APPLICABILITY OF SEQUENCE DIVERSITY AT MITOCHONDRIAL GENES ON DIFFERENT TAXONOMIC LEVELS IN GENETICS OF SPECIATION, PHYLOGENETICS AND BARCODING

1
APPLICABILITY OF SEQUENCE DIVERSITY AT
MITOCHONDRIAL GENES ON DIFFERENT TAXONOMIC LEVELS
IN GENETICS OF SPECIATION, PHYLOGENETICS AND
BARCODING

• Yuri Ph. Kartavtsev
• A.V. Zhirmunsky Institute of Marine Biology,
  Vladivostok 690041, Russia
• e-mail yuri.kartavtsev48_at_hotmail.com

2
Teacher Academician, prof. Yuri P. Altukhov,
1992-2006 director, N.Vavilov Institute of
General Genetics, Moscow (Russia)
3
MAIN GOALS

1. CBOL Fish-BOL
2. SPECIES IDENTIFICATION
3. SPECIES DEFINITIONAND SPECIES ORIGINPROBLEMS,
   RESTRICTIONS. GENETIC VIEW.

4
1. CBOL Fish-BOL
5
THE INTERNATIONAL CBOL PROJECT

• The CBOL is main global initiative. The Fish-BOL,
  its part, has over 5400 species barcoded by Co-1
  from more than 30,000 specimens what makes it
  unique. P. Hebert and B. Hanner are preparing a
  150M grant application for Genome Canada only
  for 2008. Other nations funds in CBOL are also
  big in some countries and unions USA, EU.
• B. Hanner suggests a recent Fish-BOL paper on
  Canadian freshwater fishes for your interest, as
  well as a new paper in press that demonstrates
  barcoding can identify cases of market
  substitution in North American seafood. These
  might be relevant for our meeting and ensuing
  discussions!
• In this year there will be held third world-wide
  international conference (Sept. 2008 Chindao,
  Peoples Republic of China) and many regional
  meeting like us were performed.

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THE INTERNATIONAL FISH-BOL PROJECT
Cochairmen P. Hebert B. Ward

7
Fish-BOL CURRENT STATE (2006 vs 2008)
Class Barco-ded Species Prog-ress Class Barco-ded Species Prog-ress
Actinopterygii 3623 5073 27984 27984 13 18 Cephalaspidomorphi 9 17 42 42 21 40
Elasmobranchii 259 358 968 968 27 37 Holocephali 15 15 37 37 41 41
Myxini 7 8 70 70 10 11 Sarcopterygii 0 2 11 11 0 18
8
Registration and Barcoding Utilities(BolD
www.boldsystems.org) (1)
9
Registration and Barcoding Utilities(BolD
www.boldsystems.org) (2)
10
Registration and Barcoding Utilities(BolD
www.boldsystems.org) (3)
11
Chair Masaki Miya Vice Chair Shunping He
Members Nina Bogutskaya Seinen Chow Shunping
He Yuri Kartavtsev Keiichi Matsuura Masaki
Miya Mutsumi Nishida Ekaterina Vasilieva
North East Asian Regional Working Group
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FISH-BOL. RUSSIA DEVELOPMENT
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2. SPECIES IDENTIFICATION
14
Some Objects
A
B
Fig. 1. Halibut-like flatfish, Hypoglossus
elasodon (A) and obscure flatfish,
Pseudopleuronectes obcurus (?).
15
INTRODUCTION

• Mitochondrion DNA (mtDNA) is a ring molecule of
  16-18 kilo-base pairs (kbp) in length. As
  literature data show, mtDNA of all fishes has
  similar organization (Lee et al., 2001 Kim et
  al., 2004 Kim et al., 2005 Nagase et al., 2005
  Nohara et al., 2005) and small differences among
  all vertebrate animals, including men (Anderson
  et al., 1981 Bibb et al., 1981 Wallace, 1992
  Kogelnik et al., 2005).
• The complete content of whole mitochondrial
  genome (mitogenome) includes control region (CR
  or D loop), where the site of initiation of
  replication and promoters are located, big (16S)
  and small (12S) rRNA subunits, 22 tRNA and 13
  polypeptide genes.
• Usually in phylogenetic research single gene
  sequences are used for both mtDNA and nuclear
  genome. However, recently more and more frequent
  are become complete mitogenome usage. Japanese
  scientists are leading here for water realm
  organisms.
• Most popular in phylogenetics are sequences of
  cytochrome b (Cyt-b) and cytochrome oxidase 1
  (C?-1) genes, which used for taxa comparison at
  the species - family level (Johns, Avise, 1998
  Hebert et al., 2004 Kartavtsev, Lee, 2006). Many
  sequences that bringing the phylogenetic signal
  obtained for different taxa at gene 16S rRNA as
  well.
• Sequences of separate genes can dive different
  phylogenetic signal because of differences in
  substitution rates. This is also true for
  different sections of genes. Also, under
  comparison of higher taxa there may be effects of
  homoplasy. When numerous taxa available there are
  problems of insufficient information capacity of
  sequences to cover big species diversity and
  representative taxa representation is quite
  important (Hilish et al., 1996). Nevertheless,
  for the species identification, excluding rare
  cases, fine results are available even with the
  usage of short sequences, like ??-1, with 650 bp.

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Applicability of Different DNA Types in
Phylogenetics and Taxonomy
Species Genus Family Order Class
Phylum
Most substantiated statistically results
Statistically significant results
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MATERIAL AND METHODS
18
Aligned flatfish sequences at ??-1 our and
GenBank data
19
Distance Data
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p-DISTANCES IN GROUPS OF COMPARISON,Catfish
Fig. 1. Resulting graph of mean p-distance values
at four levels of differentiation in the catfish
species (Siluriformes) for Cyt-b gene. Groups 1.
Intraspecies, among individuals of the same
species 2. Intragenus, among species of the same
genera 3. Intrafamily, among genera of the same
family 4. Intraorder, families of the order
Pleuronectiformes. There are statistically
significant variation. SE a standard error of
mean F 124.74, d.f. 3 29, p lt 0.0001
(Kartavtsev et al., 2007a, Gene).
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p-DISTANCES IN GROUPS OF COMPARISON,flatfish

• Fig. 2. Resulting graph of one factor ANOVA and
  mean p-distance values at four levels of
  differentiation in the flatfish species
  (Pleuronectiformes) for Cyt-b gene. Groups 1.
  Intraspecies, among individuals of the same
  species 2. Intragenus, among species of the same
  genera 3. Intrafamily, among genera of the same
  family 4. Intraorder, families of the order
  Pleuronectiformes. Statistically significant
  variation are shown on top of the graph. SE a
  standard error of mean (Kartavtsev et al., 2007b,
  Marine Biol.).

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p-DISTANCES IN GROUPS OF COMPARISON,turtles
Fig. 3. Resulting graph of ANOVA and mean
p-distance values at four levels of
differentiation in turtle species (Testudines)
for Cyt-b gene. Groups 1. Intraspecies, among
individuals of the same species 2. Intragenus,
among species of the same genera 3. Intrafamily,
among genera of the same family 4. Intraorder,
families of the same order. Variation among four
groups is statistically significant F 61.87,
d.f. 3 152, p lt 0.000001 (Jung et al.,
2006). ?-Distances (1) 2.330.03, (2)
3.340.48, (3) 6.410.11 ? (4) 11.920.37
(Mean SE).
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p-DISTANCES IN GROUPS OF COMPARISON,Perciformes
Fig. 3. Resulting graph of one factor ANOVA and
mean p-distance values at four levels of
differentiation in fish species (Perciformes) for
Co-1 gene sequence data. Comparison groups 1.
Intraspecies, among individuals of the same
species 2. Intragenus, among species of the same
genera 3. Intrafamily, among genera of the same
family 4. Intraorder, families of the order
Perciformes. Variation is statistically
significant. Bars are confidence intervals for
mean (95).
24
p-DISTANCES IN GROUPS OF COMPARISON,Review

• Fig. 4. Categorized plot of distribution of
  weighted mean p-distances among four groups of
  comparison at Cyt-b and Co-1 genes. Groups here
  1. Intra-species, among individuals of the same
  species 2. Intra-sibling species, 3.
  Intra-genus, among species of the same genera 4.
  Intra-family, among genera of the same family
  (Kartavtsev, Lee, 2006).

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GENETIC SIMILARITY IN TAXA OF DIFFERENT RANK
MEAN FOR THE GROUPS
Fig. 5. Genetic similarity in taxa of different
rank based on protein markers mean for the
groups. 1. Subspecies, 2. Semispecies and
sibling species, 3. Species, 4.
Genera. Intraspecies genetic distances were
measured for many groups of organisms (Lewontin,
1974, Nei, 1987, Altukhov, 1989). Mean genetic
similarity on this level is near I 0.95 (see
details in Kartavtsev, 2005). mtDNA data were
presented above. Thus, data available suggest
that in general a phyletic evolution prevail in
animal world, and so far, the Geographic
speciation events (Type 1a) prevail in nature.
Do data presented assume that speciation is
always follows the Type 1a mode? I guess, no. Few
examples below let to support this answer.
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DISTANCE VS TAXA SPLITTING

• Has punctuation an impact in species origin on
  molecular level?
• Avise, Ayala, 1976 Kartavtsev et al., 1980
  current No.
• Pegel et al., 2006 Yes.

rs 0.22, p lt 0.05
Number of Splittings
Transformed p-distance
Fig. 6. Plot of p-distance on number of
splittings at Cyt-b sequence data for catfishes
and flatfishes.
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GENETIC DISTANCES AMONG SPECIES IN SEPARATE
ANIMAL GENERA (After Avise, Aquadro, 1982)
This plot illustrate a thought that different
animal groups of the same rank are unequal in
structural gene divergence i.e. the rate of
evolution differ either at genes or at morphology
or both.
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GENETIC DISTATNCES IN TAXA OF SALMON FISHES
D
1 Populations within species, 2 Subspecies, 3
- Species
This plot support a thought that in salmon even a
very small structural gene change can create
separate biological species.
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EXAMPLES OF REGULATORY DIVERGENCE IN FISH TAXA
Comparison of chars
Table 2.1. COMPARISON OF ISOZYME ACTIVITY IN
THREE WHITEFISH FORMS (COREGONIDAE) AND GRAYLING
(THYMALLIDAE)
LEVELS OF DIFFERENCES IN ACTIVITY
LOCUS/ FORM
Ratio,
Note. Total number of loci analyzed are
Whitefish 22, Grayling 23, - Activity do
not differ significantly, Iterative activity
difference, two-fold difference,
three-fold or greater difference
30
WHAT IS MAIN OUTCOME

• Distance measure alone is not satisfactory
  descriptor.
• Data on intraspecies diversity (heterozygosity)
  at structural genes are necessary.
• Measures of regulatory genome changes should be
  necessary to describe transformative modes of
  speciation.
• Other descriptors of genomic change are required
  (e.g. chromoseme number, NF, etc.).

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3. SPECIES DEFINITIONAND SPECIES
ORIGINPROBLEMS, RESTRICTIONS.GENETIC VIEW
32
WHAT SPECIES IS?

• Species is a biological unity which
  reproductively isolated from other unities and
  consisting from one to several more or less
  stable populations of sexually reproducing
  individuals that occupy certain area in nature
  (my definition). In principal points, this is the
  definition of BSC (Biological Species Concept).
  In one of the original BSC definitions A species
  is a reproductive community of populations
  (reproductively isolated from others) that
  occupies a specific niche in nature (Mayr, 1982,
  p. 273). We will accept BSC for further
  discussion, although will keep in mind that it is
  restricted mainly to bisexual organisms (Mayr,
  1963, Timofeev-Resovsky et al., 1977, Templeton,
  1998).
• The Linnaean Species
• The Biological Species Concept (BSC) (Mayr, 1942,
  1963)
• BSC Modification II (Mayr, 1982)
• The Recognition Species Concept (Paterson, 1978,
  1985)
• The Cohesion Species Concept (Templeton, 1989)
• Evolutionary Species Concept
• Simpson (1961) Evolutionary Species Concept.
• Wileys (1978) Evolutionary Species Concept.
• The Ecological Species Concept (Van Vallen,
  1976).
• The Phylogenetic Species Concept (Crawcraft,
  1983).

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GENERAL GENETIC APPROACH ADVANCES AND
LIMITATIONS

• The problem of biological species, and
  speciation are main focus of this report. These
  problems took researchers attention since
  establishing the biology as a science. Most
  popular now among biologist is the Synthetic
  Theory of Evolution (STE), which part is
  comprised by the Biological Species Concept
  (BSC). Origin and systematic description of STE
  concept was presented in fundamental books by
  Haldane (1932), Dobzhansky (1937, 1943, 1951),
  Huxley (1954), Mayr and co-workers (Mayr, 1942,
  Mayr, 1963). A popular in Russia summary of STE
  became a book by Timofeev-Resovsky with
  co-authors (1977). Good, constructive ideas in
  STE support were developed by Vorontsov (1980).
• One of weak point in STE is absence as a rule a
  possibility to prove experimentally one of key
  criteria of BSC i.e., reproductive isolation of
  the species in nature. There are a lot of other
  criticisms that were summarized for example by
  King (1993). Nowadays, the new controversy
  between BSC and Phylogenetic Species Concept
  arise (Avise, Wollenberg, 1997). The theory of
  speciation is also not well developed in STE.
  Exactly speaking, in a quantitative meaning there
  is no theory as real matter at all.
• In should be outlined, nevertheless, that many
  directions of STE and genetics of speciation are
  developing. Thus, a diverse analysis performed to
  understand a genetic sense and conceptual
  basements of speciation (Fox, Morrow, 1981,
  Grant, 1984, King, 1992). The genetic basis for
  creation of a reproductive isolation was
  subjected to the analysis too (Leslie, 1982,
  Templeton, 1981, Nei et al., 1983, Coyne, 1992).
  As well there were considered a possibility of a
  sympatric speciation (Bush, 1975, Genermon,
  1991), the role of saltations or revolutions in
  evolution (Altukhov, Rychkov, 1970, Carson, 1974,
  Altukhov, 1985, 1997) and the genetic
  differentiation during formation of living forms
  and taxa (Avise, 1975, Avise, Aquadro, 1982,
  Nevo, Cleve, 1978, Thorpe, 1982, Nei, 1975,
  1987). What in general are the advances and
  limitations in contemporary genetic approach?

34
ADVANCES

• 1. Data reduction up to genotypic codes (values)
  give us a possibility to use genetic theory in
  the analysis.
• 2. It is possible to perform a comparative
  investigation of a variability among structural
  and regulatory elements of the genome and genetic
  divergence of taxa.
• 3. Investigations on protein and nucleotide
  divergence of species from nature discovered a
  Molecular clock.
• 4. A possibility of phylogenetic reconstruction
  occurred 1) not by similarity, but by kinship
  and 2) by in time dating of a divergence.

35
LIMITATIONS

• 1. Deduction is limited by genotypic descriptions
  and genetic theory.
• 2. Analysis is connected with preliminary
  laborious experimental investigation (with its
  own limitations).
• 3. Investigation of a species from nature is
  frequently limited by originality or rare
  repetition of an event (phenomenon).
• 4. Genotypic effects of the marker loci on
  phenotype are weak.
• 5. The theory is not sufficiently developed in
  some directions.

36
WHAT DATA ARE NECESSARY?

• Data that support (reject) central dogma of
  Neodarwinism Evolution can occurred the only on
  the base of genetic change.
• Data on variability at different levels of
  biological organization in genetic terms (by loci
  quantitative genotypic values AA, )
  single-dimensioned data tables (DT).
• Data on genotypic values of an individual at the
  set of loci (genotype AA Bb) or whole gene
  sequence set multi-dimensional DT.
• Complementary data Morphology traits, data on
  abiotic variability etc. (at least as an expert
  estimate grouping variables).

37
SCHEMATIC REPRESENTATION OF SPECIES DIVERGENCE
AND ORIGIN(From Dobzhansky, 1955)
C
The keystone of STE (Synthetic Theory of
Evolution) may be represented by Dobzhanskys
scheme (Fig. 3.1), in which the gene pool
separation is a key to speciation. If one
provides a fact that evolution is possible
without genetic change in lineages, then the
evolutionary genetic paradigm and STE in
particular can be rejected.
B
A

• Fig. 3.1. Dobzhanskys (1955) scheme of in time
  divergence.
• ? Single species population.
• B Initial phase of divergence (subspecies).
• C Different species.

38
Fig. 3.1. Main Modes of SpeciationBush, 1975)
FIG. 3.2. DIAGRAMMATIC REPRESENTATION OF BASIC
MODES OF SPECIATION (From Bush, 1975)
The gene flow breaks are able to create
Reproductive Isolating Barriers (RIB) or
Reproductive Isolation Mechanisms (RIM), which in
their turn lead to further origin of species
under different situation in nature, the
different modes of speciation acted (Fig. 3.2).
Neither, the scheme above, nor the paper itself
(Bush, 1975), answer many fundamental questions
of speciation. For instance, it is unclear, what
mode is most frequent and is a gene flow the sole
primary factor, that alter gene pools or there
are others?
In other words we have to conclude that there is
no a theory of speciation in scientific meaning
at all.
39
SPECIATION MODES (SM) POPULATION GENETIC VIEW

• ABSENCE OF QUANTITATIVE THEORY OF SPECIATION
  (QTS)
• We have mentioned in preceding section that the
  speciation theory in evolutionary genetics is
  absent in exact scientific meaning, which expects
  the ability to predict future by the theory. In
  this case this is to predict species origin, or
  at least discriminate among several speciation
  modes on the basis of some quantitative
  parameters or their empirical estimates. Attempts
  made in this direction (Avise, Wollenberg, 1997,
  Templeton, 1998) do not fit the above criteria.
  That is why we attempted to step in the
  discrimination of the speciation modes on the
  basis of main population genetic measurements
  available in literature, and that may be laid in
  the frame of a genetic speciation concept.
• BASEMENT FOR THE QTS
• As a basis for the set of evolutionary genetic
  concepts we used the descriptions made by
  Templeton (1981). As a result the classification
  scheme for 7 different modes of speciation was
  created (Fig. 3.3). This approach leads to quite
  simple experimental scheme that permits (i) to
  arrange further investigation of speciation in
  different groups of organisms, and (ii) to derive
  analytical relations for each speciation mode
  (Fig. 3.4). The approach is based on a set theory
  but it is a knowledge-based approach. I believe,
  this approach is best for such complicated matter.

40
Fig. 3.3. SPECIATION MODES (SM) POPULATION
GENETIC VIEW (Kartavtsev et al, 2002)
DIVERGENCE SM
D1. ADAPTIVE
D2. CLINAL
D3. HABITAT
DESCRIPTORS D Genetic distance at structural
genes DT in suggested parent taxa, DS
among conspecific demes, DD among subspecies or
sibling species HD Mean
heterozygosity in suggested
daughter population Hp Mean heterozygosity in
suggested parent population EP
Divergence in regulatory genes among
suggested parent taxa ED Divergence in
regulatory genes among suggested
daughter taxa TM- Test for modification
(positive) TM-- Test for modification
(negative). RIB Reproductive isolation
Barriers.
Necessary Conditions for Speciation
D1. a) Erection of extrinsic Isolating barriers
followed by gene flow break b) Pleotropic
origin of RIB (Reproductive Isolatiion
Barriers) in long time
D2. a) Selection on a cline with isolation by
distance b) Pleotropic origin of RIB
D3. a) Selection over multiple habitats with no
isolation by distance b) RIB origin by
disruptive selection at genes determined behavior
Sufficient Conditions for Speciation
Lack of efficient hybridi- zation in the zone of
contact
Lack of efficient hybridi- zation outside the
zone of contact
Lack of efficient hybridi- zation inside and
outside the zone of contact
1. DT gt DS ?1 (S) 2. ED EP 3. HD HP
4. TM-
1. DT gt DS ?2 (S) 2. ED ? EP 3. HD HP
4. TM-
1. DT DS ?3 (S) 2. ED ? EP 3. HD lt HP
4. TM-
Experimentally measurable features and possible
descriptors for the model (theory), ? (S)
41
Fig. 3.4. ANALITICAL DESCRIPTION OF SEVEN TYPES
OF SPECIATION MODES
Note. Descriptors are explained in previous
figure.
42
THE QTS EMPIRICAL PROVING

• Salmon (Kartavtsev, Mamontov, 1983, Kartavtsev et
  al., 1983),
• Cypriniformes (Kartavtsev et al., 2002),
• Turtles, flatfishes, catfishes (Jung et al.,
  2006, Kartavtsev et al., 2006, 2007).

43
PHYLOGENETICS BARCODING
44
SPECIES IDENTIFICATION AND PHYLOGENETICS
Phylogenetics Taxonomy
Identification Taxonomy
45

• Fig. 3.5. Rooted consensus (50) trees (A-B)
  showing phylogenetic interrelationships on the
  basis of Cyt-b sequence data for the analyzed
  flatfish species (Pleuronectiformes) and four
  out-group taxa. A tree based on NJ clustering
  technique with bootstrap support shown in the
  nodes (n1000), B Bayesian tree repetition
  frequencies for n106 simulated generations are
  shown () in the nodes. The tree was built based
  on the TrNIG model and was rooted with the
  sequences of four out-group species three are
  Perciformes and one is Cypriniformes. The scales
  in the left bottom corners indicate relative
  branch lengths.

46
Fig. 3.6. Consensus (50) tree showing
phylogenetic interrelationships on the basis of
Co-1 sequence data for the analyzed flatfish
species (Pleuronectiformes) and two outgroup
taxa. Rooted Bayesian tree repetition
frequencies (probabilities) for n106 simulated
generations are shown in the nodes ().The tree
was built based on the TVMIG model and rooted
with the sequences of two outgroup species,
Perciformes. The scale in the left bottom corners
indicate the relative branch lengths.
47
Fig. 3.7. Rooted consensus (50) tree showing
phylogenetic interrelationships on the basis of
Cyt-b sequence data for the analyzed flatfish
species (Pleuronectiformes) and three outgroup
taxa. Bayesian tree repetition frequencies for
n106 simulated generations are shown () in the
nodes. The trees were built based on the TrNIG
model, and rooted with the sequences of outgroup
species Perciformes. The scales in the left
bottom corners indicate the relative branch
lengths. (Kartavtsev et al., 2007, Marine Biol.).
48
Fig. 3.8. Consensus (50) trees showing
phylogenetic interrelationships on the basis of
Co-1 sequence data for 7 analyzed perch-like fish
species (Perciformes) and two outgroup sequences.
Rooted Bayesian tree was build for sample
purposes posterior probabilities for n106
simulated generations are shown in the nodes ().
The tree was built based on the HKYG model. Two
other numbers in the nodes show tree bootstrap
support based on similar clustering for NJ and ML
techniques support scores are given in the order
NJ/ML/BA. Outgroup are two sequences of a
representative of Cypriniformes. The scale in the
left bottom corner indicate the relative branch
lengths.
49
Conclusions

• Speciation mode must be specified with a set of
  descriptors not exclusively by distances
• Both Co-1 and Cyt-b are generally good barcoding
  tools for species identification
• For phylogenetic reconstructions we need to cover
  both taxa diversity and several genes sequence
  diversity

50
THANKS FOR ATTENTION!
??????? ?? ????????!
51
FEW RECENT PUBLICATIONS

• Kartavtsev YP. 2005. Molecular evolution and
  population genetics. Far Eastern State Univ.
  Press., Vladivostok, 234 p.
• Kartavtsev YP, Lee J-S. Analysis of nucleotide
  diversity at genes Cyt-b and Co-1 on population,
  species, and genera levels. Applicability of DNA
  and allozyme data in the genetics of speciation.
  Genetika, 2006. 42 437-461.
• Jung S-O, Lee Y-M, Kartavtsev YP, Park I-S, Kim
  D-S, Lee J-S. The complete mitochondrial genome
  of the Korean soft-shelled turtle Pelodiscus
  sinensis // DNA Sequence, 2006. 17(6) 471-483.
• Sasaki T, Kartavtsev YP, Uematsu T, Sviridov VV,
  Hanzawa N. Phylogenetic independence of Far
  Eastern Leuciscinae (Pisces Cyprinidae) inferred
  from mitochondrial DNA analysis. Gene and Genetic
  Systems, 2007. 82 329-340.
• Kartavtsev YP, Lee Y-M, Jung S-O, Byeon H-K, Son
  Y-, Lee J-S. Complete mitochondrial genome in the
  bullhead torrent catfish, Liobagrus obesus
  (Siluriformes, Amblycipididae) and phylogenetic
  considerations. Gene, 2007a. 396 13-27.
• Kartavtsev YP, Park T-J, Vinnikov KA, Ivankov
  VN, Sharina SN, Lee J-S. Cytochrome b (Cyt-b)
  gene sequences analysis in six flatfish species
  (Pisces, Pleuronectidae) with phylogenetic and
  taxonomic insights. Journal Marine Biology,
  2007b. 152(4) 757-773.

52
TERMS
Terminal taxa A B C D E
F G H Outgroup
53
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• 6. ????????? ?.?. (????, ?.?.?., ???.
  ????????????)
• 7. ???????? ?.?. (????, ????????)
• 8. ?????????? O.?. (???, ????????)

59
?????? ??????????? ?????????? ??

• ??? (4)
• ?????? ?.?. (?.?.?.., ???????? ?????, ???.
  ?????????)
• ??????? ?.?. (?.?.?.)
• ??????????? ?.?. (?.?.?.)
• ??????? ?.?. (?.?.?., ???. ????????????)
• ???????? ?.?. (?.?.?.) 
• ???? (4)
• ??????? ?.?. (?.?.?.)
• ????????? ?.?. (?.?.?.)
• ???????? ?.?. (????????)
• ??????? ?.?. (????????)
• ???? (3)
• ???????? ?.?. (????-????. ???, ????????)
• ???????? ?.?. (?.?.?.)
• ?????? ?.?. (????????)
•  ?????-?????, ???????, ?????????? ? ??. (7)
• ?????? ?.?. (?.?.?., ???. ????????????)
• ????????? ?.?. (?.?.?., ???. ????????????)
• ???????? ?.?. (?.?.?.)

60
???????? ??????????????????? ? GenBank (NCBI)
61
????????

• ??? ??????????? (?????) ??? ????????? ????????,
  ?????? ????? 16-18 ????? ??? ??????????? (???.).
  ??? ?????????? ???????????? ??????, ????? ????
  ??? ????? ??????? ??????????? (Lee et al., 2001
  Kim et al., 2004 Kim et al., 2005 Nagase et
  al., 2005 Nohara et al., 2005) ? ????, ???
  ?????????? ? ? ?????? ??????????? ????????,
  ??????? ???????? (Anderson et al., 1981 Bibb et
  al., 1981 Wallace, 1992 Kogelnik et al., 2005).
  
• ?????? ?????? ???????????????? ??????
• (??????????) ???????? ??????????? ??????
• (CR ??? D ?????),
• ??? ????????????? ???? ?????? ?????????? ?
• ?????????, ??????? (16S) ? ????? (12S)
• ??????????? ????, 22 ???? ?
• 13 ????????????? ?????.
• ???????????????? ???????????? ?????? ??????????
  ?????????????????? ????????? ?????, ? ??? ?????
  ? ????? ??????? ???, ???? ? ????????? ???? ???
  ???? ?????????? ??? ???? ????? ? ??????
  ?????????. ???????? ????????? ? ????????????
  ?????????????????? ????? ????????? b (Cyt-b) ?
  ???????? ???????? 1 (C?-1), ??????? ????????????
  ??? ????????? ???????? ?? ?????? ??? ?????????
  (Johns, Avise, 1998 Hebert et al., 2004
  ?????????, ??, 2006). ????? ???????????????????,
  ??????? ???????????????? ??????, ???????? ???
  ?????? ????? ????? ?? ???? 16S ????.
• ?????????????????? ????????? ????? ????? ??????
  ????????? ???????????????? ?????? ??-?? ?????????
  ?????? ????? ? ?????????? ???????????? ?/???
  ??????????????? ?????? ??????? (??????????????????
  ?????????? ???????????? ?????). ??? ????????? ?
  ? ?????? ???????? ?????? ? ???? ?? ????. ?????
  ????, ??? ????????????? ?????????????? ????????,
  ???????? ???????? ?????, ????????? ???????? ?
  ????????? ?????????? ? ?????????????
  ?????????????? ???????? ???????
  ??????????????????? ??? ???????????????? ?????
  (Hilish et al., 1996, Miya et al., 2001). ??? ??
  ?????, ??? ????????????? ?????, ?? ???????
  ????????????, ?????????? ????????? ????
  ???????????? ???????? ???????????????????,
  ???????? ???? ???????? ???????? 1 (??-1, 654 ??).