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ISSN 00268933, Molecular Biology, 2015, Vol. 49, No. 3, pp. 358–368. © Pleiades Publishing, Inc., 2015. Original Russian Text © E.A. Shubina, E.V. Ponomareva, A.V. Klimov, A.V. Klimova, O.S. Kedrova, 2015, published in Molekulyarnaya Biologiya, 2015, Vol. 49, No. 3, pp. 405–416. MOLECULAR PHYLOGENETICS UDC 597.963.576.11 Repetitive DNA Sequences as an Indicator of the Level of Genetic Isolation in Fish E. A. Shubinaa, b, E. V. Ponomarevab, A. V. Klimovc, A. V. Klimovad, and O. S. Kedrovab a Belozersky Institute of PhyicoChemical Biology, Moscow State University, Moscow, 119992 Russia; email: [email protected] b Biological Faculty, Moscow State University, Moscow, 119992 Russia c Kamchatka Federal Research Institute of Fisheries and Oceanography, Petropavlovsk Kamchatskii, 683000 Russia d Kamchatka State Technical University, Petropavlovsk Kamchatskii, 683003 Russia Received January 12, 2015; in final form, January 17, 2015 Abstract—Although the functional role is still unknown for most types of nuclear noncoding repetitive sequences, some of them proved to provide adequate phylogenetic and taxonomic markers for studying the genetic relationships of organisms at the species and withinspecies levels. Several markers were used in this work. First, microsatellite markers were used to examine populations varying in the extent of genetic subdi vision in marine and anadromous fish, including the Chilean jack mackerel Trachurus murphyi, anadromous brown trout Salmo trutta, and isolated and anadromous char populations. Locus polymorphism was propor tional to the gene flow between populations in all cases. Second, satellite DNA was used to study the phylo genetic relationships within the genera Salmo, Oncorhynchus, Salvelinus, and Coregonus. Genetic distances agreed well with the taxonomic relationships based on morphological traits and various biochemical markers and correlated with the evolutionary ages estimated for the groups by other markers. Third, RAPD PCR with a set of 20mer primers was performed to study the genus Coregonus and anadromous and isolated popula tions and species of the genus Salvelinus. The resulting phylogenetic trees may help to resolve some disputable taxonomic issues for the groups. A comparison showed that several RAPDdetected sequences contain con served fragments of coding sequences and polymorphic repeats (minisatellites) from intergenic regions or introns. The finding point to a nonrandom nature of repetitive DNA divergence and may reflect the evolution of the fish groups examined. Heterochromatic satellite repeats were assumed to contribute to generating a reproductive barrier. DOI: 10.1134/S0026893315030152 Keywords: DNA tandem repeats, microsatellite and multilocus analyses, fish, genetic isolation, speciation INTRODUCTION Studies of DNA polymorphism have been espe cially intense in the past decades and have yielded ample genomic data for key species and taxa of the evolutionary tree, but have still failed to solve the prob lem of species and speciation, which is a basic problem of biology. The failure is explained by the discrete nature of a new species as a group evolving genetically independently and the continuous nature of evolution. In the biological concept of speciation in organisms with sexual reproduction is accumulating the differ ences that suffice to ensure partial or complete repro ductive isolation. In turn, the concept suggests unlim ited genetic recombination within a species and a dis continuation of gene flow between species [1, 2]. In the case of sympatric populations, which occupy the same area, intercrossing is continuously possible, and even weak gene flow is enough to prevent speciation. Thus, a reproductive barrier must suddenly arise between populations and spread rapidly to allow sym patric speciation. The objective of this work was to study the relation ship between changes in repetitive DNA and the extent of genetic isolation in fish. In addition, we intended to demonstrate in several models that an accumulation and fixation of mutations in noncoding and nonfunctional DNA strongly agrees with phyloge netic reconstructions developed for the groups under study on the basis of morphological traits and coding sequences. EXPERIMENTAL Marine, freshwater, and anadromous fish were used as models. Sample collections were kindly provided by the Department of Ichthyology (Moscow State Uni versity), Institute of General Genetics (Russian Acad emy of Sciences), and AllRussia Institute of Fisheries and Oceanography. DNA was isolated from ethanol fixed tissues (the liver, milt, and fatty fins) according to a published protocol [3] or a Silica method with a NucleoSpin (MachereyNagel, Germany) or DiatomTM DNA Prep 100 (IzoGen, Russia) kit. To study the 358 REPETITIVE DNA SEQUENCES AS AN INDICATOR 359 (a) (b) (c) Fig. 1. Example electrophoretic profiles of microsatellite loci of marine and anadromous fish. M, molecular weight marker (20bp ladder, BioRad, United States). (a) Locus Gsp08 in the Chilean jack mackerel Trachurus murphyi, (b) locus Tch14 of the Alaska pollock Theragra halcogramma, and (c) locus SSA197 of the brown salmon Salmo trutta. population genetic structure of the brown trout Salmo trutta from rivers of the White Sea basin, scales were used to isolate DNA [4]. The samples and analytical procedures have been described in detail previously [4–8]. To study the divergence of repetitive sequences, we chose the DNA markers that differed in evolution rate, which corresponded to the taxonomic level of the model in question. A microsatellite analysis of the population structure in the Chilean jack mackerel Trachurus murphyi was based on published data [9]; genotyping at microsatellites was carried out by PAGE in 8% gel. An analysis of the population structure in the Alaska pollock Theragra halcogramma has been described previously [8]. Satellite DNA was examined by a modified restric tion enzyme analysis. Fragments obtained by digesting DNA with restriction endonucleases were endlabeled with radioactive isotopes to greatly improve the resolv ing power of the method (taxonoprint) [10]. The digestion products were resolved by PAGE in 6% gel (20 × 40 cm2) and visualized autoradiographically. Optimal arbitrary 20mer primers for multilocus RAPD PCR were selected based on the variation and the reproducibility of amplification and electrophore sis in 1.5–2% agarose gel for each primer. Because all of our experiments were variants of a fragment analysis, Nei’s genetic distances [11] were used for phylogenetic inferences and were obtained MOLECULAR BIOLOGY Vol. 49 No. 3 2015 using the TreeConw1.3 program [12] with a bootstrap analysis. The RAPD PCR products to be sequenced were cloned in Escherichia coli with an InsTAcloneTM PCR cloning kit (Fermentas, Lithuania). Homologous sequences were search in the NCBI databases using BLAST and BLASTN software [13, 14]. RESULTS AND DISCUSSION Microsatellite DNA Three fish groups of a population level were exam ined. All of the groups were conventionally considered to be marine (although salmons are known to be anadromous) and to differ in the extent of their isola tion. First, a genetic analysis was performed for the Chilean jack mackerel Trachurus murphyi from the Pacific. This is the pelagic species that is characterized by long migrations, lack of geographical or other bar riers (e.g., the velocity of population movement or hydrographic conditions), and unlimited genetic exchange between populations [15]. Interspecific and intraspecific analyses with only genetic markers have not been described in the available literature [16, 17]. Close species differ in a complex of features (morpho logical and genetic traits and obligatory parasitic spe cies), but our analysis with three highly polymorphic microsatellite loci (example, Fig. 1a) has not detected signs of genetic subdivision for Trachurus murphyi populations (unpublished data). 360 SHUBINA et al. M 1 2 3 4 5 6 7 8 M 9 10 11 12 13 1415 16 1718 19 20 22 23 M Fig. 2. Example electrophoretic profiles of the microsatellite locus OMM1070 in isolated Dolly Varden char forms (the genus Salvelinus) from the Onekotan Island. M, molecular weight marker (20bp ladder, BioRad, United States). (1, 2) Anadromous S. malma malma from Kamchatka, (3–15) resident S. m. krasheninnikivi from the Glukhoe Lake (Onekotan, North Kuril Islands), (16–24) questionable species Salvelinus gritzenkoi from the Chernoe Lake of the same island. The Alaska pollock Theragra halcogramma was another model group. The species is a commercial subarctic marine fish; is characterized by long distant feeding, wintering, and spawning migrations; and is a shelf species. A minimal extent of geographic and hydrographic isolation is commonly assumed for the species; although the egg transfer is passive, feeding stocks mix and other factors contribute to high gene flow. Microsatellite loci are highly polymorphic in Theragra halcogramma (Fig. 1b), but their comparison still reveals certain differentiation of western and east ern Bering Sea populations. Moreover, significant sex dimorphism has been observed on the basis of on one of the ten loci for the Sea of Okhotsk population (a collection of 2006), i.e., males had a higher portion of homozygotes, thus deviating from the Hardy–Wein berg proportion. The finding possibly reflects the spe cifics of demographic processes in the population [8]. Thus, the population structure can be characterized in some cases even when stock isolation is low. The brown trout Salmo trutta from the Kandalak sha Gulf was used as a third model. In this case, a tight association with its habitat (homing), high reproduc tive isolation, and a low anthropogenic influence (because creeks are poorly accessible) make it possible to study the phenetic diversity and genetic variation of the species both spatially and temporally and to ana lyze the genetic structure for small populations located only 15 km apart. In addition, microsatellite loci of this anadromous species strikingly differ from highly polymorphic loci of the marine species, improving the reliability of the analysis. Common salmon microsatel lite markers are low polymorphic (3–5 alleles) (Fig. 1c) as a result of inbreeding. Still, the populations were in equilibrium and have a certain degree of safety now. Speciation is not observed, but a high extent of genetic subdivision is maintained owing to the salmon living strategy [4]. A more illustrative example of how the structure of microsatellite loci is associated with the extent of duration of population isolation is provided by elec trophoretic patterns obtained for locus OMM1070 in two lake isolates of the Far East char Salvelinus malma krasheninnikovi from the lakes Glukhoe and Chernoe of the Onekotan Island and the anadromous Kam chatka char S. malma malma from the Avacha River. The northern anadromous form and the resident pop ulation from the Glukhoe Lake have similarly sized alleles (Fig. 2). Differentiation of the two populations is quite expectable, but a standard allele frequency analysis would be most likely necessary for a statisti cally reliable estimation of the genetic distance, although the analysis could not be considered per fectly correct because of possible homoplasia. The other resident isolate (Chernoe Lake) from the Onekotan Island strikingly differs from the other two forms. In 2000, the population was described as a new char species, S. gritzenkoi, on the basis of morpholog ical and, especially, osteological traits [18]. While RAPD markers clearly differentiate good species, we performed a RAPD analysis to verify genetically the isolation of the new species. Our results did not con firm that S. gritzenkoi is a separate species, but a high level of withinpopulation inbreeding was noted [5]. This preliminary result might provide a weighty argu ment for the independent taxonomic status of the char form from the Chernoe Lake, needing further investi gation. Satellite DNA A method detecting the restriction sites in DNA [10] was used to study how divergence of extended repetitive DNA sequences is associated with the genetic distances in species and genera of the families Salmonidae and Coregonidae. A study of approxi mately 50 animal species has shown that band distri MOLECULAR BIOLOGY Vol. 49 No. 3 2015 REPETITIVE DNA SEQUENCES AS AN INDICATOR bution patterns (taxonoprints [19]) are species spe cific, while individual, sex, and betweenpopulation polymorphism is not observed. Major bands most likely originate from high copy tandem repeats [11, 15, 20–23]. Concerted evolution via molecular drive is known to result in the high withinfamily homogeneity of repeats in individuals of a developing group. Many observations confirm that differentiation of local pop ulations due to molecular drive is possible and that, in conditions of at least partial genetic isolation, a repeat family may consequently come to mark the genomes of closely related species [13, 15, 22, 24–27]. We have previously shown that these general concepts are traceable in evolution of repetitive sequences in salmonids of the genera Salmo, Parasalmo, and Onco rhynchus [6]. Homing, which is inherent in true salmon species, results in strong reproductive isolation and subsequent divergence of populations by morpho logical and molecular characters. We compared the NJ trees of salmonids differing in evolutionary age and the extent of genetic isolation (Fig. 3). A tree of the genera Salmo, Parasalmo, and Oncorhynchus shows that each genus forms a mono phyletic cluster with a good statistical support (Fig. 3a). In particular, this is true for Pacific trout species (the genus Parasalmo), which are conventionally isolated in a genus that is in sister relationships to Oncorhyn chus by Russian researchers and erroneously included in Oncorhynchus by the American Fishery Society based on the data [28]. As for the family Coregonidae (Fig. 3c), to which the biological species concept is often thought to be hardly applicable because of free interspecific and even intergeneric crosses (for a discussion, see [29, 30]), a population genetic approach is necessary for studying the genetic structure of one of its genera (the genus Coregonus). Yet distinct clades are formed in the tree by the Coregonus species to support their morphologi cal separation (Fig. 3c), although the distances are extremely short, as is seen from the branch length in the scaled dendrogram, and internal nodes are poorly resolved and have a low statistical support. American whitefish species is the only monophyletic cluster with a high support (97%). Apart from these species, a high extent of genetic isolation is characteristic of the only species, the Siberian round whitefish Prosopium cylin draceum, which is isolated in a separate genus in the family Coregonidae and shows no intercrosses with other species. The separation of the genus Prosopium from the other cluster is even more distinct on a UPGMA tree [14]. Salmonids of the genus Salvelinus (chars) are a group that is intermediate in the extent of interspecific genetic isolation between true salmons and whitefish species. The group includes many morphological forms and shows intense speciation and form develop ment, so that the taxonomic status of a species has been assigned to many of its resident forms. Considering the MOLECULAR BIOLOGY Vol. 49 No. 3 2015 361 lengths of the internal branches in the tree (Fig. 3c), only minor genetic distances separate all questionable species of the Salvelinus alpinus–Salvelinus malma complex [31], although geographical isolation is inev itable for the majority of these forms. “Species” are supported statistically only when separated by great genetic distances in the trees. Such species include only the white spotted char Salvelinus leucomaenis, which is commonly recognized as a good species, and Salvethymus svetovidovi, whose isolation in a separate genus by morphological features [32] is in line with our phylogenetic reconstruction. The repetitive DNA variation in all of the groups considered agrees not only with the extent of repro ductive isolation, but also with the time of clade diver sification, which occurred 8–10 million years ago in the case of true salmon species, approximately 16 mil lion years ago in the case of the genus Salvelinus, and less than one million years ago in the case of the inter mediate Salvelinus alpinus–Salvelinus malma species complex. Speciation in the genus Coregonus is thought to occur in glacial refugia approximately 15000 years ago [33, 34]. Thus, the internal nodes that support the morpho logical isolation of species capable of interspecific hybridization are poorly resolved in the evolutionary young group of whitefish species of the genus Corego nus, and the genetic distances are low. The findings agree with the idea that the taxonomic ranks are unreasonably high in the group [35, 36]. In view of substantial introgression [37] and the almost total absence of genetic distances [38], the genus Coregonus is now often considered to be a network of species [39–41]. As for the genus Salvelinus of the family Salmo nidae, our findings [5] support the taxonomic status only for S. leukomaenis and the endemic species (or genus) Salvethymus svetovidovi and agree with the idea that the species status lacks validity in the case of numerous morphological forms of the typical species Salvelinus alpinus. The topology of the tree con structed for the family Salmonidae [6], which is char acterized by absolute reproductive isolation of its spe cies, testifies that the genera Parasalmo and Oncorhyn chus are wrong to combine in one genus. RAPD PCR Markers We used a modified PCR protocol [42] with com binations of long (20mer, rather than 10mer as in most studies) arbitrary primers to improve the speci ficity and increase the number of detectable DNA regions. A total of 91 primer combinations were tested, and 15 of them selected for further experiments [5, 7]. Many morphological forms and species of chars of the genus Salvelinus, including both valid and question able ones, were used as a model. Samples were col lected from 29 populations of the regions of the Kuril Islands, Sea of Okhotsk coast, Kamchatka, Chukotka, 362 SHUBINA et al. (a) 0.1 55 84 Oncorhynchus tshawytscha Oncorhynchus nerka 99 Oncorhynchus kisutch Oncorhynchus gorbusha Oncorhynchus keta Oncorhynchus masou Parasalmo clarkii 100 Parasalmo mykiss (Oregon, United States) Parasalmo irideus 97 Parasalmo mykiss (anadromous form) 100 Parasalmo mykiss (resident form) 52 Parasalmo clarkii (cutthroat trout) Salmo trutta Salmo ischchan Salmo trutta сaspius 94 61 98 100 98 76 Salmo salar 47 39 39 40 66 69 99 89 99 78 (b) Salvelinus malma Salvelinus dryagini Salvelinus alpinus Salvelinus boganidae Salvelinus elgyticus Salvelinus leucomaenis Salvelinus confluentis Salvethymus svetovidovi Oncorhynchus masou Oncorhynchus irideus Oncorhynchus mykiss Oncorhynchus clarkii Salmo salar 100 58 97 67 (c) Coregonus clupeaformis (dwarf form, Como Lake, Canada) Coregonus clupeaformis (normally sized form, Como Lake) Coregonus nasus (Como Lake) Coregonus autumnalis (Como Lake) Coregonus arthedi (Lake Huron, United States) Coregonus sardinella Coregonus muksun 74 73 Coregonus lavaretus (anadromous form) Coregonus lavaretus (resident form, Anatti Lake, Finland) Stenodus leucichthys (nelma) Coregonus albula Coregonus autumnalis migratorius Coregonus nasus Coregonus pidschiani Coregonus lavaretus montchegor Prosopium cylindraceum Osmerus eperlanus dentex Fig. 3. NJ trees constructed for salmonids on the basis of Nei’s ditances with the TreeConW 3.b program and presented at the same scale. Bootstrap indices of more than 40 are given. Salmo salar was used as an outgroup. (a) The genera Salmo, Parasalmo, and Oncorhynchus. (b) The genus Salvelinus. Species of the genera Oncorhynchus and Parasalmo are shown in the basal part of the tree for a comparison. (c) The family Coregonidae. MOLECULAR BIOLOGY Vol. 49 No. 3 2015 REPETITIVE DNA SEQUENCES AS AN INDICATOR Taimyr, Transbaikalia, Kola Peninsula, Spitsbergen, Finland, and North America. Genetic similarity was studied for 35 S. alpinus krasheninnikovi (S. malma krasheninnikovi) samples, which were collected in the regions of the Shumshu, Paramushir, Onekotan, Itu rup, and Kunashir islands. The resolving potential of our primer system is characterized in Fig. 4. Theoretically, RAPD may involve any sequences, including both conserved cod ing and variable repetitive ones. Figure 4 shows the electrophoretic patterns obtained for five char species with several combinations of 20mer primers. Pooled DNA of all individuals of a group was always used in the reaction. It is clear that polymorphism of the markers is very high and that their relative resolving potential is suitable for characterizing both species and subspecies levels. In parallel with phylogenetic recon structions, we compared Nei’s absolute genetic dis tances between commonly recognized good species and questionable ones. Our results demonstrate that Arctic char forms considered to be species by morpho logical features are not always true biological species. The distances computed using the TreeConw1.3 pro gram were far lower than those between good species. A tree combining Dolly Varden and Arctic char forms is shown in Fig. 5. Although its resolution is low, the tree has several features of interest. The tree con sists of several unsupported blocks, which can be com bined by ecological factors (the geographical region and living strategy). For instance, anadromous S. alpi nus forms from Finland, Spitsbergen, and Northwest ern Russia cluster together. Isolated and endemic forms group in a separate block, and all of the Dolly Varden char forms group similarly. Dolly Varden char forms of the Russian Far East are close to Salvethymus svetovidovi, which is isolated in a separate genus in tax onomy. Its separation is supported by our restriction enzyme analysis of satellite DNA, as discussed above. Internal nodes are supported for the lake isolates of Dolly varden char and anadromous populations from Kamchatka and Paramushir. As expected, the white spotted char Salvelinus leucomaenis forms a wellsup ported cluster, while the good American species S. fontinalis and S. namaykush group separately to form a basal branch of the tree. The clustering of the two species is not surprising because hybrids resulting from their intercrossing are traceable for up to four generations [43]. A strict conformity between the phylogenetic rela tionships and geographical localization is similarly seen in chars from five of the Kuril Islands. Separate clusters are similarly formed by different populations of resident isolates or anadromous forms living in sim ilar ecological conditions on one island [7]. Thus, our RAPD marker system is suitable for studying the phy logenetic relationships at the species and intraspecific levels. The fact suggests a variation for the markers, as is characteristic of repeats. Moreover, divergence of noncoding DNA sequences can hardly explain the MOLECULAR BIOLOGY Vol. 49 No. 3 2015 363 above tendency to clustering in agreement with eco logical conditions. To study the nature of the markers, both monomor phic and variable fragments were sequenced and tested for homology in the NCBI databases. A BLAST anal ysis revealed similar sequences mostly in EST data bases [13, 14]. A typical search result is shown in Fig. 6 (see fullcolor insert). Only single RAPD fragments were similar to expressed (i.e., coding) sequences. Homology involved mostly one or two exon regions and a region that lacked similarity to any database sequence (EST, gene, or nucleotide databases), thus belonging to introns or intergenic spacers. The pres ence of gene regions in the amplification products may reflect traces of adaptive events in RAPDbased phy logenetic reconstructions. Conserved gene sequences cannot ensure a resolution of the polymorphism forms under study, and variable regions are used to construct a tree. Thus, a possible involvement of coding sequences in adaptation does not exclude evolution of repetitive sequences in any way. Polymorphism of RAPD fragments indicates that the fragments originate from repetitive sequences, but this origin has been demonstrated experimentally for only one fragment as yet. Part of this RAPD fragment, which was amplified from Kuril Dolly Varden char S. malma krasheninnikovi, was similar to cDNA of the Danio rerio epinephrine transmitter protein. Its gene is completely sequenced, and its intron–exon structure is known. One end of our RAPD fragment coincides with the 5' end of one of the gene exons, while a major part of the fragment lacks homology to the preceeding D. rerio intron. We failed to completely sequence the char intron, but DNA reamplification from an exon primer into the intron was performed for several Dolly Varden char populations of the Kuril Islands and showed that the products were the same in size and sequence, with only one exception. One product was almost the same in size as the corresponding RAPD fragment, while the other products were 1.5fold shorter. The long and short sequences were compared using BLAST software (Fig. 6). The intron regions were identical in sequence, but with gaps of approximately 50 nt in some regions. Such a structure may suggest a minisatellite, which is a tandem repeat of up to 1000 bp in length and have repeat units of 50–70 bp. Thus, tan dem repeats, which seem to occur in introns, show the same properties as pericentric chromatin. CONCLUSIONS The main objective of this work was to examine fish forms by means of a molecular genetic analysis with noncoding DNA markers, which may reflect the extent of taxon separation at the species and intraspe cific levels and at various stages of allopatric or sympa tric speciation. The markers (microsatellites, satel lites, and DNA regions amplified with arbitrary prim MOLECULAR BIOLOGY Vol. 49 292 403 526 356 432 622 642 908 1083 1327 1296 346 1184 810 2081 306 245 392 622 M2 202 285 508 731 887 226 1084 193 535 775 1003 Reaction with 30sm 2 3 4 5 M1 M2 1448 1366 1195 1 (b) (a) 249 509 746 931 1414 1836 297 404 614 M1 M2 380 1018 2033 1602 329 457 679 1783 1305 Reaction with 15sm 1 2 3 4 5 M1 980 260 506 288 468 387 341 1190 1169 1057 1023 1001 873 786 693 620 1456 806 360 533 558 1374 309 425 Reaction with 58sm M1 1 2 3 4 5 M2 Fig. 4. RAPD PCR analysis of char forms and species of the genus Salvelinus demonstrates polymorphism of the markers used. (a) Electrophoretic patterns obtained with dif ferent primer combinations. (b) Corresponding schemes. A 8kb DNA ladder and pBR 322/HindIII digest were used as molecular weight markers. Lanes: 1, 2, Dolly Varden char Salvelinus malma krasheninnikovi from the (1) South (Iturup and Kunashir) and (2) North (Shumshu, Paramushir, and Onekotan) Kuril Islands; 3, White spotted char Salvelinus leucomaenis; 4, Salvelinus malma malma from Kamchatka; and 5, Arctic char Salvelinus alpinus. Fragment sizes (bp) are indicated. 245 496 764 956 1536 1878 Reaction with 31sm M1 M2 1 2 3 4 5 364 SHUBINA et al. No. 3 2015 REPETITIVE DNA SEQUENCES AS AN INDICATOR 90 365 Salvelinus malma malma (anadromous form, Kamchatka) Salvelinus malma krasheninnikovi (anadromous form, Paramushir) Salvelinus malma malma (anadromous form, Bering Island) Salvethymus svetovidovi Salvelinus malma krasheninnikovi (resident form, Onekotan Island) Salvelinus malma krasheninnikovi (Lake resident isolate, Onekotan Island) Salvelinus malma malma (Predatory Lake char, Kamchatka) Salvelinus alpinus (resident dwarf form, Finland) Salvelinus “boganidae” (Boganida char) Salvelinus alpinus (anadromous form, Finland) Salvelinus “dryagini”(endemic lake–river form, Taimyr) Salvelinus alpinus (Lake resident form, Finland) Salvelinus alpinus (anadromous form, Svalbard) Salvelinus alpinus (Goggleeyed char, lake resident form, Taimyr) Salvelinus alpinus neiva (lake resident form, Sea of Okhotsk coast) Salvelinus alpinus (Davatchan, resident form, Transbaikalia) Salvelinus alpinus (Lake Black char, Taimyr) Salvelinus alpinus (Murman char, anadromous form) Salvelinus alpinus (Mountain char, lake–river form, Taimyr) Salvelinus leucomaenis (Kamchatka) Salvelinus leucomaenis (South Kuril Islands) Salvelinus fontinalis (Lake Brook Trout, lake char, United States) Salvelinus namaycush (Lake Trout, lake char, United States) 42 87 95 91 78 53 57 96 Fig. 5. Unrooted UPGMA tree of char forms and species of the genus Salvelinus as constructed on the basis of the RAPD PCR analysis. Bootstrap indexes higher than 40% are shown. ers (RAPDs)) reflect the specifics of heterochromatin, whose role in reproductive isolation is still unclear and may be important. While new theoretical models were developed and empirical results obtained to characterize the genetic basis of speciation (for a review, see [44]), it remains unknown what part of the genome is responsible for reproductive isolation [45] and what is a primary fac tor that triggers rapid genome changes to generate a reproductive barrier. The majority of the models asso ciate postzygotic speciation with a situation where new alleles of coding sequences arise to cause chromo somal incompatibility in meiosis. Although repetitive DNA is no longer thought to be “selfish”or “junk” by many researchers, only a structural and, to a certain extent, regulatory roles are still commonly recognized for heterochromatin [46–48], along with maintaining the information depot and contributing to the organi zation of transferring genetic information to the next generation. However, the idea that a reproductive barrier can not arise without a cessation of gene flow between hybridizing populations is omitted in all of the models. According to Lewontin [49], if the theory of speciation has an element that might be true in all cases, it is the concept that geographical isolation and a strong MOLECULAR BIOLOGY Vol. 49 No. 3 2015 restriction of gene exchange between populations are a first essential step to speciation. With a low evolution ary rate of the majority of nuclear genes, a mutation may arise and spread through the total population only in conditions of longterm geographical isolation, so that a reproductive barrier cannot form between pop ulations that are in reproductive contact. In the con text of Mayr’s biological species concept [1], the only mechanism conceivable for reproductive isolation is based on concerted evolution of extremely simple, extended repetitive sequences. Assuming that tandem repetitive sequences are not subject to selective pres sure, they are theoretically capable of mutating and fixing the mutations independently of each other, that is, stochastically. Our study demonstrates that this is not true. All phylogenetic reconstructions based on highly repetitive noncoding DNA follow the conse quence of evolutionary events known for the model organisms in question (for a review, see [50]). While the fish groups chosen for the study substantially differ in main evolutionary stages and divergence time [33, 34, 51–55], an integral genome analysis, which simultaneously addresses many anonymous loci, makes it possible to reduce the inequality errors caused by the differences in the molecular evolution rates of macromolecules [56]. Absolute genetic dis 366 SHUBINA et al. (a) (b) r 1 60 120 180 240 300 1 100 200 300 400 500 250 300 350 400 450 500 550 600 650 700 750 826 (c) 1 150 300 450 600 750 500 400 300 |cl|8555 200 |cl|16553 Fig. 6. Graphic representation of the results of searching the NCBI databases for nucleotide sequences similar to RAPD markers. S(a) and S(b) are examples of conserved expressed sequences found in amplification fragments. S(c) is a minisatellite sequence. tances were used as a criterion in the char group, which included almost all of the species and isolated forms (questionable allopatric “species”) [57]. The approach is adequate, as evident from the fact that identical nucleotide sequences were established for RAPD fragments that were amplified with the same primers for different populations and had the same electrophoretic mobility. Our taxonoprint analysis of Atlantic and Pacific salmon (the genus Oncorhynchus) and trout (the genus Parasalmo) species showed that the set of DNA frag ments was specific for each species or even a form. The results showed additionally that Parasalmo is highly separate from the Oncorhynchus species [14]. More over, significant differences in taxonoprints were observed for sympatric species, which inhabit the same area. The set included Pacific salmons of the genus Oncorhynchus (O. keta, O. gorbuscha, O. nerka, O. tshaw ytscha, O. masou, and O. kisutsch), two Salvelinus spe cies (S. alpinus and S. leucomaenis), and two chars of the El’gygytgyn Lake (Salvethymus svetovidovi and Salvelinus alpinus). All disputable issues of the taxonomy of our model cannot be considered here in more detail because of space limitations. Yet almost all main nodes of the phylogenetic reconstructions based on taxonoprint and RAPD analyses coincide with those of wellsup ported consensus trees based on sequences of several genes [58], and allelic diversity of microsatellite and intergenic minisatellite repeats interspaced through the genome corresponds to the extent of genetic isola tion of the relevant populations and species. These findings directly implicate repetitive sequences in the establishment of reproductive isolation. A similar conclusion can be made from the data that Xchromosomal heterochromatin plays a sub stantial role in establishing the reproductive barrier between two sister Drosophila species [59, 60]. Finally, Shapiro and von Sternberg’s [46] concept of genome system architecture suggests that changes in repetitive MOLECULAR BIOLOGY Vol. 49 No. 3 2015 REPETITIVE DNA SEQUENCES AS AN INDICATOR DNA play a role in evolutionary diversifications rather than being transmitted through generations without any phenotypic expression. Following these authors, we also think that basic evolutionary events may be ini tiated in the repetitive genome portion, providing impetus toward the advent of new species. ACKNOWLEDGMENTS This work is dedicated to the memory of the prom inent Russian biologists B.M. Mednikov, K.A. Savvai tova, and O.F. Gritsenko. We are grateful to the coauthors of the studies reported previously; researchers of the Department of Ichthyology and AllRussia Institute of Fisheries and Oceanography for collecting unique samples in long range expeditions; our colleagues for interest in the study, discussions, and help in manuscript preparation for publication; and V.V. 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