Problems of Molecular Systematics of the Genus Salvelinus Based on Nucleotide Sequence Variability in the Major Noncoding Region of Mitochondrial DNA

Variability of the major noncoding mitochondrial DNA region of char (Salvelinus alpinus complex) was analyzed by the median network method on the basis of the literature data on nucleotide sequences of this region. A significant portion (21%) of the polymorphic sites of char mtDNA was shown to be evolutionarily unstable, which results in mutation homoplasia at these sites. Using median-joining analysis the chars were divided into three phylogeographical groups: Arctic, Beringian, and chars occurring in the vast region from Lake Baikal and Taimyr Peninsula to the Atlantic Ocean. The latter group includes three subgroups (Siberian, Atlantic, and Acadian), which diverged from a common ancestor. In the Arctic group, the branch of Taranetz char S. taranetzi is clearly separated. It diverged from the common ancestor earlier than S. boganidae andS. elgyticus occurring on the Chukotka Peninsula species and S. a. erythrinus occurring in Alaska. The Beringian group, which includes dolly varden char from various regions of Asia and America, is an intermediate between the two char groups. The main problems of char systematics inferred from the major noncoding mtDNA region variability arise from the high level of mutation homoplasia.     

Change in nucleotide sequence of mitochondrial DNA from cytochrome b gene in chars of Salvelinus genus

Nucleotide sequences of two (405- and 1050-bp) regions of mitochondrial DNA (mtDNA) cytochrome c gene were established in chars of the genus Salvelinus from Russian Far East and Siberia. Based on the divergence and phylogenetic analysis of nucleotide sequences of the mtDNA cytochrome c gene, S. laecomaenis was shown to carry the most ancient mitochondrial lineage, which is close to the ancestral one. The archaic mtDNA of S. levanidovi occupied an isolated position on the phylogenetic trees. The mtDNA lineage of the southern S. malma was close to the S. alpinus-S. malma complex group. Within the S. alpinus-S. malma complex, three groups of mtDNA types having particular geographic distributions were distinguished. The Kolyma-Chukotka group includes lake S. taranetzi, S. boganidae and S. elgyticus from Chukotka, lake chars from Kolyma. The Okhotsk group is represented by northern S. malma, lake chars from northern Sea of Okhotsk, and anadromous S. taranetzi. The Siberian group is close to the Okhotsk one and consists of Taimyr and Baikal region chars as well as Arctic char from Finland. The divergence of char mitochondrial lineages was dated to the Pliocene-Pleistocene.     

Variability of Nucleotide Sequences of Mitochondrial DNA Cytochrome b Gene in Chars of the Genus Salvelinus

Nucleotide sequences of two (405- and 1050-bp) regions of mitochondrial DNA (mtDNA) cytochrome c gene were established in chars of the genus Salvelinus from Russian Far East and Siberia. Based on the divergence and phylogenetic analysis of nucleotide sequences of the mtDNA cytochrome b gene, S. laecomaenis was shown to carry the most ancient mitochondrial lineage, which is close to the ancestral one. The archaic mtDNA of S. levanidovi occupied an isolated position on the phylogenetic trees. The mtDNA lineage of the southern S. malma was close to the S. alpinus–S. malma malmacomplex group. Within the S. alpinus–S. m. malma complex, three groups of mtDNA types having particular geographic distributions were distinguished. The Kolyma–Chukotka group includes lake S. taranetzi, S. boganidae, andS. elgyticus from Chukotka, lake chars from Kolyma. The Okhotsk group is represented by northernS. malma, lake chars from northern Sea of Okhotsk, and anadromous S. taranetzi. The Siberian group is close to the Okhotsk one and consists of Taimyr and Baikal region chars as well as Arctic char from Finland. The divergence of char mitochondrial lineages was dated to the Pliocene–Pleistocene.     

Variation of cytochrome b gene in sympatric chars from kronotsky lake (Kamchatka Peninsula)

In previously identified sympatric char forms (species) from Kronotsky Lake (Kamchatka Peninsula) and a number of other chars from the genus Salvelinus, nucleotide sequences of the mtDNA cytochrome b gene were determined and phylogenetic analysis of these chars was performed. The divergence estimate of the cytochrome b gene in the Kronotsky char group (0.48%) coincided with the interpopulation difference estimates in northern Dolly Varden, which indicates a relatively recent divergence of the chars from Kronotsky Lake. Haplotypes of each form of the Kronotsky Lake chars are divided into two groups, which belonged to two different phylogenetic mtDNA lineages of northern Dolly Varden from Chukotka and Kamchatka. The formation of the Kronotsky char forms proved to be unrelated to the observed variability of the mtDNA cytochrome b gene.     

Variation of cytochrome b gene in sympatric chars from Kronotsky Lake (Kamchatka oblast)

In previously identified sympatric char forms (species) from Kronotsky Lake (Kamchatka Peninsula) and a number of other chars from the genus Salvelinus, nucleotide sequences of the mtDNA cytochrome b gene were determined and phylogenetic analysis of these forms was performed. The divergence of the cytochrome b gene in the Kronotsky char group (0.48%) coincided with the interpopulation difference estimates in northern Dolly Varden, which indicates a relatively recent divergence of the chars from Kronotsky Lake. Haplotypes of each form of the Kronotsky Lake chars are divided into two groups, which belonged to two different phylogenetic mtDNA lineages of northern Dolly Varden from Chukotka and Kamchatka. The formation of the Kronotsky char forms proved to be unrelated to the observed variability of the mtDNA cytochrome b gene.     

Mitochondrial DNA data imply a stepping‐stone colonization of Beringia by arctic warbler Phylloscopus borealis

Arctic warbler Phylloscopus borealis is one of several high‐latitude Passerines which are widely distributed across one northern continent but restricted to the Beringian part of the other. Most species with such asymmetric intercontinental ranges are monomorphic across Beringia, suggesting either recent colonization of the second continent or considerable gene flow across the Bering Strait. Arctic warbler is the only migratory species in this group that has three different subspecies in Beringia: Ph. b. borealis (Scandinavia to western Beringia, south to Mongolia), Ph. b. xanthodryas (Japan, Sakhalin, Kamchatka, western Beringia), and Ph. b. kennicotti (Alaska). This polymorphism may indicate that Arctic warbler has a unique and complex phylogeographic history that differs significantly from other species with similar ranges. Our analyses of complete mtDNA ND2 sequences of 88 Arctic warblers collected across the species range showed that the clade comprised of birds breeding on Sakhalin Island and Kamchatka Peninsula diverged from the Palearctic/Beringian clade by 3.8% in ND2 sequence. Beringian birds formed a recently derived clade embedded within the Palearctic clade. Nucleotide diversity declined sharply eastward from Palearctic to western Beringia and then to eastern Beringia. Our data provided no support for currently recognized subspecies. They suggested that the barrier at the western edge of Beringia was crossed by Arctic warbler earlier than the Bering Strait resulting in a stepping‐stone colonization of Beringia by this species. Gene flow appears to be restricted across the western border of Beringia but not the Bering Strait.     

Genetic divergence of mitochondrial DNA in white char <Emphasis Type="Italic">Salvelinus albus</Emphasis> and northern Dolly Varden char <Emphasis Type="Italic">Salvelinus malma malma</Emphasis>

Comparative analysis of mitochondrial DNA variation was performed in white char Salvelinus albus Glubokovsky and in its putative ancestor species, northern Dolly Varden char Salvelinus malma malma Walbaum. Highly statistically significant differentiation of S. albus and S. m. malma in the areas of sympatric (Kamchatka River basin) and allopatric (Kronotskoe Lake and Kronotskaya River) residence was demonstrated. The mtDNA divergence between S. albus and S. m. malma did not exceed the range of intraspecific variation in the populations of northern Dolly Varden char. At the same time, clusterization pattern of the Salvelinus chars provides hypothesis on the common origin of two allopatric populations of white char. Genealogical analysis of haplotypes indicates that S. albus and S. m. malma currently demonstrate incomplete divergence of mitochondrial lineages. The low nucleotide divergence estimates between S. albus and S. m. malma reflect the short time period since the beginning of the divergence of ancestral lineages. These estimates are determined by ancestral polymorphism and haplotype exchange between the diverged phylogenetic groups as a result of introgressive hybridization.     

Toward a more accurate time scale for the human mitochondrial DNA tree

Several estimates of the time of occurrence of the most recent common mitochondrial DNA (mtDNA) ancestor of modern humans have been made. Estimates derived from noncoding regions based on a model that classifies sites into two categories (variable and invariable) have been consistently older than those derived from the third positions of codons. This discrepancy can be attributed to a violation of the assumption of rate homogeneity among variable sites when analyzing the noncoding regions. Additional data from the partial control region sequences allow us to take into account some of this further heterogeneity. By assigning the sites to three classes (highly variable, moderately variable, and invariable) and by assuming that the last common mtDNA ancestor of humans and chimpanzees lived 4 million years ago, the most recent common mtDNA ancestor of humans is estimated to have occurred 211,000 ±111,000 years ago (±1 SE), consistent with the estimate, 101,000 ± 52,000 years, made from third positions of codons and also with those proposed previously. We used the same technique to estimate when a putative expansion of modern humans out of Africa took place and estimated a time of 89,000 ± 69,000 years ago. Even though the standard errors of these estimates are large, they allow us to reject the multiregional hypothesis of modern human origin.Deceased July 21, 1991 Correspondence to: M. Hasegawa     

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

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 today's 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 divergence of chars of the genus Salvelinus from Kronotsky Lake (Kamchatka Peninsula)

Chars from the genus Salvelinus, inhabiting lakes and lake-river systems, belong to morphologically and ecologically different forms whose taxonomic status is under dispute. In the present work, we have examined genetic variation and divergence in various chars from the Kronotsky lake basin: the lacustrine chars (white, nose, and long-head) and Dolly Varden char Salvelinus malma. The study was conducted using analysis of allozyme and microsatellite loci, myogens, RAPD, and restriction analysis of two mtDNA segments. The estimates of heterozygoisty at allozyme and microsatellite loci were similar to the corresponding parameters in populations of northern Dolly Varden and Arctic char. Heterozygote deficit was recorded in both samples of individual forms, and in the combined sample of all chars from Kronotsky Lake. For both markers, appreciable genetic differentiation among the samples of different char forms was found, which was comparable to that among the spatially isolated populations of northern Dolly Varden. This result indicates reproductive isolation among the char forms examined. However, this isolation is not complete, because no fixed differences between the forms by any of the genetic systems analyzed was found. The genetic differentiation among different forms of lacustrine chars, which corresponds to the interpopulation rather than interspecies level, is thought to be explained by their comparatively recent divergence.     

Secondary contact between two divergent lineages of charrs of the genus <Emphasis Type="Italic">Salvelinus</Emphasis> in the Northwest Pacific

The present-day contact zone between the Beringian and Arctic phylogenetic lineages of charrs of the genus Salvelinus in the Northwest Pacific is documented. A comparative analysis of the genetic differentiation and divergence indices for allopatric and sympatric populations of charrs and phylogenetic and genealogical analyses of the mtDNA haplotypes indicate that Lake Achchen and the Lake Pekulineiskoe are the zones of secondary contact between S. m. malma and S. taranetzi; Lake Nachikinskoe, between S. m. malma and Salvelinus sp. 4; and Lake Dal’nee, between S. m. malma and S. krogiusae. The level of divergence between phylogenetic groups of haplotypes considerably exceeds the range of intraspecific variability of S. m. malma and could not have been achieved after colonization of the lakes in conditions of sympathy. The obtained data suggest that the territory of Kamchatka was colonized by the common ancestor of the Arctic phylogroup of Taranetz charr.     

Population structure of the Japanese eel, Anguilla japonica

Mitochondrial DNA (mtDNA) sequences that include (a) a part of the cytochrome b gene, (b) two tRNA genes, and (c) a part of the noncoding D-loop region of 31 Anguilla japonica (Japanese eel) and 1 A. marmorata collected from Taiwan, Japan, and mainland China were determined to evaluate the population structure of Japanese eel. Among 30 genotypes identified from the 31 Japanese eel mtDNAs sequenced, there are 58 variable sites, predominantly clustered at the D-loop region. The phylogenetic tree constructed by the unweighted pair-group method with arithmetic mean shows neither significant genealogical branches nor geographic clusters. Furthermore, the sequence-statistics test reveals little, if any, significant genetic differentiation. These results indicate that the 31 Japanese eels might come from a single population. Analysis of sequence variation in mtDNA by using the relationship between the number of segregating sites and the average number of nucleotide differences under the neutral mutation hypothesis reveals that neutral mutation acts as a major factor influencing the evolutionary divergence of the Japanese eel mitochondrial genome sequenced, especially in the noncoding region.      

Variability of nucleotide sequences of the mitochondrial DNA cytochrome c gene in dolly varden and taranetz char

Nucleotide sequence of the 307-bp fragment of the mitochondrial DNA cytochrome b gene was determined in representatives of the three species of the Salvelinus genus, specifically, dolly varden char (S. malma), taranetz char (S. taranetzi), and white-spotted char (S. leucomaenis). These results pointed to a high level of mitochondrial DNA (mtDNA) divergence between white-spotted char and dolly varden char, on the one hand, and taranetz char, on the other (the mean d value was 5.45%). However, the divergence between the dolly varden char and taranetz char was only 0.81%, which is comparable with the level of intraspecific divergence in the dolly varden char (d = 0.87%). It was shown that the dolly varden char mitochondrial gene pool contained DNA lineages differing from the main mtDNA pool at least in the taranetz char-specific mitochondrial lineages. One of these dolly varden char mtDNA lineages was characterized by the presence of the restriction endonuclease MspI-D variant of the cytochrome b gene. This lineage was widely distributed in the Chukotka populations but it was not detected in the Yana River (Okhotsk sea) populations. These findings suggest that dolly varden char has a more ancient evolutionary lineage, diverging from the common ancestor earlier than did taranetz char.     

Trans-Arctic dispersals and the evolution of a circumpolar marine fish species complex, the capelin (Mallotus villosus)

Trans-Arctic dispersals and population and range expansions during the Pleistocene enhanced opportunities for evolutionary diversification and contributed to the process of speciation within the capelin, a northern marine-fish complex exhibiting a circumpolar distribution. Capelin is composed of four highly divergent and geographically discrete mitochondrial DNA (mtDNA) clades (609 bp; cytochrome b). Two clades occur in the North Atlantic, one associated with Canadian Atlantic waters, including Hudson Bay, and the second distributed from West Greenland to the Barents Sea. Two additional clades occur in the Arctic and northeast Pacific Oceans, representing the most recent divergence within the capelin phylogenetic tree. Judged from mtDNA diversity, capelin populations comprising all clades experienced at least one demographic and spatial reduction-expansion episode during recent Pleistocene glaciations that imprinted their molecular architecture. The large contemporary populations in the northeast Pacific and Arctic Oceans exhibited significant genetic structure whereas no such structure was detected in the equally extensive North Atlantic clades. All clades are characterized by one or two prevalent mtDNA haplotypes distributed over the entire range of the clade. Assuming a Pacific ancestor for capelin, we infer that capelin dispersed on two separate occasions to the North Atlantic. A more recent event resulted in the isolation of eastern Pacific and Arctic clades, with the Arctic clade positioned for a potential third Atlantic invasion, as revealed by the presence of this clade in the Labrador Sea. The Labrador Sea is a potential contact zone for three of the four capelin clades.     

Comparative restriction enzyme analysis of mitochondrial DNA in several populations of lake resident chars of the genus Salvelinus

Restriction enzyme analysis was employed in studying the mitochondrial DNA (mtDNA) ATPase6/ND4L region in several Northeast Asian populations of resident lake char of the genus Salvelinus. On evidence of mitotypes, genetic similarity was assumed for populations of neiva (Ueginskoe Lake), lake resident char from the northern coast of the Sea of Okhotsk (Mak-Mak Lake and Elekchanskie lakes), and dolly varden. Mitotype AAAA proved to be common for these populations. Lake char populations of the Juliet and Maxi lakes (the basin of the Kolyma river) had mitotype DBAA, which is similar to mitotype DBAB observed earlier in Taranetz char S. taranetzi from Chukotka. The divergence between mitotypes AAA and DBAA was estimated at 0.3%. Different origins were assumed for the lake resident char populations from the basins of the Sea of Okhotsk and of the Kolyma River, the former originating in the Pacific and the latter, in the Arctic basins.     

Karyological Differentiation of Northern Dolly Varden and Sympatric Chars of the Genus <Emphasis Type="Italic">Salvelinus</Emphasis> in Northeastern Russia

In many cases the taxonomic status of sympatric chars (with exception of white-spotted char Salvelinus leucomaenis) is not clear and is actively debated. We karyotyped three pairs of sympatric chars inhabiting the Russian Far East-northern Dolly Varden, S. malma malma, and Lavanidov's char, S. levanidovi, from the Yama River (northern coast of the Sea of Okhotsk, Magadan Region), northern Dolly Varden and Taranetz's char, S. taranetzi, from Lake Achchen (east Chukotka), and northern Dolly Varden and white char, S. albus, from the Kamchatka River (Kamchatka Peninsula). Three of them had similar chromosome numbers. But all chars studied had an individual and discrete set of karyotypic characters, which enabled reliable identification each of them by chromosome number, chromosome arm number, and number and location of active nuclear organizer regions.     

Phylogeography and sympatric differentiation of the Arctic charr Salvelinus alpinus (L.) complex in Siberia as revealed by mtDNA sequence analysis

Sequence variation in the mtDNA control region of Arctic charr Salvelinus alpinus and Dolly Varden Salvelinus malma from 56 Siberian and North American populations was analysed to assess their phylogeographic relationships and the origins of sympatric forms. Phylogenetic trees confirm the integrity of phylogroups reported in previous mtDNA studies except that the Siberian group does not separate as a single cluster. Haplotype network analysis indicates the proximity of Siberian and Atlantic haplotypes. These are considered as one Eurasian group represented by the Atlantic, east Siberian (interior Siberia including Transbaikalia, Taimyr) and Eurosiberian (Finland, Spitsbergen, Taimyr) sub-groups. Salvelinus alpinus with presumably introgressed Bering group (malma) haplotypes were found along eastern Siberian coasts up to the Olenek Bay and the Lena Delta region, where they overlap with the Eurasian group and in the easternmost interior region. It is proposed that Siberia was colonized by S. alpinus in two stages: from the west by the Eurasian group and later from the east by the Bering group. The high diversity of Eurasian group haplotypes in Siberia indicates its earlier colonization by S. alpinus as compared with the European Alps. This colonization was rapid, proceeded from a diverse gene pool, and was followed by differential survival of ancestral mtDNA lineages in different basins and regions, and local mutational events in isolated populations. The results presented here support a northern origin of Transbaikalian S. alpinus , the dispersion of S. alpinus to the Lake Baikal Basin from the Lena Basin, segregation of S. alpinus between Lena tributaries and their restricted migration over the divides between sub-basins. These results also support sympatric origin of intralacustrine forms of S. alpinus .     

Karyotype and relationships of the endemic char Salvelinus krogiusae from Dal'nee Lake (Kamchatka)

Resident (lacustrine) chars, inhabiting many lakes of Kamchatka, Chukotka, and continental coast of the Sea of Okhotsk, are of particular interest for understanding the mechanisms of speciation and evolution of Salvelinus chars. Since one of these, the char from Dal'nee Lake (Paratunka River basin, southeastern Kamchatka), is substantially different from lacustrine-riverine and anadromous chars from the Paratunka River basin in several morphological traits, hemoglobin spectrum, ecology, and reproduction, it is considered an isolated species Salvelinus krogiusae. The karyotype of the resident char from Dal'nee Lake was shown to be variable in most individuals, containing 76 to 80 chromosomes in different cells, with NF = 98. This suggests that the variability of the chromosome number in this char form is explained by Robertsonian translocations. In females and males, cells with respectively 2n = 78 and 2n = 77 prevailed. Heteromorphic sex chromosomes were not found in the karyotype of this char species. Nucleolar-organizing regions (NORs) were mostly detected in the telomeric regions of short arms of large submetacentric chromosomes. These features of the Krogius char karyotype demonstrate that this species is isolated from other Kamchatka chars, being similar to Taranetz char S. taranetzi from Chukotka Peninsula, which is in good agreement with morphological data.     

Atlantic salmon colonization of the Russian Arctic coast: pioneers from North America

Previous studies of the ESTD * isozyme locus in the Atlantic salmon show the * 80 allele to be absent across the species' European range, with the exception of northern Russia, whereas the allele is nearly fixed in North American populations. The allele was found in samples from 15 out of 18 rivers on the Kola Peninsula and White sea coast and had frequencies that ranged from 0·017 to 0·363. Typing of fish in nine of these rivers for mtDNA variation in the ND1 gene region found variation characteristic of North American salmon in three Kola Peninsula populations and is the only part of Europe where such variants have been detected. The study area was completely glaciated during the late Pleistocene period and the restriction of the European distribution of these ESTD * and mtDNA variants to this area suggests that salmon that colonized the Russian Arctic coast rivers included fish of North American origin after the Pleistocene glacier had retreated.