A. G. Osinov

Holarctic phylogeography of Arctic charr (Salvelinus alpinus L.) inferred from mitochondrial DNA sequences.

Abstract This study evaluated mitochondrial DNA (mtDNA) sequence variation in a 552‐bp fragment of the control region of Arctic charr (Salvelinus alpinus) by analyzing 159 individuals from 83 populations throughout the entire range of the complex. A total of 89 (16.1%) nucleotide positions were polymorphic, and these defined 63 haplotypes. Phylogenetic analyses supported the monophyly of the complex and assigned the observed haplotypes to five geographic regions that may be associated with different glacial refugia. Most notably, a formerly defined major evolutionary lineage (S. a. erythrinus) ranging from North America across the Arctic archipelago to the Eurasian continent has now been partitioned into the Arctic group and the newly identified Siberian group. The Beringian group, formed entirely by specimens assigned to S. malma (Dolly Varden), encompassed the area formerly assigned to S. a. taranetzi. The latter, due to a unique haplotype, became the basal member of the Arctic group. Overall, the S. alpinus complex reflects divergent evolutionary groups coupled with shallow intergroup differentiation, also indicated by an analysis of molecular variance that attributed 73.7% (P < 0.001) of the total genetic variance among groups. Time estimates, based on sequence divergence, suggest a separation of the major phylogeographic groups during early to mid‐Pleistocene. In contrast, colonization of most of todays range started relatively recently, most likely late Pleistocene during the last retreat of ice sheets some 10,000–20,000 years ago. This time scale obviously is too shallow for detecting significant variation on a smaller scale using mtDNA markers. However, other studies using nuclear microsatellite DNA variation strongly suggested ongoing evolution within groups by revealing strong population‐genetic substructuring and restricted gene flow among populations. Thus, Arctic charr could serve as a model organism to investigate the linkage between historical and contemporary components of phylogeographic structuring in fish, and, with a global perspective of the distribution of genetic variation as a framework, meaningful comparisons of charr studies at a smaller geographic scale will now be possible.

Genetic diversity of trout (genus Salmo) from its most eastern native range based on mitochondrial DNA and nuclear gene variation

Russia and western Asia harbour trout populations that have been classified as distinct species and subspecies, most often on the basis of morphological and ecological variation. In order to assess their origins and to verify whether traditional taxonomy reflects their evolutionary distinctiveness, we documented their genetic relationships on the basis of mitochondrial DNA (mtDNA) RFLP, mtDNA sequence analysis, and allozyme variation. Both mtDNA and nuclear gene variation defined two ancient phylogenetic assemblages of populations distributed among northern (Baltic, White, Barents), and southern (Black, Caspian, Aral) sea basins, between which gene flow has been possible but limited in postglacial times. These results supported the traditional taxonomic differentiation between populations of these two regions. They provided weak support for the taxonomic distinction of southern brown trout (Salmo trutta) populations based on their basin of origin. They also refuted the hypothesis that L. Sevan trout (Salmo ischchan) diverged from a primitive brown trout ancestor. Nevertheless, all trout populations from southern sea basins possessed private alleles or mtDNA genotypes and were genetically distinct Therefore, they represent unique gene pools that warrant individual recognition for conservation and management.

Genetic Differentiation of Arctic Charr Salvelinus alpinus Complex from Transbaikalia Revealed by Microsatellite Markers

Variation at eight microsatellite loci was studied in Arctic charr Salvelinus alpinus complex from five Transbaikalian mountain lakes. Samples from three lakes included two sympatric charr forms (dwarf and small) differing in trophic specialization, morphology and life cycle parameters. Sympatric forms were genetically closer to each other than to charr from other lakes which evidences their independent origin in each of these lakes as the result of sympatric speciation. In each lake, gene pools of sympatric forms were segregated to a different degree (estimates of FST varying from 0.030–0.184 and those of ρST varying from 0.119–0.359). Hierarchical analysis of allelic frequencies variance (AMOVA) in Arctic charr from Lake Baikal, the Vitim, and the Olekma basins showed that variation among and within these basins accounted for 19.5% of the interpopulational variance each. In the AMOVA design, investigating differences among sympatric forms in three lakes these differences accounted for 7.1% of the total variance.

Variation of the mitochondrial DNA control region in the populations of southern form of Dolly Varden (Salvelinus malma krascheninnikovi) from Sakhalin

Analysis of a 551-bp segment of the mitochondrial DNA control region in 23 individuals from nine populations of Dolly Varden from Sakhalin and three individuals from the Shikaribetsu Lake (Hokkaido) revealed the presence of seven haplotypes of southern form, along with one haplotype of northern form of Dolly Varden. All seven haplotypes of southern Dolly Varden were earlier described in the populations from Hokkaido. Hierarchical analysis of molecular variance (AMOVA) based on the haplotype frequencies, performed using literature data, suggested that, during the glacial epoch, there were three regional population groups of Dolly Varden (from eastern and western coasts of Sakhalin, and from Southern Primorye). Population groups from Sakhalin and Primorye were clearly separated. The differences between two Sakhalin population groups in the mtDNA haplotype frequencies were not statistically significant. However, relative to the earlier obtained data on microsatellite loci, these differences were statistically significant. For the populations of Sakhalin Dolly Varden, the data on mitochondrial and microsatellite DNA variation supplement each other.

Reproduction and development in the dwarf form of Arctic charr Salvelinus alpinus from Lake Davatchan, Transbaikalia

Biological characteristics in the deepwater dwarf form of Arctic charr Salvelinus alpinus from Lake Davatchan, Transbaikalia, are described. In nine from fifteen attempts of artificial insemination of eggs from a female by sperm from a male, developing eggs are obtained. Embryonic and larval development is described at the initial temperature 1.6–3.6°C and subsequent (from early gastrulation to the transition of larvae to exogenous feeding) average temperature 4.4 and 8.6°C. To take into account the data obtained previously (Pavlov et al., 1990, 1993b), the comparison of early ontogeny is conducted between the dwarf and small forms of charr from Lake Davatchan and the charr from Lake Leprindokan (two lakes are separated by the ridge 8 km in width). A substantial similarity in early ontogeny between the dwarf and small forms of charr from Lake Davatchan suggests that genes controlling development of the embryos and prelarvae until the transition of the young to exogenous feeding have not been subjected to natural selection determining a considerable differentiation between two forms and their reproductive isolation.

Variability of microsatellite DNA and genetic differentiation of populations in the resident form of Dolly Varden Salvelinus malma krascheninnikovi from Sakhalin

Variability in 11 microsatellite loci is followed for nine populations of the resident southern form of Dolly Varden Salvelinus malma krascheninnikovi from Sakhalin. In all studied populations, the level of genetic differentiation is significant with global values of Fst and Rst 0.182 and 0.329, respectively. On the NJ tree, two clusters are separated significantly: the former is represented by the populations from southwestern Sakhalin (the Novoselka River basin), and the latter includes all populations from southern and eastern Sakhalin. Abrupt size variations (bottleneck events) are suggested for several studied populations, and 50% reductions of allelic richness and average heterozygosity are observed in two lacustrine populations of the Novoselka River basin in comparison to the riverine populations of this basin. The results suggest an occurrence of a comparatively recent gene flow between the populations of resident Dolly Varden from Sakhalin and absence of reproductive isolation between the anadromous and resident forms. The degradation of the majority of Dolly Varden populations can be expected in the near future, and conservation projects should be developed.

Blunt-snouted lenoks (genus Brachymystax: Salmoniformes, Salmonidae) from the Ob’ basin: New data on morphology and allozyme variation

Variation of 69 morphological characters and allozyme variation at 32 loci was studied in lenoks (Brachymystax) from the Biya, Tom’, and Chulym drainages (the Ob’ basin). All lenoks studied belong to the blunt-snouted form, morphological differences between their populations are low and genetic distances equal zero. At the same time they diverged substantially morphologically and, especially, genetically from populations of the blunt-snouted lenok from other river basis of Siberia and the Far East of Russia. In the Ob’-Irtysh basin populations of the blunt-and sharp-snouted lenoks are spatially segregated: the former are found in the Ob’ River basin proper and the latter, in the Irtysh River basin, the degree of their morphological and genetic (Dnei = 0.096) divergence is high. Populations of the blunt-snouted lenok in the Ob’ basin are small and the numbers of lenoks are decreasing; problems of their protection are discussed.

On the origin and phylogenetic position of Arctic charr (Salvelinus alpinus complex, Salmonidae) from Lake Cherechen’ (middle Kolyma River basin): controversial genetic data

Within the Arctic charr complex (Salvelinus alpinus complex), many different populations and forms with disputable origin and systematic status have been described. Some of them, such as the charr from Lake Cherechen’ (middle Kolyma River basin), combine characters of different phylogenetic groups, representing a possible consequence of former hybridization. The data on 32 allozyme loci and on nucleotide sequences of 501-bp fragments of the mtDNA control region as well as of 899-bp fragments of exon 2 of the RAG1 gene were used for the analysis of the origin of Lake Cherechen’ charr and their phylogenetic relationships with other representatives of the Arctic charr lineage. As was shown previously, the dwarf and large forms of charr from this lake are morphologically similar to other charr populations from the upper Kolyma River, but bear the mtDNA haplotype of northern Dolly Varden Salvelinus malma malma, not of Taranets charr Salvelinus alpinus taranetzi. The analysis of the allozymes and RAG1 gene confirms the affinity of the Lake Cherechen’ charr to the Arctic charr lineage, but it is insufficient to unambiguously attribute them to the Eurasian or Taranets group. The presence of mtDNA of northern Dolly Varden in Lake Cherechen’ Arctic charr and the replacement of their native mtDNA are the result of the introgressive hybridization with S. m. malma. An alternative explanation connected with incomplete lineage sorting seems highly improbable. Our study confirms a postglacial secondary contact of the representatives of different phylogenetic groups of the S. alpinus-S. malma species complex in the Kolyma basin and in the area from Taimyr to Chukotka. It also indicates the need for more thorough analysis of the morphological and genetic diversity of charr from this region as well as caution in taxonomic decisions.

On morphological diversity of southern Dolly Varden trout Salvelinus malma krascheninnikovi from water bodies of Sakhalin

Morphological and some biological specific features of nine samples of the southern form of Dolly Varden trout Salvelinus malma krascheninnikovi from water bodies of Sakhalin were studied. Comparative analysis was performed of our own estimates and estimates made by other authors of meristic characters of Sakhalin populations of Dolly Varden trout, as well as of samples from water bodies of the Kuril Islands and Western Kamchatka. Sakhalin populations of S. malma krascheninnikovi are characterized by a low level of morphological differentiation and the absence of geographic structure. No clinal latitudinal variation in sample averages was revealed in either of the nine studied meristic characters, including the number of vertebrae for populations of S. malma krascheninnikovi in the range from Bol’shoi Shantar Island to the south of Sakhalin. The cline in vertebral number from Kamchatka, along the Kuril Islands to Hokkaido, is most likely explained by specific features of distribution and introgressive hybridization at the secondary contact of populations of the northern and southern forms of Dolly Varden trout that originated from different glacial refugia and had a different vertebral number rather than by the temperature gradient.

Inferring phylogenetic structure, hybridization and divergence times within Salmoninae (Teleostei: Salmonidae) using RAD-sequencing.

Phylogenetic studies focusing on Salmonidae have revealed significant obstacles in trying to clarify some interspecific relationships within the Salmoninae subfamily, due to a limited number of markers typed, conflicting phylogenetic signals and ancient hybridization events. To infer reliable phylogenetic relationships, evaluate several putative scenarios of ancient hybridization, and estimate divergence times within Salmoninae, we applied restriction-site associated DNA sequencing (RAD-seq) to 43 samples, including 26 genetic lineages across 21 species, largely representing the subfamily, with an emphasis on the genus Salvelinus. We identified 28,402 loci and 28,363 putatively unlinked SNPs, which were used in downstream analyses. Using an iterative k-means partitioned dataset and a Maximum Likelihood approach; we generated a well-supported phylogeny, providing clear answers to several previous phylogenetic uncertainties. We detected several significant introgression signals, presumably ancient, in the genus Salvelinus. The most recent common ancestor of Salmonidae dates back to approximately 58.9MY ago (50.8-64 MY) and the crown age of Salmoninae was estimated to be 37.7 MY (35.2-40.8 MY) using a Bayesian molecular dating analysis with a relaxed molecular clock. The divergence among genera of the subfamily occurred between the late Eocene and middle of the Miocene (≈38-11 MY) such as the divergence between the genus Oncorhynchus and Salvelinus, which we estimated to 21.2 MY ago (95% HPD: 19.8-23.0 MY), while species diversification took place mainly during the Neogene (≈22-1.5 MY), with more than half of these events occurring in the last 10 MY.