Abstract: Akodon cursor (Winge, 1887) is widely distributed across more than 2,500 km of the Brazilian Atlantic Forest and shows diploid numbers (2n = 14, 15, 16) associated with the recognized phylogeographic clades (North, Central, and South), but unevenly distributed among populations, suggesting that A. cursor may comprise more than one taxon. We used experimental crosses to investigate how reproductive barriers emerge and accumulate in a geographically structured and chromosomally polymorphic taxon. We estimated reproductive success (RS), litter size (LS), and gestation length (GL) based on 300 intra- and interpopulation crosses founded by 55 wild-caught matrices from three populations: Pernambuco (PE; North clade; 2n = 16), Bahia (BA; North and Central clades; 2n = 14, 15, 16), and Espírito Santo (ES; Central and South clades; 2n = 14). Mean intrapopulation values (RS = 56.06%; LS = 3.7 ± 1.04) were defined as reference parameters for the species. The PE population, geographically distant by at least 1,000 km and isolated for approximately 300,000 years, was reproductively isolated from both ES and BA, supporting its recognition as an Evolutionarily Significant Unit (ESU) for research and conservation. In contrast, BA and ES were not reproductively isolated and produced fertile F₁ hybrids, with heterosis observed in F₁ females. These results show that reproductive isolation in A. cursor is mosaic, direction-dependent, sex-biased, and stage-specific, supporting its value as a model for studying speciation, chromosomal evolution, and conservation-relevant lineage divergence, as well as for understanding how geography and chromosomal architecture interact to generate reproductive isolation.

Key words: Asymmetric Sexual Isolation, Chromosomal Speciation, Haldane’s Rule, Heterosis, Reinforcement