Molecular Ecology (1999) 8, 1457–1465 © 1999 Blackwell Science Ltd Blackwell Science, Ltd A population genetic comparison of large- and small- bodied sage grouse in Colorado using microsatellite and mitochondrial DNA markers S. J. OYLER-M c CANCE,* N. W. KAHN,† K. P. BURNHAM,‡ C. E. BRAUN§ and T. W. QUINN¶ *Department of Fishery and Wildlife Biology, Colorado State University, Fort Collins, CO 80523, USA, †Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Centre, Denver, CO 80262, USA, ‡Colorado Cooperative Fish and Wildlife Research Unit, Colorado State University, Fort Collins, CO 80523 USA, §Colorado Division of Wildlife, Fort Collins, CO 80526 USA, ¶Department of Biological Science, University of Denver, Denver CO 80208, USA Abstract Sage grouse (Centrocercus urophasianus) from southwestern Colorado and southeastern Utah (United States) are 33% smaller than all other sage grouse and have obvious plumage and behavioural differences. Because of these differences, they have been tentatively recog- nized as a separate ‘small-bodied’ species. We collected genetic evidence to further test this proposal, using mitochondrial sequence data and microsatellite markers to determine whether there was gene flow between the two proposed species. Significant differences in the distribution of alleles between the large- and small-bodied birds were found in both data sets. Analysis of molecular variance (AMOVA) revealed that 65% of the variation in mitochondrial DNA (mtDNA) haplotypes could be explained by the large- vs. small-bodied distinction. Genetic distances and neighbour-joining trees based on allelic frequency data showed a distinct separation between the proposed species, although cladistic analysis of the phylogenetic history of the mitochondrial sequence haplotypes has shown a lack of reciprocal monophyly. These results further support the recognition of the small-bodied sage grouse as a distinct species based on the biological species concept, providing additional genetic evidence to augment the morphological and behavioural data. Further- more, small-bodied sage grouse had much less genetic variation than large-bodied sage grouse, which may have implications for conservation issues. Keywords: Colorado, gene flow, microsatellites, mtDNA, sage grouse, speciation Received 26 November 1998; revision received 12 March 1999; accepted 1 April 1999 Introduction Historically, sage grouse (Centrocercus urophasianus) occurred in at least 15 states and three provinces in North America (Aldrich 1963; Johnsgard 1973). They have since been extirpated from three states and one province ( Johnsgard 1973) and their range has declined markedly elsewhere (Braun et al. 1994). Declines have also occurred in Colorado where they have been extirpated from 12 of the 27 counties in which they occurred in the 1900s (Braun 1995) and populations in nine of the remaining 15 counties are thought to include fewer than 500 breeding birds. Because of this marked decline, sage grouse have become a focus of management and conservation concerns. Sage grouse have historically been classified into two subspecies: C. urophasianus urophasianus (Eastern sage grouse) and C. urophasianus phaios (Western sage grouse). This distinction was based on plumage and colouration differences (Aldrich & Duvall 1955), yet its validity has been questioned (Johnsgard 1983). Within the range of the Eastern sage grouse, Hupp & Braun (1991) and Barber (1991) found sage grouse in southwestern Colorado and southeastern Utah to be ≈ 33% smaller than those from northern Colorado and throughout the rest of the entire species’ range. These ‘small-bodied’ sage grouse have longer filoplumes, different tail banding patterns and dis- tinct ritualized strut displays compared with represent- ative ‘large-bodied’ sage grouse populations in northern Correspondence: S. J. Oyler-McCance, 201 Wagar, Department of Fishery and Wildlife Biology, Colorado State University, Fort Collins, CO 80523, USA. Fax: 970-491-1413 E-mail: soyler@lamar.colostate.edu