Forest type modulates mammalian responses to megafires

Although considered an evolutionary force responsible for shaping ecosystems and biodiversity, fires’ natural cycle is being altered by human activities, increasing the odds of destructive megafire events. Here, we show that forest type modulates the responses of terrestrial mammals, from species to assemblage level, to a catastrophic megafire in the Brazilian Pantanal. We unraveled that mammalian richness was higher 1 year after fire passage compared to a pre-fire condition, which can be attributed to habitat modification caused by wildfires, attracting herbivores and open-area tolerant species. We observed changes in assemblage composition between burned/unburned sites, but no difference in mammalian richness or relative abundance. However, by partitioning the effects of burned area proportion per forest type (monospecific vs. polyspecific), we detected differential responses of mammals at several levels of organization, with pronounced declines in species richness and relative abundance in monospecific forests. Eighty-six percent of the species presented moderate to strong negative effects on their relative abundance, with an overall strong negative effect for the entire assemblage. Wildfires are predicted to be more frequent with climate and land use change, and if events analogous to Pantanal-2020 become recurrent, they might trigger regional beta diversity change, benefitting open-area tolerant species.

Although the observed species richness was similar between burned and unburned sites, mammal assemblage composition differed, with 43% of species shared and four exclusive records in each treatment (Fig. 3A).Based on the MSOM results, the previous pattern was sustained, with no difference in species richness (Welch  28,29 and after fire passage (this study).two-sample t-test, t = 0.23, p = 0.82; Fig. 3B) or aggregated relative abundance at the site level (Wilcoxon ranksum test, W = 364, p = 0.21; Fig. 3C).
Considering the effects of variables in the MSOM over individual species, only the interactivity between the proportion of burned area and forest type showed significant moderate to strong effects on the relative abundance parameter (Fig. 5A, Supplementary Figs.3-5).We observed that mammals presented an average reduction in relative abundance in response to increasing burned area proportion in monospecific forests, with a moderate www.nature.com/scientificreports/effect for five species and a strong one for seven.A strong effect was also observed for the interactive variable on the assemblage aggregated relative abundance, which decreased in monospecific forests (Fig. 5B).

Discussion
Assessing biodiversity responses to wildfires is paramount to understanding the impacts of such events on species occurrence and abundance, especially considering that they are predicted to occur more frequently with climate change 30 .Although our study area was subject to a catastrophic megafire in 2020, a year later, the terrestrial mammalian diversity was higher than before the fire.Species previously undetected might have been attracted by habitat modification caused by fires, particularly herbivores and open-area tolerant mammals.Assemblage composition differed between burned and unburned sites, but not species richness or relative abundance.However, partitioning the effect of the proportion of burned area per forest type, we documented a differential response of mammals, from the species to the assemblage level, with an overall decrease in species richness and relative abundance as burned area proportion increased in monospecific forests.Therefore, our hypothesis that monospecific forests are more vulnerable to wildfires than polyspecific ones was confirmed, with a negative consequence for mammalian richness and relative abundance.The complementarity of camera trapping and eDNA sampling, evidenced by the reduced number of species shared between methods (22%), highlights their reliability to be combined when aiming at richness estimation 31 .Moreover, these methods used together provided a more accurate assessment of the mammalian diversity after fire passage, allowing for the detection of species that otherwise would be missed if only one method was employed.Nineteen mammal species were added to the list of TES and its surrounding areas, increasing the overall richness to 37 species.Therefore, although 38.1% of TES and 70% of its surroundings were severely burned (in a 10-km buffer 16 ), mammalian diversity increased compared to a pre-fire condition 28,29 .
These results contradict our first hypothesis, which predicted lower species richness after fire passage.Megafires can substantially alter assemblage composition via habitat modification 5 , for example, favoring the growth of herbaceous and grassy plants, which might attract herbivores 32 , such as those we detected only after fire passage (i.e., O. bezoarticus, M. cf.rufa, S. gouazoubira, and T. terrestris).For instance, in African savannas and grasslands, and other fire-prone ecosystems like Cerrado, the abundance of large herbivores was positively impacted by fires, www.nature.com/scientificreports/although responses vary widely among taxa depending on species life-history traits, habitat requirements, and refuges provided by unburned habitats (e.g., 7,8,33 ).However, other species occurring in areas surrounding TES, namely Aotus azarae, Cuniculus paca, Tamandua tetradactyla, Euphractus sexcinctus, and Tolypeutes matacus (recorded by interviews 28 ), were not detected in our study.Species with low mobility and habitat specializations are more vulnerable to megafires 8 , such as arboreal/ scansorial (i.e., A. azarae, T. tetradactyla) and semi-fossorial (i.e., E. sexcinctus and T. matacus) animals, which might explain their non-detection.Similarly, group-living animals, such as peccaries (Tayassu pecari and Dicotyles tajacu), also tend to present higher mortality in response to megafires 8 , as observed for white-lipped peccaries during data collection in other parts of Pantanal ( 13 , C. Berlinck, pers.observ.).Some species had their DNA detected only in running water samples, namely Akodon sp., C. thous, C. brachyurus, M. tridactyla, H. hydrochaeris, P. concolor, and T. pecari, with the first five classified as open-area tolerant.DNA concentrations in running water can be subject to downstream transport up to 45 km from the sampling point, considering a river flow equal to 300 m 3 /s and DNA persistence of ~ 12 h at 30 °C34 .Therefore, samples collected in running water may contain DNA from species occurring upstream of the study area, in our case, C. brachyurus, not expected to occur in this region 35 .Nonetheless, L. vetulus, a species also not expect for the region 36 , was recorded in still water.In this sense, as mentioned above, some species might be attracted to altered environments created by wildfires, particularly open-area tolerant and diet generalist ones, which might explain the presence of the aforementioned species.
It is noteworthy to mention that wildfire impacts on small mammals are yet virtually unknown.Tomas et al. 13 estimated that small rodents and marsupials summed up about 20% of the dead animals in Pantanal, which might be underestimated.Nevertheless, studies in the Cerrado showed that some generalist small mammals may experience an increase in abundance after wildfires or are poorly affected, while habitat specialists may take months or years to recover or reappear (e.g., [37][38][39] ).In this study, we detected both habitat generalist (Akodon sp., Oligoryzomys sp.) and specialist (Gracilinanus sp., Marmosa sp., Marmosops sp., Holochilus sp., Oecomys sp.) small mammal about 1 year after the fires.Nonetheless, the lack of previously published studies on small mammals in the study area prevents further comparisons.
It is difficult to uncover the aspects regarding the presence of small mammals after the megafire, particularly considering their detection by eDNA from water samples, which hinders record association to forest type or burned/unburned sites.Nevertheless, some species present mechanisms of response to wildfires 40 , allowing individuals to survive, which may play an important role in the recovery of small mammal populations locally 41 .www.nature.com/scientificreports/For example, Semedo et al. 42 reported individuals of marsh rat (Holochilus chacarius) found alive inside partially flooded underground burrows just after the megafires in Pantanal.Therefore, further studies focused on home range estimation, use of space, and genetics may contribute to a better understanding of the effects of wildfires on small mammals.Species richness was higher in polyspecific forests for both observed and estimated richness, highlighting their resistance, to some extent, to wildfires 26,27 , even during catastrophic events, something observed in other heterogeneous fire-prone ecosystems (e.g., 43 ).Despite differences in assemblage richness and composition, the aggregated relative abundance was similar between forest types, indicating a distinct landscape use by the species.Only assemblage composition differed between burned and unburned sites, confirming one aspect of our second hypothesis, which predicted changes in species richness and relative abundance, and assemblage composition after fire passage.This response was observed for forest-dependent and open-area tolerant species, strengthening that mammals may respond differently to fire conditions based on their life history traits (e.g., 20,33 ).
The strong effects of burned area proportion on monospecific forests led to pronounced reductions in mammalian richness and aggregate relative abundance at the site level and promoted significant decreases in relative abundance at the species and assemblage levels as the proportion of burned areas increased.Eighty-six percent of the species presented moderate to strong negative effects on their relative abundance, including forest-dependent (e.g., T. terrestris, P. concolor) and open-area tolerant mammals (e.g., S. gouazoubira, O. bezoarticus), although five out of seven forest-dependent species were strongly affected (e.g., D. azarae, E. barbara, L. pardalis, M. cf.rufa, P. onca).The negative effect at the assemblage level stresses the vulnerability of monospecific forests to megafires, which might result in long-term impacts on mammalian abundance besides direct mortality, that is, related to a prolonged reduction in food resources diversity and availability, and shelter opportunities, following our third hypothesis that predicted more severe impacts at this forest type.
This result might be a consequence of the habitat simplification caused by megafires of great severity 44 , which tend to reduce mammalian abundance varying with the recovery time of the vegetation 7 .Although Erythrina fusca monospecific forests seem to be favored by local environmental factors, such as low soil fertility and higher flood levels 45 , the complete mischaracterization of the environment by the megafire (Supplementary Fig. 6), slowed and even prevented their recovery.The aerial structures that allow the colonization of flooded areas by monospecific forests, also make them more susceptible to wildfire (C.Berlinck, pers.observ.).In this forest type, the 2020 megafire penetrated more than 1 m below ground, sterilizing the dry histosol, burning up the organic matter, the soil seed bank, and the underground structures of plants such as roots 46 , resulting in drastic environmental changes and, consequently, long-term negative effects for mammals and other taxa.
Seven species had their relative abundance strongly affected by increasing burned area proportion in monospecific forests, of which, two are threatened at the national level 47 , the Pampas deer (O.bezoarticus) and the jaguar (P.onca).Populations of these large species are generally small and isolated throughout their distribution, with the Pantanal being a stronghold for both species in Brazil.The Pantanal 2020 megafire highlighted the negative impacts on less mobile and habitat specialist taxa, which are expected to be more affected, and highmobile species like the jaguar 13 .A current assessment of this megafire impacts on jaguar populations indicates a disturbing scenario for the long-term conservation of the species considering the proportion of individuals and habitats potentially affected 48 .
Drastic declines in biodiversity are more associated with large fires, fire-prone vegetation not burned for long periods, and large distances between burnt and large unburnt vegetation patches 20 ; the two former aspects are observed in TES and its surrounding areas.More than one-third of the study area was affected by the 2020 megafire 16 , leaving some unburnt forest patches that possibly acted as refugia for the high-mobile mammal species in the reserve and its surroundings.Large unburnt areas and forest canopy preservation are crucial to support vertebrate survival after megafires 20,21,43 , especially for diet and habitat specialist taxa, such as those recorded in this study.
Here, we presented evidence that forest type modulates fire severity, inducing changes in mammalian richness, composition, and relative abundance in response to a catastrophic megafire.We acknowledge the limitation of not having a measure of the species' relative abundance for the period before the fire for a more meticulous comparison.However, we stress the perceived impact of this single incident at several levels of organization, from species to the assemblage, highlighting its severe and widespread negative consequences.Wildfire activity and its impacts on biodiversity are being transformed by anthropogenic drivers, such as climate and land use changes 30 , tending to increase conditions that favor wildfires in the Pantanal in the forthcoming years 49 .Cumulative effects, frequency, and severity of wildfires are responsible for reducing the diversity and abundance of organisms 4,5,7,43.If such events, analogous to the Pantanal 2020 megafire, become recurrent, they might trigger local and regional extinctions in a short time.
Management strategies such as prescribed fire become crucial to prevent and reduce the negative impacts of future wildfire events.This technique, among other management strategies, proves beneficial to fire-prone ecosystems, fostering biodiversity, nutrient cycling, and reducing the risk of catastrophic events 50 .Advancements in fire science, allied with remote sensing and climate data available in large temporal and spatial scales, and high computational power coupled with artificial intelligence, produced systems able to detect and prevent fire events, such as in the Brazilian Cerrado 51 .For the Pantanal, specifically, there is the ALARMES system (https:// alarm es.lasa.ufrj.br/), a platform that monitors fires and their spreading by combining satellite imagery, hotspots, and artificial intelligence to support environmental agencies in actions to combat wildfires.Importantly, these practices also help mitigate negative repercussions on wildlife species by creating a mosaic of habitats, offering refuges, and preventing extensive habitat alteration.These approaches acknowledge the intricate balance between natural fire regimes and human efforts to safeguard ecosystems and their biodiversity.

Study area
The study was conducted in a protected area -Taiamã Ecological Station (TES) -and its surrounding areas in northern Pantanal, Cáceres, Mato Grosso, Brazil (Fig. 6).TES is an island delimited by the Paraguay River and its branch (locally named Bracinho River), comprising an area of 115.55 km 2 .Southeast of TES lies Sararé Island (31.25 km 2 ), which is part of a proposal for creating a new protected area.TES and its surrounding areas are mainly composed of floodplains, containing a great variety of aquatic environments, such as permanent, temporary, and meander lagoons, and 'corixos' (i.e., natural connections between rivers and lagoons) (Supplementary Fig. 7).According to the Köppen classification, the climate is characterized as AW, consisting of two seasons (wet and dry) with average annual temperature ranging between 20 and 32 °C, and average precipitation of 1500 mm year-round.
The study area macro-habitats include aquatic macrophyte fields (48% of the island), flooded fields (24%), monospecific (16%) and polyspecific forests (8%), and lakes (4%) 52 .Polyspecific forests are formed by shrubs and pioneer forests along riverbanks (Supplementary Fig. 8).The monospecific forest, locally known as 'abobral' , is composed of individuals of Erythrina fusca Lour (Fabaceae), which is a dominant pioneer tree and occurs sparsely in riparian forests, mainly in Pantanal and Amazon (Supplementary Fig. 6).This forest type is associated with floating histosol 45 , which is rich in organic matter and can colonize areas that are seasonally flooded longer than polyspecific ones 53 .

Droughts and wildfire events
According to Marengo et al. 11 , the drought events observed in Pantanal between 1962-1965 and 1967-1972 registered high severity, similar to 2018-2020.Regarding the 1962-1972 period, drought intensity was higher in November 1962, while for 2018-2020, drought was higher in April 2020, the driest month since 1900.The main cause of the lack of rainfall during the consecutive summers of 2019 and 2020, was a change in the South American monsoon system, which reduced the seasonal transport of water vapor from the Amazon basin into the Pantanal during the austral summer.This reduced accumulated rainfall caused severe drought conditions, creating favorable conditions for wildfire spread in the central-western/northern Pantanal 56 .A large forest fire impacted TES and its surrounding areas in 2011 55 , and although other wildfire events occurred in the region  52 , areas burnt in 2020 54 , and other landuses 55 .Generated with QGIS 3.34.2(https:// qgis.org/).

Field data acquisition
We classified mammals in three body size categories [small (< 1 kg), medium (from 1 to 7 kg), and large (> 7 kg) 57,58 ], as forest-dependent or open-area tolerant, and according to their diet and locomotor type 25,59 .We followed the list of the Brazilian Society of Mammalogy 23 as the taxonomic authority, and assigned threat categories at national 47 and international levels 60 .We used specialized literature to identify mammal species 57,[61][62][63] .Data collection was authorized by SISBIO #79,107-1 and SisGen #ABF0E81 permits.

Camera trapping
From August to November 2021, a total of 55 sampling stations were deployed in TES and its surrounding areas, with a mean distance of 1 km from each other, following the TEAM protocol 64 (Fig. 6).A single unbaited camera trap (Bushnell, models 119949C and 119932C; Browning models Patriot and SPEC OPS ELITE HP4) was installed per station at ~ 40 cm above the ground, programmed to take three photos at 0.6 s intervals between bursts, and operating 24 h/day.We considered sampling stations independent if they were at least 500 m from each other; those within this range had their records combined, which resulted in 50 independent stations.Cameras were active from 92 to 99 days, totaling a sampling effort of 4639 trap-days.Sampling stations were distributed following two treatments: forest type, divided into monospecific (N = 19) and polyspecific (N = 31), and in burned (N = 20) and unburned sites (N = 30) (Supplementary Figs. 2 and 3).Forest type was determined according to Frota et al. 52 .Burned sites were determined by the area burned in a 30 m buffer from the sampling station location during fieldwork.We used the web platform Wildlife Insights (https:// www.wildl ifein sights.org/) to store, organize, and identify all focal species records.

Environmental DNA (eDNA)
We used water as an environmental sample to survey mammals by metabarcoding sequencing.Water collection was conducted in lentic and lotic water bodies near the camera trapping stations, with other points sampled when those were not available.We classified water samples as still water when collected in ponds or puddles (N = 7), and running water when collected in rivers or streams (N = 21), totaling 28 collection points (Fig. 6).The eDNA was obtained by water sample filtration.The collected water was poured into a syringe, and the plunger was placed and pushed manually at a flow rate of 1 mL for 10 s for filtration 65 .A volume of 45 mL was passed through a polyethersulfone membrane filter (0.22-µm pore size, 30 mm diameter, Kasvi) using a sterilized disposable syringe of 20 mL.All sampling equipment was handled with clean latex gloves, which were changed among sampling sites, and the equipment was cleaned with a 10% bleach solution after each collection.The filters were transported refrigerated at ~ 0 °C and the membranes were removed from the filters and stored in one milliliter of Longmire buffer 66 at − 20 °C.The membranes were vortexed for 30 s and 400 μL of the buffer was removed for extraction.DNA extraction was performed in a room dedicated to processing low-quantity DNA samples using the DNeasy PowerWater Kit (Qiagen).We amplified two mini-barcode regions from ribosomal mitochondrial genes (12S and 16S rRNA) using primers previously described for targeting vertebrates (12SV5F and 12SV5R 67 ) and mammals (16Smam1 and 16Smam2 68 ), respectively.We used a dual index strategy, where the product of PCR1 was cleaned using magnetic beads (Agencourt AMPure XP® -Beckman Coulter), quantified using a Qubit fluorometer (Thermo Fisher, Waltham, Massachusetts, USA), normalized to a concentration of 20 ng/µL and indexed using a Nextera Index kit® (Illumina, San Diego, California, USA).The paired-end sequencing was performed on the Illumina iSeq® platform, using an iSeq v2 300 Cycle Reagent kit (2 × 150 bp).
The bioinformatics pipeline was organized in R 4.3.1 69 .In brief, reads were initially submitted to remove undetermined bases, and quality filtering (Q-scores ≥ 30).Only reads containing the expected index sequence corresponding to each sample were kept for subsequent analysis.Error correction, read-pair merging, and chimera identification and removal were performed using the default settings of DADA2 functions 70 .No length truncation was performed since the primer removal step automatically clips uninformative regions and resulting ASVs (Amplicon Sequence Variants) out of the expected amplicon length range for each marker (135-139 bp for 12SrRNA, and 130-134 bp for 16SrRNA) were discarded.Subsequently, identified ASVs were clustered into OTUs (Operational Taxonomic Units) using SWARM v3.1.0 71, applying the fastidious option and d = 1.Taxonomic assignments were conducted using alignment of the NCBI nucleotide collection using an automated BLAST + 2.10.1 function with minimum similarity and minimum coverage (-perc_identity 90 and -qcov_hsp_ perc 90).The OTUs were also compared with sequences available in GenBank for species identification using the BLAST tool (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi).
The final dataset included only OTUs with > 90% similarity against the GenBank database and containing > 5 reads (0.5% relative abundance).All taxonomic assignments were manually curated.When a sequence had a match for two or more species with equal similarity, we selected those with expected occurrence in the studied area.When a high percentage of matches was obtained (≥ 98%), but the species is not expected to occur in the Pantanal biome, we assigned the genus.This situation generally happens when the mini-barcode sequence from the species is unavailable in GenBank and matched with other species of the same genus (e.g., small mammals).We also assumed the genus assignment for sequence matches between 90 and 97.99%.

Landscape variables
We calculated the main land uses in TES and its surrounding areas using the land use and land cover map of MapBiomas, collection 7, for year 2021 55 , and the package landscapemetrics 72 .Land uses were classified into and Wilcoxon rank-sum test were employed depending on data normality, which was assessed using the Shapiro-Wilk test.Next, using linear regressions, we assessed the relationship between species richness and aggregate relative abundance estimated by the MSOM for each sampling station, with the proportion of burned area per forest type.Finally, we assessed the magnitude and direction effects of the explanatory variables used in the MSOM on the relative abundance and detection of each species, as well as for the entire assemblage.

Figure 1 .
Figure 1.Venn diagrams comparing mammal assemblage composition in Taiamã Ecological Station (TES) and its surrounding areas in northern Pantanal, Cáceres, Mato Grosso, Brazil.(A) Comparison between camera trapping and environmental DNA (eDNA) surveys.(B) Comparison of medium and large-sized mammal assemblages before 28,29 and after fire passage (this study).

Figure 2 .
Figure 2. (A) Composition of the mammal assemblage between forest types in Taiamã Ecological Station (TES) and its surrounding areas in northern Pantanal, Cáceres, Mato Grosso, Brazil.Variation in the site level mean species richness (B) and aggregated relative abundance (C) of mammals between forest types estimated by the Bayesian multi-species occupancy model.Values in bold indicate statistical support.

Figure 3 .
Figure 3. (A) Composition of the mammal assemblage between burned and unburned sites in Taiamã Ecological Station (TES) and its surrounding areas in northern Pantanal, Cáceres, Mato Grosso, Brazil.Variation at the site level mean species richness (B) and aggregated relative abundance (C) of mammals between burned and unburned sites estimated by the Bayesian multi-species occupancy model.

Figure 4 .
Figure 4. Linear relationships of species richness and aggregated relative abundance per sampling station according to forest type [monospecific (A, C); polyspecific (B, D)] estimated by the Bayesian multi-species occupancy model, with the proportion of burned area in Taiamã Ecological Station (TES) and its surrounding areas in northern Pantanal, Cáceres, Mato Grosso, Brazil.Results in bold indicate statistical support.

Figure 5 .
Figure 5. Results of the Bayesian multi-species occupancy model, showing the magnitude and direction (Bayesian means ± credible intervals) for the posterior distributions of the interaction between the proportion of burned area and forest type on the relative abundance of mammals at the species (A) and assemblage levels (B) in Taiamã Ecological Station (TES) and its surrounding areas in northern Pantanal, Cáceres, Mato Grosso, Brazil.For effects including forest type, negative values = monospecific, positive = polyspecific.For effects including fire on site, negative = burned, positive = unburned.Black lines indicate statistical support (thin = 95%; thick = 90%).