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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
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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
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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).
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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
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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
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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.
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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
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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
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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
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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
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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
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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. Grechko for reading and
proofreading the manuscript and providing helpful
comments.
This work was supported by the Russian Scientific
Foundation (project no. 145000029).
11.
12.
13.
14.
15.
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Translated by T. Tkacheva
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