Comparative Study of Genome Divergence in Salmonids with

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2013 Article ID 629543 16 pageshttpdxdoiorg1011552013629543

Research ArticleComparative Study of Genome Divergence in Salmonids withVarious Rates of Genetic Isolation

Elena A Shubina12 Mikhail A Nikitin1 Ekaterina V Ponomareva1

Denis V Goryunov1 and Oleg F Gritsenko3

1 Belozersky Institute for Physico-Chemical Biology of Lomonosov Moscow State University Leninskie gory 1 Moscow 119991 Russia2 Biological Department of Lomonosov Moscow State University Leninskie Gory 1 Moscow 119991 Russia3 Russian Federative Research Institute of Fisheries and Oceanology 17A V Krasnoselskaya Street Moscow 107140 Russia

Correspondence should be addressed to Elena A Shubina shubinagenebeemsusu

Received 15 October 2012 Revised 22 March 2013 Accepted 15 May 2013

Academic Editor Dmitry Sherbakov

Copyright copy 2013 Elena A Shubina et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of the study is a comparative investigation of changes that certain genome parts undergo during speciation The researchwas focused on divergence of coding and noncoding sequences in different groups of salmonid fishes of the Salmonidae (SalmoParasalmo Oncorhynchus and Salvelinus genera) and the Coregonidae families under different levels of reproductive isolation Twobasic approaches were used (1) PCR-RAPDwith a 20ndash22 nt primer design with subsequent cloning and sequencing of the productsand (2) a modified endonuclease restriction analysis The restriction fragments were shown with sequencing to represent satelliteDNA Effects of speciation are found in repetitive sequencesThe revelation of expressed sequences in the majority of the employedanonymous loci allows for assuming the adaptive selection during allopatric speciation in isolated char forms

1 Introduction

In view of the biological concept of Mayr [1] the processof speciation in the organisms with the sexual reproductioninvolves accumulation of differences sufficient to set thebarrier of partial or complete incompatibility According toDobzhansky [2] it implies for the process of unlimited geneticrecombinations within the species and the lack of gene flowbetween the species Meanwhile as repeatedly noted bymany researches for example Mallet Garside and ChristieSvardson Wolf et al Gross et al and Scribner et al ([3ndash7] review [8]) hybridization between species is known tooccur both in the wild and under artificial conditions and thehybrid forms exist along with the parental speciesThe fate ofsuch interspecific hybrids sporadically occurring in the wildand their contribution in the genetic structure of populationsare still under question as Coyne and Orr and Hudson et al[9 10] showed

Repetitive DNA sequences are convenient for the studiesof the genome evolution [11ndash13] According to Ohno [14] thisfraction originates in the process of gene duplications and has

a potential for large-scale rearrangements because they arenot subjected to the pressing of the natural selection

From the directly obtained experimental data phyloge-netic reconstructions for the lower taxa on the basis of therepetitive DNA sequences yield better results than the othernuclear sequences for both animals and plants as Chase et alThompson et al and Warburton and Willard [15ndash17] wroteAs mentioned by Ohta [18] the factors of intragenomichomogenization counteract intragenomic differentiation ofthe fraction of repeatsThese sequences become peculiar spe-cific markers The process of concerted evolution has beenpreviously shown by Zimmer et al Jeffreys et al Gray etal and Elder and Turner [19ndash22] to involve highly repetitiveDNA sequences (satellite and satellite-like)

A well solution method of comparative studies of geno-mic eukaryotic DNA according to distribution of the sitesof digestion by restriction endonucleases was proposed byFedorov et al [23] The method called taxonomic finger-printing or taxonoprint is a modification of the approachinitially developed for the analysis of themitochondrial DNA[24] Investigation of about 50 animal species of various taxa

2 International Journal of Genomics

45∘

50∘

55∘

60∘

65∘

70∘

75∘

45∘

50∘

55∘

60∘

65∘

70∘

75∘

30∘

60∘

90∘

120∘

150∘

30∘

60∘

90∘

120∘

150∘

Family CoregonidaeGenus SalvelinusGenera Salmo Parasalmo and Oncorhynchus

Figure 1 Sampling localities in Russia Armenia Kazakhstan Finland and Norway

[25] has shown species specificity of the band patterns alongwith the absence of individual sexual and interpopulationpolymorphism Dominating contribution of high-copy rela-tively long tandem repetitive sequences in ldquotaxonoprintsrdquo wasrevealed by Roudykh et al [26] This method seems to us tobe sufficiently appropriate for studying the molecular aspectsof speciation

We have shown previously that these general principles ofthe concert evolution were fully pronounced in the evolutionof repetitive sequences of the salmonids of Salmo Parasalmoand Oncorhynchus generamdashMednikov et al [27] ldquoHomingrdquocommon to salmonids resulted in reliable reproductive iso-lation with the subsequent divergence of the populationsin accordance with the morphological and molecular char-acters Situation with the repetitive DNA sequences in theorganisms with the less strict genetic isolation seemed tobe worth being analyzed Whitefishes of Coregonidae familyare one of the largest groups with interspecific hybridizationaccording to many experts application of Mayrrsquos biologicalspecies concept [1] to these fishes is limited (eg see discus-sion of the problem [28ndash30])

The study was aimed at the comparative investigationof alteration of some genome fractions under differentiationof salmonid species and forms belonging to various groupsand demonstrating various rates of reproductive isolationWhitefishes of Coregonidae family true salmons of theSalmo Parasalmo (Oncorhynchus after Smith and Stearley[31]) and Oncorhynchus genera and a number of forms andspecies of Salvelinus genus were studiedThe rates of isolationin whitefish and true salmon have polar characteristics dueto extensive hybridization in some species and strict homingin the other Geographic isolates and insular populations ofanadromous chars occupy intermediate position

Twomethods of multilocus DNA analysis were applied inthe phylogenetic and taxonomic studies of the Coregonidae

and Salmonidae families The methods were based bothon the comparison of the extensive repetitive sequence[26] and collation of the anonymous PCR products (RAPDamplification in modification of Welsh and McClelland [32]and Williams [33]) and their subsequent sequencing

2 Materials and Methods

Most of the salmon tissue samples were collected by the spe-cialists of the Department of Ichthyology Moscow State Uni-versity in 1984ndash2004 and Russian Federal Research Instituteof Fisheries and Oceanography in 2000ndash2004 Arctic charsfrom the lakes of Finland were collected by M KaukkorantaWhitefish from Lake Como (Canada) were kindly providedby Yu S Reshetnikov Collecting sites are mapped onFigure 1

In the early experiments DNA samples of salmons andwhitefish were extracted from gonads at III-IV stages ofmaturity preserved in alcohol with the method of phenol-chloroform extraction [34] In some cases additional purifi-cation with CsCl in the presence of ethidium bromide [35]and additional precipitation with cetyltrimethylammoniumbromide [36] were performed The length of DNA waschecked by electrophoresis in 06 agarose gel Concentra-tions of the obtained DNA preparations were estimated onSP-800 spectrophotometer (UK) and adjusted

At a later stage either Silica method [37] or traditionalprocedure with the use of proteinase K and organic solventsof Sambrook et al [38] was applied Prior to amplificationall samples were additionally purified with PEG-8000 DNApellets were dissolved in TE buffer and stored frozen

For restriction analysis DNA aliquots were digestedby MspI TaqI Csp6I (RsaI) Tru9I (MseI) Hin6I (HhaI)andMboI tetranucleotide restriction endonucleases and twoisoschizomers sensitive to the presence of methylated bases

International Journal of Genomics 3

Table 1 The RAPD-PCR primers sequences

No Designation Sequence 51015840-31015840 Length [nucleotides]1 I CGT TGG AAG ACA GAC CTC CG 202 II ATT CCC TGT CAA AGT AGG GT 203 III GAG CAC TTT CTT GCC ATG AG 204 IV GAA GCT GCT ATG CTT CGT AT 205 VI CAT AAA TTG CTT TAA GGC G 196 VII TCA TCT TCT TCC TCT TCT TC 207 1 TGT GAC TGC TTG TAG ATG GC 208 2 TGG AGC TGT GTA AGA AGT AC 209 3 AAA AGA CAT GAA GAC TCA GG 2010 5 TGG ACA GTA CGG TGA ATG C 1911 6 CCA CAA ACC AAT ATC TCT C 1912 7 CTC AGA GTC CAA CCT GGG TAG 21

(Bsp143I and Sau3AI) as well as Cfr13I and BcnI degen-erate pentanucleotide restriction endonucleases (FermentasLithuania Sibenzym Russia) Reactions were performedovernight under conditions corresponding to the manu-facturerrsquos recommendations The fragments of hydrolysiswere labeled at cohesive 31015840 ends using Klenovrsquos fragment ofDNA-polymerase I E coli and using [a-32P] dNTPs (Insti-tute for Physics and Power Engendering Obninsk Russia)Prior to electrophoresis minor labeled oligonucleotides wereremoved from hydrolysate by gel filtration through SefadexG-50 (medium) (Sigma USA) during centrifugation accord-ing toManiatis et al [39]This procedure improved resolutionof radioautographs Electrophoresis in nondenatured 10polyacrylamide gel (20 times 40 cm) was performed manuallyaccording to the method described in Fedorov et al [23]pBR322 DNA-MspI digest was used as a marker of molecularweight

For RAPD-PCR experiments 19- or 20-mer oligonu-cleotides in various combinations were used as the primers(Tables 1 and 2)

Amplification reaction with two arbitrary primers wasperformed in 25120583L of 001M tris-HCl PH 83 buffer con-taining 005M NaCl the mixture of four dNTP (02mMeach) MgCl

2(5mM) two arbitrary primers in various com-

binations (5 120583m each) DNA-polymerase Thermus aquaticus(20 units per sample) (Dialat Russia) and appr 20 ng ofDNA PCR conditions 94∘C 20min (88∘Cmdash1min 92∘mdash1min) times 1 (94∘Cmdash45 sec 50∘Cmdash30 sec 72∘Cmdash30 sec) times 3(94∘Cmdash45 sec 60∘Cmdash30 sec 72∘Cmdash30 sec) times 35 72∘Cmdash10min NoDNAwas added to the check sample Compound-ing ingredients were prepared and mixed in accordance withpublished recommendations Electrophoretic fractionationof PCR products was performed in 2 agarose gel (15 lowmelted Sigma +05 type II Sigma) dyed with ethidiumbromide and photographed inUV light Under the describedconditions the reactions were stable and replicable Pho-tographs of gels containing DNA PCR-RAPD electrophoreticpatterns were taken in UV light in Kodak EDAS 290Electrophoresis Documentation and Analysis System AdobePhotoshop 70 and Gel-Quant (Free Trial) software wasused for further editing Subsequent compilation of gels

Table 2 Combinations of oligonucleotides used in pairwise RAPD-PCR

Pair no Combination1slv 1 + Ilowast

4sm 1 + IV9slv 2 + II11slv 2 + IVlowast

12slv 2 + V14sm 2 + VII15slv 3 + Ilowast

16sm 3 + II18sm 3 + IV30sm 5 + IIlowastlowast

31sm 5 + IIIlowastlowast

32sm 5 + IVlowastlowast

321015840sm 535slv 5 + VIIlowast

37sm 6 + IIlowastlowast

43sm 7 + I44sm 7 + II45sm 7 + III56sm 2 + 358slv 2 + 5lowast

69sm 5 + 7lowast

71sm I + II76slv I + VIIlowast

77sm II + III(lowast) Pairs used for the genus Salvelinus species and forms (lowastlowast)mdashPairs usedwith all the rest fishes DNA

and construction of the binary matrix of the characters wasperformed manually The matrix was compiled according tothe ldquopresence or absence of the fragmentrdquo principle onlystable major bands were considered

Cluster analysis with constructing dendrograms wasperformed by the distant Neighbor Joining (NJ) approach ofSaitou and Nei [40] and UPGMA Pairwise genetic distances


between patterns were calculated according to the method ofNei and Li [41] and Nei [42] Distant trees were constructedusing the TREECON 13b program of Van De Peer and DeWachter [43] Node stability was tested by bootstrap analysisaccording to Felsenstein [44] and not samples but characterswere estimated in both cases

DNA fragments were extracted following electrophoreticseparation of the PCR products in the agarose gel in columnswith GFX PCR DNA and Gel Band Purification kit (Amer-sham Biosciences Inc USA) according to the manufacturerrsquosrecommendation For sequencing of heterogeneous matricesand fragments amplifying from a single primer correspond-ing products were cloned in E coli with InsTAclone PCRCloningKit (Fermentas Lithuania) following their extractionand freeing from agarose The kit included the so-called T-vector that is pTZ57RT plasmid with the extended ldquostickyrdquoT-end The colonies of white color containing inserts weregrown after screening in liquid medium In total 160 cloneswere examined The difference between lengths ranged from30 to 50 bp in different cases

DNA sequencing was performed using ABI PRISMBigDye Terminator v 31 kit with subsequent analysis of thereaction products on ABI PRISM 3100-Avant Genetic Ana-lyzer (Life TechnologiesApplied Biosystems USA) CHRO-MAS DNA and BioEdit programs were used for inter-pretation of chromatograms Homologous sequences weresearched for in GenBank with the use of dbEST bases fromNCBI resources BLAST software was used for the search

3 Results and Discussion

31 Restriction Endonuclease Analysis Restriction analysisof salmonid repetitive DNA has revealed some peculiaritiesof electrophoretic patterns in whitefishes The first of themrefers to the total number of bands of low intensity which isextremely high The matrices are not presented in the paperbecause of their large size and low information value Wehave also found this phenomenon in the other salmonids(Atlantic and Far-Eastern salmons trout and chars) whichis probably associated with their polyploidy origin We usedsperling (Osmerus sp) as an outer group in our opinionnotably lower degree of DNA banding patterns in this speciescould testify in behalf of this assumption

The second characteristic trait is the high degree ofsimilarity of the repetitive DNA in the studied fishes whichnot only are ldquogoodrdquo morphological species but also belongto different genera On the basis of this characteristic white-fishes differed from all previously studied organisms [25]the fishes of Salmonidae family (genera Salmo ParasalmoOncorhynchus and SalvelinusmdashFigures 2 and 3) among themsee also [27] and [45] An example of whitefishesrsquo restrictionpattern is shown on Figure 4

The results obtained indicate that each whitefish speciesis characterized under chosen experimental conditions bya number of major and minor bands ranging between 600and 40 bp but only some of them are species specific Forexample major band of 230 bp appearing after digestion byTaqI restriction endonuclease is present in broad whitefish

242238

201190180

160147

123

110

90

76

67

1 2 3 5 6 7 8 9 10 11 121314151617M M

217

4

Figure 2 Electrophoretic separation of DNA TaqI digest from(1) Salmo salar (2) S trutta (3) S trutta caspius (4) S ischchan(5) Parasalmo mykiss (steelhead anadromous form) (6) P mykiss(with traits of P clarkii) (7) P mykiss (freshwater steelhead) (8) Pmykiss (introduced rainbow trout) (9) P mykiss (steelhead NorthAmerica) (10) P clarkii (11)Oncorhynchus masou (12)O keta (13)O gorbuscha (14) O nerka (15) O kisutch (16) O tshawytscha M-markerMspI-digested pBR322 DNA

Coregonus nasus the least ciscoC sardinella and three NorthAmerican species Arctic ciscoC autumnalis broadwhitefishC nasus and lake cisco C artedi This band is notably lesspronounced in MspI cutting C lavaretus from Lake AnettiFinland (predator high-gillraker form) and European ciscoC albula The rest of species under study lack this bandThe band of 150 bp is common to all whitefish species exceptfor round whitefish Prosopium cylindraceum Specific bandscorrespond to 300 bp in Canadian Arctic cisco and 80 and60 bp in Finnish high-gillraker whitefish Distinct species-specific band of 170 bp appears in round whitefish afterdigestion by MspI the rest major bands of 460 bp 310 bpand 90 bp and smaller are common for all studied DNAThe sites for Sau3AI and Tru9I restriction endonuclease arenot polymorphic at all Cfr13I (AsuI) reveals species-specificbands 180 bp and 190 bp in round whitefish the rest majorbands (350 bp double 300 bp 240 bp 150 bp 135 bp double100ndash110 bp and smaller) are identical in all studied DNA

Thus taxonoprint analysis revealed surprising homo-geneity of the fraction of high-copy DNA repeats in differentspecies and even genera of whitefish (with round whitefishbeing the single exception mentioned above) Most of themajor bands in the patterns of all used restriction endonu-cleases were absolutely identical Slightly pronounced poly-morphism was found only in the families of sequences withsmall number of copies Reliability of the relative positions


242238217201190180160147

123110

90

76

67

1 32 4 5 6 7 8 9 10 1211M M

Figure 3 Electrophoretic separation of DNA TaqI digests from(1) Salvelinus alpinus (2) S drjagini (3) S malma (4) Salvethymussvetovidovi (5) Salvelinus elgyticus (6) S boganidae (7) S con-fluentus (8) S leucomaenis (9) Parasalmo clarkii (10) P mykiss(11) Salmo irideus (12) Oncorhynchus masou Mmdashmarker MspI-digested pBR322 DNA

of the branches on computer-generated phylogenetic treesappeared to be low

Taxonoprint analysis of Salmo Parasalmo and Oncor-hynchus genera specimens (Figure 2) showed distinct divisionof the samples under study into four groups of generic rank(1) Atlantic salmon Salmo salar (2) Brown trout Salmo truttaCaspian trout Salmo trutta caspius and closely related troutSalmo ischan from Lake Sevan (3) Kamchatka rainbow troutParasalmo mykiss its American freshwater form P gairdneriand cutthroat trout P clarkii (4) other species of the Pacificsalmons Oncorhynchus genus The bands of 242 and 240 bp175 and 140 120 110 and a number of bands lower than 70 bprevealed by the use of TaqI (Figure 2) are genus specific forall Salmo sensu stricto species The bands of 240 and 150120 and 76 and short bands of 10 20 and 30 bp absent inAtlantic Salmo and in Oncorhynchys sensu stricto [27] werepresent in the American and Kamchatka trout The numberof species-specific bands appeared to be very small withcoho salmon Oncorhynchus kisutch being the sole exceptionHowever the DNA pattern of masu Oncorhynchus masouhas nothing in common with the patterns of Pacific troutand Atlantic salmons except for the family-specific bandsof 67 bp 110 bp and 510 bp (Figure 2) the rest Oncorhynchusspecies differ from them to even greater degree

NJ dendrogram constructed on the basis of taxonoprintsof repetitiveDNAof salmonid fishes of SalmoParasalmo andOncorhynchus genera is shown in Figure 5 All six species ofthe Oncorhynchus genus in its classical interpretation formwith high reliability a cluster isolated from the American andKamchatka trouts

464

309

242238217201190180160147

123110

90

76

67

1 2 M 3 5 6 7 8 9 10 11 12 13 14 15 164

Figure 4 Electrophoretic separation of DNA Taq1 digests from (1)Osmerus sp (outgroup) (2) Coregonus lavaretus lacustrine form (3)C lavaretus anadromous form (4) C autumnalis migratorius (5) Cnasus (6) C lavaretus montchegor (7) C muksun (8) C lavaretuspidschiani (9) C albula (10) C sardinella (11) Stenodus leucichthysnelma (12) Prosopium cylindraceum (13) C autumnalis (ComoLake) (14)C clupeaformis (Como Lake) (15)C nasus (Como Lake)(16) C artedi (Huron Lake)

Distance analyses of endonuclease restriction data andtrees reconstruction were done with UPGMA [43] Figure 5depicts the genetic variability within salmon (a) chars (b)and whitefishes (c)

Dendrogram (a) of the Salmonidae family represents arobust phylogeny of species with perfect reproductive iso-lation Although position of some branches is controversialthe divergence of the Pacific trout Parasalmo and the salmonOncorhynchus is firmly established The diversification ofmajor nodes is dated by 5ndash15Mya [46 47]

Within the genus Salvelinus (b) all debatable species ofthe Salvelinus alpinus minus Salvelinus malma complex (includingthe North American Dolly Varden S confluentus) are sepa-rated by small genetic distances Their phylogenetic relation-ships cannot be established as evident from low bootstrapsupport values The compact Salvelinus alpinus minus Salvelinusmalma complex separates from Salvethymus svetovidovi andthe ldquogoodrdquo species S leucomaenis although the genus rankof Salvethymus [45 48] cannot be confirmed with these dataThe genus Salvelinus diverged at least 10Mya [49] althoughthe Salvelinus alpinusminus Salvelinusmalma complex is of glacialor postglacial origin that is of age less than 1My

On the whitefish phylogeny (c) the unresolved nodecontains all European and Asian species and forms ofCoregonus The North American representatives of the genusform a robust monophyletic clade However this group isalso compact and the distance between the two containedtaxa is less than 02 scale units The basal branch leads


97

01

Parasalmo clarkii

Salmo salar

Oncorhynchus gorbuscha

Parasalmo mykiss (freshwater steelhead) Parasalmo mykiss (anadromous steelhead)

Salmo trutta

Oncorhynchus kisutch

Oncorhynchus tshawytschaOncorhynchus nerka

Oncorhynchus keta

Parasalmo mykiss (steelhead North America)

Parasalmo mykiss (clarkii-redband)

Salmo ischchanSalmo trutta caspius

97

99

98

97

100

100

99

8263

100

72

76

Parasalmo mykiss (rainbow trout)

Oncorhynchus masou

Figure 5 Salmon fishes NJ unrooted tree from restriction analysis data

to the second coregonid genus represented by Prosopiumcylindraceum Recent whitefish diverged in the Pliocene 2ndash5Mya or perhaps even earlier [50] The genus Prosopiumdiverged in the early Miocene [51] dating the origin of roundfishes to 20Mya This is the only reproductively isolatedspecies of the Coregonidae highly supported as a distinctlineage by repetitive DNA restriction data

Genetic variability of highly repetitive DNA in the threegroups (Figures 6(a)ndash6(c)) is in good agreement with dat-ing of the clades and the level of reproductive isolationhigh within true salmons (clades diverged about 15Myaspecies are strictly isolated) intermediate within chars (thegenus itself diverged about 10Mya the Salvelinus alpinus minusSalvelinus malma complexmdashless than 1Mya forms are genet-ically close albeit their hybridization is spatially prevented)and low within whitefishes (the main diversity established2ndash5Mya hybridization is commonly observed across allEurasian forms)

The presented evidence experimentally corroborates thehypothesis of concerted evolution of long satellite DNA

32 RAPD-PCR As could be expected data on PCR-RAPDof whitefish were more diversified (Figure 7) though in thiscase bootstrap indices also appeared to be not high when thephylogenetic trees of Coregonidae were constructed (Figure8)

Changes in algorithms of calculation of the geneticdistances resulted in alteration of the tree topology With allobtained dendrograms being considered we can probablydiscuss only certain tendencies in relationships of whitefishRound whitefish P cylindraceum stands apart from all otherspecies in the family Coregonidae and tends to occupythe position of the outer group It shows up in all treesconstructed by the UPGMA method when the outer group

is chosen automatically as the most distant one That is tosay remoteness of round whitefish P cylindraceum from theother studied whitefishes is comparable to that of the sperlingOsmerus sp Inconnu (nelma) Stenodus leucichthys formscluster with cisco Coregonus albula whereas Baikal omul-arctic cisco (C autumnalis migratorius) should be separatedfrom the Arctic cisco C a autumnalis as an independentspeciesThe latter is very close to the Bering ciscoC laurettaeand has probably originated from the Clupeaformis arthedigroup of numerous whitefish species inhabiting the GreatLakes This assumption matches data on allozyme analysis[52]The Baikal Arctic cisco C autumnalis have been alreadyproposed to be segregated as a separate species [53] Our datasuggest that Leucichthys is a composed diphyletic subgenus

Experiments on RAPD analysis of Salvelinus chars wereaimed at the assessment of the genetic diversity of the wildpopulations of Salvelinus malma inhabiting Paramushir andOnekotan Islands Electrophoretic pattern is shown in Figure9

Population genetics of Salvelinus is of particular interestbecause of extensive processes of form and species generationoccurring now in the genus [54] Nominal species of thegenus are polymorphic and represented by anadromous andfreshwater forms as well as the geographic isolates char-acterized by particular morphological traits Despite greatcommercial interest in salmonid fishes the rate of geneticisolation of various char forms in the wild has been poorlystudied so far DNA of five available ldquogoodrdquo morphologicalspecies (S alpinus S leucomaenis S fontinalis S namaycushand S malma) was used for selection of the primers thatallow discrimination between the Salvelinus species Thelayout of electrophoretic patterns of the reactions with thepairs of primers NN 1 9 11 12 15 30 31 32 35 37 5869 and 76 (Tables 1 and 2) was used The listed markers


Parasalmo mykiss (freshwater)

01020304050607

Parasalmo clarkii (redband trout)44

Oncorhynchus masou

Parasalmo clarkii

Oncorhynchus ketaOncorhynchus gorbuscha

Salmo salar

Parasalmo mykiss (anadromous)


Salmo trutta

Oncorhynchus nerkaOncorhynchus tshawytscha

Salmo trutta caspiusSalmo ischchan

Parasalmo mykiss (irideus)

64100

90100

5396

98

100

98

100

46

72

100Parasalmo mykiss (Oregon USA)

Oncorhynchus masou

Salvelinus leucomaenisSalvethymus svetovidoviSalvelinus drjagini

Salmo salar

Salvelinus elgyticus

Parasalmo irideus

Salvelinus malmaSalvelinus confluentus

Salvelinus alpinusSalvelinus boganidae

Parasalmo clarkiiParasalmo mykiss

7476

8797

20

79

27

89

32

30

93

Osmerus eperlanus dentexProsopium cylindraceum

Stenodus leucichthys (nelma)C autumnalis migratorius (resident)

Coregonus artedi

C sardinellaC lavaretus (resident Anatti Lake)

Coregonus lavaretus (anadromous)C muksun

C pidschianiC albulaC nasusC lavaretus montchegor

C clupeaformis (Como Lake)C clupeaformis (dwarf Como Lake)C autumnalis (resident Como Lake)C nasus (Como Lake)

100

99

5547

28

83

22

24

69

2731

35

31

100

96

(a)

(b)

(c)

Figure 6The extent of genetic variability within salmon observed from restriction endonuclease digestion data by UPGMA (a) Salmonidae(b) Salvelinus and (c) Coregonidae For (a) and (c) the proportion of acrylamidebisacrylamide in PAGE was 29 1 for (b) this proportionwas 19 1 which resulted in slightly lower numbers of detected bands and lower absolute genetic distances

had sufficient polymorphism and the difference between thespecies is evident from the pattern of the major bands aswell In the course of binary matrix compilation all bands inthe electrophoretic pattern of PCR products were taken intoconsideration

A total of 170 DNA samples representing 23 forms andspecies of chars were analyzed for the tree constructionMany analyzed isolated forms have been described [55] asspecies on the basis of various phenotypic characteristics

though some experts [54 56] consider them to represent asingle complex species S alpinus complex with circumpolardistribution and low rate of genetic distinction [57 58] Onthis particular stage of the study we attempted to use geneticdistances in a restricted system of themarkers as ameasure oftaxonomic status of the certain forms Salmo salarwas used asan outer group We took advantage of the matrix of pairwisedistances generated by TREECON for NJ tree constructionto comprise the absolute values of genetic distances between


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Figure 7 PCR-RAPD electrophoretic pattern of coregonid fishesDNAwith pairwise combination of primers no II + IV (Table 2) (1)Osmerus sp (2) Coregonus lavaretus (resident high-gillraker formAnatty Lake) (3) C lavaretus (anadromous form) (4) C lavaretus(resident form) (5)Coregonus lavaretus montchegor (6)C lavaretuspidschiani (7) C muksun (8) C autumnalis migratorius (BaykalLake resident form) (9) C autumnalis autumnalis (10) C autum-nalis (Como Lake resident form) (11)C nasus (12)C nasus (ComoLake) (13)C albula (14)C sardinella (15) Stenodus leucichthys (16)Prosopium cylindracium (17) C clupeaformis (Como Lake) (18) Cclupeaformis (dwarf) (19) C arthedi

commonly acknowledged ldquogoodrdquo as well as disputable speciesand isolates (Table 3) On the tree as well as in the table thedisputable species are designate by quotation marks

Analysis of the genetic distance dendrogram (Figure 10)allows us to draw the following conclusionsThe tree consistsof three clusters The first of them brings together all formsand species of Salvelinus alpinus complex under study Thestability of this node is 74 of bootstrapThe entire assemblyof Dolly Varden (S malma) is included into this cluster asits component It is heterogeneous in the used system ofthe markers Anadromous Dolly Varden from Bering Islandappeared to be proximal to the long-finned char Salvethymussvetovidovi from Lake Elgygytgyn though this groupingis uncertain On the whole the entire node combining themalma trout and the forms of the Arctic char seems unsolvedKamchatka predatory char (S malma) forms complex withthe other Kamchatka and Kuril Dolly Varden (40 bootstrapsupport) UPGMA method even more robustly adds pre-datory char to the cluster containing Dolly Varden (tree isnot presented) The same common cluster along with the Smalma contains also long-finned char ldquoSalvethymusrdquo des-cribed as a specimen of a separate genus [46]

Since at this stage of the work resolving power adequateto the species or close to it taxonomic level was used as acriterion for selection of the markers 46 support for theinner cluster suggests the Dolly Varden being more likelyof intraspecific status with respect to S alpinus complex asBerg supposed [59] The second supported cluster (67 ofbootstrap) is formed by white-spotted char S leucomaenisReliability of the specific status is undisputed in this caseAlthough genetic distance between the South Kuril andKamchatka white-spotted chars is relatively high they form amonophyletic tree cluster corresponding to the single speciesS leucomaenis At least two American species Salvelinusfontinalis and Salvelinus namaycush form the third branch

supported by 68 Alteration of the outer group (Salmosalar changed for Osmerus) or application of the alternativemethods to construction of the trees (UPGMAor parsimony)did not change the topology in the basal part In fact twoNorth American species represented an additional outergroup for all other members of genus Salvelinus

Within three discriminated clusters the branch points arein most cases uncertain and could hardly be used for deter-mination of the relationships of the separate populationsThedendrogram shows that anadromous Arctic char Salvelinusalpinus a type species of the genus Salvelinus forms a com-pact group that includes both anadromous and freshwaterforms from Inary and Sayma Lakes (Finland) anadromouschar from Svalbard and Drjaginrsquos char from Taimyr Accord-ing to Table 3 intrasample distances in this group rangebetween 114 and 235 Neirsquos distance units (times10minus2) and aver-aged 1803 Neirsquos units In parsimonial construction the clustershows maximal number of synapomorphies (not shown)

This group was used as a reference point for the estima-tion of the average genetic distances between chars of differ-ent taxonomic status The distances between the samples ofDolly Varden (248 times 10minus2 Neirsquos units on average) were esti-mated likewise Subsequent analysis of the correspondence ofthe genotypic data to the position of particular forms in thesystem of genus Salvelinus was performed with the use of thetable of pairwise distances Data presented in Table 3 allowscomparison of the absolute genetic distances between variousforms of chars with the disputable taxonomic status and theldquogoodrdquo species arctic char Salvelinus alpinus white-spottedchar S leucomaenis brook trout S fontinalis and lake trout Snamaycush According to the table the distances between Salpinus and S leucomaenis and S fontinalis and S namaycushexceed 40 times 10minus2 Neirsquos units The distance between all therest chars and S alpinus could be defined as correspondingto the intraspecific taxonomic level with the different rateof advance The longest distances are characteristic of theisolated forms of chars inhabiting the river and lake basinof the Taimyr Peninsula and Transbaikalian endemic species[60] Although the data of this table are by nomeans the abso-lute indicators of the taxonomic status of particular formsthey provide an idea about correspondence of the pheno-typic and genotypic data

Identification of the nucleotide sequences of the frag-ments generated in PCR in the used system of primers withsubsequent search for the homologous sequences inGenBankshowed that most of the fragments forming RAPD-PCRelectrophoretic pattern had homologies in dbEST base ofNCBI resources with the ldquoSalmonidaerdquo or ldquozebrafishrdquo as afilters (Table 4) Only extensive homological sequences withlow ldquoexpectrdquo value (low probability of the random coinci-dence) were inserted into the table

RAPD fragments for the sequencing were chosen fromNorth Kuril Islands populations Salvelinus malma DNAFrom 160 clones analyzed for 10 insert DNA the homologieswere not found (ldquojunkrdquo DNA) and for four inserts homolo-gies were found in nucleotide collection (microsatellites)The great part of the salmon fishes EST resources wherehomologies for variable RAPD fragments have been foundwas developed by Koop et al [61] The detailed analysis of


01

Osmerus sp

Prosopium cylindraceum

C autumnalis (Como Lake resident form)

C sardinella

C lavaretus (anadromous form)

C clupeaformis (dwarf)

C autumnalis migratorius (resident form)

C nasus

Coregonus muksun

C lavaretus montchegor

Stenodus leucichthysC albula

C clupeaformis (Como Lake)C nasus (Como Lake)

C arthediC autumnalis (anadromous form)

C l pidschiani

C lavaretus (Lama Lake resident form)100

78

45

42

52

87

66

51

96

C lavaretus (Anatty Lake resident form)

Figure 8 NJ tree of coregonid fishes from PCR-RAPD (more detailed description of species and forms are at the legend of Figure 6)

Table 3 The absolute values of genetic distances Nei between different forms of chars and the valid species

Species and forms S alpinuslowast S leucomaenislowast S fontinalis S namaycushGenetic distances [Nei times 10minus2]

S alpinuslowast 00 4035 430 416S alpinus (Tuulispaa Lake) 258 416 481 421S alpinus Black char (Lama Lake) 346 444 533 500ldquoS boganidaerdquo 2755 3835 403 404S alpinus (Barents Sea basin) 271 4455 500 461S alpinus Davatchan (Baikal Lake basin) 334 4635 565 533S alpinusMountain char (Lama Lake basin) 3417 444 472 496Smalma Predator (Kamchatka Peninsula) 2785 401 419 364S alpinus Goggle-eyed (Lama Lake basin) 347 49 464 488ldquoS neivardquo Neiva (Sea of Okhotsk basin) 303 453 405 483ldquoSalvethymus svetovidovirdquo (Elgigitgin Lake Chukotka Peninsula) 267 466 415 402Smalmalowast (Kuril Islands) 2926 406 403 419Thematrix of distances was generated by TREECON for construction of NJ tree For the chars that abbreviate with (lowast) the averaged pairwise distances betweenall samples analyzed are shown The more detailed description of sample is shown at Figure 10

these results is still not completed now but major patternappears to be followed It was found that themajoritymarkersused were either entire exons or contained exon fragmentsabundantly exhibited in cDNA libraries and portions ofintrons or intergenic DNA without detectable homologies inany library The sequences with open frame encoded con-servative protein domains in individual cases were found forthemolecularmarkers with exon origin For example a signi-ficant and extensive homology with the hypothetical variable

protein of D rerio with the length of 659 amino acids (accno XP 0013328301) 52 identity and very low probabilityof coincidence (2minus26) was found after the nucleotide matrixof 123 fragment had been converted into translated proteinsequence by reading from frame 3

Another fragment with the length of 905 bp was found tobe the most homologous toD rerio (zebrafish) clone DKEY-182H7 in linkage group 7 (acc no BX 66360929) andmRNAof predicted protein similar to norepinephrine (analogue of


1 2 3 4 5 6 MMMMMMM

700 bp 700 bp

Figure 9 PCR-RAPD electrophoretic pattern of chars genus Salveli-nus DNA with pairwise combination of primers II + 3 The samplesare (1) Dolly VardenmdashSalvelinus malma (Kolrsquo River KamchatkaPeninsula) (2) Dolly Varden (Chernoe Lake Onekotan Island)(3) Dolly Varden (Fontanka Stream Onekotan Island) (4) DollyVarden (Shelekhovka River Paramushir Island) (5) Dolly Varden(Golrsquotsovyi StreamOnekotan Island) and (6)white-spotted charrmdashS leucomaenis (South Kuril Islands) Mmdashmarker ladder 100 bp + 15kb

noradrenalin) carrier (acc no XM 6890463 in GenBank)All the remaining homologies found for this sequencewere tocertain extent connected with noradrenalin protein carriers

As was pointed out above all PCR-RAPD reactions wererepeated two times as minimum and reproduced absolutelyMay be it is the result of the RAPD markers connectionswith the conservative protein coding genome areas in usedprimers systemWe analyzed themonomorphous generatingthe structure of electrophoretic patterns as well as poly-morphic fragments Essentially it seems likely that intronsor repetitive ldquojunkrdquo DNA with no homologies in dbESTor nucleotide databases are most informative for estimatinggenetic distances For some of them the homologies in NCBIdbTSA were found

Only in the single cases the homological sequencesbelonged to mtDNA with high evolution rate that shouldbe able to correspond with the divergence in intraspecifictaxonomical level For example for reading from frame4 sequence of 254 bp length (Table 4) the homology wasfound with Cytb of Atlantic salmon Salmo trutta (accno ACO57211) However it is impossible to exclude theimplication of adaptive nuclear sequences in divergence ofmodel groups studied

Since the fishrsquos groups selected differ considerably in basicstages of evolution and divergence time [30 47 51 62 63]integrated genomes assessment allows reducing the errorsof disparity caused by difference in molecular evolution ofdifferent genes [64]

In the row containing almost all species and isolatedforms of chars (disputable allopatric ldquospeciesrdquo) absolutegenetic distances were used as a criterionmdashsee Thorpe [65]Correctness of this approach was confirmed by 100 homol-ogy of nucleotide sequences of RAPD fragments with identi-cal electrophoretic mobility obtained in a single reaction

According of our results the well-identified species thelake trout and the brook trout form a single cluster No infor-mation on the level of phylogenetic relationships betweenS namaycush and S fontinalis is available to us Howeverexistence of hybrid forms traced up to F

4by Berst et al [66]

and entering reproductive relations indicate close relationsbetween these two North-American species Molecular databy Westrich et al [67] also are in favor of this assumptionSister relations between S namaycush and S fontinalis oppose

the proposal to consider S namaycush as a specific genusldquoCristivomerrdquo [68] This species should be placed in genusSalvelinus

The second conclusion is connected with extremely lowvalues of genetic distances between all samples of DollyVarden and forms of the Arctic char The degree of thesedistances is the same as that between the anadromous andisolated forms of the Arctic char and even lower in somecases If we rely on the distances only we should refer allstudied populations of Dolly Varden to the Arctic char Inother words specific status of Salvelinus malma could becontested in case it is based only on the absolute values ofgenetic distances

Restriction analysis of the Atlantic and Pacific salmonshas revealed specific sets of DNA fragments in every speciesand even in the morphological forms of these fishes Alongwith it the results indicate pronounced isolation of thePacific trout from the members of the genus OncorhynchusIn addition taxonoprints considerably and reliably differ inthe species with overlapping ranges (sympatric species) It istrue for Pacific salmons of the genus Oncorhynchus (chumsalmon pink salmon sockeye salmon chinook salmoncherry salmon and coho salmon) the chars of the genusSalvelinus (S alpinus and S leucomaenis) and two chars fromLake Elgygytgyn (long-finned char Salvethymus svetovidoviand Arctic char S alpinus)

The main part of these results was received with anony-mous DNA sequences before the GenBank recourses becameavailable but they do not seem contrary to Crespi and Fulton[69] strong results with employment of a powerful tool ofgenomics (with the exception of taxonomic relations of Omasou)

Analysis of whitefishesrsquo restriction data yielded the resultsnot completely matching those generally accepted in themodern systematics of whitefishes [50 70 71] From thepoint of view of systematics Coregonidae is one of the mostcomplex and intricate groups Great variability and polymor-phism of the whitefishes are the reason for the differencesin conclusions about phylogenetic links between speciesbased on different approaches for example Bernatchez etal [72] Smith and Todd [73] Bodaly et al [52] Frolov[74] Turgeon and Bernatchez [30] Investigation of geneticstructure of the species and identification of closely relatedspecies from various sites of the range also cause difficultiesUp to a hundred intraspecific categories were described fora whitefish type species C lavaretus from the Russian waterbasins only [50]

Interspecific and even in some cases intergenerichybridization between the representatives of Coregonidaefamily yielding viable hybrids is a well-known fact describedby Garside and Christie a long time ago [4] Casual relationsbetween the diversity of the forms of whitefishes and intro-gression have been discussed more than once for exampleSvardson [75] and Bernatchez et al [76] The possibilityof exchange of genetic information in whitefish could beconsidered proved It could be due to this that the indexof genetic distances in them is usually lower than that inthe taxa of the same level in other animals as reported byBodaly et al [52] and Kartavtsev [77] Such phenomenon as


Table 4 Salvelinus malma DNA PCR-RAPD fragment sequences searched for NCBI

Pairs of primers PCR RAPD cloningfragment lengthlowastlowast

Genbank Accession Numbers of homological sequenceslowast

Microsatellites51015840 rarr 31015840 dbESTlowast Protein productlowastlowastlowast

IV + IV 555 bp EG8497461 + IV 568 bp DY73125IV + IV 463 bp EG827041 BT 0719911 + 1 304 bp EG827041IV + 1 439 bp EG930580 NP 001133464IV + 1 676 bp BX086452IV + IV 446 bp CX357234

IV + 1 395 bp EG935616DW006459 XP 003198377

1 + 1 418 bp Microsatellite(CA)n 173ndash331

1 + 1 905 bp BX663609 XM 689046

2 + VII 409 bp Microsatellite(GT)n 5ndash32

3 + 3 549 bp EG923517 NM 0011025933 + 3 384 bp DQ156149 XP 0013328303 + 3 384 bp CB511135II + 3 122 bp EU621899 XP 001336520

II + II 959 bp Microsatellite(GT)n 226ndash294 AU081124

3 + II 283 bp CX7230143 + II 453 bp CU0627333 + II 190 bp GE8281933 + II 130 bp GU552297 ADV31329IV + 3 108 bp EG911815 XP 0033850093 + IV 236 bp EG911136 ACH85273

3 + IV 389 bp Microsatellite(CA)n 276ndash329

3 + IV 413 bp EG792115 CBX111563 + IV 724 bp CA353611IV + 3 109 bp EG911815 XP 0011953783 + 3 801 bp DW556963 ACI337923 + 3 496 bp CB4860603 + 3 412 bp EG831541 NP 0011540533 + 3 322 bp BX861631 XP 003197666IV + IV 554 bp EG849746 NP 0011673053 + IV 290 bp EG915402 NP 0011352513 + 3 638 bp CB509929 ACO084363 + IV 275 bp CA388004 NP 0011333893 + 3 685 bp CK898369 NP 0011671873 + 3 414 bp FF839690

IV + IV 254 bp CB490887 ACO 57211AAP58348

3 + IV 236 bp EG911136 ACI660283 + 3 414 bp EG792114 XP 0013352243 + 3 490 bp CB486060 ACI667697 + I 330 bp EG 760735 XP 0032000233 + IV 281 bp BX861631 NP 001187967lowastOnly the first homological sequence from different libraries is exhibitedlowastlowastThe lengths of cloning PCR fragments are given without considering primerslowastlowastlowastTranslation performed by EMBOSS transeq (Sequence Translation Sites) of EBI-EMBL


01

Salmo salar

ldquoS boganidaerdquo lacustrine form (Taimyr Peninsula)

S alpinus dwarf resident form (Tuulispaa Lake Finland)

S malma predator lacustrine char (Kamchatka Peninsula)

S alpinus Black char (Lama Lake Taimyr Peninsula)

S alpinus resident form (Sayma Lake Finland)

S alpinus Davatchan resident form (Baikal Lake basin)

S alpinus anadromous form (Inari Lake Finland)

ldquoS drjaginirdquo (Sobachye Lake Taimyr Peninsula)

S alpinus anadromous form (Svalbard)

S alpinus Mountain char river-lacustrine form(Taimyr Peninsula)

S alpinus Murman char anadromous form (Barents sea basin)

ldquoS neivardquo Neiva (sea coast of Okhotsk)

S alpinus Goggle-eyed char resident form (Lama Lake)

S malma anadromous form (Bering Island)

ldquoSalvethymus svetovidovirdquo (Elgigitgin Lake Chukotka Peninsula)

S malma anadromous form (Paramushir Island)

S malma anadromous form (Kamchatka Peninsula)

ldquoS gritzenkoirdquo (Chernoe Lake Onekotan Island)

S malma resident form (Onekotan Island)

S leucomaenis white spotted char (South Kuril Islands)

S leucomaenis white spotted char (Kamchatka Peninsula)

S namaycush (North America)

S fontinalis (North America)

100

68

74

40

92

53

50

44

88

94

67

94

Figure 10 NJ tree of the g Salvelinus forms and species constructed in accordance withNei genetic distancesThe figures at the node indicatebootstrap indexes that exceed 40

ldquogenetic parasitismrdquo when the smaller and more numerousform replaces the much larger one has been found anddocumented by Svardson in whitefishes [78] Geographicisolation and isolation of other kinds as well as relativelyyoung historical age (the main diversification of Coregonusspecies dates to Pleistocene glaciationsmdashabout 15000 yearsin accordance with Behnke [51]) must have prevented variousCoregonidae species from developing specific families ofrepetitive sequences as it is common to noninterbreeding

populations On the contrary many families of repetitivesequences are homogenous within all these forms whichindicate the intensive gene flow between all these speciesallowing molecular drive to adjust them

All above mentioned enlightens us about greater rate ofthe differences in the experiments on amplification with arbi-trary primers They are the markers of the loci of expressedsequences and introns that are not subjected to molec-ular drive However no differences sufficient for reliable


discrimination of the species have been accumulated inthese fractions either Taking all the aforesaid into conside-ration we can state that all these facts summed up vividlyindicate a peculiar way of evolution and genetic structureof the Coregonidae population Contrary to the commondivergent evolution characteristic of the majority of animalswhitefishes demonstrate the elements reminding of reticulateevolution (ldquoevolution via hybridizationrdquo) as supposed byTodd and Smith [29] Turgeon Bernatchez [30] and Svard-son [79] described for many plants for example Grant [80]This pattern of evolution implies alternation of the diver-gent and hybridization (conversion) stages External pheno-typical differences in this case are determined by a smallnumber of genes acting asmorphological triggers and switch-ing morphogenesis to a certain direction the final studies ofthe process are known as discrete described forms written byRenaut and Bernatchez [81] Accumulated cross breeding ofthe hybrids with prevailing parental species could becomeone of the possible mechanisms supporting morphologicalindependence in the course of interspecies exchange ofgenetic information However any interpretation based ongenetic isolation of Coregonidae species in the wild encoun-ters the following unsolvable inconsistency In the frameworkof such traditional hypothesis one had to explain why themutations in DNA repetitive sequences no longer occurredandwere not preserved in this group which seems incredibleThus the results obtained with the use of restriction analysisof highly repetitious DNA revealed great differences betweenelectrophoretic patterns of the salmons with the high degreeof reproductive isolation and the whitefishes In the latterintrogressive hybridization has probably occurred and thespecies still hybridize retaining their independent status

Geographic isolation of the species is one of the mecha-nisms preventing interspecific mating Over a long time theview on allopatric speciation as a process of gradual accumu-lation of gene mutations of adaptive character located mostlyin the coding DNA sequences has been commonly accepted(see eg [82 83]) and noncoding repetitive sequenceswere thought to be ldquojunkrdquo DNA Now indirect evidence ofinvolvement of the repetitive sequences (mostly mobile ele-ments) in the adaptive evolution has been suggested by manyresearchesmdashsee for review Schmidt and Anderson [84] andOsada and Wu [83] Studies of two sister Drosophila specieshave shown that heterochromatic region of X chromosomeplays a great role in the establishment of the reproductivebarrier [85] Our results support this viewpoint

4 Conclusion

The families of salmon fishes with different levels of repro-ductive isolation were compared using two strategies ofmultilocus fragment analysis The compared lineages are (1)Salmonidae possessing almost perfect homing and abso-lute reproductive isolation (2) chars of g Salvelinus (fSalmonidae) possessing ldquogoodrdquo and ldquodifficultrdquo species (repro-ductive isolation is often of spatial nature) (3) g Coregonus(f Coregonidae) containing recognized taxonomic specieswith common interspecies hybridization but distinct speciesphenotypes Each sampling contained intraspecies forms

andor ldquodisputablerdquo species ldquogoodrdquo species interbreedingspecies and representatives of a sister genus

Genetic distances were compared for lineages of the sametaxonomic rank and juxtaposed with their divergence timesbootstrap support values were verified for correspondingphylogenetic clades As a markers two types of sequenceswere chosen (1) the long regions of satellite DNA and (2)anonymous loci containing in 70 cases conserved exon andintron (or intergenic) regions Genetic distances and cladesrobustness were shown to correlate well with the level ofreproductive isolation in both marker systems

The hypothesis of concerted evolution of satellite DNAis experimentally corroborated The stronger is reproductiveisolation between forms and species the more species-specific band patterns are found in satellite DNA Amongwhitefishes the round fish Prosopium cylindraceum is theonly reliably distinguished species separated from the unre-solved Coregonus clade by a genetic distance comparable tothose between individual genera in Salmonidae

Molecular markers were used to clarify particular ques-tions in salmon taxonomy and systematics (1) Salvelinusfontinalis and Salvelinus namaycush are genetically close toeach other (2) Salvethymus svetovidovi cannot be considereda separate genus (3) Dolly Varden Salvelinus malma is gene-tically identical to Salvelinus alpinus (4) all species of thegenera Salmo Parasalmo and Oncorhynchus are reliably dis-tinguished with the genus Parasalmo being sister to Oncor-hynchus

Conflict of Interests

The authors declare that they have no competing interestsrelated to any of the mentioned products

Acknowledgments

This study has been performed thanks to Boris M Mednikovideas The authors thank K A Savvaitova (the IchthyologyDepartment of Lomonosov Moscow State University) YuS Reshetnikov (AN Severtsov Institute of Ecology andEvolution Russian Academy of Sciences) M Kaukorrantafor sampling and important consultations and A Lomov forkindly placed some DNAs The authors are grateful to DrAleshin and Dr Roussine for the assistance in preparation ofthe paper The constructive remarks of two anonymous ref-erees and referee for English corrections are acknowledgedThe study was supported by Russian Federative ResearchInstitute of Fisheries and Oceanology the Russian Fond ofBasic Researches Grants 97-04-49753 and 01-04-48613 andFederal Program of the Ministry of Science and Education(the program Scientific and Scientific-Pedagogical Personnelof Innovative Russia)

References

[1] E Mayr Animal Species and Evolution Harvard UniversityPress Cambridge Mass USA 1963

[2] T Dobzhansky Genetics and the Origin of Species ColumbiaUniversity Press New York NY USA 1937


[3] J Mallet ldquoPerspectives Poulton Wallace and Jordan how dis-coveries in Papilio butterflies led to a new species concept 100years agordquo Systematics and Biodiversity vol 1 no 4 pp 441ndash4522004

[4] E T Garside and W J Christie ldquoExperimental hybridizationamong three coregonine fishesrdquo Transaction of the AmericanFishery Society vol 9 no 2 pp 196ndash200 1962

[5] G Svardson ldquoTheCoregonid problemVIIThe isolationmech-anism in sympatric speciesrdquoReports of the Institute of FreshwaterResearch vol 46 pp 95ndash123 1965

[6] J BWWolf J Lindell and N Backstrom ldquoSpeciation geneticscurrent status and evolving approachesrdquo Philosophical Transac-tions of the Royal Society B vol 365 no 1547 pp 1717ndash1733 2010

[7] R Gross B Gum R Reiter and R Kuhn ldquoGenetic introgres-sion between Arctic charr (Salvelinus alpinus) and brook trout(Salvelinus fontinalis) in Bavarian hatchery stocks inferred fromnuclear and mitochondrial DNA markersrdquo Aquaculture Inter-national vol 12 no 1 pp 19ndash32 2004

[8] K T Scribner K S Page and M L Bartron ldquoHybridization infreshwater fishes a review of case studies and cytonuclearmeth-ods of biological inferencerdquo Reviews in Fish Biology and Fish-eries vol 10 no 3 pp 293ndash323 2000

[9] J A Coyne and H A Orr ldquoThe evolutionary genetics of spe-ciationrdquo Philosophical Transactions of the Royal Society B vol353 no 1366 pp 287ndash305 1998

[10] A G Hudson P Vonlanthen R Muller and O SeehausenldquoReview the geography of speciation and adaptive radiationin coregonines Biology and management of coregonid fishesrdquoArchives of Hydrobiology Special Issue Advances in Limnologyvol 60 no 2 pp 111ndash146 2007

[11] J M Wright ldquoNucleotide sequence genomic organizationand evolution of a major repetitive DNA family in tilapia(Oreochromis mossambicuslhomorum)rdquo Nucleic Acids Researchvol 17 no 13 pp 5071ndash5079 1989

[12] J A Shapiro and R von Sternberg ldquoWhy repetitive DNA isessential to genome functionrdquo Biological Reviews of the Cam-bridge Philosophical Society vol 80 no 2 pp 227ndash250 2005

[13] M A Biscotti A Canapa E Olmo et al ldquoRepetitive DNAmolecular cytogenetics and genome organization in the Kingscallop (Pecten maximus)rdquo Gene vol 406 no 1-2 pp 91ndash982007

[14] S Ohno Evolution By Gene Duplication Springer New YorkNY USA 1970

[15] W Chase A V Cox D E Soltis et al ldquoLarge DNA sequencesmatrices phylogenetic signal and feasibility an empiricalapproachrdquo in Proceedings of the American Society of Plant Taxo-nomists Annual Meetings Montreal Canada August 1997

[16] J D Thompson J E Sylvester I Laudien Gonzalez C CConstanzi and D Gillespie ldquoDefinition of a second dimericsubfamily of human 120572 satellite DNArdquo Nucleic Acids Researchvol 17 no 7 pp 2769ndash2782 1989

[17] P Warburton and H Willard ldquoEvolution of centromeric alphasatellite DNA molecular organization within and betweenhuman and primate chromosomesrdquo in Human Genome Evo-lution M S T Jackson and G Dover Eds pp 121ndash145 BIOSScientific Publishers 1996

[18] T Ohta ldquoOn the evolution of multigene familiesrdquo TheoreticalPopulation Biology vol 23 no 2 pp 216ndash240 1983

[19] E A Zimmer S L Martin and S M Beverley ldquoRapid duplica-tion and loss of genes coding for the 120572 chains of hemoglobinrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 77 no 4 pp 2158ndash2162 1980

[20] A J Jeffreys V Wilson and S L Thein ldquoHypervariable ldquomini-satelliterdquo regions in humanDNArdquoNature vol 314 no 6006 pp67ndash73 1985

[21] KM Gray JWWhite C Costanzi et al ldquoRecent amplificationof an alpha satellite DNA in humansrdquo Nucleic Acids Researchvol 13 no 2 pp 521ndash535 1985

[22] J F Elder Jr and B J Turner ldquoConcerted evolution at the popu-lation level pupfish HindIII satellite DNA sequencesrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 91 no 3 pp 994ndash998 1994

[23] A N Fedorov L V Fedorova V V Grechko et al ldquoVariableand invariable DNA repeat characters revealed by taxonprintapproach are useful for molecular systematicsrdquo Journal ofMolecular Evolution vol 48 no 1 pp 69ndash76 1999

[24] W M Brown ldquoPolymorphism in mitochondrial DNA of hu-mans as revealed by restriction endonuclease analysisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 77 no 6 pp 3605ndash3609 1980

[25] V V Grechko L V Fedorova A N Fedorov et al ldquoRestrictionendonuclease analysis of highly repetitive DNA as a phyloge-netic toolrdquo Journal ofMolecular Evolution vol 45 no 3 pp 332ndash336 1997

[26] I A Roudykh V V Grechko D G Ciobanu D A Kramerovand I S Darevsky ldquoVariability of restriction sites in satelliteDNA as a molecular basis of taxonoprint method evidencefrom the study of Caucasian rock lizardsrdquo Russian Journal ofGenetics vol 38 no 8 pp 937ndash941 2002

[27] B M Mednikov E A Shubina M N Melrsquonikova et al ldquoTheproblem of the generic status of Pacific salmon and trout (Gen-etic Taxonomic Analysis)rdquo Journal of Ichthyology vol 39 no 1pp 10ndash17 1999

[28] L Bernatchez and J J Dodson ldquoAllopatric origin of sym-patric populations of lake whitefish (Coregonus clupeaformis)as revealed by mitochondrial-DNA restriction analysisrdquo Evolu-tion vol 44 no 5 pp 1263ndash1271 1990

[29] T N Todd andG R Smith ldquoA review of differentiation in GreatLakes ciscoesrdquo Polskie Archiwum Hydrobiologii vol 39 no 3-4pp 261ndash267 1992

[30] J Turgeon and L Bernatchez ldquoReticulate evolution and phe-notypic diversity in North American ciscoes Coregonus ssp(Teleostei Salmonidae) implications for the conservation of anevolutionary legacyrdquoConservation Genetics vol 4 no 1 pp 67ndash81 2003

[31] G R Smith and R F Stearley ldquoThe classification and scientificnames of rainbow and cutthroat troutsrdquo Fisheries vol 14 no 1pp 4ndash10 1989

[32] J Welsh and M McClelland ldquoGenomic fingerprinting usingarbitrarily primed PCR and a matrix of pairwise combinationsof primersrdquoNucleic Acids Research vol 19 no 19 pp 5275ndash52791991

[33] J G KWilliams M K Hanafey J A Rafalski and S V TingeyldquoGenetic analysis using random amplified polymorphic DNAmarkersrdquoMethods in Enzymology vol 218 pp 704ndash740 1993

[34] F E Arrighi J Bergendahl andMMandel ldquoIsolation and char-acterization of DNA from fixed cells and tissuesrdquo ExperimentalCell Research vol 50 no 1 pp 40ndash47 1968

[35] S E Saunders and J F Burke ldquoRapid isolation ofminiprepDNAfor double strand sequencingrdquo Nucleic Acids Research vol 18no 16 pp 4948ndash4950 1990

[36] M G Murray and W F Thompson ldquoRapid isolation of highmolecular weight plant DNArdquo Nucleic Acid Research vol 8 no19 pp 4321ndash4325 1980


[37] R Boom C J A Sol M M M Salimans C L Jansen P ME Wertheim-Van Dillen and J Van Der Noordaa ldquoRapid andsimplemethod for purification of nucleic acidsrdquo Journal of Clin-ical Microbiology vol 28 no 3 pp 495ndash503 1990

[38] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratoryManual Cold Spring Harbor Publishing New YorkNY USA 2nd edition 1989

[39] T Maniatis E E Fritch and J SambrookMolecular Cloning ALaboratory Manual Cold Spring Harbor Laboratory 1982

[40] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[41] M Nei andW H Li ldquoMathematical model for studying geneticvariation in terms of restriction endonucleasesrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 76 no 10 pp 5269ndash5273 1979

[42] M Nei ldquoGenetic distance between populationsrdquo AmericanNaturalist vol 106 no 949 pp 283ndash292 1972

[43] YVanDePeer andRDeWachter ldquoTreecon forwindows a soft-ware package for the construction and drawing of evolutionarytrees for the microsoft windows environmentrdquo Bioinformaticsvol 10 no 5 pp 569ndash570 1994

[44] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 no 4 pp 783ndash791 1985

[45] V AMaksimov K A Savvaitova BMMednikov et al ldquoAlpinechar-a new form of Arctic char (Salvelinus) from TaimyrrdquoJournal of Ichthyology vol 35 no 7 pp 1ndash9 1995

[46] R J Behnke Native Trout of Western North America vol 6American Fisheries Society Monograph Bethesda Md USA1992

[47] A G Osinov and V S Lebedev ldquoSalmonid fishes (SalmonidaeSalmoniformes) the systematic position in the superorder Pro-tacanthopterygii -the main stages of evolution and moleculardatingrdquo Journal of Ichthyology vol 44 no 9 pp 690ndash715 2004

[48] I A Chereshnev and M B Skopets ldquoSalvethymus svetovidovigen at sp nova a new endemic fish from subfamily Salmonidaefrom Lake Elrsquogygytgyn (the Central Chukotka)rdquo Journal ofIchthyology vol 30 no 2 pp 201ndash213 1990

[49] TM Cavender ldquoReview of the fossil history of NorthAmericanfreshwater fishesrdquo in The Zoogeography of North AmericanFreshwater Fishes C H Hocutt and E O Wiley Eds pp 700ndash724 John Wiley amp Sons NewYork NY USA 1986

[50] Yu S ReshetnikovEcology and Systematics of Coregonine FishesNauka Moscow Russia 1980

[51] R J Behnke ldquoThe systematics of salmonid fishes of recentlyglaciated lakesrdquo Journal of Fisheries Research Board of Canadavol 29 no 5 pp 639ndash671 1972

[52] R A Bodaly J Vuorinen R D Ward M Luczynski and J DReist ldquoGenetic comparisons of New and Old World coregonidfishesrdquo Journal of Fish Biology vol 38 no 1 pp 37ndash51 1991

[53] G Kh Shaposhnikova ldquoOn the taxonomy of whitefishes fromthe USSRrdquo in Biology of Coregonid Fishes C C Lindsey and CS Woods Eds pp 195ndash208 University Manitoba Press Win-nipeg Manitoba Canada 1970

[54] K A Savvaitova ldquoPatterns of diversity and processes of specia-tion in Arctic charrrdquo Nordic Journal of Freshwater Research vol71 no 1 pp 81ndash91 1995

[55] Yu S Reshetnikov Ed Atlas of Russian Freshwater Fishes vol1 Nauka Moscow Russia 2002

[56] V V Barsukov ldquoAbout systematic of the Chukotka charr of thegenus Salvelinusrdquo Journal of Ichtiology vol 14 no 1 pp 3ndash171960

[57] A G Osinov and S D Pavlov ldquoAllozyme variation and geneticdivergence between populations of Arctic char and DollyVarden (Salvelinus alpinus-Salvelinus malma complex)rdquo Journalof Ichthyology vol 38 no 1 pp 42ndash55 1998

[58] A G Osinov ldquoEvolutionary relationships between the maintaxa of the Salvelinus alpinus-Salvelinus malma complex resultsof a comparative analysis of allozyme data from differentauthorsrdquo Journal of Ichthyology vol 41 no 3 pp 192ndash208 2001

[59] L S Berg ldquoFishes of the Amur River basinrdquo Memoires delrsquoAcademie Imperial des Sciences Classe Physico-Math vol 24 no9 pp 1ndash270 1909

[60] S S Alekseev and M Y Pichugin ldquoA new form of charrSalvelinus alpinus (Salmonidae) from Lake Davatchan in Trans-baikalia and its morphological differences from sympatricformsrdquo Journal of Ichthyology vol 38 no 4 pp 292ndash302 1998

[61] B F Koop K R von Schalburg J L N Walker et al ldquoAsalmonid EST genomic study genes duplications phylogenyand microarraysrdquo BMC Genomics vol 9 p 545 2008

[62] A G Osinov and V S Lebedev ldquoGenetic divergence and phy-logeny of the Salmoninae based on allozyme datardquo Journal ofFish Biology vol 57 no 2 pp 354ndash381 2000

[63] P A Crane L W Seeb and J E Seeb ldquoGenetic relationshipsamong Salvelinus species inferred from allozyme datardquo Cana-dian Journal of Fisheries andAquatic Science vol 51 supplement1 pp 182ndash197 1994

[64] S Kumar and S B Hedges ldquoA molecular timescale for verte-brate evolutionrdquo Nature vol 392 no 6679 pp 917ndash920 1998

[65] J PThorpe ldquoThemolecular clock hypothesis biochemical evo-lution genetic differentiation and systematicsrdquo Annual Reviewof Ecology Evolution and Systematic vol 13 pp 139ndash168 1982

[66] A H Berst A R Emery and G R Spangler ldquoReproductivebehavior of hybrid charr (Salvelinus fontinalis x S namaycush)rdquoCanadian Journal of Fisheries and Aquatic Science vol 38 no 4pp 432ndash440 1981

[67] KMWestrichN RKonkolM PMatsuoka andR B PhillipsldquoInterspecific relationships among charrs based onphylogeneticanalysis of nuclear growth hormone intron sequencesrdquoEnviron-mental Biology of Fishes vol 64 no 1ndash3 pp 217ndash222 2002

[68] S U Qadri ldquoMorphological comparisons of three populationsof the lake charr Cristivomer namaykush from Ontario andManitobardquo Journal of the Fisheries Research Board of Canadavol 24 pp 1407ndash1411 1967

[69] B J Crespi and M J Fulton ldquoMolecular systematics ofSalmonidae combined nuclear data yields a robust phylogenyrdquoMolecular Phylogenetics and Evolution vol 31 no 2 pp 658ndash679 2004

[70] L N Ermolenko ldquoGenetic divergence in the family Corego-nidaerdquo Polskie ArchiwumHydrobiologii vol 39 no 3-4 pp 533ndash539 1992

[71] D V Politov N Y Gordon and A A Makhrov ldquoGenetic iden-tification and taxonomic relationships of six Siberian species ofCoregonusrdquo Advances in Limnology vol 57 pp 21ndash34 2002

[72] L Bernatchez F Colombani and J J Dodson ldquoPhylogeneticrelationships among the subfamily Coregoninae as revealedby mitochondrial DNA restriction analysisrdquo Journal of FishBiology vol 39 supplement pp 283ndash290 1991

[73] G R Smith and T N Todd ldquoMorphological cladistic study ofCoregonine fishesrdquo Polskie ArchiwumHydrobiologii vol 39 no3-4 pp 479ndash490 1992

[74] S V Frolov ldquoSome aspects of kariotype evolution in the Core-goninaerdquo Polskie Archiwum Hydrobiologii vol 39 no 3-4 pp509ndash515 1992


[75] G Svardson ldquoSignificance of introgression in coregonid evo-lutionrdquo in Biology of Coregonid Fishes C C Lindsey andC S Woods Eds pp 33ndash59 University of Manitoba PressWinnipeg Manitoba Canada 1970

[76] L Bernatchez S Renaut A RWhiteley et al ldquoOn the origin ofspecies insights from the ecological genomics of lakewhitefishrdquoPhilosophical Transactions of the Royal Society B vol 365 no1547 pp 1783ndash1800 2010

[77] Yu Kartavtsev ldquoGenetic variability and differentiation inSalmonid fishrdquo Nordic Journal of Freshwater Research vol 67pp 96ndash117 1992

[78] G Svardson ldquoThe Coregonid problem VI The palearctic spe-cies and their intergradesrdquo Reports of the Institute of FreshwaterResearch Drottningholm vol 38 pp 267ndash356 1975

[79] G Svardson ldquoPostglacial dispersal and reticulate evolutionof Nordic Coregonidsrdquo Nordic Journal of Freshwater ResearchDrottningholm vol 74 pp 3ndash32 1998

[80] V Grant Plant Speciation Columbia University Press NewYork NY USA 2nd edition 1981

[81] S Renaut and L Bernatchez ldquoTranscriptome-wide signatureof hybrid breakdown associated with intrinsic reproductiveisolation in lake whitefish species pairs (Coregonus spp Salmon-idae)rdquo Heredity vol 106 no 6 pp 1003ndash1011 2011

[82] K Johannesson ldquoParallel speciation a key to sympatric diver-gencerdquo Trends in Ecology and Evolution vol 16 no 3 pp 148ndash153 2001

[83] N Osada and C-I Wu ldquoInferring the mode of speciation fromgenomic data a study of the great apesrdquo Genetics vol 169 no 1pp 259ndash264 2005

[84] A L Schmidt and L M Anderson ldquoRepetitive DNA elementsas mediators of genomic change in response to environmentalcuesrdquo Biological Reviews of the Cambridge Philosophical Societyvol 81 no 4 pp 531ndash543 2006

[85] P M Ferree and D A Barbash ldquoSpecies-specific heterochro-matin prevents mitotic chromosome segregation to cause hy-brid lethality in Drosophilardquo PLoS Biology vol 7 no 10 ArticleID e1000234 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


45∘

50∘

55∘

60∘

65∘

70∘

75∘

45∘

50∘

55∘

60∘

65∘

70∘

75∘

30∘

60∘

90∘

120∘

150∘

30∘

60∘

90∘

120∘

150∘

Family CoregonidaeGenus SalvelinusGenera Salmo Parasalmo and Oncorhynchus

Figure 1 Sampling localities in Russia Armenia Kazakhstan Finland and Norway

[25] has shown species specificity of the band patterns alongwith the absence of individual sexual and interpopulationpolymorphism Dominating contribution of high-copy rela-tively long tandem repetitive sequences in ldquotaxonoprintsrdquo wasrevealed by Roudykh et al [26] This method seems to us tobe sufficiently appropriate for studying the molecular aspectsof speciation

We have shown previously that these general principles ofthe concert evolution were fully pronounced in the evolutionof repetitive sequences of the salmonids of Salmo Parasalmoand Oncorhynchus generamdashMednikov et al [27] ldquoHomingrdquocommon to salmonids resulted in reliable reproductive iso-lation with the subsequent divergence of the populationsin accordance with the morphological and molecular char-acters Situation with the repetitive DNA sequences in theorganisms with the less strict genetic isolation seemed tobe worth being analyzed Whitefishes of Coregonidae familyare one of the largest groups with interspecific hybridizationaccording to many experts application of Mayrrsquos biologicalspecies concept [1] to these fishes is limited (eg see discus-sion of the problem [28ndash30])

The study was aimed at the comparative investigationof alteration of some genome fractions under differentiationof salmonid species and forms belonging to various groupsand demonstrating various rates of reproductive isolationWhitefishes of Coregonidae family true salmons of theSalmo Parasalmo (Oncorhynchus after Smith and Stearley[31]) and Oncorhynchus genera and a number of forms andspecies of Salvelinus genus were studiedThe rates of isolationin whitefish and true salmon have polar characteristics dueto extensive hybridization in some species and strict homingin the other Geographic isolates and insular populations ofanadromous chars occupy intermediate position

Twomethods of multilocus DNA analysis were applied inthe phylogenetic and taxonomic studies of the Coregonidae

and Salmonidae families The methods were based bothon the comparison of the extensive repetitive sequence[26] and collation of the anonymous PCR products (RAPDamplification in modification of Welsh and McClelland [32]and Williams [33]) and their subsequent sequencing

2 Materials and Methods

Most of the salmon tissue samples were collected by the spe-cialists of the Department of Ichthyology Moscow State Uni-versity in 1984ndash2004 and Russian Federal Research Instituteof Fisheries and Oceanography in 2000ndash2004 Arctic charsfrom the lakes of Finland were collected by M KaukkorantaWhitefish from Lake Como (Canada) were kindly providedby Yu S Reshetnikov Collecting sites are mapped onFigure 1

In the early experiments DNA samples of salmons andwhitefish were extracted from gonads at III-IV stages ofmaturity preserved in alcohol with the method of phenol-chloroform extraction [34] In some cases additional purifi-cation with CsCl in the presence of ethidium bromide [35]and additional precipitation with cetyltrimethylammoniumbromide [36] were performed The length of DNA waschecked by electrophoresis in 06 agarose gel Concentra-tions of the obtained DNA preparations were estimated onSP-800 spectrophotometer (UK) and adjusted

At a later stage either Silica method [37] or traditionalprocedure with the use of proteinase K and organic solventsof Sambrook et al [38] was applied Prior to amplificationall samples were additionally purified with PEG-8000 DNApellets were dissolved in TE buffer and stored frozen

For restriction analysis DNA aliquots were digestedby MspI TaqI Csp6I (RsaI) Tru9I (MseI) Hin6I (HhaI)andMboI tetranucleotide restriction endonucleases and twoisoschizomers sensitive to the presence of methylated bases



















69sm 5 + 7lowast













242238

201190180

160147

123

110

90

76

67

1 2 3 5 6 7 8 9 10 11 121314151617M M

217

4





242238217201190180160147

123110

90

76

67

1 32 4 5 6 7 8 9 10 1211M M





464

309

242238217201190180160147

123110

90

76

67

1 2 M 3 5 6 7 8 9 10 11 12 13 14 15 164







97

01

Parasalmo clarkii

Salmo salar



Salmo trutta



Oncorhynchus keta




97

99

98

97

100

100

99

8263

100

72

76


Oncorhynchus masou












01020304050607


Oncorhynchus masou

Parasalmo clarkii


Salmo salar



Salmo trutta




64100

90100

5396

98

100

98

100

46

72


Oncorhynchus masou


Salmo salar


Parasalmo irideus




7476

8797

20

79

27

89

32

30

93



Coregonus artedi





100

99

5547

28

83

22

24

69

2731

35

31

100

96

(a)

(b)

(c)






1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19











01

Osmerus sp



C sardinella




C nasus

Coregonus muksun





C l pidschiani


78

45

42

52

87

66

51

96










1 2 3 4 5 6 MMMMMMM

700 bp 700 bp






















IV + 1 395 bp EG935616DW006459 XP 003198377


1 + 1 905 bp BX663609 XM 689046







IV + IV 254 bp CB490887 ACO 57211AAP58348



01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















69sm 5 + 7lowast













242238

201190180

160147

123

110

90

76

67

1 2 3 5 6 7 8 9 10 11 121314151617M M

217

4





242238217201190180160147

123110

90

76

67

1 32 4 5 6 7 8 9 10 1211M M





464

309

242238217201190180160147

123110

90

76

67

1 2 M 3 5 6 7 8 9 10 11 12 13 14 15 164







97

01

Parasalmo clarkii

Salmo salar



Salmo trutta



Oncorhynchus keta




97

99

98

97

100

100

99

8263

100

72

76


Oncorhynchus masou












01020304050607


Oncorhynchus masou

Parasalmo clarkii


Salmo salar



Salmo trutta




64100

90100

5396

98

100

98

100

46

72


Oncorhynchus masou


Salmo salar


Parasalmo irideus




7476

8797

20

79

27

89

32

30

93



Coregonus artedi





100

99

5547

28

83

22

24

69

2731

35

31

100

96

(a)

(b)

(c)






1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19











01

Osmerus sp



C sardinella




C nasus

Coregonus muksun





C l pidschiani


78

45

42

52

87

66

51

96










1 2 3 4 5 6 MMMMMMM

700 bp 700 bp






















IV + 1 395 bp EG935616DW006459 XP 003198377


1 + 1 905 bp BX663609 XM 689046







IV + IV 254 bp CB490887 ACO 57211AAP58348



01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









242238

201190180

160147

123

110

90

76

67

1 2 3 5 6 7 8 9 10 11 121314151617M M

217

4





242238217201190180160147

123110

90

76

67

1 32 4 5 6 7 8 9 10 1211M M





464

309

242238217201190180160147

123110

90

76

67

1 2 M 3 5 6 7 8 9 10 11 12 13 14 15 164







97

01

Parasalmo clarkii

Salmo salar



Salmo trutta



Oncorhynchus keta




97

99

98

97

100

100

99

8263

100

72

76


Oncorhynchus masou












01020304050607


Oncorhynchus masou

Parasalmo clarkii


Salmo salar



Salmo trutta




64100

90100

5396

98

100

98

100

46

72


Oncorhynchus masou


Salmo salar


Parasalmo irideus




7476

8797

20

79

27

89

32

30

93



Coregonus artedi





100

99

5547

28

83

22

24

69

2731

35

31

100

96

(a)

(b)

(c)






1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19











01

Osmerus sp



C sardinella




C nasus

Coregonus muksun





C l pidschiani


78

45

42

52

87

66

51

96










1 2 3 4 5 6 MMMMMMM

700 bp 700 bp






















IV + 1 395 bp EG935616DW006459 XP 003198377


1 + 1 905 bp BX663609 XM 689046







IV + IV 254 bp CB490887 ACO 57211AAP58348



01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


242238217201190180160147

123110

90

76

67

1 32 4 5 6 7 8 9 10 1211M M





464

309

242238217201190180160147

123110

90

76

67

1 2 M 3 5 6 7 8 9 10 11 12 13 14 15 164







97

01

Parasalmo clarkii

Salmo salar



Salmo trutta



Oncorhynchus keta




97

99

98

97

100

100

99

8263

100

72

76


Oncorhynchus masou












01020304050607


Oncorhynchus masou

Parasalmo clarkii


Salmo salar



Salmo trutta




64100

90100

5396

98

100

98

100

46

72


Oncorhynchus masou


Salmo salar


Parasalmo irideus




7476

8797

20

79

27

89

32

30

93



Coregonus artedi





100

99

5547

28

83

22

24

69

2731

35

31

100

96

(a)

(b)

(c)






1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19











01

Osmerus sp



C sardinella




C nasus

Coregonus muksun





C l pidschiani


78

45

42

52

87

66

51

96










1 2 3 4 5 6 MMMMMMM

700 bp 700 bp






















IV + 1 395 bp EG935616DW006459 XP 003198377


1 + 1 905 bp BX663609 XM 689046







IV + IV 254 bp CB490887 ACO 57211AAP58348



01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


97

01

Parasalmo clarkii

Salmo salar



Salmo trutta



Oncorhynchus keta




97

99

98

97

100

100

99

8263

100

72

76


Oncorhynchus masou












01020304050607


Oncorhynchus masou

Parasalmo clarkii


Salmo salar



Salmo trutta




64100

90100

5396

98

100

98

100

46

72


Oncorhynchus masou


Salmo salar


Parasalmo irideus




7476

8797

20

79

27

89

32

30

93



Coregonus artedi





100

99

5547

28

83

22

24

69

2731

35

31

100

96

(a)

(b)

(c)






1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19











01

Osmerus sp



C sardinella




C nasus

Coregonus muksun





C l pidschiani


78

45

42

52

87

66

51

96










1 2 3 4 5 6 MMMMMMM

700 bp 700 bp






















IV + 1 395 bp EG935616DW006459 XP 003198377


1 + 1 905 bp BX663609 XM 689046







IV + IV 254 bp CB490887 ACO 57211AAP58348



01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



01020304050607


Oncorhynchus masou

Parasalmo clarkii


Salmo salar



Salmo trutta




64100

90100

5396

98

100

98

100

46

72


Oncorhynchus masou


Salmo salar


Parasalmo irideus




7476

8797

20

79

27

89

32

30

93



Coregonus artedi





100

99

5547

28

83

22

24

69

2731

35

31

100

96

(a)

(b)

(c)






1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19











01

Osmerus sp



C sardinella




C nasus

Coregonus muksun





C l pidschiani


78

45

42

52

87

66

51

96










1 2 3 4 5 6 MMMMMMM

700 bp 700 bp






















IV + 1 395 bp EG935616DW006459 XP 003198377


1 + 1 905 bp BX663609 XM 689046







IV + IV 254 bp CB490887 ACO 57211AAP58348



01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19











01

Osmerus sp



C sardinella




C nasus

Coregonus muksun





C l pidschiani


78

45

42

52

87

66

51

96










1 2 3 4 5 6 MMMMMMM

700 bp 700 bp






















IV + 1 395 bp EG935616DW006459 XP 003198377


1 + 1 905 bp BX663609 XM 689046







IV + IV 254 bp CB490887 ACO 57211AAP58348



01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


01

Osmerus sp



C sardinella




C nasus

Coregonus muksun





C l pidschiani


78

45

42

52

87

66

51

96










1 2 3 4 5 6 MMMMMMM

700 bp 700 bp






















IV + 1 395 bp EG935616DW006459 XP 003198377


1 + 1 905 bp BX663609 XM 689046







IV + IV 254 bp CB490887 ACO 57211AAP58348



01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 MMMMMMM

700 bp 700 bp






















IV + 1 395 bp EG935616DW006459 XP 003198377


1 + 1 905 bp BX663609 XM 689046







IV + IV 254 bp CB490887 ACO 57211AAP58348



01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







IV + 1 395 bp EG935616DW006459 XP 003198377


1 + 1 905 bp BX663609 XM 689046







IV + IV 254 bp CB490887 ACO 57211AAP58348



01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


01

Salmo salar
























100

68

74

40

92

53

50

44

88

94

67

94








4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




4 Conclusion








Acknowledgments


References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Comparative Study of Genome Divergence in Salmonids with