Molecular Ecology (2003) 12, 2345 –2355 doi: 10.1046/j.1365-294X.2003.01901.x © 2003 Blackwell Publishing Ltd Blackwell Publishing Ltd. Identification of reproductively isolated lineages of Amur grayling (Thymallus grubii Dybowski 1869): concordance between phenotypic and genetic variation E. FROUFE,* , † I. KNIZHIN,‡ M. T. KOSKINEN,§ C. R. PRIMMER § and S. WEISS * , ¶ *CIBIO/ UP, Campus Agrário de Vairão, Portugal; †Faculdade de Ciências, Universidade do Porto, Portugal; ‡Irkutsk State University, Department of Vertebrate Zoology, Russia; §Department of Ecology and Systematics, Division of Population Biology, University of Helsinki, Finland; ¶Karl-Franzens University Graz, Institute of Zoology, Universitätsplatz 2, A-8010 Graz, Austria Abstract We analysed variation at maternally (mitochondrial DNA control region sequences) and bi- parentally (10 microsatellites) inherited genetic markers, as well as across 12 meristic characters in 7 populations of Amur grayling, Thymallus grubii, from eastern Siberia. All three data sets were concordant in supporting the existence of three diagnosable, reciprocally mono- phyletic, and most probably reproductively isolated, lineages of grayling within the Amur drainage. There was a significant correlation between genetic and phenotypic divergence, both within and among lineages. Two phenotypically distinct forms (with and without an orange spot on the posterior portion of the dorsal fin), found in sympatry in the lower Amur, most likely result from secondary contact, as they demonstrate 4.6% sequence divergence at the mitochondrial DNA control region. This divergence, together with the existence of at least one nearby population of orange spot grayling outside the Amur drainage (0.8% divergence) underscore the palaeo-hydrological complexity of the system, which presumably promoted genetic divergence in a shifting allopatric framework throughout the Pleistocene. Grayling from the upper Amur, corresponding to the type locality for the species, formed a sister group (1.4–1.6% divergent) to the orange spot lineage perhaps diverging in the early Pleistocene (1.4 –1.6 Ma). Keywords: Amur River, control region, grayling, microsatellites, Thymallus grubii Received 28 November 2002; revision received 7 March 2003; accepted 12 May 2003 Introduction Freshwater organisms live in isolated or fragmented habitats that restrict gene flow among demes. Fishes of the family Salmonidae exhibit life history traits such as natal hom- ing that further promote population structure, especially within the complex hydrological networks of the tem- perate ecosystems in which they are found. Such structure, in combination with the probable buffering effects and other characteristics of an autotetraploid ancestry (Allendorf & Thorgaard 1984), appears to promote intraspecific phenotypic variation. This diversity, combined with the popularity and economic value of many salmonids in commercial and sport fisheries, has attracted the attention of population geneticists and evolutionary biologists for decades. However, concordance between observed phenotypic and genetic divergence has often been illusory. Thus, one of the first broad-scale phylogeographical studies on the polytypic brown trout Salmo trutta , revealed five major matrilines, defined by primary catchment basins but wholly discordant with existing taxonomic epithets (Bernatchez et al . 1992; Osinov & Bernatchez 1996), which had historically led to the description of up to 50 species (Behnke 1986). In addition, studies evaluating sympatric forms of whitefishes Coregonus spp. (Shields et al . 1990; Turgeon et al . 1999; Lu et al . 2001; Rogers et al . 2001) often fail to reveal reproductive isolation and un- cover complex patterns of parallel evolution that under- score both the short- (within generation) and long-term (across generation) plasticity of many salmonid lineages. Phenotypic and genetic diversity find more concordance within allopatric frameworks such as for cutthroat trout Oncorhynchus clarkii in western North America where no fewer than 14 taxa are recognized, each existing in disjunct intermountain basins (Behnke 1992). But for other sub- specific designations within the genus, such as for O. Correspondence: S. Weiss. E-mail: steven.weiss@uni-graz.at