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The discovery of tetraploidy in the red viscacha rat, Tympanoctomys barrerae (4n = 102) has emphasized the evolutionary role of genome duplication in mammals. The tetraploid status of this species is corroborated here by in situ PCR and Southern blot analysis of a single-copy gene. The species meiotic configuration strongly suggests a hybrid derivation. To investigate the origin of T barrerae further, the recently described Pipanacoctomys aureus was studied. This 92-chromosome species also has a duplicated genome size, redundant gene copy number and diploid-like meiotic pairing, consistent with an event of allotetraploidization. Phylogenetic analysis of mitochondrial sequences indicates sister-group relationships between these two tetraploid rodents. The new karyotypic data and the phylogenetic relationships suggest the participation of the ancestral lineages of Octomys mimax in the genesis of R aureus. The high overall DNA similarity and shared band homology revealed by genomic Southern hybridization as well as matching chromosome numbers between O. mimax and the descendant tetraploid species support the notion of introgressive hybridization between these taxa. (C) 2004 The Linnean Society of London.