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.     

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.     

Specific features of ecology of chars of the genus <Emphasis Type="Italic">Salvelinus</Emphasis> (Salmonidae) from the basin of Lake Kronotskoe (Kamchatka) according to parasitological data

A parasitological study was performed of chars of the genus Salvelinus inhabiting Lake Kronotskoe (Kamchatka Peninsula)—S. malma, S. albus, S. schmidtii, and S. kronocius, as well as of juvenile Salvelinus spp. Twenty-three species of parasites, including six species new for the lake, Hennequya zschokkei, Protteocephalus longicollis, Diphyllobothrium dendriticum, Crepidostomum sp., Echinorhynchus salmonis, and Paracanthobdella livanowi, were found. With consideration of published data, in chars of this water body, 28 species of parasites were recorded, including seven species (N. cf. pungitius, B. luciopercae, Crepidostomum sp., Cr. fausti, Cr. cf. cooperi, Eubothrium crassium, and Proteocephalus sp.), whose presence or species identification in the lake ecosystem need confirmation. Two species (N. rutili and Diphyllobothrium sp.) are removed from the list. Parasites common for all species of chars were revealed. They include Myxobolus arcticus, E. salvelini, D. ditretum, Crepidostotum sp., Cr. farionis, Cr. metoecus, Cystidicola farionis, Cucullanus truttae, Philonema oncorhynchi, and Salmincola carpionis. Cluster analysis of the fauna of parasites of different species of chars demonstrated considerable differences in infestation, which indicates differences between them in preference for food items and occupied biotopes and thereby supports the ecological differentiation of chars in the basin of Lake Kronotskoe. S. albus and S. kronocius are most similar in parasitofauna, which is determined by their predation; S. malma as a benthophage is infected by the same species of parasites, but considerably less intensively. Extremely high indices of population numbers of some parasite species are considered as a manifestation of the Krebs cycle in parasites under the conditions of an isolated lake.     

Genetic connectivity between sympatric populations of closely related char species,Dolly Varden <Emphasis Type="Italic">Salvelinus malma</Emphasis> and white char <Emphasis Type="Italic">Salvelinus albus</Emphasis>

The closely related chars Salvelinus malma and Salvelinus albus, which sympatrically inhabit the Kamchatka River basin and Kronotsky Lake (Kamchatka), attract the attention of the researchers because of their debated origin and taxonomic status. Previous studies of sympatric populations of these chars revealed small but statistically significant genetic differences between these species at a number of molecular markers, suggesting the presence of the genetic exchange and hybridization. In this study, based on genotypic characterization of nine microsatellite loci, a considerable level of historical and contemporary genetic migration between sympatric populations of these chars was demonstrated. At the individual level, a high degree of hybridization was observed, mainly among the Dolly Varden individuals from the studied populations. The obtained evidences on the genetic connectivity between sympatric S. malma and S. albus do not support the separate species status of S. albus.     

Genetic relationships of Chukchi charr <Emphasis Type="Italic">Salvelinus andriashevi</Emphasis> and Taranetz charr <Emphasis Type="Italic">Salvelinus taranetzi</Emphasis>

On the basis of sequence analysis of the mitochondrial DNA (mtDNA) control region (CR), cytochrome b (Cytb), and cytochrome oxidase-1 (CoI) genes, the relationships of endemic species Salvelinus andriashevi Berg, 1948, represented by the only population from Lake Estikhed (Chukotka), were estimated. The data on the genealogical analysis of mtDNA haplotypes supported phylogenetic closeness of S. andriashevi and S. taranetzi. It was also demonstrated that the specimens of Chukchi charr, along with Salvelinus sp. 4 (Lake Nachikinskoe), S. krogiusae (Lake Dal’nee), S. boganidae and S. elgyticus (Lake El’gygytgyn), and S. a. erythrinus from Canada’s Northwest Territories (NWT) belonged to the Arctic group of Taranetz charr. The problem of coincidence of taxonomic differentiation of charrs of the genus Salvelinus based on morphological and genetic analyses is discussed.     

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.     

Ecological differentiation of resident Dolly Varden <Emphasis Type="Italic">Salvelinus malma</Emphasis> (Salmonidae) from Lake Dal’nee,Kamchatka

Resident Dolly Varden Salvelinus malma of Lake Dal’nee (Paratunka River basin) is represented by two stable trophic groups: one group feeds on mainly amphipods while another on gastropods. The former group is dominantly infected by Cyathocephalus truncatus and Crepidostomum metoecus, whereas the latter group is infected with Crepidostomum farionis, Ichthyocotylurus erraticus, and Diplostomum spp. The fishes of the former group grow faster and defined by higher fat content of muscular tissue. In addition, they accumulate monoenic fatty acids in the muscles. The fishes of the latter group differ in a high content of omega-3 and omega-6 fatty acids in the muscles. The amphipod foragers are characterized by a larger head with elongated upper jaw and comparatively short fins. A similar ecological differentiation is observed in the littoral benthivorous charrs from Lake Kronotskoe. The group feeding on amphipods was found in both lakes. In Lake Kronotskoe, the second group consumes the most abundant sedentary benthos, mollusks and chironomids.     

The karyotype of the great sculpin, <Emphasis Type="Italic">Myoxocephalus polyacanthocephalus</Emphasis> (Pallas, 1814) (Pisces: Cottidae), from the Russian part of the species range

The karyotype of the great sculpin, Myoxocephalus polyacanthocephalus (Pallas, 1814) (Pisces: Cottidae) from the Sea of Okhotsk and the Sea of Japan has been studied for the first time. The karyotype is stable; it consists of 40 chromosomes (4 metacentric, 2 submeta-subtelocentric, 20 subtelocentric, and 14 acrocentric chromosomes); the number of chromosomal arms is 44 + 2. Nucleolar organizer regions (NOR) are found in the telomeric region of the arm in one homologue of a pair of small metacentric chromosomes, using the Ag-NOR banding technique. A comparative analysis of the karyotype of M. polyacanthocephalus and the karyotypes of other Myoxocephalus species (M. stelleri, M. brandtii, M. jaok, M. ochotensis, and M. scorpius) has been carried out based on the main karyotype characters, as well as on the number and localization of NORs. The identified differences make it possible to differentiate the studied species, whereas the general traits indicate their taxonomic proximity.     

Molecular phylogeny of the marine <Emphasis Type="Italic">Prasiola</Emphasis> and <Emphasis Type="Italic">Rosenvingiella</Emphasis> species (Chlorophyta: Prasiolales) from southeastern Kamchatka

Molecular phylogenetic tools are often useful in distinguishing cryptic species with similar morphologies, but they can also be helpful in identifying morphotypes of a species, which displays completely different shapes. We performed molecular-phylogenetic analysis of supra-tidal green algae in the Kamchatka peninsula that belong to the order Prasiolales (Trebouxiophyceae, Chlorophyta). Based on rbcL sequences results, two new species were recorded for the first time in Kamchatka. Approximately 1.4% of the field-collected Rosenvingiella constricta had a unique uniseriate hood-like blade shape, although their rbcL sequences were 100% identical with typical multiseriate filamentous plants. Kamchatka’s population of R. constricta showed 99.4–99.6% identity in rbcL sequences with the populations from Canada and New Zealand. Another similar-looking Rosenvingiella species collected from the same locality had 93.5% identity of the rbcL gene sequence with R. constricta. Morphological and geographical analyses also suggested that this species might be a new species of the genus Rosenvingiella. Prasiola delicata was recorded for the first time in Kamchatka. The Kamchatka population of P. delicata showed 100% identity in rbcL gene sequence with the population from Vancouver, but differed from the Canadian population morphologically.     

Genetic diversity of charrs of the Commander Islands based on the analysis of mitochondrial DNA

Nucleotide sequences of the mitochondrial DNA cytochrome b (CytB) gene fragment and the control region (D-loop) of Dolly Varden (Salvelinus malma) from the Commander Islands and the Kol River of the Kamchatka Peninsula were examined. A high level of genetic variability of island populations comparable to that of the mainland population of western Kamchatka was demonstrated. The belonging of the Commander Islands chars to the genetic lineage of northern Dolly Varden Salvelinus malma malma was confirmed.     

On the status and identity of the white char, <Emphasis Type="Italic">Salvelinus albus</Emphasis> Glubokovsky, 1977, from different habitats

The white char, Salvelinus albus Glubokovsky, 1977, which was originally described from the Kamchatka River basin, and the white char from Lake Kronotsky, Kamchatka Peninsula, are believed to be identical taxonomically and represent two reproductively isolated populations of the same species. However, the data on their molecular divergence show that they may differ at a higher, probably sub-species, level. This suggests the necessity of deeper complex studies of white char and other closely related char species and forms.     

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.     

Genetic divergence of sympatric charrs of the genus <Emphasis Type="Italic">Salvelinus</Emphasis> from Nachikinskoe Lake (Kamchatka)

We studied genetic differentiation of two charr species, Dolly Varden Salvelinus malma malma Walbaum and resident lacustrine charr Salvelinus sp., which sympatrically inhabit Nachikinskoe Lake (the Bol’shaya River basin) in southwestern Kamchatka Peninsula. Using restriction analysis (RFLP), three mitochondrial DNA fragments (ND1/ND2, ND5/ND6, and Cyt b/D-loop) amplified in polymerase chain reaction (PCR) were compared. The divergence of the mtDNA sequences between Salvelinus sp. and S. malma malma was 2.8%; Salvelinus sp. and S. taranetzi, 0.36%; Salvelinus sp. and S. krogiusae, 0.21%; Salvelinus sp. and S. alpinus, 3.0%. These results point to reproductive isolation of charrs in Nachikinskoe Lake and support the earlier suggestion on a close relationship between Salvelinus sp., S. taranetzi, and S. krogiusae.     

Molecular genetic and karyological analysis of antlered sculpins of <Emphasis Type="Italic">Enophrys diceraus</Emphasis> group (Cottidae)

Molecular genetic and karyological analyses of antlered sculpin, Enophrys diceraus, from the Sea of Japan and the Sea of Okhotsk were carried out. The karyotype of this species was studied for the first time. On the basis of karyological analysis, it was established that E. diceraus from the Sea of Japan and the Sea of Okhotsk was polymorphic in terms of the number of chromosomes and their morphology (2n = 36, 35, and 37, NF = 40). It was suggested that the karyotype with 35 chromosomes could have been produced as a result of Robertsonian translocation; the karyotype with 37 chromosomes could have been produced by crossing of individuals with different number of chromosomes. On the basis of the molecular genetic analysis of the mitochondrial COI and 16S rRNA genes, considerable differences between E. diceraus from the Sea of Japan and the Sea of Okhotsk corresponding to the level of interspecies genetic variability were established. It is concluded that E. diceraus from the Sea of Japan belongs to another species, most likely, E. namiyei.     

The Karyotype of Chukotka Chars from the Estikhed Lake (Eastern Chukotka)

The karyotype of chars from the Estikhed Lake (Eastern Chukotka) was examined. This karyotype comprises 78 chromosomes, NF = 98. Marker chromosomes include one pair of submetacentrics, one pair of large acrocentrics, and one pair of large subtelocentrics with very short second arms. Nucleolus organizer regions are located in telomeric regions of short arms of marker submetacentric chromosomes. Small heterochromatin blocks are observed in centromeric regions of most chromosomes. The Chukotka char karyotype is very similar to that of Taranetz charSalvelinus taranetzi from the Achchen Lake: these karyotypes differ only in stability of the chromosome number.     

Location of low copy genes in chromosomes of <Emphasis Type="Italic">Brachiaria</Emphasis> spp.

Repetitive DNA sequences have been widely used in cytogenetic analyses. The use of gene sequences with a low-copy-number, however, is little explored especially in plants. To date, the karyotype details in Brachiaria spp. are limited to the location of rDNA sites. The challenge lies in developing new probes based on incomplete sequencing data for the genus or complete sequencing of related species, since there are no model species with a sequenced genome in Brachiaria spp. The present study aimed at the physical location of conserved genes in chromosomes of Brachiaria ruziziensis, Brachiaria brizantha, and Brachiaria decumbens using RNAseq data, as well as sequences of Setaria italica and Sorghum bicolor through the fluorescent in situ hybridization technique. Five out of approximately 90 selected sequences generated clusters in the chromosomes of the species of Brachiaria studied. We identified genes in synteny with 5S and 45S rDNA sites, which contributed to the identification of chromosome pairs carrying these genes. In some cases, the species of Brachiaria evaluated had syntenic segments conserved across the chromosomes. The use of genomic sequencing data is essential for the enhancement of cytogenetic analyses.     

Cytogenetic analyses of eight species in the genus <Emphasis Type="Italic">Leptodactylus</Emphasis> Fitzinger, 1843 (Amphibia,Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations

Background

The karyotypes of Leptodactylus species usually consist of 22 bi-armed chromosomes, but morphological variations in some chromosomes and even differences in the 2n have been reported. To better understand the mechanisms responsible for these differences, eight species were analysed using classical and molecular cytogenetic techniques, including replication banding with BrdU incorporation.

Results

Distinct chromosome numbers were found: 2n = 22 in Leptodactylus chaquensis, L. labyrinthicus, L. pentadactylus, L. petersii, L. podicipinus, and L. rhodomystax; 2n = 20 in Leptodactylus sp. (aff. podicipinus); and 2n = 24 in L. marmoratus. Among the species with 2n = 22, only three had the same basic karyotype. Leptodactylus pentadactylus presented multiple translocations, L. petersii displayed chromosome morphological discrepancy, and L. podicipinus had four pairs of telocentric chromosomes. Replication banding was crucial for characterising this variability and for explaining the reduced 2n in Leptodactylus sp. (aff. podicipinus). Leptodactylus marmoratus had few chromosomes with a similar banding patterns to the 2n = 22 karyotypes. The majority of the species presented a single NOR-bearing pair, which was confirmed using Ag-impregnation and FISH with an rDNA probe. In general, the NOR-bearing chromosomes corresponded to chromosome 8, but NORs were found on chromosome 3 or 4 in some species. Leptodactylus marmoratus had NORs on chromosome pairs 6 and 8. The data from C-banding, fluorochrome staining, and FISH using the telomeric probe helped in characterising the repetitive sequences. Even though hybridisation did occur on the chromosome ends, telomere-like repetitive sequences outside of the telomere region were identified. Metaphase I cells from L. pentadactylus confirmed its complex karyotype constitution because 12 chromosomes appeared as ring-shaped chain in addition to five bivalents.

Conclusions

Species of Leptodactylus exhibited both major and minor karyotypic differences which were identified by classical and molecular cytogenetic techniques. Replication banding, which is a unique procedure that has been used to obtain longitudinal multiple band patterns in amphibian chromosomes, allowed us to outline the general mechanisms responsible for these karyotype differences. The findings also suggested that L. marmoratus, which was formerly included in the genus Adenomera, may have undergone great chromosomal repatterning.

     

Karyotype of the sculpin <Emphasis Type="Italic">Myoxocephalus jaok</Emphasis> (Pisces: Cottidae) from Peter the Great Bay,Sea of Japan

The karyotype of the sculpin Myoxocephalus jaok from the Ussuriisky, Amursky, and Vostok Bays was studied. It was found that 2n = 24, among the chromosomes there were 16 metacentric, 4 submetacentric, and 4 acrocentric, NF = 44. There was no variation in the number of chromosomes, and no differences between male and female karyotypes were revealed. A comparison of Myoxocephalus jaok, M. brandti and M. stelleri karyotypes was performed. The pronounced distinction of Myoxocephalus jaok karyotype resulting from chromosomal rearrangements was shown.     

Karyotype of Chukotka char from Estikhed Lake (Eastern Chukotka)

The karyotype of chars from the Estikhed Lake (Eastern Chukotka) was examined. This karyotype comprises 78 chromosomes, NF = 98. Marker chromosomes include one pair of submetacentrics, one pair of large acrocentrics, and one pair of large subtelocentrics with very short second arms. Nucleolus organizer regions are located in telomeric regions of short arms of marker submetacentric chromosomes. Small heterochromatin blocks are observed in centromeric regions of most chromosomes. The Chukotka char karyotype is very similar to that of Taranetz char Salvelinus taranetzi from the Achchen Lake: these karyotypes differ only in stability of the chromosome number.     

Phylogeography of mitochondrial DNA in South Asian Dolly Varden char <Emphasis Type="Italic">Salvelinus curilus</Emphasis> Pallas, 1814 (Salmoniformes,Salmonidae): Mediated gene introgression?

In 41 individuals of South Asian Dolly Varden char Salvelinus curilus, nucleotide sequences of tRNA-Pro gene fragment (27 bp) and mtDNA control region (483–484 bp) were analyzed. The fish were collected in 20 localities covering virtually the whole range of the species: Kuril Islands, Sakhalin Island, and Primorye. In addition, six individuals of three other char species (S. albus, S. malma, and S. leucomaenis), which are closely related to S. curilus and inhabit the Russian Far East, were examined. In all, we detected 12 different variants of mtDNA haplotypes that formed three distinct groups differing in 14–20 nucleotide positions. The first group consisted of six haplotypes found in S. curilus in Kuril Islands, Sakhalin, and Primorye (mtDNA phylogroup OKHOTSKIA). The second group comprised four haplotypes representing the mtDNA phylogroup BERING, which had been described earlier (Brunner et al, 2001); they were found in S. curilus in Kuril Islands and Sakhalin, as well as in S. albus and S. malma in Kamchatka and northern Kurils. The third group included two haplotypes detected in S. leucomaenis. The existence of two mtDNA lineages (OKHOTSKIA and BERING) in S. curilus from Kurils and Sakhalin was explained by hybridization and mtDNA transfer from S. malma to S. curilus. The absence of the BERING haplotypes in S. curilus from Primorye water reservoirs is related to the physical isolation of the Sea of Okhotsk and Sea of Japan basins in past epochs. On the basis of comparing phylogenetic trees, constructed from the data on allozyme and mtDNA variation, we suggest that in this case, a mediated transfer of mtDNA in Alpinoid chars → S. malma → S. curilus chain could occur.