Patagonian forests, the southernmost temperate forests globally, are characterized by high endemism and unique genera despite low species richness. *Nothofagus pumilio*, a foundation species with wide distribution and adaptability, is crucial to this ecosystem. These forests face increasing threats from extreme climate events (droughts, warming, decreased precipitation) and anthropogenic activities (logging, fires). This study aims to define priority conservation areas and operational genetic management units for *N. pumilio* in Argentina, using genetic data to guide sustainable management and ensure long-term preservation. The study uses neutral molecular markers to understand the genetic structure across the species' vast distribution, allowing for delineation of genetically homogeneous regions (Genetic Zones, GZs) and the identification of areas with high allelic richness or exclusive alleles, crucial for conservation.

Previous research has documented the role of Quaternary climatic variability in shaping the current distribution of Patagonian forests. Recent studies highlight extreme climatic events as contributing factors to forest decline, particularly in Northern Patagonia. Furthermore, anthropogenic activities like logging and increased fire frequency further endanger these forests. Existing literature also reveals natural hybridization between *N. pumilio* and *N. antarctica*, with introgression of the chloroplast genome from *N. pumilio* to *N. antarctica*. The importance of defining operational genetic management units and priority conservation areas for *N. pumilio* is emphasized, aligning with global trends in native resource management. In Argentina, such units have been defined for other native species based on genetic criteria, highlighting the need for a similar approach for *N. pumilio*. The use of molecular markers to assess genetic diversity is established as a method for identifying priority conservation areas, particularly those with high allelic richness and/or exclusive allelic variants which serve as genetic reservoirs.

Thirty-five *N. pumilio* populations (965 adult trees) spanning its distribution in Argentina (36°S to 55°S) were sampled. A minimum of 20 trees per population were sampled, maintaining a minimum distance of 50m to avoid closely related individuals. DNA was extracted from bud tissue, and seven polymorphic loci were analyzed using microsatellite markers (nuSSRs). Genetic parameters such as number of different alleles (Na), effective number of alleles (Ne), expected heterozygosity (He), and unbiased heterozygosity were calculated using GenAlEx and Info-Gen software. The Wilcoxon test was used to compare heterozygosity among populations. Null alleles were tested with MICRO-CHECKER and FreeNA. Inbreeding coefficients (Fis) were calculated using INEST 2.2 employing Bayesian and Maximum Likelihood approaches. A bottleneck test was conducted using BOTTLENECK software to detect recent population size reductions. Genetic differentiation was measured using FST and G’ST indexes, with and without null allele exclusion. Isolation by distance (IBD) was assessed using the Mantel test and Spatial Genetic Structure (SGS) test. Population genetic structure was analyzed using BAPS 6.0 and STRUCTURE 2.2 software. CpDNA data informed the initial division of populations into northern and southern subsets. Spatial clustering in BAPS considered geographic locations. Admixture analysis in STRUCTURE helped determine the optimal number of clusters. Finally, AMOVA confirmed the number of clusters (Genetic Zones, GZs) that best grouped the populations. Genetic parameters (private alleles, locally common alleles, allelic richness) were calculated for each GZ to define management practices.

Significant differences in unbiased heterozygosity were found among populations. Twelve populations showed signs of inbreeding, with one exhibiting a significant inbreeding coefficient. A population bottleneck was identified in Sierra Colorada. Genetic differentiation (FST and G’ST) was similar to that reported in other *Nothofagus* species. An isolation-by-distance pattern was not supported. Based on cpDNA data and Bayesian clustering (BAPS) and admixture analysis (STRUCTURE), 18 GZs were delineated across the species' range. The analysis divided the populations into Northern (five GZs) and Southern groups. The Southern populations were further divided according to geographical and historical factors into groups ranging from 42°50'S to 44°S (four GZs), 44°S to 52°S (six GZs), and Tierra del Fuego (three GZs). AMOVA confirmed the optimal number of 18 GZs. Genetic diversity parameters (allelic richness, locally common alleles, private alleles) were calculated for each GZ. GZ2, GZ3, and GZ6 showed high genetic diversity, making them important for conservation. GZ2, GZ3, GZ9, GZ14, and GZ16 contained exclusive allelic variants. A bottleneck in GZ9 (near Rosario Lake) requires strict conservation. GZ7 and GZ13, exhibiting admixture patterns, also need special management consideration.

This study provides the first genetic-based management plan for *N. pumilio* across its entire distribution range in Argentina (over 2200 km). The identification of 18 GZs offers valuable operational genetic management units, minimizing the risk of altering genetic structure when transferring reproductive material within these zones. This is particularly relevant given the numerous restoration projects in *N. pumilio* forests. The identified priority conservation areas (GZ2, GZ3, GZ6, GZ9, GZ14, and GZ16) offer targets for focused in situ conservation efforts, focusing on areas with high genetic diversity and/or exclusive alleles. The findings are highly relevant for regulatory institutions responsible for enforcing national forest protection laws and for informing future conservation strategies and restoration planning. This work establishes a foundation for defining provenance regions and conservation units, enhancing the preservation of this critical southernmost forest ecosystem.

This research offers the first comprehensive, genetically informed management plan for *N. pumilio* in Argentina, encompassing its extensive distribution. Six priority conservation areas and 18 genetically defined zones (GZs) are identified, providing crucial guidance for in situ conservation and sustainable management practices. The delineated GZs serve as preliminary operational genetic management units, minimizing the risks associated with transferring reproductive materials. Future research could expand this study to include Chilean populations of *N. pumilio* and incorporate adaptive trait data to further refine management unit delineation.

This study focuses on the Argentinean distribution of *N. pumilio*. Including Chilean populations would offer a more comprehensive picture. The reliance on neutral molecular markers (SSRs) doesn't fully capture adaptive genetic diversity; incorporating adaptive trait data could strengthen the conservation strategies. The study's sampling strategy, while aiming for broad coverage, may not fully capture the fine-scale genetic structure in some areas.