More stories

  • in

    Tree species matter for forest microclimate regulation during the drought year 2018: disentangling environmental drivers and biotic drivers

    Bonan, G. B. Forests and climate change: Forcings, feedbacks, and the climate benefits of forests. Science 320, 1444–1449 (2008).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Frey, S. J. K. et al. Spatial models reveal the microclimatic buffering capacity of old-growth forests. Sci. Adv. 2, e1501392. https://doi.org/10.1126/sciadv.1501392 (2016).Article 
    ADS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Davis, K. T., Dobrowski, S. Z., Holden, Z. A., Higuera, P. E. & Abatzoglou, J. T. Microclimatic buffering in forests of the future: The role of local water balance. Ecography 42, 1–11 (2019).Article 

    Google Scholar 
    de Frenne, P. et al. Global buffering of temperatures under forest canopies. Nat. Ecol. Evol. 3, 744–749 (2019).Article 
    PubMed 

    Google Scholar 
    Zellweger, F. et al. Forest microclimate dynamics drive plant responses to warming. Science 368, 772–775 (2020).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Rahman, M. A., Moser, A., Rötzer, T. & Pauleit, S. Microclimatic differences and their influence on transpirational cooling of Tilia cordata in two contrasting street canyons in Munich, Germany. Agric. For. Meteorol. 232, 443–456 (2017).Article 
    ADS 

    Google Scholar 
    Rahman, M. A., Moser, A., Rötzer, T. & Pauleit, S. Within canopy temperature differences and cooling ability of Tilia cordata trees grown in urban conditions. Build. Environ. 114, 118–128 (2017).Article 

    Google Scholar 
    Ehbrecht, M., Schall, P., Ammer, C., Fischer, M. & Seidel, D. Effects of structural heterogeneity on the diurnal temperature range in temperate forest ecosystems. For. Ecol. Manag. 432, 860–867 (2019).Article 

    Google Scholar 
    Richter, R., Hutengs, C., Wirth, C., Bannehr, L. & Vohland, M. Detecting tree species effects on forest canopy temperatures with thermal remote sensing: The role of spatial resolution. Remote Sens. 13, 135. https://doi.org/10.3390/rs13010135 (2021).Article 
    ADS 

    Google Scholar 
    IPCC. Climate change 2021: The physical science basis. In Contribution of Working Group I to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change (eds Masson-Delmotte, V. et al.) (Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA, In Press, 2021).Rahman, M. A. et al. Traits of trees for cooling urban heat islands: A meta-analysis. Build. Environ. 170, 106606. https://doi.org/10.1016/j.buildenv.2019.106606 (2020).Article 

    Google Scholar 
    Rahman, M. A., Moser, A., Rötzer, T. & Pauleit, S. Comparing the transpirational and shading effects of two contrasting urban tree species. Urban Ecosyst. 22, 683–697 (2019).Article 

    Google Scholar 
    Joly, F.-X. et al. Tree species diversity affects decomposition through modified micro-environmental conditions across European forests. New Phytol. 214, 1281–1293 (2017).Article 
    CAS 
    PubMed 

    Google Scholar 
    Lindo, Z. & Winchester, N. Out on a limb: microarthropod and microclimate variation in coastal temperate rainforest canopies. Insect Conserv. Divers. 6, 513–521 (2013).Article 

    Google Scholar 
    Pincebourde, S., Murdock, C. C., Vickers, M. & Sears, M. W. Fine-scale microclimatic variation can shape the responses of organisms to global change in both natural and urban environments. Integr. Comp. Biol. 56, 45–61 (2016).Article 
    PubMed 

    Google Scholar 
    Janssen, P., Fuhr, M. & Bouget, C. Beyond forest habitat qualities: Climate and tree characteristics as the major drivers of epiphytic macrolichen assemblages in temperate mountains. J. Veg. Sci. 30, 42–54 (2019).Article 

    Google Scholar 
    Welti, E. A. R. et al. Temperature drives variation in flying insect biomass across a German malaise trap network. Insect Conserv. Divers. https://doi.org/10.1111/icad.12555 (2021).Article 

    Google Scholar 
    Lin, Y.-S., Medlyn, B. E. & Ellsworth, D. S. Temperature responses of leaf net photosynthesis: The role of component processes. Tree Physiol. 32, 219–231 (2012).Article 
    CAS 
    PubMed 

    Google Scholar 
    Simon, H. et al. Modeling transpiration and leaf temperature of urban trees: A case study evaluating the microclimate model ENVI-met against measurement data. Landsc. Urban Plan. 174, 33–40 (2018).Article 

    Google Scholar 
    Eamus, D., Boulain, N., Cleverly, J. & Breshears, D. D. Global change-type drought-induced tree mortality: Vapor pressure deficit is more important than temperature per se in causing decline in tree health. Ecol. Evol. 3, 2711–2729 (2013).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Eichenberg, D. et al. The effect of microclimate on wood decay is indirectly altered by tree species diversity in a litterbag study. J. Plant Ecol. 10, 170–178 (2017).Article 

    Google Scholar 
    Brockerhoff, E. G. et al. Forest biodiversity, ecosystem functioning and the provision of ecosystem services. Biodivers. Conserv. 26, 3005–3035 (2017).Article 

    Google Scholar 
    Martínez Pastur, G., Perera, A. H., Peterson, U. & Iverson, L. R. In Ecosystem Services from Forest Landscapes (eds Perera, A. H. et al.) 1–10 (Springer International Publishing, 2018).
    Google Scholar 
    Smithers, R. J. et al. Comparing the relative abilities of tree species to cool the urban environment. Urban Ecosyst. 21, 851–862 (2018).Article 

    Google Scholar 
    Shashua-Bar, L., Tsiros, I. X. & Hoffman, M. Passive cooling design options to ameliorate thermal comfort in urban streets of a Mediterranean climate (Athens) under hot summer conditions. Build. Environ. 57, 110–119 (2012).Article 

    Google Scholar 
    Song, J. & Wang, Z.-H. Impacts of mesic and xeric urban vegetation on outdoor thermal comfort and microclimate in Phoenix, AZ. Build. Environ. 94, 558–568 (2015).Article 

    Google Scholar 
    Meehl, G. A. & Tebaldi, C. More intense, more frequent, and longer lasting heat waves in the 21st century. Science 305, 994–997 (2004).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Christidis, N., Jones, G. S. & Stott, P. A. Dramatically increasing chance of extremely hot summers since the 2003 European heatwave. Nat. Clim. Change 5, 46–50 (2015).Article 
    ADS 

    Google Scholar 
    Pfleiderer, P., Schleussner, C.-F., Kornhuber, K. & Coumou, D. Summer weather becomes more persistent in a 2 °C world. Nat. Clim. Change 9, 666–671 (2019).Article 
    ADS 

    Google Scholar 
    Selten, F. M., Bintanja, R., Vautard, R. & van den Hurk, B. J. J. M. Future continental summer warming constrained by the present-day seasonal cycle of surface hydrology. Sci. Rep. 10, 4721. https://doi.org/10.1038/s41598-020-61721-9 (2020).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Gartner, K., Nadezhdina, N., Englisch, M., Čermak, J. & Leitgeb, E. Sap flow of birch and Norway spruce during the European heat and drought in summer 2003. For. Ecol. Manag. 258, 590–599 (2009).Article 

    Google Scholar 
    Speak, A., Montagnani, L., Wellstein, C. & Zerbe, S. The influence of tree traits on urban ground surface shade cooling. Landsc. Urban Plan. 197, 103748. https://doi.org/10.1016/j.landurbplan.2020.103748 (2020).Article 

    Google Scholar 
    Rahman, M. A., Armson, D. & Ennos, A. R. A comparison of the growth and cooling effectiveness of five commonly planted urban tree species. Urban Ecosyst. 18, 371–389 (2015).Article 

    Google Scholar 
    Bowden, J. D. & Bauerle, W. L. Measuring and modeling the variation in species-specific transpiration in temperate deciduous hardwoods. Tree Physiol. 28, 1675–1683 (2008).Article 
    PubMed 

    Google Scholar 
    Panferov, O. et al. The role of canopy structure in the spectral variation of transmission and absorption of solar radiation in vegetation canopies. IEEE Trans. Geosci. Remote Sens. 39, 241–253 (2001).Article 
    ADS 

    Google Scholar 
    Lin, H., Chen, Y., Zhang, H., Fu, P. & Fan, Z. Stronger cooling effects of transpiration and leaf physical traits of plants from a hot dry habitat than from a hot wet habitat. Funct. Ecol. 31, 2202–2211 (2017).Article 

    Google Scholar 
    Fauset, S. et al. Differences in leaf thermoregulation and water use strategies between three co-occurring Atlantic forest tree species. Plant Cell Environ. 41, 1618–1631 (2018).Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Chen, L., Zhang, Z. & Ewers, B. E. Urban tree species show the same hydraulic response to vapor pressure deficit across varying tree size and environmental conditions. PloS ONE 7, e47882. https://doi.org/10.1371/journal.pone.0047882 (2012).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Gallego, H. A., Rico, M., Moreno, G. & Santa Regina, I. Leaf water potential and stomatal conductance in Quercus pyrenaica Willd. forests: Vertical gradients and response to environmental factors. Tree Physiol. 14, 1039–1047 (1994).Article 
    PubMed 

    Google Scholar 
    Hölscher, D., Koch, O., Korn, S. & Leuschner, C. Sap flux of five co-occurring tree species in a temperate broad-leaved forest during seasonal soil drought. Trees 19, 628–637 (2005).Article 

    Google Scholar 
    Li, S. et al. Leaf gas exchange performance and the lethal water potential of five European species during drought. Tree Physiol. 36, 179–192 (2016).CAS 
    PubMed 

    Google Scholar 
    Schnabel, F. et al. Cumulative growth and stress responses to the 2018–2019 drought in a European floodplain forest. Glob. Change Biol. 28, 1870–1883 (2022).Article 
    CAS 

    Google Scholar 
    Sastry, A., Guha, A. & Barua, D. Leaf thermotolerance in dry tropical forest tree species: Relationships with leaf traits and effects of drought. AoB Plants 10, plx070. https://doi.org/10.1093/aobpla/plx070 (2018).Article 
    CAS 
    PubMed 

    Google Scholar 
    Banerjee, T. & Linn, R. Effect of vertical canopy architecture on transpiration, thermoregulation and carbon assimilation. Forests 9, 198. https://doi.org/10.3390/f9040198 (2018).Article 

    Google Scholar 
    Leuzinger, S. & Körner, C. Tree species diversity affects canopy leaf temperatures in a mature temperate forest. Agric. For. Meteorol. 146, 29–37 (2007).Article 
    ADS 

    Google Scholar 
    Yi, K. et al. High heterogeneity in canopy temperature among co-occurring tree species in a temperate forest. J. Geophys. Res. Biogeosci. 125, e05892. https://doi.org/10.1029/2020JG005892 (2020).Article 

    Google Scholar 
    Hagemeier, M. & Leuschner, C. Functional crown architecture of five temperate broadleaf tree species: Vertical gradients in leaf morphology, leaf angle, and leaf area density. Forests 10, 265. https://doi.org/10.3390/f10030265 (2019).Article 

    Google Scholar 
    Raabe, K., Pisek, J., Sonnentag, O. & Annuk, K. Variations of leaf inclination angle distribution with height over the growing season and light exposure for eight broadleaf tree species. Agric. For. Meteor. 214–215, 2–11 (2015).Article 

    Google Scholar 
    Kafuti, C. et al. Foliar and wood traits covary along a vertical gradient within the crown of long-lived light-demanding species of the Congo Basin semi-deciduous forest. Forests 11, 35. https://doi.org/10.3390/f11010035 (2020).Article 

    Google Scholar 
    Peiffer, M., Bréda, N., Badeau, V. & Granier, A. Disturbances in European beech water relation during an extreme drought. Ann. For. Sci. 71, 821–829 (2014).Article 

    Google Scholar 
    Stratópoulos, L. M. F. et al. Tree species from two contrasting habitats for use in harsh urban environments respond differently to extreme drought. Int. J. Biometeorol. 63, 197–208 (2019).Article 
    ADS 
    PubMed 

    Google Scholar 
    McGloin, R. et al. Available energy partitioning during drought at two Norway spruce forests and a European beech forest in Central Europe. J. Geophys. Res. Atmos. 124, 3726–3742 (2019).Article 
    ADS 

    Google Scholar 
    Schwaab, J. et al. Increasing the broad-leaved tree fraction in European forests mitigates hot temperature extremes. Sci. Rep. 10, 14153. https://doi.org/10.1038/s41598-020-71055-1 (2020).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Hari, V., Rakovec, O., Markonis, Y., Hanel, M. & Kumar, R. Increased future occurrences of the exceptional 2018–2019 Central European drought under global warming. Sci. Rep. 10, 12207. https://doi.org/10.1038/s41598-020-68872-9 (2020).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Lyon, T. L., Weil, R. R. & Brady, N. C. The Nature and Properties of Soils 15th edn. (Pearson, 2017).
    Google Scholar 
    Zweifel, R., Böhm, J. P. & Häsler, R. Midday stomatal closure in Norway spruce—reactions in the upper and lower crown. Tree Physiol. 22, 1125–1136 (2002).Article 
    CAS 
    PubMed 

    Google Scholar 
    Rahman, M. A., Moser, A., Gold, A., Rötzer, T. & Pauleit, S. Vertical air temperature gradients under the shade of two contrasting urban tree species during different types of summer days. Sci. Total Environ. 633, 100–111 (2018).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Schuldt, B. et al. A first assessment of the impact of the extreme 2018 summer drought on Central European forests. Basic Appl. Ecol. 45, 86–103 (2020).Article 

    Google Scholar 
    Hochberg, U., Rockwell, F. E., Holbrook, N. M. & Cochard, H. Iso/anisohydry: A plant-environment interaction rather than a simple hydraulic trait. Trends Plant Sci. 23, 112–120 (2018).Article 
    CAS 
    PubMed 

    Google Scholar 
    Leuschner, C., Wedde, P. & Lübbe, T. The relation between pressure–volume curve traits and stomatal regulation of water potential in five temperate broadleaf tree species. Ann. For. Sci. 76, 60. https://doi.org/10.1007/s13595-019-0838-7 (2019).Article 

    Google Scholar 
    Bartlett, M. K., Scoffoni, C. & Sack, L. The determinants of leaf turgor loss point and prediction of drought tolerance of species and biomes: A global meta-analysis. Ecol. Lett. 15, 393–405 (2012).Article 
    PubMed 

    Google Scholar 
    Hartmann, H., Link, R. M. & Schuldt, B. A whole-plant perspective of isohydry: Stem-level support for leaf-level plant water regulation. Tree Physiol. 41, 901–905 (2021).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Alonso-Forn, D. et al. Revisiting the functional basis of sclerophylly within the leaf economics spectrum of oaks: Different roads to Rome. Curr. For. Rep. 6, 260–281 (2020).
    Google Scholar 
    Hirons, A. D. & Thomas, P. A. Applied Tree Biology (John Wiley & Sons Ltd, 2017).Book 

    Google Scholar 
    Richter, R., Reu, B., Wirth, C., Doktor, D. & Vohland, M. The use of airborne hyperspectral data for tree species classification in a species-rich Central European forest area. Int. J. Appl. Earth Obs. Geoinf. 52, 464–474 (2016).ADS 

    Google Scholar 
    Qiu, G. et al. Effects of evapotranspiration on mitigation of urban temperature by vegetation and urban agriculture. J. Integr. Agric. 12, 1307–1315 (2013).Article 

    Google Scholar 
    Meier, F. & Scherer, D. Spatial and temporal variability of urban tree canopy temperature during summer 2010 in Berlin, Germany. Theor. Appl. Climatol. 110, 373–384 (2012).Article 
    ADS 

    Google Scholar 
    Landsberg, J. J. & James, G. B. Wind profiles in plant canopies: Studies on an analytical model. J. Appl. Ecol. 8, 729–741 (1971).Article 

    Google Scholar 
    Gromke, C. & Ruck, B. Aerodynamic modelling of trees for small-scale wind tunnel studies. Forestry 81, 243–258 (2008).Article 

    Google Scholar 
    Baldocchi, D. D. Turbulent transfer in a deciduous forest. Tree Physiol. 5, 357–377 (1989).Article 
    CAS 
    PubMed 

    Google Scholar 
    Derby, R. W. & Gates, D. M. The temperature of tree trunks—Calculated and observed. Am. J. Bot. 53, 580–587 (1966).
    Google Scholar 
    Jayalakshmy, M. S. & Philip, J. Thermophysical properties of plant leaves and their influence on the environment temperature. Int. J. Thermophys. 31, 2295–2304 (2010).Article 
    ADS 
    CAS 

    Google Scholar 
    Pieruschka, R., Huber, G. & Berry, J. A. Control of transpiration by radiation. Proc. Natl. Acad. Sci. U.S.A. 107, 13372–13377 (2010).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Meili, N. et al. Tree effects on urban microclimate: Diurnal, seasonal, and climatic temperature differences explained by separating radiation, evapotranspiration, and roughness effects. Urban For. Urban Green. 58, 126970. https://doi.org/10.1016/j.ufug.2020.126970 (2021).Article 

    Google Scholar 
    Oogathoo, S., Houle, D., Duchesne, L. & Kneeshaw, D. Vapour pressure deficit and solar radiation are the major drivers of transpiration of balsam fir and black spruce tree species in humid boreal regions, even during a short-term drought. Agric. For. Meteorol. 291, 108063. https://doi.org/10.1016/j.agrformet.2020.108063 (2020).Article 
    ADS 

    Google Scholar 
    Betts, M. G., Phalan, B., Frey, S. J. K., Rousseau, J. S. & Yang, Z. Old-growth forests buffer climate-sensitive bird populations from warming. Divers. Distrib. 24, 439–447 (2018).Article 

    Google Scholar 
    Pureswaran, D. S., Roques, A. & Battisti, A. Forest insects and climate change. Curr. For. Rep. 4, 35–50 (2018).
    Google Scholar 
    de Frenne, P. et al. Forest microclimates and climate change: Importance, drivers and future research agenda. Glob. Change Biol. 27, 2279–2297 (2021).Article 
    ADS 

    Google Scholar 
    Woods, C. L., Cardelús, C. L. & DeWalt, S. J. Microhabitat associations of vascular epiphytes in a wet tropical forest canopy. J. Ecol. 103, 421–430 (2015).Article 

    Google Scholar 
    Nakamura, A. et al. Forests and their canopies: Achievements and horizons in canopy science. Trends Ecol. Evol. 32, 438–451 (2017).Article 
    PubMed 

    Google Scholar 
    European State of the Climate 2020, Copernicus Climate Change Service, Full report: climate.copernicus.eu/ESOTC/2020Munzi, S. et al. Lichens as ecological indicators in urban areas: beyond the effects of pollutants. J. Appl. Ecol. 51, 1750–1757 (2014).Article 

    Google Scholar 
    Kaspari, M., Clay, N. A., Lucas, J., Yanoviak, S. P. & Kay, A. Thermal adaptation generates a diversity of thermal limits in a rainforest ant community. Glob. Change Biol. 21, 1092–1102 (2015).Article 
    ADS 

    Google Scholar 
    Baudier, K. M., Mudd, A. E., Erickson, S. C. & O’Donnell, S. Microhabitat and body size effects on heat tolerance: implications for responses to climate change (army ants: Formicidae, Ecitoninae). J. Anim. Ecol. 84, 1322–1330 (2015).Article 
    PubMed 

    Google Scholar 
    Merinero, S., Dahlberg, C. J., Ehrlén, J. & Hylander, K. Intraspecific variation influences performance of moss transplants along microclimate gradients. Ecology 101, e02999. https://doi.org/10.1002/ecy.2999 (2020).Article 
    PubMed 

    Google Scholar 
    Ben-Yakir, D. & Fereres, A. The effects of UV radiation on arthropods: A review of recent publications (2010–2015). Acta Hortic. 1134, 335–342 (2016).Vanhaelewyn, L., van der Straeten, D., de Coninck, B. & Vandenbussche, F. Ultraviolet radiation from a plant perspective: The plant-microorganism context. Front. Plant Sci. 11, 597642. https://doi.org/10.3389/fpls.2020.597642 (2020).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Jansen, E. Das Naturschutzgebiet Burgaue; Staatliches Umweltfachamt: Leipzig, Germany (1999).Sächsisches Landesamt für Umwelt, Landwirtschaft und Geologie (LFULG) & DWD Deutscher Wetterdienst (2019) [ed.]: 2018 Wetter trifft auf Klima. Dresden, Leipzig. https://www.klima.sachsen.de/download/Jahresrueckblick2018_A5_OeA.pdf.Haase, D. & Gläser, J. Determinants of floodplain forest development illustrated by the example of the floodplain forest in the District of Leipzig. For. Ecol. Manag. 258, 887–894 (2009).Article 

    Google Scholar 
    Patzak, R., Richter, R., Engelmann, R. A. & Wirth, C. Tree crowns as meeting points of diversity generating mechanisms: A test with epiphytic lichens in a temperate forest. Preprint at: https://www.biorxiv.org/content/https://doi.org/10.1101/2020.01.03.894303v1.full (2020).Meinen, C., Leuschner, C., Ryan, N. T. & Hertel, D. No evidence of spatial root system segregation and elevated fine root biomass in multi-species temperate broad-leaved forests. Trees 23, 941–950 (2009).Article 

    Google Scholar 
    van der Zande, D., Stuckens, J., Verstraeten, W. W., Muys, B. & Coppin, P. Assessment of light environment variability in broadleaved forest canopies using terrestrial laser scanning. Remote Sens. 2, 1564–1574. https://doi.org/10.3390/rs2061564 (2010).Article 
    ADS 

    Google Scholar 
    Köstner, B., Granier, A. & Cermák, J. Sapflow measurements in forest stands: Methods and uncertainties. Ann. For. Sci. 55, 13–27 (1998).Article 

    Google Scholar 
    Granier, A. Evaluation of transpiration in a Douglas-fir stand by means of sap flow measurements. Tree Physiol. 3, 309–320 (1987).Article 
    CAS 
    PubMed 

    Google Scholar 
    Metzger, J. M. & Oren, R. The effect of crown dimension on transparency and the assessment of tree health. Ecol. Appl. 11, 1634–1640 (2001).Article 

    Google Scholar 
    Lefcheck, J. S. piecewiseSEM: Piecewise structural equation modelling in r for ecology, evolution, and systematics. Methods Ecol. Evol. 7, 573–579 (2016).Article 

    Google Scholar 
    Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. & R Core Team (2020). nlme: Linear and nonlinear mixed effects models. R package version 3.1-151, https://CRAN.R-project.org/package=nlme.Dornelas, M. et al. Quantifying temporal change in biodiversity: Challenges and opportunities. Proc. Biol. Sci. 280, 20121931. https://doi.org/10.1098/rspb.2012.1931 (2013).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Shipley, B. The AIC model selection method applied to path analytic models compared using a d-separation test. Ecology 94, 560–564 (2013).Article 
    PubMed 

    Google Scholar 
    R Core Team (2021). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/. More

  • in

    Mapping tropical forest functional variation at satellite remote sensing resolutions depends on key traits

    We hypothesized that functionally distinct forest types can be mapped at moderate spatial resolutions, using a combination of canopy foliar traits and canopy structure information. Our analysis of LiDAR and imaging spectroscopy data at spatial resolutions ranging from 4 to 200 m (16 m2–40,000 m2), with an emphasis on the 30 m (900 m2) spaceborne hyperspectral spatial resolution, reveals that few remotely sensed canopy properties are needed to successfully identify ecologically distinct forest types at two diverse tropical forest sites in Malaysian Borneo. In testing our second hypothesis that mapped forest types exhibit distinct ecosystem function, we found that forest types identified using remotely sensed leaf P, LMA, Max H, and canopy cover at 20 m height (Cover20) closely align with forest types defined from field-based floristic surveys29,30,31,32,33 and inventory plot-based measurements of growth and mortality rates (Fig. 4b). Our approach, however, enables mapping of their entire spatial extent (Fig. 1) and reveals important structural and functional variation within areas characterized as a single forest type in previous studies (Fig. 3). Current and forthcoming satellite hyperspectral platforms, including PRISMA (30 m), CHIME (20–30 m), and SBG (30 m), have or will have comparable spectral resolution, higher temporal revisits, and much greater geographic coverage. The ability to conduct this type of analysis using remote sensing measurements at 30 m resolution suggests that our method can be applied to these emerging spaceborne imaging spectroscopy data to reveal important differences in structure and function across the world’s tropical forests.Nested functional forest types revealedTo test our first hypothesis, rather than making an a priori decision about the number of k-means clusters (k), we explored the capacity of remotely sensed data to reveal ecologically relevant variation in forest types. Baldeck and Asner took a similar unsupervised approach to estimating beta diversity in South Africa34. Because the choice of k directly influences analysis outcomes, careful selection of k is required. Different approaches for identifying the number of clusters, using the Gapk and Wk elbow metrics35, yielded varying optimal numbers of clusters for the Sepilok and Danum landscapes (Fig. 1, Supplementary Figs. 4 and 5). However, at both sites, a comparison of results based on different values of k revealed ecologically meaningful structural and functional differences and graduated transitions between forest types (Fig. 2, Supplementary Figs. 7 and 8), indicating that the exploration of traits that aggregate or separate forest types as k changes is a valuable exercise. Overlap between the remotely sensed forest type boundaries and inventory plots within distinct forest types indicate that the series of clustered forests align closely with forest types defined based on in situ data on species composition and ecosystem structure. In part, this type of analysis requires careful selection of the number of clusters. Additionally, however, we gained valuable insights via the exploration of varying numbers of clusters as it relates to biologically meaningful categorization of forest types. Extending this method to other parts of the tropics will require similar decision-making, which will either require user input, or the development of robust automated algorithms for selecting k.Forest types capture differences in ecosystem dynamicsWe further evaluated the canopy traits and structural attributes that were most critical for mapping distinct forest types, hypothesizing that mapped forest types exhibit distinct ecosystem function. Forest types revealed by the cluster analyses were distributed along the leaf economic spectrum, where the leaf economic spectrum characterizes a tradeoff in plant growth strategies36. LMA, which can covary strongly with leaf N and P, is a key indicator of plant growth strategies along the spectrum37. At the slow-return end of the leaf economics spectrum, plants in nutrient-poor conditions with low leaf nutrient concentrations invest in leaf structure and defense, expressed as high LMA, strategizing longer-lived, tougher leaves with slower decomposition rates. This strategy comes at the cost of slower growth. At the quick-return end of the spectrum, plants in nutrient-rich environments with higher leaf nutrient concentrations invest less in structure and defense, enabling faster growth and more rapid leaf turnover, i.e., shorter leaf lifespans. This quick-return growth strategy supports higher photosynthetic rates and more rapid carbon gain36.In this study, the principal components and clustering results yielded forest types that are indicative of community level differences associated with leaf economic spectrum differences. The nutrient rich sites (Danum1 and Danum2, Supplementary Fig. 8) show high canopy N and P and low LMA compared to the nutrient poor and acidic sites (Sandstone and Kerangas), which contributes to lower leaf photosynthetic capacity (Vcmax) and growth (Fig. 4b). Foliar N:P also increased with site fertility, confirming that tropical forests are primarily limited by phosphorus, and not nitrogen38,39, with large implications for carbon sequestration in these forests. Orthogonal differences in canopy structure and architecture between Danum forest types and Sepilok Sandstone and Alluvial forests could be indicative of ecosystem scale differences in the sensitivity of these forests to endogenous disturbance processes40.The significant differences in aboveground carbon stocks and growth and mortality rates between forest types further suggests strong differences in ecosystem dynamics. In general, growth rates varied inversely to aboveground carbon, and higher aboveground carbon corresponded to lower mortality rates. As an example, the Sepilok sandstone forests, which are largely comprised of slow-growing dipterocarp species29,33, had the highest median aboveground carbon (236 Mg C ha−1), with higher canopy P and N, and lower LMA. The taller canopy and low canopy leaf nutrient concentrations are consistent with the low growth and mortality rates found in the sandstone forest, indicating a slow-growth strategy yielding larger trees and higher aboveground carbon stocks. In contrast, alluvial forests exhibit high turnover with mortality and growth rates higher relative to Sandstone forests corresponding to lower aboveground carbon on average. Kerangas forests exhibited low aboveground carbon despite an intermediate plot-level growth rate, and mortality rates that were significantly lower than the Danum or alluvial forest types. Kerangas forests, which were characterized by the highest LMA, lowest foliar P and N (Fig. 2a), and the lowest plot-level aboveground carbon density (186 Mg C ha−1; Fig. 4a), are known to have higher stem densities, lower canopy heights, and long-lived leaves5,32,41, suggesting well-developed strategies for nutrient retention42. Interestingly, despite significantly different aboveground carbon and demography, the kerangas and sandstone forests did not differ in LAI or canopy architecture (P:H); although maximum height, Cover20, and Hpeak LAI were significantly higher in the sandstone forest, highlighting the need to account for differences beyond LAI when scaling processes from leaves to ecosystems.In addition, when three forest types were distinguished at Sepilok, the alluvial inventory plot had significantly higher aboveground carbon than the remote sensing-derived alluvial forest extent (Fig. 4a, p  More

  • in

    Diverse MarR bacterial regulators of auxin catabolism in the plant microbiome

    Bacterial strains and mediaA collection of 185 genome-sequenced bacterial isolates, described previously14, was utilized to assemble the synthetic community used in this work. These isolates were obtained from surface-sterilized Brassicaceae roots, primarily Arabidopsis thaliana, grown in two soils from North Carolina, USA35. This isolate collection includes strains V. paradoxus CL14, Arthrobacter CL28, Acinetobacter CL69 and Acinetobacter CL71, which are also used in this work in individual strain contexts. V. paradoxus CL14 ΔHS33, which has a clean deletion of genes with gene ID 2643613677 through 2643613653 was constructed previously14 and used here. Additional strains were obtained from the American Type Culture Collection (ATCC): E. soli LF7 (ATCC BAA-2102), R. pomeroyi (ATCC 700808) and B. japonicum (ATCC 10324). P. phytofirmans PsJN (DSMZ 17436) was obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures. P. putida strain 1290 was generously provided by Johan Leveau (University of California Davis). Pseudomonas strain Root 562 was generously provided by Paul Schulze-Lefert (Max-Planck-Gesellschaft). All bacteria, with exceptions noted below, were routinely grown on LB agar plates (10 g l−1 tryptone, 5 g l−1 yeast extract, 10 g l−1 NaCl, 1.5% (w/v) agar) and in 2xYT liquid medium (16 g l−1 tryptone, 10 g l−1 yeast extract, 5 g l−1 NaCl) at 28 °C. The 175-member (185-member minus 10 Variovorax strains) synthetic community (SC185-10V) was grown on KB medium as was done previously to culture this synthetic community14. B. japonicum (ATCC 10324) was routinely grown on liquid and solidified YM medium (1 g l−1 yeast extract, 10 g l−1 mannitol, 0.5 g l−1 dipotassium phosphate, 0.2 g l−1 magnesium sulfate, 0.1 g l−1 NaCl, 1 g l−1 CaCO4, pH 6.8, solidified with 1.5% agar as necessary) at 28 °C. R. pomeroyi (ATCC 700808) was routinely grown on liquid and solidified LB medium supplemented with 2% sea salt (Millipore Sigma S9883) and solidified with 1.5% (w/v) agar as necessary. M9 base medium was formulated using 1x M9 minimal salts medium (Sigma M6030) supplemented with 2 mM MgSO4, 0.1 mM CaCl2 and 10 µM FeSO4. A carbon source or sources were added to this M9 base medium to support bacterial growth. Unique strains constructed in this study are available upon request.Bacterial 16S rRNA sequencingBacterial colonization of Arabidopsis roots was assessed using a method similar to the previous study14. Roots from 8–10 plants were collected into sterilized 2 ml tubes containing three 4 mm glass beads and root fresh weight in each tube was obtained. Five such samples were collected for each bacterial treatment. The roots were washed three times with sterile distilled water and stored at −80 °C until further processing. The roots were then lyophilised for 48 h using a Labconco freeze-dry system and pulverized using an MPBio tissue homogenizer. DNA was extracted from the root samples and bacterial cell pellets saved from the bacteria for input into the experiment using the DNeasy PowerSoil HTP 96 kit (Qiagen) according to manufacturer instructions. The V3-V4 region of the bacterial 16S rRNA gene was amplified and sequenced as previously described14.16S amplicon sequence data processingThe 16S sequencing data from synthetic community experiments were processed as previously described14. Briefly, usable read output from MT-Toolbox36 (reads with 100% primer sequences that successfully merged with their pair) were filtered for quality with Sickle37 by not allowing any window with Q score under 20. The resulting sequences were globally aligned to a 16S rRNA gene sequence reference dataset from genome assemblies of the synthetic community members. For strains that do not have an intact 16S rRNA sequence in their assembly, Sanger sequencing was used to obtain the 16S rRNA gene sequence of the strains for inclusion in the reference dataset. The reference dataset also included sequences from Arabidopsis organellar sequences and known bacterial contaminants. Sequence alignment was performed with USEARCH v.7.109038 using the optional usearch_global at a 98% identity threshold. On average, 85% of read sequences matched an expected isolate. The 185 isolates of our 185-member synthetic community could not all be distinguished from one another on the basis of the V3-V4 sequence. They were thus classified into 97 unique sequences encompassing a set of identical (clustered at 100%) V3-V4 sequences coming from a single or multiple isolate strains. An isolate abundance table was created from the sequence mapping results.We estimated 16S rRNA absolute abundance using a plasmid spike-in method39. Synthetic DNA was spiked at known quantities into samples before DNA extraction and the ratio of added to recovered synthetic DNA served as a conversion factor by which the total number of 16S rRNA molecules in a given sample was estimated. We designed a plasmid which included 16S V3-V4 primer binding sequences flanking a randomly generated DNA sequence matching the most frequent length and Guanine + Cytosine (GC) content of amplicons generated using the same primer sequences from wild soil. These sequences were synthesized by Geneart (Invitrogen) and supplied cloned in plasmid pMA-T. The plasmid was transformed into E. coli and isolated using a miniprep spin kit (Qiagen). Specific volumes of this isolated plasmid were then added to individual samples before DNA extraction to spike-in approximately 20% of the predicted 16S copies occurring within the sample. We performed colony-forming units (c.f.u.) counting using similarly treated plant samples (that is, growth on SynCom-inoculated agar plates) to obtain an estimate of the 16S copy number per mg fresh weight of plant roots. We plated serial dilutions of plant root samples ground in MgCl2 on LB to perform c.f.u. counts. The c.f.u. count multiplied by a given sample’s fresh weight were used to calculate sample-specific predicted 16S copy numbers.Plant growth conditions and root growth inhibition assayA. thaliana ecotype Col-0 seeds were sterilized in 70% household bleach, 0.2% Tween-20 for 10 min with vigorous agitation and then rinsed 10 times with sterile distilled water. Seeds were stratified at 4 °C in sterile distilled water for 1–2 d. Plants were germinated for 7 d on 0.5x MS agar medium (2.22 g l−1 PhytoTech Labs M-404: Murashige & Skoog modified basal medium with Gamborg vitamins, 0.5 g l−1 MES hydrate, pH adjusted to 5.7, solidified with 1% (w/v) agar) supplemented with 0.5% (w/v) sucrose in vertical 12 ×12 cm square plates under long-day conditions (21 °C/18 °C, 16 h light/8 h dark, day/night cycle). Then 8 to 10 plants were aseptically transferred to 12 ×12 cm plates containing 0.5x MS agar medium without sucrose where the medium surface was spread with the bacterial inoculum. For assays with IAA addition, 100 nM IAA was added to the medium before pouring the plates. The plant root tip location was marked on plates after transfer to record the initial root tip position. The plates containing the plants and bacteria were incubated vertically under short-day conditions (22 °C/18 °C, 9 h light/15 h dark, day/night cycle) for an additional 11 d. Plates were imaged on a document scanner and primary root elongation was determined using imageJ to quantify the change in root tip position from the initial to the final position.Bacterial inoculation of plantsIndividual bacterial strains were grown on agar plates of the media types specified above at 28 °C. Before plant inoculation, a single colony was picked into the appropriate liquid medium and grown at 28 °C to late exponential or early stationary phase. To remove the medium from the bacteria before inoculation, strains were washed three times in sterile 10 mM MgCl2. The optical density at 600 nm (OD600) was measured for each washed strain and normalized to OD600 of 0.01 in 10 mM MgCl2. For plant experiments with mono-association of an individual strain, 100 µl of OD600 = 0.01 washed bacteria was spread on the 12 ×12 cm plate before plant transfer. For experiments in duo-association with Arthrobacter CL28, 100 µl of OD600 = 0.01 washed Arthrobacter CL28 was spread along with 100 µl of OD600 = 0.01 of the second strain.The 175-member synthetic community (SC185-10V) was prepared as described for the 185-member synthetic community used previously14 by leaving out the 10 isolates from the genus Variovorax. Briefly, 7 d before plant transfer, strains were inoculated individually into 600 µl KB medium in a 96-well plate and grown at 28 °C for 5 d. At 2 d before plant transfer, 20 ul from these 5-day-old cultures were transferred to 380 ul fresh KB medium in a new set of 96-well plates and both sets of plates were returned to the incubator for 2 d. This resulted in two cultures of each strain, one 7 d old and the other 2 d old, which were combined. The OD600 of the strains in each well was measured and the strains were combined while normalizing the OD600 of each strain in the pool. This pool was washed twice with 10 mM MgCl2 and diluted to OD600 = 0.2. For experiments with the SC185-10V SynCom, 100 µl of this OD600 = 0.2 washed pool was spread on 12 ×12 cm plates. For treatments where an additional strain was added to the SC185-10V SynCom, the individual strain was washed as described above, diluted to OD600 = 0.0034 in 10 mM MgCl2, and 100 µl of this dilution was spread on the plates with the SC185-10V SynCom. This addition of the individual strain corresponded to an OD600 three times that of a single strain in the SC185-10V SynCom (0.0034 = (0.2/175) × 3). For the addition of the 10 Variovorax strains to the SC185-10V SynCom experiment, the 10 Variovorax strains were grown individually in 2xYT medium from colonies grown on plates. The OD600 values of the 10 cultures were measured and the 10 strains were pooled while normalizing the OD600 of each strain to the same value. This mixture of the 10 Variovorax strains was then treated as the individual strains for washing and addition of 100 µl of OD600 = 0.0034 to the SC185-10V SynCom on plates.Construction of vectors with Variovorax CL14 iad gene insertsPortions of the V. paradoxus CL14 IAA degradation locus were subcloned into broad host range vector pBBR1MCS-232. Primers JMC579 through JMC604 (Supplementary Table 8) were used to amplify 3–5 kb segments of the locus by PCR using Q5 DNA polymerase (New England Biolabs). These primers were designed to amplify sections beginning and ending at gene start codons and with appropriate overlapping sequences for Gibson assembly either into the pBBR1MCS-2 backbone or to the adjacent section to make larger vector inserts, as appropriate. The pBBR1MCS-2 vector backbone was prepared for Gibson assembly by amplifying the vector by PCR using primers JMC577 and JMC578 (Supplementary Table 8) and subsequently treating with DpnI to remove circular vector template. PCR fragments were cleaned up as necessary using the QIAquick PCR purification kit (Qiagen). Appropriate fragments were mixed to construct the vectors by Gibson assembly using HiFi DNA Assembly Mastermix (New England Biolabs) according to manufacturer instructions. Gibson assembly products were transformed into NEB 10beta chemically competent E. coli (New England Biolabs) and selected on LB plates supplemented with 50 µg ml−1 kanamycin. Vectors were miniprepped using either the ZR plasmid miniprep classic kit or Zymo BAC DNA miniprep kit (Zymo Research) and confirmed via restriction mapping with PstI-HF (New England Biolabs) and Sanger sequencing (Genewiz).To construct vectors that are derivatives of pBBR1::70–66, the Q5 site-directed mutagenesis kit (New England Biolabs) was used for gene deletion. Briefly, vector pBBR1::70–66 was used as a PCR template and portions of this vector were amplified by PCR using primers JMC641 through JMC650 (Supplementary Table 8) and Q5 DNA polymerase (New England Biolabs). PCR products were cleaned up and circularized using KLD Mastermix (New England Biolabs). The product was transformed into NEB 10beta chemically competent E. coli (New England Biolabs) and selected on LB plates supplemented with 50 µg ml−1 kanamycin. Vectors were miniprepped and Sanger sequenced as described above to confirm the construction of the correct vectors.Conjugation of vectors to V. paradoxus CL14 ΔHS33Vectors were conjugated into V. paradoxus CL14 ΔHS33 using tri-parental mating. The helper E. coli strain carrying plasmid pRK201340 and donor NEB 10beta E. coli strains containing the pBBR1MCS-2-based vectors with Variovorax IAA degradation locus gene inserts were cultured in LB media containing 50 µg ml−1 kanamycin at 37 °C. V. paradoxus CL14 ΔHS33 was grown in 2xYT medium containing 100 µg ml−1 ampicillin at 28 °C. V. paradoxus CL14 wild type and derivative strains such as ΔHS33 are naturally resistant to ampicillin and this ampicillin selection allows for recovery of only Variovorax from the conjugation reaction. To prepare for conjugation, all bacteria were pelleted by centrifugation at 5,000 × g for 5 min and washed 3 times in 2xYT medium without antibiotics. For each conjugation reaction, equal volumes (100–300 µl) of each of the three washed bacteria: recipient V. paradoxus CL14 ΔHS33, donor NEB 10beta E. coli containing a pBBR1MCS-2-based vector, and helper E. coli pRK2013 were mixed. Control conjugation mixtures of each pair of strains and individual strains alone were performed in parallel to ensure successful selection of exconjugants only from mixtures of all three strains together. Conjugation mixtures were pelleted by centrifugation at 5,000 × g for 5 min, resuspended in 50 µl 2xYT media, transferred to LB media plates without antibiotics and allowed to dry in a laminar flow hood. These conjugation plates were incubated overnight at 28 °C. After 18–24 h, exconjugants were selected by streaking from the pooled conjugation mixtures on the LB plate without antibiotics to LB plates containing 50 µg ml−1 kanamycin and 100 µg ml−1 ampicillin. This selects for only V. paradoxus CL14 ΔHS33 (ampicillin resistant) containing the pBBR1MCS-2-based vector (kanamycin resistant). Individual colonies were picked into and subsequently cultured in 2xYT medium containing 50 µg ml−1 kanamycin and 100 µg ml−1 ampicillin at 28 °C.Construction of V. paradoxus CL14 gene deletionsUnmarked gene deletions in V. paradoxus CL14 were constructed as described previously14 using the suicide vector backbone pMo130 originally developed for gene knockouts in Burkholderia spp.41. Primers JMC203 and JMC204 (Supplementary Table 8) were used to amplify the pMO130 vector backbone by PCR. This product was subsequently treated with DpnI (New England Biolabs) to digest circular template DNA. Primers JMC605 through JMC612 and JMC671 through JMC677 (Supplementary Table 8) were used to amplify flanking regions for the gene deletion targets from V. paradoxus CL14 genomic DNA. All PCR was performed using Q5 DNA polymerase (New England Biolabs) and products were cleaned up, as appropriate, with the QIAquick PCR purification kit (Qiagen). These PCR products were assembled into suicide vectors using HiFi Gibson Assembly Mastermix (New England Biolabs), transformed into chemically competent NEB 5alpha E. coli (New England Biolabs), and selected on LB plates with 50 µg ml−1 kanamycin. Vectors were miniprepped using the ZR plasmid miniprep classic kit (Zymo Research) and confirmed by Sanger sequencing (Genewiz). Confirmed vectors were transformed into the chemically competent bi-parental mating strain E. coli WM3064. Transformants were selected at 37 °C on LB media supplemented with 50 µg ml−1 kanamycin and 0.3 mM diaminopimelic acid (DAP), and single colonies picked into LB medium also with 50 µg ml−1 kanamycin and 0.3 mM DAP.Bi-parental mating was performed by growing E. coli WM3064 containing the appropriate suicide vector as described above, and V. paradoxus CL14 was grown in 2xYT medium containing 100 µg ml−1 ampicillin at 28 °C. Both E. coli and Variovorax were washed separately three times using 2xYT medium, then mixed in a 1:1 ratio and pelleted. All centrifugation steps were performed at 5,000 × g for 5 min. The pelleted conjugation mixtures were resuspended in 1/10 the volume of 2xYT, plated on LB agar with 0.3 mM DAP and grown at 28 °C overnight. Exconjugants from these plates were streaked out and grown on LB agar with 100 µg ml−1 ampicillin, 50 µg ml−1 kanamycin, and no DAP at 28 °C. These strains were purified by streaking and growing on plates of the same medium once more. These strains with suicide vector integration were then grown once in liquid LB containing 100 µg ml−1 ampicillin and 1 mM isopropyl 1-thio-d-galactopyranoside (IPTG) at 28 °C and then streaked on plates containing media with 10 g l−1 tryptone, 5 g l−1 yeast extract, 100 g l−1 sucrose, 1.5% agar, 100 µg ml−1 ampicillin and 1 mM IPTG. Colonies from these plates were picked and grown in the same liquid media. These strains were then assessed for gene deletion by PCR using primers JMC657 through JMC660 and JMC697 through JMC699 (Supplementary Table 8). The Quick-DNA miniprep kit (Zymo Research) was used to isolate all genomic DNA for PCR screening. To purify the knockout strains, they were streaked and grown out three times on LB plates containing 100 µg ml−1 ampicillin before a final PCR confirmation. To check the purity of the final strains, PCR was performed with one primer outside the deletion region and one inside the deleted gene to ensure no product is produced for the knockout strain. The sequences for the primers used for this PCR reaction (JMC691, JMC717, JMC718, JMC693 and JMC694) can be found in Supplementary Table 8.Measurement of bacterial growth and IAA degradationIndividual strains were grown in 5 ml cultures in various media types supplemented with IAA at 28 °C and 250 r.p.m. To screen the V. paradoxus CL14 ΔHS33 pBBR1 vector complemented mutants, 2xYT medium supplemented with 0.1 mg ml−1 IAA was used. For comparison of other V. paradoxus CL14 mutants, M9 medium with 15 mM succinate and 0.1 mg ml−1 (0.57 mM) IAA was used. For comparison of IAA-degrading strains from diverse genera, M9 medium with 0.1% (w/v) casamino acids (Bacto) and 0.1 mg ml−1 IAA was used. For R. pomeroyi, 2% (w/v) sea salts were added to this M9 medium with casamino acids and IAA. The pBBR1 vector library in E. coli NEB 10beta was screened in LB medium supplemented with 0.04 mg ml−1 IAA and grown at 37 °C and 250 r.p.m. For all media types, IAA was solubilized in 100% ethanol at 20 mg ml−1 and diluted to 0.1 mg ml−1 in the media, resulting in 0.5% (v/v) ethanol in the media.To measure growth, a 200 µl sample was taken from the growing cultures and OD600 was determined on an Infinite M200 Pro plate reader (Tecan). Subsequently, cells were pelleted by centrifugation at 4,200 × g for 15 min and 50 µl of supernatant was transferred to a new 96-well plate and frozen at −80 °C until further analysis. IAA degradation was determined by thawing the plates containing 50 µl aliquots of culture supernatant and combining this with 100 µl of Salkowski reagent (10 mM ferric chloride and 35% perchloric acid)42. This was performed alongside mixing 50 µl of IAA standards with 100 µl of Salkowski reagent in the same 96-well plate format. Colour development was allowed to proceed for 1 h and absorbance was read at 530 nm on the Infinite M200 Pro plate reader (Tecan). The absorbances measured were converted to IAA concentration on the basis of the absorbances measured for the IAA standards.Liquid Chromatography Dual Mass Spectroscopy (LC–MS/MS) metabolomics on Variovorax IAA degradationV. paradoxus CL14 was grown in 5 ml cultures of M9 minimal medium supplemented with either 0.1 mg ml−1 IAA, 0.1 mg ml−1 13C6-IAA (with the 6 carbons of the benzene ring of the indole labelled, Cambridge Isotope Laboratories CLM-1896-PK), and/or 15 mM succinate. Cultures and parallel media controls were incubated at 28 °C with shaking at 250 r.p.m. Cultures and media controls were centrifuged (4,200 × g for 15 min at 4 °C) to pellet cells; supernatants were transferred to new tubes and both pellets (intracellular fraction) and supernatants (extracellular fraction) were stored frozen at −80 °C until extraction. All subsequent work was performed over dry ice or in chilled cold blocks. Frozen pellets from the intracellular fraction were thawed for 3 h at 4 °C, then 800 µl of cold LCMS-grade water was added to the pellets with repeated pipetting to break up the pellet until visually homogeneous. Samples were then quickly returned to −80 °C to freeze the suspension. Frozen pellet suspensions and extracellular solutions were lyophilised until dry. The cells from the dried pellet suspensions were lysed and homogenized with a bead mill (BioSpec Mini-Beadbeater-96) using one sterile 3.2 mm steel ball in each tube for 3 rounds of 5 s each with 10 s breaks in between to reduce heat production. Dried extracellular samples were concentrated by resuspension in 100 µl LCMS- grade methanol, vortexed 3 times for 10 s each, water bath sonicated for 20 min, incubated at 4 °C overnight, centrifuged (1,000 × g, 4 °C, 5 min), and the methanol supernatant was dried using a speed vacuum concentrator. On the day of LC–MS/MS analysis, homogenized dry material was suspended in LCMS-grade methanol with internal standard mix (100 µM U-13C/15N-labelled amino acids, SIGMA 767964). Intracellular samples were suspended at 11.1 µl mg−1 of original sample cell pellet wet weight; extracellular samples were suspended at 38.9 µl mg−1 of corresponding cell pellet wet weight from the culture. The solutions were vortexed 3 times for 10 s each, bath sonicated in ice water for 10 min, chilled at −20 °C for 10 min, then centrifuged (10,000 × g, 5 min, 10 °C) to pellet insoluble material. Supernatants containing the methanol extracts were filtered through 0.22 µm PVDF microcentrifuge filtration tubes (10,000 × g, 5 min, 10 °C); filtrates were transferred to glass vials and immediately capped. Filtrates were then analysed by LC–MS/MS using an Agilent 1290 UHPLC system connected to a Thermo Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer equipped with a heated electrospray ionization (HESI-II) source probe. Extracts were chromatographically separated on a ZORBAX RRHD Eclipse Plus C18, 95 Å, 2.1 × 50 mm, 1.8 µm column (Agilent) for non-polar metabolomics. Separation, ionization, fragmentation and data acquisition parameters are specified in Supplementary Table 7. Briefly, metabolites were separated by gradient elution followed by MS1 and data-dependent (top 2 most abundant MS1 ions not previously fragmented in last 7 s) MS2 collection; targeted data analysis was performed by comparison of sample peaks to a library of analytical standards analysed under the same conditions or by searching the raw data files for predicted m/z values based on structural information of compounds of interest. Three parameters were compared: matching m/z, retention time and fragmentation spectra using Metabolite Atlas (https://github.com/biorack/metatlas)43,44. Identification and standard reference comparison details are provided in Supplementary Table 6. Raw and processed data are available for download at the JGI Joint Genome Portal under ID 1340427. Statistical comparisons were performed using R version 3.6.2, using package agricolae 1.3–5 and stats 3.6.245; boxplots were generated with base R graphics using the boxplot function.Phylogenomic analysisTo guide the delineation of the IAA degradation operons across the bacterial tree of life, we constructed two Hidden Markov Model (HMM) profiles of the genes iacC and iacD by subsetting all homologous genes from the previously validated operons (Extended Data Fig. 4). In parallel, we downloaded the assembly files for all available complete genomes deposited in the NCBI RefSeq 202 repository46. For the 220,000 assembly files downloaded, we performed open reading frame (ORF) prediction using prodigal. We then used the two HMM profiles described above to query the predicted ORFs. Utilizing ad hoc scripts, we constructed a table of HMM hits along the genomes scanned and subset genomic loci where both iacC and iacD genes appeared adjacent to one another. The logic of using the iacC and iacD genes as anchor genes for our search is that the adjacent physical location of both iacC and iacD homologues is a conserved feature across all previously experimentally validated IAA-degrading operons (Extended Data Fig. 4). Next, for each region containing the adjacent iacC and iacD homologue genes, we extracted the gene neighbourhood adjacent to the anchor hit by extracting the amino acid sequence of ORFs +10 kb and −10kb with respect to the anchor hit. Using hmmscan from the Hmmer v3.1.b2 suite47, we performed HMM profiling in all ORFs extracted via our neighbourhood delineation against the COG database version 2003. Finally, we used the COG profiles across the neighbourhoods to create a matrix describing the prevalence of COGs across the regions (candidate regions) with the adjacent iacC and iacD homologue genes.For each genome containing at least one candidate region, we performed taxonomic classification using the GTDB database48. Due to the size of our estimated genomic matrix and to reduce potential biases due to over-representation of certain lineages within RefSeq, we performed principal coordinate analysis (PCoA) using a reduced matrix containing one representative candidate region per species. Species labelling was obtained from the GTDB taxonomic classification described above. PCoA was performed using the oh.pco function from the ohchibi package49, taking as input a binary version of the gene matrix described above. We classified candidate reads into the two types of IAA-degrading operon (iac-like and iad-like), utilizing a majority count-based approach using marker COGs conserved between the previously experimentally validated IAA-degrading operons (Extended Data Fig. 4). Specifically, for each potential operon, we determined the prevalence of COGs that a priori (Extended Data Fig. 4) showed differential prevalence across the two degrading operons (for example, iacA, iacB and iacI are markers of the iac operon, while iorB/iadB and iotA/iadA are exclusive markers of the iad-like operon). Hybrid gene clusters were defined as operons that exhibited the hallmark COGs of both operons.In parallel, we performed phylogenetic inference over all the genomes belonging to genera with at least one representative strain harbouring any of the two types of IAA-degrading operon. This phylogenetic tree was constructed using a super-matrix-based approach as previously described35. Finally, for each genus with at least one assembly harbouring a positive IAA-degrading operon, we estimated the prevalence of the trait across the genus by dividing the total number of isolates with detectable IAA degradation locus by the total number of isolates belonging to that genus in the dataset. In addition, to see the phylogenetic evenness of the distribution of the IAA degradation trait across each genus, we calculated the phylogenetic ratio by calculating the ratio between the average phylogenetic distance (computed via the cophenetic.phylo function from the ape R package50) of isolates with a detectable IAA degradation locus and the total average phylogenetic distance of all isolates within that genus. We constructed the MarR phylogeny using the MarR sequences from candidate regions with 100% markers of one of the two types of IAA-degrading operon. Amino acid sequences of the MarR homologues were aligned using MAFFT51 and phylogenetic inference was performed using FastTree 252.RNA-seq on Variovorax strainsV. paradoxus CL14 was grown in 5 ml cultures of M9 minimal medium supplemented with 15 mM succinate and 0.5% (v/v) ethanol alone or containing IAA. IAA was at a final concentration of 0.1 mg ml−1 in the medium to which it was added. Cultures were prepared at a starting OD600 of 0.02 and incubated at 28 °C, shaking at 250 r.p.m. Cells from all samples were collected for RNA-seq at 18 h to ensure IAA was still present in the cultures of strains that degraded IAA most rapidly. Cells were pelleted by centrifuging the culture at 4,200 × g for 15 min and removing the supernatant. Cell pellets were frozen at −80 °C before RNA extraction. To extract RNA, cells were lysed in TRIzol reagent (Invitrogen) according to manufacturer instructions for lysis and phase separation. After these steps, RNA was purified from the aqueous phase using the RNeasy mini kit (Qiagen) including the optional on-column DNase digestion with RNase-free DNase set (Qiagen). Total RNA was quantified using the Qubit 2.0 fluorometer (Invitrogen) and RNA-seq libraries were prepared using the Universal Prokaryotic RNA-Seq Prokaryotic AnyDeplete kit (Tecan) according to manufacturer instructions. The resulting libraries were pooled and sequenced on the Illumina HiSeq4000 to generate 50 bp single-end reads.RNA-seq data analysisThe V. paradoxus CL14 RNA-seq sequence data were analysed as described previously14. Briefly, the raw reads were mapped to the V. paradoxus CL14 genome (fasta file available at https://github.com/isaisg/variovoraxRGI/blob/master/rawdata/2643221508.fna) using bowtie253 with the ‘very sensitive’ flag. Hits to each individual coding sequence were counted and annotated using the function featureCounts from the R package Rsubread54, inputting the V. paradoxus CL14 gff file (available at https://github.com/isaisg/variovoraxRGI/blob/master/rawdata/2643221508.gff) and using the default parameters with the flag allowMultiOverlap = FALSE. Finally, DESeq255 was used to estimate Differentially Expressed Genes (DEGs) between treatments, with the corresponding fold-change estimates and False Discovery Rate (FDR) adjusted P values. For visualization purposes, we performed z-score standardization of each gene across samples and we visualized this standardized expression values utilizing a heat map constructed using ggplot256. These data can be found in Supplementary Table 9.MarR protein expression and purificationThe coding sequence for each gene can be found in Supplementary Table 10. MarR expression plasmids were synthesized as codon-optimized genes for E. coli expression by BioBasic in the pLIC-His N-term vector (pMCSG7) and transformed into E. coli BL21 (DE3) Gold cells for expression. Cells were grown in the presence of ampicillin in LB medium with shaking at 225 r.p.m. at 37 °C to an OD600 of 0.5, at which point the temperature was reduced to 18 °C. At an OD600 of 0.8, protein expression was induced by the addition of 0.1 mM IPTG and incubation continued overnight. Cells were collected by centrifugation at 4,500 × g for 20 min at 4 °C in a Sorvall (model RC-3B) swinging bucket centrifuge. Cell pellets were resuspended in buffer A (20 mM potassium phosphate, pH 7.4, 50 mM imidazole, 500 mM NaCl), DNase, lysozyme and a Roche Complete EDTA-free protease inhibitor tablet. Resuspended cells were sonicated and clarified via centrifugation at 17,000 × g for 60 min in a Sorvall (model RC-5B) swinging bucket centrifuge. The lysate was applied to a nickel-nitrilotriacetic acid HP column (GE Healthcare) on an Aktaxpress Fast Performance Liquid Chromatography (FPLC) system (Amersham Bioscience) and washed with buffer A. Protein was eluted with buffer B (20 mM potassium phosphate, pH 7.4, 500 mM imidazole, 500 mM NaCl). Fractions containing the protein of interest were combined and passed over a HiLoadTM 16/60 SuperdexTM 200 gel filtration column. Proteins were eluted in S200 buffer (20 mM HEPES, pH 7.4, 300 mM NaCl). Fractions were combined and concentrated for long-term storage at −80 °C.MarR mutant proteins were created by site-directed mutagenesis using primers from Integrated DNA Technologies. The mutant plasmids were sequenced to confirm the mutations. The mutants were produced and purified using E. coli BL21 (DE3) Gold as described above.Ligand binding studies by isothermal titration calorimetry (ITC)All ITC measurements were performed at 25 °C using an Auto-ITC200 microcalorimeter (MicroCal/GE Healthcare). The buffer employed was 20 mM HEPES, pH 7.4, 50 mM NaCl and 0.5% dimethly sulfoxide (DMSO) for protein/ligand binding and 20 mM HEPES, pH 7.4 and 300 mM NaCl for DNA/protein binding experiments. For ligand binding experiments, the calorimetry cell (volume 200 ml) was loaded with MarR wild-type, mutant or homologue protein at a concentration of 50 μM. The syringe was loaded with a ligand concentration of 0.5 or 2 mM. For DNA binding experiments, wild-type MarR_73 did not bind any of the DNA oligos examined; however, we hypothesized that this arose from the ability of this native receptor to remain bound to ligands retained from its recombinant expression in E. coli. Thus, we employed the MarR_73 S28A protein with reduced ligand binding capacity. Here, the calorimetry cell was loaded with duplex oligo at a concentration of 25 μM and the syringe was loaded with MarR S28A mutant protein, which was necessary to prevent ligand binding during expression and purification, at a concentration of 0.5 mM. A typical injection protocol included a single 0.2 μl first injection followed by 20 1.5 μl injections of the syringe sample into the calorimetry cell. The spacing between injections was kept at 180 s and the reference power at 8 μcal s−1. The data were analysed using Origin for ITC version 7.0 software supplied by the manufacturer and fit well to a one-site binding model. Two independent ITC measurements were performed for each condition. A non-integer N value (for example, 0.73 in Fig. 2a) indicates that some protein monomers may not be in an active conformation, and thus do not bind ligand. Additionally, small measurement errors in assessing the protein or ligand concentrations may also contribute to non-integer N values in ITC. To confirm that 300 mM NaCl did not negatively impact DNA binding, MarR_73 S28A was examined by ITC in 150 mM NaCl. In this condition, the KD for the 22 bp duplex was 0.428 ± 0.002 μM (N = 1.75 ± 0.014), while the KD for 24 bp duplex was 0.151 ± 0.025 μM (N = 2.51 ± 0.26).Protein crystallographyV. paradoxus MarR_73 was crystallized using the sitting drop vapour diffusion method at 20 °C in conditions outlined in Supplementary Table 4. Crystallization drops were set up using the Oryx4 protein crystallization robot (Douglas Instruments) and contained 0.15 μl protein and 0.15 μl well solution. For all V. paradoxus MarR_73 wild-type conditions, ligands were added at 10-fold molar excess before crystallization trials and crystals appeared within 2–5 d. V. paradoxus MarR_73 with the S28A and R46A mutations was crystallized in similar conditions as the wild-type protein. Similarly, P. putida MarR_iacR, B. japonicum MarR_Bj1, A. baumannii MarR_Ab and E. soli MarR_Es were crystallized using vapour diffusion methods in sitting drop trays at 20 °C and crystals appeared within 3–5 d. All crystallization conditions are outlined in Supplementary Table 4. Crystal specimens were cryoprotected with the well solution supplemented with glycerol to 20% (v/v) (Supplementary Table 4). X-ray diffraction data were collected at the Advanced Photon Source beamline 23-ID-D (Supplementary Table 3). Diffraction images were reduced using either XDS or Denzo and scaled with either Aimless or Scalepack57,58,59. The V. paradoxus MarR_73 structure in complex with IAA was determined by molecular replacement using the structure of 3CDH as a search model in Phaser60. All subsequent structures of V. paradoxus MarR_73 were determined using the V. paradoxus MarR_73 IAA complex structure (PDB: 7KFO) as a search model. The P. putida MarR_iacR and B. japonicum MarR_Bj1 structures were determined by molecular replacement using the structure of 3CJN as the search model. P. putida MarR_iacR (PDB: 7KUA) was subsequently used as the search model for molecular replacement to solve A. baumannii MarR_Ab and E. soli MarR_Es. A nickel ion was placed in the model of MarR_Ab. The following ions or molecules were examined and refined in this location in the MarR_Ab structure: water, Na, Mg, K, Ca, Mn, Fe, Co, Ni, Cu, Zn and Ba. Water, Na, Mg, K, Ca and Ba were deemed unacceptable in this site due to poor difference density. Of the remaining ions considered, there were no sources of Mn, Fe, Co, Cu or Zn in the protein expression media, protein purification buffers, protein storage buffer, crystallization condition or cryoprotectant solutions. Thus, we concluded that the ion present in this structure is Ni due to the use of a nickel-affinity column during the protein’s purification. It is unclear why this ion remained bound to MarR_Ab even after the subsequent size exclusion chromatography purification step, or why such an ion is only observed in this structure of the proteins examined. All structures were refined with either Phenix.refine or Refmac using iterative model building in Coot to the final parameters outlined in Supplementary Table 361,62. MarR_73 is a dimer with one protein monomer in the asymmetric unit and the dimer generated by crystallographic symmetry. PDB accession codes and associated crystallographic data are reported in Supplementary Table 3.Statistics and reproducibilityNo statistical method was used to predetermine sample size, but our sample sizes are similar to those reported in previous publications14,63,64. No data were excluded from the analyses. The experiments were randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Where not stated, data distribution was assumed to be normal, but this was not formally tested.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. More

  • in

    Multi-species occupancy modeling suggests interspecific interaction among the three ungulate species

    Study areaThe present study was conducted in Uttarkashi district, Uttarakhand, located between 38° 28′ to 31°28′ N latitude and 77°49′ to 79°25′ E longitude with an area of about 8016 km2, covering primarily hilly terrain with an altitudinal range of 715–6717 m (Fig. 3). The terrain is mountainous, consisting of undulating hill ranges and narrow valleys with temperate climatic conditions. The district lies in the upper catchment of two major rivers of India, viz., the Ganges (Bhagirathi towards upstream) and the Yamuna. The major vegetation types of the study area are Himalayan moist temperate forest, sub-alpine forest and alpine scrub59. The Uttarkashi district forests are managed under three Forest Divisions viz., (i) Uttarkashi Forest Division (ii) Upper Yamuna Badkot Forest Division and (iii) Tons Forest Division) with two Protected Areas (PAs) (i) Gangotri National Park and (ii) Govind Pashu Vihar National Park. The forested habitats of the study landscape are home to top conservation priority species, including Asiatic Black bear (Ursus thibetanus), Musk deer (Moschus spp.), Common leopard (Panthera pardus), Himalayan brown bear (Ursus arctos isabellinus) and Western Tragopan (Tragopan melanocephalus), Himalayan monal (Lophophorus impejanus). The study was conducted after a study permit issued by the Chief Wildlife Warden, Forest Department, Uttarakhand government, vide letter no. 848/5-6 dated 31/08/2019, we have not handled the species for doing research. Instead, remote camera traps have been used for collecting the data with the permission of the Chief Wildlife Warden, Government of Uttarakhand. Further, informed consent was taken before interviewing the local communities. The data was collected according to the institutional guidelines and approved by the Research Advisory and Monitoring Committee of the Zoological Survey of India.Figure 3Map of the study area Uttarkashi, Uttarakhand. ArcGIS 10.6 (ESRI, Redlands, CA) was used to create the map. (Map created using ArcGIS 10.6; http://www.esri.com).Full size imageSampling protocolThe basic sampling protocol and assumptions for multi-species occupancy modelling are identical to the single-species case7. Briefly, a set of 62 intensive sites, were randomly selected, and each site i was surveyed j times. During each survey, detection/non-detection of S focal species was recorded. Additionally, direct or indirect evidences of species presence from the different areas were also recorded.Data collectionThe complete study area was divided into 10 × 10 km grids, consisting of n = 60 grids. Based on the reconnaissance survey, out of these 60 grids, we selected 25 girds that were accessible to conduct the survey and have the species presence. Further, these grids were divided into 2 × 2 km grids to maximize our effort so that all logistically accessible grids could be covered, and we conducted intensive sampling in N = 62 grids after excluding the grids with human settlements. T The field surveys were conducted during 2018–2019, and a team of researchers systematically visited selected grids to collect data on the detection/non-detection of these ungulates. A total of 62 camera traps were deployed in selected grids, and 650 km were traversed, accounting for N = 54 trails in these sampled grids. These camera traps were visited once in every fifteen days for replacing the batteries as well as documenting the presence of the species through the sign surveys. The ultra-compact SPYPOINT FORCE-11D trail camera (SPYPOINT, GG Telecom, Canada, QC) and Browning trail camera (Defender 850, 20 MP, Prometheus Group, LLC Birmingham, Alabama, https://browningtrailcameras.com) camera traps were used to detect the presence/absence of ungulate species. The cameras were mounted 40–60 cm above ground on natural trails without lures.Data explorationWhile deploying camera traps, we also noted habitat variables through on-site observation such as distance to the village and human disturbance. We tested site covariates for collinearity and discarded one of a pair if the Pearson’s correlation was greater than 0.760. Hence, we assumed each of the site covariates could influence the occupancy and detectability of these ungulates.CovariatesWe hypothesized that habitat variables may influence these ungulates’ occupancy and detection probability. A total of 21 variables were extracted either from the field or using the ArcGIS v. 10.6 software (ESRI, Redlands, CA), and only 14 were retained after collinearity testing60 (Table 3). These covariates were classified into the following categories (Topographic variables, Habitat variables and anthropogenic variables). The topographic variables (elevation, slope and aspect) were generated using 30× resolution SRTM (Shuttle Radar Topography Mission) image downloaded from EarthExplorer (https://earthexplorer.usgs.gov/). The habitat/ land cover classification was carried out using Landsat 8 satellite imagery (Spatial resolution = 30 m) downloaded from Global Land Cover Facility by following the methodology suggested by61 using the ArcGIS v. 10.6 software (ESRI, Redlands, CA). The study area was classified into nine Land use/land cover (LULC) classes viz., West Himalayan Sub-alpine birch/fir Forest (FT 188), West Himalayan upper oak/fir forest (FT 162), West Himalayan Dry juniper forest (FT 180), Ban oak forest (FT 152), Moist Deodar Forest (FT 155), Western mixed coniferous forest (FT 156), Moist temperate Deciduous Forest (FT 157) which were used for further analysis considering their importance to species ecology and behavior60. The values for all the covariates were extracted at 30 m resolution, and a single value per site was obtained by averaging all the pixel values within each sampling site (camera trap locations).Table 3 Habitat variables used for multi species occupancy analysis of three ungulate species in Uttarkashi, Uttarakhand.Full size tableOccupancy modelling frameworkWe used multi-species occupancy modelling62 of barking deer, goral and sambar to estimate the probability of the species (s) occurred within the area (i) sampled during our survey period (j), for accounting the imperfect detection of the species8. Distinguishing the true presence/absence of a species from detection/non-detection (i.e., species present and captured or species present but not captured) requires spatially or temporally replicated data. We used camera stations to record the presence/absence of species along with sign survey in all the studied grids. The camera traps were placed along the trail/transects in the studied grids hence each grid needs to be visited once in every fifteen days to check the camera traps as well as to document the presence of the studied species. Therefore, we treated 15 trap nights as one sampling occasion at a particular camera station resulting in ~ 7 sampling occasions per camera station.Our aim was to record the presence/ absence of the species at a particular gird hence we incorporated sign survey data if the species was not detected in camera station but recorded through sign survey. We pooled the presence/absence data in a single sheet of each species following6 and fitted occupancy and detectability models using programme Mark63,64. We model the species (s) presence (ysij = 1) and absence (ysij = 0) at site i during survey j, and the sampling protocol was identical to single species case65, where the Bernoulli random variable was conditional on the presence of species s (Zs = 1) following6$${text{y}}_{sij} sim {text{ Bernoulli}}left( {{text{p}}_{sij} {text{z}}_{si} } right),$$
    where Psij represents the probability of detecting species S during replicate survey j at site i and Zsi = presence or absence of species s at site i.Furthermore, we model the latent occupancy state of species s at site i as a multivariate Bernoulli random variable:$${text{Z}}_{i} sim {text{MVB}}left( {uppsi _{i} } right)$$
    where Zi = {Z1i, Z2i….., ZSi} is an S-dimensional vector of 1’s and 0’s denoting the latent occupancy state of all S species and (ψi) is a 2S-dimensional vector denoting the probability of all possible sequences of 1’s and 0’s Zi can attain such that ∑ ψi = 1 with corresponding probability mass function (PMF) adopted from6,64.$$fleft( {{text{Z}}_{i} } right) = {text{ exp}}left( {left( {{text{Z}}_{i} {text{log}}(uppsi_{{text{i}}} {1}/uppsi_{{text{i}}} 0} right) , + {text{ log}}left( {uppsi_{{text{i}}} 0} right)} right).$$The quantity f = log (ψi1/ψi0), is the log odds species S occupies a site often referred to as a ‘natural parameter’.Since we are modeling three ungulate species (S = 3), 2S = 23 the possible encounter histories included in the dataset were eight, if neither of the two species were detected the value of ‘00’ was assigned; similarly ‘01’ indicates detection of species 1; ‘02’ indicates detection of species 2; ‘03’ indicates detection of both the species; ‘04’ indicates detection of species 3; ‘05’ indicates detection of species 1 and species 3; ‘06’ indicates detection of species 2 and species 3 and ‘07’ indicates detection of all the three species. We modelled constant occupancy and detection probability for each of the three species. Hence, we specified 6 f and p parameters, an intercept (β) for each of one-way f parameter and detection parameter p following64.$$f_{{1}}=upbeta_{{{1},}} ;;{text{p}}=upbeta_{{4}}$$$$f_{{2}} = upbeta_{{{2},}} ;{text{p }} = , upbeta 5$$$$f_{{3}} = , upbeta_{{{3},}}; {text{p }} = , upbeta_{{6}}$$We fit a set of models including the detection probability as a constant, p(.), and variable function to occupancy ψ(covariate) for site-specific covariates and models include occupancy as constant ψ(.) and variable function of the detection p(covariates) for the respective site covariates.As we have assumed the independence among all three species, the model shows marginal occupancy probabilities of species 1, species 2 and species 3 varies as a function of environmental variables. We incorporated site-level characteristics affecting species-specific occurrence (f1: occupancy of species 1, f2: occupancy of species 2, & f3: occupancy of species 3) and detection probabilities using a generalized linear modelling approach42. This requires 9 parameters: an intercept (β1, β3, β5) and slope (β2, β4, β6) coefficient for each 1-way f parameter f1, f2, f3 and an intercept parameter for each detection parameter (β7, β8, β9). Below mentioned is the model for 1-way f parameters.$$f_{{1}} = , upbeta_{{{1 } + }} upbeta_{{2}} left( {{text{Covariate}}} right),;;{text{ p }} = , upbeta_{{7}}$$$$f_{{2}} = , upbeta_{{{3 } + }} upbeta_{{4}} left( {{text{Covariate}}} right),;;{text{ p}} = , upbeta_{{8}}$$$$f_{{3}} = , upbeta_{{5}} + , upbeta_{{6}} left( {{text{Covariate}}} right),;;{text{ p }} = , upbeta_{{9}} .$$All covariates were standardized before model fitting. We fitted the most complex model to each species and considered all possible combinations of covariates using the logit link function. Our rationale for including these variables in the occupancy and detectability component of the model was that we expected these variables to influence the occupancy and detectability of the study species.Since multi-species occupancy simultaneously model environmental variables, & interspecific interactions. Further it also allows to understand the influence of environmental variables on one species occupancy, in the presence or absence of other sympatric species64. Hence, we also modeled two species occur together as a function of covariates. We examined how the variables of each camera site influenced the pair-wise interaction of the three ungulate species. This model assumes that the conditional probability of one species varies in the presence or absence of other species. We assumed f123: co-occurrence of species 1, species 2 & species 3 = 0, hence we did not include higher-order interactions in any of our models, we assumed the conditional probability of 3 species occurred together was purely a function of species-specific (f1, f2, f3) and pair-wise interaction (f12: co-occurrence of species1 & species 2, f13: co-occurrence of species 1 & species 3, f23: co-occurrence of species 2 & species 3) parameters. We modeled pair-wise interaction of species varies as a function of environmental variables keeping detection probability constant. Hence, we specified 15 f and p parameters, an intercept and slope coefficient for each of the one-way (f1, f2, f3) and the two-way f parameters (f12, f13, and f23); as well as an intercept parameter for each of the detection models. The model equation below implies for 2-way f parameters:$$f_{{{12}}} = , upbeta_{{{7 } + }} upbeta_{{8}} left( {{text{Covariate}}} right),;;{text{ p }} = , upbeta_{{{13}}}$$$$f_{{{13}}} = , upbeta_{{{9 } + }} upbeta_{{{1}0}} left( {{text{Covariate}}} right),;;{text{ p }} = , upbeta_{{{14}}}$$$$f_{{{23}}} = , upbeta_{{{11 } + }} upbeta_{{{12}}} left( {{text{Covariate}}} right),;;{text{ p }} = , upbeta_{{{15}}} .$$We also fitted models including co-occurrence and detection probability of a species varies as a function of environmental variables. Hence, we specified 18 f and p parameters, an intercept and slope coefficient for each of one-way (f1, f2, f3) and two-way f parameters (f12, f13, f23); and an intercept as well as the slope parameters for each of the detection models. The model equation below implies for 2-way f parameters:$$f_{{{12}}} = , upbeta_{{{7 } + }} upbeta_{{8}} left( {{text{Covariate}}} right),{text{ p }} = , upbeta_{{{13 } + }} upbeta_{{{14}}} left( {{text{covariate}}} right)$$$$f_{{{13}}} = , upbeta_{{{9 } + }} upbeta_{{{1}0}} left( {{text{Covariate}}} right),{text{ p }} = , upbeta_{{{15}}} + , upbeta_{{{16}}} left( {{text{covariate}}} right)$$$$f_{{{23}}} = , upbeta_{{{11 } + }} upbeta_{{{12}}} left( {{text{Covariate}}} right),{text{ p }} = , upbeta_{{{17}}} + , upbeta_{{{18}}} left( {{text{covariate}}} right)$$A total of 38 models were run to test the influence of environmental variables on occupancy and detection probability of species-specific (f1, f2, f3) and pair-wise interaction of the three ungulate species. The best-supported model was identified by selecting the model with the lowest AICc value and highest model weights66, where higher model weights indicate a better fit of the model to the data. Second-Order Information Criterion (AICc)67 values were used to rank the occupancy models, and all the models whose ΔAICc  More

  • in

    Citizen science plant observations encode global trait patterns

    Sakschewski, B. et al. Leaf and stem economics spectra drive diversity of functional plant traits in a dynamic global vegetation model. Glob. Change Biol. 21, 2711–2725 (2015).Article 

    Google Scholar 
    Berzaghi, F. et al. Towards a new generation of trait-flexible vegetation models. Trends Ecol. Evol. 35, 191–205 (2020).Article 
    PubMed 

    Google Scholar 
    Bruelheide, H. et al. Global trait–environment relationships of plant communities. Nat. Ecol. Evol. 2, 1906–1917 (2018).Article 
    PubMed 

    Google Scholar 
    Joswig, J. S. et al. Climatic and soil factors explain the two-dimensional spectrum of global plant trait variation. Nat. Ecol. Evol. 6, 36–50 (2021).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    van Bodegom, P. M., Douma, J. C. & Verheijen, L. M. A fully traits-based approach to modeling global vegetation distribution. Proc. Natl Acad. Sci. USA 111, 13733–13738 (2014).PubMed Central 

    Google Scholar 
    Moreno Martínez, A. et al. A methodology to derive global maps of leaf traits using remote sensing and climate data. Remote Sens. Environ. 218, 69–88 (2018).Article 

    Google Scholar 
    Pérez-Harguindeguy, N. et al. New handbook for standardized measurment of plant functional traits worldwide. Aust. J. Bot. 23, 167–234 (2013).Article 

    Google Scholar 
    Kattge, J. et al. TRY—a global database of plant traits. Glob. Change Biol. 17, 2905–2935 (2011).Article 

    Google Scholar 
    Kattge, J. et al. TRY plant trait database-enhanced coverage and open access. Glob. Change Biol. 26, 119–188 (2020).Article 

    Google Scholar 
    Jetz, W. et al. Monitoring plant functional diversity from space. Nat. Plants 2, 16024 (2016).Article 
    PubMed 

    Google Scholar 
    Butler, E. E. et al. Mapping local and global variability in plant trait distributions. Proc. Natl Acad. Sci. USA 114, E10937–E10946 (2017).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Boonman, C. C. et al. Assessing the reliability of predicted plant trait distributions at the global scale. Glob. Ecol. Biogeogr. 29, 1034–1051 (2020).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Madani, N. et al. Future global productivity will be affected by plant trait response to climate. Sci. Rep. 8, 2870 (2018).Vallicrosa, H. et al. Global distribution and drivers of forest biome foliar nitrogen to phosphorus ratios (N:P). Glob. Ecol. Biogeogr. 31, 861–871 (2022).Article 

    Google Scholar 
    Meyer, H. & Pebesma, E. Predicting into unknown space? Estimating the area of applicability of spatial prediction models. Methods Ecol. Evol. 12, 1620–1633 (2021).Article 

    Google Scholar 
    Schiller, C. et al. Deep learning and citizen science enable automated plant trait predictions from photographs. Sci. Rep. 11, 16395 (2021).Aguirre-Gutiérrez, J. et al. Pantropical modelling of canopy functional traits using sentinel-2 remote sensing data. Remote Sens. Environ. 252, 112–122 (2021).Article 

    Google Scholar 
    Homolova, L. et al. Review of optical-based remote sensing for plant trait mapping. Ecol. Complex. 15, 1–16 (2013).Article 

    Google Scholar 
    Van Cleemput, E. et al. The functional characterization of grass-and-shrubland ecosystems using hyperspectral remote sensing: trends, accuracy and moderating variables. Remote Sens. Environ. 209, 747–763 (2018).Article 

    Google Scholar 
    Kattenborn, T., Fassnacht, F. E. & Schmidtlein, S. Differentiating plant functional types using reflectance: which traits make the difference? Remote Sens. Ecol. Conserv. 5, 5–19 (2019).Article 

    Google Scholar 
    Hauser, L. T. et al. Explaining discrepancies between spectral and in-situ plant diversity in multispectral satellite earth observation. Remote Sens. Environ. 265, 112684 (2021).Article 

    Google Scholar 
    Wäldchen, J. & Mäder, P. Plant species identification using computer vision techniques: a systematic literature review. Arch. Comput. Methods Eng. 25, 507–543 (2018).Article 
    PubMed 

    Google Scholar 
    Jones, H. G. What plant is that? Tests of automated image recognition apps for plant identification on plants from the British flora. AoB Plants 12, plaa052 (2020).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Hampton, S. E. et al. Big data and the future of ecology. Front. Ecol. Environ. 11, 156–162 (2013).Article 

    Google Scholar 
    WÜest, R. O. et al. Macroecology in the age of big data—where to go from here? J. Biogeogr. 47, 1–12 (2020).Article 

    Google Scholar 
    Mäder, P. et al. The Flora Incognita app—interactive plant species identification. Methods Ecol. Evol. 12, 1335–1342 (2021).Article 

    Google Scholar 
    Di Cecco, G. J. et al. Observing the observers: how participants contribute data to iNaturalist and implications for biodiversity science. BioScience 71, 1179–1188 (2021).Article 

    Google Scholar 
    Mahecha, M. D. et al. Crowd-sourced plant occurrence data provide a reliable description of macroecological gradients. Ecography 44, 1131–1142 (2021).Article 

    Google Scholar 
    Botella, C. et al. Jointly estimating spatial sampling effort and habitat suitability for multiple species from opportunistic presence-only data. Methods Ecol. Evol. 12, 933–945 (2021).Article 

    Google Scholar 
    iNaturalist Research-Grade Observations (GBIF, accessed 5 January 2022); https://www.gbif.org/dataset/50c9509d-22c7-4a22-a47d-8c48425ef4a7Callaghan, C. T. et al. Three frontiers for the future of biodiversity research using citizen science data. BioScience 71, 55–63 (2020).
    Google Scholar 
    Dickinson, J. L., Zuckerberg, B. & Bonter, D. N. Citizen science as an ecological research tool: challenges and benefits. Ann. Rev. Ecol. Evol. Syst. 41, 149–172 (2010).Article 

    Google Scholar 
    Kosmala, M. et al. Assessing data quality in citizen science. Front. Ecol. Environ. 14, 551–560 (2016).Article 

    Google Scholar 
    Boakes, E. H. et al. Patterns of contribution to citizen science biodiversity projects increase understanding of volunteers’ recording behaviour. Sci. Rep. 6, 33051 (2016).Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Bowler, D.E. et al. Temporal trends in the spatial bias of species occurrence records. Ecography 2022, e06219 (2022). https://doi.org/10.1111/ecog.06219GBIF Occurrence Download (GBIF, 4 January 2022); https://doi.org/10.15468/dl.34tjreBruelheide, H. et al. sPlot—a new tool for global vegetation analyses. journal of vegetation science. J. Veg. Sci. 30, 161–186 (2019).Article 

    Google Scholar 
    Sabatini, F. et al. sPlotOpen—an environmentally balanced, open access, global dataset of vegetation plots. Glob. Ecol. Biogeogr. 30, 1740–1764 (2021).Article 

    Google Scholar 
    Whittaker, R.H. et al. Communities and Ecosystems (Macmillan/Collier Macmillan, 1970).Olson, D. M. et al. Terrestrial ecoregions of the world: a new map of life on Earth: a new global map of terrestrial ecoregions provides an innovative tool for conserving biodiversity. BioScience 51, 933–938 (2001).Article 

    Google Scholar 
    Joswig, J., Wirth, C. & Schuman, M. Climatic and soil factors explain the two-dimensional spectrum of global plant trait variation. Nat. Ecol. Evol. 6, 36–50 (2022).Article 
    PubMed 

    Google Scholar 
    Díaz, S. et al. The global spectrum of plant form and function. Nature 529, 167–171 (2016).Article 
    PubMed 

    Google Scholar 
    Ploton, P. et al. Spatial validation reveals poor predictive performance of large-scale ecological mapping models. Nat. Commun. 11, 4540 (2020).Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Meyer, H. & Pebesma, E. Spatial validation reveals poor predictive performance of large-scale ecological mapping models. Methods Ecol. Evol. 12, 1620–1633 (2021).Article 

    Google Scholar 
    Schrodt, F. et al. Bhpmf—a hierarchical Bayesian approach to gap filling and trait prediction for macroecology and functional biogeography. Glob. Ecol. Biogeogr. 24, 1510–1521 (2015).Article 

    Google Scholar 
    Kuppler, J. et al. Global gradients in intraspecific variation in vegetative and floral traits are partially associated with climate and species richness. Glob. Ecol. Biogeogr. 29, 992–1007 (2020).Article 

    Google Scholar 
    Scheiter, S., Langan, L. & Higgins, S. I. Next-generation dynamic global vegetation models: learning from community ecology. New Phytol. 198, 957–969 (2013).Article 
    PubMed 

    Google Scholar 
    Taubert, F. et al. Confronting an individual-based simulation model with empirical community patterns of grasslands. PLoS ONE 15, e0236546 (2020).Roger, E. & Klistorner, S. (2016) Bioblitzes help science communicators engage local communities in environmental research. J. Sci. Commun. https://doi.org/10.22323/2.15030206 (2016).Legendre, P. & Legendre, L. Numerical Ecology 3rd edn (Elsevier, 2012).Warton, D. I. et al. Smatr 3—an R package for estimation and inference about allometric lines. Methods Ecol Evol 3, 257–259 (2012).Article 

    Google Scholar 
    Wolf, S. et al. iNaturalist_traits: iNaturalist trait maps version 1 (January 5, 2022) Zenodo https://doi.org/10.5281/zenodo.6671891 (2022). More

  • in

    Assessing Müllerian mimicry in North American bumble bees using human perception

    Bates, H. W. XXXII. Contributions to an insect fauna of the Amazon Valley. Lepidoptera: Heliconidæ. Trans. Linn. Soc. Lond 23, 495–566 (1862).Article 

    Google Scholar 
    Müller, F. Ituna and thyridia: A remarkable case of mimicry in butterflies. Trans. Entomol. Soc. Lond. 1879, 20–29 (1879).
    Google Scholar 
    Baxter, S. W. et al. Convergent evolution in the genetic basis of Müllerian mimicry in Heliconius butterflies. Genetics 180, 1567–1577 (2008).Article 
    CAS 

    Google Scholar 
    Sheppard, P. M., Turner, J. R. G., Brown, K., Benson, W. & Singer, M. Genetics and the evolution of Muellerian mimicry in Heliconius butterflies. Philos. Trans R. Soc. Lond. B, Biol. Sci. 308, 433–610 (1985).Article 
    ADS 

    Google Scholar 
    Mallet, J. & Gilbert, L. E. Jr. Why are there so many mimicry rings? Correlations between habitat, behaviour and mimicry in Heliconius butterflies. Biol. J. Lin. Soc. 55, 159–180 (1995).
    Google Scholar 
    Brower, A. V. Parallel race formation and the evolution of mimicry in Heliconius butterflies: A phylogenetic hypothesis from mitochondrial DNA sequences. Evolution 50, 195–221 (1996).Article 
    CAS 

    Google Scholar 
    Wilson, J. S. et al. North American velvet ants form one of the world’s largest known Müllerian mimicry complexes. Curr. Biol. 25, R704–R706. https://doi.org/10.1016/j.cub.2015.06.053 (2015).Article 
    CAS 
    PubMed 

    Google Scholar 
    Wilson, J. S., Williams, K. A., Forister, M. L., Von Dohlen, C. D. & Pitts, J. P. Repeated evolution in overlapping mimicry rings among North American velvet ants. Nat. Commun. 3, 1272 (2012).Article 
    ADS 

    Google Scholar 
    Wilson, J. S., Pan, A. D., Limb, E. S. & Williams, K. A. Comparison of African and North American velvet ant mimicry complexes: Another example of Africa as the ‘odd man out’. PLoS ONE 13, e0189482. https://doi.org/10.1371/journal.pone.0189482 (2018).Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Plowright, R. & Owen, R. E. The evolutionary significance of bumble bee color patterns: A mimetic interpretation. Evolution 34, 622–637 (1980).Article 
    CAS 

    Google Scholar 
    Williams, P. The distribution of bumblebee colour patterns worldwide: Possible significance for thermoregulation, crypsis, and warning mimicry. Biol. J. Lin. Soc. 92, 97–118 (2007).Article 

    Google Scholar 
    Hines, H. M. & Williams, P. H. Mimetic colour pattern evolution in the highly polymorphic Bombus trifasciatus (Hymenoptera: Apidae) species complex and its comimics. Zool. J. Linn. Soc. 166, 805–826 (2012).Article 

    Google Scholar 
    Koch, J. B., Rodriguez, J., Pitts, J. P. & Strange, J. P. Phylogeny and population genetic analyses reveals cryptic speciation in the Bombus fervidus species complex (Hymenoptera: Apidae). PLoS ONE 13, e0207080 (2018).Article 

    Google Scholar 
    Ezray, B. D., Wham, D. C., Hill, C. E. & Hines, H. M. Unsupervised machine learning reveals mimicry complexes in bumblebees occur along a perceptual continuum. Proc. R. Soc. B 286, 20191501 (2019).Article 

    Google Scholar 
    Bateson, W. The alleged “Aggressive Mimicry” of volucellæ. Nature 46, 585 (1892).Article 
    ADS 

    Google Scholar 
    Poulton, E. B. The volucellœ as alleged examples of variation “almost unique among animals”. Nature 47, 126 (1892).Article 
    ADS 

    Google Scholar 
    Cockerell, T. D. New social bees. Psyche A J. Entomol. 24, 120–128 (1917).Article 

    Google Scholar 
    Koch, J., Strange, J. & Williams, P. In: Bumble bees of the western United States (US Forest Service, San Francisco California, 2012).
    Google Scholar 
    Williams, P. H., Thorp, R. W., Richardson, L. L. & Colla, S. R. In: Bumble bees of North America: An identification guide Vol. 87 (Princeton University Press, Princeton, 2014).
    Google Scholar 
    Ruxton, G. D., Franks, D. W., Balogh, A. C. & Leimar, O. Evolutionary implications of the form of predator generalization for aposematic signals and mimicry in prey. Evol Int. J. Org. Evol. 62, 2913–2921 (2008).Article 

    Google Scholar 
    Rowe, C., Lindström, L. & Lyytinen, A. The importance of pattern similarity between Müllerian mimics in predator avoidance learning. Proc. R. Soc. Lond. Ser. B Biol. Sci. 271, 407–413 (2004).Article 

    Google Scholar 
    Beatty, C. D., Beirinckx, K. & Sherratt, T. N. The evolution of Müllerian mimicry in multispecies communities. Nature 431, 63 (2004).Article 
    ADS 
    CAS 

    Google Scholar 
    Chittka, L. & Osorio, D. Cognitive dimensions of predator responses to imperfect mimicry. PLoS Biol. 5, e339 (2007).Article 

    Google Scholar 
    Dittrigh, W., Gilbert, F., Green, P., McGregor, P. & Grewcock, D. Imperfect mimicry: A pigeon’s perspective. Proc. R. Soc. Lond. Ser. B Biol. Sci. 251, 195–200 (1993).Article 
    ADS 

    Google Scholar 
    Sherratt, T. N., Whissell, E., Webster, R. & Kikuchi, D. W. Hierarchical overshadowing of stimuli and its role in mimicry evolution. Anim. Behav. 108, 73–79 (2015).Article 

    Google Scholar 
    Beatty, C. D., Bain, R. S. & Sherratt, T. N. The evolution of aggregation in profitable and unprofitable prey. Anim. Behav. 70, 199–208 (2005).Article 

    Google Scholar 
    Kazemi, B., Gamberale-Stille, G., Tullberg, B. S. & Leimar, O. Stimulus salience as an explanation for imperfect mimicry. Curr. Biol. 24, 965–969 (2014).Article 
    CAS 

    Google Scholar 
    Kikuchi, D. W., Dornhaus, A., Gopeechund, V. & Sherratt, T. N. Signal categorization by foraging animals depends on ecological diversity. Elife. 8, e43965 (2019).Article 

    Google Scholar 
    Rapti, Z., Duennes, M. A. & Cameron, S. A. Defining the colour pattern phenotype in bumble bees (Bombus): A new model for evo devo. Biol. J. Lin. Soc. 113, 384–404 (2014).Article 

    Google Scholar 
    Wilson, J. S., Sidwell, J. S., Forister, M. L., Williams, K. A. & Pitts, J. P. Thistledown velvet ants in the desert mimicry ring and the evolution of white coloration: Müllerian mimicry, camouflage and thermal ecology. Biol. Lett. 16, 20200242 (2020).Article 

    Google Scholar 
    Ascher, J. & Pickering, J. Discover Life bee species guide and world checklist (Hymenoptera: Apoidea: Anthophila) (2019).iNaturalist. Available from https://www.inaturalist.org. Accessed [2022].Bombus Latreille, 1802 in GBIF Secretariat (2021). GBIF Backbone Taxonomy. Checklist dataset https://doi.org/10.15468/39omei accessed via GBIF.org on 2021-12-03. More

  • in

    Chemical forms of cadmium in soil and its distribution in French marigold sub-cells in response to chelator GLDA

    Sarwar, N. et al. Phytoremediation strategies for soils contaminated with heavy metals: Modifications and future perspectives. Chemosphere 171, 710–721 (2017).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Lin, H. M. et al. Cadmium-stress mitigation through gene expression of rice and silicon addition. Plant Growth Regul.: Int. J. Nat. Synthetic Regul. 81(1), 91–101 (2017).Article 
    CAS 

    Google Scholar 
    Pan, F. S. et al. Enhanced Cd extraction of oilseed rape (Brassica napus) by plant growth-promoting bacteria isolated from Cd hyperaccumulator Sedum alfredii Hance. Int. J. Phytorem. 19(1/6), 281–289 (2017).Article 
    CAS 

    Google Scholar 
    Puangprasert, S. & Prueksasit, T. Health risk assessment of airborne Cd, Cu, Ni and Pb for electronic waste dismantling workers in Buriram Province, Thailand. J. Environ. Manag. 252, 109601 (2019).Article 
    CAS 

    Google Scholar 
    Tipu, M. I. et al. Growth and physiology of maize (Zea mays L.) in a nickel-contaminated soil and phytoremediation efficiency using EDTA. J. Plant Growth Regul. 40(2), 774–786 (2021).Article 
    CAS 

    Google Scholar 
    Chaturvedi, N., Dhal, N. K. & Patra, H. K. EDTA and citric acid-mediated phytoextraction of heavy metals from iron ore tailings using Andrographis paniculata: A comparative study. Int. J. Min. Reclam. Environ. 29(1), 33–46 (2015).Article 
    CAS 

    Google Scholar 
    Wang, G. Y. et al. Heavy metal removal by GLDA washing: Optimization, redistribution, recycling, and changes in soil fertility. Sci. Total Environ. 569–570, 557–568 (2016).Article 
    ADS 
    PubMed 

    Google Scholar 
    Kołodyńska, D. Cu(II), Zn(II), Co(II) and Pb(II) removal in the presence of the complexing agent of a new generation. Desalination 267(2–3), 175–183 (2011).Article 

    Google Scholar 
    Guo, X. F. et al. Mixed chelators of EDTA, GLDA, and citric acid as washing agent effectively remove Cd, Zn, Pb, and Cu from soils. J. Soils Sediments 18(2), 835–844 (2017).
    Google Scholar 
    Wang, X. et al. Subcellular distribution and chemical forms of cadmiun in Bechmeria nivea L. Gaud. Environ. Exp. Bot. 62(3), 389–395 (2008).Article 
    CAS 

    Google Scholar 
    Gallego, S. M. et al. Unravelling cadmium toxicity and tolerance in plants: Insight into regulatory mechanisms. Environ. Exp. Bot. 83, 33–46 (2012).Article 
    CAS 

    Google Scholar 
    Clemens, S., Aarts, M. G. M., Thomine, S. & Verbruggen, N. Plant science: The key to preventing slow cadmium poisoning. Trends Plant Sci. 18(2), 92–99 (2013).Article 
    CAS 
    PubMed 

    Google Scholar 
    Zhou, J. T. et al. Integration of cadmium accumulation, subcellular distribution, and physiological responses to understand cadmium tolerance in apple rootstocks. Front. Plant Sci. 8, 966 (2017).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Yang, L. P., Zhu, J., Wang, P., Lyu, D. G. & Li, H. F. Effect of Cd on growth, physiological response, Cd subcellular distribution and chemical forms of Koelreuteria paniculata. Ecotoxicol. Environ. Saf. 160, 10–18 (2018).Article 
    CAS 
    PubMed 

    Google Scholar 
    Wang, W. J., Zhang, M. Z. & Liu, J. N. Subcellular distribution and chemical forms of Cd in Bougainvillea spectabilis Willd. as an ornamental phytostabilizer: An integrated consideration. Int. J. Phytorem. 20(11), 1087–1095 (2017).Article 

    Google Scholar 
    Weigel, H. J. & Jäger, H. J. Subcellular distribution and chemical form of cadmium in bean plants. Plant Physiol. 65(3), 480–482 (1980).Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Khanna, K., Kohli, S. K., Ohri, P., Bhardwaj, R. & Ahmad, P. Agroecotoxicological aspect of Cd in soil–plant system: Uptake, translocation and amelioration strategies. Environ. Sci. Pollut. Res. 29, 30908–30934 (2022).Article 
    CAS 

    Google Scholar 
    Wei, Z. B., Chen, X. H., Wu, Q. T. & Tan, M. Biodegradable chelator GLDA induced remediation of heavy metal contaminated soil in Southeast Jingtian. Environ. Sci. 36(5), 1864–1869 (2015).CAS 

    Google Scholar 
    Wang, K., Liu, Y. H., Song, Z. G., Wang, D. & Qiu, W. W. Chelator complexes enhanced Amaranthus hypochondriacus L. phytoremediation efficiency in Cd-contaminated soils. Chemosphere 237, 124480 (2019).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Meng, N., Wang, M., Chen, L., Zheng, H. & Chen, S. B. Remediation effects of different herbaceous plants intercropping on Cd-contaminated soil. China Environ. Sci. 38(7), 2618–2624 (2018).CAS 

    Google Scholar 
    Jones, D. & Willett, V. Experimental evaluation of methods to quantify dissolved organic nitrogen (don) and dissolved organic carbon (doc) in soil. Soil Biol. Biochem. 38(5), 991–999 (2006).Article 
    CAS 

    Google Scholar 
    Su, F. L. et al. The distribution and enrichment characteristics of copper in soil and Phragmites australis of Liao River estuary wetland. Environ. Monit. Assess.: Int. J. 190(6), 1–9 (2018).Article 
    CAS 

    Google Scholar 
    Shahid, M., Dumat, C. & Khalid, S. Reviews of Environmental Contamination and Toxicology Vol. 241, 3–137 (Springer, 2016).
    Google Scholar 
    Yuliya, V. et al. Comparison of soil-to-root transfer and translocation coefficients of trace elements in vines of Chardonnay and Muscat white grown in the same vineyard. Sci. Hortic. 192, 89–96 (2015).Article 

    Google Scholar 
    Liu, Q. Q., Chen, Y. H., Shen, Z. G. & Zheng, L. Q. Roles of cell wall in plant heavy metal tolerance. Plant Physiol. J. 50(5), 605–611 (2014).
    Google Scholar 
    Zhen, S. et al. Foliar application of Zn reduces Cd accumulation in grains of late rice by regulating the antioxidant system, enhancing Cd chelation onto cell wall of leaves, and inhibiting Cd translocation in rice. Sci. Total Environ. 770, 145302 (2021).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Shi, Y. X. et al. Simulation of the absorption, migration and accumulation process of heavy metal elements in soil-crop system. Environ. Sci. 37(10), 3996–4003 (2016).
    Google Scholar 
    Yan, X. X. et al. Effect of foliar application of different manganese fertilizers on cadmium accumulation and subcellular distribution in pak choi. J. Agro Environ. Sci. 38(8), 1872–1881 (2019).
    Google Scholar 
    He, S., Wu, Q. & He, Z. Effect of DA-6 and EDTA alone or in combination on uptake, subcellular distribution and chemical form of Pb in Lolium perenne. Chemosphere 93(11), 2782–2788 (2013).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Li, C. C. et al. Integration of metal chemical forms and subcellular partitioning to understand metal toxicity in two lettuce (Lactuca sativa L.) cultivars. Plant Soil 384(1/2), 201–212 (2014).Article 
    CAS 

    Google Scholar 
    Li, D., He, T., Saleem, M. & He, G. Metalloprotein-specific or critical amino acid residues: Perspectives on plant-precise detoxification and recognition mechanisms under cadmium stress. Int. J. Mol. Sci. 23(3), 1734 (2022).Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Perriguey, J., Sterckeman, T. & Morel, J. L. Effect of rhizosphere and plantrelated factors on the cadmium uptake by maize(Zea mays L.). Environ. Exp. Bot. 63(1/3), 333–341 (2008).Article 
    CAS 

    Google Scholar 
    Dai, S. et al. Effects of biochar amendments on speciation and bioavailability of heavy metals in coal-mine-contaminated soil. Hum. Ecol. Risk Assess. Int. J. 24(7), 1887–1900 (2018).Article 
    CAS 

    Google Scholar 
    Hou, S., Zheng, N., Tang, L., Ji, X. F. & Li, Y. Y. Effect of soil pH and organic matter content on heavy metals availability in maize (Zea mays L.) rhizospheric soil of non-ferrous metals smelting area. Environ. Monit. Assess. 191(10), 634 (2019).Article 
    PubMed 

    Google Scholar 
    Wu, H. J. et al. Effects of Astragalus smicuson cadmium effectiveness in paddy soil and cadmium accumulation in rice plant. Chin. Agric. Sci. Bull. 33(16), 105–111 (2017).ADS 

    Google Scholar 
    Jin, P. K., Liu, K. J. & Wang, X. B. Conversion and utilization of slowly biodegradable organic matter. Chin. J. Environ. Eng. 10(5), 2168–2174 (2016).CAS 

    Google Scholar 
    Kopáček, J. et al. Factors affecting the leaching of dissolved organic carbon after tree dieback in an unmanaged European mountain forest. Environ. Sci. Technol. 52(11), 6291–6299 (2018).Article 
    ADS 
    PubMed 

    Google Scholar 
    Anwar, S. et al. Impact of chelator-induced phytoextraction of cadmium on yield and ionic uptake of maize. Int. J. Phytorem. 19(6), 505–513 (2017).Article 
    CAS 

    Google Scholar 
    Wu, J. M., Xi, M. & Kong, F. L. Review of researches on the factors influencing the dynamics of dissolved organic carbon in soils. Geol. Rev. 59(5), 953–961 (2013).CAS 

    Google Scholar 
    AkzoNobel. Dissolvine GL® Technichal Brochure 1–5 (AkzoNobel Amsterdam, 2010).
    Google Scholar 
    Beygi, M. & Jalali, M. Assessment of trace elements (Cd, Cu, Ni, Zn) fractionation and bioavailability in vineyard soils from the Hamedan, Iran. Geoderma 337, 1009–1020 (2019).Article 
    ADS 
    CAS 

    Google Scholar 
    Gul, I. et al. Comparative effectiveness of organic and inorganic amendments on cadmium bioavailability and uptake by Pelargonium hortorum. J. Soils Sediments 19(5), 2346–2356 (2019).Article 
    CAS 

    Google Scholar 
    Wang, H., Sun, L. N., Li, H. B. & Sun, T. Y. Effect of different chelators application on Cd accumulation in metal polluted soils by Beta vulgaris var. cicla L. Ecol. Environ. 17(6), 2249–2252 (2008).
    Google Scholar 
    Zhang, G. X. et al. Effects of biochars on the availability of heavy metals to ryegrass in an alkaline contaminated soil. Environ. Pollut. 218, 513–522 (2016).Article 
    CAS 
    PubMed 

    Google Scholar 
    Gu, M. H. et al. Effects of manganese application on the formation of manganese oxides and cadmium fixation in soil. Ecol. Environ. Sci. 229(2), 360–368 (2020).
    Google Scholar 
    Bradl, H. B. Adsorption of heavy metal ions on soils and soils constituents. J. Colloid Interface Sci. 277(1), 1–18 (2004).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar  More

  • in

    Protected area personnel and ranger numbers are insufficient to deliver global expectations

    Data collectionIn phase 1 (2017), we first circulated a comprehensive multi-language questionnaire and associated guidelines on protected area personnel numbers to major national protected area agencies, focusing on the 50 countries listed in the WDPA as having the most protected areas. The questionnaire requested information on personnel numbers, type of employers and management levels (from executive to skilled practical workers). Protected area personnel were defined as those spending at least 50% of their work time on protected area-related tasks. The questionnaire also requested information about job titles used for personnel equivalent to rangers. This phase produced usable data for 28 countries/territories.In phase 2 (2018 onwards), we conducted online searches for published data on protected area personnel numbers in the countries/territories not included in the questionnaire survey or where questionnaire responses were incomplete or unclear. The resulting information came from official organizational reports (10 countries/territories), published external studies, project documents and journal papers (35 countries/territories) and websites of protected area organizations or individual sites (9 countries/territories).In phase 3 (2018–2021), we directly requested personal contacts to locate or supply information from official sources both for the remaining countries/territories and to improve or verify data from phases 1 and 2. The minimum data requested were the overall number of protected area personnel, the number of those personnel that could be categorized as rangers, the terrestrial area of protected areas managed by the listed personnel and the source of the information. This phase contributed usable data for 68 countries and territories. Data for a further 17 countries/territories were assembled from multiple sources.The final dataset covered 176 countries/territories: 167 surveyed countries/territories and a further 9 countries/territories that have no WDPA-listed protected areas (Supplementary Table 1), with contributions from more than 150 individuals.Initial data processingTo assess and, where necessary, improve the reliability of data obtained in a wide range of formats and levels of detail and from multiple sources, we scored the data for each country/territory from 0 to 5 for each of four criteria—detail, accuracy, source and age of the data—with a maximum score of 20 (Supplementary Table 1 and Supplementary Fig. 1). For all low-scoring records (a score of less than 15), we sought more-reliable sources in later phases of the study, rejecting any final scores of less than 10.On reviewing the data, we excluded from the analysis protected areas identified in the WDPA as predominantly or entirely marine, Antarctica and countries/territories categorized in the WDPA as polar (Greenland, French Southern Territories, Bouvet Island, Heard Island and McDonald Islands, South Georgia and the South Sandwich Islands). These large, remote and/or largely uninhabited areas are likely to have quite different management models and scales of staffing from terrestrial protected areas (although marine protected areas are also widely understaffed11). For example, in 2012 the 972,000 km2 of Northeast Greenland Protected Area (categorized by the WDPA as polar) was only periodically visited by six two-person teams of naval personnel47, and the 2008 management plan of the 1.51 million km2 Papahānaumokuākea Marine National Monument (Hawai’i, USA) specifies just nine personnel, working in conjunction with several other agencies48. Data for one country were supplied by officials on the agreement that the country was not specifically identified in publications (the country is given the three-letter code ZZZ in relevant tables and figures).Because the format, completeness and level of detail of the data varied widely, from comprehensive personnel lists to single figures, we restricted our raw dataset to six variables that could be consistently extracted from data obtained for each country/territory:

    1.

    Total number of non-ranger personnel (if known)

    2.

    Total number of rangers (if known)

    3.

    Total number of protected area personnel (either the sum of 1 and 2 or provided as an undifferentiated total)

    4.

    Terrestrial area of protected areas covered by surveyed personnel (km2)

    5.

    Total terrestrial area of protected areas of the country/territory (km2)

    6.

    Year of the data

    We used the WDPA, official publications and websites to determine (or verify) the area of terrestrial protected areas covered by the personnel listed for each country/territory, using WDPA data if there were discrepancies. Total national terrestrial protected area coverage was taken from the WDPA, with the exception of Turkey, where the area officially reported to the WDPA is significantly less than the nationally published area.The raw data from the survey are shown in Supplementary Table 1.Candidate predictorsTo predict the number of rangers and non-rangers in countries and territories for which we had no data (Statistical analysis), we collected information on the following set of variables, hereafter referred to as candidate predictors:Location dataThe WGS84 latitude and longitude of the centroid of the largest land mass associated with each country/ territory (to obtain the polygons defining the land masses, we used the R package rnaturalearth version 0.1.0; https://github.com/ropensci/rnaturalearth)2020 data from the World Bank (https://data.worldbank.org/indicator)

    Area of the country/territory

    Population density: the mid-year population divided by land area

    Gross domestic product (GDP) in US dollars

    GDP per capita in US dollars (GDP divided by mid-year population)

    Growth rate of GDP

    The proportion of rural inhabitants

    The proportion of unemployed inhabitants

    The forested proportion of the country/territory

    2020 data for each country/territory from the WDPA (https://www.protectedplanet.net/)

    The total terrestrial area of WDPA-listed protected areas

    The proportion of the terrestrial area of all IUCN-categorized protected areas (Categories I–VI) that falls within protected areas in Category I or II

    The proportion of the terrestrial area of all IUCN-categorized protected areas (Categories I–VI) that falls within protected areas in Categories I–IV

    2020 data from the Yale Center for Environmental Law and Policy Environmental Performance Index (https://epi.yale.edu/)

    Environmental Performance Index (EPI): a composite index using 32 performance indicators across 11 categories

    Ecosystem Vitality Index (EVI): an indicator of how well countries preserve, protect and enhance ecosystems and the services they provide

    Species Protection Index (SPI): an indicator of the species-level ecological representativeness of each country’s/territory’s protected area network

    Not all this information was available for all countries/territories. Most of the missing data were for small territories that account for only a very small proportion of the total area of protected areas worldwide (Supplementary Table 2c).Statistical analysisOur primary objective was to estimate the total number of all personnel engaged in managing all the world’s WDPA-listed terrestrial protected areas and the number categorized as rangers. Our raw data collection yielded full, partial or no information on total personnel and ranger numbers for each country/territory (Supplementary Table 1 shows the completeness of all the data collected). Our first task, therefore, was (1) to impute the information for unsurveyed protected areas on the basis of information from surveyed protected areas within the same countries/territories and (2) to predict those numbers for countries/territories where no information was available on overall personnel numbers and/or ranger numbers on the basis of relationships we could establish between available information and candidate predictors in other countries/territories (Supplementary Table 7). A brief description of these two approaches follows, and full details on the analysis are provided in Supplementary Information.Data imputationFor countries/territories where we had obtained information about numbers of personnel and/or rangers for only some protected areas, our strategy was to populate the unsurveyed protected areas in proportion to the densities of personnel or rangers from the surveyed protected areas of the same countries/territories. For example, for Spain we obtained evidence that there are 619 rangers responsible for protected areas covering 44,328 km2, out of a national total protected area system covering 142,573 km2. To impute the number of rangers for the remaining 98,245 km2, we used the density of rangers in the surveyed area (one ranger per 44,328/619 = 71.6 km2) and applied that to the unsurveyed area, giving a total of 1,991 rangers (619 + (98,245/71.6)). This imputation assumes that unsurveyed areas are staffed at the same density as surveyed areas, whereas in reality the relative densities are likely to vary in unknown ways within different countries/territories. To study the sensitivity of our results to the assumed proportion, we repeated our analysis using the following proportions of the observed densities: 0, 0.25, 0.50, 0.75 and 1.00. This provided a range of personnel numbers from a minimum (based on a proportion of 0) to a presumed maximum (based on a proportion of 1.00). From the data obtained, it was not possible to calculate the actual proportions, but based on the experience of the practitioners in the author team, the unsurveyed areas are highly unlikely to be staffed at higher densities than surveyed areas and, on average, are very likely to be staffed at lower densities. After all, most survey respondents were national or subnational agencies responsible for protected areas subject to stronger formal requirements for protection and management and therefore likely to have larger workforces. Unsurveyed protected areas are more likely to be managed by local entities, with fewer resources, less-stringent management obligations and therefore fewer personnel. The range of proportions we considered to populate unsurveyed areas should therefore yield predictions encompassing the actual (unknown) numbers of rangers and non-rangers with a conservative margin of error. In the main text, we have reported the results of imputation assuming a proportion of 1, which is probably the most optimistic assessment of the current workforce in protected areas within the proportions of the observed densities considered. Results using lower proportions are shown in Extended Data Fig. 2 and Supplementary Tables 4 and 5.Data predictionOur imputation approach was not possible for countries/territories where (1) zero ranger or personnel data had been obtained and (2) specific data had not been obtained that allowed imputation either for rangers or for total personnel (where only total personnel numbers or only ranger numbers had been obtained). To predict the missing information, we used two different statistical approaches: linear mixed models (LMMs)49 and a general implementation of random forests, which we term RF/ETs because it encompasses both random forests sensu stricto (RFs)50 and a variant called extremely randomized trees (ETs)51. LMMs and RFs have been extensively discussed and reviewed in the literature49,52,53. We adopted these approaches because both have proved successful in producing accurate predictions for a wide range of applications and because both are well suited to our data since they both produce predictions from a set of predictors and allow for the consideration of spatial effects54,55. Furthermore, comparing predictions generated through very different methods informs us about the robustness of our results with respect to key statistical assumptions. LMMs come from the ‘data modelling culture’56 and belong to parametric statistics; RF/ETs come from the ‘algorithmic modelling culture’ and belong to non-parametric statistics.We followed the same workflow for both statistical approaches, comprising eight steps: (1) general data preparation; (2) preparation of initial training datasets; (3) selection of predictor variables and of the method used for handling spatial autocorrelation; (4) preparation of final training datasets; (5) fine tuning; (6) final training; (7) preparation of datasets for predictions and simulations; and (8) predictions and simulations (see Supplementary Information for details).Both approaches yielded very similar results with our data. We chose to present the LMM results in the main text, but we provide and compare the results obtained by both approaches in Supplementary Information.SoftwareWe performed all the data analyses using the free open-source statistical software R version 4.157. We used the R package spaMM version 3.9.13 to implement LMMs58 and the R package ranger version 0.13.1 to implement RF/ETs59. To reformat and plot the data, we used the Tidyverse suite of packages60. Details are provided in an R package we specifically developed so that findings presented in this paper can readily be reproduced (see Code availability). Using a workstation with an AMD Ryzen Threadripper 3990 × 64-core processor and 256 GB of RAM, our complete workflow ran in ~3,000 CPU hours.Estimation of required numbers and densities of personnelTo estimate the numbers of personnel and rangers required for effective management of existing protected areas, we referred to ref. 25. This estimates that the minimum budget needed to adequately manage the existing protected area system is US$67.6 billion per year and that current annual expenditure is US$24.3 billion. From these figures, we can calculate that resources invested in the current global system of protected areas are approximately 36% of what is required. We consulted data from https://ourworldindata.org to determine that the proportion of global public expenditure on employee compensation has remained between 21.01% and 23.33% in the years from 2006 to 2019. We obtained these figures from the ‘Government Spending’ section of the site, consulting the chart ‘Share of employee compensation in public spending, 2002 to 2019’ and selecting data for ‘World’. On the basis of this broadly constant proportion and the assumption that total employee compensation is an indicator of total employee numbers, we inferred that current numbers of protected area employees are also around 36% of what is required. We therefore multiplied our estimations of personnel and ranger numbers by 1/0.36 and recalculated the densities on this basis (current requirement = 1/0.36 × current estimate).To estimate staffing requirements for 30% global coverage of protected areas—the global target intended to be reached by 2030—we used the mean personnel and ranger densities calculated as being required at present to ‘populate’ a global area of terrestrial protected areas if increased from the percentage at the time of our study (15.7%) to 30% (current requirement × (0.300/0.157)).Economic calculationsWe based our calculations on published data from 202025, which estimate that expanding the protected areas to 30% would generate higher overall output (revenues) than non-expansion (an extra US$64–454 billion per year by 2050). This figure is only an indicative, partial estimate, generated for the purposes of comparison and to illustrate the substantial return on investment that protected area staff investments imply. Using these figures and our estimates of personnel requirements to ensure effective management of 30% coverage, we calculated the range of sums that each additional protected area staff member has the potential to generate (Supplementary Table 8). For clarity, we rounded these figures to the nearest hundred US dollars in the main text.Our estimates of the gross value added per worker in forestry and agriculture (sectors responsible for similar proportions of the world as protected areas) are included to provide a point of comparison for the figures showing the economic benefit generated per protected area personnel member (see the preceding). The data for the gross annual value of world agricultural production (US$3,550,231,736,000) and the number of workers employed in agriculture (343,527,711) come from the Food and Agriculture Organization of the United Nations30, providing an average gross value of annual agricultural production per worker of US$10,335. We adjusted these 2018 data to 2020 price levels using a deflator based on the US consumer price index (CPI) from the World Economic Outlook database61 (Supplementary Table 9). This ensures that all the economic value data we present are directly comparable for protected area, agricultural and forestry workers. We calculated the gross value of forest production per worker on the basis of direct contribution of forestry of more than US$539 billion to world GDP in 201162 and total forest-sector employment of 11.881 million full-time-equivalent jobs in 201032. These were the most up-to-date global estimates we could locate from credible sources that presented comparable estimates of forest-sector employment and contribution to GDP. This gives an average gross value of forest production per worker of US$45,367 per year. We used the same method as for agriculture to bring these figures to 2020 price levels (Supplementary Table 9). These figures are rounded to the nearest hundred US dollars in the main text. More