More stories

  • in

    Experimental analysis of roasted and raw turtle butchery and implications for early human cognition and behaviour

    AbstractChelonid exploitation – including tortoises and freshwater turtles – has been increasingly recognised as a significant element of Palaeolithic subsistence in the Mediterranean and Iberian Peninsula. This study offers an experimental assessment of fire’s role in processing these reptiles, contrasting raw and roasted specimens to evaluate impacts on butchery efficiency, surface modifications, skeletal representation and lithic use-wear. The roasting process markedly reduced disarticulation effort and time, irrespective of the operator’s experience. Cut marks and percussion traces were more frequent in raw-processed individuals, while burnt specimens displayed extensive thermal damage, particularly on carapace plates. However, Fourier-Transform Infrared Spectroscopy (FTIR) revealed limited diagnostic potential for low-intensity thermal exposure. Conversely, lithic tools used in processing exhibited macroscopic edge damage and minor polishes, paralleling wear patterns documented in the butchery of other small fauna. These results align with archaeological evidence from multiple Iberian and Mediterranean sites, suggesting a culturally structured practice of in-shell roasting and anatomical disarticulation. The finds highlight fire’s role in labour optimisation and knowledge transmission, supporting broader discussions on small game exploitation and cognitive planning in early human behaviour.

    Data availability

    The datasets generated during and/or analysed during the current study are available in the CORA repository and can be accessed through the following links https:/dataverse.csuc.cat/dataset.xhtml?persistentId = doi%3A10.34810%2Fdata2413 and https:/dataverse.csuc.cat/dataset.xhtml?persistentId = doi%3A10.34810%2Fdata2412 Our study does not involve human participants in the usual sense. All individuals who appear in the images are the authors of the study and no other identifiable persons or patient data are included. For this reason, we consider a formal informed-consent statement from study participants not applicable. All authors explicitly agree to the publication of these images and any associated information in an online open-access format.
    ReferencesBiton, R., Sharon, G., Oron, M., Steiner, T. & Rabinovich, R. Freshwater turtle or tortoise? The exploitation of testudines at the Mousterian site of Nahal mahanayeem outlet, hula valley, israel. J. Archaeol. Sci. Rep. 14, 409–419. https://doi.org/10.1016/j.jasrep.2017.05.058 (2017).
    Google Scholar 
    Blasco, R. et al. The earliest evidence for human consumption of tortoises in the european early pleistocene from sima del elefante, sierra de atapuerca, spain. J. Hum. Evol. 61, 503–509. https://doi.org/10.1016/j.jhevol.2011.06.002 (2011).
    Google Scholar 
    Boneta I. Chelonians in the Iberian Peninsula Archaeological Record: Approximation to its study from the ensemble of the Chalcolithic site of Camino de las Yeseras. Unpublished PhD thesis submitted to Universidad Autónoma de Madrid, (Spain, 2022).Martin, L., Edwards, Y. & Garrard, A. Broad spectrum or specialised activity? Birds and tortoises at the epipalaeolithic site of wadi jilat 22 in the eastern jordan steppe. Antiquity 87, 649–665. https://doi.org/10.1017/S0003598X00049371 (2013).
    Google Scholar 
    Morales, J. V. & Sanchis, A. The quaternary fossil record of the genus Testudo in the Iberian Peninsula. Archaeological implications and diachronic distribution in the western Mediterranean. J. Archaeol. Sci. 36, 1152–1162. https://doi.org/10.1016/j.jas.2008.12.019 (2009).
    Google Scholar 
    Moya, R., Sanchis, A. & Fernández-López, J. Freshwater turtles and mesolithic human subsistence: Taxonomic and taphonomic study of the assemblages from El Collado (Oliva, Eastern Iberian Peninsula). J. Archaeol. Sci. Rep. 66, 105234. https://doi.org/10.1016/j.jasrep.2025.105234 (2025).
    Google Scholar 
    Real, C. et al. Abrigo de la quebrada level IV (Valencia, Spain): Interpreting a middle palaeolithic palimpsest from a zooarchaeological and lithic perspective. J. Paleolit. Archaeol. 3, 187–224. https://doi.org/10.1007/s41982-018-0012-z (2020).
    Google Scholar 
    Real, C. et al. Estudio de la fauna del nivel IV del Abrigo de la Quebrada y su aportación al conocimiento de la economía y el comportamiento humano en el Paleolítico medio de la vertiente Mediterránea Ibérica. Spal 28(2), 17–49. https://doi.org/10.12795/spal.2019.i28.13 (2019).
    Google Scholar 
    Sanchis, A., Morales, J.V., Pérez, L.J., Hernández, C.M., Galván, B. La tortuga mediterránea en yacimientos valencianos del Paleolítico medio: Distribución, origen de las acumulaciones y nuevos datos procedentes del Abric del Pastor (Alcoi, Alacant). In Preses petites i grups humans en el passat. II Jornades d’arqueozologia del Museu de Prehistòria de València. València: Museu de Prehistòria de València, pp. 97-120 (2015).Blasco, R. et al. Tortoises as a dietary supplement: A view from the middle pleistocene site of qesem cave, israel. Quatern. Sci. Rev. 133, 165–182. https://doi.org/10.1016/j.quascirev.2015.12.006 (2016).
    Google Scholar 
    Nabais, M. & Zilhão, J. The consumption of tortoise among Last Interglacial Iberian Neanderthals. Quatern. Sci. Rev. 217, 225–246. https://doi.org/10.1016/j.quascirev.2019.03.024 (2019).
    Google Scholar 
    Speth, J. D. & Tchernov, E. Middle Paleolithic Tortoise Use at Kebara Cave (Israel). J. Archaeol. Sci. 29, 471–483. https://doi.org/10.1006/jasc.2001.0740 (2002).
    Google Scholar 
    Stiner M. The Faunas of the Hayonim Cave Israel A 20.000-Year Record of Paleolithic Diet, Demography and Society. In Peabody Museum of Archaeology and Ethnology. (Harvard University, Cambridge, Massachusetts, 2005)Munro, N. D. & Grosman, L. Early evidence (ca. 12,000 B.P.) for feasting at a burial cave in Israel. Proc. Natl. Acad. Sci. U.S.A. 107, 15362–15366. https://doi.org/10.1073/pnas.1001809107 (2010).
    Google Scholar 
    Radu, V., Mărgărit, M., Voinea, V., Boroneant, A. & Dulama, I. D. Processing the Testudo carapace in prehistoric romania (8th and 5th millennia BC). Archaeol. Anthropol. Sci. 14, 60. https://doi.org/10.1007/s12520-022-01523-4 (2022).
    Google Scholar 
    Barkai, R., Rosell, J., Blasco, R. & Gopher, A. Fire for a reason: barbecue at middle pleistocene qesem Cave, israel. Curr. Anthropol. 58, S314–S328. https://doi.org/10.1086/691211 (2017).
    Google Scholar 
    Angelucci, D. E., Nabais, M. & Zilhão, J. Formation processes, fire use, and patterns of human occupation across the Middle Palaeolithic (MIS 5a–5b) of gruta da oliveira (Almonda karst system, Torres Novas, Portugal). PLoS ONE 18, e0292075. https://doi.org/10.1371/journal.pone.0292075 (2023).
    Google Scholar 
    MacDonald, K., Scherjon, F., Van Veen, E., Vaesen, K. & Roebroeks, W. Middle Pleistocene fire use: The first signal of widespread cultural diffusion in human evolution. Proc. Natl. Acad. Sci. U.S.A. 118, e2101108118. https://doi.org/10.1073/pnas.2101108118 (2021).
    Google Scholar 
    Roebroeks, W. & Villa, P. On the earliest evidence for habitual use of fire in Europe. Proc. Natl. Acad. Sci. U.S.A. 108, 5209–5214. https://doi.org/10.1073/pnas.1018116108 (2011).
    Google Scholar 
    Blasco, R. Human consumption of tortoises at Level IV of Bolomor Cave (Valencia, Spain). J. Archaeol. Sci. 35, 2839–2848. https://doi.org/10.1016/j.jas.2008.05.013 (2008).
    Google Scholar 
    Boneta, I., Pérez-García, A. & Liesau, C. Chelonians from the middle palaeolithic site of mealhada (Coimbra, Portugal): An update. Diversity 15, 243. https://doi.org/10.3390/d15020243 (2023).
    Google Scholar 
    Rhodin, A.G.J., Iverson, J.B., Bour, R., Fritz, U., Georges, A., Shaffer, H.B., van Dijk P.P. Turtles of the world: Annotated checklist and atlas of taxonomy, synonymy, distribution, and conservation status. In Conservation Biology of Freshwater Turtles and Tortoises: A Compilation Project of the IUCN/SSC Tortoise and Freshwater Turtle Specialist Group Chelonian Research Monographs 9th edn, Vol. 8 (Eds. Rhodin, A.G.J., Iverson, J.B., van Dijk, P.P., Stanford, C.B., Goode, E.V., Buhlmann, K.A., & Mittermeier, R.A.) 1–472 (2021).Nicholson, R. A morphological investigation of burnt animal bone and an evaluation of its utility in archaeology. J. Archaeol. Sci. 20, 411–428. https://doi.org/10.1006/jasc.1993.1025 (1993).
    Google Scholar 
    Shipman, P., Foster, G. & Schoeninger, M. Burnt bones and teeth: An experimental study of color, morphology, crystal structure and shrinkage. J. Archaeol. Sci. 11, 307–325. https://doi.org/10.1016/0305-4403(84)90013-X (1984).
    Google Scholar 
    Simonsen, K. P., Rasmussen, A. R., Mathisen, P., Petersen, H. & Borup, F. A fast preparation of skeletal materials using enzyme maceration. J. Forensic Sci. 56, 480–484. https://doi.org/10.1111/j.1556-4029.2010.01668.x (2011).
    Google Scholar 
    Kamminga, J. The Nature of Use Polish and Abrasive Smoothing on Stone Tools. In Lithic Use-wear Analysis (ed. Hayden, B.) 143–158 (Academic Press, 1979).
    Google Scholar 
    Keeley, L. H. Experimental Determination of Stone Tool Uses (University of Chicago Press, 1980).
    Google Scholar 
    Plisson, H. Etude fonctionnelle d’outillages lithiques prehistoriques par l’analyse des micro-usures: Recherche méthodologique et archéologique. PhD thesis, Université Paris I (1985).Tringham, R., Cooper, G., Odell, G., Voytek, B. & Whitman, A. Experimentation in the formation of edge damage: A new approach to lithic analysis. J. Field Archeol. 1, 171–196. https://doi.org/10.1179/jfa.1974.1.1-2.171 (1974).
    Google Scholar 
    Andrefsky W. Lithics: Macroscopic approaches to analysis. In 2nd edition (Cambridge University Press, 2005).Claud, E. et al. Le référentiel des outils lithiques. Palethnologie. https://doi.org/10.4000/palethnologie.5142 (2019).
    Google Scholar 
    Claud, E. et al. The practices used by the neanderthals in the acquisition and exploitation of plant and animal resources and the function of the sites studied: Summary and discussion. Palethnologie https://doi.org/10.4000/palethnologie.4179 (2019).
    Google Scholar 
    McPherron, S. et al. An experimental assessment of the influences on edge damage to lithic artifacts: a consideration of edge angle, substrate grain size, raw material properties, and exposed face. J. Archaeol. Sci. 49, 70–82. https://doi.org/10.1016/j.jas.2014.04.003 (2014).
    Google Scholar 
    González Urquijo, J. E. & Ibáñez Estévez, J. J. Metodologia de análisis functional de instrumentos tallados en sílex (Universidad de Deusto, 1994).
    Google Scholar 
    Semenov, S. A. Prehistoric Technology: An Experimental Study of the Oldest Tools and Artefacts from Traces of Manufacture and Wear (Cory, Adams & Mackay, London, 1964).
    Google Scholar 
    Costamagno, S., Claud, E., Thiébaut, C., Chacón, M. & Soulier, M. C. L’exploitation des ressources végétales et animales au paléolithique: quels outils méthodologiques pour quelles questions?. Palethnologie https://doi.org/10.4000/palethnologie.3866 (2019).
    Google Scholar 
    Greiner, M. et al. Bone incineration: An experimental study on mineral structure, colour and crystalline state. J. Archaeol. Sci. Rep. 25, 507–518. https://doi.org/10.1016/j.jasrep.2019.05.009 (2019).
    Google Scholar 
    Lebon, M. et al. New parameters for the characterization of diagenetic alterations and heat-induced changes of fossil bone mineral using Fourier transform infrared spectrometry. J. Archaeol. Sci. 37, 2265–2276. https://doi.org/10.1016/j.jas.2010.03.024 (2010).
    Google Scholar 
    Jia, X. & Sit, M. M. Laminated tortoise scepter. Gems Gemology 52, 419–420 (2016).
    Google Scholar 
    Alashwal, B.Y., Gupta, A., Husain, M.S.B., Characterization of dehydrated keratin protein extracted from chicken feather. In IOP Conference Series: Materials Science and Engeneering, 702 012033 https://doi.org/10.1088/1757-899X/702/1/012033 (2019).Hainschwang, T. & Leggio, L. The characterization of tortoise shell and its imitations. Gem. Gemol. 42, 36–52 (2006).
    Google Scholar 
    Hua, K. et al. Assessment of the defatting efficacy of mechanical and chemical treatment for allograft cancellous bone and its effects on biomechanics properties of bone. Orthop. Surg. 12, 617–630. https://doi.org/10.1111/os.12639 (2020).
    Google Scholar 
    Mattiello, S. et al. Physico-Chemical characterization of keratin from wool and chicken feathers extracted using refined chemical methods. Polymers (Basel) 15, 181. https://doi.org/10.3390/polym15010181 (2022).
    Google Scholar 
    Scaggion, C. et al. An FTIR-based model for the diagenetic alteration of archaeological bones. J. Archaeol. Sci. 161, 105900. https://doi.org/10.1016/j.jas.2023.105900 (2024).
    Google Scholar 
    Koon, H. E. C., Nicholson, R. A. & Collins, M. J. A practical approach to the identification of low temperature heated bone using TEM. J. Archaeol. Sci. 30, 1393–1399. https://doi.org/10.1016/S0305-4403(03)00034-7 (2003).
    Google Scholar 
    Mamede, A. et al. The potential of bioapatite hydroxyls for research on archaeological burned bone. Anal. Chem. https://doi.org/10.1021/acs.analchem.8b02868 (2018).
    Google Scholar 
    Alibardi, L. & Toni, M. Skin structure and cornification proteins in the soft-shelled turtle Trionyx spiniferus. Zoology (Jena) 109, 182–195. https://doi.org/10.1016/j.zool.2005.11.005 (2006).
    Google Scholar 
    Rosa, J. et al. The effects of exogenous substances on the color of heated bones. Am. J. Biol. Anthropol. 184, e24905. https://doi.org/10.1002/ajpa.24905 (2024).
    Google Scholar 
    Asaikkutti, A., Bhavan, P. S., Vimala, K., Karthik, M. & Cheruparambath, P. Dietary supplementation of green synthesized manganese-oxide nanoparticles and its effect on growth performance, muscle composition and digestive enzyme activities of the giant freshwater prawn Macrobrachium rosenbergii. J. Trace Elem. Med. Biol. 35, 7–17. https://doi.org/10.1016/j.jtemb.2016.01.005 (2016).
    Google Scholar 
    Meza, E., Ferreira, L M. Agencia Indígena y Colonialismo Una arqueología de contacto sobre la producción de aceite de tortuga en el Orinoco Medio, Venezuela (siglos XVIII y XIX). Amazônica: Revista de Antropologia 7 377 402 (2015).Guevara Chumacero, M., Pichardo Fragoso, A. & Martínez, Cornelio M. La tortuga en tabasco: comida, identidad y representación. Estud. De Cultura Maya 49(97), 122. https://doi.org/10.19130/iifl.ecm.2017.49.758 (2016).
    Google Scholar 
    Ferronato, B. & Georges, A. Distribution of freshwater turtle rock art and archaeological sites in australia: A glimpse into aboriginal use of chelonians. Herpetol. Conserv. Biol. 18, 374–391 (2023).
    Google Scholar 
    Sudiana, I.G. N., Ardika, I. W., Parimartha, I. G., Titib, I. M. Exploitation and protection of turtles at Serangan and Tanjung Benoa villages South Bali in the perspective of cultural studies. E-J. Cult. Stud. ISSN 2338–2449. Available at: https://ojs.unud.ac.id/index.php/ecs/article/view/3577 (2012).Susilo, E., Isdianto, A., Parawangsa, I. N. Y., Fathah, A. L. & Putri, B. M. Balancing tradition and conservation: The use of turtles in Balinese ceremonies and its environmental implications. Int. J. Environ. Impact. 7, 233–243. https://doi.org/10.18280/ijei.070208 (2024).
    Google Scholar 
    Charnov, E. L. Optimal foraging, the marginal value theorem. Theor. Popul. Biol. 9, 129–136. https://doi.org/10.1016/0040-5809(76)90040-X (1976).
    Google Scholar 
    Stephens, D. W. & Krebs, J. R. Foraging Theory Monographies in Behaviour and Ecology (Princeton University Press, 1987).
    Google Scholar 
    Stiner, M. Thirty years on the “Broad Spectrum Revolution” and paleolithic demography. PNAS 98, 6993–6996. https://doi.org/10.1073/pnas.121176198 (2001).
    Google Scholar 
    Stiner, M. & Munro, N. Approaches to prehistoric diet breadth, demography, and prey ranking systems in time and space. J. Archaeol. Method. Theor. 9, 181–214. https://doi.org/10.1023/A:1016530308865 (2002).
    Google Scholar 
    Stiner, M. C., Munro, N. D., Surovell, T. A., Tchernov, E. & Bar-Yosef, O. Paleolithic population growth pulses evidenced by small animal exploitation. Science 283, 190–194. https://doi.org/10.1126/science.283.5399.190 (1999).
    Google Scholar 
    Laland, K., Matthews, B. & Feldman, M. W. Ahn introduction to niche construction theory. Evol. Ecol. 30, 191–202. https://doi.org/10.1007/s10682-016-9821-z (2016).
    Google Scholar 
    Odling-Smee, F. J., Laland, K. N., Feldman, M. W., Niche construction: The neglected process in evolution. In Monographies in Population Biology Vol. 37 (Princeton University Press 2003).Zeder, M. The broad spectrum revolution at 40: Resource diversity, intensification, and an alternative to optimal foraging explanations. J. Anthropol. Archaeol. 31, 241–264. https://doi.org/10.1016/j.jaa.2012.03.003 (2012).
    Google Scholar 
    Quintanilha, L. et al. Carcass traits of amazon turtles (Podocnemis Expansa) reared in captivity in Brazil. Int. J. Plant Anim. Environ. Sci. 3, 60–67 (2013).
    Google Scholar 
    Sorensen, A. C., Claud, E. & Soressi, M. Neanderal fire-making technology inferred from microwear analysis. Sci. Rep. 8, 10065. https://doi.org/10.1038/s41598-018-28342-9 (2018).
    Google Scholar 
    Stout, D. & Khreisheh, N. Skill learning and human brain evolution: An experimental approach. Camb. Archaeol. J. 25, 867–875. https://doi.org/10.1017/S0959774315000359 (2015).
    Google Scholar 
    Gowlett, J. The discovery of fire by humans: A long and convoluted process. Philos. Trans. Royal Soc. B: Biol. Sci. https://doi.org/10.1098/rstb.2015.0164 (2016).
    Google Scholar 
    Lew-Levy, S., Reckin, R., Lavi, N., Cristóbal-Azkarate, J. & Ellis-Davies, K. How do hunter-gatherer children learn subsistence skills? A meta-ethnographic review. Hum. Nat. 28, 367–394. https://doi.org/10.1007/s12110-017-9302-2 (2017).
    Google Scholar 
    MacDonald, K. Cross-cultural comparison of learning in human hunting. Hum. Nat. 18, 386–402. https://doi.org/10.1007/s12110-007-9019-8 (2007).
    Google Scholar 
    Benson-Amram, S., Dantzer, B., Stricker, G., Swanson, E. & Holekamp, K. Brain size predicts problem-solving ability in mammalian carnivores. Proc. Natl. Acad. Sci. 113, 2532–2537. https://doi.org/10.1073/pnas.1505913113 (2016).
    Google Scholar 
    Download referencesAcknowledgementsWe acknowledge the use of the Vertebrate Reference Collection (Osteoteca) of the Archaeosciences Laboratory of the Património Cultural I.P, in Lisbon, Portugal. We also extend our gratitude to the PRISC infrastructure (Portuguese Research Infrastructure of Scientific Collections) for their support.FundingFinancial support has been provided by the research project “PALAEO.WEST.IBERIA – Contrasting Dietary Patterns and Adaptive Responses among Modern Humans, Neanderthals and Pre-Neanderthals in the Atlantic Façade of Iberia” funded through the Fundação para a Ciência e a Tecnologia (FCT) Scientific Research and Technological Development (IC&DT) call across all scientific domains, Call Reference MPr-2023–12, project number 2023.16301.ICDT. This research was also funded by Mariana Nabais’ postdoc contract for project “SMALLPREY – Neanderthal and Anatomically Modern Human interactions with small prey in Atlantic Iberia throughout the changing environments of the Pleistocene”, as part of the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 101034349, and from the State Research Agency of the Spanish Ministry of Science and Innovation through the Program Maria de Maeztu Unit of Excellence (CEX2019-000945-M). Additional support has been given by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia in the framework of the project “UID/00698/2025 (doi.org/https://doi.org/10.54499/UID/00698/2025): Centre for Archaeology. University of Lisbon”. Ruth Blasco develops her work within the project PID 2022-138590NB-C41 funded by MCIN/AEI/https://doi.org/10.13039/501100011033/FEDER, UE and the projects 2021-SGR-01237 and CLT009/22/000045 funded by the Generalitat de Catalunya. Ruth Blasco is supported by a Ramon y Cajal research contract by the Spanish Ministry of Science and Innovation (RYC 2019–026386-I). Valentina Lubrano is beneficiary of a FCT Doctoral Grant (reference: 2021.05263.BD). Anna Rufà is currently a beneficiary of a CEEC – 3rd Edition research contract promoted by the Portuguese FCT (reference: 2020. 00877.CEECIND) and her research is also funded in part by the Fundação para a Ciência e Tecnologia, I.P. (FCT, https://ror.org/00snfqn589) under the grant UID/0421. Mariana Nabais, Ruth Blasco, Valentina Lubrano and Anna Rufà also participate in the Spanish MICINN project NEANDIVERSITY2, PID2022-138590NB-C41.Author informationAuthors and AffiliationsUNIARQ – Centre for Archaeology, University of Lisbon, Lisbon, PortugalMariana Nabais & Marina IgrejaSWAD – South-West Archaeology Digs, Moura, PortugalMariana NabaisIPHES-CERCA – Catalan Institute of Human Palaeoecology and Social Evolution, Tarragona, SpainRuth BlascoDepartment of History and Art History, Área de Prehistòria, Rovira i Virgili University, Tarragona, SpainRuth BlascoAutónoma University of Madrid, Madrid, SpainIratxe BonetaArqueozoo S.L, Madrid, SpainIratxe BonetaLARC – Archaeosciences Laboratory (LARC), Património Cultural I.P, Lisbon, PortugalDavid Gonçalves & Marina IgrejaResearch Centre for Anthropology and Health, Department of Life Sciences, University of Coimbra, Coimbra, PortugalDavid GonçalvesCentre for Functional Ecology, Laboratory of Forensic Anthropology, Department of Life Sciences, University of Coimbra, Coimbra, PortugalDavid GonçalvesCIBIO – Research Centre in Biodiversity and Genetic Resources, University of Porto, Porto, PortugalMarina IgrejaICArEHB – Interdisciplinary Center for Archaeology and the Evolution of Human Behaviour, University of Algarve, Faro, PortugalValentina Lubrano & Anna RufàPACEA UMR 5199, University of Bordeaux, Bordeaux, FranceAnna RufàAuthorsMariana NabaisView author publicationsSearch author on:PubMed Google ScholarRuth BlascoView author publicationsSearch author on:PubMed Google ScholarIratxe BonetaView author publicationsSearch author on:PubMed Google ScholarDavid GonçalvesView author publicationsSearch author on:PubMed Google ScholarMarina IgrejaView author publicationsSearch author on:PubMed Google ScholarValentina LubranoView author publicationsSearch author on:PubMed Google ScholarAnna RufàView author publicationsSearch author on:PubMed Google ScholarContributionsConceptualisation: MN Data curation: MN Formal analysis: MN, MI, DG Funding acquisition: MN, AR, RB Investigation: MN, MI, DG Methodology: MN, IB, MI, DG Resources: MN, AR, MI, DG Visualisation: MN, MI, DG Writing – original draft: MN, MI, DG Writing – review & editing: MN, AR, IB, MI, VL, RB, DG.Corresponding authorCorrespondence to
    Mariana Nabais.Ethics declarations

    Competing interests
    The authors declare no competing interests.

    Informed consent
    Our study does not involve human participants in the usual sense. All individuals who appear in the images are the authors of the study and no other identifiable persons or patient data are included. For this reason, we consider a formal informed-consent statement from study participants not applicable. All authors explicitly agree to the publication of these images and any associated information in an online open-access format.

    Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Rights and permissions
    Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
    Reprints and permissionsAbout this articleCite this articleNabais, M., Blasco, R., Boneta, I. et al. Experimental analysis of roasted and raw turtle butchery and implications for early human cognition and behaviour.
    Sci Rep (2025). https://doi.org/10.1038/s41598-025-31738-zDownload citationReceived: 03 July 2025Accepted: 04 December 2025Published: 24 December 2025DOI: https://doi.org/10.1038/s41598-025-31738-zShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
    Provided by the Springer Nature SharedIt content-sharing initiative
    KeywordsExperimental archaeologyTaphonomySubsistence strategiesLithic use-wearFTIRChelonids More

  • in

    Thermal evolution of light hydrocarbon fingerprints in biodegraded oils from Ordovician reservoirs, Tabei Uplift, Tarim Basin

    AbstractWithin the Tabei Uplift of the Tarim Basin, Ordovician reservoirs in both the northern Halahatang (N-Halahatang) and western Lunnan (W-Lunnan) areas experienced extensive biodegradation during the Late Hercynian (Permian). Subsequent Himalayan (Neogene–Quaternary) tectonism induced divergent burial-thermal histories: the N-Halahatang reservoirs underwent intensive maturation (> 6,500 m depth; 1.02–1.22% Ro), while the W-Lunnan reservoirs experienced milder maturation (< 5,800 m depth; 0.70–0.85% Ro). Despite similar δ13Coil values indicating genetic affinity, the relatively deeply buried biodegraded oils from the N-Halahatang area contain abundant C6–C8 light hydrocarbons (LHs), while the biodegraded oils from the W-Lunnan area exhibit only trace amounts of C6–C8 LHs. To elucidate the evolution of LHs compositions and fingerprints in biodegraded oils under thermal maturation, and to determine whether the more enriched C6–C8 LHs in the N-Halahatang oils can be attributed to enhanced burial-thermal maturation, two relatively shallower-burial biodegraded oils (Well LG40: slight to moderate biodegradation‌; Well LG7: heavy to severe biodegradation) from the W-Lunnan area were artificially pyrolyzed to various maturities. Subsequently, LH parameters of the pyrolyzed oils were compared with those of the naturally matured, deeply buried oils (heavy to severe biodegradation) from the N-Halahatang area. The results indicated that both biodegraded oils generated C6–C8 LHs through thermal cracking, and the more severely biodegraded oil (Well LG7) exhibited a lower LH maximum yield than that from Well LG40. Certain parameters for organic matter type classification (n-C7–DMCP–MCH and 3RP–5RP–6RP diagrams) generally remained applicable during thermal maturation, whereas most parameters for secondary alteration identification and maturity assessment were significantly compromised. Additionally, LH parameters of the N-Halahatang oils (1.02–1.22% Ro) matched those of the LG7 pyrolyzed oils at EasyRo = 1.00–1.20%, confirming that the enriched C6–C8 LHs in the N-Halahatang oils can be attributed to cracking of biodegraded oils (with ‌biodegradation levels equivalent to Well LG7‌) under intense burial-thermal maturation. Furthermore, the potential C6–C13 LHs derived from biodegraded oil cracking constitute 11–16 wt% of N-Halahatang’s liquid hydrocarbon resources.

    Data availability

    Datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
    ReferencesChang, X. C., Wang, T. G., Li, Q. M., Cheng, B. & Tao, X. W. Geochemistry and possible origin of petroleum in palaeozoic reservoirs from Halahatang depression. J. Asian Earth Sci. 74, 129–141. https://doi.org/10.1016/j.jseaes.2013.03.024 (2013).
    Google Scholar 
    Chang, X. C., Wang, T. G., Li, Q. M. & Ou, G. X. Charging of ordovician reservoirs in the Halahatang depression (Tarim Basin, NW China) determined by oil geochemistry. J. Petrol. Geol. 36, 383–398. https://doi.org/10.1111/jpg.12562 (2013).
    Google Scholar 
    Wang, Y. F. et al. Phase change of the ordovician hydrocarbon in the Tarim basin: A case study from the Halahatang-Shunbei area. Open. Geosci. 16, 20220629. https://doi.org/10.1515/geo-2022-0629 (2024).
    Google Scholar 
    Yang, P. et al. Petroleum accumulation history of deeply buried carbonate reservoirs in the Northern Tarim Basin, Northwestern China. AAPG Bull. 108, 1193–1229. https://doi.org/10.1306/06212321210 (2024).
    Google Scholar 
    Zhang, S. C. et al. Geochemistry of paleozoic marine oils from the Tarim Basin, NW China. Part 4: paleobiodegradation and oil charge mixing. Org. Geochem. 67, 41–57. https://doi.org/10.1016/j.orggeochem.2013.12.008 (2014).
    Google Scholar 
    Li, M. J. et al. Paleo-heat flow evolution of the Tabei uplift in Tarim Basin, Northwest China. J. Asian Earth Sci. 37, 52–66. https://doi.org/10.1016/j.jseaes.2009.07.007 (2010).
    Google Scholar 
    Wang, T. G. et al. Stratigraphic thermohistory and its implications for regional geoevolution in the Tarim Basin, NW China. Sci. China Earth Sci. 53, 1495–1505. https://doi.org/10.1007/s11430-010-4069-x (2010).
    Google Scholar 
    He, D. F., Jia, C. Z., Liu, S. B., Pan, W. Q. & Wang, S. J. Dynamics for multistage pool formation of Lunnan low uplift in Tarim basin. Chin. Sci. Bull. 47, 128–138. https://doi.org/10.1007/bf02902829 (2002).
    Google Scholar 
    Cai, Z. X., Wu, N., Yang, H. J., Gu, Q. Y. & Han, J. F. Mechanism of evaporative fractionation in condensate gas reservoirs in Lunnan low salient. Natur. Gas Ind. 29, 21–24 (2009).
    Google Scholar 
    Wu, N. et al. Hydrocarbon charging of the ordovician reservoirs in Tahe-Lunnan area, China. Sci. China Earth Sci. 56, 763–772. https://doi.org/10.1007/s11430-013-4598-1 (2013).
    Google Scholar 
    Zhu, G. Y., Liu, X. W., Zhu, Y. F., Su, J. & Wang, K. The characteristics and the accumulation mechanism of complex reservoirs in the Hanicatam area, Tarim basin. Bull. Mineral. Petrol. Geochem. 32, 231–242 (2013).
    Google Scholar 
    Zhu, G. Y., Wang, M. & Zhang, T. W. Identification of polycyclic sulfides hexahydrodibenzothiophenes and their implications for heavy oil accumulation in ultra-deep strata in Tarim basin. Mar. Pet. Geol. 78, 439–447. https://doi.org/10.1016/j.marpetgeo.2016.09.027 (2016).
    Google Scholar 
    Zhu, G. Y. et al. Alteration and multi-stage accumulation of oil and gas in the ordovician of the Tabei Uplift, Tarim Basin, NW china: implications for genetic origin of the diverse hydrocarbons. Mar. Pet. Geol. 46, 234–250. https://doi.org/10.1016/j.marpetgeo.2013.06.007 (2013).
    Google Scholar 
    Zhu, G. Y. et al. The complexity, secondary geochemical process, genetic mechanism and distribution prediction of deep marine oil and gas in the Tarim Basin, China. Earth Sci. Rev. 198, 102930. https://doi.org/10.1016/j.earscirev.2019.102930 (2019).
    Google Scholar 
    Chang, X. C., Wang, T. G., Li, Q. M., Cheng, B. & Zhang, L. P. Maturity assessment of severely biodegraded marine oils from the Halahatang depression in Tarim basin. Energy Explor. Exploit. 30, 331–350. https://doi.org/10.1260/0144-5987.30.3.331 (2012).
    Google Scholar 
    Li, M. J. et al. Practical application of reservoir geochemistry in petroleum exploration: case study from a paleozoic carbonate reservoir in the Tarim basin (Northwestern China). Energy Fuels. 32, 1230–1241. https://doi.org/10.1021/acs.energyfuels.7b03186 (2018).
    Google Scholar 
    Wang, J. B., Guo, R. T., Xiao, X. M., Liu, Z. F. & Shen, J. G. Timing and phases of hydrocarbon migration and accumulation of the formation of oil and gas pools in Lunnan low uplift of Tarim basin. Acta Sedimentol. Sin. 20, 320–325 (2002).
    Google Scholar 
    Zhao, W. Z., Zhu, G. Y., Su, J., Yang, H. J. & Zhu, Y. F. Study on multi stage filling and accumulation model of marine oil and gas reservoirs: A case study of the Lungudong area, Tarim basin. Acta Petrol. Sin. 28, 709–721 (2012).
    Google Scholar 
    Cheng, B., Wang, T. G. & Chang, X. C. Application of C5–C7 light hydrocarbons in geochemical studies: A case study of ordovician crude oils from the Halahatang depression, Tabei uplift. Nat. Gas Geosci. 24, 398–405 (2013).
    Google Scholar 
    Wang, Y. P., Chang, X. C., Cheng, B. & Shi, S. B. Comparison of C5-C7 light hydrocarbons in Halahatang ordovician oil analyzed by comprehensive 2-D and conventional gas chromatography. Nat. Gas Geosci. 26, 1814–1822 (2015).
    Google Scholar 
    Wu, X. Q., Liu, Q. Y., Tao, X. W. & Hu, G. Y. Geochemical characteristics of natural gas from Halahatang Sag in the Tarim basin. Geochimica 43, 477–488. https://doi.org/10.19700/j.0379-1726.2014.05.006 (2014).
    Google Scholar 
    Tissot, B. P. & Welte, D. H. Petroleum Formation and Occurence (Springer-Verlag, 1984).Hossain, M. A., Suzuki, N., Matsumoto, K., Sakamoto, R. & Takeda, N. In-reservoir fractionation and the accumulation of oil and condensates in the surma Basin, NE Bangladesh. J. Petrol. Geol. 37, 269–286. https://doi.org/10.1111/jpg.12583 (2014).
    Google Scholar 
    Thompson, K. F. M. Fractionated aromatic petroleums and the generation of gas-condensates. Org. Geochem. 11, 573–590. https://doi.org/10.1016/0146-6380(87)90011-8 (1987).
    Google Scholar 
    Thompson, K. F. M. Gas-condensate migration and oil fractionation in deltaic systems. Mar. Pet. Geol. 5, 237–246. https://doi.org/10.1016/0264-8172(88)90004-9 (1988).
    Google Scholar 
    Zhang, S. C. et al. Geochemistry of palaeozoic marine petroleum from the Tarim Basin, NW china: part 3. Thermal cracking of liquid hydrocarbons and gas washing as the major mechanisms for deep gas condensate accumulations. Org. Geochem. 42, 1394–1410. https://doi.org/10.1016/j.orggeochem.2011.08.013 (2011).
    Google Scholar 
    Zhu, X. J. et al. Effects of evaporative fractionation on diamondoid hydrocarbons in condensates from the Xihu Sag, East China sea shelf basin. Mar. Pet. Geol. 126, 104929. https://doi.org/10.1016/j.marpetgeo.2021.104929 (2021).
    Google Scholar 
    Cai, J., Lü, X. X. & Li, B. Y. Tectonic fracture and its significance in hydrocarbon migration and accumulation: a case study on middle and lower ordovician in Tabei uplift of Tarim Basin, NW China. Geol. J. 51, 572–583. https://doi.org/10.1002/gj.2656 (2016).
    Google Scholar 
    Chen, J. Q., Ma, K. Y., Pang, X. Q. & Yang, H. J. Secondary migration of hydrocarbons in ordovician carbonate reservoirs in the Lunnan area, Tarim basin. J. Petrol. Sci. Eng. 188, 106962. https://doi.org/10.1016/j.petrol.2020.106962 (2020).
    Google Scholar 
    Li, S. M., Zhang, B. S., Xing, L. T., Sun, H. & Yuan, X. Y. Geochemical feathers of deep hydrocarbon migration and accumulation in Halahatang-Yingmaili area of the Northern Tarim basin. Acta Petrol. Sin. 36, 92–101. https://doi.org/10.7623/syxb2015S2008 (2015).
    Google Scholar 
    Hill, R. J., Tang, Y. C. & Kaplan, I. R. Insights into oil cracking based on laboratory experiments. Org. Geochem. 34, 1651–1672. https://doi.org/10.1016/s0146-6380(03)00173-6 (2003).
    Google Scholar 
    Peters, K. E. & Moldowan, J. M. The Biomarker Guide: Interpreting Molecular Fossils in Petroleum and Ancient Sediments (Prentice Hall, 1993).Jiang, B., Liu, W. M., Liao, Y. H. & Peng, P. A. Molecular transformations of heteroatomic organic compounds in crude oils caused by biodegradation and subsequent thermal maturation: insights from ESI FT-ICR MS. Org. Geochem. 188, 104741. https://doi.org/10.1016/j.orggeochem.2024.104741 (2024).
    Google Scholar 
    Liao, Y. H., Shi, Q., Hsu, C. S., Pan, Y. H. & Zhang, Y. H. Distribution of acids and nitrogen-containing compounds in biodegraded oils of the Liaohe basin by negative ion ESI FT-ICR MS. Org. Geochem. 47, 51–65. https://doi.org/10.1016/j.orggeochem.2012.03.006 (2012).
    Google Scholar 
    Pan, Y. H., Liao, Y. H. & Peng, X. Z. Variations in chemical and stable carbon isotopic compositions of Liaohe crude oil during aerobic biodegradation simulation. Geochimica 44, 581–589. https://doi.org/10.19700/j.0379-1726.2015.06.007 (2015).
    Google Scholar 
    Huang, Y. Y., Liao, Y. H., Xu, T., Wang, Y. P. & Peng, P. A. Characteristics of light hydrocarbons under the superimposed influence of biodegradation and subsequent thermal maturation. Org. Geochem. 177, 104557. https://doi.org/10.1016/j.orggeochem.2023.104557 (2023).
    Google Scholar 
    Liao, Y. H. et al. Superimposed secondary alteration of oil reservoirs. Part I: influence of biodegradation on the gas generation behavior of crude oils. Org. Geochem. 142, 103965. https://doi.org/10.1016/j.orggeochem.2019.103965 (2020).
    Google Scholar 
    Liu, W. M. et al. Superimposed secondary alteration of oil reservoirs. Part II: the characteristics of biomarkers under the superimposed influences of biodegradation and thermal alteration. Fuel 307, 121721. https://doi.org/10.1016/j.fuel.2021.121721 (2022).
    Google Scholar 
    Chai, Z. & Chen, Z. H. Biomarkers, light hydrocarbons, and diamondoids of petroleum in deep reservoirs of the Southeast Tabei Uplift, Tarim basin: implication for its origin, alteration, and charging direction. Mar. Pet. Geol. 147, 106019. https://doi.org/10.1016/j.marpetgeo.2022.106019 (2023).
    Google Scholar 
    Li, F. et al. The disputes on the source of paleozoic marine oil and gas and the determination of the cambrian system as the main source rocks in Tarim basin. Acta Petrol. Sin. 42, 1417–1436. https://doi.org/10.7623/syxb202111002 (2021).
    Google Scholar 
    Liu, Z. et al. The hydrocarbon generation process of the deeply buried cambrian yuertusi formation in the Tabei uplift, Tarim Basin, Northwestern china: constraints from calcite veins hosting oil inclusions in the source rock. Geol. Soc. Am. Bull. 136, 3810–3824. https://doi.org/10.1130/b37295.1 (2024).
    Google Scholar 
    Wu, L. et al. Long-lived paleo-uplift controls on Neoproterozoic-Cambrian black shales in the Tarim basin. Mar. Pet. Geol. 155, 106343. https://doi.org/10.1016/j.marpetgeo.2023.106343 (2023).
    Google Scholar 
    Wang, S. et al. Genetic mechanism of multiphase States of ordovician oil and gas reservoirs in Fuman oilfield, Tarim Basin, China. Mar. Pet. Geol. 157, 106449. https://doi.org/10.1016/j.marpetgeo.2023.106449 (2023).
    Google Scholar 
    Xiao, X. M., Liu, Z. F., Liu, D. H., Shen, J. G. & Fu, J. M. A new method to reconstruct hydrocarbon-generating histories of source rocks in a petroleum-bearing basin – the method of geological and geochemical sections. Chin. Sci. Bull. 45, 35–40. https://doi.org/10.1007/bf02893782 (2000).
    Google Scholar 
    Zhan, Z. W., Tian, Y. K., Zou, Y. R., Liao, Z. W. & Peng, P. A. De-convoluting crude oil mixtures from palaeozoic reservoirs in the Tabei Uplift, Tarim Basin, China. Org. Geochem. 97, 78–94. https://doi.org/10.1016/j.orggeochem.2016.04.004 (2016).
    Google Scholar 
    Tian, F. et al. Multiscale geological-geophysical characterization of the epigenic origin and deeply buried paleokarst system in Tahe Oilfield, Tarim basin. Mar. Pet. Geol. 102, 16–32. https://doi.org/10.1016/j.marpetgeo.2018.12.029 (2019).
    Google Scholar 
    Zhu, G. Y. et al. Hydrocarbon accumulation mechanisms and industrial exploration depth of large-area fracture-cavity carbonates in the Tarim Basin, Western China. J. Petrol. Sci. Eng. 133, 889–907. https://doi.org/10.1016/j.petrol.2015.03.014 (2015).
    Google Scholar 
    Zhu, Z. J. et al. Analysis of the filling patterns and reservoir development models of the ordovician paleokarst reservoirs in the Tahe oilfield. Mar. Pet. Geol. 161, 106690. https://doi.org/10.1016/j.marpetgeo.2024.106690 (2024).
    Google Scholar 
    Jacob, H. Classification, structure, genesis and practical importance of natural solid oil bitumen (‘migrabitumen’). Int. J. Coal Geol. 11, 65–79. https://doi.org/10.1016/0166-5162(89)90113-4 (1989).
    Google Scholar 
    Parnell, J., Monson, B. & Geng, A. Maturity and petrography of bitumens in the carboniferous of Ireland. Int. J. Coal Geol. 29, 23–38. https://doi.org/10.1016/0166-5162(95)00023-2 (1996).
    Google Scholar 
    Karweil, J. Die metamorphose der Kohlen vom standpunkt der physikalischen chemie. Z. Dtsch. Geol. Ges. 107, 132–139 (1956).
    Google Scholar 
    Liao, Y. H., Geng, A. S. & Huang, H. P. The influence of biodegradation on resins and asphaltenes in the Liaohe basin. Org. Geochem. 40, 312–320. https://doi.org/10.1016/j.orggeochem.2008.12.006 (2009).
    Google Scholar 
    Sweeney, J. J. & Burnham, A. K. Evaluation of a simple model of vitrinite reflectance based on chemical kinetics. AAPG Bull. 74, 1559–1570 (1990).
    Google Scholar 
    Huang, Y. Y. et al. Improved light hydrocarbons collection method for the pyrolysis of crude oil in gold tube closed system experiments. Org. Geochem. 168, 104432. https://doi.org/10.1016/j.orggeochem.2022.104432 (2022).
    Google Scholar 
    Odden, W., Patience, R. L. & van Graas, G. W. Application of light hydrocarbons (C4-C13) to oil/source rock correlations: a study of the light hydrocarbon compositions of source rocks and test fluids from offshore Mid-Norway. Org. Geochem. 28, 823–847. https://doi.org/10.1016/s0146-6380(98)00039-4 (1998).
    Google Scholar 
    Leythaeuser, D., Schaefer, R. G., Cornford, C. & Weiner, B. Generation and migration of light hydrocarbons (C2–C7) in sedimentary basins. Org. Geochem. 1, 191–204. https://doi.org/10.1016/0146-6380(79)90022-6 (1979).
    Google Scholar 
    Thompson, K. F. M. Classification and thermal history of petroleum based on light hydrocarbons. Geochim. Cosmochim. Acta. 47, 303–316. https://doi.org/10.1016/0016-7037(83)90143-6 (1983).
    Google Scholar 
    Dai, J. X. Identification of various alkane gases. Sci. China-Chem‌. 35, 1246–1257 (1992).
    Google Scholar 
    ten Haven, H. L. Applications and limitations of Mango’s light hydrocarbon parameters in petroleum correlation studies. Org. Geochem. 24, 957–976. https://doi.org/10.1016/s0146-6380(96)00091-5 (1996).
    Google Scholar 
    Mango, F. D. An invariance in the isoheptanes of petroleum. Science 237, 514–517. https://doi.org/10.1126/science.237.4814.514 (1987).
    Google Scholar 
    Mango, F. D. The origin of light cycloalkanes in petroleum. Geochim. Cosmochim. Acta. 54, 23–27. https://doi.org/10.1016/0016-7037(90)90191-m (1990).
    Google Scholar 
    Mango, F. D. The origin of light hydrocarbons in petroleum: A kinetic test of the steady-state catalytic hypothesis. Geochim. Cosmochim. Acta. 54, 1315–1323. https://doi.org/10.1016/0016-7037(90)90156-f (1990).
    Google Scholar 
    Mango, F. D. The origin of light hydrocarbons in petroleum: ring preference in the closure of carbocyclic rings. Geochim. Cosmochim. Acta. 58, 895–901. https://doi.org/10.1016/0016-7037(94)90513-4 (1994).
    Google Scholar 
    Mango, F. D. The light hydrocarbons in petroleum: a critical review. Org. Geochem. 26, 417–440. https://doi.org/10.1016/s0146-6380(97)00031-4 (1997).
    Google Scholar 
    Akinlua, A., Ajayi, T. R. & Adeleke, B. B. Niger delta oil geochemistry: insight from light hydrocarbons. J. Petrol. Sci. Eng. 50, 308–314. https://doi.org/10.1016/j.petrol.2005.12.003 (2006).
    Google Scholar 
    Obermajer, M., Osadetz, K. G., Fowler, M. G. & Snowdon, L. R. Light hydrocarbon (gasoline range) parameter refinement of biomarker-based oil-oil correlation studies: an example from Williston basin. Org. Geochem. 31, 959–976. https://doi.org/10.1016/s0146-6380(00)00114-5 (2000).
    Google Scholar 
    Smith, M. & Bend, S. Geochemical analysis and Familial association of red river and Winnipeg reservoired oils of the Williston Basin, Canada. Org. Geochem. 35, 443–452. https://doi.org/10.1016/j.orggeochem.2004.01.008 (2004).
    Google Scholar 
    Philippi, G. T. The deep subsurface temperature controlled origin of the gaseous and gasoline-range hydrocarbons of petroleum. Geochim. Cosmochim. Acta. 39, 1353–1373. https://doi.org/10.1016/0016-7037(75)90115-5 (1975).
    Google Scholar 
    Thompson, K. F. M. Light hydrocarbons in subsurface sediments. Geochim. Cosmochim. Acta. 43, 657–672. https://doi.org/10.1016/0016-7037(79)90251-5 (1979).
    Google Scholar 
    Mackenzie, A. S. & McKenzie, D. Isomerization and aromatization of hydrocarbons in sedimentary basins formed by extension. Geol. Mag. 120, 417–470. https://doi.org/10.1017/s0016756800027461 (1983).
    Google Scholar 
    Peters, K. E. & Moldowan, J. M. Effects of source, thermal maturity, and biodegradation on the distribution and isomerization of Homohopanes in petroleum. Org. Geochem. 17, 47–61. https://doi.org/10.1016/0146-6380(91)90039-m (1991).
    Google Scholar 
    Peters, K. E., Walters, C. C. & Moldowan, J. M. The Biomarker Guide (Cambridge University Press, 2005).van Graas, G. W. Biomarker maturity parameters for high maturities: calibration of the working range up to the oil/condensate threshold. Org. Geochem. 16, 1025–1032. https://doi.org/10.1016/0146-6380(90)90139-q (1990).
    Google Scholar 
    Kissin, Y. V. Catagenesis and composition of petroleum: origin of n-alkanes and isoalkanes in petroleum crudes. Geochim. Cosmochim. Acta. 51, 2445–2457. https://doi.org/10.1016/0016-7037(87)90296-1 (1987).
    Google Scholar 
    Behar, F., Lorant, F. & Mazeas, L. Elaboration of a new compositional kinetic schema for oil cracking. Org. Geochem. 39, 764–782. https://doi.org/10.1016/j.orggeochem.2008.03.007 (2008).
    Google Scholar 
    Chang, C. T., Lee, M. R., Lin, L. H. & Kuo, C. L. Application of C7 hydrocarbons technique to oil and condensate from type III organic matter in Northwestern Taiwan. Int. J. Coal Geol. 71, 103–114. https://doi.org/10.1016/j.coal.2006.06.011 (2007).
    Google Scholar 
    Li, F. L. et al. Fault system dynamics and their impact on ordovician carbonate karst reservoirs: outcrop analogs and 3D seismic analysis in the Tabei region, Tarim Basin, NW China. Mar. Pet. Geol. 167, 106923. https://doi.org/10.1016/j.marpetgeo.2024.106923 (2024).
    Google Scholar 
    Download referencesAcknowledgementsThe authors thank Dr. Jinzhong Liu, Mr. Yong Li, Dr. Zewen Liao, and Dr. Yankuan Tian for their assistance in laboratory analyses. The authors are also grateful to the anonymous reviewers for their constructive suggestions.FundingThis work was supported by the National Natural Science Foundation of China (Grant Nos. 42173056 and 42572184), the project Theory of Hydrocarbon Enrichment under Multi-Spheric Interactions of the Earth (Grant No. THEMSIE04010104). This is also a contribution to the Special Fund for the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA14010103).Author informationAuthors and AffiliationsState Key Laboratory of Deep Earth Processes and Resources, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou, 510640, ChinaYuwei Yang, Yuhong Liao, Yueyi Huang, Bin Cheng, Huanyu Lin & Yunpeng WangState Key Laboratory of Advanced Environmental Technology, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou, 510640, ChinaYijun Zheng & Ping’An PengUniversity of Chinese Academy of Sciences, Yuquan Road, Beijing, 100049, ChinaYuwei Yang, Yuhong Liao, Yijun Zheng, Bin Cheng, Huanyu Lin, Yunpeng Wang & Ping’An PengCAS Center for Excellence in Deep Earth Science, Guangzhou, 510640, ChinaYuwei Yang, Yuhong Liao, Yijun Zheng, Bin Cheng, Huanyu Lin, Yunpeng Wang & Ping’An PengBiogas Institute of Ministry of Agriculture and Rural Affairs, Chengdu, 610041, ChinaYueyi HuangAuthorsYuwei YangView author publicationsSearch author on:PubMed Google ScholarYuhong LiaoView author publicationsSearch author on:PubMed Google ScholarYueyi HuangView author publicationsSearch author on:PubMed Google ScholarYijun ZhengView author publicationsSearch author on:PubMed Google ScholarBin ChengView author publicationsSearch author on:PubMed Google ScholarHuanyu LinView author publicationsSearch author on:PubMed Google ScholarYunpeng WangView author publicationsSearch author on:PubMed Google ScholarPing’An PengView author publicationsSearch author on:PubMed Google ScholarContributionsYuwei Yang: Investigation, Methodology, Formal analysis, Writing-Original Draft; Yuhong Liao: Supervision, Conceptualization, Funding acquisition, Validation, Writing-Reviewing and Editing; Yueyi Huang: Data Curation; Yijun Zheng: Writing-Reviewing and Editing; Bin Cheng: Resources; Huanyu Lin: Visualization; Yunpeng Wang: Project administration, Funding acquisition; Ping’An Peng: Funding acquisition.Corresponding authorCorrespondence to
    Yuhong Liao.Ethics declarations

    Competing interests
    The authors declare no competing interests.

    Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationBelow is the link to the electronic supplementary material.Supplementary Material 1Rights and permissions
    Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
    Reprints and permissionsAbout this articleCite this articleYang, Y., Liao, Y., Huang, Y. et al. Thermal evolution of light hydrocarbon fingerprints in biodegraded oils from Ordovician reservoirs, Tabei Uplift, Tarim Basin.
    Sci Rep (2025). https://doi.org/10.1038/s41598-025-33256-4Download citationReceived: 28 October 2025Accepted: 17 December 2025Published: 24 December 2025DOI: https://doi.org/10.1038/s41598-025-33256-4Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
    Provided by the Springer Nature SharedIt content-sharing initiative
    KeywordsBiodegradationBurial-thermal maturationLight hydrocarbonsOrdovician reservoirsTabei UpliftTarim Basin More

  • in

    Integrated geographical and ecological analysis reveals environmental drivers of Gardenia jasminoides distribution and chemical variation

    AbstractGardenia jasminoides, a widely distributed resource rich in Crocin, has generated substantial market demand due to its potential value as a saffron substitute. This necessitates the exploration of efficient and sustainable cultivation strategies to obtain target compounds for specific purposes. To enhance cultivation efficiency and secure supply chains, we integrated MaxEnt modeling, spatial interpolation, and geodetector analysis. This framework aimed to predict suitable habitats for G. jasminoides across China, map spatial variation in bioactive compounds including Crocin, Gardenia Yellow, and Geniposide, and identify environmental drivers influencing their distribution. MaxEnt achieved high predictive accuracy (AUC = 0.960), identifying Jiangxi, Zhejiang, and Guangdong as key high-suitability regions. Precipitation of the driest month and human population density emerged as dominant factors shaping species distribution. Spatial gradients revealed that Crocin and Gardenia Yellow decrease from southwest to northeast, whereas Geniposide exhibits latitudinal differentiation characterized by higher concentrations in northern regions. Geodetector analysis highlighted vegetation type as the primary driver of compound variation, with q values of 0.618 for Crocin, 0.606 for Gardenia Yellow, and 0.639 for Geniposide. These results indicate that the accumulation of target compounds is strictly modulated by ecological niches, where specific vegetation types drive metabolic differentiation through microclimate regulation and interspecific competition. Based on these findings, we advocate for an industry-oriented divergent cultivation strategy. Southwestern China should be prioritized for Crocin-rich germplasm to support the natural pigment industry, whereas northern regions are designated as premium zones for pharmaceutical-grade Geniposide sourcing. Furthermore, recognizing vegetation type as a critical driver facilitates the implementation of targeted habitat management techniques. These findings provide a direct guide for designating priority cultivation zones and optimizing harvest timing to maximize the yield of target compounds for specific industrial uses.

    Data availability

    The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
    ReferenceState Pharmacopoeia Committee. Pharmacopoeia of the People’s Republic of China (China Medical Science and Technology Press, 2020).
    Google Scholar 
    Tian, J., Qin, S., Han, J., Meng, J. & Liang, A. A review of the ethnopharmacology, phytochemistry, pharmacology and toxicology of Fructus Gardeniae (Zhi-zi). J. Ethnopharmacol. 289, 114984. https://doi.org/10.1016/j.jep.2022.114984 (2022).
    Google Scholar 
    Li, Z., Shen, J., Bi, W., He, C. & Peng, Y. Progress of research on the resources and utilization of Gardenia jasminoides in China. J. Chin. Med. Mater. 40, 498–503. https://doi.org/10.13863/j.issn1001-4454.2017.02.055 (2017).
    Google Scholar 
    Sommano, S. R., Suppakittpaisarn, P., Sringarm, K., Junmahasathien, T. & Ruksiriwanich, W. Recovery of crocins from floral tissue of Gardenia jasminoides ellis. Front. Nutr. 7, 106. https://doi.org/10.3389/fnut.2020.00106 (2020).
    Google Scholar 
    Liu, Q., Huang, L., Fu, C., Gu, Z. & Yang, C. Effects of environmental factors on crocin accumulation and related genes in Gardenia jasminoides. J. Chinese Med. Mater. 45, 516–523. https://doi.org/10.13863/j.issn1001-4454.2022.03.002 (2022).
    Google Scholar 
    Pironon, S. et al. The global distribution of plants used by humans. Science 383, 293–297. https://doi.org/10.1126/science.adg8028 (2024).
    Google Scholar 
    Namgung, H., Kim, M.-J., Baek, S., Lee, J.-H. & Kim, H. Predicting potential current distribution of Lycorma delicatula (Hemiptera: Fulgoridae) using MaxEnt model in South Korea. J. Asia-Pacific Entomol. 23, 291–297. https://doi.org/10.1016/j.aspen.2020.01.009 (2020).
    Google Scholar 
    Wang, G. et al. Integrating Maxent model and landscape ecology theory for studying spatiotemporal dynamics of habitat: Suggestions for conservation of endangered Red-crowned crane. Ecol. Indic. 116, 106472. https://doi.org/10.1016/j.ecolind.2020.106472 (2020).
    Google Scholar 
    Elith, J. et al. A statistical explanation of MaxEnt for ecologists. Divers. Distrib. https://doi.org/10.1111/j.1472-4642.2010.00725.x (2010).
    Google Scholar 
    Phillips, S. J. & Dudík, M. Modeling of species distributions with Maxent: new extensions and a comprehensive evaluation. Ecography https://doi.org/10.1111/j.0906-7590.2008.5203.x (2008).
    Google Scholar 
    Miao, Q. et al. Study on ecological suitability of Gardenia jasminoides based on ArcGIS and Maxent model. China J. Chin. Materia Medica 41, 3181–3185. https://doi.org/10.4268/cjcmm20161711 (2016).
    Google Scholar 
    Inderjit, Catford, J. A., Kalisz, S., Simberloff, D., and Wardle, D. A. A framework for understanding human-driven vegetation change. Oikos 126 1687–1698. https://doi.org/10.1111/oik.04587 (2017)Fristoe, T. S. et al. Evolutionary imbalance, climate and human history jointly shape the global biogeography of alien plants. Nat. Ecol. Evol. 7, 1633–1644. https://doi.org/10.1038/s41559-023-02172-z (2023).
    Google Scholar 
    Cao, Q. et al. Phenotypic diversity of Gardenia jasminoides and Its correlations with environmental fators. J. Yunnan Agric. Univ. (Nat. Sci.) 38(587), 596. https://doi.org/10.12101/j.issn.1004-390X(n).202202009 (2023).
    Google Scholar 
    Zhang, Y. X., Peng S. W., Wang, C. H., and Zhu X. Q. Standardized cultivation of a traditional typical medicinal Gardenia jasminoides Ellis in Hunan Province (2016). Modern Agricultural Science and Technology 106-107 109. https://doi.org/10.3969/j.issn.1007-5739.2016.12.067Hu, Y., Lei, X., Li, H., Zhang, Y. & Li, L. Determination of effective components of Gardenia jasminenoides. Chin. Agric. Sci. Bullet. 39, 126–130 (2023).
    Google Scholar 
    Wu, W., Zhao, Y. & Liu, Y. Determination of the content of Gardenia jasminoides Ellis from different producing areas by HPLC. China Surfactant Deterg. Cosmet. 52, 451–456. https://doi.org/10.3969/j.issn.1001-1803.2022.04.016 (2022).
    Google Scholar 
    Wang, L. & Jackson, D. A. Effects of sample size, data quality, and species response in environmental space on modeling species distributions. Landsc. Ecol. 38, 4009–4031. https://doi.org/10.1007/s10980-023-01771-2 (2023).
    Google Scholar 
    Majkowska-Gadomska, J., Kaliniewicz, Z., Francke, A., Sałata, A. & Jadwisieńczak, K. K. An evaluation of the biometric parameters and chemical composition of the florets, leaves, and stalks of broccoli plants grown in different soil types. Appl. Sci. 14, 4411. https://doi.org/10.3390/app14114411 (2024).
    Google Scholar 
    Ogundola, A. F., Bvenura, C., Ehigie, A. F. & Afolayan, A. J. Effects of soil types on phytochemical constituents and antioxidant properties of Solanum nigrum. S. Afr. J. Bot. 151, 325–333. https://doi.org/10.1016/j.sajb.2022.09.048 (2022).
    Google Scholar 
    Dimoudi, A. & Nikolopoulou, M. Vegetation in the urban environment: microclimatic analysis and benefits. Energy Build. 35, 69–76. https://doi.org/10.1016/S0378-7788(02)00081-6 (2003).
    Google Scholar 
    Gao, J. et al. Study of the effects of ten-year microclimate regulation based on different vegetation type combinations in a city riparian zone. Water 14, 1932. https://doi.org/10.3390/w14121932 (2022).
    Google Scholar 
    Wang, C. et al. Effects of vegetation restoration on local microclimate on the loess plateau. J. Geogr. Sci 32, 291–316. https://doi.org/10.1007/s11442-022-1948-y (2022).
    Google Scholar 
    Wang, D. et al. Global assessment of the distribution and conservation status of a key medicinal plant (Artemisia annua L.): The roles of climate and anthropogenic activities. Sci. Total Environ. 821, 153378 (2022).
    Google Scholar 
    Giaccone, E. et al. Influence of microclimate and geomorphological factors on alpine vegetation in the Western Swiss Alps. Earth Surf. Process. Landf. 44, 3093–3107. https://doi.org/10.1002/esp.4715 (2019).
    Google Scholar 
    Li, K. et al. Unveiling molecular mechanisms of pigment synthesis in gardenia (gardenia jasminoides) fruits through integrative transcriptomics and metabolomics analysis. Food Chem.: Sci. 9, 100209. https://doi.org/10.1016/j.fochms.2024.100209 (2024).
    Google Scholar 
    Shahrajabian, M. H., Kuang, Y., Cui, H., Fu, L. & Sun, W. Metabolic changes of active components of important medicinal plants on the basis of traditional chinese medicine under different environmental stresses. Curr. Org. Chem. 27, 782–806. https://doi.org/10.2174/1385272827666230807150910 (2023).
    Google Scholar 
    Bouwmeester, H., Dong, L., Wippel, K., Hofland, T. & Smilde, A. The chemical interaction between plants and the rhizosphere microbiome. Trends Plant Sci. 30, 1002–1019. https://doi.org/10.1016/j.tplants.2025.06.001 (2025).
    Google Scholar 
    Philippot, L., Raaijmakers, J. M., Lemanceau, P. & van der Putten, W. H. Going back to the roots: the microbial ecology of the rhizosphere. Nat. Rev. Microbiol. 11, 789–799. https://doi.org/10.1038/nrmicro3109 (2013).
    Google Scholar 
    Wardle, D. A. et al. Ecological linkages between aboveground and belowground biota. Science 304, 1629–1633. https://doi.org/10.1126/science.1094875 (2004).
    Google Scholar 
    Ou, Y. Y. et al. Effects of plants-associated microbiota on cultivation and quality of chinese herbal medicines. Chin. Herb. Med. 16, 190–203. https://doi.org/10.1016/j.chmed.2022.12.004 (2024).
    Google Scholar 
    Pang, Z. et al. Linking plant secondary metabolites and plant microbiomes: a review. Front. Plant Sci. https://doi.org/10.3389/fpls.2021.621276 (2021).
    Google Scholar 
    Tullus, A. et al. Climate and competitive status modulate the variation in secondary metabolites more in leaves than in fine roots of betula pendula. Front. Plant Sci. https://doi.org/10.3389/fpls.2021.746165 (2021).
    Google Scholar 
    Zeng, X. et al. Differences in response of tree species at different succession stages to neighborhood competition. Forests 15, 435. https://doi.org/10.3390/f15030435 (2024).
    Google Scholar 
    Peng, Z. et al. Interspecific rhizosphere interactions enhance secondary metabolite synthesis and volatile oil content in atractylodes lancea through root exudates. Ind. Crops Prod. 236, 122084. https://doi.org/10.1016/j.indcrop.2025.122084 (2025).
    Google Scholar 
    Divekar, P. A. et al. Plant secondary metabolites as defense tools against herbivores for sustainable crop protection. Int. J. Mol. Sci. 23, 2690. https://doi.org/10.3390/ijms23052690 (2022).
    Google Scholar 
    Warren, D. L., Glor, R. E. & Turelli, M. ENMTools: a toolbox for comparative studies of environmental niche models. Ecography 33, 607–611. https://doi.org/10.1111/j.1600-0587.2009.06142.x (2010).
    Google Scholar 
    Glor, R. E. & Warren, D. Testing ecological explanations for biogeographic boundaries. Evolution 65, 673–683. https://doi.org/10.1111/j.1558-5646.2010.01177.x (2011).
    Google Scholar 
    Fick, S. E. & Hijmans, R. J. WorldClim 2: new 1-km spatial resolution climate surfaces for global land areas. Int. J. Climatol. https://doi.org/10.1002/joc.5086 (2017).
    Google Scholar 
    Xu, X. China population spatial distribution kilometer grid dataset. https://doi.org/10.12078/2017121101 (2017)FAO, and IIASA Harmonized world soil database version 2.0. FAO International Institute for Applied Systems Analysis (IIASA) Accessed January 14 2025 https://openknowledge.fao.org/handle/20.500.14283/cc3823en (2023)Abdelaal, M., Fois, M., Dakhil, M. A., Bacchetta, G. & El-Sherbeny, G. A. Predicting the potential current and future distribution of the endangered endemic vascular plant primula boveana Decne. ex Duby in Egypt. Plants 9, 957. https://doi.org/10.3390/plants9080957 (2020).
    Google Scholar 
    Jenks, G. F. The data model concept in statistical mapping. Int. Yearb. Cartogr. 7, 186–190 (1967).
    Google Scholar 
    Venne, S. & Currie, D. J. Can habitat suitability estimated from MaxEnt predict colonizations and extinctions?. Diversity Distrib. 27, 873–886. https://doi.org/10.1111/ddi.13238 (2021).
    Google Scholar 
    Zhou, C. et al. Study on the determination of the contents of fourteen terpenoids in gardeniae fructus by UPLC-MS/MS. J. Chin. Med. Mater. 45, 1644–1649. https://doi.org/10.13863/j.issn1001-4454.2022.07.022 (2022).
    Google Scholar 
    Mollalo, A., Alimohammadi, A. & Khoshabi, M. Spatial and spatio-temporal analysis of human brucellosis in Iran. Trans. R. Soc. Trop. Med. Hyg. 108, 721–728. https://doi.org/10.1093/trstmh/tru133 (2014).
    Google Scholar 
    Tang, F. et al. Spatio-temporal trends and risk factors for Shigella from 2001 to 2011 in Jiangsu Province People’s Republic of China. PLOS ONE 9, e83487. https://doi.org/10.1371/journal.pone.0083487 (2014).
    Google Scholar 
    Peng, J., Yang, M., Liang, H., Wang, S. & Dai, C. The trend-surface analysis of childhood tuberculosis in Maoxian based on Geographic Information System(GIS). Chinese J. Child Health Care 17(422–424), 427. https://doi.org/10.3969/j.issn.1672-0504.2005.04.024 (2009).
    Google Scholar 
    Cha, W. et al. Spatial autocorrelation and trend surface analysis of bacillary dysentery by geographic information system (GIS) in Hunan Province in 2013. Chin. J. Dis. Control Prev. 19, 1096–1100. https://doi.org/10.16462/j.cnki.zhjbkz.2015.11.005 (2015).
    Google Scholar 
    Li, X., Cheng, G. & Lu, L. Comparison of spatial interpolation methods. Adv. Earth Sci. 15, 260–265. https://doi.org/10.3321/j.issn:1001-8166.2000.03.004 (2000).
    Google Scholar 
    Wang, J. & Xu, C. Geodetector: Principle and prospective. Acta Geographica Sinica 72, 116–134. https://doi.org/10.11821/dlxb201701010 (2017).
    Google Scholar 
    Wang, J., Zhang, T. & Fu, B. A measure of spatial stratified heterogeneity. Ecol. Indic. 67, 250–256. https://doi.org/10.1016/j.ecolind.2016.02.052 (2016).
    Google Scholar 
    Song, Y., Wang, J., Ge, Y. & Xu, C. An optimal parameters-based geographical detector model enhances geographic characteristics of explanatory variables for spatial heterogeneity analysis: cases with different types of spatial data. GISci. Remote Sens. 57(5), 593–610. https://doi.org/10.1080/15481603.2020.1760434 (2020).
    Google Scholar 
    Download referencesAcknowledgmentsWe sincerely thank all the scholars and farmers who have helped us during the sample collection process. We would like to express our gratitude to all previous researchers who helped and references for this study.FundingThis research was supported by the National Natural Science Foundation of China (No. 82274052), CACMS Innovation Fund (No.CI2023E002, CI2024E003), Special Project on Survey of Scientific and Technological Basic Resources (No. 2022FY101000), National Key R&D Program: Intergovernmental Cooperation in International Science and Technology Innovation (No. 2022YFE0119300).Author informationAuthors and AffiliationsState Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, ChinaMingxu Zhang, Cong Zhou, Suhua Huang, Hui Wang, Tingting Shi, Meng Li, Zhixian Jing & Xiaobo ZhangAuthorsMingxu ZhangView author publicationsSearch author on:PubMed Google ScholarCong ZhouView author publicationsSearch author on:PubMed Google ScholarSuhua HuangView author publicationsSearch author on:PubMed Google ScholarHui WangView author publicationsSearch author on:PubMed Google ScholarTingting ShiView author publicationsSearch author on:PubMed Google ScholarMeng LiView author publicationsSearch author on:PubMed Google ScholarZhixian JingView author publicationsSearch author on:PubMed Google ScholarXiaobo ZhangView author publicationsSearch author on:PubMed Google ScholarContributionsM.Z.: Writing – original draft, Conceptualization, Methodology, Validation, Data curation, Writing – review & editing; C.Z.: Writing – original draft, Conceptualization, Methodology, Validation, Data curation, Writing – review & editing; S.H.: Conceptualization, Writing – review & editing; H.W.: Writing-review and editing, Methodology; T.S.: Writing – review & editing, Data curation; Z.J.: Writing – review & editing, Data curation; M.L.: Writing – review & editing; X.Z.: Writing – review & editing, Conceptualization, Methodology.Corresponding authorCorrespondence to
    Xiaobo Zhang.Ethics declarations

    Competing interests
    The authors declare no competing interests.

    Ethics
    Information on the voucher specimens, including the deposition location, deposition number, and specimen identifier, is provided in Additional Table 1. We confirm that all research involving field studies and the collection of Gardenia jasminoides was conducted in strict compliance with relevant institutional, national, and international guidelines and legislation. Given the Least Concern status of Gardenia jasminoides and that collection occurred outside of protected areas, specific collection licenses were not required; all collection adhered strictly to local regulations. Furthermore, we adhere to the principles outlined in the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. We note that Gardenia jasminoides was most recently assessed for The IUCN Red List of Threatened Species in 2023 and is listed as Least Concern. This classification is consistent with its national assessment in China, where the species is also categorized as Least Concern.

    Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationSupplementary Information.Rights and permissions
    Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
    Reprints and permissionsAbout this articleCite this articleZhang, M., Zhou, C., Huang, S. et al. Integrated geographical and ecological analysis reveals environmental drivers of Gardenia jasminoides distribution and chemical variation.
    Sci Rep (2025). https://doi.org/10.1038/s41598-025-32876-0Download citationReceived: 01 September 2025Accepted: 12 December 2025Published: 24 December 2025DOI: https://doi.org/10.1038/s41598-025-32876-0Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
    Provided by the Springer Nature SharedIt content-sharing initiative
    Keywords
    Gardenia jasminoides
    Suitable distributionSpatial differentiationQuality variationEnvironmental drivers More

  • in

    Structure and community assembly of rare bacterial community in sediments of Sancha Lake

    AbstractTo explore the structure and assembly of the rare bacterial community within sediment samples, as well as their responses their responses to environmental influencing factors, we collected surface sediment and overlying water samples from Sancha Lake across four seasons. MiSeq high-throughput sequencing was applied to the V3-V4 hypervariable regions of the 16 S rRNA genes, and the β – Nearest Taxon index (βNTI) was utilized to analyze the bacterial community assembly in the sediment samples. Our findings uncovered abundant bacterial diversity within the sediment samples of Sancha Lake, with 9314 operational taxonomic units (OTUs) identified, encompassing 59 phyla, 198 classes, 279 orders, 447 families, and 758 genera of bacteria. Proteobacteria and Chloroflexi were the dominant rare bacteria at the phylum level, whereas Coxiella and hgcl_clade were the principal rare bacteria at the genus level. The variety index of rare communities across diverse seasons was notably higher than that of abundant ones (P < 0.01). Bacterial community structure differed between spring and other seasons, and the rare bacterial community exhibited substantial seasonal alterations during non-spring periods. pH, dissolved oxygen (DO), total phosphorus (TP), and soluble reactive phosphorus (SRP) were the predominant environmental factors, exerting an even greater influence on rare bacteria. Within the co-occurrence network, rare bacteria constituted the majority of nodes and connections and were the dominant key species throughout all seasons. The assembly of their community was chiefly deterministic in autumn and random in other seasons. This study indicated that rare bacteria in Sancha Lake were diverse. They were keystone taxa for maintaining community interactions and stable operation, and their assembly process was influenced by both stochastic and deterministic factors.

    Similar content being viewed by others

    Discovery of a novel bacterial class with the capacity to drive sulfur cycling and microbiome structure in a paleo-ocean analog

    Article
    Open access
    18 August 2023

    Seasonal changes of prokaryotic microbial community structure in Zhangjiayan Reservoir and its response to environmental factors

    Article
    Open access
    06 March 2024

    A long-read sequencing approach to high-resolution profiling of bacterioplankton diversity in a shallow freshwater lake

    Article
    Open access
    10 April 2025

    Data availability

    The datasets analysed during the current study are available in the NCBI repository (https://www.ncbi.nlm.nih.gov/). The BioProject accession number is PRJNA1336117.
    ReferencesAlexander, T. J., Vonlanthen, P. & Seehausen, O. Does eutrophication-driven evolution change aquatic ecosystems? Philos. Trans. R. Soc. B-Biol. Sci. 372 (1712), 20160041 (2017).
    Google Scholar 
    Yang, Y., Gao, B., Hao, H., Zhou, H. & Lu, J. Nitrogen and phosphorus in sediments in China: a national-scale assessment and review. Sci. Total Environ. 576, 840–849 (2017).
    Google Scholar 
    Jin, X. et al. Environmental factors influencing the spatial distribution of sediment bacterial community structure and function in Poyang lake. Res. Environ. Sci. 30 (4), 529–536 (2017).
    Google Scholar 
    Zhong, M. et al. Spatial-temporal distribution of bacterial communities and main nutrients driver of sediment in a shallow lake. Earth Environ. Sci. 51 (4), 377–387 (2023).
    Google Scholar 
    Shao, K., Gao, G., Wang, Y., Tang, X. & Qin, B. Vertical diversity of sediment bacterial communities in two different trophic States of the eutrophic lake Taihu, China. J. Environ. Sci. 25 (6), 1186–1194 (2013).
    Google Scholar 
    Xue, Y. et al. The diversity of bacterial communities in the sediment of different lake zones of lake Taihu in winter. China Environ. Sci. 38 (2), 719–728 (2018).
    Google Scholar 
    Gilbert, J. A. et al. Defining seasonal marine microbial community dynamics. ISME J. 6 (2), 298–308 (2012).
    Google Scholar 
    Zhou, J. & Ning, D. Stochastic community assembly: does it matter in microbial ecology? Microbiol. Mol. Biol. Rev. 81 (4), 1–32 (2017).
    Google Scholar 
    Sloan, W. T. et al. Quantifying the roles of immigration and chance in shaping prokaryote community structure. Environ. Microbiol. 8 (4), 732–740 (2006).
    Google Scholar 
    Qi, R. et al. Distinct composition and assembly processes of bacterial communities in a river from the arid area: ecotypes or habitat types? Microb. Ecol. 84 (3), 769–779 (2021).
    Google Scholar 
    Zeng, J. et al. Patterns and assembly processes of planktonic and sedimentary bacterial community differ along a trophic gradient in freshwater lakes. Ecol. Indic. 106, 105491 (2019).
    Google Scholar 
    Jia, X., DiniAndreote, F. & Salles, J. F. Unravelling the interplay of ecological processes structuring the bacterial rare biosphere. ISME Commun. 2 (1), 1–11 (2022).
    Google Scholar 
    Qiu, Z. et al. Large scale exploration reveals rare taxa crucially shape microbial assembly in alkaline lake sediments. Npj Biofilms Microbiomes. 10 (1), 62 (2024).
    Google Scholar 
    Ren, Z., Luo, W. & Zhang, C. Rare bacterial biosphere is more environmental controlled and deterministically governed than abundant one in sediment of thermokarst lakes across the Qinghai-Tibet plateau. Front. Microbiol. 13, 944646 (2022).
    Google Scholar 
    Song, Y. et al. Antibiotic pollution and its effects on the Spatiotemporal variation in microbial community structure and functional genes in sediment of Baiyangdian lake. Environ. Sci. 45 (8), 4904–4914 (2024).
    Google Scholar 
    Haglund, A. L., Lantz, P., Törnblom, E. & Tranvik, L. Depth distribution of active bacteria and bacterial activity in lake sediment. FEMS Microbiol. Ecol. 46 (1), 31–38 (2003).
    Google Scholar 
    Zhou, T. X. et al. Spatial distribution of composition and diversity of aquatic bacterial communities in lake fuxian during vertical stratification period. J. Lake Sci. 34 (5), 1642–1655 (2022).
    Google Scholar 
    Jones, I. D., Winfield, I. J. & Carse, F. Assessment of long-term changes in habitat availability for Arctic charr(Salvelinus alpinus) in a temperate lake using oxygen profiles and hydroacoustic surveys. Freshw. Biol. 53 (2), 393–402 (2008).
    Google Scholar 
    Chen, Y. et al. Spatiotemporal variation characteristics of mixed layer depth and hypoxic zone during the thermal stratification decay period in a Southern Chinese reservoir: a case study of Tianshuiku reservoir in Nanning City. J. Lake Sci. 35 (5), 1623–1634 (2023).
    Google Scholar 
    Jia, B., Fu, W., Yu, J., Zhang, C. & Tang, Y. Relationship among sediment characteristics, eutrophication process and human activities in the Sancha Lake, Sichuan, Southwestern China. China Environ. Sci. 33 (9), 1638–1644 (2013).
    Google Scholar 
    Ruban, V., Brigault, S., Demare, D. & Philippe, A. M. An investigation of the origin and mobility of phosphorus in freshwater sediments from Bort-Les-Orgues Reservoir, France. J. Environ. Monit. 1 (4), 403–407 (1999).
    Google Scholar 
    Ruban, V. et al. Harmonized protocol and certified reference material for the determination of extractable contents of phosphorus in freshwater sediments — a synthesis of recent works. Fresenius J. Anal. Chem. 370 (2), 224–228 (2001).
    Google Scholar 
    Xu, N., Tan, G. C., Wang, H. Y. & Gai, X. Effect of Biochar additions to soil on nitrogen leaching, microbial biomass and bacterial community structure. Eur. J. Soil. Biol. 74, 1–8 (2016).
    Google Scholar 
    Schloss, P. D., Gevers, D. & Westcott, S. L. Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS One 6 (12), e27310 (2011).Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41 (D1), D590–D596 (2013).
    Google Scholar 
    Jiao, S. & Lu, Y. Abundant fungi adapt to broader environmental gradients than rare fungi in agricultural fields. Glob. Chang. Biol. 26 (8), 4657–4668 (2020).
    Google Scholar 
    Hair, J. F. Multivariate Data Analysis: An Overview (Springer, 2011).Wu, P. et al. Unraveling the spatial-temporal distribution patterns of soil abundant and rare bacterial communities in china’s subtropical mountain forest. Front. Microbiol. 15, 1323887 (2024).
    Google Scholar 
    Ling, N. et al. Insight into how organic amendments can shape the soil Microbiome in long-term field experiments as revealed by network analysis. Soil. Biol. Biochem. 99, 137–149 (2016).
    Google Scholar 
    Chen, C., Shen, Q., Zhong, J., Liu, C. & Fan, C. Distribution characteristics of phosphorus in surface sediments during seasons of algae blooms in bloom-accumulation area in the Taihu lake. Resour. Environ. Yangtze River Basin 23 (9), 1258–1264 (2014).
    Google Scholar 
    Zhang, X., Zhao, Y. & Jin, Y. Diversity and phylogenetic analysis of bacteria in sediments of Xiaokou sites in lake Wuliangsuhai. J. Inner Mongolia Agric. Univ. (Nat. Sci. Ed.) 32 (4), 206–212 (2011).
    Google Scholar 
    Welch, D. B. M. & Huse, S. M. Microbial diversity in the deep sea and the underexplored rare biosphere. In Handbook of Molecular Microbial Ecology II: Metagenomics in Different Habitats. 243–252 (2011).Li, Y. et al. Divergent adaptation strategies of abundant and rare bacteria to salinity stress and metal stress in polluted Jinzhou Bay. Environ. Res. 245 (Mar.), 118030 (2024).
    Google Scholar 
    Chu, B. T. T. et al. Metagenomics reveals the impact of wastewater treatment plants on the dispersal of microorganisms and genes in aquatic sediments. Appl. Environ. Microbiol. 84 (5), e02168–e02117 (2018).
    Google Scholar 
    Yuan, W. et al. The vertical distribution of bacterial and archaeal communities in the water and sediment of lake Taihu. FEMS Microbiol. Ecol. 71 (2), 263–276 (2010).
    Google Scholar 
    Keshri, J., Pradeep Ram, A. S. & Sime-Ngando, T. Distinctive patterns in the taxonomical resolution of bacterioplankton in the sediment and pore waters of contrasted freshwater lakes. Microb. Ecol. 75 (3), 662–673 (2017).
    Google Scholar 
    Cheng, H. et al. Changes of bacterial communities in response to prolonged hydrodynamic disturbances in the eutrophic water-sediment systems. Int. J. Environ. Res. Public. Health. 16 (20), 3868 (2019).
    Google Scholar 
    Liu, L., Yang, J., Yu, Z. & Wilkinson, D. M. The biogeography of abundant and rare bacterioplankton in the lakes and reservoirs of China. ISME J. 9 (9), 2068–2077 (2015).
    Google Scholar 
    Jiao, C. et al. Abundant and rare bacterioplankton in freshwater lakes subjected to different levels of tourism disturbances. Water 10 (8), 16 (2018).
    Google Scholar 
    Jiao, S., Chen, W. & Wei, G. Biogeography and ecological diversity patterns of rare and abundant bacteria in oil-contaminated soils. Mol. Ecol. 26 (19), 5305–5317 (2017).
    Google Scholar 
    Zhang, W. et al. The diversity and biogeography of abundant and rare intertidal marine microeukaryotes explained by environment and dispersal limitation. Environ. Microbiol. 20 (2), 462–476 (2018).
    Google Scholar 
    Dang, C. et al. Rare biosphere regulates the planktonic and sedimentary bacteria by disparate ecological processes in a large source water reservoir. Water Res. 216, 118296 (2022).
    Google Scholar 
    Yu, X. et al. Seasonal changes of prokaryotic microbial community structure in Zhangjiayan reservoir and its response to environmental factors. Sci. Rep. 14 (1), 1–14 (2024).
    Google Scholar 
    Wan, Y., Ruan, X., Zhang, Y. & Li, R. Illumina sequencing-based analysis of sediment bacteria community in different trophic status freshwater lakes. Microbiol. Open 6(4), e00450 (2017).Yin, X. et al. Composition and predictive functional analysis of bacterial communities in surface sediments of the Danjiangkou reservoir. J. Lake Sci. 30 (4), 1052–1053 (2018).
    Google Scholar 
    Winters, A. D., Marsh, T. L., Brenden, T. O. & Faisal, M. Molecular characterization of bacterial communities associated with sediments in the Laurentian great lakes. J. Gt Lakes Res. 40 (3), 640–645 (2014).
    Google Scholar 
    Goberna, M. & Verdú, M. Cautionary notes on the use of co-occurrence networks in soil ecology. Soil. Biol. Biochem. 166, 108534 (2022).
    Google Scholar 
    De Vries, F. T. et al. Soil bacterial networks are less stable under drought than fungal networks. Nat. Commun. 9 (1), 1–12 (2018).
    Google Scholar 
    Lynch, M. D. J. & Neufeld, J. D. Ecology and exploration of the rare biosphere. Nat. Rev. Microbiol. 13 (4), 217 (2015).
    Google Scholar 
    Li, X. et al. Comparing diversity patterns and processes of microbial community assembly in water column and sediment in lake Wuchang, China. PeerJ 11, e14592 (2023).
    Google Scholar 
    Download referencesAcknowledgementsThis research was funded by Student Research Training Program(242005).FundingThis research was funded by Student Research Training Program(242005).Author informationAuthors and AffiliationsSchool of Environmental Science and Engineering, Southwest Jiaotong University, Chengdu, 610031, ChinaYong Li, Yajie Li, Yihan Wu, Zuguang Liu, Shiqi Luo & Sidan GongAuthorsYong LiView author publicationsSearch author on:PubMed Google ScholarYajie LiView author publicationsSearch author on:PubMed Google ScholarYihan WuView author publicationsSearch author on:PubMed Google ScholarZuguang LiuView author publicationsSearch author on:PubMed Google ScholarShiqi LuoView author publicationsSearch author on:PubMed Google ScholarSidan GongView author publicationsSearch author on:PubMed Google ScholarContributionsY.L. writing – review & editing, investigation, conceptualization. Y.L. and Y.W. writing – original draft, visualization, project administration. Z.L. and S.L. writing – original draft, visualization. S.G. and Y.L. methodology, investigation. All authors read and approved the final manuscript.Corresponding authorCorrespondence to
    Yong Li.Ethics declarations

    Competing interests
    The authors declare no competing interests.

    Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationBelow is the link to the electronic supplementary material.Supplementary Material 1Rights and permissions
    Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
    Reprints and permissionsAbout this articleCite this articleLi, Y., Li, Y., Wu, Y. et al. Structure and community assembly of rare bacterial community in sediments of Sancha Lake.
    Sci Rep (2025). https://doi.org/10.1038/s41598-025-31889-zDownload citationReceived: 08 April 2025Accepted: 05 December 2025Published: 24 December 2025DOI: https://doi.org/10.1038/s41598-025-31889-zShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
    Provided by the Springer Nature SharedIt content-sharing initiative
    KeywordsSancha Lake sedimentsEutrophicationRare bacteriaDiversityCommunity assembly More

  • in

    Social familiarity shapes collective decision-making in response to looming stimuli in Medaka fish

    AbstractSocial familiarity within groups promotes behavioural synchrony and facilitates information transfer. Whether it shapes collective decision-making under predator threat is unknown. Here groups of six medaka (Oryzias latipes) familiarised for one month were used to test whether familiarisation promotes instantaneous collective decision-making in response to a looming stimulus (LS) mimicking a predator attack. First, we analysed behavioural transitions, defined as changes among three behavioural states: high-speed, normal and freezing-like before, during, and after LS in groups of six individuals. Individuals showing high-speed state in response to LS typically tended to shift to freezing-like state afterwards, whereas non-responders were more likely to maintain normal state. Group-level analysis revealed a bimodal distribution in the number of individuals exhibiting freezing-like state, with peaks at zero and six individuals, corresponding to ‘all non-freezing’ and ‘all-freezing’. Clustering analysis further identified three consistent group profiles: ‘freezing-dominant’, ‘non-freezing-dominant’, and ‘mixed-type’ based on behavioural tendencies across 10 trials. In contrast, in unfamiliar groups assembled immediately before testing, the ‘freezing-dominant’ profile was absent, and the distribution in the number of individuals exhibiting freezing-like state shifted to unimodal. In these groups, even at the individual level, responses more often showed a transition from high-speed state to normal state rather than freezing-like state. The results indicate that social familiarity promotes synchronous freezing-like state and consensus decisions under looming threat. Our study presents a behavioural assay for predator-evoked collective decision-making in a genetic model fish, providing a framework for future efforts to link behavioural ethology with neuroscience.

    Similar content being viewed by others

    Optimization, purification, and characterization of xylanase production by a newly isolated Trichoderma harzianum strain by a two-step statistical experimental design strategy

    Article
    Open access
    22 October 2022

    IoT and ML approach for ornamental fish behaviour analysis

    Article
    Open access
    05 December 2023

    Comparison of anxiety-like and social behaviour in medaka and zebrafish

    Article
    Open access
    28 June 2022

    IntroductionAnimals living in groups often exhibit synchronous behaviour in contexts such as migration, foraging, and predator avoidance. The emergence of coordinated behavioural patterns through interactions among individuals is termed collective behaviour, where group-level order and synchrony are thought to arise from local rules at the individual level, including attraction, alignment, and repulsion1. Collective behaviour includes collective decision-making, in which a group selects a single option from among multiple alternatives, and this phenomenon is observed in various contexts such as movement2,3, foraging4, and predator evasion5,6,7.In the context of predator avoidance, consensus formation during collective decision-making has been studied across various species. For instance, in sticklebacks, once the number of individuals escaping in a particular direction exceeds a threshold, the remaining group members tend to follow6. Similarly, simulations involving humans have shown that when a critical number of escape responses are observed, the group tends to adopt avoidance behaviour5. In elephants, the oldest female has been reported to be particularly sensitive to predator vocalisations and to influence the group’s decision to flee7. However, few studies have quantitatively investigated collective decision-making as an instantaneous group response under emergency conditions. In particular, how dynamic group-level responses to a rapidly approaching predator emerge remains poorly understood. While mechanisms underlying rapid individual decision-making in response to visual looming stimuli have been demonstrated8, systematic analyses of dynamic group-level responses remain scarce.Social familiarity has also been shown to affect individual recognition and interaction patterns, thereby influencing behaviour and information transmission9. At the dyadic level, social familiarisation enhances responsiveness to predators in predatory mites and reduces encounter frequency10. In sticklebacks, familiarisation reduced leadership tendencies in bold individuals, leading to more balanced coordination11. In cichlids, social familiarity promotes exploratory behaviour and reduces fear responses to novel stimuli12, while in zebrafish, the transmission of social fear is enhanced among familiar individuals13. At the group level, wild female guppies tend to associate with familiar individuals14, avoidance frequency in response to predator odour increases in fathead minnows15, and the latency to initiate avoidance of a predator model is reduced in brown trout16. In tropical damselfish, both responsiveness to fear stimuli and inter-individual information transmission are enhanced through familiarisation17. Overall, the literature indicates that social familiarity enhances alignment coordination and information transmission; however, how these factors influence collective decision-making remains unclear.Furthermore, most empirical research in this field has relied on observations in natural environments or on wild individuals6,15, yet relatively few studies have been conducted in controlled experimental settings. Although collective decision-making regarding movement direction has been demonstrated in zebrafish18, reports of such decisions in response to predators are lacking. Moreover, integrative frameworks that link decision-making mechanisms at both the individual and group levels with molecular and neural analysis, particularly in genetically tractable model organisms, are still lacking.To address these gaps, we focused on medaka Oryzias latipes, a well-established model organism in molecular genetics. Medaka are known to exhibit coordinated behaviour with conspecifics19 and to improve foraging efficiency through visual social learning20. Preliminary observations revealed that small groups of medaka responded synchronously to a human approach, either by showing ‘freezing after escape’ or by maintaining continuous movement without escape. Motivated by these findings, we aimed to develop a behavioural assay capable of quantitatively assessing instantaneous collective decision-making using a looming stimulus (LS) mimicking the sudden approach of a predator. LS has been used to elicit individual avoidance responses in mice21, zebrafish22, and fruit flies23. However, most previous studies have focused on individual-level decision-making8.Even in group contexts, the focus has remained on how individual responses are influenced by conspecifics23,24, with little attention paid to whether the group as a whole converges on a single collective choice.In this study, we optimised LS parameters and established a quantitative behavioural system capable of replicating the distinct response patterns during preliminary observations. We then tested whether medaka groups exhibit instantaneous collective decision-making in response to LS and examined the effects of social familiarity on the decision-making patterns. Our findings establish an experimental model for examining collective decisions under acute threat. They also lay the foundation for elucidating how familiarisation modulates synchrony and group-level decision-making processes in a genetically accessible model organism.MethodsEthics statementAll the methods in this study were carried out in accordance with relevant guidelines and regulations. The work in this paper was conducted using protocols specifically approved by the Animal Care and Use Committee of Tohoku University (permit number: 2022LsA-003). All efforts were made to minimise suffering following the NIH Guide for the Care and Use of Laboratory Animals. Fish and breeding conditions are described above. The study was carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org/arrive-guidelines).AnimalsMedaka (Oryzias latipes, fading strain) were obtained from Dr Tetsuro Takeuchi (Fig. 1a)25. This strain gradually loses body pigmentation in different body parts and at different timing among individuals, which initially appeared suitable for individual identification based on body colour. However, we later found that distinguishing individuals within a group of six remained difficult. During group observations, we consistently noted characteristic and reproducible collective behavioural patterns. As this strain has been maintained as a closed colony with limited genetic variation, we considered it an appropriate model for investigating the mechanisms underlying such stable group-level behavioural patterns. All individuals were hatched and bred in our laboratory. Medaka fish were maintained in groups of six individuals in plastic aquariums (22.6 cm× 14.6 cm× 14.5 cm, Sanko) or custom-made acrylic aquariums(22 cm × 14.5 cm × 14.5 cm) under controlled temperature (26 ± 1 °C) and light (14 h: 10 h light: dark) conditions. Every day, fish were fed brine shrimp between 12:00 and 13:00 and solid bait Otohime β−2 (Marubeni Nissin Feed, Tokyo, Japan) at least twice around 10:00 and 17:00 on weekdays. This study used individuals aged 2–9 months post hatch.Fig. 1Experimental design, state transition analysis, and statistical evaluation of freezing-like behaviour in medaka after looming stimulus. (a) Medaka fish (fading strain) (b) Groups of medaka were transferred, with their tanks, from the circulating water system to the apparatus after feeding. One hour later, the looming stimulus (LS) was presented five times at 30-minute intervals. The experiment was conducted over two consecutive days, giving ten LS. For analysis, three 10-second periods (before, during, and after each LS) were used, totalling 30 s per trial. (c) A state transition diagram visualises individual-level states (FS: freezing-like state, NS: normal state, and HS: high-speed state) across three intervals: before, during, and after LS. Node size represents the proportion of individuals, and numbers within nodes indicate counts. Node colours are red for HS, light blue for NS, and grey for FS. Numbers on edges indicate the transition probabilities, and edge thickness corresponds to the number of individuals. Edge colours indicate the originating state: red for transitions from HS, blue from NS, and grey from FS. (d) A bar plot showing the frequencies of transition patterns across the three intervals. Each pattern is categorised according to the sequence of states. Red bars indicate transitions to HS during LS followed by FS after LS. Blue bars indicate individuals that remained NS during and after LS. Grey bars represent all other patterns. To assess statistical significance, a binomial test with false discovery rate (FDR) correction was applied. Patterns with q < 0.001 are marked with ***. (e) The X-axis shows the number of individuals in FS after LS, and the Y-axis shows its frequency. The blue and red lines represent the observed data (17 groups) and the simulated data (17 groups × 1000 trials, seed = 1, …, 1000), respectively. A chi-square test result is shown in the graph (χ² (6) = 147, p < 0.001).Full size imageBehavioural experiments under familiar conditionsMedaka (Oryzias latipes) aged either one month or nine months were randomly selected to form groups. Ten groups of six individuals each were formed from one-month-old fish (N = 60), and seven groups were formed from nine-month-old fish (N = 42). These groups were formed without regard to sex, because the sex ratio of one-month-old fish could not be reliably determined. In contrast, the nine-month-old groups were adjusted to achieve a 1:1 sex ratio. Each group of six individuals was maintained separately for a month. In total, 17 groups of sexually mature individuals aged either two months or ten months that had undergone familiarisation, were used for experiments.Behavioural experiments under unfamiliar conditionsMedaka aged 4 or 9 months, reared in a recirculating aquaculture system, were randomly selected. From the 4-month-old fish (N = 36), six groups were formed, and from the 9-month-old fish (N = 36), another six groups, each consisting of six individuals, yielded a total of 12 groups. The sex ratio in each group was adjusted to 1:1. Group formation took place in the morning, and experiments were conducted 1 to 2 h after the afternoon feeding. Subsequent procedures were conducted in accordance with those described in the familiarisation experiments.Looming stimulus (LS)The looming stimulus (LS) was a visual stimulus mimicking an approaching predator, created using the “Zoom” animation function in Microsoft PowerPoint (Figure S1). The stimulus expanded to a width of 14.5 cm over 5.5 s, gradually darkened over 10 s, and remained black for 3 min (Figure S1). It was presented on an LCD monitor (EXLDH271DB, I-ODATA) mounted on the side of the aquarium (Figure S2). Both plastic and acrylic aquaria, also used as breeding tanks, were employed. Approximately 1–2 h after daytime feeding (12:00–13:00), each group was transferred in its breeding tank directly to the behavioural testing apparatus and the water level was adjusted to 6 cm. The LS was presented five times at 30-minute intervals for two consecutive days, beginning 1 h after transferring the aquarium from the rearing system to the testing apparatus (Fig. 1b).Behavioural recording and trackingBehaviour was recorded from above using an action camera (M80 Air, Apexcam, or HERO8, GoPro) at a resolution and frame rate of 4 K (30 fps) or 2.7 K (50 fps). Recordings lasted 5 min, beginning 2 min before the LS and ending 3 min after. For analysis, a 30-s segment was extracted for each trial: 10 s before, during, and after LS (Fig. 1b). Video files were extracted using QuickTime Player, converted to JPEG format using FFMPEG (v4.4.1), and subsequently converted to MP4 format at 5 fps. Tracking was performed using UMATracker26, and coordinate data were obtained with the UMATracker-Tracking tool, applying either the Pochi-Pochi (manual positioning) or Group Tracker GMM algorithm. Tracking errors, such as identity swaps, were manually corrected using UMATracker-TrackingCorrector.To convert pixel values to centimetres, the number of pixels along the centre of the long side of the aquarium was measured in ImageJ, which was based on the actual inner length (20.0–20.5 cm). Velocity (cm s⁻¹) was calculated from coordinate data (5 fps). A velocity matrix (6 individuals × 10 trials × 17 groups; 1020 × 150 frames) was compiled, and a moving average was applied (window size = 5) using pandas v1.4.0 to smooth short-term fluctuations.Definition of behavioural types and statesTo capture how individual fish responded to LS (looming stimulus) in terms of state transitions, we expressed behavioural responses as transition patterns across three intervals (before, during, and after LS). In total, 17 groups of six individuals each (N = 102) were tested. For each group, fish were transferred to the experimental arena and habituated for one hour, after which the looming stimulus was presented five times at 30-minute intervals over two consecutive days (Fig. 1b). This protocol yielded a total of 6 individuals × 10 trials × 17 groups of individual-level datasets for subsequent analyses. For this purpose, we first defined behavioural types for each interval based on velocity data. Using histograms and kernel density estimation (KDE) curves of velocity, we categorised behaviour into three types: ‘freezing-like behaviour’, ‘normal swimming’, and ‘high-speed swimming’. The rationale for these definitions is as follows. Some individuals exhibited freezing-like behaviour after LS. The velocity histogram for the post-LS interval showed a bimodal distribution with a trough at approximately 0.2 cm/s (Figure S3a). Therefore, frames with speeds below 0.2 cm/s were defined as ‘freezing-like behaviour’. During LS, escape-like responses characterised by high-speed swimming were observed. Such behaviours were rarely seen before the LS onset. Comparison of KDE curves for the pre-LS and LS intervals revealed minimal overlap above 6 cm/s (Figure S3b). Thus, frames with speeds of 6 cm/s or higher were defined as ‘high-speed swimming’. Frames with velocities between 0.2 cm/s and 6 cm/s were categorised as ‘normal swimming’. All histograms, KDE curves, and heat maps were generated using Python v3.8 and matplotlib v3.7.5.Based on speed-based behavioural types, we then defined the behavioural state for each 10-second interval (before LS, during LS, and after LS). An interval was categorised as a ‘freezing-like state (FS)’ if freezing-like behaviour persisted for ≥ 8 s, and as a ‘normal state (NS)’ if freezing-like behaviour lasted for < 2 s. During LS, escape behaviour occurred rapidly. Therefore, if high-speed swimming (≥ 6 cm/s) was sustained for 0.2 s (equivalent to one frame at 5 fps), we defined this as a ‘high-speed state (HS)’. This threshold reflects the minimum temporal resolution required to identify continuous motion.Statistical analysisCharacterisation of state transition patterns at the individual levelTo calculate the state transition probabilities for behavioural transitions before, during, and after LS, we constructed state transition matrices by counting the number of transitions between states and normalising each row, following established methods using Markov chain analysis27,28. To visualise the transition dynamics, we created state transition diagrams using python-graphviz v0.20.3, where each state (NS, FS, and HS) was represented by a node, with edges indicating transition probabilities.Furthermore, we used a binomial test to compare whether there were significantly more specific state transition patterns in the series of flows from before LS to after LS. In the binomial test, we set the null hypothesis that ‘the 27 behavioural patterns occur with equal probability (1/27)’ and performed a one-sided test. To control for type I errors due to multiple comparisons, we applied FDR correction to the binomial test results.Analysis of group-level freezing-like states after LSBased on the analysis of individual-level behavioural patterns, we next examined group-level freezing-like states (FS) after LS. For each trial, the number of individuals in the FS after LS was counted. To test whether synchronous FS occurred, virtual datasets were generated by randomly shuffling the states of each trial among groups (17 groups × 1,000 trials; seeds = 1, 2, …, 1,000). The proportions of individuals in each state across all trials were compared between the virtual datasets and the observational data using a chi-square test (scipy v1.10.1). The null hypothesis was defined as: “The presence or absence of the FS for each individual is independent, and synchronous FS for the entire group occur at random.”Classification of group response profilesTo classify these characteristics, we performed a principal component analysis (PCA) on 27 individual-level behavioural patterns from before to after the LS intervention. We then calculated the cumulative contribution rate and reduced the number of dimensions to the minimum required to explain > 95% of the variance. To visualise the cluster structure, we further projected the PCA-reduced data using UMAP29 (umap-learn v0.5.7). Classification was performed using spectral clustering (scikit-learn v1.2.2), and the optimal number of clusters was determined based on the silhouette coefficient. This coefficient approaches 1 when intra-cluster cohesion and inter-cluster separation are high; therefore, the number of clusters yielding the highest silhouette coefficient was selected.Comparison of state transition patterns at the individual level between clustersDifferences in the frequency of state transition patterns between clusters were evaluated using binomial tests with FDR correction, as described above.Analysis of group-level freezing-like statesTo examine differences in group-level freezing-like states (FS) across clusters and between familiar and unfamiliar groups after LS, we applied a generalized linear mixed model (GLMM)30. The dependent variable was the number of individuals exhibiting the FS (0–6) within each group. Cluster identity and the presence or absence of familiarisation were included as fixed effects. Experimental group identity, trial number, and group identity were incorporated as random effects to account for repeated measurements and inter-group variability. The model assumed a binomial distribution with a logit link function, which is appropriate for categorical or count data with hierarchical structure. Analyses were performed in Python v3.8.12. We used pyper v1.1.2 to call R v4.1.2, and the lme4 and multcomp packages for model fitting and post hoc tests. Tukey’s method was applied for multiple comparison correction.ResultsDetection of individual-level state transition characteristicsTo examine how individuals responded to the looming stimulus (LS), we analysed behavioural transitions across three intervals: before, during, and after LS. Fish behaviour was first classified into three types based on swimming velocity: freezing-like (< 0.2 cm/s), normal (0.2–6 cm/s), and high-speed (≥ 6 cm/s). Each 10-second interval was then categorized into one of three behavioural states—freezing-like, normal, or high-speed—according to duration thresholds (≥ 8 s freezing, < 2 s freezing, and ≥ 0.2 s high-speed). These definitions enabled consistent identification of state transitions across trials. Using these thresholds, each of the three temporal intervals was classified into one of the three behavioural states: ‘freezing-like state (FS)’, ‘normal state (NS)’, or ‘high-speed state (HS)’ (Figure S4).To examine how the behavioural states of individuals transitioned from before LS to during LS, and from during LS to after LS, we calculated state transition probabilities using a Markov chain and visualised them as a state transition diagram (Fig. 1c). Between the pre-LS and LS intervals, 39% of individuals transitioned from NS to HS, whereas 60% remained in NS. Among individuals that entered HS during LS, 70% transitioned to FS after LS. In contrast, individuals that remained in the NS during LS had a 76% probability of continuing in that state after LS. These findings suggest that individuals showing escape-like behaviour during LS tended to transition into FS after LS, whereas those unresponsive to LS generally maintained NS.To statistically evaluate trends in state transition patterns, we extracted behavioural state sequences across the three intervals (before, during, and after LS). Each sequence was expressed as a combination of the three defined behavioural states: HS, NS, and FS, resulting in 27 possible transition patterns. We quantified the frequency of each pattern across trials (Fig. 1d).The most frequent transition pattern was the maintenance of NS throughout the three intervals (NS→NS→NS; blue; q < 0.001). The second most frequent pattern was NS→HS→FS (red; q < 0.001), and the third was FS→HS→FS (red; q < 0.001), both involving a transition to HS during LS followed by FS: NS→HS→FS (red; q < 0.001) and FS→HS→FS (red; q < 0.001). Additional significantly overrepresented patterns were NS→HS→NS (grey; q < 0.001), NS→HS→FS (grey; q < 0.001), and FS→NS→NS (blue; q < 0.001), all of which exceeded the expected frequency under a uniform distribution (1/27).Among these six prominent transition patterns (Fig. 1d), NS→NS→NS and FS→NS→NS represent non-reactive behaviours where individuals maintained or returned to the NS during and after LS (blue). In contrast, NS→HS→FS and FS→HS→FS represent reactive responses, characterised by HS during LS followed by FS (red). These two reactive patterns accounted for 41% and 30% of all observations, respectively, and thus constitute the typical individual-level responses to LS stimulation. Notably, escape without subsequent FS (NS→HS→NS; approx. 9%) and no initial response followed by FS after LS (NS→NS→FS; approx. 8%) were also observed at appreciable frequencies.Population polarisation into synchronous freezing-like and non-freezing-like states after LSTo investigate whether medaka groups exhibited synchronous responses (either FS or non-FS) after LS, we counted the number of individuals exhibiting FS in each trial. The distribution was bimodal, with peaks at 0 and 6 individuals (Fig. 1e, blue line), suggesting that entire groups tended to respond uniformly.To determine whether this distribution could be explained by chance, we generated a virtual dataset by randomly shuffling the individual-level FS and non-FS classifications within groups of the same size (Fig. 1e, red line). This reconstructed the expected distribution under the assumption that individuals responded independently of one another. A chi-square test comparing the observed and expected distributions revealed a significant difference (χ² (6) = 147, p < 0.001). This result suggests that the strong bias towards either ‘all-freezing’ or ‘all non-freezing’ within groups after LS is unlikely to have occurred by chance alone. Instead, it indicates that individuals within a group reacted in a synchronous manner through social interaction.Group response profiles to LS classified into three typesDuring the behavioural experiments, we observed groups in which all individuals synchronously exhibited FS, as well as groups in which all individuals remained unresponsive and continued swimming. Moreover, the same groups tended to display similar response tendencies across repeated trials. Based on these preliminary observations, we hypothesised that groups exhibit consistent and characteristic behavioural tendencies, which we define as group response profiles. To evaluate this hypothesis, we classified groups according to their individual-level behavioural patterns. Specifically, behavioural data from 10 trials per group were aggregated, dimensionality reduction was performed using principal component analysis (PCA) followed by UMAP, and spectral clustering was applied to classify the groups.PCA indicated that 16 dimensions were required to exceed a cumulative variance contribution of 95%, and this was adopted as the optimal dimensionality (Figure S5). The reduced data were then projected into two dimensions using UMAP, revealing a clear distinct group-level structures (Fig. 2g). Spectral clustering, guided by the silhouette coefficient identified three as the optimal number of clusters (Figure S6). Accordingly, groups were classified into three clusters (Figure S7).Fig. 2State transition diagrams and frequencies of behavioural state transition patterns for each cluster. (a–c) State transition diagrams for each cluster were generated to represent individual-level behavioural states (FS: freezing-like state, NS: normal state, and HS: high-speed state) before, during, and after LS. All the visual elements are consistent with those in Fig. 1c. (a) State transition diagram for Cluster 0. b) State transition diagram for Cluster (1) (c) State transition diagram for Cluster (2) (d–f) Bar graphs showing the frequency of occurrence for each behavioural state transition pattern in each cluster. Details of colour coding and statistical tests are as described in Fig. 1d. (d) Distribution of transition pattern frequencies in Cluster 0. (e) Distribution of transition pattern frequencies in Cluster (1) (f) Distribution of transition pattern frequencies in Cluster (2) (g) The X-axis represents the first UMAP component and the Y-axis the second. Each point shows the group centroid, obtained by reducing the original 23 dimensions to 16 dimensions using PCA and further to two dimensions using UMAP. Colours indicate classification results based on spectral clustering. (h) The X-axis represents the number of individuals in the freezing-like state after LS, and the Y-axis represents the frequency of these counts across all trials. The lines correspond to the IDs of the three clusters. Tukey’s post hoc test based on a GLMM was performed, and the results of the cluster comparisons are shown within the graph.Full size imageTo analyse state transitions of individuals across the pre-, during-, and post-LS intervals in each cluster, we calculated state transition probabilities using a Markov chain and visualised them as state transition diagrams (Fig. 2a-c).In Cluster 0 (Fig. 2a), the probability of transitioning from NS to HS from before LS to during LS was 72.5%, while the transition probability from FS to HS was 76%. Individuals that exhibited HS during LS had an 89% probability of subsequently transitioning to the FS. In addition, even individuals that remained in NS during LS had an 80.4% probability of transitioning to FS afterwards. These results suggest that in Cluster 0, both responsive (HS) and unresponsive (NS) individuals tended to synchronise into a FS after LS. The most frequent pattern was NS→HS→FS (red; q < 0.001), and the second was FS→HS→FS (red; q < 0.001), both involving a transition to HS during LS followed by FS (Fig. 2d). These findings suggest that Cluster 0 corresponds to groups in which all individuals synchronise and exhibit FS after LS.In Cluster 1 (Fig. 2b), the probability of maintaining the NS from before LS to during LS was 86.2%, and individuals that remained unresponsive during LS continued NS after LS with a probability of 94.2%. The dominant patterns were those where individuals maintained NS throughout (NS→NS→NS and FS→NS→NS, q < 0.001; Fig. 2e, blue). These results suggest that Cluster 1 corresponds to groups in which all individuals synchronise and maintain NS after LS.In Cluster 2 (Fig. 2c, f), NS→NS→NS remained the most frequent pattern (q < 0.001). However, various other transitions were also observed, including NS→HS→NS (q < 0.001; grey), NS→HS→FS (q < 0.001; red), and NS→NS→FS (q < 0.001; grey). This diversity of transitions indicates that Cluster 2 represents a heterogeneous group response profile, reflecting a mixture of multiple individual-level response types rather than a single dominant pattern.Synchronisation of freezing-like and non-freezing-like states across the group profilesIndividual-level state transition analysis revealed that in Cluster 0, individuals frequently responded to the looming stimulus (LS) and then entered FS, whereas in Cluster 1, transitions in which individuals did not respond to LS and continued NS were predominant. We next examined whether all individuals in Cluster 0 synchronised to exhibit FS, and whether all individuals in Cluster 1 synchronised to continue NS. To this end, we counted the number of individuals in FS after LS for each group and compared these counts across clusters. A significant difference was observed in the number of individuals exhibiting FS (Fig. 2h, p < 0.001). In Cluster 0, the most frequent outcome was that all six individuals showed FS, whereas in Cluster 1, the most likely outcome was that no individual showed FS. In Cluster 2, the number of individuals exhibiting FS ranged mostly from zero to three, yielding a distribution distinct from both Clusters 0 and 1. These results indicate that in Cluster 0, individuals tended to synchronise to FS, whereas in Cluster 1 they synchronised to non-FS (continued NS). In contrast, Cluster 2 showed no clear synchronisation, with only a subset of individuals exhibiting FS after LS.Individual-level differences in behavioural transition patterns between familiar and unfamiliar groupsWe determined the behavioural patterns of individuals in the unfamiliar group (Figure S7-8), constructed a state transition diagram (Fig. 3a), and classified individual-level transition patterns into 27 categories, comparing their frequencies of occurrence (Fig. 3b). As in the familiar groups, the most frequent pattern was the non-reactive type (NS→NS→NS, blue; q < 0.001). The pattern (NS→HS→NS, grey) in which fish transitioned to HS during LS and returned to NS afterwards also appeared at a significantly high frequency (q < 0.001). In addition, the pattern in which fish transitioned to HS during LS and then entered FS after LS (NS→HS→FS, red) occurred significantly more often (q < 0.05). However, the patterns in which individuals entered FS after LS (NS→HS→FS, red; NS→NS→FS, grey), which were significantly enriched in the familiar groups, did not reach significance in the unfamiliar group. These findings suggest that individual-level transition patterns differed between the two conditions.Fig. 3State transition diagram of individual-level behaviour (Unfamiliar group). (a) For individual-level behaviours classified as FS: freezing-like state, NS: normal state, or HS: high-speed state, the state transition probabilities were shown from before to during LS and from during to after LS using a Markov chain. Details are as described in Fig. 1c. (b) Bar graphs showing transition patterns and their frequencies from before LS to during and after LS for FS, NS, and HS. Statistical significance is denoted as follows: ***q < 0.001, **q < 0.01, *q < 0.05, and no notation for q > 0.05. Details are as described in Fig. 1d.Full size imageAbsence of group-level synchronous freezing-like state in the unfamiliar groupTo test whether individuals exhibited synchronous responses after LS, we counted the number of individuals in FS per trial for each group and compared the observed data with control data generated by virtual shuffling, as in the familiar groups (Figure S9). In the observed data, the number of freezing-like individuals peaked at zero, and there was a significant difference in both the number and frequency of freezing-like individuals between the observed and virtual data (χ² (6) = 22.1, p < 0.01) (Figure S9). These results indicate that the peak at zero was not coincidental, but rather that all individuals within the unfamiliar group tended to exhibit synchronous non-FS, continuing to NS after LS.Differences in synchronous freezing-like states between familiar and unfamiliar groupsTo examine whether the occurrence of FS at the group level after LS differed depending on familiarisation, the number of individuals exhibiting FS per trial was counted for each group. The aggregated group-level data were then compared between the familiar and unfamiliar groups using GLMM (Figure S10). The results showed that, following LS, the familiar groups tended to have a higher number of individuals exhibiting FS (Figure S10, β = 2.02, p < 0.05) and displayed a bimodal distribution. In contrast, the unfamiliar group showed fewer freezing-like individuals and exhibited a unimodal distribution. This indicates that, unlike the familiar groups, the unfamiliar group lacked the peak where all six individuals exhibited FS after LS.Disappearance of the collective freezing in the unfamiliar groupTo clarify similarities and differences in collective behavioural patterns between familiar and unfamiliar groups, we integrated and analysed data from both conditions. Specifically, we performed dimensionality reduction and clustering based on 27 individual-level behavioural transition patterns. Principal component analysis (PCA) revealed that 17 dimensions were required to explain 95% of the variance, which was therefore set as the optimal number (Figure S11). The silhouette coefficient indicated that the optimal number of clusters was three (Figure S12). The clustering results were visualised using a two-dimensional UMAP embedding derived from the 17 principal components and classified into three clusters by spectral clustering (Fig. 4a). Groups in the familiar condition were distributed across all three clusters, whereas the unfamiliar groups were absent from Cluster 0, indicating a clear bias (Fig. 4b). We next verified that in Cluster 0, all individuals tended to exhibit synchronous FS, while in Cluster 1 they tended to exhibit synchronous non-FS (continued NS). In Cluster 2, synchrony was absent, with only a subset of individuals showing FS after LS. Importantly, the classification showed that the unfamiliar groups were not represented in Cluster 0, showing that the ‘freezing-dominant’ cluster was absent from their collective behavioural profiles (Fig. 4c). To evaluate whether this disappearance of the freezing-dominant response could be attributed to the immediate formation of unfamiliar groups, we compared the distributions of the number of freezing-like individuals between groups tested on the first and the following day. Although a significant difference was detected (χ² (6) = 14.2, p = 0.028), this was mainly due to a slight increase in groups with two or four freezing-like individuals, whereas the frequency of ‘all-freezing’ remained almost unchanged (Figure S13). These results suggest that handling or grouping stress immediately after formation had little effect, and that the disappearance of freezing-dominant is a robust feature of unfamiliar groups.Fig. 4Visualisation of dimensionality reduction using PCA and UMAP, and comparison of freezing-like states across clusters. (a–b) The X-axis represents the first UMAP component and the Y-axis the second. Each point corresponds to the group centroid. (a) Colours indicate cluster IDs obtained by spectral clustering. (b) Colours indicate familiarisation status: familiar groups (blue) and unfamiliar groups (orange). (c) Distribution of freezing-like states (FS) after LS exposure in the integrated dataset combining familiar and unfamiliar groups. Details are as described in Fig. 2h.Full size imageDiscussionIn this study, we established a quantitative behavioural assay to analyse collective decision-making in medaka (Oryzias latipes) in response to a looming stimulus (LS). In this system, small groups of medaka were presented with an LS that mimicked an approaching predator, and their collective behavioural choices were examined. Two dichotomous collective response patterns consistently emerged at the group level: ‘all-freezing’ and ‘all non-freezing’. Furthermore, the distribution of the number of FS individuals per trial was bimodal, with clear peaks at either zero or six individuals. These results demonstrate the presence of a dichotomous collective behavioural choice in medaka and validate as a robust tool for investigating collective decision-making under controlled laboratory conditions. Moreover, this assay will provide a platform for elucidating the genetic and neural bases of collective decision-making in vertebrates.Previous studies of collective decision-making under laboratory conditions have primarily used small fish species, such as sticklebacks and golden shiners2,4,6, often focusing on wild populations in ecological contexts. By contrast, our study employed medaka, a well-established genetic model organism, thereby enabling experimental systems in which genetic and environmental factors can be controlled. This approach enables the establishment of highly reproducible behavioural assays and allows for long-term monitoring of behavioural development, from the individual to the group level.Most previous studies of collective decision-making have focused on gradual responses to predators6,7. In contrast, our findings revealed a novel phenomenon: rapid collective responses to sudden visual threats. In coral reef fishes, escape responses of individuals can be predicted from the expansion rate of looming stimuli or the behaviour of neighbours24. However, how these individual responses converge into synchronous group-level behaviour remains unclear. Our results demonstrate that under time-constrained predatory threat, rapid collective decision-making can emerge, thereby complementing existing models of gradual escape behaviour.Our findings further suggest that a certain period of familiarisation is required for collective behavioural choices in response to the LS. In particular, in familiar groups, many individuals transitioned from HS during LS to FS afterwards, and entire groups tended to enter the FS. Such consistent behavioural synchrony was mainly observed in groups that had undergone sufficient familiarisation, suggesting that social familiarity may contribute to coordinated collective decisions. In our experiments, social familiarity increased the proportion of individuals that exhibited a FS after escape-like HS, indicating a change in behavioural regularity at the individual level. However, these individual-level changes alone cannot fully explain the emergence of dichotomous collective outcomes (all-freezing versus all non-freezing). It remains unclear whether familiarisation (1) enhanced each individual’s social sensitivity, making them more likely to be influenced by others, or (2) homogenised behavioural traits within groups. Distinguishing between these two possibilities was beyond the scope of this study. Future approaches incorporating longitudinal tracking of individually identified fish and quantitative measures of behavioural synchrony will be necessary to address this question.If explanation (1) is correct, repeated interactions during familiarisation may allow individuals to recognise and predict the behaviour of conspecifics, thereby strengthening group-level properties such as polarisation and alignment. Previous studies have reported various effects of social familiarity in fish. For example, in female guppies, 12 days of familiarisation led to preferential associations with familiar conspecifics31. Social familiarity has also been shown to promote group cohesion and alignment in guppies14 and to enhance information transfer under social threat in damselfish17. In addition, familiarisation may also induce social fear contagion. In zebrafish, individuals are known to switch from high-speed swimming to freezing when exposed to alarm cues from conspecific skin extracts13,32, suggesting that this behavioural pattern may be widespread among fishes. Moreover, zebrafish exhibit similar freezing-like responses when observing familiar conspecifics or groups displaying fear responses13,33.On the other hand, explanation (2), that familiarisation homogenises behavioural traits within groups, cannot be excluded. Previous studies have shown that bold individuals tend to maintain stable behavioural traits, whereas shy individuals are more plastic and influenced by social context. For instance, in guppies, bold individuals rely on their own information and explore independently, whereas shy individuals adjust their behaviour according to social information34. In sticklebacks, bold individuals also show stable exploratory behaviour, while shy individuals display behavioural plasticity and can change over time35. However, to our knowledge, no studies have directly demonstrated long-term homogenisation of behavioural traits caused by familiarisation in any animal species. Thus, we consider explanation (1) to be the more plausible mechanism underlying our findings.Our study therefore extends previous fish familiarity research, which has reported average increases in shoal cohesion, by showing that long-term social familiarity also structures the variability of collective decisions. Familiar groups not only became cohesive; they also differentiated into groups that reached full consensus (freezing-dominant or non-freezing-dominant) and groups that failed to do so (mixed-type), revealing that familiarity regulates the probability—rather than the inevitability—of consensus formation under threat.In summary, our results indicate that social familiarity promotes the dichotomisation of collective behavioural choices in medaka, and that factors such as social familiarity or changes in social sensitivity may contribute to this process. Although further investigation will be required to directly verify these mechanisms, our study provides a foundation for exploring how social experience shapes collective decision-making under time-constrained predatory threats in vertebrates.

    Data availability

    All data generated or analysed during this study are available from the corresponding author on reasonable request.
    ReferencesKrause, J. & Ruxton, G. D. in Living in Groups. (eds Krause, J. & Ruxton, G. D.) (Oxford University Press, 2002). https://doi.org/10.1093/oso/9780198508175.002.0001Couzin, I. et al. Uninformed individuals promote democratic consensus in animal groups. Science 334, 1578–1580 (2011).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Strandburg-Peshkin, A., Farine, D. R., Couzin, I. D. & Crofoot, M. C. Shared decision-making drives collective movement in wild baboons. Science 348, 1358–1361 (2015).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Ward, A. J., Krause, J. & Sumpter, D. J. Quorum decision-making in foraging fish shoals. PloS One. 7, e32411 (2012).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Clément, R. J. G., Wolf, M., Snijders, L., Krause, J. & Kurvers, R. H. J. M. Information transmission via movement behaviour improves decision accuracy in human groups. Anim. Behav. 105, 85–93 (2015).Article 

    Google Scholar 
    Ward, A. J. W., Sumpter, D. J. T., Couzin, I. D., Hart, P. J. B. & Krause, J. Quorum decision-making facilitates information transfer in fish shoals. Proc. Natl. Acad. Sci. 105, 6948–6953 (2008).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    McComb, K. et al. Leadership in elephants: the adaptive value of age. Proc. R Soc. B Biol. Sci. 278, 3270–3276 (2011).Article 

    Google Scholar 
    Shang, C. et al. Divergent midbrain circuits orchestrate escape and freezing responses to looming stimuli in mice. Nat. Commun. 9, 1232 (2018).Article 
    ADS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Ward, A. J. W. & Hart, P. J. B. The effects of kin and familiarity on interactions between fish. Fish. Fish. 4, 348–358 (2003).Article 

    Google Scholar 
    Strodl, M. A. & Schausberger, P. Social familiarity reduces reaction times and enhances survival of group-living predatory mites under the risk of predation. PLOS ONE. 7, e43590 (2012).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Riley, R. J., Kwon, Y. M., Manica, A. & Savage, J. L. Familiarity dampens the effect of boldness on coordination in three-spined sticklebacks. Behaviour 162, 191–206 (2025).Article 

    Google Scholar 
    Galhardo, L., Vitorino, A. & Oliveira, R. F. Social familiarity modulates personality trait in a cichlid fish. Biol. Lett. 8, 936–938 (2012).Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Fernandes Silva, P., Garcia de Leaniz, C. & Luchiari, A. C. Fear contagion in zebrafish: a behaviour affected by familiarity. Anim. Behav. 153, 95–103 (2019).Article 

    Google Scholar 
    Davis, S., Lukeman, R., Schaerf, T. M. & Ward, A. J. W. Familiarity affects collective motion in shoals of guppies (Poecilia reticulata). R Soc. Open. Sci. 4, 170312 (2017).Article 
    ADS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Chivers, D., Brown, G. & Smith, J. Familiarity and shoal cohesion in Fathead minnows (Pimephales promelas): implications for antipredator behaviour. Can. J. Zool. 73, 955–960 (1995).Article 
    ADS 

    Google Scholar 
    Griffiths, S., Brockmark, S., Höjesjö, J. & Johnsson, J. Coping with divided attention: the advantage of familiarity. Proc. Biol. Sci. 271, 695–699 (2004).Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Nadler, L. E., McCormick, M. I., Johansen, J. L. & Domenici, P. Social familiarity improves fast-start escape performance in schooling fish. Commun. Biol. 4, 897 (2021).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Kadak, K. & Miller, N. Follow the straggler: zebrafish use a simple heuristic for collective decision-making. Proc. R. Soc. B Biol. Sci. 287:20202690 (2020).Imada, H. et al. Coordinated and cohesive movement of two small conspecific fish induced by eliciting a simultaneous optomotor response. PLoS One. 5, e11248 (2010).Article 
    ADS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Ochiai, T., Suehiro, Y., Nishinari, K., Kubo, T. & Takeuchi, H. A new data-mining method to search for behavioral properties that induce alignment and their involvement in social learning in Medaka fish (Oryzias latipes). PLoS One. 8, e71685 (2013).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Yilmaz, M. & Meister, M. Rapid innate defensive responses of mice to looming visual stimuli. Curr. Biol. 23, 2011–2015 (2013).Article 
    CAS 
    PubMed 

    Google Scholar 
    Temizer, I., Donovan, J. C., Baier, H. & Semmelhack, J. L. A visual pathway for looming-evoked escape in larval zebrafish. Curr. Biol. 25, 1823–1834 (2015).Article 
    CAS 
    PubMed 

    Google Scholar 
    Ferreira, C. H. & Moita, M. A. Behavioral and neuronal underpinnings of safety in numbers in fruit flies. Nat. Commun. 11, 4182 (2020).Article 
    ADS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Hein, A. M., Gil, M. A., Twomey, C. R., Couzin, I. D. & Levin, S. A. Conserved behavioral circuits govern high-speed decision-making in wild fish shoals. Proc. Natl. Acad. Sci. 115, 12224–12228 (2018).Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Takeuchi, T. & Manabe, E. Genetical study on the new mutant of the fading medaka, Oryzias latipes. Res. Bull. Shujitsu Women’s Coll. Shujitsu Jr Coll. 14, 1–18 (1984).
    Google Scholar 
    Yamanaka, O. & Takeuchi, R. UMATracker: an intuitive image-based tracking platform. J. Exp. Biol. 221, jeb182469 (2018).Article 
    PubMed 

    Google Scholar 
    Tuqan, M. & Porfiri, M. Mathematical modeling of zebrafish social behavior in response to acute caffeine administration. Front. Appl. Math. Stat. 7, 751351. https://doi.org/10.3389/fams.2021.751351 (2021).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Syme, J., Kiszka, J. J. & Parra, G. J. Behavioural variation facilitates coexistence and explains the functions of mixed-species groups of sympatric delphinids. Anim. Behav. 210, 395–408 (2024).Article 

    Google Scholar 
    McInnes, L., Healy, J. & Melville, J. U. M. A. P. Uniform Manifold Approximation and Projection for dimension reduction. arXiv:1802.03426v3 (2020).Bolker, B. M. et al. Generalized linear mixed models: a practical guide for ecology and evolution. Trends Ecol. Evol. 24, 127–135 (2009).Article 
    PubMed 

    Google Scholar 
    Griffiths, S. W. & Magurran, A. E. Familiarity in schooling fish: how long does it take to acquire? Anim. Behav. 53, 945–949 (1997).Article 

    Google Scholar 
    Masuda, M. et al. Identification of olfactory alarm substances in zebrafish. Curr. Biol. 34, 1377–1389e7 (2024).Article 
    CAS 
    PubMed 

    Google Scholar 
    Akinrinade, I. et al. Evolutionarily conserved role of Oxytocin in social fear contagion in zebrafish. Science 379, 1232–1237 (2023).Article 
    ADS 
    CAS 
    PubMed 

    Google Scholar 
    Trompf, L. & Brown, C. Personality affects learning and trade-offs between private and social information in guppies, Poecilia reticulata. Anim. Behav. 88, 99–106 (2013).Article 

    Google Scholar 
    Jolles, J., Briggs, H., Araya, Y. & Boogert, N. Personality, plasticity and predictability in sticklebacks: bold fish are less plastic and more predictable than shy fish. Anim. Behav. 154, 193–202 (2019).Article 

    Google Scholar 
    Download referencesAcknowledgementsWe thank Dr. Tetsuro Takeuchi for sharing the fading strain. We thank Drs. Masahiro Daimon and Masayuki Koganezawa for their advice on the development of the behavioural assay. We thank Drs. Ken-Ichiro Tsutsui, Hiromu Tanimoto, Jamie M. Kass and Towako Hiraki-Kajiyama for comments on the manuscript.FundingThis work was supported by the National Institute for Basic Biology Priority Collaborative Research Project 10–104 (to H.T.), 19–347 (to H.T.), and 21–335 (to H.T.); a grant for Joint Research (#01111904) by the National Institutes of Natural Sciences (to H.T.); Japan Society for the Promotion of Science (JSPS) KAKENHI Grants 21H04773 (to H.T.), 20H04925 (to H.T.), 18H02479 (to H.T.), 22H05483 (to H.T.), 23K27205 (to H.T.), 24H01216 (to H.T.) and 24K21957 (to H.T.). Takeda Science Foundation (to H.T.), and the natural science grant of the Mitsubishi Foundation (to H.T.); Japan Science and Technology Agency (JST) SPRING, Grant Number JPMJSP2114(to R.N.).Author informationAuthors and AffiliationsMolecular Ethology Laboratory, Graduate School of Life Science, Tohoku University, Sendai, 980-8577, JapanRyohei Nakahata & Hideaki TakeuchiDepartment of Cardiac Regeneration Biology, National Cerebral and Cardiovascular Centre, Osaka, 564-8565, JapanRyohei NakahataAuthorsRyohei NakahataView author publicationsSearch author on:PubMed Google ScholarHideaki TakeuchiView author publicationsSearch author on:PubMed Google ScholarContributionsR.N. and H.T. designed experiments. R.N. conducted experiments, wrote code and analysed data. R.N. and H.T. co-wrote and edited the paper and supervised the project.Corresponding authorsCorrespondence to
    Ryohei Nakahata or Hideaki Takeuchi.Ethics declarations

    Competing interests
    The authors declare no competing interests.

    Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationBelow is the link to the electronic supplementary material.Supplementary Material 1Rights and permissions
    Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
    Reprints and permissionsAbout this articleCite this articleNakahata, R., Takeuchi, H. Social familiarity shapes collective decision-making in response to looming stimuli in Medaka fish.
    Sci Rep 15, 43650 (2025). https://doi.org/10.1038/s41598-025-30656-4Download citationReceived: 14 September 2025Accepted: 26 November 2025Published: 23 December 2025Version of record: 23 December 2025DOI: https://doi.org/10.1038/s41598-025-30656-4Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
    Provided by the Springer Nature SharedIt content-sharing initiative More

  • in

    Exploring the social-ecological potential for indigenous agroforestry in peri-urban areas: a participatory mapping approach

    AbstractPeri-urban agroforestry can provide affordable, fresh, and nutritious food and a departure from conventional forms of cropping. Indigenous foods are well-adapted to local conditions, and may hold cultural and economic value for peri-urban residents. Social, ecological, and economic variables influence the feasibility of indigenous agroforestry in peri-urban areas. This study uses participatory mapping and geographic information systems (GIS) to assess these variables and to map suitable spaces and species for peri-urban indigenous agroforestry at three peri-urban sites in Durban, South Africa. We find that: land tenure, livelihood opportunities, and indigenous food perceptions factor into socioeconomic preferences; topography and soil quality influence ecological feasibility; access to water and roads influences perceived economic viability. Although GIS techniques can identify land suitability, participatory mapping adds local fine-scale context to enhance decision-making. Based on the social-ecological conditions at the three sites, we suggest specific configurations of locally adapted foods and farm designs for peri-urban agroforestry. Our study demonstrates how agroforestry is more feasible in places where basic living conditions are fulfilled, and how co-design can improve recognition of local needs, accessibility to services, and balancing urban green equity.

    Similar content being viewed by others

    The impact of multiple agricultural land uses in sustaining earthworm communities in agroecosystems – A global meta-analysis

    Article
    Open access
    04 December 2024

    Analysis of food system drivers of deforestation highlights foreign direct investments and urbanization as threats to tropical forests

    Article
    Open access
    16 July 2024

    Impacts of commodity prices and governance on the expansion of tropical agricultural frontiers

    Article
    Open access
    22 April 2024

    IntroductionUrban and peri-urban food production can improve the resilience of food systems in multiple ways1. From a logistic viewpoint, it can reduce the risk of supply chain failure and subsequent food and nutritional insecurity. Ecologically, it can reduce impacts from large-scale farming and transportation2, and socioeconomically, it can provide urban residents accessible, affordable, and nutritious alternatives to mass-produced and processed foods3. However, availability of land, labour, and materials are the main determinants of urban and peri-urban food production, in the global North and South4. In the face of densification and development, green space is a critical yet contested component of the urban landscape5,6. Cities worldwide have various legislations and allocations for food production in urban and peri-urban areas in communal gardens, private farms, and food forests7. These allocations help city planning to balance local economies, development interests, and urban environments, often in collaboration with local residents8. City-level adaptations are often crucial for successful implementation of national and regional food policies9,10.Urban and peri-urban agriculture is an emerging and potent response to provisioning fresh and nutritious food, closing nutrient loops, creating circular economies, and reducing carbon footprints11. It can take various forms, from intensive indoor vertical farms, to communal agroecological (including agroforestry) spaces, with several intermediate configurations of social, ecological, and technological variables12. In this article, we focus on urban agroforestry as the proposed intervention to improve food and nutritional security among urban and peri-urban dwellers. Urban agroforestry systems, defined as urban landscapes combining crops and trees are increasingly recognised as productive landscapes with greater allied cultural and ecological benefits than conventional agriculture13. The cultural and recreational values associated with urban agroforestry systems can facilitate more equitable and widespread uptake of the food and nutritional produce yielded by these landscapes (ibid).The feasibility of urban and peri-urban agroforestry could vary across different urban contexts. For example, in densely populated or historically established sections of cities, it could involve planting fruit trees and/or food crops along verges of transportation and utility lines that provide substantial nutrient yields14,15,16. In some cases, urban and peri-urban brownfields (previously developed land) may be reclaimed by municipalities or citizen collectives to grow food17,18,19. Urban parks and gardens established primarily for recreation may also be a significant and legitimate source of food and nutrition20,21,22. Structural constraints to urban and peri-urban agroforestry include the availability of contiguous land and arable soil4,11 and also resident and developer preferences for gentrified forms of nature, neighbourhoods, and greenspaces23. While biophysical and infrastructural variables can help determine suitability for urban agroforestry, social structures and perceptions are crucial to its long-term sustainability24.Indigenous crops and trees are important components of agroecological systems. They are often resilient to local ecological stresses and shocks25, as well as human disturbance and extraction26,27. On farms, indigenous crops and trees provide pollination services, alternative income, and nutrition for farmers28. In urban and peri-urban areas, they can also provide habitat connectivity to wildlife29,30, including pollinators important to rural and urban food production31. This makes them ideal candidates for fragmented landscapes of high-intensity human use, such as urban and peri-urban areas, where large-scale farming is impractical. Foods from indigenous crops and trees are rich in high-quality micronutrients32,33, which are generally deficient in urban diets due to constrained accessibility and affordability. Recent research on indigenous crops has focussed on nutritional yields and land suitability for annual crops such as grains and tubers34,35. Although the potential of indigenous food-bearing tree species has been recognised36,37,38, the research and application of these in agroforestry is still nascent114,115. Therefore, in this study, we also attempt to identify the feasibility of planting indigenous crops and trees, comprising indigenous agroforestry, in urban and peri-urban areas, identifying synergies and constraints as applicable.Participatory mapping was employed to document the cultural, economic, and social values of peri-urban agroforestry, seeking to establish its potential in enhancing livelihoods and promoting environmental sustainability. This involved mapping the variables favouring indigenous food production at three study sites. Thus, a suite of social science methods were used to elicit spatial and temporal data, trends, and preferences in landscapes and land uses39. The participatory mapping was conducted to promote democratic, inclusive, and locally appropriate decision-making when combined with GIS modelling techniques40. This is especially important in urban and peri-urban areas, where land use and land cover are fast-changing, and can often leave under-resourced communities impoverished41. People’s values for landscape features and uses can play an important role in successful landscape governance42, including the implementation and observance of regulations43. Peri-urban areas in the Global South differ from many in the Global North, in that the regulations and infrastructure in the former are not as organised and developed as urban areas44. This situation underscores the need for participatory mapping and GIS modelling to enable better planning and service provisioning in peri-urban areas in the Global South45. Our study follows a mixed methods approach giving equal importance to communities and experts in the mapping process46.In this study, we seek to design locally appropriate indigenous agroforestry systems for urban and peri-urban areas with the aim of improving their food and nutritional security47. We combine data on social perceptions, spatial modelling, and indigenous agroforestry species to generate these designs, which are intended to inform local communities and municipal departments on feasible agroforestry and food security initiatives. Our study demonstrates how government policies and programmes, e.g.55,56,115 can be operationalised at local scale, e.g.9,93,96,116 by combining participatory research and different forms of knowledge. This study is part of the Durban Research Action Partnership between the local metropolitan eThekwini Municipality and University of KwaZulu-Natal, which aims to generate knowledge and learning to address the gap between scientific research, policy development and management within a local government113.The local contextAs in the case of many developing nations, households in South Africa experience the triple burden of malnutrition, which includes undernutrition (stunting and wasting), micronutrient deficiencies (often termed hidden hunger), and overnutrition (overweight and obesity)48. Urbanising and westernising lifestyles influence the preference for cheap, convenient, ultra-processed and packaged food over traditional, nutritious, and fresh, diverse farm-based food49. Post-apartheid market liberalisation has facilitated the penetration of cheap and calorie-dense low-nutrient foods into local markets for consumers and incentivised the export of high-quality foods such as fruit and vegetables to foreign markets for producers50. Smallholder farmers who cannot export or sell to mainstream domestic markets often struggle with a lack of infrastructure and institutional support to improve yields and sales51.In the broader socioeconomic sense, unemployment and economic inequality result in income poverty and food poverty, and limited opportunities for people experiencing poverty to engage in either primary production or secondary activities to secure an income52. Particularly in cities, legacy spatial planning also constrains access to greenfields (previously undeveloped land) and greenspace, which can often be a source of food or materials to support the household economy37,53 As a result of apartheid-era policies, green infrastructure and public access to it is well-developed in affluent neighbourhoods, but is severely lacking in poorer (often racially differentiated) neighbourhoods54. Allocating and enriching urban and peri-urban spaces for food production are a priority on the National Development Plan for South Africa55, and could also contribute to national-level cross-cutting initiatives like the Integrated Food Security and Nutrition Programme and the Natural Resources Management Programme56. In this study three communities in the peri-urban areas of the eThekwini Metropolitan Municipality (that houses Durban, hereafter eThekwini) were consulted to identify spaces where food production can be undertaken, to enhance food and nutritional security.MethodsConceptual framingThe study aimed to identify the most compatible configurations of peri-urban food production given the social-ecological conditions at each site. The study used an overarching landscape ecology approach57 to define the landscape configuration, land use, land cover change, and landscape management guidelines. The study combined participatory mapping and GIS suitability analyses to identify suitable areas for peri-urban food production. The research objective was achieved by answering three questions (Fig. 1) in both the participatory mapping and suitability analysis approaches. The methodology characterised local social-ecological factors and existing and potential land use for food production at each site (Fig. 1). These were analysed to produce socioeconomic and land use guidelines suggesting configurations of peri-urban food production suitable to each site. Landscape configuration was determined using spatial datasets (Table 1). The land use and cover change were determined from social data, to account for socio-polictical nuance about these changes. The management guidelines emerging from the analysis of these datasets were combined to propose locally appropriate landscape management guidelines that included biophysical suitability maps, social aspirations and needs. The conceptual framing and resulting management guidelines resonate with recent calls for agriculture to incorporate indigenous species as ‘trade-ons’, through appropriate design for ‘land maxing’20,114. The management guidelines resulting from this study are applicable examples of mainstreaming biodiversity and indigenous knowledge into productive and adaptive peri-urban agroforestry115.Fig. 1Conceptual framing of the study, research questions, data collected, and analyses.Full size imageTable 1 Influence percentage for each factor used to produce the final suitability maps.Full size tableStudy areaThe eThekwini municipality is host to a population of 3.9 million people, in its urban centre of Durban, as well as several peri-urban areas58. Due to legacy planning and diverse tenure systems, the city centre and suburbs have designated greenspace, whereas the peri-urban areas are more informal and sporadic in structure. About 44% of the land in eThekwini falls under the Ingonyama Trust, governed by traditional chiefs, and is not subject to the same planning requirements as municipal land37. The Durban Metropolitan Open Space System (DMOSS) was instituted in the 1990s to plan and govern land use across formal, informal, protected, and indigenous greenspace in urban and peri-urban areas59,60,61. Under this system, land use is restricted in areas of ecological importance, ecological restoration offsets are required where feasible, and urban greening and agroforestry are promoted in collaboration with municipal departments and NGOs62. The municipality routinely undertakes reforestation and restoration across these open spaces, with the dual intention of improving biodiversity and supporting local bio-economy livelihoods63. There is also a strong emphasis on removing and controlling invasive alien species and planting endemic and indigenous species in greenspaces37. Given this background, our research questions consider the diversity of land tenure and indigenous species, especially trees, that are of interest at each study site (Fig. 2). The three study sites were suggested by eThekwini Municipality as areas of interest for rolling out peri-urban agroforestry interventions, as part of their municipal agroecology programme9,116.Fig. 2Location of study sites in the local context and South Africa (inset). Some sites are fragmented because of dual land tenure—traditional and municipal.Full size image

    Maphephetheni: situated on the mountainous area surrounding the Inanda reservoir on the Umgeni River, built in the 1980s, contiguous with the suburb of Inanda. The peri-urban settlement consists of savanna and grassland vegetation. Areas degraded by invasive species (e.g. Acacia spp.) and fire are being replanted with useful food and forage species (e.g. Canthium spp., Ficus spp., Searsia spp.) to encourage sustainable land use64. The municipality’s erstwhile Environmental Planning and Climate Protection Department (EPCPD), now the Biodiversity Management Department, engages members within these communities to nurture saplings for restoration, increase awareness and stewardship and prevent cyclical degradation and restoration. The area has a population density of 344 persons per km2 as per the 2011 census.

    Ntshongweni: situated along the ridge of the Shongweni dam on the Mlazi River, built in the 1920s, with vegetation consisting of riparian forest, grassland, and some wetland. Rail and road connections to the urban centres of Durban and Pietermaritzburg have attracted investment in transport and logistics centres in the vicinity, and more recently, in commercial retail and residential development. The EPCPD also runs invasive alien control and reforestation programmes at Ntshongweni. The area has a population density of 399 persons per km2 as per the 2011 census.

    Osindisweni: situated along the ridge of the Hazelmere dam on the Mdloti River, built in the 1970s, with vegetation consisting of riparian forest and grassland. It is adjacent to Buffelsdraai, a site historically degraded by intensive sugarcane farming, and currently serving (since 2008) as a suburban landfill ring-fenced by indigenous forest fragments65. These fragments are gradually expanded and connected by ongoing planting, and although most of the forest is protected, the periphery and a small section of the site have been earmarked to grow indigenous food-bearing tree species for surrounding communities. The EPCPD does not yet run restoration programmes at Osindisweni. The area has a population density of 439 persons per km2 as per the 2011 census.

    Participatory mapping workshopsOne participatory mapping workshop was conducted in each community between September 2021 and March 2022. Local chiefs and councillors were approached for their consent to engage with the community, and for assistance in recruiting community members to participate in the workshops. We acknowledge that this recruitment strategy may have resulted in a representational bias, but assert that we communicated to each chief and councillor the need to engage with all sections of the community including youth, elders, employed, unemployed, and women. The aim of the research was introduced at the beginning, and informed consent was obtained from all participants to record their responses and take photographs for research purposes only. The study was ethically reviewed and approved by the Humanities and Social Sciences Research Ethics Committee of the University of KwaZulu-Natal in June 2021 (Protocol Reference Number HSS/1971/017D). All methods were performed in accordance with the Economic and Social Research Council guidelines on ethical scientific research.The outline map (Online Appendix Fig. A1) of the community with key features, namely, rivers, roads, schools, and hospitals, was presented to the participants. They were asked: (i) What are the various greenspaces in the community, and what are their tenure and access terms? (ii) What are the resources and uses associated with each of these greenspaces? (iii) What are the positive and negative characteristics of these greenspaces? (iv) What changes have these greenspaces undergone in the past 10 years? (v) What changes, if any, would the community like to see in these greenspaces? (vi) What species of food, especially indigenous trees, grow or are grown in the community, and where? (vii) What food species would the community want growing in their greenspaces, and where? We used the most open and commonly accepted definition of greenspace, implying undeveloped land that harbours some form (cultivated or wild) of vegetation, and is used for one or more of the purposes of: agriculture and food cultivation, cultural and recreational activities, foraging, fishing, and grazing66.A native isiZulu speaker interpreted the questions and responses, and all responses were recorded on the map during the discussion. Names of places and indigenous plants were recorded in isiZulu. “Tree” spaces were recorded as a separate category overlapping with other types of spaces. They included home gardens, sports fields, and open spaces, as they may be fragmented yet productive in their food and non-food yields (e.g. fibre, fuel, medicine, wood). Food production was recorded as a use only when explicitly mentioned by participants (e.g. home gardens or open spaces where food was gathered from plants but not grown as crops were not deemed used for food production). The data collected were analysed qualitatively using reflexive grounded theory, e.g.67 for emergent themes and descriptions in MS Word. Quotes from participants were anonymised using the monikers ‘Respondent n’, and presented to illustrate examples, claims, and arguments.Species selectionSocial-ecological attributes of land use and cover change at each site were derived from the data shared by respondents. Landscape design configurations were suggested in response to these attributes, with functions such as biophysical tolerance and cultural importance. The landscape management guidelines recommended biome-appropriate indigenous food species from36,68, and69. The underlying philosophy of this species allocation was to increase land productivity for local food and nutritional security, regenerating natural capital for human and environmental health, and enabling local communities to generate human, social, physical and financial capital through the planting of useful indigenous species114.Suitability analysesSeveral factors influence land suitability for urban agricultural farming. Biophysical, socio-economic, and technical aspects are some of the primary factors. The principal purpose of land suitability for urban crop farming is to predict the potential and limitation of land for crop production70. Generally, determining suitable areas for crop farming in urban areas revolves around making the most sustainable use of land resources while avoiding depleting other resources71. Crop farming land suitability analysis requires an efficient decision support system to analyse and interpret the related ecological, environmental and spatial information. GIS and participatory GIS are combined with multicriteria decision analysis (MCDA) methods to deliver a better spatial decision72.This study determined suitable areas for peri-urban food production in three stages. First, the factors affecting the agricultural uses were set up as criterion maps. Secondly, all the factors were scored in the suitability range based on expert opinion and the results from the participatory mapping workshops. Finally, GIS spatial analysis modelling techniques were used to generate suitability maps for the three sites.The study adopted six factors, as suggested by73, to set up criterion maps. The factors include land cover, agricultural land capability, dominant soils, slope, proximity to water sources and proximity to the main road. The weighted overlay in ArcGIS Pro was used to generate the final suitability maps based on the percentage of influence for each geographic factor. Here, the influence of each factor (weights) was arbitrarily chosen based on the results of the interviews and experts’ knowledge. Thus, each layer contributes to the influence based on the type of agricultural land use (Table 1). In this study, the final suitability maps were reclassified into five classes with suitability scales ranging from highly suitable to not suitable.Results and discussionCharacterising greenspace attributes, perceptions, preferences and potential for peri-urban agroforestry through participatory mappingThe workshops lasted about 90 min at each site, and involved between 11 and 29 participants. Participants included but were not limited to, representatives of the ward, workers with different departments of the municipality such as community services, education, environment, and health and sanitation, local smallholder farmers, part-time employed and unemployed youth and elders, private sector employees, and representatives of local NGOs, churches, and cooperatives. The participation in the workshops was variable and limited due to the ongoing Covid-19 restrictions at the time. Nevertheless, we believe the depth and diversity of the discussions are representative of the sites.Current land use of greenspaces included provisioning and recreation, although the former was reported as significant only at Maphephetheni and Ntshongweni (Table 2). Greenspaces are an important avenue for urban and peri-urban foraging at all three sites, providing residents with resources and recreational opportunities76,77. Food production was intentionally undertaken in communal and public greenspaces at the aforementioned sites, but not at Osindisweni. Tenure over greenspaces also varied across the sites, and areas under the traditional authority, i.e. the local chief, were used for recreation and grazing, but not to grow food. Land tenure is an important driver of land use and stewardship, and traditional authority tenure can deter land-based livelihoods such as food production and agroforestry. For example, lack of accountability and definition in spatial allocation in communal areas can result in violation of land use agreements78, reducing certainty of long-term land use, and subsequent investment of labour and capital in food production79. It may also result in rent appropriation by powerful stakeholders at the expense of the community80 and undemocratic development on land intended for food production, especially in urban and peri-urban areas81. This may partly explain why at Osindisweni, where most greenspaces are under communal tenure, participants expressed low interest in food production and agroforestry.Table 2 Existing land use: types of greenspaces, their tenure, and uses at the three study sites. Species information is listed in Online Appendix Table A1.Full size tableThe productivity of greenspaces varied across sites, with Ntshongweni residents earning and saving money from the sale of food produced in home and public greenspaces (Table 3). Participants at Ntshongweni expressed an interest in diversifying their food production by including indigenous crop and animal species.Table 3 Perceived land use potential and food production feasibility in greenspaces at the three study sites.Full size table“The municipality [representative] tells us that there is a market for indigenous crops and chickens. We would like to learn about how to farm these so that we can sell not just within our communities, but also to the urban market.”—Respondent 1.Natural greenspaces were “far away” for residents of Maphephetheni and Osindisweni.“[That place] is far away, so we visit only on some weekends, maybe once or twice a year. When we go there, it is with family and friends. We can take our time and be one with nature.”—Respondent 2.These observations make a case for the development of more accessible parks and gardens for residents closer to residential areas. Planning for such should consider local perceptions of safety and environmental quality to minimise unintended consequences such as dereliction or gentrification82. Across all three sites, lack of plant material, stable water supply, livestock predation, and know-how were reported as hindrances to food production in community food and school gardens.“There are times in the summer when we don’t have water [on tap] for some ten, twenty days. This is when the plants also need water, and we also need [drinking] water. That’s why our [community food] gardens are not successful. The crops die.”—Respondent 3.“What we need to know is how to grow crops and trees properly. Both common and indigenous ones. We need to learn how to water them care for them, how to harvest them at the right time.”—Respondent 4.Participants made different site-specific recommendations to improve food productivity. For example, in Maphephetheni, home gardens were considered more effective than public gardens, as protecting them from water shortages, flooding, and livestock and human predation was easier. On the other hand, Osindisweni respondents prioritised shops and soup kitchens as means to improve food security, as they believed their land to be no longer viable for food production due to pollution associated with the landfill.“We live close to the city. We do not need to grow our own food. What we need is more shops to buy our food from. We need schools and soup kitchens to support our people with meals for food security. This is the support we need from the government.”—Respondent 5.“The soil here is very degraded. There is so much dumping, so many fires. People suffer from respiratory problems because of this environment. Crops and trees will never grow here. If the municipality wants to help us, they should collect our garbage more regularly.”—Respondent 6.Suggesting locally occurring, useful indigenous species suited to respective site attributes for peri-urban agroforestryBased on the pros, cons, and potential identified in the previous stages, we characterise seven site attributes and six response functions that can be served by greening for urban food production, in addition to improving food and nutritional security (Table 4). We suggest using thorny plants as fencing structures to prevent livestock predation while simultaneously maintaining biomass for humans and non-humans in the form of fruits and fodder. Given the use of greenspaces for non-food and non-timber products and the need for invasive alien replacement, indigenous trees with multiple uses can be planted in various greenspaces. Some of these species already grow in greenspaces across these sites (Table A1) but were not referred to as serving the proposed functions. None of the herbs or crops were specifically mentioned by participants during the elicitation at the workshops.Table 4 Design configurations based on synthesised site attributes, desired response functions, reviewed literature on indigenous food species, and participatory mapping locations, for Maphephetheni (M), Ntshongweni (N), and Osindisweni (O). (Y = Yes, N = No, indicating species suitability at site).Full size tableWe acknowledge that cultivation of some of the trees and crops suggested in Table 4 may require significant investment in technical training and infrastructure. For example, Carissa, Dovyalis, and Harpephyllum are dioecious species, requiring careful selection and planting of sufficient male and female plants in close proximity to ensure fruiting. Cultivation of crops, especially grains, may require knowledge of seed accessions, and access to postharvest facilities for processing and storage110,111,112. The local-scale matching of trees and crops to sites undertaken in this study is validated by species distribution based on biophysical parameters35,37. Findings from our research present the first step towards operationalising national policy on indigenous knowledge and agriculture115, and further directions for developing the required physical and social infrastructure by the local municipality.A number of these indigenous trees serve as a significant conduit to the intergenerational transfer of ecological knowledge and a connection to nature69, which in turn forms an important part of biocultural diversity and landscape stewardship83. Fast-growing herbs that require little input can be grown in marginal areas where the terrain poses difficulties, or where land tenure induces uncertainty. Crops that can resist waterlogging, enrich soil, and improve local productivity are also suggested where appropriate. Similarly, choices of crops exist for areas that are more prone to drought or heatwaves, or for marginal soils or shaded or windy areas68. Spatiotemporal intercropping of these with conventional crops can help remediate soil84,85. Surplus production of indigenous crops and trees can feed into short and high value supply chains to urban centres, e.g.38,86.Determining biophysical land suitability for peri-urban agroforestry using geospatial analysesFigure 3 shows the maps produced using expert-derived weights and value functions in each area. According to experts` knowledge, a higher weight was suggested for land cover than for agricultural land capability, dominant soils, slope, proximity to water sources and proximity to the main road. It should be noted that bare land plays a major role in delineating suitable urban areas for food production. Based on the results, a final weight of 0.35 was assigned to land cover. The final suitability maps for each area were divided into five agriculture suitability quality classes defined at discrete levels, allowing for comparisons between the three maps. The classes include suitable, moderately suitable, marginally suitable and not suitable areas for peri-urban agriculture. A simple visual comparison of the suitability patterns revealed by the three maps shows that Osindisweni has the greatest proportion of highly suitable and suitable areas for peri-urban agriculture. The Osindisweni area has suitable areas such as land cover, agricultural suitability, and open spaces, which favour the area’s suitability for agriculture. Also, this area has a good road and river network.Fig. 3Peri-urban agriculture suitability maps for (a) Osindisweni, (b) Ntshongweni and (c) Maphephetheni.Full size imageFor further analysis, the highly suitable, suitable and moderately Suitable areas were combined and overlaid with PGIS-identified suitable areas. Areas identified in the participatory mapping workshops tended to overlap with the high- to moderately-suitable classes of land identified in the GIS (biophysical) model (Fig. 4). This shows an agreement between the two methods used in this study to identify areas suitable for peri-urban food production at the three sites. Using both approaches strengthens the estimates of suitable areas by identifying the areas for which both approaches identify while minimising the number of wrongly identified areas. These maps will significantly value future land use and land cover change analysis for urban crop production.Fig. 4The suitable areas for peri-urban agriculture after overlaying both the PGIS and GIS layers in Osindisweni, Maphephetheni and Ntshongweni districts.Full size imageTable 5 shows that 75% of the study area in Maphephetheni, 4.53% in Ntshongweni and 0.21% in Osindisweni is permanently unsuitable for peri-urban crop production. These areas have unsuitable land cover and steep slopes, far from the road and river network. With a 10.74% suitability rate, the Osindisweni area has the highest potential for peri-urban food production, followed by Maphephetheni (1.2%) and Ntshongweni (0.84%). Generally, a small portion of the total area in all three study areas is suitable for urban crop production. For successful and effective peri-urban food production, growing crops with high production over a small piece of land, such as onions, herbs, garlic and leaf vegetables, is advisable.Table 5 The distribution of land suitability for each site from the PGIS land suitability analysis model.Full size tableStatistics comparing the number of cells assigned to each suitability class for the three maps are presented in Table 5 and Fig. 3. The difference between the areas of the site was up to 50 km2, with Ntshongweni being the smallest (167.61 km sq.), followed by Maphephetheni (183.83 km sq.) and Osindisweni (217.95 km sq.). The GIS suitability analysis indicated that Osindisweni has the largest absolute area of suitable land and the largest ratio of suitable to unsuitable land, with over 99% of its area being suitable for greening for food (Table 5). Conversely, Maphephetheni had the smallest suitable land area, accounting for about 25% of its total area. Ntshongweni also had a high ratio of 96% of its land suitable for greening for food.Osindisweni’s proximity to erstwhile sugarcane fields65 corroborates the finding that it has a greater proportion of suitable to marginally suitable agricultural land. However, more recent social-political developments, such as rapid urbanisation and the expansion of landfills have resulted in food production being perceived as untenable in the area. Indeed, expanding industrial and urban activities can accelerate a shift from land-based livelihoods and a decline in soil and water quality87,88. Our findings reiterate the importance of triangulating land use planning across large to fine scales through participatory methods. Alongside soil depth and nutrients, the slope is an important landscape determinant of land suitability for conventional food production89. Notwithstanding, results from our participatory and synthesis process offer options to reinforce local food and nutritional security through innovative design elements12. Despite the proximity to water sources, last-mile connectivity to arable land emerged as a significant limitation for agroforestry at the three sites. Plans to promote food production should consider strategies to manage nutrient flows in soil and water90,91. Excess runoff of agricultural enrichment materials may threaten water quality and safety. This is especially important given the immediate dependence of peri-urban and urban dwellers on surface and groundwater92. Agroecological strategies, including organic and circular inputs, are likely to alleviate environmental and food safety issues93. We acknowledge that our model considers arable land suitability in general, but that this may vary depending upon crop and tree species. Future research on multi-species indigenous agroforestry could be more species-specific, e.g.94.LimitationsOur study has two main limitations, namely the sample frame, and analytical depth. Our strategy to enlist participatory mapping workshop participants relied mainly on the local ward councillors and traditional chiefs, as this is standard practice to demonstrate respect of local authorities and build trust with local communities in the area. Participatory mapping work was carried out during a period when pandemic lockdown restrictions were in effect to varying degrees. Under these conditions, the participation in workshops was variable across the three sites, and it is possible that certain sections of communities that are less socially empowered may have been under-represented in the sample. Secondly, the scope of this study was to combine basic social, spatial, and species data to suggest locally appropriate peri-urban agroforestry design. Therefore, detailed evaluations of equitable access, agroclimatic variability, agronomic feasibility, etc. were not possible at this stage. We suggest that future research can delve into these specificities at site and regional scale. We posit that findings from this study provide a valuable baseline for research and implementation.Policy, practice, and research implicationsThis study highlights the role of participatory co-design in developing urban agriculture configurations. It combines a social-ecological systems lens95 with a landscape ecology approach57 to derive locally appropriate designs using locally adapted species. The communities expressed their aspirations for local food and nutrition security, which took on different forms. The participatory mapping outcomes demonstrate how local social-ecological and political situations influence preferences and feasibility of urban food production. Where food production is ongoing, diversification is welcomed, but where basic living conditions such as water supply and environmental quality are compromised, food production becomes secondary to expectations of urban living standards. This reiterates the need for participatory planning in the development of urban agriculture as a sustainable and citizen-driven enterprise in South Africa96. Our study demonstrates an interdisciplinary and participatory design approach to designing urban green infrastructure for ecosystem services97. Co-design has the benefits of recognising local needs, making services more accessible, balancing environmental regulatory frameworks with land use guidelines, and improving local resilience for urban green equity98,99. Further work in these communities has included the planting of a community agroforestry trial100 and an agroecology demonstration hub101 using indigenous species. These sites will serve as living learning laboratories for indigenous urban and peri-urban agroforestry. Communities will be involved in research related to assessing biodiversity and ecological implications such as species richness and plant biomass102,103, and also with development of market linkages for urban and peri-urban agroforestry, e.g.24,114.ConclusionThis study finds that while GIS tools can generate detailed information on land use suitability, the participatory process allows for the democratic exchange of knowledge, particularly in fast-changing socioeconomic landscapes like peri-urban areas. Specifically, the site where people have secure land tenure, service delivery, and existing involvement in agroforestry was found to be more suited to diversified multifunctional agroforestry configurations. Conversely, the site with uncertain land tenure and service delivery could support fewer configurations despite having the most are of suitable land according to GIS modelling. The site with most area of non-suitable land could also be matched with a high number of agroforestry configurations. This integrated approach can aid the development of site-specific solutions, forming dynamic governance co-produced by communities and institutions8,83. The participatory research component also helps to build an adaptive, responsive community of practice around each site by engaging with relevant stakeholders in non-political terms104,105,106. Through innovative design considerations, our findings aim to enable synergistic improvements in food and nutritional security, agroecology, and multifunctional urban green infrastructure107,108. The outputs contribute to achieving the sustainable development goals (SDGs) 2 (reducing hunger), 3 (promoting health and wellbeing), 9 (infrastructure innovation), 11 (sustainable cities and communities), 12 (responsible consumption and production), 13 (climate action), 15 (life on land), and 17 (partnerships) (SDG 2015)109. Policymakers and planners can draw from this partnership template using participatory research to feed into programmatic implementation at local scale8,9,10,113,114,115,116. In turn, participatory action research can be a conduit to intervention scaling, and to generating social-ecological evidence through living laboratories. This form of exchange is especially applicable to developing the field of indigenous local ecological knowledge28,35,38,86,94,115.

    Data availability

    All data generated or analysed during this study are included in this published article [and its supplementary information files].
    ReferencesAugstburger, H., Käser, F. & Rist, S. Assessing food systems and their impact on common pool resources and resilience. Land 8(4), 71 (2019).Article 

    Google Scholar 
    Sarkodie, S. A., Owusu, P. A. & Leirvik, T. Global effect of urban sprawl, industrialization, trade and economic development on carbon dioxide emissions. Environ. Res. Lett. 15(3), 034049 (2020).Article 
    ADS 

    Google Scholar 
    Bergius, M. & Buseth, J. T. Towards a green modernization development discourse: the new green revolution in Africa. J. Polit. Ecol. 26(1), 57–83 (2019).
    Google Scholar 
    Follmann, A., Willkomm, M. & Dannenberg, P. As the city grows, what do farmers do? A systematic review of urban and peri-urban agriculture under rapid urban growth across the Global South. Landsc. Urban Plan. 215, 104186 (2021).Article 

    Google Scholar 
    Haaland, C. & van den Bosch, C. K. Challenges and strategies for urban green-space planning in cities undergoing densification: A review. Urban For. Urban Green. 14(4), 760–771 (2015).Article 

    Google Scholar 
    Kabisch, N., Qureshi, S. & Haase, D. Human–environment interactions in urban green spaces—A systematic review of contemporary issues and prospects for future research. Environ. Impact Assess. Rev. 50, 25–34 (2015).Article 

    Google Scholar 
    Hajzeri, A. & Kwadwo, V. O. Investigating integration of edible plants in urban open spaces: Evaluation of policy challenges and successes of implementation. Land Use Policy 84, 43–48 (2019).Article 

    Google Scholar 
    Buijs, A. et al. Mosaic governance for urban green infrastructure: Upscaling active citizenship from a local government perspective. Urban For. Urban Green. 40, 53–62 (2019).Article 

    Google Scholar 
    Greenberg, S., Drimie, S., Losch, B. & May, J. From local initiatives to coalitions for an effective agroecology strategy: Lessons from South Africa. Sustainability 15(21), 15521 (2023).Article 
    ADS 

    Google Scholar 
    Sardeshpande, M., Vanak, A., Ashwini, A., Casiker, C. V., Hariya, H., Mukherjee, R., Chatterjee, S., Lepcha, P. Y., Biswas, D., Devy, M. S., Hiremath, A. J., Jamwal, P., Khaling, S., & Setty, S. (In Press). Harnessing synergies across networks to drive actionable change for sustainable and healthy food systems in India. Journal of Agriculture and Food Research.Opitz, I., Berges, R., Piorr, A. & Krikser, T. Contributing to food security in urban areas: differences between urban agriculture and peri-urban agriculture in the Global North. Agric. Hum. Values 33(2), 341–358 (2016).Article 

    Google Scholar 
    Armanda, D. T., Guinée, J. B. & Tukker, A. The second green revolution: Innovative urban agriculture’s contribution to food security and sustainability—A review. Glob. Food Sec. 22, 13–24 (2019).Article 

    Google Scholar 
    Taylor, J. R. & Lovell, S. T. Designing multifunctional urban agroforestry with people in mind. Urban Agric. Regional Food Syst. 6(1), e20016 (2021).Article 

    Google Scholar 
    Botzat, A., Fischer, L. K. & Kowarik, I. Unexploited opportunities in understanding liveable and biodiverse cities. A review on urban biodiversity perception and valuation. Glob. Environ. Chang. 39, 220–233 (2016).Article 

    Google Scholar 
    Larondelle, N. & Strohbach, M. W. A murmur in the trees to note: Urban legacy effects on fruit trees in Berlin, Germany. Urban For. Urban Green. 17, 11–15 (2016).Article 

    Google Scholar 
    Säumel, I., Weber, F. & Kowarik, I. Toward livable and healthy urban streets: Roadside vegetation provides ecosystem services where people live and move. Environ. Sci. Policy 62, 24–33 (2016).Article 

    Google Scholar 
    Bonthoux, S., Brun, M., Di Pietro, F., Greulich, S. & Bouché-Pillon, S. How can wastelands promote biodiversity in cities? A review. Landsc. Urban Plan. 132, 79–88 (2014).Article 

    Google Scholar 
    Rupprecht, C. D., Byrne, J. A., Garden, J. G. & Hero, J. M. Informal urban green space: A trilingual systematic review of its role for biodiversity and trends in the literature. Urban For. Urban Green. 14(4), 883–908 (2015).Article 

    Google Scholar 
    Sardeshpande, M., Rupprecht, C. & Russo, A. Edible urban commons for resilient neighbourhoods in light of the pandemic. Cities 109, 103031 (2021).Article 

    Google Scholar 
    Hurley, P. T. & Emery, M. R. Locating provisioning ecosystem services in urban forests: Forageable woody species in New York City, USA. Landsc. Urban Plan. 70, 266–275 (2018).Article 

    Google Scholar 
    Colinas, J., Bush, P. & Manaugh, K. The socio-environmental impacts of public urban fruit trees: A Montreal case-study. Urban For. Urban Green. 45, 126132 (2019).Article 

    Google Scholar 
    Bunge, A., Diemont, S. A., Bunge, J. A. & Harris, S. Urban foraging for food security and sovereignty: quantifying edible forest yield in Syracuse, New York using four common fruit-and nut-producing street tree species. J. Urban Ecol. 5(1), juy028 (2019).Article 

    Google Scholar 
    Zhu, J., He, B. J., Tang, W. & Thompson, S. Community blemish or new dawn for the public realm? Governance challenges for self-claimed gardens in urban China. Cities 102, 102750 (2020).Article 

    Google Scholar 
    Wiek, A. & Albrecht, S. Almost there: On the importance of a comprehensive entrepreneurial ecosystem for developing sustainable urban food forest enterprises. Urban Agric. Reg. Food Syst. 7(1), e20025 (2022).Article 

    Google Scholar 
    Mabhaudhi, T., O’Reilly, P., Walker, S. & Mwale, S. Opportunities for underutilised crops in southern Africa’s post–2015 development agenda. Sustainability 8(4), 302 (2016).Article 
    ADS 

    Google Scholar 
    Gaoue, O. G., Jiang, J., Ding, W., Agusto, F. B. & Lenhart, S. Optimal harvesting strategies for timber and non-timber forest products in tropical ecosystems. Thyroid Res. 9(3), 287–297 (2016).
    Google Scholar 
    Lankoandé, B., Ouédraogo, A., Boussim, J. I. & Lykke, A. M. Natural stands diversity and population structure of Lophira lanceolata Tiegh. ex Keay, a local oil tree species in Burkina Faso, West Africa. Agroforestry Syst. 91(1), 85–96 (2017).Article 

    Google Scholar 
    Leakey, R. R. A re-boot of tropical agriculture benefits food production, rural economies, health, social justice and the environment. Nature Food 1(5), 260–265 (2020).Article 

    Google Scholar 
    Champness, B. S., Palmer, G. C. & Fitzsimons, J. A. Bringing the city to the country: relationships between streetscape vegetation type and bird assemblages in a major regional centre. J. Urban Ecol. 5(1), juz018 (2019).Article 

    Google Scholar 
    Zietsman, M. Y., Montaldo, N. H. & Devoto, M. Plant–frugivore interactions in an urban nature reserve and its nearby gardens. J. Urban Ecol. 5(1), juz021 (2019).Article 

    Google Scholar 
    Bennett, A. B. & Lovell, S. Landscape and local site variables differentially influence pollinators and pollination services in urban agricultural sites. PLoS ONE 14(2), e0212034 (2019).Article 
    PubMed 
    PubMed Central 
    CAS 

    Google Scholar 
    Broegaard, R. B. et al. Wild food collection and nutrition under commercial agriculture expansion in agriculture-forest landscapes. Forest Policy Econ. 84, 92–101 (2017).Article 

    Google Scholar 
    Bvenura, C. & Sivakumar, D. The role of wild fruits and vegetables in delivering a balanced and healthy diet. Food Res. Int. 99, 15–30 (2017).Article 
    PubMed 

    Google Scholar 
    Mugiyo, H. et al. Multi-criteria suitability analysis for neglected and underutilised crop species in South Africa. PLoS ONE 16(1), e0244734 (2021).Article 
    PubMed 
    PubMed Central 
    CAS 

    Google Scholar 
    Fredenberg, E., Karl, K., Passarelli, S., Porciello, J., Rattehalli, V., Auguston, A., Chimwaza, G., Grande, F., Holmes, B., Kozlowski, N., Laborde, D., MacCarthy, D., Masikati, P., McMullin, S., Mendez Leal, E., Valdivia, R., Van Deynze, A., van Zonneveld, M. Vision for adapted crops and soils (VACS) research in action: Opportunity crops for Africa (2024). https://doi.org/10.7916/3hd1-8t86Sardeshpande, M. & Shackleton, C. Wild edible fruits: A systematic review of an under-researched multifunctional NTFP. Forests 10(6), 467 (2019).Article 

    Google Scholar 
    Sardeshpande, M. & Shackleton, C. Fruits of the veld: Ecological and socioeconomic patterns of natural resource use across South Africa. Hum. Ecol. 48, 665–677. https://doi.org/10.1007/s10745-020-00185-x (2020).Article 

    Google Scholar 
    Nkosi, N. N., Mostert, T. H. C., Dzikiti, S. & Ntuli, N. R. Prioritization of indigenous fruit tree species with domestication and commercialization potential in KwaZulu-Natal, South Africa. Genet. Resour. Crop Evol. 67, 1567–1575 (2020).Article 

    Google Scholar 
    Rich, K. M., Rich, M. & Dizyee, K. Participatory systems approach for urban and peri-urban agriculture planning: The role of system dynamics and spatial group model building. Agric. Syst. 160, 110–123. https://doi.org/10.1016/j.agsy.2016.09.022 (2018).Article 

    Google Scholar 
    Bilgilioglu, S. et al. A GIS-based multi-criteria decision-making method for the selection of potential municipal solid waste disposal sites in Mersin, Turkey. Environ Sci Pollut Res 29, 5313–5329. https://doi.org/10.1007/s11356-021-15859-2 (2022).Article 

    Google Scholar 
    Schubert, H., Rauchecker, M., Caballero Calvo, A. & Schütt, B. Land use changes and their perception in the Hinterland of Barranquilla, Colombian Caribbean. Sustainability 11(23), 6729. https://doi.org/10.3390/su11236729 (2019).Article 
    ADS 

    Google Scholar 
    Kirby, M. G., Scott, A. J. & Walsh, C. L. Translating policy to place: exploring cultural ecosystem services in areas of Green Belt through participatory mapping. Ecosyst. People 19(1), 2276752. https://doi.org/10.1080/26395916.2023.2276752 (2023).Article 

    Google Scholar 
    Depietri, Y. & Orenstein, D. E. Managing fire risk at the wildland-urban interface requires reconciliation of tradeoffs between regulating and cultural ecosystem services. Ecosyst. Serv. 44, 101108 (2020).Article 

    Google Scholar 
    Carrilho, J. & Trindade, J. Sustainability in Peri-Urban Informal Settlements: A Review. Sustainability 14(13), 7591. https://doi.org/10.3390/su14137591 (2022).Article 
    ADS 

    Google Scholar 
    Sahana, M., Ravetz, J., Patel, P. P., Dadashpoor, H. & Follmann, A. Where is the peri-urban? A systematic review of peri-urban research and approaches for its identification and demarcation worldwide. Remote Sens 15(5), 1316. https://doi.org/10.3390/rs15051316 (2023).Article 
    ADS 

    Google Scholar 
    Johnson, M. S. et al. The benefits of Q + PPGIS for coupled human-natural systems research: A systematic review. Ambio 51, 1819–1836. https://doi.org/10.1007/s13280-022-01709-z (2022).Article 
    ADS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Sardeshpande, M., Bangira, T., Matongera, T. N., Azong Cho, M., & Mabhaudhi, T. (2023). Appraising peri-urban food production in Durban, South Africa, with Participatory Geographic Information Systems (PGIS), 15 Nov 2023, PREPRINT (Version 1). Accessed on 08 Dec 2023, at Research Square.Department of Health (DoH). (2013). Roadmap for nutrition in South Africa 2013–2017. http://www.adsa.org.za/Portals/14/Documents/DOH/Nutrition%20Road%20Map%20 2013–2017.pdf (Accessed on 30/07/2017).Van der Hoeven, M. et al. Indigenous and traditional plants: South African parents’ knowledge, perceptions and uses and their children’s sensory acceptance. J. Ethnobiol. Ethnomed. 9, 1–12 (2013).
    Google Scholar 
    Porkka, M., Kummu, M., Siebert, S. & Varis, O. From food insufficiency towards trade dependency: a historical analysis of global food availability. PLoS ONE 8, e82714 (2013).Article 
    ADS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Bizikova, L. et al. A scoping review of the contributions of farmers’ organizations to smallholder agriculture. Nature Food 1(10), 620–630 (2020).Article 
    PubMed 

    Google Scholar 
    StatsSA. (2017). Poverty trends in South Africa: An examination of absolute poverty between 2006 and 2015. Report 031006, Statistics South Africa. Pretoria, South Africa, 138pp.Sardeshpande, M. & Shackleton, C. Urban foraging: Land management policy, perspectives, and potential. PLoS ONE 15(4), e0230693 (2020).Article 
    PubMed 
    PubMed Central 
    CAS 

    Google Scholar 
    Venter, Z. S., Shackleton, C. M., Van Staden, F., Selomane, O. & Masterson, V. A. Green Apartheid: Urban green infrastructure remains unequally distributed across income and race geographies in South Africa. Landsc. Urban Plan. 203, 103889 (2020).Article 

    Google Scholar 
    NDP. (2013). National Development Plan 2030. Our Future – make it work. Republic of South Africa. ISBN: 978–0–621–41180–5. 498pp. https://www.gov.za/documents/national-development-plan-2030-our-future-make-it-work accessed 08/04/2020NDA. (2021). National Department of Agriculture, Republic of South Africa. Programmes webpage. National Department of Agriculture, Land Reform, and Rural Development, Republic of South Africa. Available online at https://www.nda.agric.za/Programmes accessed on 17/08/2021.Hersperger, A. M., Grădinaru, S. R., Pierri Daunt, A. B., Imhof, C. S., & Fan, P. (2021). Landscape ecological concepts in planning: review of recent developments. Landscape Ecology, 1–17.eThekwini website. (2023). About us (eThekwini Municipality). Available online at https://www.durban.gov.za/pages/government/about-ethekwini accessed on July 13 2023Roberts, D. C. The design of an urban open-space network for the city of Durban (South Africa). Environ. Conserv. 21(1), 11–17 (1994).Article 

    Google Scholar 
    Davids, R., Rouget, M., Boon, R. & Roberts, D. Identifying ecosystem service hotspots for environmental management in Durban South Africa. Bothalia-African Biodiver. Conserv. 46(2), 1–18 (2016).Article 

    Google Scholar 
    Davids, R. et al. Civic ecology uplifts low-income communities, improves ecosystem services and well-being, and strengthens social cohesion. Sustainability 13(3), 1300 (2021).Article 
    ADS 
    PubMed 
    PubMed Central 

    Google Scholar 
    Bisaga, I., Parikh, P. & Loggia, C. Challenges and opportunities for sustainable urban farming in South African low-income settlements: A case study in Durban. Sustainability 11(20), 5660 (2019).Article 
    ADS 

    Google Scholar 
    Moyo, H. et al. Adaptive management in restoration initiatives: Lessons learned from some of South Africa’s projects. S. Afr. J. Bot. 139, 352–361 (2021).Article 

    Google Scholar 
    eThekwini Municipality. (2021). iNanda Mountain Community Reforestation Project Available online: http://www.durban.gov.za/City_Services/development_planning_management/environmental_planning_climate_protection/Projects/Pages/Inanda-Mountain-Community-Reforestation.aspx (accessed on August 13, 2021).Douwes, E. et al. The buffelsdraai landfill site community reforestation project. Unasylva 67, 12–19 (2016).
    Google Scholar 
    Taylor, L. & Hochuli, D. F. Defining greenspace: Multiple uses across multiple disciplines. Landsc. Urban Plan. 158, 25–38 (2017).Article 

    Google Scholar 
    Braun, V. & Clarke, V. Reflecting on reflexive thematic analysis. Qualitative Res. Sport Exercise Health 11(4), 589–597 (2019).Article 

    Google Scholar 
    Modi, A. T., & Mabhaudhi, T. M. (2018). Underutilised crop species in South Africa. Report to the water research commission of South Africa as part of WRC Project K5/2603//4 on developing a research agenda for promoting underutilised, indigenous and traditional crops.Sardeshpande, M. & Shackleton, C. Fruits of the city: The nature, nurture and future of urban foraging. People Nat. 5, 213–227. https://doi.org/10.1002/pan3.10428 (2023).Article 

    Google Scholar 
    El Baroudy, A. A. Mapping and evaluating land suitability using a GIS-based model. CATENA 140, 96–104 (2016).Article 

    Google Scholar 
    Kapoor, N., Jain, M. & Bansal, V. K. A methodological approach for weighting factors in land suitability assessment: a tool for facilitating spatial planning. J. Mt. Sci. 17(3), 724–739 (2020).Article 

    Google Scholar 
    Kazemi, H. & Akinci, H. A land use suitability model for rainfed farming by multi-criteria decision-making analysis (MCDA) and geographic information system (GIS). Ecol. Eng. 116, 1–6 (2018).Article 

    Google Scholar 
    Taherdoost, H. & Madanchian, M. Multi-criteria decision making (MCDM) methods and concepts. Encyclopedia 3(1), 77–87 (2023).Article 

    Google Scholar 
    Mazahreh, S., Bsoul, M. & Hamoor, D. A. GIS approach for assessment of land suitability for different land use alternatives in semi arid environment in Jordan: Case study (Al Gadeer Alabyad-Mafraq). Inform. Process. Agric. 6(1), 91–108 (2019).
    Google Scholar 
    Rstudio Team (2020). Rstudio: Integrated Development for R. Rstudio, PBC, Boston, MA URL http://www.rstudio.com/.Shackleton, C. M. et al. How important is green infrastructure in small and medium-sized towns? Lessons from South Africa. Landsc. Urban Plan. 180, 273–281 (2017).Article 

    Google Scholar 
    Shackleton, C. M., Hurley, P. T., Dahlberg, A. C., Emery, M. R. & Nagendra, H. Urban foraging: A ubiquitous human practice overlooked by urban planners, policy, and research. Sustainability 9(10), 1884 (2017).Article 
    ADS 

    Google Scholar 
    Bennett, J., Ainslie, A. & Davis, J. Contested institutions? Traditional leaders and land access and control in communal areas of Eastern Cape Province, South Africa. Land Use Policy 32, 27–38 (2013).Article 

    Google Scholar 
    Shackleton, C. M. et al. Deactivation of field cultivation in communal areas of South Africa: Patterns, drivers and socio-economic and ecological consequences. Land Use Policy 82, 686–699 (2019).Article 

    Google Scholar 
    Olofsson, M. Expanding commodity frontiers and the emergence of customary land markets: A case study of tree-crop farming in Venda, South Africa. Land Use Policy 101, 105203 (2021).Article 

    Google Scholar 
    Lidzhegu, Z. & Kabanda, T. Declining land for subsistence and small-scale farming in South Africa: A case study of Thulamela local municipality. Land Use Policy 119, 106170 (2022).Article 

    Google Scholar 
    Combrinck, Z., Cilliers, E. J., Lategan, L. & Cilliers, S. Revisiting the proximity principle with stakeholder input: Investigating property values and distance to urban green space in 26otchefstroom. Land 9(7), 235 (2020).Article 

    Google Scholar 
    Elands, B. H. M. et al. Biocultural diversity: A novel concept to assess human-nature interrelations, nature conservation and stewardship in cities. Urban For. Urban Green. 40, 29–34 (2019).Article 

    Google Scholar 
    Tiroesele, B., Obopile, M. & Karabo, O. Insect diversity and population dynamics of natural enemies under sorghum–legume intercrops. Trans. Royal Soc. South Africa 74(3), 258–267 (2019).Article 

    Google Scholar 
    Malviya, M. K. et al. Sugarcane-legume intercropping can enrich the soil microbiome and plant growth. Front. Sustain. Food Syst. 5, 606595 (2021).Article 

    Google Scholar 
    Pfukwa, T. M. et al. Southern African indigenous fruits and their byproducts: Prospects as food antioxidants. J. Funct. Foods 75, 104220 (2020).Article 
    CAS 

    Google Scholar 
    Ahmad, W. et al. Impact of land use/land cover changes on water quality and human health in district Peshawar Pakistan. Sci. Rep. 11(1), 16526 (2021).Article 
    ADS 
    PubMed 
    PubMed Central 
    CAS 

    Google Scholar 
    Ozsahin, E. & Ozdes, M. Agricultural land suitability assessment for agricultural productivity based on GIS modeling and multi-criteria decision analysis: the case of Tekirdağ province. Environ. Monit. Assess. 194(1), 41 (2022).Article 

    Google Scholar 
    Seyedmohammadi, J. & Navidi, M. N. Applying fuzzy inference system and analytic network process based on GIS to determine land suitability potential for agricultural. Environ. Monit. Assess. 194(10), 712 (2022).Article 
    PubMed 
    CAS 

    Google Scholar 
    Chang, N. B., Imen, S. & Vannah, B. Remote sensing for monitoring surface water quality status and ecosystem state in relation to the nutrient cycle: a 40-year perspective. Crit. Rev. Environ. Sci. Technol. 45(2), 101–166 (2015).Article 
    CAS 

    Google Scholar 
    Zhang, Q. R. et al. Spatial distribution and quantitative source identification of nutrients and beneficial elements in the soil of a typical suburban area, Beijing. Environ. Monit. Assess. 195(1), 1–14 (2023).Article 
    CAS 

    Google Scholar 
    Calijuri, M. L., Castro, J. D. S., Costa, L. S., Assemany, P. P. & Alves, J. E. M. Impact of land use/land cover changes on water quality and hydrological behavior of an agricultural subwatershed. Environ. Earth Sci. 74, 5373–5382 (2015).Article 
    ADS 

    Google Scholar 
    Greenberg, S., & Drimie, S. (2021). The state of the debate on agroecology in South Africa A scan of actors, discourses and policies. Report to CIRAD, available online at https://foodsecurity.ac.za/wp-content/uploads/2022/11/TAFS-project_The-state-of-the-debate-on-agroecology-in-South-Africa-A-scan-of-actors-discourses-and-policies.pdf Accessed on 19/06/2023Wotlolan, D. L., Lowry, J. H., Wales, N. A. & Glencross, K. Land suitability evaluation for multiple crop agroforestry planning using GIS and multi-criteria decision analysis: A case study in Fiji. Agrofor. Syst. 95(8), 1519–1532 (2021).Article 

    Google Scholar 
    Andersson, E. et al. What are the traits of a social-ecological system: Towards a framework in support of urban sustainability. NPJ Urban Sustain. 1(1), 14 (2021).Article 

    Google Scholar 
    Cilliers, E. J., Lategan, L., Cilliers, S. S. & Stander, K. Reflecting on the potential and limitations of urban agriculture as an urban greening tool in South Africa. Front. Sustain. Cities 2, 43 (2020).Article 

    Google Scholar 
    Kamjou, E., Scott, M. & Lennon, M. A bottom-up perspective on green infrastructure in informal settlements: Understanding nature’s benefits through lived experiences. Urban For. Urban Green. 94, 128231 (2024).Article 

    Google Scholar 
    Nesbitt, L., Meitner, M. J., Girling, C. & Sheppard, S. R. Urban green equity on the ground: Practice-based models of urban green equity in three multicultural cities. Urban For. Urban Green. 44, 126433 (2019).Article 

    Google Scholar 
    Vargas-Hernández, J. G. & Zdunek-Wielgołaska, J. Urban green infrastructure as a tool for controlling the resilience of urban sprawl. Environ. Dev. Sustain. 23(2), 1335–1354 (2021).Article 

    Google Scholar 
    Burgdorf, R., Sardeshpande, M. & Cooper, G. Indigenous tree forestry: Economic potential or potential fantasy?. Timber Indus. Presents Mag. 4(2), 14–20 (2023).
    Google Scholar 
    Green Trust (2024). Woza Nami: Trees combating hunger in South Africa. Available online at: https://www.greentrust.org.za/2024/09/03/woza-nami-phase-ii-trees-combating-hunger-in-south-africa/ Accessed on 27/11/2024Rockwell, C. A., Crow, A., Guimarães, É. R., Recinos, E. & La Belle, D. Species richness, stem density, and canopy in food forests: contributions to ecosystem services in an urban environment. Urban Plan. 7(2), 139–154 (2022).Article 

    Google Scholar 
    Balima, L. H. et al. Higher diversity, denser stands and greater biomass in peri-urban forests than in adjacent agroforestry systems in Western Burkina Faso: implications for urban sustainability. Environ. Monit. Assess. 195(9), 1077 (2023).Article 
    PubMed 

    Google Scholar 
    Fischer, J. et al. Advancing sustainability through mainstreaming a social–ecological systems perspective. Curr. Opinion Environ. Sustain. 14, 144–149 (2015).Article 

    Google Scholar 
    Wyborn, C. Co-productive governance: a relational framework for adaptive governance. Glob. Environ. Chang. 30, 56–67 (2015).Article 

    Google Scholar 
    Goodwin, G. The problem and promise of coproduction: Politics, history, and autonomy. World Dev. 122, 501–513 (2019).Article 

    Google Scholar 
    Isaac, M. E., Sinclair, F., Laroche, G., Olivier, A. & Thapa, A. The ties that bind: how trees can enhance agroecological transitions. Agrofor. Syst. 98, 2369–2383 (2024).Article 

    Google Scholar 
    Shackleton, C. M. et al. Embedding opportunities for poverty alleviation in urban green infrastructure design and management using South Africa as a case example. Cities 155, 105442 (2024).Article 

    Google Scholar 
    Hák, T., Janoušková, S. & Moldan, B. Sustainable Development Goals: A need for relevant indicators. Ecol. Ind. 60, 565–573 (2016).Article 

    Google Scholar 
    Alemneh, S. T., Emire, S. A., Hitzmann, B. & Zettel, V. Comparative study of chemical composition, pasting, thermal and functional properties of teff (Eragrostis tef) flours grown in Ethiopia and South Africa. Int. J. Food Prop. 25(1), 144–158 (2022).Article 
    CAS 

    Google Scholar 
    Bultosa, G. Teff: Overview. In 2015 (eds Wrigley, C. W. et al.) 209–220 (Academic Press, 2016).
    Google Scholar 
    Heuzé V., Thiollet H., Tran G., Lebas F., 2017. Tef (Eragrostis tef) hay. Feedipedia, a programme by INRAE, CIRAD, AFZ and FAO. https://www.feedipedia.org/node/22768 Last updated on March 6, 2017, 14:38 Accessed on 15 October 2025.Cockburn, J., Rouget, M., Slotow, R., Roberts, D., Boon, R., Douwes, E., & Willows-Munro, S. (2016). How to build science-action partnerships for local land-use planning and management: lessons from Durban, South Africa. Ecology and Society, 21(1).Leakey, R. R. & Harding, P. E. ‘Land maxing’: Regenerative, remunerative, productive and transformative agriculture to harness the six capitals of sustainable development. Sustainability 17(13), 5876 (2025).Article 
    ADS 

    Google Scholar 
    Republic of South Africa. (2019). Act No. 6 of 2019: Protection, Promotion, Development and Management of Indigenous Knowledge Act, 2019. Government Gazette 650: 42647.Greenberg, S., Drimie, S., Losch, B., & Jila, N. (2022). Agroecological initiatives in eThekwini Metropolitan Municipality, KwaZulu-Natal. TAFS phase 2. Final site report. Report to CIRAD, available online at https://agritrop.cirad.fr/605098/1/TAFS%20eThekwini%20research%20report_July%202022%20final%20.pdf Accessed on 22 October 2025Download referencesAcknowledgementsMS is grateful to all workshop participants at the three sites and the local chiefs and ward councillors for their time and efforts. MS thanks Kuhlekonke Mathenjwa for isiZulu and English interpretation at the workshops, and Nathi Ngcobo, Linda Mhlotshwa, and their associates for organising permissions and logistics. Thanks also to Errol Douwes, Mbali Goge, and Nomzamo Mncube for their support through the Durban Research Action Partnership. This research was funded in part by the Wellcome Trust [Grant number: 205200/Z/16/Z]. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. Fieldwork was funded in part by the WoodRIGHTS Project, which the University of KwaZulu-Natal Strategic Flagships funds.FundingThis research was funded in part by the Wellcome Trust [Grant number: 205200/Z/16/Z]. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. Fieldwork was funded in part by the WoodRIGHTS Project, which the University of KwaZulu-Natal Strategic Flagships funds.Author informationAuthors and AffiliationsCentre for Transformative Agricultural and Food Systems, University of KwaZulu-Natal, Scottsville, 3209, South AfricaMallika Sardeshpande, Tsitsi Bangira, Matilda Azong Cho, Trylee Nyasha Matongera & Tafadzwanashe MabhaudhiDepartment of Geography, School of Agriculture and Science, University of KwaZulu-Natal, Scottsville, 3209, South AfricaMatilda Azong Cho & Trylee Nyasha MatongeraCentre on Climate Change and Planetary Health, London School of Hygiene and Tropical Medicine, London, UKTafadzwanashe MabhaudhiAuthorsMallika SardeshpandeView author publicationsSearch author on:PubMed Google ScholarTsitsi BangiraView author publicationsSearch author on:PubMed Google ScholarMatilda Azong ChoView author publicationsSearch author on:PubMed Google ScholarTrylee Nyasha MatongeraView author publicationsSearch author on:PubMed Google ScholarTafadzwanashe MabhaudhiView author publicationsSearch author on:PubMed Google ScholarContributionsAll authors have read and consented to publication of this manuscript. Conceptualisation, Investigation, Data Curation, Writing—original: Mallika Sardeshpande; Methodology, Formal Analyses, Visualisation, Writing—review and editing: Mallika Sardeshpande, Tsitsi Bangira, Trylee Matongera, Matilda Azong Cho; Funding Acquisition, Supervision: Tafadzwanashe Mabhaudhi.Corresponding authorCorrespondence to
    Mallika Sardeshpande.Ethics declarations

    Competing interests
    The authors declare no competing interests.

    Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationBelow is the link to the electronic supplementary material.Supplementary Material 1Rights and permissions
    Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
    Reprints and permissionsAbout this articleCite this articleSardeshpande, M., Bangira, T., Azong Cho, M. et al. Exploring the social-ecological potential for indigenous agroforestry in peri-urban areas: a participatory mapping approach.
    Sci Rep 15, 44344 (2025). https://doi.org/10.1038/s41598-025-27864-3Download citationReceived: 25 June 2025Accepted: 06 November 2025Published: 23 December 2025Version of record: 23 December 2025DOI: https://doi.org/10.1038/s41598-025-27864-3Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
    Provided by the Springer Nature SharedIt content-sharing initiative
    KeywordsAgroecologyFarm designIndigenous foodsPeri-urban agricultureSuitability analysis More

  • in

    Environmental heterogeneity and its influence on fern diversity in a low-altitude mountain forest in central Taiwan

    AbstractEnvironmental heterogeneity plays a crucial role in shaping the distribution and composition of natural vegetation, including understory ferns. This study investigated the influence of environmental variation on understory fern communities within a one-hectare permanent plot in a low-altitude mountain forest in central Taiwan. Twenty-two environmental factors, including topographic, soil, and biotic variables, were recorded. Multiple regression, cluster (two-way indicator species analysis, TWINSPAN), and ordination (detrended correspondence analysis, DCA, and canonical correspondence analysis, CCA) analyses were conducted. A total of 51 fern species (including Lycophytes) belonging to 20 families and 30 genera were recorded. Among these, 43 were terrestrial, and eight were epiphytic; however, only terrestrial species were analyzed because of the limited representation of epiphytes. Multiple regression analyses revealed that environmental variables significantly affected fern richness, abundance, and community composition. Specifically, stream distance and the importance value (IV) of the saplings significantly influenced fern richness; herb/vine IV affected abundance; and the carbon-to-nitrogen ratio (C/N), manganese concentration (Mn), and herb/vine IV impacted the first axis of the DCA. Furthermore, the elevation, curvature, slope, and topographic wetness index (TWI) significantly influenced the second axis of the DCA. In all the models, topographic variables—particularly stream distance—were one of the most influential drivers. TWINSPAN categorized the ferns into four distinct groups (Diplazium donianum var. donianum [DIPLDO], D. donianum var. aphanoneuron [DIPLAP], Blechnopsis orientalis [BLECOR], and Angiopteris lygodiifolia [ANGILY]), and CCA revealed that environmental factors structured the community compositions in line with the TWINSPAN grouping. The DIPLDO and DIPLAP groups were associated with ridges and upper slope habitats characterized by higher elevations and drier conditions. In contrast, the BLECOR and ANGILY groups were associated with lower elevations, stream proximity, steeper slopes, and higher humidity. This study highlights the role of topographic and soil C/N heterogeneity in structuring fern communities in fine-scale plots for future ecological monitoring in subtropical forest ecosystems.

    Similar content being viewed by others

    Species diversity and spatial pattern of heritage trees in Taiyuan

    Article
    Open access
    21 May 2025

    Eco-systematic assessment of the spring herbaceous vegetation under edaphic and topographic effects

    Article
    Open access
    23 July 2024

    Changes in taxonomic and functional diversity of plants in a chronosequence of Eucalyptus grandis plantations

    Article
    Open access
    24 May 2021

    IntroductionEnvironmental gradients strongly influence the diversity and spatial distribution of plant communities. At regional scales, climate variables such as temperature and precipitation are dominant drivers1,2,3. In contrast, at local scales, fine-scale topography, soil characteristics, and biological interactions play significant roles4,5,6,7,8,9. Among biotic factors, canopy structure and density are widely recognized for their influence on understory plant communities10,11. Ferns, the second largest group of vascular plants, are a dominant component of understory vegetation in tropical and subtropical forests3,12 and are particularly sensitive to environmental heterogeneity.Topography—such as elevation, slope, aspect, and stream proximity—shapes microhabitats by altering light, temperature, and moisture regimes13,14. Topographic variation at the local scale is known to affect both fern diversity10 and abundance15. Stream proximity, in particular, is a strong predictor of fern assemblages16.Soil characteristics are also closely tied to fern distribution. Soil moisture (or humidity) is critical for the growth and development of ferns5,17. Variables such as nutrient content (e.g., N, P, K, Ca, and Mg), pH, and organic matter significantly influence fern performance5,16,18. The carbon-to-nitrogen (C/N) ratio, in particular, serves as a proxy for soil fertility and has been linked to fern richness in several tropical studies19,20. However, soil properties are partially influenced by topographic variations21,22, which in turn may affect the distribution of ferns.Understory ferns depend on canopy-mediated light availability for growth and reproduction23,24. Canopy openness not only alters photosynthetically active radiation but also modulates temperature and humidity in the understory25. Research in Southeast Asia and Taiwan suggests that canopy openness is an important factor influencing fern richness and cover10,26. Furthermore, ferns interact with other plant groups. Dense fern layers can suppress tree seedling recruitment27,28, whereas the diversity of co-occurring understory taxa appears to influence fern richness in varying ways29,30. These biotic interactions may result in mutual inhibition or facilitation depending on local environmental conditions.Ferns reproduce via spores that are readily dispersed by wind. Despite the fact that many ferns may produce spores capable of travelling long distances, chances of establishing new populations are low31. Allopatric differentiation may be associated with gametophytes that are highly sensitive to microclimatic and edaphic parameters32,33. In addition, environmental factors at the mesoscale—such as soil moisture, humidity, temperature, wind speed, rainfall, vegetation type, and canopy openness—significantly influence fern distribution by affecting sporophytes’ water requirements, temperature tolerance, and photosynthetic capacity5,26,34,35,36. Therefore, the diversity of forest microenvironments varies across regions and is reflected in corresponding differences in fern diversity and composition. We surveyed the relationship between environmental heterogeneity and fern diversity within a one-hectare plot embedded in a broader 25-hectare permanent plot in the Lienhuachih region, central Taiwan. We addressed the following questions: (1) Which environmental factors most strongly influence fern richness, abundance, and composition? (2) How do ferns cluster into ecological groups on the basis of these factors?Materials and methodsStudy areaThe study site is located in the Lienhuachih Experimental Forest (23°55’N, 120°52’E), which is located in a low-altitude mountainous area of central Taiwan (Fig. 1). A one-hectare permanent plot was established within a natural forest and represents the northwestern section of a broader 25-hectare forest dynamics plot initiated in 2007. The plot encompasses both mid-slope and riparian habitats, with an elevation range spanning from 755 m to 814 m (Table S1). On the basis of earlier tree surveys (DBH ≥ 1 cm), two forest types were identified: one dominated by Diospyros morrisiana and Cryptocarya chinensis and the other by Machilus japonica var. kusanoi and Helicia formosana37. The region experiences a mean annual temperature of 21.2℃ and receives approximately 2,178 mm of precipitation annually, with rainfall concentrated from March to September and a dry season from October to November26. The soil properties of the 25-hectare plot are partially influenced by topography21.Fig. 1Map of Taiwan (left) and a topographic map (right) of the one-hectare natural forest plot in Lienhuachih within the Houloun stream catchment, a mountainous region in central Taiwan.Full size imageSampling designThe one-hectare plot (100 m × 100 m) was divided into 100 subplots (10 m × 10 m) used as survey units. Vegetation data were collected per subplot, within which all ferns, herbs, vines, and tree saplings (< 1 cm in DBH, > 30 cm in height) were recorded. Fern abundance was assessed by counting individual clumps (treating each clump as one individual) and by estimating percent cover. Epiphytic ferns and vines were measured by the horizontal projection of their canopy cover. Tree data (DBH ≥ 1 cm) were also recorded. Ferns were categorized as either terrestrial or epiphytic, with the former defined as those growing on soil or rocks and the latter as those occurring on tree trunks. Taxonomy follows Volume 6 of the Flora of Taiwan38 and the classification by Kuo et al. (2019)39. Surveys were conducted from July 13 to 15, 2023.Environmental variablesThe topographic variables included elevation, plan curvature, slope, aspect, topographic wetness index (TWI), and distance to the stream. These data were collected from the center of each subplot. Soil variables (data were collected by Chang et al. (2013)40 (2023)41 ) included pH, the carbon-to-nitrogen ratio (C/N), nitrogen, phosphorus, potassium, calcium, magnesium, manganese, zinc, iron, and copper. Soil data were collected at a 20 m × 20 m resolution, with each 10 m × 10 m subplot assigned the values of its nearest soil sample point. Soil moisture was measured at a depth of 5 cm using a RiXEN M-700 S meter between February 26 and March 3, 2024, and the data were averaged from three diagonal points per subplot. Canopy openness was measured from March 15 to 22, 2024, using spherical crown densiometers at a height of 1.3 m at the center of each 10 m × 10 m subplot.Statistical analysisSince the soil properties of the 25-hectare plot are influenced by topography, a two-factor Pearson correlation coefficient analysis was conducted to examine the relationships between topography and soil properties in this study plot. Forward stepwise multiple regression (FSMR) with Poisson and linear models was employed to examine the effects of environmental factors on fern diversity. The abundance data for each species were quantified using the importance value (IV), which was calculated as the sum of its relative density (individuals per species/total individuals, unit: %) and relative cover (cover per species/total cover, unit: %). Twenty-two environmental variables—spanning topography (elevation, plan curvature, slope, aspect, TWI, and stream distance), soil properties (pH, C/N, N, P, K, Ca, Mg, Mn, Zn, Fe, Cu, and soil moisture), and biotic factors (canopy openness, tree density, sapling IV, and herb/vine IV)—were included in the analyses (Supplementary Table S1). The dependent variables included fern richness (species count), abundance (IV), and community composition (first two DCA axes). FSMR was performed for model selection using the “MuMIn” package in R. Significant predictors were selected (p < 0.05, chi-square test [Poisson] for fern richness and F test for fern abundance and composition [linear]), and then collinearity was assessed using the variance inflation factor (VIF). Variables with a VIF > 5 were iteratively excluded. Finally, Poisson regression was used to analyze fern richness and its selected predictor variables, whereas linear regression was applied to abundance, composition, and their respective selected predictors.Community classification was performed using two-way indicator species analysis (TWINSPAN) (dissimilarity metric = total inertia)42. Detrended correspondence analysis (DCA)43 was used to ordinate species and subplots. Canonical correspondence analysis (CCA)44 related species distributions to environmental gradients. The raw data utilized in the aforementioned analysis were derived from the species-subplot matrix, with the data comprising the previously described importance value (IV). All analyses were performed in R v4.3.1.ResultsA total of 51 fern species representing 20 families and 30 genera were recorded within the one-hectare plot. Of these, 43 species were terrestrial, and eight were epiphytic. Diplazium dilatatum was the most abundant species, with 1,011 individuals observed in 98 subplots, followed by Pleocnemia winitii, with 567 individuals in 90 subplots. These two species accounted for 55.8% of the total abundance of terrestrial ferns (Table 1). In contrast, ten terrestrial species were found in only one subplot (Supplementary Table S2), representing 23.3% of the total terrestrial fern richness.Table 1 Number of subplots, relative density, and relative coverage for terrestrial species of fern in the one-hectare plot of the low-altitude natural forest of central Taiwan. The species did not occur in fewer than 2 subplots.Full size tableIn addition to ferns, 37 herb, 43 vine and 76 sapling species were recorded, resulting in a total of 207 understory species (including 8 epiphytic fern species). Owing to their limited abundance and patchy distribution, epiphytic ferns were excluded from further analyses but are documented in Supplementary Table S2. Among the environmental variables, elevation was most significantly correlated with soil properties (11), followed by slope (nine) and stream distance (seven) (Supplementary Table S3).The regression models (Table 2) revealed that among the six selected variables, fern richness was significantly influenced by stream distance (negatively) and sapling abundance (positively). Fern abundance was most strongly associated with herb/vine IVs. With respect to fern composition, DCA1 was associated with stream distance, the C/N ratio, manganese, and herb/vine IV—with only stream distance being not significant. The best model of DCA2 included eight variables, among which elevation, curvature, slope, and TWI were significant.Table 2 Environmental factor models in a one-hectare natural forest plot in Lienhuachih used Poisson regression for fern richness and linear regression for fern abundance and composition (two DCA axes). “VIF” indicates the variance inflation factor test. *: p < 0.05; **: p < 0.01; and ***: p < 0.001.Full size tableTWINSPAN classified the fern community into four groups (Fig. 2a; Supplementary Figure S1): the Diplazium donianum var. donianum group (DIPLDO; n = 52), the D. donianum var. aphanoneuron group (DIPLAP; n = 21), the Blechnopsis orientalis group (BLECOR; n = 8), and the Angiopteris lygodiifolia group (ANGILY; n = 19). The mean fern richness was lowest in DIPLDO (4.2 ± 1.9) and highest in ANGILY (6.4 ± 2.5) (Supplementary Figure S2). Fern abundance (log-transformed) was positively correlated with richness (r = 0.46, p < 0.001), a pattern that was consistent across groups (Fig. 3). However, significant correlations were observed only in the DIPLDO and ANGILY groups, whereas the other two groups showed no significant correlation.Fig. 2TWINSPAN and CCA from a one-hectare natural forest plot in the Lienhuachih area of central Taiwan. (a) TWINSPAN identified four fern groups: DIPLDO (□), DIPLAP (△), BLECOR (○), and ANGILY (●). (b) The figure of the first two axes from the CCA; the words beside the lines represent environmental and biological factors, and the direction indicates the trend in which the value increases. (c) The same analysis as in b, with the letters representing the fern species (see Table 1).Full size imageFig. 3Relationships between fern abundance (log-transformed) and richness (r = 0.46, p < 0.001). The Pearson correlation in the four fern groups was r = 0.48 (p < 0.001, DIPLDO), 0.26 (p = 0.264, DIPLAP), 0.50 (p = 0.205, BLECOR), and 0.65 (p = 0.002, ANGILY).Full size imageThe cumulative explained variance of the first three CCA axes was 9.7%, 16.5%, and 21.2%, respectively. On the first axis of the CCA, herb/vine IV had the highest absolute score (0.80), followed by C/N (0.54), elevation (–0.50), and stream distance (–0.46); on the second axis of the CCA, stream distance (–0.61) had the highest absolute score, followed by Ca (–0.58), elevation (–0.50), and slope (0.43). In addition, herb/vine IV, stream distance, Ca and C/N were the most important determinants of one of the first two CCA axes (Table 3). As shown in Fig. 2b, the BLECOR group is more distinct, whereas the ANGILY group somewhat overlaps with the other groups, and the DIPLDO and DIPLAP groups exhibit greater overlap. Our results showed that these fern groups have adapted to different environments (Fig. 4).Table 3 Scores of the first two CCA axes with the environmental factors in the 1 ha plot of Lienhuachih in the low-altitude natural forest of central Taiwan. * shows the significance test (p < 0.05) for Pearson correlation between these factors and the CCA axes.Full size tableFig. 4Variation in elevation (a), stream distance (b), slope (c), and C/N (d) among different fern communities. Different letters denote statistically significant differences among the different types of fern vegetation (p < 0.05).Full size imageThe species ordination (Fig. 2c) revealed dominant ferns (Diplazium dilatatum and Pleocnemia winitii) near the plot center, whereas the species of named TWINSPAN groups aligned with the environmental characteristics of their respective groups. For example, DIPLDO’s D. donianum var. donianum was located in a topographic and edaphic space that is indicative of drier, upland sites.DiscussionTopographic effectsTopography has long been recognized as a key determinant of forest vegetation patterns8,45,46. In this study, elevation and stream distance emerged as primary predictors of fern richness and composition, despite the modest elevation range (~ 59 m) within the plot. These gradients reflect moisture availability: ridges with higher elevations and well-drained soils tend to be drier, whereas lower streamside zones retain more moisture. The strong correlation between elevation and stream distance (r = 0.40, p < 0.001) reinforces this interpretation. Our findings align with those of previous studies5,10,16 in montane forests where even fine-scale topographic variation influences fern diversity. For the other plant taxa in the 25-ha plot (of which our 1-ha plot was a part), topography was the most important factor affecting the changes in the plant community and species composition37.Soil effectsSoil properties such as nutrient concentrations and organic matter content often covary with topography because of erosion, leaching, and deposition21,47. While some studies have indicated a positive correlation between soil fertility and fern richness49,50, others have shown that lower fertility results in more fern species15,20. In our plot, C/N was significantly associated with fern composition, particularly along the first two CCA axes. Stream-adjacent soils, which are rich in organic matter and nitrogen, presented elevated carbon-to-nitrogen (C/N) ratios because of the greater accumulation of organic matter than that associated with decomposition in moist areas. Although fern richness was not directly correlated with C/N, its indirect effects via topographic mediation were evident. Calcium and manganese were also included in the regression models, although their contributions were relatively modest.Water serves as a critical determinant of both fern richness and distribution4,5,24. Water availability, inferred through the topographic wetness index (TWI), slope, and stream distance, likely exerts a dominant control on fern distributions. While soil moisture in the 25-ha plot (6 transects) was an important factor for the understory plants, including fern species30, it did not emerge as a significant predictor in the models in this study plot. The strong influence of hydrologically relevant topographic variables suggests their overriding importance in determining local fern composition.Biotic influencesLight availability is a well-documented driver of fern performance and affects morphology, abundance, and richness10,24. Although canopy openness was not retained in the final regression models, its significant correlation with tree density (r = − 0.22, p < 0.05) implies indirect effects. Denser tree canopies may reduce understory light, thus constraining fern growth.Interestingly, this study revealed positive associations between fern richness (or abundance) and both sapling and herb/vine cover, contrary to previous findings that emphasized competitive suppression28,30,48. The factors contributing to this outcome are likely multifaceted. The underlying mechanism may involve moisture availability, which is influenced by proximity to the stream. Although herb and vine cover are strongly correlated with fern abundance and composition, the primary determinant appears to be the distance from streams, as areas closer to streams generally exhibit higher moisture levels. Tuomisto et al. (2002) similarly reported that fern and Melastomataceae diversity co-occurred with tree richness in fertile tropical soils29.Fern community grouping and habitat differentiationTWINSPAN and CCA revealed clear compositional differentiation among the four fern groups, corresponding to distinct habitat types. The DIPLDO and DIPLAP groups were associated with ridges and upper slope habitats characterized by higher elevations and drier conditions. In contrast, the BLECOR and ANGILY groups were associated with lower elevations, stream proximity, steeper slopes, and higher humidity. These habitat preferences support the role of environmental filtering in fern assembly and align with prior vegetation classifications within the same forest37.In terms of the correlation between fern richness and abundance, compared with the DIPLDO group, the ANGILY group exhibited communities with greater evenness. These findings suggest that the environment inhabited by the ANGILY group is more conducive to the survival of a diverse range of ferns.ConclusionThis study highlights the significant role of environmental heterogeneity in shaping fern diversity and community composition in a low-altitude subtropical forest. Among the examined factors, topographic variables—particularly stream distance—exerted one of the most influential drivers of fern richness, abundance, and species assemblage. Soil properties, especially C/N, further mediated these relationships and reflected microhabitat variation. The classification of ferns into four ecological groups on the basis of environmental gradients highlights the structuring effect of habitat differentiation. The DIPLDO and DIPLAP groups occupied ridges and upper slope habitats characterized by higher elevations and drier conditions, whereas the BLECOR and ANGILY groups were associated with lower elevations, greater proximity to streams, steeper slopes, and higher humidity. This study highlights the role of topographic and soil-related heterogeneity in structuring fern communities in fine-scale plots. Long-term monitoring incorporating both abiotic and biotic variables is essential for understanding how fern communities respond to environmental change and for informing conservation strategies in subtropical forest ecosystems.

    Data availability

    The datasets utilized and/or analyzed during the current study are available from the first author upon reasonable request.
    ReferencesAntonelli, A. et al. Geological and Climatic influences on mountain biodiversity. Nat. Geosci. 11, 718–725 (2018).Article 
    ADS 
    CAS 

    Google Scholar 
    Lin, H. Y. et al. Climate-based approach for modeling the distribution of montane forest vegetation in Taiwan. Appl. Veg. Sci. 23, 239–253 (2020).Article 

    Google Scholar 
    Suissa, J. S., Sundue, M. A. & Testo, W. L. Mountains, climate and niche heterogeneity explain global patterns of fern diversity. J. Biogeogr. 48, 1296–1308 (2021).Article 

    Google Scholar 
    Richard, M., Bernhardt, T. & Bell, G. Environmental heterogeneity and the Spatial structure of fern species diversity in one hectare of old-growth forest. Ecography 23, 231–245 (2000).Article 
    ADS 

    Google Scholar 
    Karst, J., Gilbert, B. & Lechowicz, M. J. Fern community assembly: the roles of chance and the environment at local and intermediate scales. Ecology 86, 2473–2486 (2005).Article 

    Google Scholar 
    Bohlman, A. S. et al. Importance of soils, topography and geographic distance in structuring central Amazonian tree communities. J. Veg. Sci. 19, 863–874 (2008).Article 

    Google Scholar 
    Da Silva, K. E. et al. Tree species community Spatial structure in a Terra firme Amazon forest, Brazil. Bosque (Valdivia). 35, 347–355 (2014).Article 

    Google Scholar 
    Zhou, T. et al. Biodiversity of Jinggangshan mountain: the importance of topography and geographical location in supporting higher biodiversity. PLoS ONE. 10, e0120208 (2015).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Zuleta, D. et al. Importance of topography for tree species habitat distributions in a Terra firme forest in the Colombian Amazon. Plant. Soil. 450, 133–149 (2020).Article 
    CAS 

    Google Scholar 
    Jones, M. M., Cicuzza, D., Straaten, O., Veldkamp, E. & Kessler, M. Determinants of fern and angiosperm herb community structure in lower montane rainforest in Indonesia. J. Veg. Sci. 25, 1216–1224 (2014).Article 

    Google Scholar 
    Mendes, C. N., Diniz, E. S., Terra, M. C. N. S., Jeannot, K. K. & Fontes, M. A. L. Light conditions imposed by canopy: allometric strategies of an understorey palm (Geonoma schottianamart) in Atlantic forest. J. Trop. Sci. 31, 332–342 (2019).
    Google Scholar 
    Weigand, A. et al. Global fern and lycophyte richness explained: how regional and local factors shape plot richness. J. Biogeogr. 47, 59–71 (2020).Article 

    Google Scholar 
    Chao, K. J., Chao, W. C., Chen, K. M. & Hsieh, C. F. Vegetation dynamics of a lowland rainforest at the Northern border of the paleotropics at Nanjenshan, Southern Taiwan. Taiwan. J. Sci. 25, 29–40 (2010).
    Google Scholar 
    Moeslund, J. E., Arge, L., Bøcher, P. K., Dalgaard, T. & Svenning, J. C. Topography as a driver of local terrestrial vascular plant diversity patterns. Nord J. Bot. 31, 129–144 (2013).Article 

    Google Scholar 
    Cicuzza, D. & Mammides, C. Soil, topography and forest structure shape the abundance, richness and composition of fern species in the fragmented tropical landscape of Xishuangbanna, Yunnan, China. Forests 13, 1453 (2022).Article 

    Google Scholar 
    Bonari, G. et al. Fine-scale fern ecological responses inform on riparian forest habitat conservation status. Biodivers. Conserv. 31, 2141–2161 (2022).Article 

    Google Scholar 
    Poulsen, A. D., Tuomisto, H. & Balslev, H. Edaphic and floristic variation within a 1-ha plot of lowland Amazonian rain forest. Biotropica 38, 468–478 (2006).Article 

    Google Scholar 
    Viana, J. L., Turner, B. L. & Dalling, J. W. Compositional variation in understorey fern and palm communities along a soil fertility and rainfall gradient in a lower montane tropical forest. J. Veg. Sci. 32, 12947 (2021).Article 

    Google Scholar 
    Lwanga, J. S., Balmford, A. & Badaza, R. Assessing fern diversity: relative species richness and its environmental correlates in Uganda. Biodivers. Conserv. 7, 1387–1398 (1998).Article 

    Google Scholar 
    Zuquim, G. et al. Predicting environmental gradients with fern species composition in Brazilian Amazonia. J. Veg. Sci. 25, 1195–1207 (2014).Article 

    Google Scholar 
    Chang, L. W. et al. Lienhuachih Subtropical Evergreen Broadleaf Forest Dynamics Plot Tree Species Characteristics and Distribution Patterns 334–335 (Taiwan Forestry Research Institute, 2012).Wei, J. B., Xiao, D. N., Zeng, H. & &Fu, Y. K. Spatial variability of soil properties in relation to land use and topography in a typical small watershed of the black soil region, Northeastern China. Environ. Geol. 53, 1663–1672 (2008).Article 
    ADS 
    CAS 

    Google Scholar 
    Messier, C., Parent, S. & Bergeron, Y. Effects of overstory and understory vegetation on the understory light environment in mixed boreal forests. J. Veg. Sci. 9, 511–520 (1998).Article 

    Google Scholar 
    Hill, J. D. & Silander, J. A. Distribution and dynamics of two ferns: Dennstaedtia punctilobula (Dennstaedtiaceae) and Thelypteris noveboracensis (Thelypteridaceae) in a Northeast mixed hardwoods–hemlock forest. Amer. J. Bot. 88, 894–902 (2001).Article 
    CAS 

    Google Scholar 
    Angelini, A., Corona, P., Chianucci, F. & Portoghesi, L. Structural attributes of stand overstory and light under the canopy. Ann. Silvic Res. 39, 23–31 (2015).
    Google Scholar 
    Lee, P. H., Huang, Y. M., Chiou, W. L., Tseng, Y. H. & Tzeng, H. Y. The correlation between fern diversity and environmental factors-case study in a Chinese fir plantation in Lienhuachih, central Taiwan. Taiwan J. For. Sci. 39, 271–289 (2024).
    Google Scholar 
    George, L. O. & Bazzaz, F. A. The fern understory as an ecological filter: growth and survival of canopy-tree seedlings. Ecology 80, 846–856 (1999).Article 

    Google Scholar 
    Royo, A. A. & Carson, W. P. On the formation of dense understory layers in forests worldwide: consequences and implications for forest dynamics, biodiversity, and succession. Can. J. Res. 36, 1345–1362 (2006).Article 
    ADS 

    Google Scholar 
    Tuomisto, H. et al. Distribution and diversity of pteridophytes and Melastomataceae along edaphic gradients in Yasuní National Park, Ecuadorian Amazonia. Biotropica 34, 516–533 (2002).Article 

    Google Scholar 
    Chang, L. W., Huang, J. L., Luo, S. F. & Lee, P. H. Understory plant composition and its relations with environmental factors of the Lienhuachih forest dynamics plot at a subtropical evergreen broadleaf forest in central Taiwan. Taiwan J. For. Sci. 30, 245–257 (2015).
    Google Scholar 
    Wolf, P. G., Schneider, H. & Ranker, T. A. Geographic distributions of homosporous ferns: does dispersal obscure evidence of vicariance? J. Biogeogr. 28, 263–270 (2001).Article 

    Google Scholar 
    Page, C. N. The diversity of ferns: an ecological perspective. In: The Experimental Biology of Ferns (ed. Dyer, A. F.) 10–56 (1979).Leake, J. R. The biology of myco-heterotrophic (saprophytic) plants. New. Phytol. 127, 171–216 (1994).Article 
    PubMed 

    Google Scholar 
    Sanginés-Franco, C., Luna-Vega, I., Ayala, O. A. & Contreras-Medina, R. Distributional patterns and biogeographic analysis of ferns in the Sierra madre Oriental, Mexico. Amer Fern J. 101, 81–104 (2011).Article 

    Google Scholar 
    Chiu, T. Y. et al. Ecophysiological characteristics of three Cyathea species in Northeastern Taiwan. Taiwan J. For. Sci. 30, 147–155 (2015).
    Google Scholar 
    Silva, V. L., Mehltreter, K. & Schmitt, J. L. Ferns as potential ecological indicators of edge effects in two types of Mexican forests. Ecol. Indic. 93, 669–676 (2018).Article 

    Google Scholar 
    Chang, L. W. et al. Changes of plant communities classification and species composition along the micro-topography at the Lienhuachih forest dynamics plot in the central Taiwan. Taiwania 57, 359–371 (2012).CAS 

    Google Scholar 
    Boufford, D. E. et al. A Checklist of the Vascular Plants of Taiwan. in Flora of Taiwan, 2nd edition, Vol. 6 (ed. Editorial Committee of the Flora of Taiwan, S. E.) 15–139 (2003).Kuo, L. Y. et al. Updating Taiwanese pteridophyte checklist: A new phylogenetic classification. Taiwania 64, 367–395 (2019).
    Google Scholar 
    Chang, L. W., Zelený, D., Li, C. F., Chiu, S. T. & Hsieh, C. F. Better environmental data May reverse conclusions about niche- and dispersal-based processes in community assembly. Ecology 94, 2145–2151 (2013).Article 
    PubMed 

    Google Scholar 
    Chang, L. W., Chiu, S. T. & Hsieh, C. F. Contrasting Spatial distribution of pioneer versus non-pioneer saplings in a Taiwanese forest: A multiple scale approach. Ecol. Res. 38, 604–616 (2023).Article 

    Google Scholar 
    Rolecěk, J., Tichy, L., Zeleny, D. & Chytry, M. Modified TWINSPAN classification in which the hierarchy respects cluster heterogeneity. J. Veg. Sci. 20, 596–602 (2009).Article 

    Google Scholar 
    Hill, M. O. & Gauch, H. G. Detrended correspondence analysis: an improved ordination technique. Vegetatio 42, 47–58 (1980).Article 

    Google Scholar 
    Ter Braak, C. J. F. Canonical correspondence analysis: A new eigenvector technique for multivariate direct gradient analysis. Ecology 67, 1167–1179 (1986).Article 

    Google Scholar 
    Itoh, A. et al. Importance of topography and soil texture in the Spatial distribution of two sympatric dipterocarp trees in a Bornean rainforest. Ecol. Res. 18, 307–320 (2003).Article 

    Google Scholar 
    Jucker, T. et al. Topography shapes the structure, composition and function of tropical forest landscapes. Ecol. Lett. 21, 989–1000 (2018).Article 
    PubMed 
    PubMed Central 

    Google Scholar 
    Wilcke, W. et al. Soil properties and tree growth along an altitudinal transect in Ecuadorian tropical montane forest. J. Plant. Nutr. Soil. Sci. 171, 220–230 (2008).Article 
    CAS 

    Google Scholar 
    Liang, M. et al. Research progress on the ecology of dense fern understory. J. Trop. Subtrop Bot. 30, 219–300 (2022).ADS 

    Google Scholar 
    Ruokolainen, K., Tuomisto, H., Macía, M. J., Higgins, M. A. & Yli-Halla, M. Are floristic and edaphic patterns in Amazonian rain forests congruent for trees, pteridophytes and melastomataceae? J. Trop. Ecol. 23, 13–25 (2007).Article 

    Google Scholar 
    Jones, M. M., Ruokolainen, K., Martinez, N. C. L. & Tuomisto, H. Differences in topographic and soil habitat specialization between trees and two understorey plant groups in a Costa Rican lowland rain forest. J. Trop. Ecol. 32, 482–497 (2016).Article 

    Google Scholar 
    Download referencesAcknowledgementsWe thank the students of the Department of Forestry at National Chung Hsing University for their assistance in the wild investigation.Author informationAuthors and AffiliationsDepartment of Forestry, National Chung Hsing University, No. 145, Xingda Rd., South Dist, Taichung, 402202, TaiwanPei-Hsuan Lee, Yen-Hsueh Tseng & Hsy-Yu TzengTaiwan Forestry Research Institute, No. 53, Nanhai Rd, Taipei, 100051, TaiwanPei-Hsuan Lee, Yao-Moan Huang, Li-Wan Chang, Jian-Hong Yang, Wen-Liang Chiou & Yen-Hsueh TsengAuthorsPei-Hsuan LeeView author publicationsSearch author on:PubMed Google ScholarYao-Moan HuangView author publicationsSearch author on:PubMed Google ScholarLi-Wan ChangView author publicationsSearch author on:PubMed Google ScholarJian-Hong YangView author publicationsSearch author on:PubMed Google ScholarWen-Liang ChiouView author publicationsSearch author on:PubMed Google ScholarYen-Hsueh TsengView author publicationsSearch author on:PubMed Google ScholarHsy-Yu TzengView author publicationsSearch author on:PubMed Google ScholarContributionsConception or design of the work: Pei-Hsuan Lee, Hsy-Yu Tzeng. Data collection: Pei-Hsuan Lee, Li-Wan Chang, Jian-Hong Yang. Data analysis and interpretation: Pei-Hsuan Lee. Drafting the article: Pei-Hsuan Lee. Critical revision of the article: Wen-Liang Chiou, Yao-Moan Huang, Hsy-Yu Tzeng, Yen-Hsueh Tseng. All the authors reviewed the manuscript.Corresponding authorCorrespondence to
    Hsy-Yu Tzeng.Ethics declarations

    Competing interests
    The authors declare no competing interests.

    Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationBelow is the link to the electronic supplementary material.Supplementary Material 1Rights and permissions
    Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
    Reprints and permissionsAbout this articleCite this articleLee, PH., Huang, YM., Chang, LW. et al. Environmental heterogeneity and its influence on fern diversity in a low-altitude mountain forest in central Taiwan.
    Sci Rep 15, 44383 (2025). https://doi.org/10.1038/s41598-025-28048-9Download citationReceived: 29 May 2025Accepted: 07 November 2025Published: 23 December 2025Version of record: 23 December 2025DOI: https://doi.org/10.1038/s41598-025-28048-9Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
    Provided by the Springer Nature SharedIt content-sharing initiative
    KeywordsFern communityTopographySoil propertiesBiotic factors More

  • in

    Functional morphology of the leg musculature in the marine seal louse: adaptations for high-performance attachment to diving hosts

    AbstractThe seal louse (Echinophthirius horridus) is a remarkable example of evolutionary adaptation, thriving as an obligate ectoparasite on deep-diving marine mammals under extreme environmental conditions, including high hydrostatic pressure, extreme drag force, salinity, and fluctuating temperatures. To investigate the anatomical and functional specializations enabling this lifestyle, we compared the leg morphology and musculature of E. horridus with its terrestrial relative, the human head louse (Pediculus humanus capitis), using synchrotron-based 3D microtomography and confocal laser scanning microscopy. Our findings reveal that the seal louse has developed a highly compact and robust leg structure with a fused tibiotarsus, an additional set of leg muscles, and a shortened claw tendon—an unprecedented adaptation among insects. These features allow for greater force transmission and reduced metabolic cost during sustained attachment. Behavioral assays further show that E. horridus can only move effectively on hair-like substrates, underscoring its complete reliance on host fur. These findings suggest a highly specialized muscular control system enabling strong, reliable, and reversible attachment in a challenging aquatic environment.

    Similar content being viewed by others

    Attachment performance of the ectoparasitic seal louse Echinophthirius horridus

    Article
    Open access
    05 January 2024

    The ectoparasitic seal louse, Echinophthirius horridus, relies on a sealed tracheal system and spiracle closing apparatus for underwater respiration

    Article
    Open access
    03 June 2025

    The deeper the rounder: body shape variation in lice parasitizing diving hosts

    Article
    Open access
    09 September 2024

    Data availability

    All data is provided in the Supplementary Material of the manuscript. Synchrotron data and histological sectioning series90 can be provided upon request or online under: [https://doi.org/10.6084/m9.figshare.28596953.v2] (https:/doi.org/https://doi.org/10.6084/m9.figshare.28596953.v2).
    ReferencesGorb, S.N. (2001) Attachment Devices of Insect Cuticle. 1st edn, Springer Netherlands. 1st edn. Dordrecht, Netherlands: Springer, Dordrecht. https://doi.org/10.1007/0-306-47515-4.Beutel, R. G. & Gorb, S. N. Ultrastructure of attachment specializations of hexapods (Arthropoda): Evolutionary patterns inferred from a revised ordinal phylogeny. J. Zool. Syst. Evol. Res. 39(4), 177–207. https://doi.org/10.1046/j.1439-0469.2001.00155.x (2001).
    Google Scholar 
    Gorb, S. N. Biological attachment devices: exploring nature’s diversity for biomimetics. Philos. Trans. R. Soc. A: Math., Phys. Eng. Sci. 366(1870), 1557–1574. https://doi.org/10.1098/rsta.2007.2172 (2008).
    Google Scholar 
    Gorb, S. N. & Beutel, R. Evolution of locomotory attachment pads of hexapods. Naturwissenschaften 88(12), 530–534. https://doi.org/10.1007/s00114-001-0274-y (2001).
    Google Scholar 
    Bajerlein, D. et al. To attach or not to attach? The effect of carrier surface morphology and topography on attachment of phoretic. Naturwissenschaften 103(7–8), 61. https://doi.org/10.1007/s00114-016-1385-9 (2016).
    Google Scholar 
    Federle, W. & Labonte, D. Dynamic biological adhesion: Mechanisms for controlling attachment during locomotion. Philos. Trans. R. Soc. B: Biol. Sci. 374(1784), 20190199. https://doi.org/10.1098/rstb.2019.0199 (2019).
    Google Scholar 
    Li, D., Huson, M. G. & Graham, L. D. Proteinaceous adhesive secretions from insects, and in particular the egg attachment glue of Opodiphthera sp. moths. Arch. Insect Biochem. Physiol.: Publ. Collab. Entomol. Soc. Am. 69(2), 85–105. https://doi.org/10.1002/arch.20267 (2008).
    Google Scholar 
    Autumn, K. et al. Adhesive force of a single gecko foot-hair. Nature 405(6787), 681–685. https://doi.org/10.1038/35015073 (2000).
    Google Scholar 
    Autumn, K. et al. Evidence for van der Waals adhesion in gecko setae. Proc. Natl. Acad. Sci. 99(19), 12252–12256. https://doi.org/10.1073/pnas.192252799 (2002).
    Google Scholar 
    Autumn, K. & Hansen, W. Ultrahydrophobicity indicates a non-adhesive default state in gecko setae. J. Comp. Physiol. A. 192, 1205–1212. https://doi.org/10.1007/s00359-006-0149-y (2006).
    Google Scholar 
    Autumn, K. & Peattie, A. M. Mechanisms of adhesion in geckos. Integr. Comp. Biol. 42(6), 1081–1090. https://doi.org/10.1093/icb/42.6.1081 (2002).
    Google Scholar 
    Badge, I. et al. The role of surface chemistry in adhesion and wetting of gecko toe pads. Sci. Rep. 4(1), 6643. https://doi.org/10.1038/srep06643 (2014).
    Google Scholar 
    Maderson, P. F. A. Keratinized epidermal derivatives as an aid to climbing in gekkonid lizards. Nature 203(4946), 780–781. https://doi.org/10.1038/203780a0 (1964).
    Google Scholar 
    Mitchell, C. T. et al. The effect of substrate wettability and modulus on gecko and gecko-inspired synthetic adhesion in variable temperature and humidity. Sci. Rep. 10(1), 19748. https://doi.org/10.1038/s41598-020-76484-6 (2020).
    Google Scholar 
    Ruibal, R. & Ernst, V. The structure of the digital setae of lizards. J. Morphol. 117(3), 271–293. https://doi.org/10.1002/jmor.1051170302 (1965).
    Google Scholar 
    Williams, E. E. & Peterson, J. A. Convergent and alternative designs in the digital adhesive pads of scincid lizards. Science 215(4539), 1509–1511. https://doi.org/10.1126/science.215.4539.1509 (1982).
    Google Scholar 
    Higham, T. E. & Russell, A. P. Geckos running with dynamic adhesion: Towards integration of ecology, energetics and biomechanics. J. Exp. Biol. 228, 247980. https://doi.org/10.1242/jeb.247980 (2025).
    Google Scholar 
    Russell, A. P. The morphological basis of weight-bearing in the scansors of the tokay gecko (Reptilia: Sauria). Can. J. Zool. 64(4), 948–955. https://doi.org/10.1139/z86-144 (1986).
    Google Scholar 
    Chen, Y. et al. Underwater attachment using hairs: The functioning of spatula and sucker setae from male diving beetles. J. R. Soc. Interface 11(97), 20140273. https://doi.org/10.1098/rsif.2014.0273 (2014).
    Google Scholar 
    Kampowski, T. et al. Exploring the attachment of the Mediterranean medicinal leech (Hirudo verbana) to porous substrates. J. R. Soc. Interface 17(168), 20200300. https://doi.org/10.1098/rsif.2020.0300 (2020).
    Google Scholar 
    Kier, W. M. & Smith, A. M. The structure and adhesive mechanism of octopus suckers. Integr. Comp. Biol. 42(6), 1146–1153. https://doi.org/10.1093/icb/42.6.1146 (2002).
    Google Scholar 
    Smith, A. M. Negative pressure generated by octopus suckers: a study of the tensile strength of water in nature. J. Exp. Biol. 157(1), 257–271. https://doi.org/10.1242/jeb.157.1.257 (1991).
    Google Scholar 
    Smith, A. M. Cephalopod sucker design and the physical limits to negative pressure. J. Exp. Biol. 199(4), 949–958. https://doi.org/10.1242/jeb.199.4.949 (1996).
    Google Scholar 
    Busshardt, P. & Gorb, S. N. Walking on smooth and rough ground: Activity and timing of the claw retractor muscle in the beetle Pachnoda marginata peregrina (Coleoptera, Scarabaeidae). J. Exp. Biol. 216(2), 319–328. https://doi.org/10.1242/jeb.075614 (2013).
    Google Scholar 
    Dunlop, J. A. Movements of scopulate claw tufts at the tarsus tip of a tarantula spider. Netherlands J. Zool. 45(3–4), 513–520 (1994).
    Google Scholar 
    Federle, W. et al. Biomechanics of the movable pretarsal adhesive organ in ants and bees. Proc. Natl. Acad. Sci. 98(11), 6215–6220. https://doi.org/10.1073/pnas.111139298 (2001).
    Google Scholar 
    Frantsevich, L. & Gorb, S. Structure and mechanics of the tarsal chain in the hornet, Vespa crabro (Hymenoptera: Vespidae): implications on the attachment mechanism. Arthropod Struct. Dev. 33(1), 77–89. https://doi.org/10.1016/j.asd.2003.10.003 (2004).
    Google Scholar 
    Frazier, S. F. et al. Elasticity and movements of the cockroach tarsus in walking. J. Comp. Physiol. – A Sensory, Neural, Behav. Physiol. 185(2), 157–172. https://doi.org/10.1007/s003590050374 (1999).
    Google Scholar 
    Heming, B. S. Functional morphology of the thysanopteran pretarsus. Can. J. Zool. 49(1), 91–108. https://doi.org/10.1139/z71-014 (1971).
    Google Scholar 
    Niederegger, S. & Gorb, S. N. Tarsal movements in flies during leg attachment and detachment on a smooth substrate. J. Insect Physiol. 49(6), 611–620. https://doi.org/10.1016/S0022-1910(03)00048-9 (2003).
    Google Scholar 
    Gorb, S. N. et al. The insect unguitractor plate in action: Force transmission and the micro CT visualizations of inner structures. J. Insect Physiol. 117, 103908. https://doi.org/10.1016/j.jinsphys.2019.103908 (2019).
    Google Scholar 
    Gorb, S. N., Gorb, E. V. & Kastner, V. Scale effects on the attachment pads and friction forces in syrphid flies (Diptera, Syrphidae). J. Exp. Biol. 204(8), 1421–1431. https://doi.org/10.1242/jeb.204.8.1421 (2001).
    Google Scholar 
    Büscher, T. H. et al. The exceptional attachment ability of the ectoparasitic bee louse Braula coeca (Diptera, Braulidae) on the honeybee. Physiol. Entomol. 47(2), 83–95. https://doi.org/10.1111/PHEN.12378 (2022).
    Google Scholar 
    Petersen, D. S. et al. Holding tight to feathers – structural specializations and attachment properties of the avian ectoparasite Crataerina pallida (Diptera, Hippoboscidae). J. Exp. Biol. 221(13), jeb179242. https://doi.org/10.1242/jeb.179242 (2018).
    Google Scholar 
    Preuss, A. et al. Attachment performance of the ectoparasitic seal louse Echinophthirius horridus. Commun. Biol. 7(1), 36. https://doi.org/10.1038/s42003-023-05722-0 (2024).
    Google Scholar 
    Bush, A. O. et al. Parasitism: The diversity and ecology of animal parasites (Cambridge University Press, Cambridge, 2001).
    Google Scholar 
    Kim, K. C. Coevolution of parasitic arthropods and mammals (Wiley-Interscience, 1985).
    Google Scholar 
    Anderson, R. C. ‘Host-parasite relations and evolution of the Metastrongyloidea (Nematoda)’, Memoires du Museum National d’Histoire Naturelle. Serie A. Zoologie 123, 129–132. https://doi.org/10.5281/zenodo.16007555 (1982).
    Google Scholar 
    Raga, J. A. et al. Parasites. In Encyclopedia of Marine Mammals (eds Perrin, W. F. et al.) 821–830 (Academic Press, London, UK, 2009). https://doi.org/10.1016/B978-0-12-373553-9.00193-0.
    Google Scholar 
    Rybczynski, N., Dawson, M. R. & Tedford, R. H. A semi-aquatic Arctic mammalian carnivore from the Miocene epoch and origin of Pinnipedia. Nature 458(7241), 1021–1024. https://doi.org/10.1038/nature07985 (2009).
    Google Scholar 
    Durden, L. A. & Musser, G. G. The sucking lice (Insecta, Anoplura) of the world – a taxonomic checklist with records of mammalian hosts and geographical distributions. Bull. Am. Mus. Nat. Hist. 218, 1–90 (1994).
    Google Scholar 
    Grzimek, B. Grzimek’s encyclopedia of mammals (McGraw-Hill Publishing Company, 1990).
    Google Scholar 
    Leonardi, M. S. & Palma, R. L. Review of the systematics, biology and ecology of lice from pinnipeds and river otters (Insecta: Phthiraptera: Anoplura: Echinophthiriidae). Zootaxa 3630(3), 445–466. https://doi.org/10.11646/zootaxa.3630.3.3 (2013).
    Google Scholar 
    Eguchi, T. & Harvey, J. ‘Diving behavior of the Pacific harbor seal (Phoca vitulina richardii) in Monterey Bay California. Mar. Mamm. Sci. 21, 283–295. https://doi.org/10.1111/j.1748-7692.2005.tb01228.x (2006).
    Google Scholar 
    Frost, K. J., Simpkins, M. A. & Lowry, L. F. Diving behavior of subadult and adult harbor seals in Prince William Sound, Alaska. Mar. Mamm. Sci. 17(4), 813–834. https://doi.org/10.1111/j.1748-7692.2001.tb01300.x (2001).
    Google Scholar 
    Gjertz, I., Lydersen, C. & Wiig, Ø. Distribution and diving of harbour seals (Phoca vitulina) in Svalbard. Polar Biol. 24(3), 209–214. https://doi.org/10.1007/s003000000197 (2001).
    Google Scholar 
    Hastings, K. K. et al. Regional differences in diving behavior of harbor seals in the Gulf of Alaska. Can. J. Zool. 82(11), 1755–1773. https://doi.org/10.1139/z04-145 (2004).
    Google Scholar 
    Kolb, P. M. A harbor seal Phoca vitulina richardsi, taken from a sablefish trap. California Fish Game 68, 123–124 (1982).
    Google Scholar 
    Rosing-Asvid, A. et al. Deep diving harbor seals (Phoca vitulina) in South Greenland: movements, diving, haul-out and breeding activities described by telemetry. Polar Biol. 43(4), 359–368. https://doi.org/10.1007/s00300-020-02639-w (2020).
    Google Scholar 
    Dehnhardt, G., Mauck, B. & Hyvärinen, H. Ambient temperature does not affect the tactile sensitivity of mystacial vibrissae in harbour seals. J. Exp. Biol. 201(22), 3023–3029. https://doi.org/10.1242/jeb.201.22.3023 (1998).
    Google Scholar 
    Mauck, B. et al. Thermal windows on the trunk of hauled-out seals: Hot spots for thermoregulatory evaporation?. J. Exp. Biol. 206(10), 1727–1738. https://doi.org/10.1242/jeb.00348 (2003).
    Google Scholar 
    Hansen, S. & Lavigne, D. M. Ontogeny of the thermal limits in the harbor seal (Phoca vitulina). Physiol. Zool. 70(1), 85–92. https://doi.org/10.1086/639549 (1997).
    Google Scholar 
    Watts, P. Thermal constraints on hauling out by harbor seals (Phoca vitulina). Can. J. Zool. 70, 553–560. https://doi.org/10.1139/z92-083 (2011).
    Google Scholar 
    Leonardi, M. S. et al. Under pressure: the extraordinary survival of seal lice in the deep sea. J. Exp. Biol. 223(17), jeb226811. https://doi.org/10.1242/jeb.226811 (2020).
    Google Scholar 
    Williams, T. M. & Kooyman, G. L. Swimming performance and hydrodynamic characteristics of harbor seals Phoca vitulina. Physiol. Zool. 58(5), 576–589. https://doi.org/10.1086/physzool.58.5.30158584 (1985).
    Google Scholar 
    Preuss, A., Gorb, S.N., et al. (2025) ‘Role of the Setae in an Ectoparasitic Seal Louse in Reducing Surface Drag: Numerical Modeling Approach’, Advanced Theory and Simulations, p. e00429. https://doi.org/10.1002/adts.202500429.Herzog, I. et al. Heartworm and seal louse: Trends in prevalence, characterisation of impact and transmission pathways in a unique parasite assembly on seals in the North and Baltic Sea. Int. J. Parasitol.: Parasites Wildlife 23, 100898. https://doi.org/10.1016/j.ijppaw.2023.100898 (2024).
    Google Scholar 
    Herzog, I., Siebert, U. & Lehnert, K. High prevalence and low intensity of Echinophthirius horridus infection in seals revealed by high effort sampling. Sci. Rep. 14(1), 14258. https://doi.org/10.1038/s41598-024-64890-z (2024).
    Google Scholar 
    Siebert, U. et al. Pathological findings in harbour seals (Phoca vitulina): 1996–2005. J. Comp. Pathol. 137(1), 47–58. https://doi.org/10.1016/j.jcpa.2007.04.018 (2007).
    Google Scholar 
    Michels, J. & Gorb, S. N. Detailed three-dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy. J. Microsc. 245(1), 1–16. https://doi.org/10.1111/j.1365-2818.2011.03523.x (2012).
    Google Scholar 
    Andersen, S. O. Biochemistry of insect cuticle. Annu. Rev. Entomol. 24(1), 29–59. https://doi.org/10.1146/annurev.en.24.010179.000333 (1979).
    Google Scholar 
    Büsse, S. & Gorb, S. N. Material composition of the mouthpart cuticle in a damselfly larva (Insecta: Odonata) and its biomechanical significance. R. Soc. Open Sci. 5(6), 172117. https://doi.org/10.1098/rsos.172117 (2018).
    Google Scholar 
    Josten, B., Gorb, S. N. & Büsse, S. The mouthparts of the adult dragonfly Anax imperator (Insecta: Odonata), functional morphology and feeding kinematics. J. Morphol. 283(9), 1163–1181. https://doi.org/10.1002/jmor.21497 (2022).
    Google Scholar 
    Vincent, J. F. V. Arthropod cuticle: a natural composite shell system. Compos. A Appl. Sci. Manuf. 33(10), 1311–1315. https://doi.org/10.1016/S1359-835X(02)00167-7 (2002).
    Google Scholar 
    Cecilia, A. et al. The IMAGE beamline at the KIT light source. J. Synchrotron Radiat. 32(4), 1036–1051. https://doi.org/10.1107/s1600577525003777 (2025).
    Google Scholar 
    Douissard, P. A. et al. A versatile indirect detector design for hard X-ray microimaging. J. Instrum. 7(9), P09016. https://doi.org/10.1088/1748-0221/7/09/P09016 (2012).
    Google Scholar 
    Vogelgesang, M. et al. Real-time image-content-based beamline control for smart 4D X-ray imaging. J. Synchrotron Radiat. 23(5), 1254–1263. https://doi.org/10.1107/S1600577516010195 (2016).
    Google Scholar 
    Vogelgesang, M. et al. (2012) ‘UFO: A Scalable GPU-based Image Processing Framework for On-line Monitoring’, in Proceedings of HPCC-ICESS., pp. 824–829. https://doi.org/10.1109/HPCC.2012.116.Faragó, T. et al. Tofu: A fast, versatile and user-friendly image processing toolkit for computed tomography. J. Synchrotron Radiat. 29, 916–927. https://doi.org/10.1107/S160057752200282X (2022).
    Google Scholar 
    Gray, P. T. A., Mill, P. J. & Dodd, J. M. ‘The musculature of the prothoracic legs and its innervation in Hierodula membranacea (Mantidea)’. Philos. Trans. R. Soc. London B, Biol. Sci. 309(1140), 479–503. https://doi.org/10.1098/rstb.1985.0094 (1997).
    Google Scholar 
    Preuss, A. et al. The ectoparasitic seal louse, Echinophthirius horridus, relies on a sealed tracheal system and spiracle closing apparatus for underwater respiration. Commun. Biol. 8(1), 1–14. https://doi.org/10.1038/s42003-025-08285-4 (2025).
    Google Scholar 
    Leonardi, M. S. et al. The deeper the rounder: body shape variation in lice parasitizing diving hosts. Sci. Rep. 14(1), 1–10. https://doi.org/10.1038/s41598-024-71541-w (2024).
    Google Scholar 
    Appel, E. et al. Ultrastructure of dragonfly wing veins: Composite structure of fibrous material supplemented by resilin. J. Anat. 227(4), 561–582. https://doi.org/10.1111/joa.12362 (2015).
    Google Scholar 
    Vaughan, J. A. & Azad, A. F. Patterns of erythrocyte digestion by bloodsucking insects: constraints on vector competence. J. Med. Entomol. 30(1), 214–216. https://doi.org/10.1093/jmedent/30.1.214 (1993).
    Google Scholar 
    Waniek, P. J. The digestive system of human lice: Current advances and potential applications. Physiol. Entomol. 34(3), 203–210. https://doi.org/10.1111/j.1365-3032.2009.00681.x (2009).
    Google Scholar 
    Leonardi, M. S. et al. Host-parasite coevolution leads to underwater respiratory adaptations in extreme diving insects, seal lice (Lepidophthirus macrorhini). Communications Biology 8(1), 1–11. https://doi.org/10.1038/s42003-025-08306-2 (2025).
    Google Scholar 
    Soler Cruz, M. D. & Martín Mateo, M. P. Scanning electron microscopy of legs of two species of sucking lice (Anoplura: Phthiraptera). Micron 40(3), 401–408. https://doi.org/10.1016/j.micron.2008.10.001 (2009).
    Google Scholar 
    Wolfram, L. J. Human hair: A unique physicochemical composite. J. Am. Acad. Dermatol. 48(6), S106–S114. https://doi.org/10.1067/mjd.2003.276 (2003).
    Google Scholar 
    Aibekova, L. et al. The skeletomuscular system of the mesosoma of Formica rufa Workers (Hymenoptera: Formicidae). Insect Systematics and Diversity 6(2), 1–26. https://doi.org/10.1093/isd/ixac002 (2022).
    Google Scholar 
    Woodworth, C. W. The leg tendons of insects. Am. Nat. 42(499), 452–456. https://doi.org/10.1086/278953 (1908).
    Google Scholar 
    Lee, S. S. M. & Piazza, S. J. Built for speed: Musculoskeletal structure and sprinting ability. J. Exp. Biol. 212(22), 3700–3707. https://doi.org/10.1242/jeb.031096 (2009).
    Google Scholar 
    Baxter, J. R. et al. Ankle joint mechanics and foot proportions differ between human sprinters and non-sprinters. Proc. R. Soc. B: Biol. Sci. 279(1735), 2018–2024. https://doi.org/10.1098/rspb.2011.2358 (2012).
    Google Scholar 
    Fletcher, J. R. & MacIntosh, B. R. Achilles tendon strain energy in distance running: consider the muscle energy cost. J. Appl. Physiol. 118(2), 193–199. https://doi.org/10.1152/japplphysiol.00732.2014 (2015).
    Google Scholar 
    MacIntosh, B.R. and Holash, R.J. (2000) ‘Power output and force-velocity properties of muscle’, In: B.M. Nigg, B.R. MacIntosh, and J. Mester (Eds) Biomechanics and biology of movement. Human Kinetics. Champaign, Illinois (US): Human Kinetics, pp. 193–210.Kim, K. C. Ecology and morphological adaptations of the sucking lice on the nothern fur seal. J. Cons. Int. Explor. Mer. 169, 504–515 (1975).
    Google Scholar 
    Koštál, V. Eco-physiological phases of insect diapause. J. Insect Physiol. 52(2), 113–127. https://doi.org/10.1016/j.jinsphys.2005.09.008 (2006).
    Google Scholar 
    Leonardi, M. S. & Lazzari, C. R. Uncovering deep mysteries: The underwater life of an amphibious louse. J. Insect Physiol. 71, 164–169. https://doi.org/10.1016/j.jinsphys.2014.10.016 (2014).
    Google Scholar 
    Gorb, S. N. Design of insect unguitractor apparatus. J. Morphol. 230(2), 219–230. https://doi.org/10.1002/(SICI)1097-4687(199611)230:2%3c219::AID-JMOR8%3e3.0.CO;2-B (1996).
    Google Scholar 
    Hörger, V., Labisch, S. & Dirks, J.-H. Biomimetic tag attachment inspired by the seal louse. Bioinspiration Biomimetics 20(6), 066015. https://doi.org/10.1088/1748-3190/adfbb8 (2025).
    Google Scholar 
    Preuss, A., Schwaha, T., et al. (2025a) ‘Nano-CT data and histological sections of Pediculus humanus capitis and Echinophthirius horridus’, figshare. https://doi.org/10.6084/m9.figshare.28596953.v2.Download referencesAcknowledgementsWe express our profound gratitude to Dr. Thies Büscher, Dr. Helen Gorges, Fabian Bäumler, Julian Thomas, Simon Züger, and Benedikt Josten for their invaluable guidance and assistance throughout this study. We also extend our appreciation to Esther Appel and Dr. Alexander Kovalev for their technical support during the experiments. We would also like to thank the TiHo Hannover, especially Dr. Insa Herzog and Dr. Kristina Lehnert, for supplying us with seal louse samples. Additionally, we acknowledge Marta Tischer for generously supplying head louse samples preserved in ethanol. We are indebted to Dr. Angelica Cecilia for her assistance during beamtime and to Dr. Tomáš Faragó for his work on tomographic reconstructions. We express our gratitude to the KIT Light Source for providing instruments at their beamlines and to the Institute for Beam Physics and Technology (IBPT) for operating the storage ring, the Karlsruhe Research Accelerator (KARA). Furthermore, we acknowledge the funding provided to S.N.G. by the grant GO 995 46-1 from the German Science Foundation (DFG) within the Special Priority Program (SPP 2332) “Physics of Parasitism.” The funders had no involvement in the study design, data collection and analysis, publication decisions, or manuscript preparation.FundingOpen Access funding enabled and organized by Projekt DEAL. Funding for this study was granted to S.N.G by the German Science Foundation (DFG) under grant number GO 995 46–1, as part of the Special Priority Program (SPP 2332) known as “Physics of Parasitism”.Author informationAuthors and AffiliationsDepartment of Functional Morphology and Biomechanics, Zoological Institute, Kiel University, Am Botanischen Garten 1–9, 24118, Kiel, GermanyAnika Preuss, Lina Ornowski & Stanislav N. GorbInstitute for Photon Science and Synchrotron Radiation (IPS), Karlsruhe Institute of Technology (KIT), Hermann-Von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, GermanyThomas van de Kamp, Elias Hamann & Marcus ZuberLaboratory for Applications of Synchrotron Radiation (LAS), Karlsruhe Institute of Technology (KIT), Kaiserstr.12, 76131, Karlsruhe, GermanyThomas van de KampAuthorsAnika PreussView author publicationsSearch author on:PubMed Google ScholarThomas van de KampView author publicationsSearch author on:PubMed Google ScholarElias HamannView author publicationsSearch author on:PubMed Google ScholarMarcus ZuberView author publicationsSearch author on:PubMed Google ScholarLina OrnowskiView author publicationsSearch author on:PubMed Google ScholarStanislav N. GorbView author publicationsSearch author on:PubMed Google ScholarContributionsA.P., S.N.G.—Conceptualization; A.P., T.v.d.K., S.N.G.—Data curation; A.P., T.v.d.K., S.N.G.—Formal analysis; S.N.G.—Funding acquisition; A.P., T.v.d.K., L.O.—Investigation; A.P., T.v.d.K., E.H., M.Z., S.N.G.—Methodology; S.N.G.—Project administration; A.P.—Software; S.N.G.—Supervision; A.P., S.N.G.—Validation; A.P., L.O.—Visualization; A.P.—Writing—original draft; T.v.d.K., E.H., M.Z., L.O., S.N.G.—Writing – review & editing.Corresponding authorCorrespondence to
    Anika Preuss.Ethics declarations

    Competing interests
    The authors declare no competing interests.

    Ethical approval
    Ethical review and approval were not required for this study, as all host animals were either found dead, died naturally, or were euthanized on welfare grounds, with none being killed specifically for this research. The authors were not involved in the euthanasia of the hosts, which was carried out by certified seal rangers for reasons unrelated to this study. All regulations regarding animal use were strictly followed.

    Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7Supplementary Material 8Supplementary Material 9Rights and permissions
    Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
    Reprints and permissionsAbout this articleCite this articlePreuss, A., van de Kamp, T., Hamann, E. et al. Functional morphology of the leg musculature in the marine seal louse: adaptations for high-performance attachment to diving hosts.
    Sci Rep (2025). https://doi.org/10.1038/s41598-025-32804-2Download citationReceived: 16 September 2025Accepted: 12 December 2025Published: 23 December 2025DOI: https://doi.org/10.1038/s41598-025-32804-2Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
    Provided by the Springer Nature SharedIt content-sharing initiative
    KeywordsParasitismSeal louseHuman head louseMarine mammalsBiomechanicsExtremitiesSkeleton-muscle organizationSupplementary Material 4Supplementary Material 5Supplementary Material 6 More