The Diatoms: Biology and Morphology of the Genera. 747p (Cambridge University Press, 1990).Medlin, L. K. Evolution of the diatoms: major steps in their evolution and a review of the supporting molecular and morphological evidence. Phycologia 55, 79–103 (2016).CAS 

Google Scholar 
McMurdie, P. J. & Holmes, S. phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8, e61217 (2013).CAS 

Google Scholar 
Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer Verlag, 2016).Oksanen, J. et al. vegan: Community Ecology Package. R package version 2.5-7. https://CRAN.R-project.org/package=vegan (2020).Stief, P. Intracellular Nitrate Storage by Diatoms-Source data. figshare. Dataset. https://doi.org/10.6084/m9.figshare.19790176.v1 (2022). More

An experiment in secondary forests in the Democratic Republic of the Congo finds that calcium, an overlooked soil nutrient, is scarcer than phosphorus, and represents a potentially greater limitation on tropical forest growth.Ecology can reveal distributional patterns and dynamics in nature. One approach used is studying the elemental composition of plants, which has been linked to ecological processes such as growth, diversity or water use efficiency. More recently, elemental composition has been detected as a cofactor in governing the carbon sink capacity of plants, and therefore climate change mitigation1,2,3. This discovery has added an extra layer of urgency to the field, which now aims to better understand and predict global change. The study of nitrogen and/or phosphorus has until now received most of the attention of plant ecologists: nitrogen is the most abundant element in dry leaves after hydrogen and carbon, forms the main structure of proteins and is strongly linked to photosynthesis4. Phosphorus represents around one-tenth of nitrogen abundance in leaves and is key in energy storage and nucleic acids. However, although these represent only two of the many chemical elements that are in flux throughout ecosystems, whether others may have a dominant role in ecosystem dynamics is an open question. Writing in Nature Ecology & Evolution, Bauters et al.5 share some evidence to motivate broadening out from the dominant focus on nitrogen and phosphorus in terrestrial ecology, by revealing a crucial limiting role of calcium in the dynamics of tropical forest succession. More

Sabkha soil analysisSoil biochemical analysis shows higher concentration of salt in the middle (average of 200 ppt) which decreases to 20 ppt outside of the sabkha (Fig. 1B). Soil conductivity and total dissolved solids (TDS) were positively correlated with salinity (r = 0.8, p  More

Gabor transformThe Gabor transform has rarely been used as a feature in a landscape classification OBIA approach but has been used in other OBIA processes such as fingerprint enhancement and human iris detection and for data dimensionality reduction24,29,30,31,32,33,34,35. Gabor filters are a bandpass filter applied to an image to identify texture. The different Gabor bandpass filters mathematically model the visual cortical cells of mammalian brains and thus is expected to improve segmentation and classification accuracy when compared to a human delineated and classified image26,27.Samiappan et al.36 compared Gabor filters to other texture features (grey-level co-occurrence matrix, segmentation-based fractal texture analysis, and wavelet texture analysis) within the GEOBIA process, of a wetland, using sub-meter resolution multispectral imagery. These Gabor filters performed comparably, in overall classification accuracy and Kappa coefficients, with other texture features. However, they were still outperformed by all other texture features. This study did not use any other data for analysis for determining the performance of Gabor filters when paired with data sources such as spectral, NDVI, or LiDAR36,37. Wang et al.38 paired a Gabor transformation with a fast Fourier transformation for edge detection on an urban landscape image that contained uniform textures with promising results. Su30 used the textural attributes derived from Gabor filters for classification but had similar results to Samiappan et al.36 where they found that Gabor features were one of the least useful/influential that contributed to the classification of a mostly agricultural landscape.Gabor filters are a Fourier influenced wavelet transformation, or bandpass filter, that identifies texture as intervals in a 2-D Gaussian modulated sinusoidal wave. This modulation differentiates the Gabor transform from the Fourier transform23,26. These Gabor transformed wavelets are parameterized by the angle at which they alter the image and the frequency of the wavelet. Rather than smoothing an image at the cost of losing detail through Fourier transforms or median filters, Gabor transformed images identify the repeated pattern of localized pixels and gives them similar values if they are a part of the same repeated sequence. Gabor features can closely emulate the visual cortex of mammalian brains that utilize texture to identify objects26,27. This is based on the evaluation of neurons associated with the cortical vertex that respond to different images or light profiles39. Marcelja27 identified that cortical cells responded to signals that are localized frequencies of light like what is represented by the Gabor transformations. Within the frequency domain, the Gabor transform can be defined by Eq. (1):$$Gleft(u, v;f, theta right)= {e}^{-frac{{pi }^{2}}{{f}^{2}} ({gamma }^{2}({u}^{{prime}}-f{)}^{2}+{n}^{2}{v}^{{{prime}}2})}$$
(1)

where (f) is the user-determined frequency (or wavelength); (theta) is the user-determined orientation at which the wavelet is applied to the image; (gamma) and (n) are the standard deviations of the Gaussian function in either direction23,38. These parameters define the shape of the band pass filter and determines its effect on one-dimensional signals. Daugman26, created a 2-D application of this filter in Eq. (2);$$gleft(u,vright)= {e}^{-{pi }^{2}/{f}^{2}[{gamma }^{2}{left({u}^{{prime}}-fright)}^{2}+{n}^{2}{{v}^{{prime}}}^{2}]}$$
(2)

where u’ = ucos − vsin θ θ and v’ = usin − vcos θ.In order to implement Gabor filters on multi-band spectral images, we used Matlab’s Gabor feature on the University of Iowa’s Neon high performance computer (HPC)40 which has up to 512 GB of RAM, which was necessary for processing these images. The first implementation of Gabor filters was performed on a 1610 × 687 single band pixel array (a small subset of the study area), a filter bank of 4 orientations and 8 wavelengths, on a 32 GB RAM computer, and took approximately 8 h to complete. Filter banks are a set of Gabor filters with different parameters that is applied to the spectral image and are required to identify different textures with different orientations and frequencies. By lowering the number of wavelengths from 8 to 4 on an 8128 × 8128 single band pixel array on the same machine 32 GB RAM, the processing was reduced to an hour. Using the HPC, this was further reduced to approximately 90 s using the same filter bank. Before implementing on the HPC, the original spectral image was divided into manageable subsets with overlap in order to prevent ‘edge-effect.’ These images were converted to greyscale by averaging values across all three bands33. When wavelengths become too long, they no longer attribute the textural information desired from the image and therefore add unnecessary computing time. The wavelengths that were used for the filter bank were selected as increasing powers of two starting from 2.82842712475 ((24/sqrt{2})) up to the pixel length of the hypotenuse of the input image. From this, we used only 2.82842712475, 7.0710678, 17.6776695, and 44.19417382. The directional orientation was selected as 45° intervals, from 0 to 180: 0, 45, 90, 135. These parameters were based on the reasoning outlined within Jain and Farrokhina25. More directional orientations could have been included but four were used for computational efficiency. The radial frequencies were selected so that they could capture the different texture in the landscape represented by consistent changes in pixels values within each landcover class. When frequencies are too wide or fine of a width they no longer represent the textures of the different landcover classes and thus are not included. This selection of filter bank parameters are similar or the same as other studies that look into the use of Gabor features for OBIA25,30,31.From the different combinations of parameters (four directions and four frequencies) in the Gabor Transform filter bank, sixteen magnitude response images were created from the converted greyscale three band average image. To limit high local variance within the output Gabor texture images, a Gaussian filter was applied. The magnitude response values were normalized across the 16 different bands so that a Principal Component Analysis (PCA) could be applied. The first principal component of the PCA, from these Gabor transformed images, was used for this study since it limits the computation time to process 16 separate Gabor features, in addition to the other data sources, while still retaining the most amount of information from the different Gabor response features. The Gabor band that was used for this study can be viewed in Fig. 2.Figure 2Gabor transformation. Gabor transformed image of study area derived from original image using the first principal component of all gabor outputs using the filter bank parameters. Software: ArcMap (10.x).Full size imageSegmentationFor this study, we used the watershed algorithm for the segmentation of GEOBIA, implemented by ENVI version 5.0 Feature Extraction tool, due to its ubiquitous use within GEOBIA, its ability to create a hierarchy of segmented objects, and support within the literature as a reliable algorithm37,41,39,43. The watershed algorithm can either use a gradient image or intensity image for segmentation. Based on the observed results, this study used the intensity method. The intensity method averages the value of pixels across bands. Scale, a user-defined parameter, is selected to identify the threshold that decides if a given intensity value within the gradient image can be a boundary. This allows the user to decide the size of the objects created. A secondary, user-defined, parameter defines how similar, adjacent, objects need to be before they are combined or merged. The user arbitrarily selects the parameter value based on how it reduces both under and over segmentation. The parameters selected for this study were visually chosen based on a compromise between over and under segmentation relative to the hand demarcated objects.The merging of two separate objects was based on the full lambda schedule where the user selects a merging threshold ({t}_{i, j}) which is defined by Eq. (3):$${t}_{i, j}= frac{frac{left|{O}_{i}right|cdot left|{O}_{j}right|}{left|{O}_{i}right|+ left|{O}_{j}right|}cdot {Vert {u}_{i}-{u}_{j}Vert }^{2}}{mathrm{length}(mathrm{vartheta }left({O}_{i},{O}_{j}right))}$$
(3)

where ({O}_{i}) is the object of the image, (left|{O}_{i}right|) is the area of (i), ({u}_{i}) is the average of object (i), ({u}_{j}) is the average of object (j), (Vert {u}_{i}-{u}_{j}Vert) is the Euclidean distance between the average values of the pixel values in regions (i) and (j), and (mathrm{length}left(mathrm{vartheta }left({O}_{i},{O}_{j}right)right)) is the length of the shared boundary of ({O}_{i}) and ({O}_{j}).To compare the segmentation of a riparian landscape, with and without Gabor features, we conducted segmentation on two separate sets of data. One dataset was a normalized stacked layer of NDVI and CHM (see Fig. 3) with the original multispectral image used as ancillary data; the other dataset differed only by the inclusion of the Gabor feature. For both instances, the bands were converted to an intensity image by averaging across bands rather than being converted into a gradient image for segmentation. The dataset that included the Gabor features had a scale parameter set at 30 with merge settings at 95 and 95.7 for the sub and super-objects, respectively. The dataset that did not include the Gabor features had a scale parameter of 10 with merge settings at 95.6 and 98.5 for the sub and super-objects, respectively. This resulted in the creation of 87,198 and 62,905 segments for the sub and super objects, respectively, that were created when the Gabor feature was included. 191,050 and 51,664 segments were created for the sub and super objects when the Gabor features, respectively, were not included within the segmentation process. As you will see in the next section, these segments also represent the number of training data that will be included within the supervised classification.Figure 3CHM and NDVI. LiDAR derived canopy height model (top) and normalized difference vegetation index derived from original spectral image. Software: ArcMap (10.x).Full size imageTo create a hierarchy of land cover classes, two sets of segmentation parameters needed to be selected for each dataset. One set of parameters would be used for the sub-objects within the hierarchy and the other set would be used to create super-objects. All parameters used the intensity and full lambda schedule algorithms for the watershed method. The only setting that changed between the sub and super-objects, for either dataset, was the merge parameter which helped maintain similar boundaries as much as possible. Despite this, boundaries could moderately change due to the Euclidean distance, between the pixel values of (i) and (j), changing from the merging of objects; causing ({t}_{i, j}) to cross the threshold which results in a new boundary being drawn. A representation of these results can be viewed and visually compared to the hand demarcated objects in Fig. 4.Figure 4Automated and manual segmented comparison. Juxtaposition of hand delineated, sub-objects, and super-objects for segments generated using the Gabor features. Software: ArcMap (10.x).Full size imageTraining dataThe training data, used for this study, is the transfer of class attributes from hand demarcated and classified segments to automatically segmented objects based on the majority overlap of the hand demarcated segments. Experts identified them using two different classification schemes referenced from the General Wetland Vegetation Classification System44. The 7-class scheme within this system identified objects of either being forest, marsh, agriculture, developed, open water, grass/forbs, or sand/mud. The 13-class scheme identified objects of either being agriculture, developed, grass/forbs, open water, road/levee, sand/mud, scrub-shrub, shallow marsh, submerged aquatic vegetation, upland forest, wet forest, wet meadow, and wet shrub. Not every class from the 7-class scheme will have a sub-class (i.e. developed, open water) but some do for example wet and upland forest are sub-objects of the forest class and wet meadow and shallow marsh are sub-objects of marsh. Figure 5 visually illustrates both classification schemes across the study area.Figure 5Hand delineated objects of both scales. Software: ArcMap (10.x).Full size imageENVI’s feature extraction tool calculates several landscape, spectral, and textural metrics. These attributes were used for each random forest classifier. The Gabor and Hierarchical features will be included selectively to be able to compare their contributions to the (out-of-bag) OOB classification errors. When Gabor features are included within the classification, they are computed the same way as the other image bands.Random forestThe random forest classifier was implemented in R using the random forest module45. The number of trees, that were randomly generated, was large enough (n = 250) to where the Strong law of large numbers would take effect as indicated by the decrease in the change of accuracy. The default number of variables randomly sampled as candidates at each split variable (mtry parameter) was the total number of variables divided by 3 for each dataset. R also generates two separate variable indices: mean decrease in accuracy and mean decrease Gini. Mean decrease in accuracy refers to the accuracy change in the random forest when a single variable is left out. This is a practical metric to determine the usefulness of a variable. The Gini index measures the purity change within a dataset when it is split based upon a given variable within a decision tree.The random forest classification accuracy will be based on the OOB error. The random forest algorithm trains numerous decision trees on random subsets of the training set leaving out a number of training samples when training each decision tree. The samples that are left out of each decision tree are then classified by the decision tree that they were not included within during the training step. The OOB error is the average error of each predicted bootstrapped sample across the ensemble of decision trees within the random forest algorithm.Figure 6 illustrates how the Gabor and hierarchal features were included within the classification of the super and sub-objects.Figure 6Classification procedure. Schematic flow chart illustrating how the Gabor and hierarchal features were included within the classification of the super and sub-objects. OOB classification error included in parenthesis.Full size imageHierarchical schemeTo attribute the hierarchical structure to the sub-objects, we first classified the larger segments that were created with and without the Gabor features using the broader 7-class scheme. These classified super objects were then converted to raster to calculate the majority overlap with the smaller sub-objects. This gave the sub-objects an attribute, the broader 7-class scheme, that could be used to contribute to the classification of the sub-objects with the finer 13-class scheme. This builds the hierarchical relationship between the two class schemes into the supervised classification of the sub-objects. Figure 6 illustrates how the hierarchal structure was included within two of the four sub-object’s list of features used within classification. This methodological approach aligns with O’Neill et al.21 landscape ecology principle that a super-object’s class could be a useful property in defining or predicting a sub-object. This is also different than the more common rule-based approach of iteratively classifying the landscape into smaller and smaller sub-classes22.Segmentation assessmentMost studies rely upon the accuracy assessment of their classifiers to provide support for their analysis results. However, this does not provide evidence whether a new data fusion technique improves the ability to delineate objects of interest within an image. To assess the performance of our segmented polygons, this study evaluated the segments created with and without the Gabor feature using a method highlighted in Xiao et al.37.Our segmentation results were evaluated using an empirical discrepancy measure, used frequently in image segmentation evaluation37,46,47. Discrepancy measures utilize ground truth images that represent the “correct” delineated/classified image to compare the semi-automated image results. In our study, the objects that were delineated and classified by experts from the U.S. Fish and Wildlife Service, were used as training data for our random forest classifier and as ground truth for the discrepancy measure. The discrepancy measure used the percentage of right segmented pixels (PR) in the whole image. To calculate PR, we converted the classified segmented and ground truth polygons to raster and measured the ratio of incorrect pixels to total amount of pixels which was converted to a percentage.Additionally, landscape metrics were calculated using FRAGSTATS48, an open source program commonly used for calculating landscape metrics. FRAGSTATS computed these metrics from thematic raster maps that represent the land cover types of interest. These thematic classes, used for analysis, were the classified objects at both the super and sub-object level. Since we are not attempting to compare the segmentation results for any specific class or area, we calculated metrics on a landscape level. Landscape metrics will represent the segmentation patterns for the entire study area.FRAGSTATS can calculate various metrics representing different aspects of the landscape. The metrics for analysis attempts to understand object geometry. The metrics calculated, for these analyses, were the average and standard deviation for the area (AREA), the fractal dimension index (FRAC), and the perimeter area ratio (PARA). The number of patches (NP) was also included in each result. To take a more landscape centric approach, the area weighted mean was chosen over a simple average. More

Virtual hydrostatic model parametersVarious morphological characteristics were held constant in order to isolate and manipulate the variable of conch shape. A CT-scanned Nautilus pompilius conch was essentially morphed into ammonoid-like conch shapes, populating the Westermann morphospace22 while holding constant septal morphology, septal spacing, and shell/septal thicknesses (Fig. 9). Furthermore, body chamber proportions were determined by iteratively computing soft body volumes that yield Nautilus-like chamber liquid (~ 12% of the phragmocone volume retained)67,68. Septal spacing was measured as the angle from the ventral attachment of the current and previous septa, and the spiraling axis of the conch. Because septal spacing differs in early ontogeny (Fig. S11), only measurements from the 7th to 33rd (terminal) septum were considered. The average angle of 23.46° ± 3.32° (standard deviation) was rounded to 23° and held constant throughout the ontogeny of the hydrostatic models.Figure 9Hydrostatic models of theoretical planispiral cephalopods. These models were constructed by morphing a Nautilus pompilius conch into ammonoid shapes (see “Methods”): (a) oxycone, (b) serpenticone, (c) sphaerocone, and (d) morphospace center. The centers of buoyancy and mass are denoted by the tips of the blue (upper) and red (lower) cones. Prime symbols (′) refer to transparent, transverse views of each respective conch shape. (e) Westermann morphospace22 showing relative positions of these conch shapes. All models were rendered in MeshLab76.Full size imageShell and septal thicknesses were measured with digital calipers from a physical specimen of Nautilus pompilius (Table S13). These measurements were recorded as a ratio of inner whorl height (measured from the ventral point on the current whorl to the ventral point on the previous whorl). These ratios were used in the theoretical models to define shell and septum thicknesses (3.1% of inner whorl height for shell thickness and 2.1% of inner whorl height for septal thickness; Table S13).Hydrostatic model constructionThe near-endmember models were constructed from representative ammonoid specimens (Sphenodiscus lobatus and S. lenticularis—oxycone; Dactylioceras commune—serpenticone; Goniatites crenistria—sphaerocone). Lateral and transverse views were measured from figured specimens for the oxycone (Fig. 5 of Kennedy et al.69), serpenticone (Fig. 2 of Kutygin and Knyazev70), and sphaerocone (Figs. 17 and 20 of Korn and Ebbighausen71). These models were constructed with array algorithms similar to earlier hydrostatic models9,35,72, which were used in a piecewise manner to account for allometric changes in coiling throughout ontogeny (Table S14). These arrays replicated the adult whorl section backwards and translated, rotated, and scaled each successive one. These whorl sections were bridged together to create a single tessellated surface representing the outer interface of the shell. Shell thickness was defined by shrinking the original whorl section so that the thickness between the two was equal to 3.1% of the inner whorl height (Table S13), then using the same array to build the internal interface of the shell. The morphospace center was constructed from previously used conch measurements18 and averaging the whorl section shape in blender (Fig. S12). The corresponding Westermann morphospace parameters (Fig. S13) for each morphology are reported in Table S15.Virtual models of the septa were derived from the CT-scan of Nautilus pompilius (Fig. S14). A single septum was isolated from the adult portion of the phragmocone then smoothed to delete the siphuncular foramen. This septum was placed within the whorl section of each theoretical model and stretched in the lateral directions until it approximately fit. The “magnetize” tool in Meshmixer (Autodesk Inc.) was used to attach the septal margin to the new whorl section so that the Nautilus suture was transferred to the new whorl section. The septum was then smoothed to reconcile the first order curves with the new location of the septal margin. The respective septum for each theoretical model was then replicated with the same array instructions used to build the shell. Because each replicated object was rotated one degree (Table S14), 22 septa were deleted in between every two so that the septal spacing was equal to 23° (Fig. S11).For each theoretical model, the septa were unified with the model of the shell using Boolean operations in Netfabb (Autodesk Inc.). To perform hydrostatic calculations, virtual models must be created for each material of unique density. The virtual model of the shell constrains the shape of the soft body (within the body chamber) and chamber volumes (within the phragmocone). These internal interfaces were isolated from the model of the shell, then their faces inverted for proper, outward-facing orientations of their normals. A conservative soft body estimate was created, aligning with previously published reconstructions64,65,73. The profile shape of this soft body was scaled and maintained between each model. External interfaces of the shell and soft body were also isolated to create a model of the water displaced by each theoretical cephalopod. Each of these models are necessary for hydrostatic calculations (buoyancy and the distribution of organismal mass).Each hydrostatic model is stored in an online repository (Dataset S1; https://doi.org/10.5281/zenodo.5684906). The hydrostatic centers of each virtual model and their volumes and masses are listed in Tables S16 and S17.Hydrostatic calculationsEach theoretical model was scaled to have equal volume (near one kilogram; 0.982 kg–a result of arbitrarily scaling the sphaerocone model to 15 cm in conch diameter). An object is neutrally buoyant when the sum of organismal mass is equal to the mass of water displaced (the principle of Archimedes). The percentage of chamber liquid can be computed to satisfy this condition.$${Phi } = frac{{left( {frac{{{text{V}}_{{{text{wd}}}} {uprho }_{{{text{wd}}}} – {text{V}}_{{{text{sb}}}} {uprho }_{{{text{sb}}}} – {text{V}}_{{{text{sh}}}} {uprho }_{{{text{sh}}}} }}{{{text{V}}_{{{text{ct}}}} }}} right) – left( {{uprho }_{{{text{cl}}}} } right)}}{{left( {{uprho }_{{{text{cg}}}} – {uprho }_{{{text{cl}}}} } right)}}$$
(1)
where Vwd and ρwd are the volume and density of the water displaced, Vsb and ρsb are the volume and density of the soft body, Vsh and ρsh are the volume and density of the shell, ρcl is the density of cameral liquid, ρcg is the density of cameral gas, and Vct is the total volume of all chambers. A soft body density of 1.049 g/cm3 is used based on bulk density calculations of Nautilus-like tissues74, a seawater-filled mantle cavity, and thin calcitic mouthparts21. A shell density of 2.54 g/cm374, cameral liquid density of 1.025 g/cm375, and cameral gas density of 0.001 g/cm3 are adopted from recent hydrostatic studies.Other hydrostatic properties depend on the relative positions of the centers of buoyancy and mass. The center of buoyancy is equal to the center of volume of water displaced. This center and the centers of each virtual model of unique density were computed in the program MeshLab76. The individual centers for each organismal model (soft body, shell, cameral liquid and cameral gas) were used to compute the total center of mass, with an average weighted by material density:$$M = frac{{sum left( {L*m_{o} } right)}}{{sum m_{o} }}$$
(2)
where M is the total center of mass in a principal direction, L is the center of mass of a single object measured with respect to an arbitrary datum in each principal direction, and (m_{o}) is the mass of each object with unique density. Equation 2 was used in the x, y, and z directions to compute the 3D coordinate position of the center of mass. The centers of mass for the chamber contents (liquid and gas) were set equal to the center of volume of all chambers, a minor assumption considering the capillary retention of liquid around the septal margins in the living animals62.The hydrostatic stability index (St) is computed from the relative location of the centers of buoyancy (B) and mass (M), normalized by the cube root of volume (V) for a dimensionless metric that is independent of scale:$$S_{t} = frac{{ sqrt {left( {B_{x} – M_{x} } right)^{2} + left( {B_{y} – M_{y} } right)^{2} + left( {B_{z} – M_{z} } right)^{2} } }}{{sqrt[3]{V}}}$$
(3)
where the subscripts correspond to the x, y, and z components of each hydrostatic center.Apertural orientations were measured in blender after orienting each model so that the center of buoyancy was vertically aligned above the center of mass. Apertural angles of 0° correspond to a horizontally facing soft body, while angles of + 90° and − 90° correspond to upward- and downward-facing orientations, respectively.Thrust angles were measured from the hyponome location (ventral edge of the aperture) to the midpoint of the hydrostatic centers, with respect to the horizontal. Thrust angles of 0° infer idealized horizontal backward transmission of energy into movement, while thrust angles of + 90° and − 90° infer more efficient transmission of energy into downward and upward vertical movement, respectively.Biomimetic robot constructionTo isolate the variable of shell shape on swimming capabilities, only the external shape, and static orientation of each virtual hydrostatic model were used to build physical, 3D printed robots. That is, each model has artificially high hydrostatic stability (Tables S3) to nullify the effect of the thrust angle (the angle at which thrust energy passes through the hydrostatic centers and most efficiently transmits energy into movement; Table S4). Less stable morphotypes (e.g., serpenticones and sphaerocones) are more sensitive to the constraints imposed by this hydrostatic property.Space constraints inside each model were determined by first constructing a propulsion system and electronic components that operate the motor. The models use impeller-based water pumps (Figs. 1d and 10a) driven by a brushed DC motor. This system creates a partial vacuum by centrifugal acceleration, drawing water from a “mantle cavity” and expelling it out of a “hyponome”. This system was optimized by iteratively designing models in Blender77, then testing 3D-printed, stand-alone water pumps. After three iterations, a four-blade impeller and gently tapering hyponome (inner diameter at distal end = 6.7 mm) were chosen. The electronic components used to drive the motor consist of an Arduino Pro Micro microcontroller, a motor driver, and two batteries (Fig. 10). A 3.7 V battery operates the microcontroller, and a larger 7.4 V battery supplies power to the motor. Communication is achieved via infrared, allowing specification of the jet pulse duration, number of pulses, and the power level of the motor (using pulse-width modulation; PWM). Each of these electronic components fold into a compact cartridge capable of being plugged into 3D-printed models of each investigated shell shape (Figs. 2 and 10). Each model was designed with brackets to hold the electronics cartridge in place. The sphaerocone had the most severe space constraints, with low conch diameter to volume ratio. After determining the space required for the electronics (Fig. 10) this model was scaled to 15 cm, and all other models were scaled to have similar volumes (with subtle volume differences due to minor differences in soft body shape compared to the hydrostatic models).Figure 10Biomimetic cephalopod robot components. (a) Ventral view of the sphaerocone biomimetic robot (before covering the pump and mantle cavities) with assembled electronics cartridge to the right. (b) View of electronic components that fit into the cartridge. (c) Electronics cartridge placed in robot. These two halves are fit together with wax to create a water-tight seal. Each model component is denoted by letters in circles: A = Arduino microcontroller, B = microcontroller charger / voltage regulator, C = motor driver, D = infrared sensor, E = indicator LED, F = microcontroller battery (3.7 V), G = motor battery (7.4 V), H = brushed motor, I = impeller and water pump cavity, J = electronics cartridge. The colors of annotations correspond to components depicted in Figs. 1 and 2.Full size imageIn addition to having a propulsion system, biomimetic cephalopod robots must also be capable of neutral buoyancy, while assuming the proper orientation in the water. These robots, and their once-living counterparts, each have differing material densities and associated mass distributions for each component. To reconcile these differences, the total mass and total centers of mass for each model were manipulated by controlling the volume and 3D distribution of the 3D-printed PETG (polyethylene terephthalate glycol) thermoplastic. That is, the shape of this material holds each model component in place while correcting for these differences in hydrostatics. The PETG mass required for neutral buoyancy was found by subtracting the mass of every other model component from the mass of the water displaced by the model (i.e., electronics cartridge, bismuth counterweight, liquid, motor, batteries, electronic components, and self-healing rubber; Table S1). This model configuration also allows buoyancy to be fine-tuned in water, compensating for potential density differences between the virtual water and the actual water in the experimental settings. That is, each virtual model accounts for ~ 9 g of internal liquid, but the actual volume of this liquid can be adjusted in the physical robot with a syringe through a self-healing rubber valve (Table S1; Fig. 1).The 3D position of the total center of mass was manipulated by accounting for the local centers of mass of each material of unique density. Materials like the batteries, motor, and electronic components were each assigned bulk density values because they are made up of composite materials. While this is an approximation, their contributions to the total center of mass are low because they account for small fractions of the total model mass (Tables S1 and S2). These components, like all others, were digitally modeled in Blender77 and their volumes and centers of mass were computed in the program MeshLab76. A dense, bismuth counterweight was also modeled, and positioned to artificially stabilize each model (pulling the z component of the total center of mass downward, while maintaining the horizontal components). The virtual model of this counterweight was used to make a 3D-printed mold, allowing a high heat silicone mold to be casted. The bismuth counterweight was cast from this silicone mold and filed/sanded to the dimensions of its virtual counterpart. Hyponomes were oriented horizontally, to yield movement in this direction. To maintain the same static orientation as the virtual model (same x and y center of mass components), the PETG center of mass was computed with the following equation:$$D_{PETG} = frac{{Mmathop sum nolimits_{i = 1}^{n} m_{i} – mathop sum nolimits_{i = 1}^{n} (D_{i} m_{i} )}}{{left( {m_{PETG} } right)}}$$
(4)
where DPETG is the location of the PETG center of mass from an arbitrary datum in each principal direction. M is the total center of mass in a particular principal direction, mi is the mass of each model component, Di is the local center of mass of each model component in a particular principal direction and mPETG is the mass of the PETG required for a neutrally buoyant condition. See Tables S1 and S2 for a list of model components and measurements.Each model was 3D printed with an Ultimaker S5 3D printer using clear (natural) PETG in separate parts, allowing the internal components to be implanted (i.e., brushed DC motors and bismuth counterweights). Each model part was chemically welded together with 100% dichloromethane, with minor amounts of cyanoacrylate glue used to fill seams (e.g., the water pump lid; Fig. 10a). Each final model consists of the main body (housing the water pump, motor, and counterweight), and a “lid” with brackets that house the electronics cartridge (Figs. 2 and 10). The main body and lid were fused together before each experiment by placing wax (paraffin-beeswax blend) along a tongue and groove seam, heating it with a hairdryer, then vigorously squeezing each part together. Surplus wax extruded from the seam was removed and smoothed, producing a water-tight seal.Thrust calibrationEven though each model was designed to have equal mantle cavity and pump cavity volumes, they produced slightly different thrusts. These differences were likely due to variable degrees of friction between the impellers and the surrounding water pumps. To correct for these differences, the thrust produced by each model was measured with a Vernier Dual-Range Force Sensor (0.01 N resolution). Each robot was attached at the hyponome location, through a series of pulleys, and to the sensor with fishing line (Fig. S1; similar to the methods used for living cephalopods78). Force was recorded for 30-s intervals at a sample rate of 0.05 s. During this time, each model was recorded jetting with a 6-s pulse for 15 trials (Fig. S2A). Each trial had initial noise from setting up the model, then peaked randomly when the fishing line became taught, then stabilized after some period of oscillation. Only the stabilized portion of the thrust profile was used to record thrust at 100% voltage for each model (Fig. S2B). The true zero datum was also subtracted from each of these trials. The lowest thrust from each of the models was used as a baseline (serpenticone and oxycone). Each model was recorded again for 15 trials by lowering the motor voltage in increments of 5% until they yielded similar thrusts (0.3 N) to the original serpenticone and oxycone trials (Fig. S2C). The final power levels were then determined for each model and adjusted with pulse-width modulation (PWM) through the microcontroller: serpenticone (100%), oxycone (100%), sphaerocone (95%), and morphospace center (85%).The peak thrust measured for 1 kg extant Nautilus is around 2 N16. The time-averaged thrust during each pulse is around 23% of this value (0.46 N16). This computed value slightly overpredicts observed maximum velocities for this animal (33 cm/s instead of 25 cm/s), so the appropriate time-averaged thrust is probably slightly lower. The motor in the robots quickly reaches its maximum thrust (~ 0.3 N) once initiated then quickly declines after shutting off (Fig. S2). Therefore, the thrust produced by the robots can be treated as a conservative Nautilus-like jet thrust close to the behavior of escape jetting. One-second pulse and refill intervals are also on par with values reported for extant Nautilus16.Robot buoyancyEach of the models were made near neutrally buoyant by adjusting the allotted ~ 9 g of internal liquid with a syringe through a self-healing rubber valve. The single-pulse experiments were performed in an external pool (ranging ~ 23.5 to 26.5 °C). The three-pulse and maneuverability experiments were performed in an internal pool (the Crimson Lagoon at the University of Utah). This internal pool had slightly higher temperatures (~ 28 °C), yielding lower ambient water densities than the virtual water. These conditions required slightly less internal liquid (~ 2–5 g). These differences in internal liquid masses produced negligibly small shifts in mass distributions because they are very small proportions of total robot masses (Table S1).Perfect neutral buoyancy cannot be practically achieved, but this condition can be closely approached. Each of the biomimetic robots experience subtle upward or downward movements of the course of their 5–15 s long trials due to slightly positive or negative buoyancies. Because these differences in buoyancy influence the vertical component of movement, only the horizontal components are considered for discussion. However, a comparison of velocities computed from full, 3D movement (Eq. 5) and restricted 2D components (Eq. 6) reveals that these differences are minor (Figs. S7 and S8). These comparisons demonstrate that model buoyancy did not substantially influence kinematics other than gross trajectories (Figs. 4 and S9).3D motion trackingAfter adjusting buoyancy, each model was positioned underwater with a grabber tool. This tool was fitted with a bundle of fiber-optic cable (Fig. S4) attached to an infrared remote control. Arduino code (Dataset S2) was uploaded to the microcontroller in the robot allowing jet pulse duration, number of pulses, and power to be adjusted with this remote control. After an infrared pulse is received, the motor activates, and activity is indicated by a green LED that illuminates the model from the inside. This light is used to determine time-zero for each trial of motion tracking.After sending an infrared signal, the movement of each model was recorded with a submersible camera rig fitted with two waterproof cameras (Fig. 3). Each of the four models were monitored during a single, one-second jet for at least 9 trials each. Additionally, the laterally compressed morphotypes (serpenticone and oxycone) were monitored during three, one-second pulses for 10 trials each. The inflated morphotypes (sphaerocone and morphospace center) were not able to be monitored over longer distances because they had the tendency to rotate about the vertical axis, obscuring views of the tracking points. In addition to horizontal movement, turning efficiency (maneuverability about the vertical axis) was monitored by directing the cameras with a top-down view of each model. A 90° elbow attachment for the hyponome was fit to each model to investigate the ease or difficulty of rotation. Each model was designed to spin counter-clockwise when viewed from above so that the influence of the motor’s angular momentum was consistent between models.Footage was recorded with two GoPro Hero 8 Black cameras at 4K resolution and 24 (23.975) frames per second, with linear fields of view. Motion tracking was performed with the software DLTdv879 to record the pixel locations of each tracking point (Figs. 1c and S4). These coordinates were transformed into 3D coordinates in meters using the program easyWand580. The tracking points on each model were used for wand calibration because the distances between these sets of points were fixed. Standard deviations of the reproduced tracking point distances of less than 1 cm were considered suitable.The 3D position datasets allowed velocity, acceleration, rocking, to be computed for each experiment. Additionally angular displacement and angular velocity was of interest for the rotation experiments about the vertical axis. Velocity was computed under two scenarios: (1) using the 3D movement direction between each timestep (Eq. 5), and (2) only considering the horizontal movement direction between each time step (Eq. 6). The latter scenario was preferred to nullify the influences of model buoyancies, which were not perfectly neutral and caused some degree of vertical movement.$$V_{i} = frac{{sqrt {left( {x_{i} – x_{i – 1} } right)^{2} + left( {y_{i} – y_{i – 1} } right)^{2} + left( {z_{i} – z_{i – 1} } right)^{2} } }}{{left( {t_{i} – t_{i – 1} } right)}}$$
(5)
$$V_{i} = frac{{sqrt {left( {x_{i} – x_{i – 1} } right)^{2} + left( {y_{i} – y_{i – 1} } right)^{2} } }}{{left( {t_{i} – t_{i – 1} } right)}}$$
(6)
where V and t are velocity and time, and the subscripts i and i −1 refer to the current and previous time steps, respectively. Coordinate components are denoted by x, y, and z at each timestep. The averaged 3D location of both tracking points was used for each model (i.e., midpoints). Note that Eq. (5) uses the 3D form of the Theorem of Pythagoras, whereas Eq. (6) uses the 2D version. Time zero for each trial was defined as the frame where the robot was illuminated by the internal LED, indicating motor activity. Acceleration was modeled by fitting a linear equation to the datapoints during the one-second pulse interval(s) using the curve fitting toolbox in MATLAB R2020A.The artificially high hydrostatic stability of each model was designed to nullify rocking during movement. This behavior was computed for each model during the one-pulse experiments with the following equation:$$theta_{dv} = cos^{ – 1} left( {frac{{left( {z_{2} – z_{1} } right)}}{{sqrt {left( {x_{2} – x_{1} } right)^{2} + left( {y_{2} – y_{1} } right)^{2} + left( {z_{2} – z_{1} } right)^{2} } }}} right) – theta_{tp}$$
(7)
where (theta_{dv}) is the angle deviated from true vertical and (theta_{tp}) is the angle of the tracking points measured from the vertical in a static setting. The subscripts 1 and 2 of the x, y, and z coordinates refer to the anterior and posterior tracking points, respectively.Maneuverability about the vertical axis was determined by computing the angle between the horizontal components of each tracking point. The net angle from the starting angle for each trial was tabulated. Angular velocity was determined by dividing the change in angle between each frame by the frame duration (1/23.975 fps).Links to example motion tracking footage, and robotic models are deposited in an online repository60,61,63 (Dataset S2; https://doi.org/10.5281/zenodo.6180801). More

Constant contacts modelWe assume the affected population is composed of two risk-groups, a fraction p of the population is composed of risk-takers (RT) and the remaining fraction (1-p) are risk-evaders (RE). We differentiate the RT and RE subpopulations by assuming the RE population face a reduced likelihood of infection due to adopting precautionary behaviors. On the other hand, we assume RT do not follow public health recommendations, thus facing a higher risk of infection, relative to the RE population. Political or ideological reasons, economic stress, the lack of reasonable alternatives, epidemic politicization or the lack of trust in public health authorities are some of the documented factors that potentially lead the population to risk the dangers of COVID-19 infection44, 45.Previous mathematical models consider complex within-host disease dynamics46 or the impact of exogenous factors on the COVID-19 transmission dynamics47. In this study, we focus on incorporating individual heterogeneous adaptive behavioral responses, based on group-specific infection risk perceptions. Our model of disease progression assumes that individuals in each behavioral group may show the following health status: Susceptible (S), infectious Exposed (E), Infectious symptomatic (I), infectious Asymptomatic (A), and Recovered (R). We consider a pre-symptomatic infectious health status (E), following evidence suggesting that exposed individuals exhibit a period of viral shedding38, 48,49,50,51. RT susceptible individuals ((S_1)) can get infected by making contacts with either: symptomatic ones (I) with a baseline per-contact likelihood of disease transmission (beta), exposed individuals ((E_1) and (E_2)) with reduced per-contact likelihood of infection (rho beta) , or asymptomatic individuals ((A_1) and (A_2)) with reduced per-contact likelihood of infection (alpha beta). Similarly RE susceptible individuals ((S_2)) may get infected by making contacts with symptomatic, exposed or asymptomatic individuals at respective likelihoods, (epsilon beta), (rho epsilon beta), and (alpha epsilon beta), where (0 More

Chileshe, M. N. et al. Physico-chemical characteristics and heavy metal concentrations of copper mine wastes in Zambia: Implications for pollution risk and restoration. J. For. Res. https://doi.org/10.1007/s11676-019-00921-0 (2019).Article 

Google Scholar 
Sracek, O. Formation of secondary hematite and its role in attenuation of contaminants at mine tailings: Review and comparison of sites in Zambia and Namibia. Front. Environ. Sci. 2, 1–11 (2015).ADS 
Article 

Google Scholar 
Kayika, P., Siachoono, S., Kalinda, C. & Kwenye, J. An investigation of concentrations of copper, cobalt and cadmium minerals in soils and mango fruits growing on Konkola copper mine tailings dam in Chingola, Zambia. Arch. Sci. 1, 2–5 (2017).
Google Scholar 
Nazir, R. et al. Accumulation of heavy metals (Ni, Cu, Cd, Cr, Pb, Zn, Fe) in the soil, water and plants and analysis of physico-chemical parameters of soil and water collected from Tanda Dam Kohat. J. Pharm. Sci. Res. 7, 89–97 (2015).CAS 

Google Scholar 
Surbakti, E. P., Iswantari, A., Effendi, H. & Sulistiono. Distribution of dissolved heavy metals Hg, Pb, Cd, and As in Bojonegara Coastal Waters, Banten Bay. IOP Conf. Ser. Earth Environ. Sci. 744, 012085 (2021).Article 

Google Scholar 
Van Nguyen, T. et al. Arsenic and heavy metal contamination in soils under different land use in an estuary in northern Vietnam. Int. J. Environ. Res. Public Health 13, 1091 (2016).Article 
CAS 

Google Scholar 
Yabe, J. et al. Uptake of lead, cadmium, and other metals in the liver and kidneys of cattle near a lead-zinc mine in Kabwe, Zambia. Environ. Toxicol. Chem. 30, 1892–1897 (2011).CAS 
PubMed 
Article 

Google Scholar 
Salem, M. A., Bedade, D. K., Al-ethawi, L. & Al-waleed, S. M. Heliyon Assessment of physiochemical properties and concentration of heavy metals in agricultural soils fertilized with chemical fertilizers. Heliyon 6, e05224 (2020).PubMed 
PubMed Central 
Article 

Google Scholar 
Tuakuila, J. et al. Worrying exposure to trace elements in the population of Kinshasa, Democratic Republic of Congo (DRC). Int. Arch. Occup. Environ. Health 85, 927–939 (2012).CAS 
PubMed 
Article 

Google Scholar 
Setia, R. et al. Phytoavailability and human risk assessment of heavy metals in soils and food crops around Sutlej river, India. Chemosphere 263, 128321 (2021).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Burga, D. & Saunders, K. Understanding and Mitigating Lead Exposure in Kabwe: A One Health Approach (S. Afr. Inst. Policy Res, 2019).
Google Scholar 
Ikenaka, Y., Nakayama, S. M. M., Muzandu, K. & Choongo, K. Heavy metal contamination of soil and sediment in Zambia. Afr. J. Environ. Sci. Technol. https://doi.org/10.4314/ajest.v4i11.71339 (2010).Article 

Google Scholar 
Taylor, A. A. et al. Critical review of exposure and effects: Implications for setting regulatory health criteria for ingested copper. Environ. Manag. 65, 131–159 (2020).Article 

Google Scholar 
Gummow, B., Botha, C. J., Basson, A. T. & Bastianello, S. S. Copper toxicity in ruminants: Air pollution as a possible cause. Onderstepoort J. Vet. Res. 58, 33–39 (1991).CAS 
PubMed 

Google Scholar 
Cheng, S. Effects of heavy metals on plants and resistance mechanisms. Environ. Sci. Pollut. Res. 10, 256–264 (2003).CAS 
Article 

Google Scholar 
Olobatoke, R. & Mathuthu, M. Heavy metal concentration in soil in the tailing dam vicinity of an old gold mine in Johannesburg, South Africa. Can. J. Soil Sci. 96, 299–304 (2008).Article 
CAS 

Google Scholar 
Peša, I. Between waste and profit: Environmental values on the Central African Copperbelt. Extr. Ind. Soc. https://doi.org/10.1016/j.exis.2020.08.004 (2020).Article 

Google Scholar 
Trevor, M. et al. Statistical and spatial analysis of heavy metals in soils of residential areas surrounding the Nkana Copper Mine Site in Kitwe District, Zambia. Am. J. Environ. Sustain. Dev. 4, 26–37 (2019).
Google Scholar 
Nalishuwa, L. Investigation on Copper Levels in and Around Fish Farms in Kitwe, Copperbelt Province, Zambia (Sokoine University of Agriculture, 2015).
Google Scholar 
Ikenaka, Y. et al. Heavy metal contamination of soil and sediment in Zambia. Afr. J. Environ. Sci. Technol. 4, 109–128 (2014).
Google Scholar 
Sracek, O., Mihaljevič, M., Kříbek, B., Majer, V. & Veselovský, F. Geochemistry and mineralogy of Cu and Co in mine tailings at the Copperbelt, Zambia. J. Afr. Earth Sci. 57, 14–30 (2010).ADS 
CAS 
Article 

Google Scholar 
Manchisi, J. et al. Potential for bioleaching copper sulphide rougher concentrates of Nchanga Mine, Chingola, Zambia. J. S. Afr. Inst. Min. Metall. 112, 1051–1058 (2012).
Google Scholar 
Fernández-Caliani, J. C., Barba-Brioso, C., González, I. & Galán, E. Heavy metal pollution in soils around the abandoned mine sites of the Iberian Pyrite Belt (Southwest Spain). Water Air Soil Pollut. 200, 211–226 (2009).ADS 
Article 
CAS 

Google Scholar 
Prasad, R. & Chakraborty, D. Phosphorus Basics: Understanding Phosphorus Forms and Their Cycling in the Soil 1–4 (Alabama Coop. Ext. Syst, 2019).
Google Scholar 
Verma, F. et al. Appraisal of pollution of potentially toxic elements in different soils collected around the industrial area. Heliyon 7, e08122 (2021).PubMed 
PubMed Central 
Article 

Google Scholar 
Hermans, S. M., Buckley, H. L., Case, B. S., Curran-cournane, F. & Taylor, M. Bacteria as emerging indicators of soil condition. Appl. Environ. Microbiol. 83, 1–13 (2017).Article 

Google Scholar 
Ndeddy Aka, R. J. & Babalola, O. O. Identification and characterization of Cr-, Cd-, and Ni-tolerant bacteria isolated from mine tailings. Bioremediat. J. 21, 1–19 (2017).Article 
CAS 

Google Scholar 
Hassan, A., Pariatamby, A., Ahmed, A., Auta, H. S. & Hamid, F. S. Enhanced bioremediation of heavy metal contaminated landfill soil using filamentous fungi consortia: A demonstration of bioaugmentation potential. Water Air Soil Pollut. 230, 1–20 (2019).Article 
CAS 

Google Scholar 
Zhou, L. et al. Restoration of rare earth mine areas: organic amendments and phytoremediation. Environ. Sci. Pollut. Res. 22, 17151–17160 (2015).CAS 
Article 

Google Scholar 
Kapungwe, E. M. Heavy metal contaminated water, soils and crops in peri urban wastewater irrigation farming in Mufulira and Kafue towns in Zambia. J. Geogr. Geol. 5, 55–72 (2013).
Google Scholar 
Sandell, E. Post-Mining Restoration in Zambia (Swedish University of Agricultural Sciences, 2020).
Google Scholar 
Kumar, V., Pandita, S. & Setia, R. A meta-analysis of potential ecological risk evaluation of heavy metals in sediments and soils. Gondwana Res. 103, 487–501 (2022).ADS 
CAS 
Article 

Google Scholar 
Kumar, V., Sihag, P., Keshavarzi, A., Pandita, S. & Rodríguez-Seijo, A. Soft computing techniques for appraisal of potentially toxic elements from Jalandhar (Punjab), India. Appl. Sci. 11, 8362 (2021).CAS 
Article 

Google Scholar 
Setia, R. et al. Assessment of metal contamination in sediments of a perennial river in India using pollution indices and multivariate statistics. Arab. J. Geosci. 14, 1–9 (2021).Article 
CAS 

Google Scholar 
Kumar, V. et al. Pollution assessment of heavy metals in soils of India and ecological risk assessment: A state-of-the-art. Chemosphere 216, 449–462 (2019).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Environmental Council of Zambia. Environment Outlook Report in Zambia (2008).Kasali, G. Clacc Capacity Strengthening in the Least Developed Countries. CLACC Working Paper (2008).Ettler, V., Mihaljevič, M., Kříbek, B., Majer, V. & Šebek, O. Tracing the spatial distribution and mobility of metal/metalloid contaminants in Oxisols in the vicinity of the Nkana copper smelter, Copperbelt province, Zambia. Geoderma 164, 73–84 (2011).ADS 
CAS 
Article 

Google Scholar 
Cook, J. M. et al. The comparability of sample digestion techniques for the determination of metals in sediments. Mar. Pollut. Bull. 34, 637–644 (1997).CAS 
Article 

Google Scholar 
Güven, D. E. & Akinci, G. Comparison of acid digestion techniques to determine heavy metals in sediment and soil samples. Gazi Univ. J. Sci. 24, 29–34 (2011).
Google Scholar 
Jha, P. et al. Predicting total organic carbon content of soils from Walkley and Black analysis. Commun. Soil Sci. Plant Anal. 45, 713–725 (2014).CAS 
Article 

Google Scholar 
Walkley, A. & Black, I. A. A critical examination of rapid method for determining organic carbon in soil. Soil Sci. 63, 251–254 (1974).ADS 
Article 

Google Scholar 
Ure, A. M. Methods of analysis for heavy metals in soils. In Heavy Metals Soils (ed. Alloway, B. J.) 58–102 (Springer, 1995).Chapter 

Google Scholar 
Staniland, S. et al. Cobalt uptake and resistance to trace metals in comamonas testosteroni isolated from a heavy-metal contaminated site in the Zambian Copperbelt. Geomicrobiol. J. 27, 656–668 (2010).CAS 
Article 

Google Scholar 
Ajmone-Marsan, F. & Biasioli, M. Trace elements in soils of urban areas. Water Air Soil Pollut. 213, 121–143 (2010).ADS 
CAS 
Article 

Google Scholar 
Adriano, D. C. Trace elements in terrestrial environments. J. Environ. Qual. 32, 374 (2003).
Google Scholar 
Adriano, D. C. Trace Elements in Terrestrial Environments: Biogeochemistry, Bioavailability and Risks of Metals (Springer, 2001).Book 

Google Scholar 
R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.r-project.org/ (2020).Wickham, H. Ggplot2: Elegant Graphics for Data Analysis. Springer, New York, NY, USA, (2009).Hakanson, L. Ecological risk index for aquatic pollution control. A sedimentological approach. Water Res. 14, 975–1001 (1980).Article 

Google Scholar 
Muller, G. Index of geoaccumulation in sediments of the Rhine River. Geojournal 2, 108–118. (1969).
Google Scholar 
Usero, J., A. Garcia and J. Fraidias, 2000. Andalicia Board, Environmental Counseling. 1st Edn., Seville, Editorial, pp: 164.Sikamo, J., Mwanza, A. & Mweemba, C. Copper mining in Zambia—history and future. J. S. Afr. Inst. Min. Metall. 116, 6–8 (2016).Article 
CAS 

Google Scholar 
DR Congo: copper production 2010–2020|Statista. https://www.statista.com/statistics/1276790/copper-production-in-democratic-republic-of-the-congo/.Lydall, M. I. & Auchterlonie, A. The Southern African Institute of Mining and Metallurgy 6th Southern Africa base metals conference 2011. The Democratic Republic of Congo and Zambia: A growing global ‘Hotspot’ for copper-cobalt mineral investment and explo. In The Southern African Institute of Mining and Metallurgy 25–38 (2011).Worlanyo, A. S. & Jiangfeng, L. Evaluating the environmental and economic impact of mining for post-mined land restoration and land-use: A review. J. Environ. Manag. 279, 111623 (2021).CAS 
Article 

Google Scholar 
Shengo, M. L., Kime, M. B., Mambwe, M. P. & Nyembo, T. K. A review of the beneficiation of copper-cobalt-bearing minerals in the Democratic Republic of Congo. J. Sustain. Min. 18, 226–246 (2019).Article 

Google Scholar 
Tembo, B. D., Sichilongo, K. & Cernak, J. Distribution of copper, lead, cadmium and zinc concentrations in soils around Kabwe town in Zambia. Chemosphere 63, 497–501 (2006).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Tveitnes, S. Soil productivity research programme in the high rainfall areas in Zambia. Agricultural University of Norway (1981).Esshaimi, M., El Gharmali, A., Berkhis, F., Valiente, M. & Mandi, L. Speciation of heavy metals in the soil and the mining residues, in the Zinclead Sidi Bou Othmane Abandoned mine in Marrakech area. Linnaeus Eco-Tech https://doi.org/10.15626/eco-tech.2010.102 (2017).Article 

Google Scholar 
Vítková, M. et al. Primary and secondary phases in copper-cobalt smelting slags from the Copperbelt Province, Zambia. Mineral. Mag. 74, 581–600 (2010).Article 
CAS 

Google Scholar 
Van Brusselen, D. et al. Metal mining and birth defects: A case-control study in Lubumbashi, Democratic Republic of the Congo. Lancet Planet. Health 4, e158–e167 (2020).PubMed 
Article 

Google Scholar 
Peša, I. Between waste and profit: Environmental values on the Central African Copperbelt. Extr. Ind. Soc. 8, 100793 (2021).
Google Scholar 
Muleya, F. et al. Investigating the suitability and cost-benefit of copper tailings as partial replacement of sand in concrete in Zambia: An exploratory study. J. Eng. Des. Technol. 19, 828–849 (2020).
Google Scholar 
Namweemba, M. G. Mining Induced Heavy Metal Soil and Crop Contamination in Chililabombwe on the Copperbelt of Zambia (University of Zambia, 2017).
Google Scholar 
Colombo, C., Palumbo, G., He, J.-Z., Pinton, R. & Cesco, S. Review on iron availability in soil: Interaction of Fe minerals, plants, and microbes. J. Soils Sediments 14, 538–548 (2014).CAS 
Article 

Google Scholar 
Barsova, N., Yakimenko, O., Tolpeshta, I. & Motuzova, G. Current state and dynamics of heavy metal soil pollution in Russian Federation—A review. Environ. Pollut. 249, 200–207 (2019).CAS 
PubMed 
Article 

Google Scholar 
WHO/FAO. Food additives and contaminants. Joint FAO. WHO Food Stand. Program. ALINORM 1, 1–289 (2001).
Google Scholar 
Sracek, O. et al. Mining-related contamination of surface water and sediments of the Kafue River drainage system in the Copperbelt district, Zambia: An example of a high neutralization capacity system. J. Geochem. Explor. 112, 174–188 (2012).CAS 
Article 

Google Scholar 
Hasimuna, O. J., Chibesa, M., Ellender, B. R. & Maulu, S. Variability of selected heavy metals in surface sediments and ecological risks in the Solwezi and Kifubwa Rivers, Northwestern province, Zambia. Sci. Afr. 12, e00822 (2021).
Google Scholar 
Kříbek, B. Mining and the environment in Africa. Conserv. Lett. 7, 302–311 (2011).
Google Scholar 
Crommentuijn, T., M.D.Polder & Plassche, E. J. van de. Maximum Permissible Concentrations and Negligible Concentrations for metals, taking background concentrations into account. National Institute of Public Health and the Environment Bilthoven, The Netherlands (1997).Maboeta, M. S., Oladipo, O. G. & Botha, S. M. Ecotoxicity of mine tailings: Unrehabilitated versus rehabilitated. Bull. Environ. Contam. Toxicol. 100, 702–707 (2018).CAS 
PubMed 
Article 

Google Scholar 
Festin, E. S., Tigabu, M., Chileshe, M. N., Syampungani, S. & Odén, P. C. Progresses in restoration of post-mining landscape in Africa. J. For. Res. 30, 381–396 (2019).Article 

Google Scholar 
Volk, J. & Yerokun, O. Effect of application of increasing concentrations of contaminated water on the different fractions of Cu and Co in sandy loam and clay loam soils. Agriculture 6, 64 (2016).Article 
CAS 

Google Scholar 
Pietrini, F. et al. Effect of different copper levels on growth and morpho-physiological parameters in giant reed (Arundo donax L.) in semi-hydroponic mesocosm experiment. Water (Switzerland) 11, 1837 (2019).CAS 

Google Scholar 
EPA. Ecological Soil Screening Level for Iron Interim Final 211 (US Environ. Prot. Agency – Off. Solid Waste Emerg., 2005).
Google Scholar  More