Fire and Herbivory as Architects of Mediterranean Biodiversity

Fire and Herbivory as Architects of Mediterranean Biodiversity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fire and Herbivory as Architects of Mediterranean Biodiversity Marion Lestienne, Pauline Saurat, Gwendal Mouden, Andy Hennebelle, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6163168/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This research breaks new ground by showing that, contrary to generally accepted key role of fire in shaping plant communities, herbivores shaped both landscape heterogeneity and fire-prone habitats in the Mediterranean area. The interconnected roles of fire and herbivores predisposed habitat resilience, highlighting the need to integrate grazing for fire risk mitigation and biodiversity conservation. The recent decline in pastoralism coincides with the expansion of highly flammable vegetation, exacerbating fire risks under current climate conditions. These insights advocate for conservation strategies that balance fire management and sustainable herbivory to preserve Mediterranean biodiversity in the face of increasing anthropogenic and climatic pressures. Earth and environmental sciences/Ecology/Fire ecology Earth and environmental sciences/Ecology/Ecosystem ecology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Mediterranean region is among the most species-rich biomes on Earth ( 1 ). Its remarkable biodiversity reflects a long history of interactions between disturbances ( i.e. fires and herbivores), climate variability, anthropogenic activities and land uses such as sylvo-pastoralism ( 2 , 3 ). However, Mediterranean ecosystems are increasingly under pressure from climatic shifts and human impacts, making the understanding of their resilience and dynamics critical ( 4 , 5 ). Fire is a dominant ecological force in the Mediterranean, where hot, dry summers and dense vegetation sustain frequent, high-intensity fires that have long shaped plant communities ( 6 , 7 ). Fire-adapted species and open habitats owe their persistence to the fire regime, which has helped maintain the mosaic of landscapes characteristic of the region ( 8 , 9 ). Although fire is a well-established disturbance acting on Mediterranean vegetation dynamics and landscape structure, herbivores have been demonstrated to play a key role on Mediterranean landscape dynamics ( 10 , 11 ). Both wild herbivores and domesticated livestock have contributed to shaping vegetation patterns over millennia, influencing plant diversity and preventing the dominance of woody species ( 3 , 12 , 13 ). While the role of grazing in pastoral contexts is relatively well-documented ( 3 , 14 ), the ecological role of wild herbivores remains understudied despite their long-term influence on vegetation structure and ecosystem resilience ( 15 ). In addition, the cumulative impacts of both herbivores and fire is poorly reported due to the lack of records for both disturbances in this area ( 16 ). This oversight hampers our ability to fully understand the interactions between fire and herbivory and their cumulative impacts on vegetation and biodiversity. Addressing this knowledge gap requires integrating evidence from both wild and domestic herbivores to assess their combined effects on Mediterranean ecosystems. This study seeks to address these gaps by reconstructing the long-term dynamics of vegetation habitats and herbivores densities in the Crau Plain, where a unique landscape of Mediterranean steppe and mixed oak forest co-exist today. By employing a multi-millennial record of both vegetation and herbivore types and densities, we aim at disentangling the cumulative effects of fire and herbivores on biodiversity and vegetation composition over millennia. This approach will enhance our understanding of the processes underpinning Mediterranean biodiversity and inform strategies for sustainable ecosystem management under future environmental pressures. To better understand habitat dynamics and vegetation combustibility in Mediterranean ecosystems, we use Trabaud's classification, which focuses on habitat types and flammability rather than species ( 17 ). This functional approach highlights structural and ecological traits influencing fire behavior, offering insights into how fire regimes and herbivores shape habitat resilience and biodiversity over millennia ( 18 , 19 ). Results and discussion Fire and human influence on habitat dynamics The Crau Plain's vegetation was classified into five main habitats: white oak coppice, pine forest, garrigue/heathland, grassland, and green oak coppice, following Trabaud's (1971) classification ( 17 ) (Figure 2; see SI Figures S2 for details). These habitats exhibit varying levels of combustibility, with open landscapes such as grasslands and garrigue/heathland being more prone to fire, while closed forests such as white oak coppice and beech forest exhibit lower combustibility. The dominance and shifts in these habitats reflect a complex interplay between climate, fire regimes, and human activities throughout the last 12,000 years of climate variations ( i.e. the Holocene). Fire is recognized as a key ecological driver ( 6 ), and its influence on the Crau Plain is evident in the higher combustibility of habitats observed before 6,500 years cal. BP and after 1,000 years cal. BP. The mean combustibility of habitats during these periods reached 3.12 and 3.30, respectively, compared to lower values of 2.71 and 2.69 during intermediate periods (Figure 2). Significant differences were determined using a non-parametric Mann-Whitney U test, with the following p-values: periods 1 vs. 2 (p-value = 1.754e -08 ), periods 1 vs. 3 (p-value = 1.004e -08 ), periods 2 vs. 4 (p-value = 1.281e -06 ), and periods 3 vs. 4 (p-value = 1.677e -06 ). Periods 1 vs. 4 (p-value = 0.1036) and periods 2 vs. 3 (p-value = 0.9324) show no significant differences. During warmer and drier periods ( 5 ), fire-adapted species thrived in open and dry environments ( 20 ). Fire acts as a selective pressure favoring pyrophytic species but also plays a critical role in shaping plant communities composition by suppressing fire-sensitive species and promoting open vegetation ( 21 ). This process leads to a mosaic of habitats that supports high biodiversity, particularly in grassland and shrubland ecosystems ( 20 ). Conversely, phases of reduced fire activity coincided with wetter and colder climatic conditions ( 22 ), favoring habitats like white oak coppice and beech forests, which suppress fire spread ( 23 ). Human influence emerged as a significant factor in shaping habitats composition and fire regimes ( 24 , 25 ). Declines in wild herbivore densities (Figure 3E, G) point to early hunting pressure and land-use changes ( 26 ). Then, grazing by domestic livestock transformed the landscape by promoting open habitats such as grasslands and garrigue/heathland ( 11 , 27 ). This transformation increased the prevalence of highly combustible environments, particularly after the introduction of these pastoral systems ( 19 ). In transitional periods, shifts in habitats composition highlight the interplay between natural and anthropogenic forces. For example, the gradual replacement of beech and white oak coppice by green oak coppice and pine forest reflects a combination of human-driven deforestation, selective tree clearance, and climatic fluctuations ( 20 ). These habitats are less fire-resistant and mark the adaptive response of Mediterranean ecosystems to changing environmental pressures. The recent landscape dynamics, characterized by forest expansion following land abandonment ( 11 ), further demonstrate the evolving relationships between humans and the environment. Increased forest cover, particularly of combustible species, has increased fire risks in recent centuries ( 28 , 29 ). This trend confirms the unique situation of the area as of today, which reveals the necessity for stakeholders to account for ecological drivers such as both herbivores and human land-use to mitigate future fire hazards ( 30 , 31 ). As such, while fire and human activities have played pivotal roles in shaping habitat dynamics, herbivores act as a parallel force, maintaining high plant diversity and preventing the dominance of woody species, thereby reinforcing the resilience of Mediterranean ecosystems. Herbivores have promoted high plant diversity. Herbivores emerge as a key ecological driver of biodiversity, fostering both species richness and heterogeneity across Mediterranean landscapes (Figs. 2 and 3). Grazing reduces woody plant dominance, preventing forest encroachment and enabling diverse plant communities to thrive ( 32 , 33 ). Despite significant changes in habitats and combustibility, community evenness remains relatively stable throughout millennia, with values consistently above 0.80 (Figure 3A), indicating that landscape dynamics did not lead to homogenization. Palynological richness reach its peaks in open, high-herbivore-density landscapes during the warm, dry climatic phase ( 5 ) and periods of high combustibility (Figure 2), highlighting the role of herbivores in sustaining diverse grasslands and shrublands in Mediterranean ecosystems ( 34 ). During these periods, palynological richness values were approximately 20% higher than during forested phases (Figure 3B). Forest densification and canopy closure reduce light availability and limit the establishment of herbaceous species, resulting in a homogenization of plant communities ( 21 , 35 ). However, the onset of livestock grazing in the region reopened the landscape, restoring herbivore density and promoting biodiversity recovery. Grazing by domestic livestock plays a dual role, maintaining open habitats by reducing the fuel accumulation and mitigating fire risk. In contrast, the decline in grazing intensity over recent centuries has increased combustible biomass, promoting woody species encroachment and elevating fire risk ( 32 ). This highlights the complex interplay between herbivores density, vegetation structure, and fire regimes, where grazing historically contributed to maintaining open, fire-resilient landscapes ( 33 , 36 ). Turnover peaks (Figure 3C, F) during transitional periods indicate shifts in communities structure driven by changes in grazing intensity and habitat openness. There is a strong positive correlation between herbivores density and palynological richness (Figure 4B, p-value < 0.0001***, 𝑟 = 0.38), as well as between coprophilous fungal spores’ diversity and palynological richness (Figure 4C, p-value < 0.0001***, 𝑟 =0.44). These patterns emphasize the critical role of herbivores in maintaining vegetation diversity, as higher herbivore densities are consistently associated with increased richness and more open landscapes. This relationship highlights how grazing regulates vegetation structure, preventing woody plant encroachment and fostering heterogeneous plant communities ( 3 ). By limiting the dominance of competitive species, herbivores play a role similar to fire, fostering the coexistence of a wide range of species in the plant community ( 37 ). Similar mechanisms have been documented across Mediterranean ecosystems, where herbivores play a pivotal role in shaping plant community structure and promoting diversity ( 38 ). For instance, in regions with long grazing histories, such as the Iberian Peninsula, grazing has been linked to the persistence of species-rich grasslands and the prevention of shrub encroachment ( 21 ). Our findings align with those of the Iberian Peninsula dynamics, with the addition that wild herbivores have driven both biodiversity and habitat heterogeneity for the last 12,000 years. Conclusion This study underscores the intricate interplay between fires and herbivores in shaping Mediterranean landscapes and sustaining biodiversity. Fire has historically played a pivotal role in maintaining open habitats ( 39 ) and favoring fire-adapted species ( 21 ). Herbivores (both wild and human managed through pastoral practices) emerge as a complementary ecological force, limiting the encroachment of woody species, fostering ecological heterogeneity, and enhancing habitat resilience ( 34 , 38 ). The trends observed during the late Holocene highlight the dual influence of natural processes and human activities, particularly grazing by both wild fauna and livestock, in preserving the mosaic of Mediterranean habitats ( 11 , 20 ). However, the recent decline in grazing intensity and the associated expansion of closed habitats underscore the urgent need for proactive ecosystem management. Reintroducing sustainable grazing practices, coupled with strategies to mitigate woody vegetation encroachment, could play a crucial role in preserving the ecological balance, biodiversity, and diminishing fire risks of Mediterranean ecosystems ( 33 , 36 ). These findings emphasize the importance of integrating fire and herbivores dynamics into holistic conservation and land-use planning, particularly in the context of accelerating climate change and anthropogenic pressures. By embracing such integrated approaches, we can ensure the long-term sustainability and resilience of these unique landscapes. Materials and Methods Study area. This study examines a pond located in the Crau plain, the Étang des Aulnes (43° 35′ 33″ N, 4° 47′ 23″ E), situated in the Saint-Martin-de-Crau area in southern France (Fig. 1). The Crau plain is characterized by a topography of plateaus and hills, with a maximum altitude of 142 meters. This plain is a fossilized riverbed, with 50% of its surface covered by siliceous stones. A limestone layer overlays the soil, which has a thickness of approximately 40 cm and lies above an impermeable conglomerate layer that ranges from one to five meters deep. This conglomerate prevents plant roots from accessing the alluvial water table ( 40 ). The Crau plain experiences a Mediterranean climate, known for its dry conditions, characterized by warm summers and mild, wet winters ( 22 ). This climate associated with human activities and soil type have shaped the vegetation dynamics and ecosystems of the Crau plain. Today, the Crau plain hosts diverse ecosystems, including an alluvial forest and a steppe composed by Poaceae species such as Brachypodium retusum and Thymus vulgaris ( 41 ). Chronology. A core of 4 meters of sediments was extracted on the Aulnes pond in 2023 and 9 14 C dates from macroplant remains and bulk sediments helped establishing the depth-age relationships, modeled with a Bayesian model using the ‘rbacon’ package ( 42 ) (for details, see SI Figure S1 and Table S1 ). For the entire sequence, the reconstruction spans ca. 7,959 years (min: 7,605; max: 8,538 years cal. BP), given a median resolution of 103 years per sample. Pollen and fungal spore’s analysis. Pollen analysis provides valuable insights into past vegetation dynamics ( 43 ), while coprophilous fungal spores serve as proxies for herbivore presence and abundance ( 44 ). Subsamples (2 cm 3 ) were collected at intervals of 4 cm, providing temporal resolution between ca -72- and 7,959 years cal. BP (min: 6,737; max: 7,762 years cal. BP). Lycopodium marker tablets ( 45 ) were added to each subsample for estimation of the pollen concentration (grains.cm − 3 ) and influx (grains.cm − 2 .year − 1 ) Pollen grains and spores were extracted following standard techniques ( 46 ), in which successive suspensions in 40% HF, 10% HCl and acetolysis were used to remove silicates, carbonates and cellulose respectively. The pollen grains and spores were counted under a light microscope at x400 magnification. Pollen identification followed using pollen keys and photo collections ( 47 – 49 ). Coprophilous fungal spores were identified from the Non-Pollen Palynomorph Image Database ( 50 ). An average of 450 pollen grains of terrestrial species was counted for each sample, with a maximum of 788 grains and a minimum of 92 grains for the deepest sample. Pollen percentage was calculated based on the pollen sum excluding Alnus, (semi)aquatic pollen and coprophilous fungal spores. Pollen diagram was constructed using the R package ‘rioja’ ( 51 ). A constrained sum-of-squares cluster analysis (CONISS) ( 52 , 53 ) was used to identify pollen assemblage zones, and the significance of each zone was tested with the broken stick model ( 54 ) using the ‘rioja’ R package ( 51 ). Each pollen taxon was associated with a specific species linked to a habitat type based on Trabaud’s classification ( 17 ), vegetation surveys (personal communication from Vidaller, unpublished) and Mediterranean plant lists established for the area with Word Flora online ( 55 ). This approach allowed the grouping of pollen taxa into ecological categories reflecting the combustibility and vegetation dynamics of the identified habitats. When a species could not be directly assigned to a habitat in Trabaud’s classification, it was grouped into a separate "other" category (28.52% of the total pollen influx). The majority of the taxa in this category represents species associated with riparian or floodplain vegetation (e.g., Alnus sp. , Abies sp. , Ulmus sp. ), which do not align directly with the habitats defined by Trabaud. Diversity analysis. To assess the biodiversity changes in the surroundings of the sample site, pollen diversity has been measured using richness ( 56 ) and evenness ( 57 ) to encompass all diversity dimensions. Palynological richness (PRI) represents the α-diversity corresponding to the expected number of taxa found in samples of equal size as estimated by rarefaction analysis. It is a robust method used in many palaeoecological studies in Europe ( 3 , 5 , 58 ). PRI was calculated by using the ‘Vegan’ package on all pollen taxa ( 59 ) of the statistical software R ( 52 ) and a constant pollen sum, which was standardized on the minimum pollen sum (n = 92). Although the representativeness of pollen to portray the plant richness has been questioned over decades overall pollen richness is considered a good indicator of vegetation richness ( 60 ). While it is a valuable measure of long-term biodiversity changes ( 3 , 5 ), these results must be interpreted carefully ( 61 ). The probability of interspecific encounters (PIE) was used as an index of evenness ( 57 ). This index gives the probability of two randomly sampled pollen grains from a given habitat type representing two different sets of species. To assess possible alteration of PRI due to evenness influence, we also calculated evenness-detrended palynological richness (DPRI) ( 5 ). PRI is regarded to be unaffected by palynological evenness if both PRI and DPRI show similar trends ( 5 ). The palynological turnover has been computed based on an improved algorithm (R package ‘R-Ratepol’) for estimating rates of change (RoCs) for palaeoecological time series ( 62 ). RoCs were calculated as the compositional dissimilarity between consecutive time intervals, using the chi-squared coefficient (DC = "chisq") to quantify dissimilarity. The results were standardized to represent changes per 500 years (time_standardisation = 500) to facilitate temporal comparability. The smoothing was performed using moving average (smooth_method = "m.avg"). To preserve the resolution of the original dataset, RoCs were computed for individual levels (Working_Units = "levels") with data standardized to the lowest pollen count detected or a default value of 150 (standardise = TRUE). A total of 10000 randomizations (rand = 10000) were used to account for stochasticity in the reconstruction. LOESS (locally estimated scatterplot smoothing) curves were applied to detect temporal trends in diversity and herbivory proxies over millennia to capture non-linear dynamics without assuming a specific functional form, providing a clear visualization of long-term ecological patterns (Cleveland, 1979; Colombaroli et al., 2007). Linear models were used to assess relationships between biodiversity metrics (e.g., detrended pollen richness) and herbivory indicators (e.g., coprophilous fungal spores’ influx), enabling the identification of significant correlations. Declarations Acknowledgments: We thank Damien Rius for conducting the master core, Julien Didier for his contribution to the coring operations, Élodie Brisset for her assistance in coring and sediment analysis, and the District of Bouches-du-Rhône for granting access to the study site. We also thank Cécile Latapy for her help with preliminary analyses and Christel Vidaller for providing vegetation surveys from the Crau Plain, which assisted us in characterizing the habitats. This work was supported by the French government under the France 2030 investment plan, as part of the French national funding agency (ANR-22-CE02-0008-01), the Initiative d'Excellence d'Aix-Marseille Université - A*MIDEX (AMX-19-IET-012, ITEM funding EV22_IMP); and by District of Bouches du Rhone (D2022/10059). Funding: ANR-22-CE02-0008-01 A*MIDEX (AMX-19-IET-012, ITEM funding EV22_IMP) District of Bouches du Rhone (D2022/10059) Author contributions: Conceptualization: BL, ML Methodology: ML, BL, PS, GM Investigation: ML, BL, PS, GM, AH Visualization: ML Funding acquisition: BL Project administration: BL Supervision: BL Writing – original draft: ML, BL Writing – review & editing: ML, BL, PS, GM, AH Competing interests: The authors declare no competing interests. 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Li, Indication of coprophilous fungal spores for monitoring grazing intensity in the Horqin Sandy Land, Northern China. Progress in Physical Geography: Earth and Environment , 03091333241258892 (2024). M. J. Richardson, Coprophilous fungi from Brazil. Braz. arch. biol. technol. 44 , 283–289 (2001). A. Bell, Dung Fungi: An Illustrated Guide to Coprophilous Fungi in New Zealand (Victoria University Press, 1983). B. Dietre, É. Gauthier, F. Gillet, Modern pollen rain and fungal spore assemblages from pasture woodlands around Lake Saint-Point (France). Review of Palaeobotany and Palynology 186 , 69–89 (2012). H. Wei, R. Duan, Q. Xu, S. Yang, Q. Fan, G. Hou, Y. Du, Z. Qin, J. Gao, Fungal spore indicators of vegetation and highland pastoralism in modern topsoil and dung, eastern Tibetan Plateau. Catena 202 , 105231 (2021). 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Basumatary, “Non-Pollen Palynomorphs from the Late-Holocene Sediments of Majuli Island, Assam (Indo-Burma Region): Implications to Palaeoenvironmental Studies” in Climate Change and Environmental Impacts: Past, Present and Future Perspective (Springer, 2023), pp. 63–81. A. G. Perrotti, E. Van Asperen, Dung fungi as a proxy for megaherbivores: opportunities and limitations for archaeological applications. Vegetation History and Archaeobotany 28 , 93–104 (2019). C. Cugny, F. Mazier, D. Galop, Modern and fossil non-pollen palynomorphs from the Basque mountains (western Pyrenees, France): the use of coprophilous fungi to reconstruct pastoral activity. Veget Hist Archaeobot 19 , 391–408 (2010). B. van Geel, J. Buurman, O. Brinkkemper, J. Schelvis, A. Aptroot, G. van Reenen, T. Hakbijl, Environmental reconstruction of a Roman Period settlement site in Uitgeest (The Netherlands), with special reference to coprophilous fungi. Journal of Archaeological Science 30 , 873–883 (2003). A. G. Baker, S. A. Bhagwat, K. J. Willis, Do dung fungal spores make a good proxy for past distribution of large herbivores? Quaternary Science Reviews 62 , 21–31 (2013). H. A. Raja, C. A. Shearer, Arnium gigantosporum, a new ascomycete species from fresh water in Florida. Fungal Diversity 22 , 219–225 (2006). Md. F. Quamar, N. Stivrins, Modern pollen and non-pollen palynomorphs along an altitudinal transect in Jammu and Kashmir (Western Himalaya), India. Palynology 45 , 669–684 (2021). Additional Declarations There is NO Competing Interest. Supplementary Files SILestienneetalCEE.pdf Supplementary Materials Figs. S1 to S2 Tables S1 to S3 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6163168","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":425002869,"identity":"2b1c73aa-fc9f-46c1-af80-065e665ef06b","order_by":0,"name":"Marion Lestienne","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIie2PsWrDMBRFn9HQRUWrAyH+gsALAUPA0H5KisEds3arS0CeQlbnM0KhdJR4a4pXDxkSCp6dsUOhamIvJaozFqoDgsuDw9UFcDj+KMq8fhtAXHlP6hKFA7CT0pt7aacCrXIEqamzMcze9urwClyIQpuwHYyJpaqGaBbcnlfCzT3q1QZ4L4/BhGockpfqHJLJ2tIVqgToWgLHkn0HunsxCnEgHKUWpahOyk1BQJ+SHp/nXUrZtkAM5EmaImuUwDI/LCszQfrcL2PUC0mj/LgFE0SbUiSsPshoIJb6vf6QFIgs2+/qhwgDy8ca/J8HU4HqV+UcHS0Oh8Pxf/gC+vxggvG3lbwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-7229-2300","institution":"IMBE, Aix Marseille Université, Avignon Université, CNRS, IRD, Aix-en-Provence, France","correspondingAuthor":true,"prefix":"","firstName":"Marion","middleName":"","lastName":"Lestienne","suffix":""},{"id":425002870,"identity":"0d078d45-b2c8-4367-a853-89e9776b5d8f","order_by":1,"name":"Pauline Saurat","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Pauline","middleName":"","lastName":"Saurat","suffix":""},{"id":425002871,"identity":"5484f528-3a08-4e39-b660-57c8801ed102","order_by":2,"name":"Gwendal Mouden","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Gwendal","middleName":"","lastName":"Mouden","suffix":""},{"id":425002872,"identity":"4d931df1-a4b0-48f9-a4f7-5301e66e8281","order_by":3,"name":"Andy Hennebelle","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Andy","middleName":"","lastName":"Hennebelle","suffix":""},{"id":425002873,"identity":"db5693bd-ee91-47f2-9d9a-70b926c8d4fc","order_by":4,"name":"Lisa Bajolle","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"Bajolle","suffix":""},{"id":425002874,"identity":"dfb89af6-2789-46a2-90f3-19c65a4210ff","order_by":5,"name":"Bérangère Leys","email":"","orcid":"","institution":"Aix Marseille Univ","correspondingAuthor":false,"prefix":"","firstName":"Bérangère","middleName":"","lastName":"Leys","suffix":""}],"badges":[],"createdAt":"2025-03-05 13:45:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6163168/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6163168/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78497310,"identity":"69598b89-4ca8-4568-a774-c569b79ba2aa","added_by":"auto","created_at":"2025-03-14 04:56:34","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":492305,"visible":true,"origin":"","legend":"\u003cp\u003eStudy area and land cover classification. (A) Location of the study area in France, with the department highlighted in red. (B) Land cover classification of the department based on the CORINE Land Cover 2018 dataset, showing the main land cover groups: Artificial surfaces (e.g., urban fabric, industrial and commercial areas, road networks), Agricultural areas (e.g., arable land, permanent crops, pastures, heterogeneous agricultural landscapes), Forest and semi-natural areas (e.g., broad-leaved and coniferous forests, natural grasslands, shrublands), Wetlands (e.g., inland marshes, peat bogs), and Water bodies (e.g., rivers, lakes, reservoirs). The study site is indicated by a red rectangle. (C) Detailed land cover map of the study area, providing a finer-scale classification of habitat types within the site and their association with the broader CORINE Land Cover categories, allowing for a more precise understanding of local landscape heterogeneity.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6163168/v1/1dfe2c0600596237f29bb8c5.jpeg"},{"id":78497511,"identity":"7425ecfa-fc6d-4676-99cb-3d509e98cfba","added_by":"auto","created_at":"2025-03-14 05:04:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":263581,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal evolution of habitats in the Crau plain, grouped according to Trabaud's (1971) habitat classification. Boxplots and violins represent combustibility across four periods, as inferred from the pollen data. Below the boxplots, the influx of pollen taxa is shown for each habitat type: Beech Forest, White Oak Coppice, Green Oak Coppice, Pine Forest, Garrigue/Heathland, and Grassland. Pollen percentages values illustrate the relative abundance of taxa for each habitat. Habitat groupings were established based on their ecological and combustibility characteristics as defined by Trabaud's classification (see Table S2 for details). Combustibility is classified into five levels based on vegetation characteristics and fire behavior. Class 1 (Very Low) represents habitats with low flammability and limited fire propagation, typically found in humid or temperate environments like beech forests or white oak coppices. Class 2 (Low) includes vegetation that burns under specific conditions, such as prolonged drought, with moderate fire spread, as seen in green oak coppices. Class 3 (Moderate) corresponds to habitats with higher flammability and faster fire spread, such as pine forests, where resinous materials enhance combustion. Class 4 (High) is characterized by highly flammable vegetation, rich in volatile compounds like essential oils or resins, leading to intense fires, typical of garrigue and heathlands. Class 5 (Very High) includes vegetation that is extremely flammable and burns explosively, with rapid fire propagation, as seen in dry grasslands dominated by fine, dry biomass.\u0026nbsp; A non-parametric Mann-Whitney U test was conducted to compare mean combustibility between two periods. Results are displayed within the figure as pairwise comparisons using letters to indicate statistically significant differences. Groups sharing the same letter are not significantly different. The mean combustibility for periods 1 to 4 is 3.12, 2.71, 2.69, and 3.30, respectively. Data do not follow a normal distribution. Significant differences were determined using a non-parametric Mann-Whitney U test, with the following p-values: periods 1 vs. 2 (p-value = 1.754e\u003csup\u003e-08\u003c/sup\u003e), periods 1 vs. 3 (p-value = 1.004e\u003csup\u003e-08\u003c/sup\u003e), periods 2 vs. 4 (p-value = 1.281e\u003csup\u003e-06\u003c/sup\u003e), and periods 3 vs. 4 (p-value = 1.677e\u003csup\u003e-06\u003c/sup\u003e). Periods 1 vs. 4 (p-value = 0.1036) and periods 2 vs. 3 (p-value = 0.9324) show no significant differences.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6163168/v1/2fb128a4df008166f6c7aa2c.png"},{"id":78497321,"identity":"55f98edf-8e58-4cd9-aab1-0b98c18496dc","added_by":"auto","created_at":"2025-03-14 04:56:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":441656,"visible":true,"origin":"","legend":"\u003cp\u003ePollen and Coprophilous Fungal Spores diversity: A) Palynological Evenness (probability of interspecific encounters (PIE)); B) the palynological richness (PRI) represents the α-diversity corresponding to the expected number of taxa found in samples of equal size as estimated by rarefaction analysis and has been detrended to assess the evenness influence; C) Palynological turnover (rate of change has been computed based on an improved algorithm (R package R-Ratepol) for estimating rates of change for palaeoecological time series; D) Palynological Influx (#.cm\u003csup\u003e-2\u003c/sup\u003e.yr); E) Coprophilous Fungal Spores Richness (The specnumber function from the R package ”vegan” was used because rarefaction analysis was not applicable due to the low influx); F) Coprophilous Fungal Spores turnover estimated by the same method than pollen; and G) Coprophilous Fungal Spores influx (#.cm\u003csup\u003e-2\u003c/sup\u003e.yr). The light red represents all the coprophilous spores used for the study and the dark red section represents spore species well-documented in the scientific literature as reliable indicators of herbivores density or pastoral activities. Details of species and references are provided in the Supplementary Information (Table S3). Icons are from the noun project (https://thenounproject.com/).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6163168/v1/103d8a1ea5494414982408ce.png"},{"id":78497313,"identity":"83062590-02ec-4a42-93d9-f4992ad57398","added_by":"auto","created_at":"2025-03-14 04:56:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":257196,"visible":true,"origin":"","legend":"\u003cp\u003eA) Palynological Influx as a function of coprophilous fungal spores’ influx. A Spearman test highlighted significant positive correlation between these two variables (p-value \u0026lt; 0.0001***, rho = 0.61); B) Palynological Detrended Richness as a function of coprophilous fungal spores’ influx. A Spearman test highlighted a significantly positive correlation between these two variables (p-value \u0026lt; 0.0001***, rho = 0.48); and C) Palynological Detrended Richness as a function of coprophilous fungal spores Richness. A Pearson test (based on the normality of residuals) highlighted a significantly positive correlation between these two variables (p-value \u0026lt; 0.0001***, 𝑟= 0.52).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6163168/v1/99c0af151d03f7bbd2b00743.png"},{"id":79543557,"identity":"7189f3af-a74b-41d8-ad01-e4716b2473e3","added_by":"auto","created_at":"2025-03-31 04:33:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1875540,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6163168/v1/56d59c4f-1dff-42e5-903b-507cc6ec6d57.pdf"},{"id":78497513,"identity":"5015f7c7-05cb-4a8d-9e23-4391ad2d65f3","added_by":"auto","created_at":"2025-03-14 05:04:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":393148,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Materials\u003c/p\u003e\n\u003cp\u003eFigs. S1 to S2\u003c/p\u003e\n\u003cp\u003eTables S1 to S3\u003c/p\u003e","description":"","filename":"SILestienneetalCEE.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6163168/v1/258ddd650595b2c537518ced.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Fire and Herbivory as Architects of Mediterranean Biodiversity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMediterranean region is among the most species-rich biomes on Earth (\u003cem\u003e1\u003c/em\u003e). Its remarkable biodiversity reflects a long history of interactions between disturbances (\u003cem\u003ei.e.\u0026nbsp;\u003c/em\u003efires and herbivores), climate variability, anthropogenic activities and land uses such as sylvo-pastoralism (\u003cem\u003e2\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e). However, Mediterranean ecosystems are increasingly under pressure from climatic shifts and human impacts, making the understanding of their resilience and dynamics critical (\u003cem\u003e4\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eFire is a dominant ecological force in the Mediterranean, where hot, dry summers and dense vegetation sustain frequent, high-intensity fires that have long shaped plant communities (\u003cem\u003e6\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e). Fire-adapted species and open habitats owe their persistence to the fire regime, which has helped maintain the mosaic of landscapes characteristic of the region (\u003cem\u003e8\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e). Although fire is a well-established disturbance acting on Mediterranean vegetation dynamics and landscape structure, herbivores have been demonstrated to play a key role on Mediterranean landscape dynamics (\u003cem\u003e10\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e). Both wild herbivores and domesticated livestock have contributed to shaping vegetation patterns over millennia, influencing plant diversity and preventing the dominance of woody species (\u003cem\u003e3\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e). While the role of grazing in pastoral contexts is relatively well-documented (\u003cem\u003e3\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e), the ecological role of wild herbivores remains understudied despite their long-term influence on vegetation structure and ecosystem resilience (\u003cem\u003e15\u003c/em\u003e). In addition, the cumulative impacts of both herbivores and fire is poorly reported due to the lack of records for both disturbances in this area (\u003cem\u003e16\u003c/em\u003e). This oversight hampers our ability to fully understand the interactions between fire and herbivory and their cumulative impacts on vegetation and biodiversity. Addressing this knowledge gap requires integrating evidence from both wild and domestic herbivores to assess their combined effects on Mediterranean ecosystems.\u003c/p\u003e\n\u003cp\u003eThis study seeks to address these gaps by reconstructing the long-term dynamics of vegetation habitats and herbivores densities in the Crau Plain, where a unique landscape of Mediterranean steppe and mixed oak forest co-exist today. By employing a multi-millennial record of both vegetation and herbivore types and densities, we aim at disentangling the cumulative effects of fire and herbivores on biodiversity and vegetation composition over millennia. This approach will enhance our understanding of the processes underpinning Mediterranean biodiversity and inform strategies for sustainable ecosystem management under future environmental pressures. To better understand habitat dynamics and vegetation combustibility in Mediterranean ecosystems, we use Trabaud\u0026apos;s classification, which focuses on habitat types and flammability rather than species (\u003cem\u003e17\u003c/em\u003e). This functional approach highlights structural and ecological traits influencing fire behavior, offering insights into how fire regimes and herbivores shape habitat resilience and biodiversity over millennia (\u003cem\u003e18\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e).\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eFire and human influence on habitat dynamics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Crau Plain\u0026apos;s vegetation was classified into five main habitats: white oak coppice, pine forest, garrigue/heathland, grassland, and green oak coppice, following Trabaud\u0026apos;s (1971) classification (\u003cem\u003e17\u003c/em\u003e) (Figure 2; see SI Figures S2 for details). These habitats exhibit varying levels of combustibility, with open landscapes such as grasslands and garrigue/heathland being more prone to fire, while closed forests such as white oak coppice and beech forest exhibit lower combustibility. The dominance and shifts in these habitats reflect a complex interplay between climate, fire regimes, and human activities throughout the last 12,000 years of climate variations (\u003cem\u003ei.e.\u0026nbsp;\u003c/em\u003ethe Holocene). Fire is recognized as a key ecological driver (\u003cem\u003e6\u003c/em\u003e), and its influence on the Crau Plain is evident in the higher combustibility of habitats observed before 6,500 years cal. BP and after 1,000 years cal. BP. The mean combustibility of habitats during these periods reached 3.12 and 3.30, respectively, compared to lower values of 2.71 and 2.69 during intermediate periods (Figure 2). Significant differences were determined using a non-parametric Mann-Whitney U test, with the following p-values: periods 1 vs. 2 (p-value = 1.754e\u003csup\u003e-08\u003c/sup\u003e), periods 1 vs. 3 (p-value = 1.004e\u003csup\u003e-08\u003c/sup\u003e), periods 2 vs. 4 (p-value = 1.281e\u003csup\u003e-06\u003c/sup\u003e), and periods 3 vs. 4 (p-value = 1.677e\u003csup\u003e-06\u003c/sup\u003e). Periods 1 vs. 4 (p-value = 0.1036) and periods 2 vs. 3 (p-value = 0.9324) show no significant differences. During warmer and drier periods (\u003cem\u003e5\u003c/em\u003e), fire-adapted species thrived in open and dry environments (\u003cem\u003e20\u003c/em\u003e). Fire acts as a selective pressure favoring pyrophytic species but also plays a critical role in shaping plant communities composition by suppressing fire-sensitive species and promoting open vegetation (\u003cem\u003e21\u003c/em\u003e). This process leads to a mosaic of habitats that supports high biodiversity, particularly in grassland and shrubland ecosystems (\u003cem\u003e20\u003c/em\u003e). Conversely, phases of reduced fire activity coincided with wetter and colder climatic conditions (\u003cem\u003e22\u003c/em\u003e), favoring habitats like white oak coppice and beech forests, which suppress fire spread (\u003cem\u003e23\u003c/em\u003e). Human influence emerged as a significant factor in shaping habitats composition and fire regimes (\u003cem\u003e24\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e). Declines in wild herbivore densities (Figure 3E, G) point to early hunting pressure and land-use changes (\u003cem\u003e26\u003c/em\u003e). Then, grazing by domestic livestock transformed the landscape by promoting open habitats such as grasslands and garrigue/heathland (\u003cem\u003e11\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e). This transformation increased the prevalence of highly combustible environments, particularly after the introduction of these pastoral systems (\u003cem\u003e19\u003c/em\u003e). In transitional periods, shifts in habitats composition highlight the interplay between natural and anthropogenic forces. For example, the gradual replacement of beech and white oak coppice by green oak coppice and pine forest reflects a combination of human-driven deforestation, selective tree clearance, and climatic fluctuations (\u003cem\u003e20\u003c/em\u003e). These habitats are less fire-resistant and mark the adaptive response of Mediterranean ecosystems to changing environmental pressures. The recent landscape dynamics, characterized by forest expansion following land abandonment (\u003cem\u003e11\u003c/em\u003e), further demonstrate the evolving relationships between humans and the environment. Increased forest cover, particularly of combustible species, has increased fire risks in recent centuries (\u003cem\u003e28\u003c/em\u003e, \u003cem\u003e29\u003c/em\u003e). This trend confirms the unique situation of the area as of today, which reveals the necessity for stakeholders to account for ecological drivers such as both herbivores and human land-use to mitigate future fire hazards (\u003cem\u003e30\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e). As such, while fire and human activities have played pivotal roles in shaping habitat dynamics, herbivores act as a parallel force, maintaining high plant diversity and preventing the dominance of woody species, thereby reinforcing the resilience of Mediterranean ecosystems.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHerbivores have promoted high plant diversity.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHerbivores emerge as a key ecological driver of biodiversity, fostering both species richness and heterogeneity across Mediterranean landscapes (Figs. 2 and 3). Grazing reduces woody plant dominance, preventing forest encroachment and enabling diverse plant communities to thrive (\u003cem\u003e32\u003c/em\u003e, \u003cem\u003e33\u003c/em\u003e). Despite significant changes in habitats and combustibility, community evenness remains relatively stable throughout millennia, with values consistently above 0.80 (Figure 3A), indicating that landscape dynamics did not lead to homogenization. Palynological richness reach its peaks in open, high-herbivore-density landscapes during the warm, dry climatic phase (\u003cem\u003e5\u003c/em\u003e) and periods of high combustibility (Figure 2), highlighting the role of herbivores in sustaining diverse grasslands and shrublands in Mediterranean ecosystems (\u003cem\u003e34\u003c/em\u003e). During these periods, palynological richness values were approximately 20% higher than during forested phases (Figure 3B). Forest densification and canopy closure reduce light availability and limit the establishment of herbaceous species, resulting in a homogenization of plant communities (\u003cem\u003e21\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e). However, the onset of livestock grazing in the region reopened the landscape, restoring herbivore density and promoting biodiversity recovery. Grazing by domestic livestock plays a dual role, maintaining open habitats by reducing the fuel accumulation and mitigating fire risk. In contrast, the decline in grazing intensity over recent centuries has increased combustible biomass, promoting woody species encroachment and elevating fire risk (\u003cem\u003e32\u003c/em\u003e). This highlights the complex interplay between herbivores density, vegetation structure, and fire regimes, where grazing historically contributed to maintaining open, fire-resilient landscapes (\u003cem\u003e33\u003c/em\u003e, \u003cem\u003e36\u003c/em\u003e). Turnover peaks (Figure 3C, F) during transitional periods indicate shifts in communities structure driven by changes in grazing intensity and habitat openness. There is a strong positive correlation between herbivores density and palynological richness (Figure 4B, p-value \u0026lt; 0.0001***,\u0026nbsp;𝑟\u0026nbsp;= 0.38), as well as between coprophilous fungal spores\u0026rsquo; diversity and palynological richness (Figure 4C, p-value \u0026lt; 0.0001***,\u0026nbsp;𝑟\u0026nbsp;=0.44). These patterns emphasize the critical role of herbivores in maintaining vegetation diversity, as higher herbivore densities are consistently associated with increased richness and more open landscapes. This relationship highlights how grazing regulates vegetation structure, preventing woody plant encroachment and fostering heterogeneous plant communities\u0026nbsp;(\u003cem\u003e3\u003c/em\u003e). By limiting the dominance of competitive species, herbivores play a role similar to fire, fostering the coexistence of a wide range of species in the plant community\u0026nbsp;(\u003cem\u003e37\u003c/em\u003e). Similar mechanisms have been documented across Mediterranean ecosystems, where herbivores play a pivotal role in shaping plant community structure and promoting diversity\u0026nbsp;(\u003cem\u003e38\u003c/em\u003e). For instance, in regions with long grazing histories, such as the Iberian Peninsula, grazing has been linked to the persistence of species-rich grasslands and the prevention of shrub encroachment\u0026nbsp;(\u003cem\u003e21\u003c/em\u003e). Our findings align with those of the Iberian Peninsula dynamics, with the addition that wild herbivores have driven both biodiversity and habitat heterogeneity for the last 12,000 years.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study underscores the intricate interplay between fires and herbivores in shaping Mediterranean landscapes and sustaining biodiversity. Fire has historically played a pivotal role in maintaining open habitats (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) and favoring fire-adapted species (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Herbivores (both wild and human managed through pastoral practices) emerge as a complementary ecological force, limiting the encroachment of woody species, fostering ecological heterogeneity, and enhancing habitat resilience (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The trends observed during the late Holocene highlight the dual influence of natural processes and human activities, particularly grazing by both wild fauna and livestock, in preserving the mosaic of Mediterranean habitats (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, the recent decline in grazing intensity and the associated expansion of closed habitats underscore the urgent need for proactive ecosystem management. Reintroducing sustainable grazing practices, coupled with strategies to mitigate woody vegetation encroachment, could play a crucial role in preserving the ecological balance, biodiversity, and diminishing fire risks of Mediterranean ecosystems (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). These findings emphasize the importance of integrating fire and herbivores dynamics into holistic conservation and land-use planning, particularly in the context of accelerating climate change and anthropogenic pressures. By embracing such integrated approaches, we can ensure the long-term sustainability and resilience of these unique landscapes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eStudy area.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis study examines a pond located in the Crau plain, the \u0026Eacute;tang des Aulnes (43\u0026deg; 35\u0026prime; 33\u0026Prime; N, 4\u0026deg; 47\u0026prime; 23\u0026Prime; E), situated in the Saint-Martin-de-Crau area in southern France (Fig.\u0026nbsp;1). The Crau plain is characterized by a topography of plateaus and hills, with a maximum altitude of 142 meters. This plain is a fossilized riverbed, with 50% of its surface covered by siliceous stones. A limestone layer overlays the soil, which has a thickness of approximately 40 cm and lies above an impermeable conglomerate layer that ranges from one to five meters deep. This conglomerate prevents plant roots from accessing the alluvial water table (\u003cem\u003e40\u003c/em\u003e). The Crau plain experiences a Mediterranean climate, known for its dry conditions, characterized by warm summers and mild, wet winters (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). This climate associated with human activities and soil type have shaped the vegetation dynamics and ecosystems of the Crau plain. Today, the Crau plain hosts diverse ecosystems, including an alluvial forest and a steppe composed by Poaceae species such as \u003cem\u003eBrachypodium retusum\u003c/em\u003e and \u003cem\u003eThymus vulgaris\u003c/em\u003e (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eChronology.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA core of 4 meters of sediments was extracted on the Aulnes pond in 2023 and 9 \u003csup\u003e14\u003c/sup\u003eC dates from macroplant remains and bulk sediments helped establishing the depth-age relationships, modeled with a Bayesian model using the \u0026lsquo;rbacon\u0026rsquo; package (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) (for details, see SI Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For the entire sequence, the reconstruction spans ca. 7,959 years (min: 7,605; max: 8,538 years cal. BP), given a median resolution of 103 years per sample.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePollen and fungal spore\u0026rsquo;s analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePollen analysis provides valuable insights into past vegetation dynamics (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), while coprophilous fungal spores serve as proxies for herbivore presence and abundance (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Subsamples (2 cm\u003csup\u003e3\u003c/sup\u003e) were collected at intervals of 4 cm, providing temporal resolution between ca -72- and 7,959 years cal. BP (min: 6,737; max: 7,762 years cal. BP). Lycopodium marker tablets (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) were added to each subsample for estimation of the pollen concentration (grains.cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) and influx (grains.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e.year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) Pollen grains and spores were extracted following standard techniques (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), in which successive suspensions in 40% HF, 10% HCl and acetolysis were used to remove silicates, carbonates and cellulose respectively. The pollen grains and spores were counted under a light microscope at x400 magnification. Pollen identification followed using pollen keys and photo collections (\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Coprophilous fungal spores were identified from the Non-Pollen Palynomorph Image Database (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). An average of 450 pollen grains of terrestrial species was counted for each sample, with a maximum of 788 grains and a minimum of 92 grains for the deepest sample. Pollen percentage was calculated based on the pollen sum excluding Alnus, (semi)aquatic pollen and coprophilous fungal spores. Pollen diagram was constructed using the R package \u0026lsquo;rioja\u0026rsquo; (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). A constrained sum-of-squares cluster analysis (CONISS) (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) was used to identify pollen assemblage zones, and the significance of each zone was tested with the broken stick model (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e) using the \u0026lsquo;rioja\u0026rsquo; R package (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Each pollen taxon was associated with a specific species linked to a habitat type based on Trabaud\u0026rsquo;s classification (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), vegetation surveys (personal communication from Vidaller, unpublished) and Mediterranean plant lists established for the area with Word Flora online (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). This approach allowed the grouping of pollen taxa into ecological categories reflecting the combustibility and vegetation dynamics of the identified habitats. When a species could not be directly assigned to a habitat in Trabaud\u0026rsquo;s classification, it was grouped into a separate \"other\" category (28.52% of the total pollen influx). The majority of the taxa in this category represents species associated with riparian or floodplain vegetation (e.g., \u003cem\u003eAlnus sp.\u003c/em\u003e, \u003cem\u003eAbies sp.\u003c/em\u003e, \u003cem\u003eUlmus sp.\u003c/em\u003e), which do not align directly with the habitats defined by Trabaud.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiversity analysis.\u003c/b\u003e To assess the biodiversity changes in the surroundings of the sample site, pollen diversity has been measured using richness (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e) and evenness (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e) to encompass all diversity dimensions. Palynological richness (PRI) represents the α-diversity corresponding to the expected number of taxa found in samples of equal size as estimated by rarefaction analysis. It is a robust method used in many palaeoecological studies in Europe (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). PRI was calculated by using the \u0026lsquo;Vegan\u0026rsquo; package on all pollen taxa (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e) of the statistical software R (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) and a constant pollen sum, which was standardized on the minimum pollen sum (n\u0026thinsp;=\u0026thinsp;92). Although the representativeness of pollen to portray the plant richness has been questioned over decades overall pollen richness is considered a good indicator of vegetation richness (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). While it is a valuable measure of long-term biodiversity changes (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), these results must be interpreted carefully (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). The probability of interspecific encounters (PIE) was used as an index of evenness (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). This index gives the probability of two randomly sampled pollen grains from a given habitat type representing two different sets of species. To assess possible alteration of PRI due to evenness influence, we also calculated evenness-detrended palynological richness (DPRI) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). PRI is regarded to be unaffected by palynological evenness if both PRI and DPRI show similar trends (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The palynological turnover has been computed based on an improved algorithm (R package \u0026lsquo;R-Ratepol\u0026rsquo;) for estimating rates of change (RoCs) for palaeoecological time series (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). RoCs were calculated as the compositional dissimilarity between consecutive time intervals, using the chi-squared coefficient (DC = \"chisq\") to quantify dissimilarity. The results were standardized to represent changes per 500 years (time_standardisation\u0026thinsp;=\u0026thinsp;500) to facilitate temporal comparability. The smoothing was performed using moving average (smooth_method = \"m.avg\"). To preserve the resolution of the original dataset, RoCs were computed for individual levels (Working_Units = \"levels\") with data standardized to the lowest pollen count detected or a default value of 150 (standardise\u0026thinsp;=\u0026thinsp;TRUE). A total of 10000 randomizations (rand\u0026thinsp;=\u0026thinsp;10000) were used to account for stochasticity in the reconstruction. LOESS (locally estimated scatterplot smoothing) curves were applied to detect temporal trends in diversity and herbivory proxies over millennia to capture non-linear dynamics without assuming a specific functional form, providing a clear visualization of long-term ecological patterns (Cleveland, 1979; Colombaroli et al., 2007). Linear models were used to assess relationships between biodiversity metrics (e.g., detrended pollen richness) and herbivory indicators (e.g., coprophilous fungal spores\u0026rsquo; influx), enabling the identification of significant correlations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Damien Rius for conducting the master core, Julien Didier for his contribution to the coring operations, \u0026Eacute;lodie Brisset for her assistance in coring and sediment analysis, and the District of Bouches-du-Rh\u0026ocirc;ne for granting access to the study site. We also thank C\u0026eacute;cile Latapy for her help with preliminary analyses and Christel Vidaller for providing vegetation surveys from the Crau Plain, which assisted us in characterizing the habitats. This work was supported by the French government under the France 2030 investment plan, as part of the French national funding agency (ANR-22-CE02-0008-01), the Initiative d\u0026apos;Excellence d\u0026apos;Aix-Marseille Universit\u0026eacute; - A*MIDEX (AMX-19-IET-012, ITEM funding EV22_IMP); and by District of Bouches du Rhone (D2022/10059).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eANR-22-CE02-0008-01\u003c/p\u003e\n\u003cp\u003eA*MIDEX (AMX-19-IET-012, ITEM funding EV22_IMP)\u003c/p\u003e\n\u003cp\u003eDistrict of Bouches du Rhone (D2022/10059)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: BL, ML\u003c/p\u003e\n\u003cp\u003eMethodology: ML, BL, PS, GM\u003c/p\u003e\n\u003cp\u003eInvestigation: ML, BL, PS, GM, AH\u003c/p\u003e\n\u003cp\u003eVisualization: ML\u003c/p\u003e\n\u003cp\u003eFunding acquisition: BL\u003c/p\u003e\n\u003cp\u003eProject administration: BL\u003c/p\u003e\n\u003cp\u003eSupervision: BL\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: ML, BL\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: ML, BL, PS, GM, AH\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e LESTIENNE, Marion (2025). [Dataset] Fire and Herbivory as Architects of Mediterranean Biodiversity. Lestienne et al. submitted. figshare. Dataset. https://doi.org/10.6084/m9.figshare.28351808.v2 . Made immediately available upon request by the editor or reviewers.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eN. Myers, R. A. Mittermeier, C. G. Mittermeier, G. A. B. da Fonseca, J. Kent, Biodiversity hotspots for conservation priorities. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e403\u003c/strong\u003e, 853\u0026ndash;858 (2000).\u003c/li\u003e\n\u003cli\u003eD. Colombaroli, A. Marchetto, W. Tinner, Long‐term interactions between Mediterranean climate, vegetation and fire regime at Lago di Massaciuccoli (Tuscany, Italy). \u003cem\u003eJournal of Ecology\u003c/em\u003e \u003cstrong\u003e95\u003c/strong\u003e, 755\u0026ndash;770 (2007).\u003c/li\u003e\n\u003cli\u003eM. Lestienne, I. Jouffroy-Bapicot, D. Leyssenne, P. Sabatier, M. Debret, P.-J. Albertini, D. 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Stivrins, Modern pollen and non-pollen palynomorphs along an altitudinal transect in Jammu and Kashmir (Western Himalaya), India. \u003cem\u003ePalynology\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 669\u0026ndash;684 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6163168/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6163168/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis research breaks new ground by showing that, contrary to generally accepted key role of fire in shaping plant communities, herbivores shaped both landscape heterogeneity and fire-prone habitats in the Mediterranean area. The interconnected roles of fire and herbivores predisposed habitat resilience, highlighting the need to integrate grazing for fire risk mitigation and biodiversity conservation. The recent decline in pastoralism coincides with the expansion of highly flammable vegetation, exacerbating fire risks under current climate conditions. These insights advocate for conservation strategies that balance fire management and sustainable herbivory to preserve Mediterranean biodiversity in the face of increasing anthropogenic and climatic pressures.\u003c/p\u003e","manuscriptTitle":"Fire and Herbivory as Architects of Mediterranean Biodiversity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-14 04:56:29","doi":"10.21203/rs.3.rs-6163168/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d02ff4c7-f186-472b-b606-211f73a9bd5e","owner":[],"postedDate":"March 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45295658,"name":"Earth and environmental sciences/Ecology/Fire ecology"},{"id":45295659,"name":"Earth and environmental sciences/Ecology/Ecosystem ecology"}],"tags":[],"updatedAt":"2025-03-31T13:13:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-14 04:56:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6163168","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6163168","identity":"rs-6163168","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}