Matías E. Mac Allister ^1,2* Carlos E. Figueroa ^1,2 Regina Mazzei ¹ Ramino G. Tintorelli ^2,3 Diana Acosta ^1,2 Orlando Gallo ⁴ Diego Castillo ^2,5 Emiliano Pinardi ^1,2 Virginia D. Zelada Perrone ^1,2 Alejandro Rodríguez ⁶ Juan I. Zanón Martínez ^2,7,8 Mariano Lisandro Merino ^1,9 Juan I. Túnez ^10,2 Alejandro Travaini ^11,2 Gabriela P. Fernández ^1*

• ¹ CONICET Northwest Research and Transfer Center of the Province of Buenos Aires (CIT NOBA), Pergamino, Argentina
• ² National Scientific and Technical Research Council (CONICET), Buenos Aires, Buenos Aires, Argentina
• ³ Instituto Tecnológico de Buenos Aires, Buenos Aires, Buenos Aires, Argentina
• ⁴ Department of Biology and Biotechnology Charles Darwin, Faculty of Mathematics, Physics, and Natural Sciences, Sapienza University of Rome, Rome, Lazio, Italy
• ⁵ Instituto de investigaciones Biológicas y Biomédicas del Sur (CONICET), Bahía Blanca, Buenos Aires, Argentina
• ⁶ Department of Conservation Biology, Doñana Biological Station, Spanish National Research Council (CSIC), Sevilla, Spain
• ⁷ Instituto Multidisciplinario sobre Ecosistemas y Desarrollo Sustentable, Facultad de Ciencias Exactas, Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil, Buenos Aires, Argentina
• ⁸ National University of La Pampa, Santa Rosa, La Pampa, Argentina
• ⁹ Comisión de Investigaciones Científicas, La Plata, Buenos Aires, Argentina
• ¹⁰ Instituto de Ecología y Desarrollo Sustentable (INEDES-CONICET-UNLu), Departamento de Ciencias Básicas, Universidad Nacional de Luján, Luján, Buenos Aires, Argentina
• ¹¹ Centro de Investigaciones Puerto Deseado (UNPA), Universidad Nacional de la Patagonia Austral (UNPA), Deseado, Argentina

The puma (Puma concolor Linnaeus, 1771) is the top predator with the widest distribution in America. Since the establishment of European settlers on the American continent, puma populations have experienced significant contractions and reductions in their original distribution. In Argentina, the management of the conflict between humans and pumas (direct persecution and habitat modification) focused on reduction or elimination methods, leading to a drastic contraction, even total eradication, of puma distribution as seen in Patagonia and the eastern part of the country. Despite the lack of knowledge about puma population demographic trends, there are taxonomic issues that remain controversial and need to be resolved to implement appropriate management and conservation measures. Therefore, the aim of this study was to genetically characterize puma populations in the central-southern region of Argentina using two mitochondrial markers, evaluate their demographic history, compare our results at a macro-regional level, and discuss our findings in a conservation and management context. A total of 203 individuals were used, and a fragment of the control region and another of ND5 were sequenced. The genetic variability obtained was moderate. Substitution rates for each locus and the tMRCA were calculated from calibrated trees. In a concatenated tree, two main phylogenetic clades were identified (posterior probability = 1), although a reciprocal monophyly was not observed, with a divergence time of 235 thousand years and a 95% CI [120-368 thousand years]. When evaluating population structuring, three genetic clusters were found, one characteristic of the Patagonian region and the others in the central part of the country. Calculating the ФST values for pairs resulted in significant structuring between Patagonia and the rest of the populations, suggesting the arid diagonal as a possible barrier to gene flow. When evaluating the demographic history, neutrality tests would support a recent expansion in Patagonia. These findings are crucial in defining two distinct Management Units (MUs) in the southern part of their distribution and providing valuable information for management and conservation measures for the species.