Molecular Ecology (1999) 8, S79 – S94 © 1999 Blackwell Science Ltd Blackwell Science, Ltd Disparate phylogeographic patterns of molecular genetic variation in four closely related South American small cat species WARREN E. JOHNSON,* JILL PECON SLATTERY,* EDUARDO EIZIRIK,* JAE-HEUP KIM,* MARILYN MENOTTI RAYMOND,* CRISTIAN BONACIC,† RICHARD CAMBRE,‡ PETER CRAWSHAW,§ ADAUTO NUNES,¶ HÉCTOR N. SEUÁNEZ,** MIGUEL ANGELO MARTINS MOREIRA,** KEVIN L. SEYMOUR,†† FAIÇAL SIMON,‡‡ WILLIAM SWANSON§§ and STEPHEN J. O’BRIEN* *Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD 21702–1201, USA, †Departamento de Ingenieria Forestal, Universidad Catolica, Chile, ‡Department of Animal Health, National Zoological Park, Washington DC, USA, §CENAP/ IBAMA, Sorocaba, Brazil, ¶Zoo Municipal de Sorocaba, Brazil, **Instituto Nacional de Cancer, Serviço de Pesquisa Básica, Rio de Janeiro, Brazil, ††Department of Palaeobiology, Royal Ontario Museum, 100 Queen’s Park, Toronto, Ontario, Canada M5S 2C6, ‡‡Parque Zoologico de Sao Paulo, Sao Paulo, Brazil, §§Cincinnati Zoo and Botanical Garden, Cincinnati, OH, USA Abstract Tissue specimens from four species of Neotropical small cats ( Oncifelis geoffroyi, N = 38; O. guigna, N = 6; Leopardus tigrinus, N = 32; Lynchailurus colocolo, N = 22) collected from throughout their distribution were examined for patterns of DNA sequence variation using three mitochondrial genes, 16S rRNA, ATP8, and NADH-5. Patterns between and among O. guigna and O. geoffroyi individuals were assessed further from size variation at 20 microsatellite loci. Phylogenetic analyses using mitochondrial DNA sequences revealed monophyletic clustering of the four species, plus evidence of natural hybridization between L. tigrinus and L. colocolo in areas of range overlap and discrete population subdivisions reflecting geographical isolation. Several commonly accepted subspecies partitions were affirmed for L. colocolo, but not for O. geoffroyi. The lack of geographical substructure in O. geoffroyi was recapitulated with the microsatellite data, as was the monophyletic cluster- ing of O. guigna and O. geoffroyi individuals. L. tigrinus forms two phylogeographic clusters which correspond to L.t. oncilla (from Costa Rica) and L.t. guttula (from Brazil) and which have mitochondrial DNA (mtDNA) genetic distance estimates comparable to inter- specific values between other ocelot lineage species. Using feline-specific calibration rates for mitochondrial DNA mutation rates, we estimated that extant lineages of O. guigna diverged 0.4 million years ago (Ma), compared with 1.7 Ma for L. colocolo, 2.0 Ma for O. geoffroyi, and 3.7 Ma for L. tigrinus. Keywords: conservation genetics, evolution, microsatellites, mitochondrial DNA, phylogeography, South American felids Received 11 February 1999; revision received 10 June 1999; accepted 15 July 1999 Introduction Creation of the land bridge between North and South America 3 – 4 million years ago (Ma) (Coates & Obando 1996) facilitated one of the most impressive faunal migra- tions in mammal history, the great American interchange (Webb 1976, 1978; Stehli & Webb 1985; Webb & Rancy 1996). Utilizing molecular genetic markers, the evolutionary history of such a large-scale invasion can be reconstructed to provide insights into patterns of speciation and be utilized for the identification and management of gen- etic diversity. One of the consequences of the interchange was the introduction and rapid diversification of Carni- vora into South America, as is hypothesized to have Correspondence: W. E. Johnson. E-mail: johnsonw@ncifcrf.gov