Pre-Planting Epigenetic Diversity Assessment in Mangrove Species for Reforestation Management via epiGBS 2

Anthropogenic fragmentation impairs genetic variability, threatening the evolutionary potential of mangrove ecosystems. Cytosine DNA methylation serves as a vital epigenetic mechanism for rapid, inheritable environmental responses without primary sequence alterations. This study aimed to assess the pre-planting epigenetic diversity of Avicennia schaueriana, Laguncularia racemosa, and Rhizophora mangle across potential source populations in Rio de Janeiro to optimize regional ecological restoration. Sixty individuals per species (180 total samples) were collected from six localities within the Guapimirim Environmental Protection Area (APA) and Guanabara Ecological Station (ESEC). Reduced-representation bisulfite libraries were generated and sequenced using the de novo epiGBS 2 pipeline to concurrently track single methylation polymorphisms (SMPs) in the absence of a reference genome. Differentially methylated cytosines (DMCs) in the CpG context were identified using methylKit (q-value < 0.01, methylation difference > 25%), utilizing the most geographically distant site (Guaraí) as the baseline control. High-depth sequencing (ranging from 30x to over 78x) yielded robust epigenomic profiling across species. A. schaueriana displayed low global epigenetic structuring and small numbers of total DMCs (38–72), indicating high individual plasticity but baseline profile stability. Conversely, L. racemosa (105–166 DMCs) and R. mangle (195–295 DMCs) exhibited higher site-specific divergence and distinct geographic clustering, indicating a robust local environmental signature and inheritable epigenetic memory. Across all species, a high percentage of unique DMCs (up to 59.7% in specific river mouth sites) was recorded. Epigenetic trajectories vary significantly among these co-occurring mangrove species. The distinct epigenetic profiles and high proportions of exclusive DMCs highlight the need to transition from traditional restoration to molecularly informed management. We propose an evidence-based technical protocol that incorporates composite, multi-origin propagule collection and localized epigenetic biomarker monitoring to maximize functional forest resilience in changing environments.