Bruno Ruiz Brandão da Costa1, Fernanda Anselmo-Moreira2, Alex Nascimento2, Eduardo Luís Martins Catharino2, Michael Staudt3, Agnès Borbon4, Adalgiza Fornaro5, Silvia Ribeiro de Souza2, Cláudia Maria Furlan1
Introduction
The Atlantic Forest is recognized as a biodiversity hotspot. Yet, the effects of urbanization and seasonality on plant metabolism in its fragmented remnants are still poorly understood. In this study, we compare two forest fragments in the Metropolitan Area of São Paulo: Reserva Florestal Morro Grande (RMG), located 40 km from the city center, and Matão-IAG, a forest adjacent to major roads and urban infrastructure. These locations represent distinct gradients of anthropogenic influence and provide a useful contrast for examining environmental stress. Previous dry-season morphoanatomical and physiological analyses of two native tree species—Alchornea sidifolia (AS) and Guarea macrophylla (GM)—revealed species-specific responses, with AS showing greater resilience to urban stress and GM displaying higher sensitivity. To build on these results, we employed untargeted metabolomics to assess how seasonality and site affect the metabolic profiles of both species.
Material and Methods
Leaves were collected in August 2023 (dry) and February 2024 (rainy). Dried tissues (20 mg) were extracted with chloroform:methanol:water (12:5:1, v/v/v). The polar phase, which is the focus of this study, was derivatized and analyzed by gas chromatography–mass spectrometry (GC-MS). Deconvolution was performed using the Global Natural Products Social Molecular Networking (GNPS) platform. Compound annotation was conducted using both GNPS and the National Institute of Standards and Technology (NIST) Mass Spectral Library 2.0. Multivariate analysis via principal component analysis (PCA) was performed in MetaboAnalyst 6.0. Univariate differences were assessed using Mann–Whitney U tests in R (v4.5).
Results and discussion
Metabolomics revealed distinct species responses. In AS, 99 compounds (94.3% of peak area) and in GM, 101 (80.9%) were annotated, across 10 chemical families. PCAs showed weak clustering for AS, except for seasonal shifts at Matão-IAG. GM displayed clearer separations: seasonal differences at RMG and site divergence in the dry season. These patterns match earlier morphoanatomical data, supporting AS’s resilience and GM’s urban-stress sensitivity. Univariate analysis revealed context-dependent shifts. In AS, rainy-season Matão-IAG samples had higher disaccharides; dry-season samples had more amino acids and alkaloids. In GM, RMG dry-season leaves had more organic acids and polyols; rainy-season samples had increased trisaccharides. At Matão-IAG, GM showed fewer significant changes, consistent with weaker PCA separation.
Conclusion
This study highlights species-specific metabolic strategies in response to seasonality and urbanization. AS demonstrated metabolic resilience, while GM showed greater sensitivity, consistent with previous morphoanatomical findings. Our results emphasize the complex metabolic adaptations of native trees to anthropogenic and seasonal stresses in fragmented urban forest ecosystems.
Agradecimentos: Research supported by the State of São Paulo Research Foundation (FAPESP) (Grant numbers: 20/07141-2, 22/13213-1, 2022/07326-8, and 2021/06227-3).