Native tree species in silvopastoral system outperforms Eucalyptus in enhancing soil healthABSTRACT
Soil health is critical for sustainable development in the Amazon, where microbial communities play a pivotal role. This study assessed soil health in integrated livestock-forestry systems (ILFSs) with Samanea tubulosa and Eucalyptus pellita compared with native forest (NF) in Porto Velho, Rondônia, Brazil. Soil chemical, physical, and biological properties were evaluated, including bacterial and archaeal community composition through 16S rRNA gene sequencing. Soil chemical and microbiological properties in the S. tubulosa ILFS (ILFS-ST) were significantly enhanced compared with E. pellita ILFS (ILFS-EP). In the soil of ILFS-ST, there was higher microbial diversity and enriched functional groups, particularly those involved in nitrogen cycling, such as ammonia-oxidizing archaea (AOA), aromatic compound degradation and photoautotrophy. The only negative aspects observed in the ILFSs compared with the NF were increased soil penetration resistance and reduced K availability. Integrated livestock–forestry systems enhance soil health, particularly when S. tubulosa is used. Therefore, the use of these systems is recommended to promote sustainable cattle ranching in the Western Amazon. Also, new perspectives for investigating the interaction between native tree species and soil microorganisms are emerging, underscoring the importance of considering species diversity in selecting silvopastoral systems and highlighting the need for further research on the topic. The results also show that NF fragments serve as crucial reservoirs of native soil microbiota, which is essential for maintaining a healthy, resilient ecosystem.
Keywords
Bordão-de-Velho; differential abundance analysis; FAPROTAX; soil microbiome; sustainable livestock
INTRODUCTION
Soil health is a cornerstone of sustainable agricultural and forestry systems, playing a critical role in ecosystem function and productivity (Lehmann et al., 2020). In the Amazon, where soils are inherently nutrient-poor and prone to degradation, maintaining healthy soil is paramount for long-term sustainability (He et al., 2021; Nunes et al., 2022; Sanquetta et al., 2022; Wright, 2022). To address this challenge, integrated livestock-forestry systems (ILFSs), or silvopastoralism, have emerged as a promising management strategy.
By integrating livestock production with tree components, these systems offer a multifaceted approach to enhancing soil health through several mechanisms, notably improving land cover, which protects against erosion and compaction (Feitosa et al., 2019; Zolin et al., 2021). Additionally, these integrated systems are essential for enhancing nutrient cycling, as trees contribute to soil organic matter through leaf, branch, and fruit litter as well as root turnover (Sileshi et al., 2020; Solanki et al., 2020; Zago et al., 2020). They are also key to promoting biodiversity, as these systems support a wider range of plants, animals, and microorganisms that are essential for soil function (Bartz et al., 2014; Selari et al., 2021; Santos et al., 2024).
Research on livestock-forest integration has grown significantly in Brazil over the last few decades (Lopes et al., 2021; Silva et al., 2021), along with the area adopting such systems (Rede ILPF, 2021). However, relatively few studies and sites focus on native tree species, despite the large number of potential native tree species in the Amazon and other Brazilian biomes (Andrade et al., 2012; Martínez et al., 2019; Melotto et al., 2019).
One of the most promising native species for ILFSs in the Amazon is the nitrogen-fixing tree Samanea tubulosa (Carvalho, 2006). Besides its ability to improve forage quality, this species possesses other favorable traits for ILFSs, such as high dispersion, local acceptance, edible fruits, and easy establishment (Andrade et al., 2012; Oliveira and Luz, 2012; Olival et al., 2021, 2022). Research has even shown that sheep fed with S. tubulosa fruits emit less methane (Pérez-Márquez et al., 2025), which could further improve the sustainability of the system.
However, a complete understanding of integrated systems requires comparison with widely adopted exotic alternatives. Eucalyptus pellita, like other eucalyptus species, is a fast-growing tree widely planted in Brazil for wood production, both in monoculture and in integrated systems (Behling et al., 2021; Oliveira et al., 2022a; Hutapea et al., 2023; IBÁ, 2023).
While the roles of different tree species are gaining attention, the influence of these systems on soil biota is increasingly being studied using modern molecular techniques. The first studies on soil microbiota in integrated systems in the Amazon using next-generation sequencing (NGS) have been published recently (Carmo et al., 2024; Santos et al., 2024). These studies highlight that the richness and diversity of soil bacterial communities are influenced by different cultivation systems, with integrated systems helping to maintain the biodiversity and stability of the soil microbial communities.
However, there are still significant gaps in the literature. No studies using NGS in integrated systems with native Amazonian tree species have been found. Also, more studies are needed to elucidate the regional and temporal dynamics of soil microbiota in Amazonian livestock production systems.
We hypothesized that: (i) ILFSs (both S. tubulosa and E. pellita) would maintain soil health indicators at levels statistically closer to the NF reference area than typically observed in converted lands; (ii) ILFS with the native species S. tubulosa would promote soil health, particularly in microbial community diversity and functionality, compared with the ILFS with exotic species E. pellita; and (iii) the proximity to the tree component (0.00 m) would result in superior soil chemical, physical and biological properties compared with the intermediate zone (5.25 m), reflecting the concentrated input and localized influence of the tree canopy and root system. This study aimed to assess soil health, considering chemical, physical, and biological (microbial) indicators, in ILFSs utilizing the native species Samanea tubulosa and the exotic species Eucalyptus pellita.
MATERIALS AND METHODS
Site description
Samples were taken from two four-year-old integrated livestock-forestry systems (ILFSs) experiments and a native forest (NF) fragment (Table S1) located within the Empresa Brasileira de Pesquisa Agropecuária (Embrapa) experimental field in Porto Velho, Rondônia State, Brazil (8° 48’ S, 63° 51’ W; 95 m altitude) (Figure 1). According to the Köppen climate classification system, the climate is Am, or tropical monsoon (Alvares et al., 2013). The area has an average annual rainfall of 2,216 mm, with average annual maximum and minimum temperatures of 30.4 and 23.3 °C, respectively (Climate-Data.org, 2022) (Figure S1). The soil is classified as a Latossolo Vermelho-Amarelo distrófico (LVAd), according to the Brazilian Soil Classification System (Valente et al., 1998; Santos et al., 2025), which corresponds to a Ferralsol (IUSS Working Group WRB, 2022), or an Hapludox (Soil Survey Staff, 2022). The soil has a clay texture (Table S2).
Study area location and photographs. ILFS-EP: integrated livestock-forestry system with Eucalyptus pellita; ILFS-ST: integrated livestock-forestry system with Samanea tubulosa; NF: native forest. In ILFS-EP and ILFS-ST, soil samples were collected at 0.00 m (planting line) and 5.25 m from the trees (arrows are illustrative and do not indicate the exact sampling points). At all locations, samples were taken from the 0.00–0.10, 0.10–0.20, and 0.20–0.40 m soil layers. Satellite image source: Google Earth V 7.3.6.10441 (64-bit) (2025-09-03). Embrapa Rondônia, Porto Velho. 8° 48’ S, 63° 51’ W, eye altitude 1.46 km. Imagery date: 2025-05-19. Maxar Technologies, 2025. http://www.earth.google.com [visualized on 2025-11-28]. Photographs were taken on 2022-11-22.
Soil sampling
Soil samples were collected using a cylindrical auger in July 2022 (dry season) from the layers of 0.00–0.10, 0.10–0.20, and 0.20–0.40 m. In the ILFSs, samples were taken at two specific distances from the tree lines: 0.00 m (the planting line) and 5.25 m (Figure 1). The 5.25 m distance was chosen for standardization, as it corresponds to one-eighth of the 42 m inter-row spacing used in other studies conducted within the same experimental field (Feitosa et al., 2019, 2022). Furthermore, this distance is representative of an intermediate zone among those reported in studies assessing the influence of trees on pasture and soil properties in integrated crop-livestock-forestry systems.
Spatial distribution of sampling points was planned to ensure representativeness and minimize edge effects within the experimental areas. In NF, the five sampling points were established along a transect parallel to the boundary road, with each point located more than 30 m from the edge/road to avoid external influences. Successive samples were collected approximately every 30 m along this transect. In the ILFSs, the five replicate sampling points for each distance (0.00 and 5.25 m) were positioned along the tree rows. A 30 m buffer zone was maintained from both the start and the end of the rows. Samples were collected at approximately 30 m intervals along the row, alternating between the NE and SW sides of the planting line.
A total of 75 samples were collected. Specifically, the NF area contributed with 15 samples (1 system × 3 layers × 5 replicates), while the two ILFSs areas contributed with 30 samples each, factoring in the two distance levels (2 ILFSs × 2 distances × 3 layers × 5 replicates). Subsamples for DNA extraction were stored at -20 °C, while soil samples for chemical and granulometric analysis were air-dried, ground, sieved (2 mm), and stored at room temperature.
Chemical and physical properties
Soil chemical and gravimetric moisture analyses were conducted following the methodology described in Teixeira et al. (2017). Soil pH was measured in a 1:2.5 soil:water suspension. Exchangeable aluminum (Al), calcium (Ca), and magnesium (Mg) were extracted with 1 mol L-1 KCl. Exchangeable Ca and Mg were determined by atomic absorption spectrometry, and Al by acid-base titration. Soil phosphorus and potassium were extracted using the Mehlich-1 method, with available P determined by visible spectrophotometry and K by flame atomic emission spectrometry. Potential acidity (H+Al) was determined using the 0.5 mol L-1 calcium acetate method at pH 7.0. Exchangeable bases (EB) are the sum of Ca2+, Mg2+, and K+; cation exchange capacity (CEC) is the sum of EB and H+Al; base saturation (BS) is the ratio of EB to CEC; and aluminum saturation (m%) is the ratio of exchangeable Al to CEC. Soil organic matter (OM) was measured using the Walkley–Black method, as modified by Jackson (1982). Gravimetric moisture (θw) was determined as the mass of water per mass of soil oven-dried at 105 °C for 24 h. These properties were analyzed at the soil and plant laboratory at Embrapa Rondônia in Porto Velho, Brazil.
Soil resistance to penetration (RP) was measured in the field with an impact penetrometer, Model IAA-Planalsucar-Stolf (Stolf et al., 1983). The penetrometer data were converted to MPa for the 0.00–0.10, 0.10–0.20, and 0.20–0.40 m layers using equations in Excel spreadsheets (Stolf et al., 2014). Five measurements were taken per area, close to the soil sampling points.
Soil DNA extraction and sequencing
The total DNA from the soil samples was extracted using the DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. First, the soil sample was homogenized, and the microbial cells were lysed using mechanical and chemical methods. The total genomic DNA was then captured on a silica membrane in a spin column format, washed, and eluted to obtain the extract. Content and quality of DNA were measured using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA).
The V3-V4 region of the 16S rRNA gene was amplified to assess the abundance of prokaryotes (Archaea and Bacteria) using updated primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′). Paired-end sequencing with 2 × 250 bp reads was performed on an Illumina NovaSeq 6000 platform. Both amplification and sequencing were performed by Novogene Bioinformatics Technology, adhering to standard approaches defined by the Earth Microbiome Project (Gilbert et al., 2014). The raw reads were deposited in the Sequence Read Archive (SRA) under Bioproject PRJNA1215384.
Raw reads were analyzed using the DADA2 pipeline (Callahan et al., 2016), with acceptable sequences having a quality score greater than 20, a maximum of 2 expected errors per read, and a minimum length of 50. Filtered reads were grouped into amplicon sequence variants (ASVs) and matched to taxonomy using the SILVA database v. 138.2 (Quast et al., 2013). The resulting ASV table was imported into a phyloseq object for downstream analysis with the microeco package for R (Liu et al., 2021). The ASVs not assigned to the Bacteria or Archaea kingdoms, or classified as “chloroplast” or “mitochondria,” were removed. Count data were normalized using the scaling with ranked subsampling (SRS) method (Beule and Karlovsky, 2020), with a minimum of 76,376 reads across samples (Figure S2).
Data analysis
Soil properties and alpha diversity
Statistical analyses were performed using the following experimental design: two ILFSs (ILF-EP and ILF-ST) by two distances from the trees (0 and 5.25 m), plus one additional treatment (NF), with five replicates. Each soil layer (0.00-0.10, 0.10-0.20, and 0.20-0.40 m) was analyzed separately. Richness and Shannon Index were calculated from the taxonomic relative abundance matrix at the ASV level. After verifying homoscedasticity and normality, removing outliers, and applying the Box–Cox transformation when necessary, the means of alpha diversity (number of distinct ASVs and Shannon index) as well as soil physical and chemical properties were compared among areas using ANOVA or Dunnett’s test (for comparisons with NF), with the aid of Minitab 22 software and the Tratamentos.ad package in R (Azevedo, 2022).
Differential abundance analysis (DAA)
Principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) were used to test the effects of distance from trees and areas on the taxonomic structure of microbial communities, based on a Bray-Curtis dissimilarity matrix at the ASV level. The differential expression analysis using the Negative Binomial distribution (DESeq2) method (Love et al., 2014) was used to identify ASVs that were distinctly abundant across areas.
To identify functional groups, taxonomic groups were compared with the dataset from the automated functional classifier FAPROTAX v. 1.2.6 (Louca et al., 2016). The output functional table was generated with the "abundance_weighted" and "dec" arguments set to "TRUE" and "10", respectively. Therefore, the functional percentage was calculated considering the abundance of each taxon and 10 remaining decimal places.
The linear discriminant analysis (LDA) effect size (LEfSe) test (Segata et al., 2011) was used to identify differential functional groups specific to each area. The parameters for LEfSe analysis included a minimum effect size of 2.0 and 1,000 permutations. A 5 % significance level was adopted for all analyses, with FDR correction for multiple comparisons using the Benjamini-Hochberg procedure when necessary.
RESULTS
Soil chemical and physical properties
Except for gravimetric moisture (θw), the soil chemical and physical properties showed significant differences among areas, at least for one layer. The integrated livestock-forestry systems (ILFSs) had better soil chemical properties than the native forest (NF), except for available K, which was higher in the NF; and available P, which was similar between the NF and ILFSs (Tables 1 and 2).
Soil pH, exchangeable Al3+, potential acidity (H+Al), aluminum saturation, and base saturation under integrated livestock–forestry systems and native forest across three soil layers
Mean (± standard error) of available P and K, and exchangeable Ca, and Mg in three soil layers
The largest differences among the ILFSs were observed for pH, Ca2+, Al3+, and m%, with better values for ILFS-ST. Available K and BS showed no significant differences among ILFSs or across soil layers. The effect of distance from the trees was more pronounced for pH and available P (at 0.00-0.10 m), and H+Al (at 0.00-0.10 and 0.20-0.40 m), with higher acidity and P closer to the eucalyptus trees (0.00 m).
Soil organic matter (OM) differed significantly between ILFS-EP at 0.00 and 5.25 m in the 0.10-0.20 m layer, between NF and ILFS-EP at 5.25 m, and between ILFS-EP at 5.25 m and ILFS-ST at 5.25 m, with lower values in the eucalyptus system (Table 3).
Soil resistance to penetration (RP) was significantly lower in the NF than in the ILFSs at all depths. No difference in RP was observed among the integrated systems at 0.00-0.10 m. However, at a depth of 0.10-0.20 m, RP was higher in the ILFS-ST than in ILFS-EP and closer to the trees. Additionally, at 0.20-0.40 m, RP was higher in ILFS-ST at 5.25 m than in ILFS-EP at 5.25 m (Table 3).
Mean (± standard error) of organic matter (OM), gravimetric moisture (θw), and resistance to penetration (RP) in three soil layers
Microbial community diversity and structure
The ASV richness and Shannon index values were higher in ILFS-ST, followed by ILFS-EP and NF, regardless of depth (Table 4). Significant differences for both richness and the Shannon index were observed between NF and ILFS-ST in the 0.00-0.10 and 0.10-0.20 m layers. Furthermore, a significant difference was found between ILFS-ST and ILFS-EP in the 0.10-0.20 m layer for the Shannon index and in the 0.20-0.40 m layer for richness.
Richness (number of ASVs) and Shannon Index calculated at the ASV level for Archaea and Bacteria in soil (mean ± standard error)
Principal Coordinate Analysis (PCoA) and Permutational Analysis of Variance (PERMANOVA) revealed significant differences in soil microbial community composition between the native forest (NF) and both integrated livestock-forestry systems (ILFS-ST and ILFS-EP) across all soil layers (Figure 2).
With respect to distance from the trees (0.00 and 5.25 m), no significant differences were observed for ILFS-ST at any layer or for ILFS-EP in the 0.00–0.10 m layer. At 5.25 m from the trees, the ILFS-ST microbial community differed significantly from ILFS-EP in all layers. However, at 0.00 m, the difference between the two ILFSs was significant only in the first and second layers.
Beta diversity of soil microbial communities among areas and soil layers. Principal Coordinate Analysis (PCoA) plots are based on Bray-Curtis dissimilarity at the ASV level. The proximity of points indicates the similarity between samples, while greater distance indicates dissimilarity. Ellipses encompass all samples from the same area. The accompanying table presents significant pairwise comparisons for the corresponding soil layers based on PERMANOVA (p<0.05; FDR-adjusted). ILFS-ST: integrated livestock-forestry system with Samanea tubulosa; ILFS-EP: integrated livestock-forestry system with Eucalyptus pellita; 0.00 and 5.25 m: distances from the trees in the ILFSs; NF: native forest.
Microbial taxonomic distinction and functional potential
The overall most abundant genera were Conexibacter, Acidothermus, Acidiferrimicrobium, Bradyrhizobium, and Candidatus Solibacter. These genera accounted for over 40 % of total prokaryotic abundance, excluding unclassified ASVs (Figure S3). The mean relative abundance of Archaea was lower than that of Bacteria, at 0.34 %. Crenarchaeota (now Thermoproteota) were predominant in the 0.00-0.10 and 0.10-0.20 m layers, while Thermoplasmatota (now Methanobacteriota) dominated the 0.20-0.40 m layer. Notably, the ILFS-ST 0.00 m plot showed the highest mean relative abundance of Archaea (Figure S4).
Using the DESeq2 method, 18 to 420 significantly differentially abundant ASVs were identified among the areas (Figure S5). The highest number of distinct ASVs was observed in comparisons between the native forest (NF) and the ILFSs, especially ILFS-ST, within the 0.00-0.10 m layer. In contrast, comparisons of distances from the trees (0.00 and 5.25 m) yielded the fewest distinct features. The number of differential features also tended to decrease with increasing depth. ASV_652 (phylum WPS-2, now Vulcanimicrobiota) was the most significantly different in 6 out of 30 possible pairwise comparisons, all within the 0.00-0.10 m soil layer. It was followed by ASV_217 (genus Sinomonas), ASV_74 (family Streptomycetaceae), and ASV_184 (family Xanthobacteraceae), each of which was significantly different in four pairwise comparisons.
The LEfSe analysis revealed 22, 25, and 12 distinct functions among the areas at the 0.00-0.10, 0.10-0.20, and 0.20-0.40 m layers, respectively (Figure 3). In general, chemoheterotrophy, aerobic chemoheterotrophy, dark hydrogen oxidation, cellulolysis, anaerobic chemoheterotrophy, and nitrogen fixation were the most abundant functional groups across all areas and layers, with relative abundances ranging from approximately 2 to 4 % (Figure S6). The LEfSe analysis showed these groups were predominant in the native forest (NF) across all soil layers.
In the 0.00-0.10 and 0.10-0.20 m layers, the ILFS-ST 0.00 m plot had the most highlighted functions. Conversely, NF showed more improved functional groups in the 0.20-0.40 m layer. Most of the enhanced functional groups in the ILFS-ST 0.00 m plot were related to the nitrogen cycle, though sulfur cycling, aromatic compound degradation, and photoautotrophy functions also showed improvement. Functions for aromatic and aliphatic non-methane hydrocarbon degradation were improved in the ILFS-EP 0.00 m plot (0.10-0.20 m layer) and in the ILFS-EP plot (0.20-0.40 m layer).
The LEfSe analysis of putative functional groups (FAPROTAX) of Archaea and Bacteria in soil. The plots show the most significant functional groups for each area and soil layer. ILFS-ST: integrated livestock-forestry system with Samanea tubulosa; ILFS-EP: integrated livestock-forestry system with Eucalyptus pellita; 0.00 m and 5.25 m: distance from the trees in the ILFSs; NF: native forest.
DISCUSSION
Soil chemical and physical properties
The implementation of integrated livestock-forestry systems (ILFSs) led to a significant improvement in soil chemical properties and an increase in soil hardness. The observed differences in chemical properties can be partially attributed to the legacy effect of the previous pasture management and the agricultural practices of the ILFSs (such as fertilization and liming), which rapidly modify chemical properties. Simultaneously, the higher soil hardness is attributed to the effects of machinery and animal traffic, which are absent in the NF (Álvarez et al., 2021; Lima et al., 2021). These findings are consistent with previous studies, such as that by Souza et al. (2023), who also found improved chemical properties but increased soil compaction in a conventional tillage area compared with a native forest fragment in the same experimental field.
Compared with Eucalyptus pellita (ILFS-EP), the system with Samanea tubulosa (ILFS-ST) exhibited significantly more favorable soil acidity parameters (pH, Ca2+, Mg2+, Al3+, and m%). This difference is likely attributable to the faster growth of Eucalyptus, which demands greater nutrient uptake and consequently promotes soil acidification (Berthrong et al., 2009; Rwibasira et al., 2021; Wang et al., 2023). While many studies have shown that well-managed integrated systems can improve soil health (La Scala Júnior et al., 2012; Bermeo et al., 2022; Oliveira et al., 2022b; Silva-Olaya et al., 2022; Rego et al., 2023; Anésio et al., 2024), the present results further highlight the superior performance of native tree species in this regard.
The high soil resistance to penetration (RP) values observed in this study, especially in the ILFSs, were notably greater than those reported in other studies with similar soil types and management practices (Passos et al., 2017; Feitosa et al., 2019; Lima et al., 2021; Valani et al., 2022). It should be explained due to sampling during the dry season, as RP is inversely related to soil moisture (Mome Filho et al., 2014; Duarte et al., 2022). Sampling during the dry season may also explain the negligible differences in soil moisture among the areas, although θw values remained within the range reported in other studies with similar soil textures (Rangel-Vasconcelos et al., 2015; Feitosa et al., 2019; Lima et al., 2021). Moreover, the lack of significant differences in θw across areas confirms that the observed differences in RP are primarily due to land-use and management effects, rather than variations in soil water content.
High RP negatively impacts sustainability by creating a physical barrier that restricts root growth and development for both the pasture and the trees, potentially limiting nutrient and water uptake, which is critical in drier periods. Furthermore, increased soil hardness can reduce water infiltration and gas exchange, leading to poor soil aeration and increased runoff (Shah et al., 2017). Although an increase in RP is virtually unavoidable when converting native forest areas to livestock production systems, sustainable management practices such as stocking rate control, already adopted in the area, help alleviate these issues by reducing compaction effects over time (Hamza and Anderson, 2005; Valani et al., 2022).
The higher RP observed in ILFS-ST at certain depths compared with ILFS-EP suggests that the tree species directly influences localized compaction patterns. This difference may be a consequence of differential animal traffic patterns induced by the tree species. It is plausible that livestock concentrate more closely near the S. tubulosa trees, perhaps due to superior shade or canopy characteristics, leading to greater localized trampling and compaction, whereas traffic might be more dispersed under Eucalyptus (Sartor et al., 2020; Cipriani et al., 2023). Additionally, the difference could reflect variations in root architecture and exudate effects that influence soil mechanical stability (Clemente et al., 2005; Kumi et al., 2023). Further investigation into specific animal behavior and root density is required to elucidate these localized RP differences fully.
Furthermore, superior OM content was observed specifically at the 0.10-0.20 m soil layer when comparing the ILFS-ST and ILFS-EP at the 5.25 m distance. This localized difference likely stems from the distinct quality and distribution of root inputs. Being a N-fixing legume, S. tubulosa provides N-rich fine root turnover and exudates, which promote the stabilization of organic carbon in the subsurface soil aggregates, a process that might be less effective under the eucalyptus system (Alberti et al., 2015; Ridgeway et al., 2024). This specialized accumulation in the intermediate zone (5.25 m) highlights the localized, biologically-driven benefit of the native species. In fact, PCoA results further confirmed the higher microbiota modulation potential of S. tubulosa in this zone.
The native forest showed significantly higher levels of available K. This is characteristic of mature forest ecosystems, where K is efficiently cycled from the subsoil, accumulated in the biomass, and rapidly returned to the topsoil through litter decomposition, maintaining a concentrated nutrient pool near the surface. In contrast, the ILFSs represent an open system where K is continually exported via grazing and the subsequent removal of animal products and, potentially, tree biomass (Ferreira et al., 2009; Das et al., 2019; Santos et al., 2021). Since whole-area pasture fertilization was rarely performed, this constant export interrupts the natural closed cycle, creating a nutrient deficit that is not adequately replenished. Therefore, the higher K in the NF highlights the need for targeted fertilization strategies within the ILFSs to sustain long-term fertility.
Soil microbiota and functional traits
The establishment of Integrated Livestock-Forestry Systems (ILFSs) not only affected soil chemical and physical properties but also significantly influenced the soil microbiota and its ecological traits. Tree species used in the ILFSs strongly modulate the soil microbiome. The silvopastoral systems increased soil prokaryotic diversity, particularly in the upper layers, compared with the native forest (NF). Furthermore, diversity was higher in the S. tubulosa system than in the E. pellita system. The PCoA analysis confirmed that the ILFSs clustered distinctly from the NF in all layers, but only in some layers was ILFS-ST separated from ILFS-EP. These findings, corroborated by DESeq2 analysis, indicate that the influence of trees and management on the soil microbiota diminishes with increasing depth. This is a common observation in other studies (Pereira et al., 2017; Sarto et al., 2020), which reported that with increasing depth, the influence of plant roots, moisture, and temperature is reduced, while pedogenic features become more important in modulating the microbiome (Leewis et al., 2022; Byers et al., 2023).
The increase in microbial diversity in managed systems is a well-documented phenomenon, often linked to reduced soil acidity and increased nutrient availability (Mendes et al., 2015; Merloti et al., 2019; Pedrinho et al., 2023; Tomazelli et al., 2023). Barros et al. (2018) observed an increase in the copy numbers of total bacteria, ammonium-oxidizing bacteria, and diazotrophic bacteria in silvopastoral systems planted with leguminous trees, compared with pasture monoculture. Similarly, in our study, the higher proportion of nitrogen cycling groups near the S. tubulosa trees, a nitrogen-fixing species, may be related to increased N availability or plant-recruitment of these microbes (Merloti et al., 2019; Pedrinho et al., 2020; Freitas et al., 2023; Tomazelli et al., 2023; Philippot et al., 2024). This suggests improved soil fertility, but it also raises concerns about potential N₂O emissions (Grassmann et al., 2022; Mori et al., 2023; Yin et al., 2023), which warrants further investigation.
Beyond N and sulfur cycling, S. tubulosa also increased the abundance of photoautotrophs, which can enhance autotrophic carbon fixation and improve nutrient cycling, ultimately affecting agroecosystem productivity (Liu et al., 2023). Predatory or exoparasitic bacteria can have both positive and negative effects on soil health. They are considered beneficial if they prey on pathogenic microorganisms, helping to suppress diseases in the soil. However, they can be detrimental if they target and consume bacteria that promote plant growth. The presence of these bacteria might also indicate a high abundance of the microorganisms they prey on, which can be linked to higher overall microbial diversity (Wang et al., 2020; Su et al., 2024). It is required to investigate further the specific roles and long-term persistence of these groups within integrated livestock-forestry systems to fully understand their implications for soil health.
Degradation functions for aromatic and aliphatic non-methane hydrocarbons, improved in the ILFSs, are associated with improved soil health. Because these compounds are significant environmental pollutants, a greater soil capacity to degrade them suggests a higher potential for bioremediation (Fuchs et al., 2011).
In the native forest (NF), the higher abundance of chemoheterotrophic and cellulolytic functional groups indicates enhanced microbial activity and greater capacity for cellulose breakdown. This suggests a more active cellulolytic bacterial community, reflecting the soil's ability to decompose organic matter and cycle nutrients in its characteristically acidic, nutrient-poor environment. This finding is also consistent with the higher abundance of iron-respiration groups, which are often found under such conditions (Gao et al., 2019; Liu et al., 2024).
The higher relative abundance of archaea in the ILFS-ST 0.00 m plot may be related to nitrogen fixation by S. tubulosa, as archaea play a significant role in nitrogen cycling (Wright and Lehtovirta-Morley, 2023; Coelho et al., 2025). The higher relative abundance of Thermoplasmatota (Methanobacteriota) at a layer of 0.20-0.40 m is likely due to lower oxygen availability, as these archaea are associated with methanogenesis (Bräuer et al., 2020; Liu et al., 2024).
Samanea tubulosa appears to modulate the soil microbiota over a greater distance than E. pellita, as no significant difference was found between ILFS-ST at 0.00 and 5.25 m in the PERMANOVA analysis. However, individual DAA showed significant differences. The ASV_214, identified as the ammonia-oxidizing archaea (AOA) Nitrosopumilus, had the highest logarithmic fold change (LFC) in the ILFS-ST 0.00 m plot. As a member of the Thermoproteota phylum, this archaeon plays a critical role in soil nitrogen cycling (Meyer et al., 2014; Wright and Lehtovirta-Morley, 2023), which supports the findings from the potential functional profile analysis. A similar increase in AOA abundance has been reported for Acacia mangium, an exotic legume (Pereira et al., 2019; Santana et al., 2021), though no comparable studies with native leguminous trees were found.
In the 0.00-0.10 m layer, the most distinct features in the native forest (NF), ILFS-EP 0.00 m, and ILFS-EP 5.25 m were from the family Xanthobacteraceae (phylum Pseudomonadota). This family is the most abundant overall in the studied areas and plays a significant role in soil ecosystems due to its diverse metabolic capabilities, including nitrogen fixation and chemolithoautotrophy (Soltysiak et al., 2025).
In the 0.10-0.20 m layer, ASV_74 (family Streptomycetaceae) was the least relatively abundant among the significant features in the ILFS-ST 0.00 m plot. The Streptomycetaceae family is well known for its role in organic matter decomposition, antibiotic production, and phosphorus solubilization (Kämpfer et al., 2014; Chouyia et al., 2022). While a significant difference in relative abundance at the family level was found between ILFS-ST 0.00 m and NF (Table S3), the reduction in this specific ASV's abundance may be a result of compensation by other Streptomycetaceae ASVs, which warrants further investigation.
At the 0.20-0.40 m soil layer, the most differentially abundant ASVs were from the AD3 class (Dormibacteraeota phylum) and the genus Sinomonas. The AD3 bacteria are often found in Brazilian savannah soils during the dry season and are linked to higher root density (Castro et al., 2016; Billings et al., 2018; Silva et al., 2019; Selari et al., 2021). The genus Sinomonas (Micrococcaceae family) consists mainly of soil-dwelling bacteria with plant growth-promoting abilities (Fu et al., 2025). The strong differences in ASV relative abundance within the same taxonomic ranks across the different areas emphasize the metabolic specificity of ASVs and highlight the need for more in-depth studies on their characterization and relationship to soil attributes and management.
While the findings provide significant evidence regarding the differential performance of the studied tree species, certain methodological limitations limit the broad generalizability of the conclusions. The assessment of seasonal and temporal dynamics, as well as the extrapolation of results to other native species or distinct environmental conditions, is limited by the study design, which features two ILFSs and one NF reference, sampled at a single time point during the dry season. However, the four-year timeframe was considered adequate to detect rapid and significant shifts in soil properties and the microbial community, especially given the site's long-term pasture history. It is suggested that future research should prioritize multisite studies with broader temporal sampling to validate these findings and fully characterize the resilience and stability of ILFSs across different biomes.
CONCLUSIONS
The silvopastoral system with S. tubulosa showed better soil health indicators than the Eucalyptus system. This suggests that promoting biodiversity through integrated systems with native species can enhance soil health and ecosystem resilience, especially when wood production is not a goal. Understanding the key players in microbial communities and their ecological functions can guide sustainable land use practices and conservation efforts. Future research should focus on unraveling the mechanisms driving microbial interactions in ILFSs and keystone features. Moreover, maintaining native forest fragments on rural properties is critical for preserving the native soil microbiota. Land-use changes, even for conservation-focused production systems, can significantly alter this microbiota. Therefore, these fragments serve as vital reservoirs of native microorganisms, helping conserve biodiversity beneath the soil.
ACKNOWLEDGEMENTS
To Paulo Humberto Marcante (Embrapa Rondônia) for the photographs in Figure 1 and support on field work. To Guilherme Lucio Martins and Juan Andrés de Domini, for their aid in Figure 1 construction. To the RBCS editorial board and the anonymous reviewers for their constructive comments.
How to cite:
FUNDING
DATA AVAILABILITY
The soil property data are available at Redape (https://doi.org/10.48432/QC7QW0). The 16S rRNA gene sequences are available in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1215384.
SUPPLEMENTARY MATERIALS
Supplementary data to this article can be found online at https://www.rbcsjournal.org/wp-content/uploads/articles_xml/1806-9657-rbcs-50-e0250164/1806-9657-rbcs-50-e0250164-suppl01.pdf
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