FISH BARCODING IN RUSSIA: REGIONAL PERSPECTIVE Yuri Ph. Kartavtsev, S.D. Institute of Marine Biology

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FISH BARCODING IN RUSSIA REGIONAL
PERSPECTIVEYuri Ph. Kartavtsev, S.D.Institute
of Marine Biology,Vladivostok, Russia
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GREETINGS
Academician, Altukhov sending to the participants
of Fish BOL meeting great success and performing
an exiting plans in fish barcoding!
Academician, prof. Yuri P. Altukhov, director,
N.Vavilov Institute of General Genetics, Moscow
(Russia)
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MAIN GOALS

• 1. Area Covered and Problems in Data Base
  Gathering.
• 2. General Approach Advances and Limitations.
• 3. Concluding Remarks.

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1. AREA COVERED AND PROBLEMS IN DATA BASE
GATHERING
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AREA COVERED

• We are establishing two groups centered one in
  Moscow and second in Vladivostok.
• Area 1 Barents Sea, White Sea, Baltic Sea, and
  Black Sea. Main participating institutions in
  Moscow center will be Vavilov Institute of
  General Genetics (VIGG, Moscow), Institute of
  Fisheries Research and Oceanography (VNIRO,
  Moscow), Polar Institute of Fisheries Research
  and Oceanography (PINRO, Murmansk), Moscow State
  University (MSU, Moscow), and others.
• Area 2 Sea of Japan, Okhotsk Sea, Bering Sea and
  North-Western Pacific. Major participating
  institutions in the Far East will be Institute of
  Marine Biology (IMB, Vladivostok), Far Eastern
  State University (FESU, Vladivostok), Pacific
  Institute of Fisheries Research and Oceanography
  (TINRO-center, Vladivostok), Institute of
  Biological Problems of North (IBPN, Magadan), and
  others.

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NUMBER OF FISH SPECIES

• Recent catalogue of Agnathans and fishes of
  fresh and brackish waters in Russia includes 557
  species and other taxa (Bogutskaya, Naseka,
  2004). Truly freshwater are 367 species (Froesy,
  Pauly, 2005 World Wide Web Fishbase).
• Approximate number of marine species in Russian
  waters is 568 (Froesy, Pauly, 2005 World Wide
  Web Fishbase).
• The figures above are of course incomplete and
  future research may change this number.

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BRIEF SUMMARY OF PLANS FOR FACILITATING AREA
COVERAGE

• Agreements are accomplished with principal
  researches of the institutions participating in
  gathering of specimens, species identification,
  and digital camera photo documentation.
• This spring-summer sampling will start at the
  Far East at Sakhalin Island and Primorye Area
  (I.e. Sea of Japan and Okhotsk Sea).

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HAS COI SEQUENCED STARTED IN THIS REGION YET?

• To our knowledge based on SCI-search and also
  judging on WEB-site on cytochrome c oxidase I
  gene (COI) created by Dr. Hebert there are no
  papers publish prior this date on Russian marine
  fish species sequence analysis at COI. However,
  skilled scientists are available in Vladivostok
  and other places in Russia. At Vladivostok
  science center we have two sequencing machines,
  which are used mainly for biotechnology needs.
  Also at three institutions there is a staff that
  dealing with DNA (RFLPs, RAPD analyses). At VIGG
  in Moscow there is equipment and staff dealing
  with for micro-satellite research.
• This season, for example, we have initiated the
  investigation for Cyt b and COI gene sequences of
  4 flounder species in collaboration with Korean
  researchers (Dr. J.-S. Lee group, Hanyang
  University, Seoul). Also, several years before we
  have started DNA sequencing at Cyt b gene and
  phylogenetic research on Cyprinid fishes, focused
  on Leuciscinae subfamily jointly with Japanese
  scientists (Dr. N. Hanzawa group, Yamagata
  University, Yamagata).

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FISH COLLECTORS IN REGION, FISH TAXONOMISTS IN
REGION (1)

• I have indicated above that contacts have been
  made. In our country usually it is the same
  person who collect data and identify species. At
  least those persons are key persons who request
  collection and species identification be made.
• Personally we will be able to gather minimum up
  to 16 ichtyologists and fishery biologists in the
  Far East of Russia. In Europe their number will
  be not less and in Siberia no special contacts
  was established yet.
• Specific taxonomic groups that we are qualified
  to deal with right now listed below.
• Far East Group Engraulidae, Salmonidae,
  Osmeridae, Salangidae, Cyprinidae,
  Scomberesocidae, Hemirhamphidae, Belonidae,
  Gadidae, Blennioidai Pholidae, Cottoidei
  Scorpanidae, Hexagrammidae, Cottidae,
  Cyclopteridae, Liparidae (IMB staff),
  Bathymasteridae, Zoarcidae, Liparidae,
  Cyclopteridae, Stichaeidae,Pholididae, Cottidae,
  Hexagrammidae, Psychrolutidae, Agonidae,Trichodont
  idae, Pleuronectidae (IBPS staff). Total that is
  22 different families.
• Moscow Group No info yet.

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DEPOSITION PLANS FOR VOUCHER SPECIMENS

• There are two ways to gather specimens. The
  first is to pay for collections to fishery
  institutes holders. The second is to attract
  collections by exchange on the basis of mutual
  benefit between certain researchers. We cant
  avoid sampling by the first way. Reasonable price
  here is 10-50 US dollars per specimen in a
  collection. Thus, calculation per 600 marine fish
  species, per 4 individuals gives 24,000, if
  based on lowest price, 10. The second way seems
  is more cheap. However, in the conditions of our
  country we need some payments here too. For
  example, some fishery biologist said that they
  are ready to collect specimens but they have no
  digital camera for the zoological documentation.
• Voucher specimens is special task in which we
  have some experience. At IMB we have alcohol
  tissue collection for some 100 species, although
  there is no basically any special photo
  documentation. We have at IMB biological
  collection too and skilled museum staff. Also
  there are plans to develop special storage
  facilities for keeping specimens in low
  temperatures (deep freezer and liquid nitrogen)
  and PC data base managment.

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PLANS FOR COI SEQUENCE ANALYSIS

• At IMB we have started sequencing (Hanzawa et
  al., 2000, Sasaki, Kartavtsev et al., 2005, in
  press) mainly using Cyt b gene for Leuciscinae
  (Cyprinidae). Such researches will be continued
  in the collaboration, including also Korean team.
  Since next year we will be able to develop mass
  analysis in Russia based on COI, providing that
  funding will be available. At IMB we will get
  next year the sequencing machine and chemicals
  for pilot research. We will have Amersham
  Biosciences MEGA BASE 750 or 1000 (48-96
  specimens set and up to 1,200 bp in one run). At
  VIGG in Moscow they have possible access to a
  good sequencer at other institution (VNIRO).
  However, the following progress in both groups in
  Russia will be dependent on financial sources.
  Approximate sequencing cost may comprise US
  1,569,600. In the calculation we accepted 1
  per 1 bp per 1 specimen to be sequenced, multiply
  by 654 bp, multiply by 600 marine fish species,
  and by 4 individuals.
• Who personally will be involved.
• In Moscow the research group will be leaded by
  academician Y.P. Altukhov and Dr. E.A.
  Salmenkova. Available fish geneticists staff at
  starting point is 7-10 scientists.
• In Vladivostok the research group will be leaded
  by Dr. Y.Ph. Kartavtsev. Available fish
  geneticists staff at starting point is 9-10
  scientists.
• Other persons may be involved and other centers
  are also possible to establish in Russia under
  resonable funding. Among the first attracted may
  be MSU, IBPN, VNIRO, TINRO.

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2. GENERAL APPROACH ADVANCES AND LIMITATIONS
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ADVANCES

• I am agree with general approach declared by Drs.
  P. Hebert and B. Ward in their proposals to this
  meeting. 1. The activation of an international
  research network that will assemble a
  comprehensive DNA barcode library for fishes and
  to contribute to two international initiatives -
  The Census of Marine Life and The Consortium for
  the Barcode of Life. 2. Focusing on barcode
  acquisition for some 20,000 marine fish species
  within 5 years and concentration later on
  freshwater fishes and, I hope, in future on
  shellfishes too.
• The success of pilot projects which combine data
  for some 600 fish species has convinced that
  mitochondrial DNA and choice of gene COI is also
  very reasonable. Quick divergence of this mtDNA
  gene and species specificity is well established
  (Hebert et al., 2003 a,b, Proposal to Fish BOL,
  2005).
• Combining molecular genetic approach with
  traditional taxonomy which seen in the intention
  to gather qualitative zoological identification
  is also very promising and fine agrees with the
  goal number one above.
• Thus, the approaches, which will be applied in
  fish BOL project, on one hand, are based on
  precise and substantiated molecular genetic and
  classical techniques, and on the other hand will
  comprise an original complex approach. This
  approach will extends first of all on main theme,
  i.e. proving the unambiguous discrimination and
  systematic status of species and other taxa in
  question and also the evaluation of phylogenetic
  relationships in the groups of taxa.

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LIMITATIONS

• There are several limitations of which we should
  remember and Id like to stress three of them.
• The first constrain is that COI is short DNA
  sequence of some 600 bp. This will preclude the
  construction of reliable phylogenetic trees, if
  OTU (Operation Taxonomic Units) number is large
  enough (Nei, 1987 Naylor, Brown, 1998 Nei,
  Kumar, 2000).
• The second limitation connected with
  mitochondrial origin of COI gene. It is known for
  fishes (Campton, 1987 Avise, 1994 Glemet et
  al., 1998 Hebert et al., 2003) that sometimes
  there is gene introgression at mtDNA markers.
  Interspecies transfer of mtDNA was find for
  several genera in Drosophila, Mus and Rana
  (Powell, 1983 Ferris et al., 1983 Spolsky,
  Uzzell, 1984 Yonekawa et al., 1984 2000). As
  well heteroplasy is known for some fish species
  (Magoulas, Zouros, 1993) and for many
  invertebrates.
• The third, the investigation should be organized
  in a special mode to cover, firstly, different
  levels of variability (sex, ontogenetic, intra-,
  interpopulation) and, secondly, to maximize
  representation of a different species or other
  OTU in the taxa that are analyzed. Technically
  coverage of a small number of representatives of
  each taxa (4-5 individuals) is substantiated with
  the intension to investigate maximum of fish
  diversity. However, such approach will be limited
  to the species-specific labelling with no
  variation that likely do exist at COI. We may
  avoid this kind of problem by exploring one
  species per taxon as a specific sample to test
  different levels of diversity.
• Major limitation of course is not mentioned above
  but time (5 years) and money.

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3. CONCLUDING REMARKS
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FUNDING SOURCES

• At IMB unfortunately we have no any funding now
  for sequencing program. Both the sequencing
  machine at IMB and funding could be available
  only next year, providing successful application.
  Same situation is at other institutions. The
  amount of big state grant from budget in Russia
  is some 30,000 annually. Even if we will get
  such special grant for fish barcoding it is
  obviously hardly enough to cover mass sample
  collection. Thus, a prompt following progress in
  Russia will be dependent on financial income from
  other sources.
• What kinds of sources can be involved in my
  view.
• Internal. Although small, two other sources are
  available potentially in Russia. One is Creating
  special states or Academy of Science program for
  Fish BOL in Russia. Another is Fishery Ministry
  special fundings. Also there are very small
  research grants from RFFR (Russian Foundation for
  Basic Research). However, actually in Russia
  everything is dependent on budget. If probability
  to take big states grant in my estimate is as
  low as 10, the realization of two other
  possibilities is much less.
• External. The funding sources here are out of my
  expertise. However, we may estimate necessary
  amount. The amount of donations is such an
  approximation. We have estimated above sampling
  and sequencing amounts 24,000 1,569,600,
  thus total is 1,593,600. Also, some equipment,
  chemicals and salary must be planned for
  laboratory job. It will comprise for two initial
  research groups 70,000. Thus we expect that
  amount of funding must be at least US 332,720
  annually. So far, for five year program we are
  expecting US 1,663,600. Russian side donations
  may cove certain equipment (Mega Base cost
  e250,000), development of necessary lab
  infrastructure and links (PC, data base managing,
  Internet, etc.).

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CONCLUSIONS

• FISH BOL INITIATIVE IS MOST WELCOME AND AN
  ORGANIZING INSTITUTION ESTABILISHING IS A STRICT
  NECESSITY.
• UNDER APPROACH, SUGGESTING BY ORGANIZERS, MOST
  FISH TAXA REPRESENTATIVES COULD BE BARCODED AND
  INCLUDED IN DATA BASE WITH NECESSARY ZOOLOGICAL
  BACKGROUND.
• RUSSIA IS READY TO PARTICIPATE IN FISH BOL,
  HOWEVER, HELP OF INTERNATIONAL INSTITUTIONS IS
  EXTREMELY SUBSTANTIAL.

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GENETIC DISTANCES AMONG SPECIES IN SEPARATE
ANIMAL GENERA (After Avise, Aquadro, 1982
Hebert et al., 2003 Kartavtsev, 2005)
These plots illustrate 1. 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. 2. Evolution in major groups
is gradual, i.e. follows geographic SM. Taxa 1
Between populations, 2 Between Subspecies, 3
Within Genus, 4 - Within Family.
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Yuri KartavtsevMolecular Evolution and
Population Genetics. Vladivostok, FESU Publ.,
2005.
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LIST OF ICHTIOLOGISTS AND FISH BIOLOGISTS

• IMB (4)
• Pitruk D.L. (Ph.D., Museum curator)
• Yakovlev Y.M. (Ph.D.)
• Sokolovski A.M. (Ph.D.)
• Balanov A.A. (Ph.D.)
•  
• FESU (3)
• Ivankov V.N. (S.D., RNSA academician)
• Platoshina L.K. (Ph.D.)
• Vinnikov K.V. (Ph.D. student)
• Rutenko O.A. (Ph.D. student)
• IBPN (3)
• Chereshnev I.A. (S.D., RAS corresponding member)
• Shestakov A.V. (Ph.D.)
• Grunin S.I. (Ph.D. student)
•  
• TINRO, SakhalinNIRO, AmurNIRO, others (7)
• Shuntov V.N. (S.D., RNSA academician)

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LIST OF GENETICISTS

• Moscow
• 1. Altukhov Y.P. (S.D., RAS academician)
• 2. Salmenkova E.A. (Ph.D.)
• 3. Gordeeva N.V. (Ph.D.)
• 4. Afanasiev K.N. (Ph.D.)
• 5. Politov D.V. (Ph.D.)
• 6. Mahrov A.A. (Ph.D.)
• 7. Barmintsev A.A. (Ph.D.)
• Vladivostok
• 1. Kartavtsev Y.Ph. (S.D.)
• 2. Chichvarkhin A.Y. (Ph.D. student)
• 3. Chichvarkhina O.V. (Ph.D. student)
• 4. Brykov V.A. (S.D.)
• 5. Polyakova N.E. (Ph.D.)
• 6. Podlesnikh A.V. (Ph.D.)
• 7. Kuhlevski A.N. (Ph.D.)
• 8. Vinnikov K.V. (Ph.D. student)
• 9. Polyakova A.V. (Postgraduate student)