Molecular Ecology (2007) 16, 4335–4355 doi: 10.1111/j.1365-294X.2007.03527.x© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing LtdBlackwell Publishing LtdExtreme population subdivision throughout a continuous range: phylogeography of Batrachoseps attenuatus (Caudata: Plethodontidae) in western North AmericaIÑIGO MARTÍNEZ–SOLANO,*† ELIZABETH L. JOCKUSCH† and DAVID B. WAKE**Museum of Vertebrate Zoology, University of California, Berkeley, 3101 VLSB, Berkeley, California 94720, USA, †Ecology and Evolutionary Biology, University of Connecticut, Storrs, 75 N Eagleville Road, Unit 3043, Storrs, Connecticut 06269, USAAbstractLow-vagility species with deep evolutionary histories are key to our understanding of thebiogeographical history of geologically complex areas, such as the west coast of NorthAmerica. We present a detailed study of the phylogeography of the salamander Batra-choseps attenuatus (Caudata: Plethodontidae) using sequences of the mitochondrial genecob from 178 individuals sampled from throughout the species’ range. Sequences of three othermitochondrial genes (16S, cox1, nad4) and a nuclear gene (RAG-1) were used to investigatethe deeper evolutionary history of the species. We found high levels of genetic diversityand deep divergences within a mostly continuous distribution, with five genetically well-differentiated and geographically structured mitochondrial DNA clades. Significantassociation between geographical and genetic distances within these clades suggestsdemographic stability, whereas Fu’s FS tests suggest demographic expansions in three ofthem. Mantel tests identify two biogeographical barriers, the San Andreas Fault and theSacramento–San Joaquin Delta, as important in the diversification of lineages. The timingof the main splitting events between intraspecific lineages was estimated by applyingrelaxed molecular clock methods combining several mutation rates and a fossil calibration.The earliest splitting events are old (Pliocene/Miocene), with more recent (Pleistocene) sub-divisions in some clades. Disjunct populations distributed along the western foothills ofthe Sierra Nevada colonized this area relatively recently from a single refugium east of SanFrancisco Bay. The combination of fine-scale, comprehensive sampling with phylogenetic,historical demographic and hypothesis-based tests allowed delineation of a complexbiogeographical scenario with general implications for the study of codistributed taxa.Keywords: Batrachoseps attenuatus, mtDNA, phylogeography, RAG-1, Sacramento–San JoaquinDelta, San Andreas FaultReceived 22 May 2007; revision received 10 July 2007; accepted 1 August 2007IntroductionThe study of patterns of genetic structure in natural popu-lations has increased dramatically since the late 1960s,when the first studies based on allozyme data sets werepublished (Avise 1974; Larson et al. 1984). These studieswere followed by the generalized use of mitochondrialDNA (mtDNA) and, more recently, of nuclear introns andmicrosatellites in resolving patterns of intraspecific geneticdiversification in different taxa across their geographicalranges (Hare 2001; Zhang & Hewitt 2003). Large amountsof data are now available, setting the stage for comparativestudies and, ultimately, for the emergence of new discip-lines, such as comparative phylogeography (Bermingham& Moritz 1998; Avise 2000). The aim of comparative phylo-geography is to infer the existence of common biogeographicalevents through the concordance of independent gene treesfrom a variety of taxa in a shared geographical background.Comparative studies have been used, for instance, to identifymajor historical events affecting a variety of organisms(Schneider et al. 1998; Riddle et al. 2002), to infer thelocation of refugial areas during cold periods in theCorrespondence: Iñigo Martínez-Solano, Fax: 860 4866364; E-mail:[email protected] I. MARTÍNEZ-SOLANO, E. L. JOCKUSCH and D. B. WAKE© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing LtdPleistocene as well as potential recolonization routes duringwarmer periods (Hewitt 1999), and to inform managementpolicies concerned with the maintenance of evolutionarypatterns and processes (Moritz et al. 2001).The last decade has produced an increasing number ofpublications focused on phylogeographic patterns in westernNorth America (Brunsfeld et al. 2001; Calsbeek et al. 2003;Swenson & Howard 2005). The complex geological historyof this region makes it challenging for comparative studies,and despite the identification of some general patterns (ref-erences above; Feldman & Spicer 2006; Rissler et al. 2006),many studies have also uncovered significant differencesbetween taxa, suggesting considerable variation associatedwith historical factors in shaping present-day communities.Thus, species with similar distributions may have experi-enced very different evolutionary histories (Wake 2006).Salamanders in the genus Batrachoseps provide an excel-lent system for testing the influence of biogeographicalbarriers for several reasons. First, they constitute an oldclade, which probably has been native to western NorthAmerica for over 40 million years (Myr) (Yanev 1980; Wake2006). Second, they are extremely sedentary, with homeranges among the smallest described within vertebrates(typically < 10 m, Hendrickson 1954). Third, while some speciespresent restricted distributions, making them valuable fordelimitation of areas of endemism, other species haverelatively large distributions, providing opportunities to testbiogeographical scenarios at broader geographical scales.Such is the case of Batrachoseps attenuatus (Eschscholtz 1883),which ranges from southwestern Oregon to central California,north of Monterey Bay (Stebbins 2003). The distribution ismostly continuous along the coast; there are also severalisolated populations in the Great Central Valley, and a dis-junct group of populations distributed along the westernSierra Nevada foothills (Fig. 1). This species is the sistertaxon to a clade comprised of the 15 other species in theBatrachoseps (sensu stricto) clade (Jockusch & Wake 2002),and Late Miocene fossil remains attributed to Batrachosepshave been reported from east of San Francisco Bay (Clark1985), suggesting a long presence of the genus in this area.Fig. 1 Sampling localities and distribution of the main clades identified within Batrachoseps attenuatus. ID numbers as in the Appendix.Shading