Leaf-Cutting Insects are Dominant Drivers of Juvenile Plant Herbivory in the Cerrado Savanna

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Cardoso, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8311461/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Herbivory is a key ecological filter for plant recruitment, structuring plant communities through distinct insect feeding strategies commonly categorized as feeding guilds. However, current classifications overlook non-consumptive tissue removal behaviors that can strongly limit regeneration. This gap is critical in biodiversity hotspots such as the Brazilian Cerrado, the world’s most biodiverse and threatened savanna, where contrasting open and closed vegetation formations host distinct insect communities and regeneration dynamics. We quantified herbivory over six months in 276 juvenile individuals of 22 woody species across open and closed savanna formations, distinguishing six insect-herbivore guilds: mining, galling, sucking, rasping, chewing, and a newly defined ‘cutting guild’ representing leaf-cutter ants ( Atta and Acromyrmex ) and bees ( Megachile ). Herbivory frequency varied with savanna formation and guild, being greater in the open for rasping and chewing, and in the closed for cutting. Mining and galling were less frequent in both formations. Although the closed savanna formation had 1.4-fold higher overall leaf damage, the cutting guild caused the most severe impact, removing on average 95% of leaf area and severing the shoot in ~ 27% of juveniles through a single stem cut, a previously unquantified non-consumptive behavior. In comparison, other guilds caused ~ 10% damage. Recognizing the cutting guild reveals that non-consumptive tissue removal can dominate herbivory budgets not only in the Cerrado but also across the broader Neotropics where these insects occur. This addition advances herbivory classification and predictive frameworks for plant recruitment and vegetation recovery. feeding guilds ants herbivory intensity Neotropical vegetation plant regeneration Figures Figure 1 Figure 2 Figure 3 1. INTRODUCTION Understanding the functional diversity of insect herbivory is essential for predicting how plant communities assemble and persist (Anurag A. Agrawal and Maron, 2022). Herbivory is broadly characterized as the removal of plant tissue by animals, irrespective of ingestion, because physical damage to plant structures has ecological effects similar to consumptive feeding (Müller et al., 2024 ; Swanson et al., 2019 ). However, current frameworks largely overlook forms of non-consumptive damage, as they are conceptually or quantitatively grouped within existing feeding guilds. Herbivores are commonly classified into guilds such as sucking, mining, galling, rasping, and chewing, based on feeding mode, abundance, and host specificity (Andrade et al., 2020 ; Novotny et al., 2010 ; Vidal and Murphy, 2018 ). Guilds such as sucking, mining, and galling comprise mostly specialized species that feed on a narrow range of hosts and cause minimal visible damage, contributing to their underrepresentation in the literature (Kozlov and Zvereva, 2017 ; Oliveira et al., 2020 ). In contrast, rasping and chewing herbivores tend to be generalists capable of removing large fractions of leaf biomass across diverse hosts, and are therefore more frequently quantified (Kozlov and Zvereva, 2017 ; Oliveira et al., 2020 ). However, a distinct form of tissue removal remains clumped within the chewing guild: cutting, in which insects excise leaf or stem segments for purposes other than feeding. Leaf-cutter ants ( Atta and Acromyrmex ) harvest leaves to cultivate fungal gardens, and leaf-cutter bees ( Megachile spp.) remove segments for nest construction (Michener, 2007 ; Pitts-Singer and Cane, 2011 ). These interactions can cause severe defoliation and the removal of entire shoots in juvenile plants (Raupp et al., 2025 ). In some systems, leaf-cutting ants alone may remove more plant biomass than all other herbivores combined (Costa et al., 2008 ; Pialle Urbas et al., 2007). Grouping cutting within chewing masks key functional differences in how herbivore guilds influence plant performance and community structure. Here, we address this conceptual gap by proposing a distinct cutting guild within herbivory classification schemes. Herbivory patterns also vary widely across ecosystems and environmental gradients due to differences in vegetation structure, resource availability, plant community composition, and microclimate (Andrew et al., 2012 ; Martini et al., 2021 ; Salgado-Luarte and Gianoli, 2010 ). However, guild-based herbivory studies of juvenile plants (often the most vulnerable life stage) are largely restricted to forest environments (e.g., de la Cruz and Dirzo, 1987 ; Martini and Goodale, 2020 ; Souza et al., 2013 ; Stiegel and Mantilla-Contreras, 2018 ). This leaves a major knowledge gap in open ecosystems, where plant-insect interactions unfold under contrasting structural and microclimatic conditions. The Brazilian Cerrado, the world’s most biodiverse savanna and a global biodiversity hotspot facing extensive habitat conversion (Cardoso et al., 2025 ; Myers et al., 2000 ), provides an ideal system to address this gap. Its mosaic of open to closed savanna formations (Cardoso et al., 2025 ) supports a diverse insect fauna (Robert J. Marquis et al., 2001), with leaf-cutter ants recognised as the most influential herbivores in terms of biomass removal and diet breadth (Costa et al., 2019 ). Yet, no study has quantified how different insect guilds, including the proposed cutting guild, contribute to herbivory in young woody plants across these savanna formations. In this study, we tested how insect herbivory in young plants varies across savanna formation and herbivore guilds. For that, we quantified the frequency and intensity of herbivory in 276 juvenile individuals (< 30 cm tall) of 22 native woody species across open and closed Cerrado savanna formations, distinguishing damage from six guilds: mining, galling, sucking, rasping, chewing, and cutting. We hypothesised that (i) herbivory frequency and intensity vary between formations due to differences in vegetation structure, resource availability, and microclimatic conditions; (ii) specialized guilds (sucking, mining, and galling) cause less frequent and intense damage than more generalist guilds (rasping, chewing, and cutting), which exploit a wider range of hosts; and (iii) cutting insects, particularly leaf-cutter ants, excert disproportionately high impacts on young plants given their foraging behavior and biomass demands. By explicitly distinguishing cutting from chewing guild, we aim to refine functional classifications of herbivory and evaluate how this guild contributes to variation in early herbivory across contrasting savanna formations. 2. MATERIAL AND METHODS 2.1. Study area This study was conducted at the Panga Ecological Reserve (19°10’ S, 48°23’ W), a 405-ha reserve managed by the Federal University of Uberlândia (UFU) in Minas Gerais state, Brazil. The climate is AW under the Köppen-Geiger system, with a rainy season from October to April and a dry season from May to September (Alvares et al., 2013 ). The mean annual temperature is 23°C, and the annual precipitation is 1650 mm (Cardoso et al., 2009 ). The reserve has various plant formations characteristic of the Brazilian Cerrado (Gonçalves et al., 2021 ). Our fieldwork focused on two structurally contrasting savanna formations: (i) cerrado ralo (open savanna), with less than 20% canopy cover, scattered shrubs and small trees (2–4 m), and a prominent, diverse understory layer, and (ii) cerrado denso (closed savanna), with approximadetely 70% canopy cover, trees from 5 to 8 m height, and sparse herbaceous understory vegetation (Ribeiro and Walter, 2008 ). 2.2. Experimental design We established ten 4 m 2 plots in each savanna formation, spaced at least 30 m apart. Within each plot, we tagged all juvenile shrubs and trees (< 30 cm tall) and identified them to species or genus levels. To avoid overrepresentation of common species and maintain comparable sampling effort among species, we randomly selected up to three individuals per species for herbivory assessment. Sample size per species ranged from nine to 14 individuals, depending on abundance in each savanna, resulting in 276 woody juveniles from 22 species, with higher species richness in the closed (n = 20) than in the open (n = 6) formation ( Online Resource 1 ). To enable temporal herbivory monitoring, two to three leaves per individual were permanently marked from apex to base using an indelible pen, a non-destructive method that does not damage the leaves or prevent herbivory (Lowman, 1984 ). Each marked leaf was photographed monthly from March to August 2020, capturing the wet-to-dry seasonality in the Cerrado, allowing us to encompass fluctuations in herbivore activity and plant vulnerability that typify this seasonal biome (Alvares et al., 2013 ). All photographs were processed in ImageJ v.1.48 (Rasband, 2018 ) to quantify the proportion of damaged or removed leaf area (sensu Calixto et al., 2015 ). In total, we assessed six-month cumulative herbivory across 814 leaves. Each damage event was assigned to one of six insect herbivore guilds: mining, galling, sucking, rasping, chewing, and cutting (Fig. 1 ). The cutting guild, introduced here, groups damage caused by leaf-cutter ants ( Atta and Acromymex ), which remove plant tissues to cultivate mutualistic fungi as their primary food source (De Fine Licht and Boomsma, 2010 ), and leaf-cutter bees ( Megachile ), which use leaf fragments for nest construction (Michener, 2007 ; Pitts-Singer and Cane, 2011 ). In addition to removing circular or semicircular leaf fragments, leaf-cutter ants can also sever juvenile shoots entirely by cutting at the stem base (Fig. 1 ). These behaviors produce distinctive and easily diagnosable damage patterns, justifying their recognition as a separate functional category. During herbivory monitoring, plants with missing marked leaves without evidence of herbivory (n = 89 leaves) were attributed to natural senescence (Dent, 2004 ), and their previously recorded damage was retained. A single observer performed all herbivory assessments to ensure consistency across samples and time points. 2.3. Statistical analyses We investigated the frequency of accumulated herbivory at the community level across open and closed formations and insect-herbivore guilds by converting the data to presence-absence and fitting a generalized linear mixed-effects model (GLMM), with a binomial error distribution and logit link. Fixed effects included savanna formation, herbivore guild, and their interaction. Random effects accounted for the sampling hierarchy, with plots crossed with leaves nested within individuals, nested within species (Schielzeth and Nakagawa, 2013 ). To investigate the intensity of accumulated herbivory across savanna formation and herbivore guild, we used proportional herbivory data in a GLMM with the same fixed and random factors described above, but with a beta error distribution and logit link, which accommodates values between 0 and 1 (Stroup et al., 2024 ). Observed proportions ranged from 0.0001 to 1 (i.e., total leaves removed), but we rescaled values to 0.0001–0.9999 using the scales R-package v.1.1.1 to preserve relative differences while meeting distributional requirements. All models were fitted in R v.4.0.3 using glmmTMB (v.1.0.2.1; Brooks et al., 2017 ). Models’ fit was assessed using the QQ-plots and residuals vs. predicted plots using residuals simulated 1000 times in DHARMa (v.0.3.3.0; Hartig, 2016 ). The significance of fixed effects was assessed using type II Wald chi-square tests ( car v.3.0.10; Fox and Weisberg, 2018 ). When the interaction was significant, we conducted Tukey-adjusted post-hoc contrasts among all combinations of guild and formation using emmeans (v.1.5.3; Lenth, 2020 ). Estimated marginal means (EMMs) and standard errors were back-transformed for interpretation and plotting ( RVAideMemoire v.0.9.78; Hervé, 2020 ). 3. RESULTS 3.1. Frequency of herbivory Overall, 69.5% of leaves showed herbivory damage (566 leaves from 238 plants), while 30.5% remained undamaged (248 leaves from 38 plants). Among damaged leaves, 40.0% (n = 228) were attacked by more than one herbivore guild. Herbivory frequency varied strongly among herbivore guilds and depended on savanna formation (χ 2(5) = 71.21, p < 0.001; Fig. 2 ). In the closed formation, cutting was the most frequent guild (~ 0.30), while rasping, chewing, and sucking occurred at similar intermediate levels. In the open formation, rasping and chewing were the most frequent guilds, followed by sucking, which was also more frequent than cutting. Mining and galling were consistently the least frequent guilds in both formations and did not vary across environments. Across savanna formations, mean frequency of herbivory did not differ between open and closed sites (~ 0.10; χ 2(1) = 2.72, p = 0.099), but guild identity strongly structured herbivory incidence (χ 2(5) = 257.75, p = < 0.001). 3.2. Intensity of herbivory Total leaf herbivory across juvenile plant communities was 30.4% ± 27.6 (mean ± SD). Herbivory per leaf ranged from 0.01% to 100%, with a highly right-skewed distribution (Q1 = 1.95%, median = 12.25%, Q3 = 100%), indicating that while most leaves experienced minor damage, a substantial fraction exhibited complete tissue loss. The interaction between savanna formation and herbivore guild was not significant for herbivory intensity (χ 2(5) = 8.47, p = 0.132). Formation alone had a significant main effect (χ 2(1) = 5.33, p = 0.021, Fig. 3 A), with herbivory 1.4-fold higher in the closed formation (21%) than in the open formation (15%). When cutting damage was exploratorily excluded, mean herbivory became similar between formations (~ 5.2%; Online Resource 2 ), indicating that this guild strongly contributed to the observed difference. Herbivore guild had a strong effect on intensity (χ 2(5) = 2053.83, p < 0.001, Fig. 3 B). Cutting caused the highest tissue loss (95%), often removing the entire shoot system (n = 74 plants). This was followed by chewing (12%), mining (11%), rasping (9%), galling (8%), and sucking (5%). Across most plant species, cutting damage exceeded all other guilds combined, averaging 6.9-fold higher in the closed formation and 1.9-fold higher in the open ( Online Resource 2 ). 4. DISCUSSION Herbivory is a central filter in plant recruitment, but current feeding-guild frameworks largely overlook non-consumptive tissue removal (Andrew et al., 2012 ), limiting our ability to evaluate early herbivore impacts across ecosystems. We addressed this gap by quantifying cumulative leaf loss in juvenile Cerrado plants and distinguishing the cutting guild from established feeding modes. Although we hypothesized lower herbivory in the open formation due to reduced vegetation complexity, weaker impacts from specialist guilds relative to generalists, and disproportionate effects from cutting insects, our results only partially aligned with these expectations. Specialist guilds caused low and infrequent damage as predicted, and cutting produced the strongest impacts, but spatial patterns of damage reflected guild composition rather than vegetation structure. These findings underscore the importance of functionally resolving herbivore strategies to clarify how herbivory shapes early plant-stage dynamics and to accurately interpret herbivory patterns in juvenile communities. Across savanna formations, herbivory frequency was comparable, but plants in the closed savanna experienced greater total tissue loss, driven by the higher incidence of cutting damage. When cutting was excluded, herbivory levels converged between formations, indicating that vegetation complexity or canopy cover alone did not explain spatial patterns of herbivory. Instead, the presence and activity of cutting insects dominated the herbivory budget, a result consistent with studies showing that leaf-cutter ant nests can occur widely across the Cerrado regardless of physiognomy and that colony aggregation is not strongly tied to vegetation structure (Costa and Vieira-Neto, 2016 ). This suggests that herbivory in heterogeneous savannas is best understood through the spatial distribution of key generalist guilds rather than simply vegetation complexity, with cutting insects acting as locally intense ecological filters wherever their colonies or foraging trails intersect juvenile plant cohorts. Guild-specific patterns further clarified the distinct ecological roles driving community-level herbivory. As expected, generalist guilds (chewing, rasping, and cutting) accounted for much of the observed frequency and intensity of damage, reflecting their capacity to exploit a broader range of hosts and microhabitats (Oliveira et al., 2020 ). In contrast, sucking, and particularly mining and galling guilds, were less frequent and did not vary across savanna formations. However, their low frequency did not always correspond to the lowest herbivory intensity. These patterns are consistent with their narrow host specificity: by repeatedly exploiting the same plant individuals or tissues, specialist guilds can generate localized but sustained damage despite their limited abundance (Brezzi et al., 2017 ; Novotny et al., 2010 ; Stiegel and Mantilla-Contreras, 2018 ). Overall, these differences suggest that generalists drive the spatial heterogeneity in herbivory, while specialists supply a consistent underlying layer of damage that supports the overall community-level signal. Recognising cutting as a distinct functional guild revealed that non-consumptive tissue removal is the dominant pathway of biomass loss in Cerrado juveniles. Cutting damage exceeded all other guilds combined and frequently resulted in complete shoot removal, an outcome recently linked to nearly threefold increases in seedling mortality (Raupp et al., 2025 ). While moderate defoliation may not affect seedling survival, the complete loss of photosynthetic tissue sharply reduces establishment potential and growth trajectories (Barton and Shiels, 2020 ; Hinman and Fridley, 2020 ; Raupp et al., 2025 ). These results highlight that the cutting guild operates not merely as high-intensity herbivores but as potent demographic filters capable of excluding species from recruitment windows (Costa et al., 2017 ; Meyer et al., 2011 ; Vasconcelos and Cherrett, 1997 ). Importantly, the ecological reach of these insects extends far beyond the Cerrado: Atta and Acromyrmex ants occur throughout the Neotropics, inhabiting tropical forests, savannas, drylands, and agroecosystems (Fowler, 1983 ; Gómez-Díaz et al., 2023 ). Their broad biogeographic distribution means that non-consumptive cutting is likely a dominant and under-recognised filter in many plant communities, not only in South American savannas but across diverse ecosystems where leaf-cutting insects are abundant. Cumulative herbivory in our study (~ 30%) was at least twice that reported for seedling communities in tropical (9.2 to 10.5%; Benítez-Malvido and Lemus-Albor, 2005 ; de la Cruz and Dirzo, 1987 ), subtropical (14%; Martini et al., 2021 ), and temperate forests (2.7%; Murphy et al., 2016 ), as well as for adult plants in other Neotropical savannas (4.6 to 9.3%; Fowler and Duarte, 1991 ; Marquis et al., 2001). This elevated herbivory likely reflects not only the higher vulnerability of juvenile plants due to smaller size, nutrient-rich tissue, and reduced defences (Hanley et al., 2004 ), but also the prevalence of cutting insects in this biome (Costa et al., 2019 ). Yet, beyond the dominance of cutting, the remaining five guilds should not be dismissed, as even seemingly minor herbivory can accumulate significant impacts on plant growth and reproduction over time (Kozlov and Zvereva, 2017 ; Zvereva et al., 2010 ). For example, as little as 10% leaf loss has been linked to long-term reductions of ~ 45% in vertical growth (Zvereva et al., 2012 ), potentially constraining the competitive ability of juvenile plants and influencing future community composition. Thus, while cutting insects caused the strongest defoliation and likely immediate plant effects, background herbivory from other guilds contributes to sustained demographic filtering across plant cohorts. Although our monitoring encompassed the seasonal transition from wet to dry periods, and thus major shifts in insect activity and plant phenology (Calixto et al., 2021 ), our estimates likely remain conservative. We used continuous, non-inflated measures of cumulative leaf loss, avoiding categorical scoring systems that often underestimate or obscure non-consumptive removal (Getman-Pickering et al., 2020 ; Kozlov and Zvereva, 2017 ). Moreover, plants remained exposed to herbivores throughout six months, and continuous foraging by herbivores may amplify cumulative tissue loss (Filip et al., 1995 ). These methodological considerations reinforce that cumulative herbivory, particularly from cutting insects, may be even more pervasive across Cerrado landscapes than our estimates suggest. Taken together, our results reveal that non-consumptive cutting represents an ecologically consequential and previously under-appreciated herbivore strategy capable of dominating herbivory budgets and driving recruitment bottlenecks in the world’s most biodiverse and threatened savanna (Myers et al., 2000 ). By incorporating cutting into herbivore guild frameworks, we not only refine functional classifications of herbivory but also enhance predictive understanding of how insect communities shape plant regeneration. Because leaf-cutting insects are widespread throughout the Neotropics, the implications extend well beyond the Cerrado, underscoring the need to integrate non-consumptive tissue removal into models of plant population dynamics, community assembly, and ecosystem recovery across diverse global regions where these insects occur. Declarations CONFLICT OF INTEREST The authors declare no competing interests. FUNDING Grants and MSc scholarship of PPR and RVS from the Brazilian Council for Research and Scientific Development (CNPq, grant projects PELD 441225/2016-0, 441142/2020-6, and 445542/2024-1), the Research Foundation of Minas Gerais (FAPEMIG, grant projects APQ-03372-21, APQ-03249-22, and RED-00039-23), and Coordination for Improvement of Higher Education Personnel (CAPES) supported this research. AUTHOR CONTRIBUTION PPR and ANC formulated the idea and designed the methodology. PPR, RVG, and ANC performed experiments and data collection. PPR and JCFC analyzed the data. PPR, ESC, and ANC wrote the manuscript. All authors contributed critically to the drafts and gave final approval for publication. ACKNOWLEDGEMENTS We thank Jaqueline Vaz for the support in identifying plant species. AVAILABILITY OF DATA AND MATERIALS Available from the corresponding author upon request. 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Nature 403:853–858. https://doi.org/10.1038/35002501 Novotny V, Miller SE, Baje L, Balagawi S, Basset Y, Cizek L, Craft KJ, Dem F, Drew RAI, Hulcr J, Leps J, Lewis OT, Pokon R, Stewart AJA, Allan Samuelson G, Weiblen GD (2010) Guild-specific patterns of species richness and host specialization in plant–herbivore food webs from a tropical forest. J Anim Ecol 79:1193–1203. https://doi.org/10.1111/j.1365-2656.2010.01728.x Oliveira J b. b. s., Faria ML, Borges MAZ, Fagundes M, de Araújo WS (2020) Comparing the plant–herbivore network topology of different insect guilds in Neotropical savannas. Ecological Entomology 45, 406–415. https://doi.org/10.1111/een.12808 Pialle Urbas, Araújo Jr MV, Leal IR, Wirth R (2007) Cutting More from Cut Forests: Edge Effects on Foraging and Herbivory of Leaf-Cutting Ants in Brazil. Biotropica 39:489–495. https://doi.org/10.1111/j.1744-7429.2007.00285.x Pitts-Singer TL, Cane JH (2011) The Alfalfa Leafcutting Bee, Megachile rotundata: The World’s Most Intensively Managed Solitary Bee*. Ann Rev Entomol 56:221–237. https://doi.org/10.1146/annurev-ento-120709-144836 Rasband W (2018) ImageJ Raupp PP, Gonçalves RVS, Cardoso JCF, Calixto ES, Costa AN (2025) Herbivory intensity and plant species traits interact with tree canopy cover to drive seedling survival in Neotropical savannas. Plant Ecol 226:473–484. https://doi.org/10.1007/s11258-025-01509-x Ribeiro JF, Walter BMT (2008) Fitofisionomia do bioma Cerrado, in: Cerrado: ecologia e flora. Embrapa CPAC Robert J, Marquis, Diniz IR, Morais HC (2001) Patterns and correlates of interspecific variation in foliar insect herbivory and pathogen attack in Brazilian cerrado. J Trop Ecol 17:127–148. https://doi.org/10.1017/S0266467401001080 Salgado-Luarte C, Gianoli E (2010) Herbivory on Temperate Rainforest Seedlings in Sun and Shade: Resistance, Tolerance and Habitat Distribution. PLoS ONE 5:e11460. https://doi.org/10.1371/journal.pone.0011460 Schielzeth H, Nakagawa S (2013) Nested by design: model fitting and interpretation in a mixed model era. Methods Ecol Evol 4:14–24. https://doi.org/10.1111/j.2041-210x.2012.00251.x Souza DG, Santos BA, Wirth R, Leal IR, Tabarelli M (2013) Community-Level Patterns of Insect Herbivory in a Fragmented Atlantic Forest Landscape. Environ Entomol 42:430–437. https://doi.org/10.1603/EN12273 Stiegel S, Mantilla-Contreras J (2018) Environment vs. Plant Ontogeny: Arthropod Herbivory Patterns on European Beech Leaves along the Vertical Gradient of Temperate Forests in Central Germany. Insects 9:9. https://doi.org/10.3390/insects9010009 Stroup WW, Ptukhina M, Garai J (2024) Generalized Linear Mixed Models: Modern Concepts, Methods and Applications, 2nd edn. Chapman and Hall/CRC, New York. https://doi.org/10.1201/9780429092060 Swanson AC, Schwendenmann L, Allen MF, Aronson EL, Artavia-León A, Dierick D, Fernandez-Bou AS, Harmon TC, Murillo-Cruz C, Oberbauer SF, Pinto-Tomás AA, Rundel PW, Zelikova TJ (2019) Welcome to the Atta world: A framework for understanding the effects of leaf-cutter ants on ecosystem functions. Funct Ecol 33:1386–1399. https://doi.org/10.1111/1365-2435.13319 Vasconcelos HL, Cherrett JM (1997) Leaf-cutting ants and early forest regeneration in central Amazonia: effects of herbivory on tree seedling establishment. J Trop Ecol 13:357–370. https://doi.org/10.1017/S0266467400010567 Vidal MC, Murphy SM (2018) Bottom-up vs. top-down effects on terrestrial insect herbivores: a meta-analysis. Ecol Lett 21:138–150. https://doi.org/10.1111/ele.12874 Zvereva EL, Lanta V, Kozlov MV (2010) Effects of sap-feeding insect herbivores on growth and reproduction of woody plants: a meta-analysis of experimental studies. Oecologia 163:949–960. https://doi.org/10.1007/s00442-010-1633-1 Zvereva EL, Zverev V, Kozlov MV (2012) Little strokes fell great oaks: minor but chronic herbivory substantially reduces birch growth. Oikos 121:2036–2043. https://doi.org/10.1111/j.1600-0706.2012.20688.x Supplementary Files SuplementaryinformationRaupp2025.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 20 Jan, 2026 Reviewers invited by journal 20 Jan, 2026 Editor assigned by journal 10 Dec, 2025 First submitted to journal 08 Dec, 2025 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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Cardoso","email":"","orcid":"","institution":"Universidade Federal de Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"Custódio F.","lastName":"Cardoso","suffix":""},{"id":577540582,"identity":"8a8e24b2-26cd-4154-9367-3c6a19f6031f","order_by":3,"name":"Eduardo Soares Calixto","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"Soares","lastName":"Calixto","suffix":""},{"id":577540583,"identity":"12baeb38-bca7-4875-a3d2-cb4020224ed3","order_by":4,"name":"Alan Nilo Costa","email":"","orcid":"","institution":"Universidade Federal de Uberlandia","correspondingAuthor":false,"prefix":"","firstName":"Alan","middleName":"Nilo","lastName":"Costa","suffix":""}],"badges":[],"createdAt":"2025-12-08 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10:25:56","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114694,"visible":true,"origin":"","legend":"","description":"","filename":"OECOD25006740structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8311461/v1/e9e678578fa3515150f16714.xml"},{"id":100876541,"identity":"618cccd3-4669-4e1f-b5d8-33a9dc43723e","added_by":"auto","created_at":"2026-01-22 10:26:00","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":123641,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8311461/v1/cf4e6424eef3323020a568eb.html"},{"id":100876498,"identity":"3457d6ae-a10c-43d0-b620-68b8df85b5b3","added_by":"auto","created_at":"2026-01-22 10:25:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":580590,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic leaf damage patterns associated with major insect herbivore guilds. Photographs illustrate representative field-collected examples taken during this study, alongside descriptions of characteristic damage and typical herbivore taxa responsible for each guild. Guild definitions and classification criteria were adapted from established frameworks (Andrade et al., 2020; de la Cruz and Dirzo, 1987; Dyer et al., 2010).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8311461/v1/b15df6138cbfd0044660d9f8.png"},{"id":100876519,"identity":"28609f1b-92f6-4178-85f6-2265495104bb","added_by":"auto","created_at":"2026-01-22 10:25:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":324335,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency of leaf herbivory across the interaction between insect-herbivore guild and savanna formation (open and closed). Points and error bars show back-transformed marginal model-adjusted means ± standard errors. Jittered dots represent raw data. Different letters indicate Tukey-adjusted pairwise differences among guild and formation combinations (p ≤ 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8311461/v1/6dd5c9ee536556f889021edd.png"},{"id":100876515,"identity":"3bdf13d1-7f22-47e5-9d54-f08ec06f75e6","added_by":"auto","created_at":"2026-01-22 10:25:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210704,"visible":true,"origin":"","legend":"\u003cp\u003eIntensity of herbivory (% leaf area damage) according to (A) savanna formation (open and closed) and (B) insect-feeding guild. Points and error bars represent back-transformed model-adjusted means ± standard errors. Jittered dots show raw data. Different letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e ≤ 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8311461/v1/df3cf8e496d5e181e001d7eb.png"},{"id":100876596,"identity":"b5d79be2-10c3-4f50-a041-a8af8f2c496c","added_by":"auto","created_at":"2026-01-22 10:26:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1587096,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8311461/v1/9453eb28-0e13-41c9-acc3-133a4632b415.pdf"},{"id":100876501,"identity":"ade3c91c-2b7d-4da3-8ade-c4adf29b97b4","added_by":"auto","created_at":"2026-01-22 10:25:56","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":292621,"visible":true,"origin":"","legend":"","description":"","filename":"SuplementaryinformationRaupp2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8311461/v1/bb93a2e59271a95e06e06f58.pdf"}],"financialInterests":"","formattedTitle":"Leaf-Cutting Insects are Dominant Drivers of Juvenile Plant Herbivory in the Cerrado Savanna","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eUnderstanding the functional diversity of insect herbivory is essential for predicting how plant communities assemble and persist (Anurag A. Agrawal and Maron, 2022). Herbivory is broadly characterized as the removal of plant tissue by animals, irrespective of ingestion, because physical damage to plant structures has ecological effects similar to consumptive feeding (M\u0026uuml;ller et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Swanson et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, current frameworks largely overlook forms of non-consumptive damage, as they are conceptually or quantitatively grouped within existing feeding guilds.\u003c/p\u003e \u003cp\u003eHerbivores are commonly classified into guilds such as sucking, mining, galling, rasping, and chewing, based on feeding mode, abundance, and host specificity (Andrade et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Novotny et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Vidal and Murphy, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Guilds such as sucking, mining, and galling comprise mostly specialized species that feed on a narrow range of hosts and cause minimal visible damage, contributing to their underrepresentation in the literature (Kozlov and Zvereva, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Oliveira et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, rasping and chewing herbivores tend to be generalists capable of removing large fractions of leaf biomass across diverse hosts, and are therefore more frequently quantified (Kozlov and Zvereva, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Oliveira et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, a distinct form of tissue removal remains clumped within the chewing guild: cutting, in which insects excise leaf or stem segments for purposes other than feeding. Leaf-cutter ants (\u003cem\u003eAtta\u003c/em\u003e and \u003cem\u003eAcromyrmex\u003c/em\u003e) harvest leaves to cultivate fungal gardens, and leaf-cutter bees (\u003cem\u003eMegachile\u003c/em\u003e spp.) remove segments for nest construction (Michener, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Pitts-Singer and Cane, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These interactions can cause severe defoliation and the removal of entire shoots in juvenile plants (Raupp et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In some systems, leaf-cutting ants alone may remove more plant biomass than all other herbivores combined (Costa et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Pialle Urbas et al., 2007). Grouping cutting within chewing masks key functional differences in how herbivore guilds influence plant performance and community structure. Here, we address this conceptual gap by proposing a distinct cutting guild within herbivory classification schemes.\u003c/p\u003e \u003cp\u003eHerbivory patterns also vary widely across ecosystems and environmental gradients due to differences in vegetation structure, resource availability, plant community composition, and microclimate (Andrew et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Martini et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Salgado-Luarte and Gianoli, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, guild-based herbivory studies of juvenile plants (often the most vulnerable life stage) are largely restricted to forest environments (e.g., de la Cruz and Dirzo, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Martini and Goodale, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Souza et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Stiegel and Mantilla-Contreras, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This leaves a major knowledge gap in open ecosystems, where plant-insect interactions unfold under contrasting structural and microclimatic conditions. The Brazilian Cerrado, the world\u0026rsquo;s most biodiverse savanna and a global biodiversity hotspot facing extensive habitat conversion (Cardoso et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Myers et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), provides an ideal system to address this gap. Its mosaic of open to closed savanna formations (Cardoso et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) supports a diverse insect fauna (Robert J. Marquis et al., 2001), with leaf-cutter ants recognised as the most influential herbivores in terms of biomass removal and diet breadth (Costa et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Yet, no study has quantified how different insect guilds, including the proposed cutting guild, contribute to herbivory in young woody plants across these savanna formations.\u003c/p\u003e \u003cp\u003eIn this study, we tested how insect herbivory in young plants varies across savanna formation and herbivore guilds. For that, we quantified the frequency and intensity of herbivory in 276 juvenile individuals (\u0026lt;\u0026thinsp;30 cm tall) of 22 native woody species across open and closed Cerrado savanna formations, distinguishing damage from six guilds: mining, galling, sucking, rasping, chewing, and cutting. We hypothesised that (i) herbivory frequency and intensity vary between formations due to differences in vegetation structure, resource availability, and microclimatic conditions; (ii) specialized guilds (sucking, mining, and galling) cause less frequent and intense damage than more generalist guilds (rasping, chewing, and cutting), which exploit a wider range of hosts; and (iii) cutting insects, particularly leaf-cutter ants, excert disproportionately high impacts on young plants given their foraging behavior and biomass demands. By explicitly distinguishing cutting from chewing guild, we aim to refine functional classifications of herbivory and evaluate how this guild contributes to variation in early herbivory across contrasting savanna formations.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Study area\u003c/h2\u003e \u003cp\u003eThis study was conducted at the Panga Ecological Reserve (19\u0026deg;10\u0026rsquo; S, 48\u0026deg;23\u0026rsquo; W), a 405-ha reserve managed by the Federal University of Uberl\u0026acirc;ndia (UFU) in Minas Gerais state, Brazil. The climate is AW under the K\u0026ouml;ppen-Geiger system, with a rainy season from October to April and a dry season from May to September (Alvares et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The mean annual temperature is 23\u0026deg;C, and the annual precipitation is 1650 mm (Cardoso et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The reserve has various plant formations characteristic of the Brazilian Cerrado (Gon\u0026ccedil;alves et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our fieldwork focused on two structurally contrasting savanna formations: (i) \u003cem\u003ecerrado ralo\u003c/em\u003e (open savanna), with less than 20% canopy cover, scattered shrubs and small trees (2\u0026ndash;4 m), and a prominent, diverse understory layer, and (ii) \u003cem\u003ecerrado denso\u003c/em\u003e (closed savanna), with approximadetely 70% canopy cover, trees from 5 to 8 m height, and sparse herbaceous understory vegetation (Ribeiro and Walter, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental design\u003c/h2\u003e \u003cp\u003eWe established ten 4 m\u003csup\u003e2\u003c/sup\u003e plots in each savanna formation, spaced at least 30 m apart. Within each plot, we tagged all juvenile shrubs and trees (\u0026lt;\u0026thinsp;30 cm tall) and identified them to species or genus levels. To avoid overrepresentation of common species and maintain comparable sampling effort among species, we randomly selected up to three individuals per species for herbivory assessment. Sample size per species ranged from nine to 14 individuals, depending on abundance in each savanna, resulting in 276 woody juveniles from 22 species, with higher species richness in the closed (n\u0026thinsp;=\u0026thinsp;20) than in the open (n\u0026thinsp;=\u0026thinsp;6) formation (\u003cb\u003eOnline Resource 1\u003c/b\u003e). To enable temporal herbivory monitoring, two to three leaves per individual were permanently marked from apex to base using an indelible pen, a non-destructive method that does not damage the leaves or prevent herbivory (Lowman, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Each marked leaf was photographed monthly from March to August 2020, capturing the wet-to-dry seasonality in the Cerrado, allowing us to encompass fluctuations in herbivore activity and plant vulnerability that typify this seasonal biome (Alvares et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll photographs were processed in ImageJ v.1.48 (Rasband, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) to quantify the proportion of damaged or removed leaf area (sensu Calixto et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In total, we assessed six-month cumulative herbivory across 814 leaves. Each damage event was assigned to one of six insect herbivore guilds: mining, galling, sucking, rasping, chewing, and cutting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The cutting guild, introduced here, groups damage caused by leaf-cutter ants (\u003cem\u003eAtta\u003c/em\u003e and \u003cem\u003eAcromymex\u003c/em\u003e), which remove plant tissues to cultivate mutualistic fungi as their primary food source (De Fine Licht and Boomsma, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and leaf-cutter bees (\u003cem\u003eMegachile\u003c/em\u003e), which use leaf fragments for nest construction (Michener, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Pitts-Singer and Cane, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In addition to removing circular or semicircular leaf fragments, leaf-cutter ants can also sever juvenile shoots entirely by cutting at the stem base (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These behaviors produce distinctive and easily diagnosable damage patterns, justifying their recognition as a separate functional category. During herbivory monitoring, plants with missing marked leaves without evidence of herbivory (n\u0026thinsp;=\u0026thinsp;89 leaves) were attributed to natural senescence (Dent, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and their previously recorded damage was retained. A single observer performed all herbivory assessments to ensure consistency across samples and time points.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Statistical analyses\u003c/h2\u003e \u003cp\u003eWe investigated the frequency of accumulated herbivory at the community level across open and closed formations and insect-herbivore guilds by converting the data to presence-absence and fitting a generalized linear mixed-effects model (GLMM), with a binomial error distribution and logit link. Fixed effects included savanna formation, herbivore guild, and their interaction. Random effects accounted for the sampling hierarchy, with plots crossed with leaves nested within individuals, nested within species (Schielzeth and Nakagawa, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo investigate the intensity of accumulated herbivory across savanna formation and herbivore guild, we used proportional herbivory data in a GLMM with the same fixed and random factors described above, but with a beta error distribution and logit link, which accommodates values between 0 and 1 (Stroup et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Observed proportions ranged from 0.0001 to 1 (i.e., total leaves removed), but we rescaled values to 0.0001\u0026ndash;0.9999 using the \u003cem\u003escales\u003c/em\u003e R-package v.1.1.1 to preserve relative differences while meeting distributional requirements.\u003c/p\u003e \u003cp\u003eAll models were fitted in R v.4.0.3 using \u003cem\u003eglmmTMB\u003c/em\u003e (v.1.0.2.1; Brooks et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Models\u0026rsquo; fit was assessed using the QQ-plots and residuals vs. predicted plots using residuals simulated 1000 times in \u003cem\u003eDHARMa\u003c/em\u003e (v.0.3.3.0; Hartig, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The significance of fixed effects was assessed using type II Wald chi-square tests (\u003cem\u003ecar\u003c/em\u003e v.3.0.10; Fox and Weisberg, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). When the interaction was significant, we conducted Tukey-adjusted post-hoc contrasts among all combinations of guild and formation using \u003cem\u003eemmeans\u003c/em\u003e (v.1.5.3; Lenth, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Estimated marginal means (EMMs) and standard errors were back-transformed for interpretation and plotting (\u003cem\u003eRVAideMemoire\u003c/em\u003e v.0.9.78; Herv\u0026eacute;, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Frequency of herbivory\u003c/h2\u003e \u003cp\u003eOverall, 69.5% of leaves showed herbivory damage (566 leaves from 238 plants), while 30.5% remained undamaged (248 leaves from 38 plants). Among damaged leaves, 40.0% (n\u0026thinsp;=\u0026thinsp;228) were attacked by more than one herbivore guild. Herbivory frequency varied strongly among herbivore guilds and depended on savanna formation (χ\u003csup\u003e2(5)\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;71.21, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the closed formation, cutting was the most frequent guild (~\u0026thinsp;0.30), while rasping, chewing, and sucking occurred at similar intermediate levels. In the open formation, rasping and chewing were the most frequent guilds, followed by sucking, which was also more frequent than cutting. Mining and galling were consistently the least frequent guilds in both formations and did not vary across environments. Across savanna formations, mean frequency of herbivory did not differ between open and closed sites (~\u0026thinsp;0.10; χ\u003csup\u003e2(1)\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;2.72, p\u0026thinsp;=\u0026thinsp;0.099), but guild identity strongly structured herbivory incidence (χ\u003csup\u003e2(5)\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;257.75, p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Intensity of herbivory\u003c/h2\u003e \u003cp\u003eTotal leaf herbivory across juvenile plant communities was 30.4% \u0026plusmn; 27.6 (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). Herbivory per leaf ranged from 0.01% to 100%, with a highly right-skewed distribution (Q1\u0026thinsp;=\u0026thinsp;1.95%, median\u0026thinsp;=\u0026thinsp;12.25%, Q3\u0026thinsp;=\u0026thinsp;100%), indicating that while most leaves experienced minor damage, a substantial fraction exhibited complete tissue loss. The interaction between savanna formation and herbivore guild was not significant for herbivory intensity (χ\u003csup\u003e2(5)\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;8.47, p\u0026thinsp;=\u0026thinsp;0.132). Formation alone had a significant main effect (χ\u003csup\u003e2(1)\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;5.33, p\u0026thinsp;=\u0026thinsp;0.021, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with herbivory 1.4-fold higher in the closed formation (21%) than in the open formation (15%). When cutting damage was exploratorily excluded, mean herbivory became similar between formations (~\u0026thinsp;5.2%; \u003cb\u003eOnline Resource 2\u003c/b\u003e), indicating that this guild strongly contributed to the observed difference. Herbivore guild had a strong effect on intensity (χ\u003csup\u003e2(5)\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;2053.83, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Cutting caused the highest tissue loss (95%), often removing the entire shoot system (n\u0026thinsp;=\u0026thinsp;74 plants). This was followed by chewing (12%), mining (11%), rasping (9%), galling (8%), and sucking (5%). Across most plant species, cutting damage exceeded all other guilds combined, averaging 6.9-fold higher in the closed formation and 1.9-fold higher in the open (\u003cb\u003eOnline Resource 2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eHerbivory is a central filter in plant recruitment, but current feeding-guild frameworks largely overlook non-consumptive tissue removal (Andrew et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), limiting our ability to evaluate early herbivore impacts across ecosystems. We addressed this gap by quantifying cumulative leaf loss in juvenile Cerrado plants and distinguishing the cutting guild from established feeding modes. Although we hypothesized lower herbivory in the open formation due to reduced vegetation complexity, weaker impacts from specialist guilds relative to generalists, and disproportionate effects from cutting insects, our results only partially aligned with these expectations. Specialist guilds caused low and infrequent damage as predicted, and cutting produced the strongest impacts, but spatial patterns of damage reflected guild composition rather than vegetation structure. These findings underscore the importance of functionally resolving herbivore strategies to clarify how herbivory shapes early plant-stage dynamics and to accurately interpret herbivory patterns in juvenile communities.\u003c/p\u003e \u003cp\u003eAcross savanna formations, herbivory frequency was comparable, but plants in the closed savanna experienced greater total tissue loss, driven by the higher incidence of cutting damage. When cutting was excluded, herbivory levels converged between formations, indicating that vegetation complexity or canopy cover alone did not explain spatial patterns of herbivory. Instead, the presence and activity of cutting insects dominated the herbivory budget, a result consistent with studies showing that leaf-cutter ant nests can occur widely across the Cerrado regardless of physiognomy and that colony aggregation is not strongly tied to vegetation structure (Costa and Vieira-Neto, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This suggests that herbivory in heterogeneous savannas is best understood through the spatial distribution of key generalist guilds rather than simply vegetation complexity, with cutting insects acting as locally intense ecological filters wherever their colonies or foraging trails intersect juvenile plant cohorts.\u003c/p\u003e \u003cp\u003eGuild-specific patterns further clarified the distinct ecological roles driving community-level herbivory. As expected, generalist guilds (chewing, rasping, and cutting) accounted for much of the observed frequency and intensity of damage, reflecting their capacity to exploit a broader range of hosts and microhabitats (Oliveira et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, sucking, and particularly mining and galling guilds, were less frequent and did not vary across savanna formations. However, their low frequency did not always correspond to the lowest herbivory intensity. These patterns are consistent with their narrow host specificity: by repeatedly exploiting the same plant individuals or tissues, specialist guilds can generate localized but sustained damage despite their limited abundance (Brezzi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Novotny et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Stiegel and Mantilla-Contreras, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Overall, these differences suggest that generalists drive the spatial heterogeneity in herbivory, while specialists supply a consistent underlying layer of damage that supports the overall community-level signal.\u003c/p\u003e \u003cp\u003eRecognising cutting as a distinct functional guild revealed that non-consumptive tissue removal is the dominant pathway of biomass loss in Cerrado juveniles. Cutting damage exceeded all other guilds combined and frequently resulted in complete shoot removal, an outcome recently linked to nearly threefold increases in seedling mortality (Raupp et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). While moderate defoliation may not affect seedling survival, the complete loss of photosynthetic tissue sharply reduces establishment potential and growth trajectories (Barton and Shiels, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hinman and Fridley, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Raupp et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These results highlight that the cutting guild operates not merely as high-intensity herbivores but as potent demographic filters capable of excluding species from recruitment windows (Costa et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Meyer et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Vasconcelos and Cherrett, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Importantly, the ecological reach of these insects extends far beyond the Cerrado: \u003cem\u003eAtta\u003c/em\u003e and \u003cem\u003eAcromyrmex\u003c/em\u003e ants occur throughout the Neotropics, inhabiting tropical forests, savannas, drylands, and agroecosystems (Fowler, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; G\u0026oacute;mez-D\u0026iacute;az et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Their broad biogeographic distribution means that non-consumptive cutting is likely a dominant and under-recognised filter in many plant communities, not only in South American savannas but across diverse ecosystems where leaf-cutting insects are abundant.\u003c/p\u003e \u003cp\u003eCumulative herbivory in our study (~\u0026thinsp;30%) was at least twice that reported for seedling communities in tropical (9.2 to 10.5%; Ben\u0026iacute;tez-Malvido and Lemus-Albor, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; de la Cruz and Dirzo, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), subtropical (14%; Martini et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and temperate forests (2.7%; Murphy et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), as well as for adult plants in other Neotropical savannas (4.6 to 9.3%; Fowler and Duarte, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Marquis et al., 2001). This elevated herbivory likely reflects not only the higher vulnerability of juvenile plants due to smaller size, nutrient-rich tissue, and reduced defences (Hanley et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), but also the prevalence of cutting insects in this biome (Costa et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Yet, beyond the dominance of cutting, the remaining five guilds should not be dismissed, as even seemingly minor herbivory can accumulate significant impacts on plant growth and reproduction over time (Kozlov and Zvereva, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zvereva et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). For example, as little as 10% leaf loss has been linked to long-term reductions of ~\u0026thinsp;45% in vertical growth (Zvereva et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), potentially constraining the competitive ability of juvenile plants and influencing future community composition. Thus, while cutting insects caused the strongest defoliation and likely immediate plant effects, background herbivory from other guilds contributes to sustained demographic filtering across plant cohorts.\u003c/p\u003e \u003cp\u003eAlthough our monitoring encompassed the seasonal transition from wet to dry periods, and thus major shifts in insect activity and plant phenology (Calixto et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), our estimates likely remain conservative. We used continuous, non-inflated measures of cumulative leaf loss, avoiding categorical scoring systems that often underestimate or obscure non-consumptive removal (Getman-Pickering et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kozlov and Zvereva, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, plants remained exposed to herbivores throughout six months, and continuous foraging by herbivores may amplify cumulative tissue loss (Filip et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). These methodological considerations reinforce that cumulative herbivory, particularly from cutting insects, may be even more pervasive across Cerrado landscapes than our estimates suggest.\u003c/p\u003e \u003cp\u003eTaken together, our results reveal that non-consumptive cutting represents an ecologically consequential and previously under-appreciated herbivore strategy capable of dominating herbivory budgets and driving recruitment bottlenecks in the world\u0026rsquo;s most biodiverse and threatened savanna (Myers et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). By incorporating cutting into herbivore guild frameworks, we not only refine functional classifications of herbivory but also enhance predictive understanding of how insect communities shape plant regeneration. Because leaf-cutting insects are widespread throughout the Neotropics, the implications extend well beyond the Cerrado, underscoring the need to integrate non-consumptive tissue removal into models of plant population dynamics, community assembly, and ecosystem recovery across diverse global regions where these insects occur.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eGrants and MSc scholarship of PPR and RVS from the Brazilian Council for Research and Scientific Development (CNPq, grant projects PELD 441225/2016-0, 441142/2020-6, and 445542/2024-1), the Research Foundation of Minas Gerais (FAPEMIG, grant projects APQ-03372-21, APQ-03249-22, and RED-00039-23), and Coordination for Improvement of Higher Education Personnel (CAPES) supported this research.\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTION\u003c/h2\u003e \u003cp\u003ePPR and ANC formulated the idea and designed the methodology. PPR, RVG, and ANC performed experiments and data collection. PPR and JCFC analyzed the data. PPR, ESC, and ANC wrote the manuscript. All authors contributed critically to the drafts and gave final approval for publication.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eWe thank Jaqueline Vaz for the support in identifying plant species.\u003c/p\u003e\u003ch2\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/h2\u003e \u003cp\u003eAvailable from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlvares CA, Stape JL, Sentelhas PC, De Moraes Gon\u0026ccedil;alves JL, Sparovek G (2013) K\u0026ouml;ppen\u0026rsquo;s climate classification map for Brazil. metz 22, 711\u0026ndash;728. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1127/0941-2948/2013/0507\u003c/span\u003e\u003cspan address=\"10.1127/0941-2948/2013/0507\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndrade JF, Alvarado F, Carlos Santos J, Santos BA (2020) Rainfall reduction increases insect herbivory in tropical herb communities. 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Oikos 121:2036\u0026ndash;2043. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1600-0706.2012.20688.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1600-0706.2012.20688.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oecologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"oeco","sideBox":"Learn more about [Oecologia](https://www.springer.com/journal/442)","snPcode":"442","submissionUrl":"https://submission.nature.com/new-submission/442/3","title":"Oecologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"feeding guilds, ants, herbivory intensity, Neotropical vegetation, plant regeneration","lastPublishedDoi":"10.21203/rs.3.rs-8311461/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8311461/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHerbivory is a key ecological filter for plant recruitment, structuring plant communities through distinct insect feeding strategies commonly categorized as feeding guilds. However, current classifications overlook non-consumptive tissue removal behaviors that can strongly limit regeneration. This gap is critical in biodiversity hotspots such as the Brazilian Cerrado, the world\u0026rsquo;s most biodiverse and threatened savanna, where contrasting open and closed vegetation formations host distinct insect communities and regeneration dynamics. We quantified herbivory over six months in 276 juvenile individuals of 22 woody species across open and closed savanna formations, distinguishing six insect-herbivore guilds: mining, galling, sucking, rasping, chewing, and a newly defined \u0026lsquo;cutting guild\u0026rsquo; representing leaf-cutter ants (\u003cem\u003eAtta\u003c/em\u003e and \u003cem\u003eAcromyrmex\u003c/em\u003e) and bees (\u003cem\u003eMegachile\u003c/em\u003e). Herbivory frequency varied with savanna formation and guild, being greater in the open for rasping and chewing, and in the closed for cutting. Mining and galling were less frequent in both formations. Although the closed savanna formation had 1.4-fold higher overall leaf damage, the cutting guild caused the most severe impact, removing on average 95% of leaf area and severing the shoot in ~\u0026thinsp;27% of juveniles through a single stem cut, a previously unquantified non-consumptive behavior. In comparison, other guilds caused\u0026thinsp;~\u0026thinsp;10% damage. Recognizing the cutting guild reveals that non-consumptive tissue removal can dominate herbivory budgets not only in the Cerrado but also across the broader Neotropics where these insects occur. This addition advances herbivory classification and predictive frameworks for plant recruitment and vegetation recovery.\u003c/p\u003e","manuscriptTitle":"Leaf-Cutting Insects are Dominant Drivers of Juvenile Plant Herbivory in the Cerrado Savanna","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 10:25:37","doi":"10.21203/rs.3.rs-8311461/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-01-20T18:10:25+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-20T14:31:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-10T06:36:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oecologia","date":"2025-12-08T18:36:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oecologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"oeco","sideBox":"Learn more about [Oecologia](https://www.springer.com/journal/442)","snPcode":"442","submissionUrl":"https://submission.nature.com/new-submission/442/3","title":"Oecologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"683b256a-d754-436c-80b3-3c38ffe96536","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T20:31:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 10:25:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8311461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8311461","identity":"rs-8311461","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}