Phylogeography of Douglas-fir based on mitochondrial and chloroplast DNA sequences: testing hypotheses from the fossil record PAUL F. GUGGER,* SHINYA SUGITA† and JEANNINE CAVENDER-BARES* *Department of Ecology, Evolution and Behavior, University of Minnesota, Saint Paul, MN 55108, USA, †Institute of Ecology, Tallinn University, 10120 Tallinn, Estonia Abstract The integration of fossil and molecular data can provide a synthetic understanding of the ecological and evolutionary history of an organism. We analysed range-wide maternally inherited mitochondrial DNA and paternally inherited chloroplast DNA sequence data with coalescent simulations and traditional population genetic methods to test hypoth- eses of population divergence generated from the fossil record of Douglas-fir (Pseudotsuga menziesii), an ecologically and economically important western North American conifer. Specifically, we tested (i) the hypothesis that the Pliocene orogeny of the Cascades and Sierra Nevada caused the divergence of coastal and Rocky Mountain Douglas-fir varieties; and (ii) the hypothesis that multiple glacial refugia existed on the coast and in the Rocky Mountains. We found that Douglas-fir varieties diverged about 2.11 Ma (4.37 Ma–755 ka), which could be consistent with a Pliocene divergence. Rocky Mountain Douglas-fir probably resided in three or more glacial refugia. More variable molecular markers would be required to detect the two coastal refugia suggested in the fossil record. Comparison of mitochondrial DNA and chloroplast DNA variation revealed that gene flow via pollen linked populations isolated from seed exchange. Postglacial colonization of Canada from coastal and Rocky Mountain refugia near the ice margin at the Last Glacial Maximum produced a wide hybrid zone among varieties that formed almost exclusively by pollen exchange and chloroplast DNA introgression, not seed exchange. Postglacial migration rates were 50–165 m ⁄ year, insufficient to track projected 21st century warming in some regions. Although fossil and genetic data largely agree, each provides unique insights. Keywords: coalescent simulations, glacial refugia, introgression, isolation with migration, muta- tion rate, postglacial migration, Pseudotsuga menziesii, SAMOVA Received 6 December 2009; Revision received 22 February 2010; accepted 3 March 2010 Introduction The integration of fossil and molecular genetic data is critical to understanding the ecological and evolution- ary history of an organism (Petit et al. 2008; Hu et al. 2009). Fossil data offer direct, dated evidence of species presence (and sometimes abundance), but suffer from lack of population (or even species) resolution and lim- ited sampling. In contrast, inferences of population his- tory from molecular data from modern forest trees can provide population and species level resolution from across an entire distribution, but yield estimates of pop- ulation divergence that are imprecise. Hypotheses from the fossil record can be tested with molecular data, which in turn produce new insights that can be tested with fossil data as more sites are analysed. In this way, fossil and molecular data offer complementary informa- tion that can converge on the major geological, climato- logical or ecological causes of population divergence. Range-wide syntheses of the fossil record in Europe and North America have revealed that temperate and Correspondence: Paul F. Gugger, Fax: 612-624-6777; E-mail: gugg0030@umn.edu Ó 2010 Blackwell Publishing Ltd Molecular Ecology (2010) 19, 1877–1897 doi: 10.1111/j.1365-294X.2010.04622.x