Journal of Fish Biology (2000) 57 (Supplement A), 51–71doi:10.1006/jfbi.2000.1608, available online at http://www.idealibrary.com on

Identification of palearctic coregonid fish species usingmtDNA and allozyme genetic markers

D. V. P*†‡, N. Y. G*, K. I. A*,Y. P. A* J. W. B†

*Vavilov Institute of General Genetics, 3 Gubkin St., GSP-1, Moscow 117809, Russiaand †Department of Wildlife and Fisheries Sciences, Texas A&M University,

College Station TX, 77843-2258, U.S.A.

Two types of molecular genetic markers were used for genetic identification of species and localstocks of palearctic coregonids (Coregonidae, Salmoniformes, Teleostei). Seven nominatespecies of whitefishes and ciscoes Coregonus spp. of Eurasia Arctic Sea basin and inconnuStenodus leucichthys nelma represented by specimens from North America were studied. Usingrestriction analysis of PCR-amplified products of the ND-1 gene of mitochondrial DNA(mtDNA) and allelic composition at several allozyme loci discrimination was successfulbetween C. lavaretus pidschian Siberian whitefish, C. nasus broad whitefish, C. autumnalis Arcticcisco, C. migratorius Baikal omul, C. peled peled, and C. sardinella least cisco. Muksun C.muksun was indistinguishable from Siberian whitefish. Creatine kinase (CK) isozyme patternsand Rsa I restriction patterns of ND-1 were the most effective markers allowing discriminationamong species. Intra-specific differentiation in mtDNA was found in all species but was muchless pronounced than inter-species variation. In several specimens composite haplotypes typicalof another species were found that reflect probable gene introgression by hybridization. Acombination of mtDNA and nuclear genetic markers is suggested for reliable identification ofboth typical species representatives and hybrids. � 2000 The Fisheries Society of the British Isles

Key words: coregonid; Coregonus; mtDNA; isozyme; identification; species.

‡Author to whom correspondence should be addressed. Tel.: (095) 135 5067; fax: (095) 132 8962, email:dvp@vigg.ru

INTRODUCTION

Coregonid fishes (Coregonidae, Salmoniformes) are core elements of estuarineand freshwater ecosystems of the Arctic Sea basin (Reshetnikov, 1980; Novikovet al., 2000), especially in Siberia and North-east European Russia. Along withArctic charr Salvelinus alpinus (L.) and Arctic grayling Thymallus arcticus(Pallas), coregonids play the principal role in food chains of the tundra zone andhave great economic importance in subsistence and commercial fisheries.Besides its practical importance, the identification and differentiation of speciesand local stocks of coregonid fishes is important for understanding the evolu-tionary mechanisms of speciation at high latitudes and the development of Arcticaquatic faunas. Phenotypic identification of coregonid species is complicated byhigh plasticity of body features and meristic characters (Lindsey, 1981). Existingkeys are often misleading or provide ambiguous diagnoses especially forindividuals with intermediate values in traits that are supposed to be discrimi-native. It is not unusual to encounter situations in which individuals possess acombination of character states thought to be typical of different species.Greater efficiency and reliability of morphological identification of coregonids

51

0022–1112/00/57A051+21 $35.00/0 � 2000 The Fisheries Society of the British Isles

52 . . .

can be achieved by careful prior analysis of traits in large samples to selectdiagnostic ones (Lindsey, 1962), but this was not always the case when theexisting identification keys for coregonids were created. Discrimination andphylogenetic inferences based on anatomical, especially osteological, traits ofcoregonids have been successful (GTsowska, 1960; Shaposhnikova, 1968, 1970;Smith & Todd, 1992; Chereshnev, 1994). However, these traits are not useful forfield identification. Given these problems, genetic markers such as isozyme lociand mitochondrial DNA (mtDNA) have been employed widely in populationand phylogenetic studies of coregonids. However, until recently most studiesfocused on populations inhabiting North America, Central Europe andFennoscandia (Bernatchez & Dodson, 1991, 1994; Bernatchez et al., 1991; Reistet al., 1998; Vuorinen et al., 1998; Douglas et al., 1999). Genetic data oncoregonids from Russian territories are relatively scarce (Bodaly et al., 1991,1994; Ermolenko, 1992; Bernatchez & Dodson, 1994) and represent poorlythe vast ranges of these fishes in Siberia and North European Russia. Identitiesof European, Siberian and North American coregonid forms known underthe same scientific and common names are often questionable. Even theconspecificity of populations of some Russian coregonids from different parts ofthe species ranges needs to be tested by genetic data.

This paper presents information about genetic identification of coregonidspecies distributed in the Russian part of the Arctic Sea basin and continues aseries of publications on the population genetic structure and phylogeny ofPalearctic (Politov & Gordon 1998; Politov et al., 2000) and Nearctic (Bickhamet al., 1989, 1997; Morales et al., 1993) coregonids.

MATERIALS AND METHODS

Tissue samples were obtained during fieldwork conducted in 1994–1998 (Table I).Most samples were collected during the Swedish–Russian Expedition ‘ Tundra Ecology-94 ’ (Grønlund & Melander, 1995; Novikov et al., 1995; Politov et al., 2000; Novikovet al., 2000). Baikal omul Coregonus migratorius (Georgi) and Baikal whitefishC. lavaretus baicalensis Dybowski were collected from the southern coast of BolshoiUshkaniy Island in the east central part of Baikal Lake (Politov et al., 2000). PeledC. peled Gmelin was collected from Turukhan River (a tributary of the Yenisey River,Table I). In addition, several specimens of circumpolarly distributed inconnu Stenodusleucichthys nelma Guldenstadt were collected from the North American Arctic coastbasin (the same sub-species is known from the Palearctic rivers).

Specimens were identified in the field using traditional morphological keys by Berg(1948) and Reshetnikov (1980). Taxonomy follows Reshetnikov (1998), except forBaikal omul, that is treated here as a separate species according to its first description.This is supported by osteological (Shaposhnikova, 1970) and previous isozyme (Politovet al., 2000) data. Reference specimens of each species from several localities were fixedin formalin and later deposited in collections of the Department of Ichthyology, MoscowState University and the Swedish Museum of Natural History (Stockholm). Tissuestaken for genetic analysis (skeletal muscle, heart, liver and eye) are stored frozen at�80� C at the Laboratory of Population Genetics, Vavilov Institute of General Genetics(Moscow, Russia).

Isozyme loci nomenclature followed Shaklee et al. (1990). Interpretation of isozymepatterns with respect to number of coding loci and enzyme sub-unit structure generallycorresponded to previous reports for coregonids (Vuorinen, 1984; Vuorinen & Piironen,1984). Other details of the isozyme techniques used are to be found in Politov et al.(1998, 2000).

53

T I. Studied samples of coregonid fishes

Species/location Sample size* Dates ofcollection

Arctic cisco—Coregonus autumnalis Pallas n=64 (63)W. to Pechora River delta (68�48�40� N; 53�42� E) 2 (2) August 1994W. Yamal Peninsula (70�20� N; 67�50� E) 3 (3) June and

August 1994N. Yamal Peninsula (72�51�30� N; 70�55� E) 8 (8) August 1994E. to Yana River mouth (72�24� N; 140�57�30� E) 50 (49) August 1994Indigirka River delta (71�26�30� N; 149�45� E) 1 (1) July 1994

Least cisco—Coregonus sardinella Valenciennes n=67 (17)W. to Pechora River delta (68�48�40�; N 53�42� E) 3 (4) June–August

1994W. Yamal Peninsula (70�20� N; 67�50� E) 16 (4) June and

August 1994N. Yamal Peninsula (72�51�30� N; 70�55� E) 3 (1) August 1994Kotelny Island (75�03�30� N; 140�10� E) 9 (1) July–August

1994Indigirka River delta (71�26�30� N; 149�45� E) 32 (4) July 1994Kolyma River delta (69�32�30� N; 160�42� E) 4 (3) July 1994

Peled—Coregonus peled Gmelin n=0 (12)Yenisey River basin (65�47� N; 87�56� E) 0 (12) July 1998

Muksun—Coregonus muksun Pallas n=47 (17)W. Yamal Peninsula (70�20� N; 67�50� E) 36 (8) June and

August 1994Indigirka River delta (71�26�30� N; 149�45� E) 7 (6) July 1994Kolyma River delta (69�32�30� N; 160�42� E) 4 (4) July 1994

Broad whitefish—Coregonus nasus Pallas n=106 (33)W. Yamal Peninsula (70�20� N; 67�50� E) 15 (8) June and

August,1994Olenekskii Bay (73�28�30�N 118�01� E) 1 (1) July 1994E. to Yana River mouth (72�24� N; 140�57�30� E) 11 (10) August 1994Indigirka River delta (71�26�30� N; 149�45� E) 23 (7) July 1994Kolyma River delta (69�32�30� N; 160�42� E) 60 (7) July 1994

Siberian whitefish—Coregonus lavaretus pidschian Pallas n=100 (20)W. to Pechora River delta (68�48�40�; N 53�42� E) 27 (5) August 1994W. Yamal Peninsula (70�20� N; 67�50� E) 17 (4) June and

August 1994Indigirka River delta (71�26�30� N; 149�45� E) 31 (4) July 1994Kolyma River delta (69�32�30� N; 160�42� E) 25 (8) July 1994

Baikal Lake whitefish—Coregonus lavaretusbaicalensis Dybowski

n=0 (2)

Lake Baikal (53�50� N; 108�37�) 0 (2) August 1995

Baikal omul—Coregonus migratorius (Georgi) n=42 (40)Lake Baikal (53�50� N; 108�37�) 42 (40) August 1995

Inconnu—Stenodus leucichthys nelma Guldenstadt n=0 (4)MacKenzie River basin, Canada (66�58� N; 135�08�W) 0 (4) August 1988

*Numbers of fish analysed by isozymes, sample sizes for mtDNA analysis are given in parentheses.Sample sizes for each species are given according to field morphological diagnoses and may differ from

those inferred from genetic data.

54 . . .

Deep-frozen or ethanol preserved tissues were used for DNA analysis. Prior to DNAextraction frozen tissues (heart, skeletal muscle or liver) were transferred to 20% DMSOin saturated NaCl as recommended by Maiers et al. (1998) to enhance DNA recovery.Total DNA was extracted by lysis of tissues for 1 h at 60� C in 0·65 ml extraction buffer(540 �l 0·1 Tris pH 7·8, 70 �l 10% SDS, 40 �l 1% proteinase K). One extraction withphenol: chloroform: isoamyl alcohol (24:24:1) and one extraction with methylenechloride: isoamyl alcohol (24:1) were followed by ethanol precipitation of the aqueousphase, centrifugation for 5 min at 12 000 rpm and resuspension of the DNA pellet in100 �l of TE buffer (Sambrook et al., 1989). Polymerase chain reaction (PCR) was usedto amplify the NADH-dehydrogenase-1 gene of the mtDNA together with flankingregions using primers LGL381: 5�-ACCCCGCCTGTTTACCAAAAACAT-3� andLGL563: 5�-GGTTCATTAGTGAGGGAAGG-3� (Cronin et al., 1993). PCR reactionswere performed in 60 �l volumes using a Perkin-Elmer DNA Thermal Cycler (PerkinElmer Applied Biosystems, Foster City, CA). PCR was carried out in 32 cycles ofdenaturation at 94� C for 1 min, annealing at 52� C for 1 min and extension at 72� C for1 min 45 s. PCR product (hereafter named ‘ ND-1 fragment ’) was c. 2100 bp in length.Aliquots (2·5 �l) of these products were cut by 18 restriction enzymes (Ase I, Ava II, BsaJI, Bsp1286 I, BstN I, BstU I, Dde I, Dpn II, Hae III, Hinc II, Hinf I, Hha I, Hph I, MspI, Nci I, Rsa I, Ssp I, Taq I). Restriction digests were incubated for at least 2 h underconditions specified by the manufacturer (New England BioLabs, Beverly, MA) and runon 2–2·5% agarose gels, stained with ethidium bromide and screened under UV light.Fragment size was determined using GIBCO BRL 100 bp DNA size marker (LifeTechnologies, Gaithersburg, MD). Restriction banding patterns were transformed into amatrix of presence or absence of particular cutting sites. Numbers of non-sharedrestriction sites (hereafter called pairwise differences) were counted among haplotypesand a minimum-spanning network was constructed (Schneider et al., 2000).

RESULTS AND DISCUSSION

Allelic frequencies at polymorphic allozyme loci are given in Table II.Restriction fragment length polymorphism (RFLP) patterns of ND-1 mtDNAhaplotypes are summarized in Table III. Their distributions among differentspecies and species diagnoses based on different types of data are presented inTable IV.

GENERA COREGONUS AND STENODUSFour specimens of Stenodus leucichthys nelma were analyzed by mtDNA only.

Unique restriction patterns were produced with nine enzymes (Table IV). Theinconnu Nci I pattern occurred only in a rare Baikal omul haplotype O31. Witha minimum of 23 pairwise differences with the closest Coregonus (Table IV),inconnu was the most easily identifiable among the species studied. All geneticstudies have shown inconnu to be closer to Coregonus than the third coregonidgenus Prosopium (Hamada et al., 1998). However, estimations of its positionwithin this clade have been inconsistent. For example, S. leucichthys wassuggested to be the sister taxon to: (1) all species of Coregonus (Ermolenko, 1992;Vuorinen et al., 1998), (2) C. autumnalis (Pallas)–C. artedii (Lesueur) speciescomplex (Bernatchez et al., 1991), (3) all Coregonus excluding C. albula (L.)(Sajdak & Phillips, 1997) and (4) C. albula–C. sardinella (Valenciennes)–C. peledspecies complex (Lockwood et al., 1993; Reist et al., 1998). The observed highgenetic differentiation of inconnu makes it possible to use it as an outgroup forphylogenetic reconstructions among species of genus Coregonus.

55

SUBGENERA COREGONUS AND LEUCICHTHYSNo alleles or mtDNA haplotypes delimited the commonly recognized sub-

genera Coregonus (whitefishes) and Leucichthys (ciscoes). Instead, at least threemajor groups of species were observed. Allozymes indicate equal levels ofdivergence among least cisco C. sardinella, Arctic cisco C. autumnalis andwhitefishes (Politov et al., 2000). Virtually the same result was obtained byanalysis of the mtDNA ND-1 fragment (Table V). Arctic cisco and least ciscohaplotypes were even more differentiated (a minimum of 15 pairwise differences)than least cisco and whitefishes (11–13). Therefore, the traditional sub-divisionof the genus Coregonus into sub-genera based on mouth position (sub-terminalin whitefishes and terminal or supra-terminal in ciscoes) is not supported (TablesII–V). Previous isozyme (Bodaly et al., 1991, 1994; Ermolenko, 1992; Vuorinenet al., 1998) and DNA-based (Bernachez et al., 1991; Reist et al., 1998) studiesalso have not found evidence for the validity of these sub-genera.

IDENTIFICATION OF SPECIES IN THE GENUS COREGONUS

C. sardinella

Least cisco can be identified by a combination of the following allozymealleles: fixed AH-1*B, AH-2*C, nearly fixed alleles GPI-A1* and PGM-1*, andalso private alleles at polymorphic frequencies in CK-1,2* (Fig. 1) and someother loci (Table II). Siberian populations have a unique ND-1 restrictionpattern with Rsa I (Fig. 2), while European least cisco shares type P with peled(differing in Dpn II). Six different haplotypes were observed among 17 studiedleast cisco individuals (Table IV).

C. peledPeled was not analysed by allozymes. However, mtDNA data provide enough

resolution to discriminate it clearly from other Coregonus species. Peled has aunique Dpn II pattern and shares Rsa I P type (Fig. 2) only with European leastcisco. Peled differs from Arctic cisco, Baikal omul and whitefishes by severalother restriction enzymes (Table IV), being most closely related to least cisco(Table V, Fig. 3). All restriction sites except for Dpn II were shared betweenpeled haplotype P5 and the closest least cisco haplotype CS966 (Tables IV, V;and Fig. 3). Earlier genetic studies also demonstrated that peled is phylogeneti-cally close to least cisco (Bernatchez et al., 1991; Bodaly et al., 1994; Vuorinenet al., 1998) and to its closest relative from Europe, vendace C. albula (Reistet al., 1998). Four haplotypes were found among 12 peled specimens (Tables IV,V, Fig. 3).

C. autumnalisArctic cisco has CK-1,2*A allele at creatine kinase major isoloci (Fig. 1)

that differentiates it reliably from other species except for Baikal omul. Arcticcisco IDH-4*B allele otherwise occurs only in Baikal omul and broad whitefishC. nasus (Pallas) at very low frequencies. Arctic cisco can also be identified byprivate alleles mMEP-1,2*A and GPI-B1*E, and by high (0·98) frequency ofPGM-1*A.

Restriction enzymes Hae III, Hinf I and Rsa I (Fig. 2) produced patternsspecific to Arctic cisco. A minimum of nine pairwise differences has been found

56 . . .

T II. Allelic frequencies at variable loci in six Coregonus species (alleles suggested forspecies identification are marked by different fonts*

Allele nasus muksun l. pidschian migratorius autumnalis sardinella

AH-1*B 0 0 0 0 0 1AH-1*A 1 1 1 1 1 0

AH-2*B 0·25 0 0 0 0 0AH-2*A 0·75 1 1 1 1 0AH-2*C 0 0 0 0 0 1

CK-1,2*A 0 0 0 1 1 0CK-1,2*B 1 1 1 0 0 0·20CK-1,2*C 0 0 0 0 0 0·45CK-1,2*D 0 0 0 0 0 0·35

CK-3*A 0 0 0 0 1 0CK-3*B 0 1 1 1 0 0CK-3*C 0 0 0 0 0 1CK-3*D 1 0 0 0 0 0

EST-1*A 0 0 0 0·08 0 0EST-1*B 0·12 0 0·10 0·29 0 0EST-1*C 0·83 0·06 0·26 0·58 0 0EST-1*D 0·05 0·63 0·53 0·04 1 0·91EST-1*null 0 0·32 0·11 0 0 0·09

G3PDH-1*A 0 0 0 0 0·03 0G3PDH-1*B 1 0·81 1 1 0·18 0·86G3PDH-1*C 0 0·06 0 0 0·55 0G3PDH-1*D 0 0 0 0 0 0·08G3PDH-1*E 0 0·125 0 0 0·24 0·06

GPI-A1,2*A 0 0 0·01 0 0 0·01GPI-A1,2*B 0·64 0·99 0·98 1 1 0·98GPI-A1,2*C 0·36 0·01 0·01 0 0 0·01

GPI-B1*A 0 0 0 0 0 0·04GPI-B1*B 0 0 0 0 0 0·95GPI-B1*C 0 0·04 0·005 0 0 0GPI-B1*D 1 0·95 0·99 0·99 0·54 0·01GPI-B1*E 0 0 0 0 0·45 0GPI-B1*F 0 0·01 0·005 0 0·01 0GPI-B1*G 0 0 0 0·01 0 0

GPI-B2*A 0 0 0 0 0 0·03GPI-B2*B 0 0·02 0·01 0 0 0GPI-B2*C 1 0·97 0·89 1 1 0·97GPI-B2*D 0 0·01 0·10 0 0 0

IDDH-1,2*A 0 0 0 0 0·01 0IDDH-1,2*B 0·47 0·78 0·67 0·49 0·49 0·20IDDH-1,2*C 0·53 0·23 0·32 0·50 0·50 0·55IDDH-1,2*D 0 0 0·01 0·01 0 0·25

IDHP-1*A 0 0·01 0 0 0 0IDHP-1*B 1 0·99 1 1 1 1IDHP-2*A 0 0·16 0 0 0 0IDHP-2*B 1 0·84 1 1 1 1

Continued

57

T II. Continued

Allele nasus muksun l. pidschian migratorius autumnalis sardinella

IDHP-4*A 0 0 0 0 0 0·01IDHP-4*B 0·95 1 1 0·97 0 0·99IDHP-4*C 0·05 0 0 0·03 0·95 0IDHP-4*D 0 0 0 0 0·05 0

mMEP-1,2*A 0 0 0 0 0·27 0mMEP-1,2*B 0·38 0 0 0 0 0mMEP-1,2*C 0·62 1 1 1 0·73 1

PGDH*A 0 0 0 0 0·02 0PGDH*B 0·95 1 1 0·16 0·94 1PGDH*C 0·05 0 0 0·84 0·04 0

PGM-1*A 0·005 0 0·015 0 0·98 0PGM-1*B 0·005 0 0 0 0 0PGM-1*C 0·99 0·90 0·94 0·88 0·01 0·01PGM-1*D 0 0·10 0·02 0·12 0 0PGM-1*E 0 0 0·02 0 0 0PGM-1*F 0 0 0·005 0 0·01 0·97PGM-1*G 0 0 0 0 0 0·01PGM-1*H 0 0 0 0 0 0·01

PGM-2*A 0 0 0 0·86 0 0PGM-2*B 0 0·30 0·27 0·14 0·01 0PGM-2*C 1 0·70 0·73 0 0·99 0·98PGM-2*D 0 0 0 0 0 0·02

sSOD*A 0·04 0·14 0·01 0 0 0·21sSOD*B 0·96 0·86 0·99 1 1 0·79

*Fixed private alleles are given in bold italics; other private alleles in italics; fixed alleles specific to agroup of species in bold; alleles at polymorphic frequencies (>0·1) that are also useful for speciesidentification are underlined.

between Arctic cisco haplotypes and haplotypes of other species making thisspecies most differentiated and easily identified among Coregonus. Arctic ciscohad the least pronounced intraspecific variation in mtDNA with 58 out of 64individuals represented by a single haplotype A1 and an average of two pairwisedifferences within species (Table V).

C. migratoriusArctic cisco and Baikal omul are fixed for the same allele at the major creatine

kinase isoloci CK-1,2* (Fig. 1) that was not observed in any other species.However, these two forms can be differentiated by position of a minor CK3 band(Fig. 1), by alleles EST-1*C and PGM-1*D (not found in Arctic cisco), andPGDH*C and PGM-1*C (rare in Arctic cisco), IDH-4*C (rare in Baikal omul),G3PDH-1*C and GPI-B1*E (absent in Baikal omul). Baikal omul has aspecies-specific allele (frequency 0·86) at PGM-2*A.

Five restrictases produced different RFLP patterns in Arctic cisco and Baikalomul. In addition to Hae III, Hinf I and Rsa I (Fig. 2) A haplotypes which arespecific to C. autumnalis, BstN I and Hph I A haplotypes are absent in Baikalomul. Alternatively, O and O1 patterns produced by BstN I are specific to

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61

Baikal whitefishC. lavaretus baicalensis has haplotype O1 which is the most frequent haplotype

in Baikal omul and absent from any other whitefishes. This suggests that theBaikal whitefish is a form of omul, C. migratoruis, at least with respect tomtDNA. Further examination of nuclear markers and morphology is needed todetermine whether its sub-terminal mouth, typical to whitefishes, evolvedindependently or through introgression with true whitefishes (e.g. C. lavaretuspidschian, which is also present in Baikal Lake). Sukhanova et al. (2000) haveshown extremely high sequence similarity among Baikal omul and Baikal lakewhitefish in both the cytochrome b gene and the control region of mtDNA.

C. nasus and other whitefishesAs a group, the whitefishes (Coregonus with sub-terminal mouths) possess

allozyme alleles that are alternative to those that distinguish other species.Clustering based on allozyme data revealed a clade that included muksun C.muksun (Pallas), Siberian and broad whitefish that had high bootstrap support.However, mtDNA haplotypes of broad whitefish do not cluster together withmuksun and Siberian whitefish on the MST (Fig. 3), this fact raising a questionabout the monophyletic origin of this group. Introgression also might accountfor closer similarity among whitefishes at allozyme loci than for mtDNAhaplotypes.

Among the other whitefishes, reliable identification is possible only for thebroad whitefish. This species can be differentiated clearly from both C. muksun

Baikal omul. This species can be identified reliably by Hae III O and O1haplotypes and Rsa I O, O1 and O2 haplotypes as well (Fig. 2). The most similarhaplotypes in Arctic cisco (A1) and Baikal omul (O5) differed by nine restrictionsites. For comparison, a minimum of 12 pairwise differences were observedbetween Baikal omul and Siberian whitefish C. lavaretus pidschian Gmelin, 11with least cisco and 13 with peled. It is noteworthy that often Baikal omul istreated currently as a sub-species of C. autumnalis (Berg, 1948; Nikolsky &Reshetnikov, 1970; Reshetnikov, 1980). An alternative classification assumes itsstatus as a full species C. migratorius (GTsowska, 1960; Shaposhnikova, 1968,1970). Present data indicate that it is as distinct genetically in both isozymes andmtDNA (Table II–IV, see also Politov et al., 2000) as other commonlyrecognized coregonid species, so its status as a sub-species of C. autumnalisshould be rejected. Likewise, Sukhanova et al. (2000) found sequences of D-loopand cytochrome b regions of mtDNA in Baikal omul to be much more similar towhitefishes than Arctic cisco. It is noteworthy that Baikal omul was the mosthighly variable species among those studied (Table IV); 10 haplotypes werefound among 40 individuals. Considering these facts, a currently popularhypothesis of the origin of this form from late-Pleistocene Arctic cisco migrantsfrom the Lena River or the Yenisey River seems to be unrealistic. It is veryunlikely that such a great intraspecific diversity appeared in postglacial time.The MST indicates Arctic cisco to be a terminal taxon, and if Baikal omul andArctic cisco do form a monophyletic clade (which is also questionable) it is morelikely that Baikal omul is a pre-Pleistocene ancestor of Arctic cisco rather thanits descendant.

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64 . . .

F. 1. Creatine kinase zymogram showing characteristic alleles of CK-1,2* isoloci (A–D) and position ofminor CK3 band in different species (AU, C. autumnalis; O, C. migratorius; P, C. lavaretuspidschian; M, C. muksun; N, C. nasus; CS, C. sardinella).

T V. Mean (below diagonal) and minimum (above diagonal) number of non-sharedrestriction sites among OTUs

Species AU O P+M N CS PE SN

C. autumnalis (AU) 2·00 9 20 20 15 17 23C. migratorius (O) 13·60 3·96 12 15 11 13 23C. l. pidschian+C. muksun (P+M) 22·19 15·50 4·56 12 8 9 23C. nasus (N) 23·96 19·45 16·68 4·39 10 12 31C. sardinella (CS) 17·11 14·50 13·03 14·54 2·76 1 26C. peled (P E) 17·83 15·20 12·80 15·08 5·75 3·50 26S. l. nelma (SN) 25·17 25·10 26·80 33·88 27·17 27·00 1·00

and C. lavaretus pidschian by the absence of the CK� band (Fig. 1), that isotherwise always present in muksun and Siberian whitefish. It has species-specific alleles at ACO-2* and mMDHP-1,2*. Broad whitefish also carries afixed allele C at sMDHP-3,4* isoloci (coding for supernatant malic enzyme),while C. muksun has different fixed alleles at sMDHP-3* and sMDHP-4*, andthe Siberian whitefish is polymorphic for three alleles. Frequencies of theEST-1*B allele and GPI-B1,2*C in broad whitefish are significantly higher thanin other whitefishes.

ND-1 RFLP patterns also separate broad whitefish clearly from othercoregonids. All studied specimens demonstrate a unique haplotype N when thefragment was digested by Hae III and Rsa I (Fig. 2). Patterns produced by MspI and Nci I (N haplotypes, Tables III and IV) are different from all other speciesand occur only as rare variants in least cisco and Baikal omul, respectively. Ingeneral, the distinctness of C. nasus in allozymes and mtDNA agrees well withthe results of comparative morphological (Lindsey, 1962) and karyological(Viktorovsky & Ermolenko, 1982) studies. Earlier allozyme (Bodaly et al., 1991,1994; Ermolenko, 1992; Vuorinen et al., 1998) and mtDNA studies showed this

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66 . . .

species to be a part of the whitefish clade. Intraspecific variation in broadwhitefish was relatively high (Tables IV and V).

F. 3. Minimum spanning network among haplotypes represented in Tables III and IV. Number ofpairwise differences is given for some interspecific connections. Inconnu haplotypes are connectedto main network at four different points by 23 pairwise differences.

C. muksun and C. lavaretus pidschianAlthough these two whitefish forms are recognized currently as different

species they shared the most frequent alleles in all allozyme loci. Differencesbetween them were restricted to presence or absence of particular low-frequencyalleles (Table II). For instance, allele EST-1* B is absent in muksun but reachesa frequency of 0·10 in Siberian whitefish. Alleles mIDHP-1*A (P=0·01) andmIDH-2*A (P=0·16) were found exclusively in muksun. Allele sSOD*Ais common in muksun but was found in just one heterozygote in the Siberianwhitefish sample. Principal component analysis (PCA) of allozyme variationdid not reveal distinct clusters corresponding to the nominate species (Politovet al., 2000). This contrasted with other inter-species comparisons where PCAdiscriminated individuals of different species efficiently (Politov et al., 2000).

67

RFLP analysis revealed three haplotypes (P3, P97 and M27) found in bothspecies and some low frequency haplotypes specific to either muksun or toSiberian whitefish (Table IV). Present results (Fig. 3) correspond well withearlier allozyme (Ermolenko, 1992) and mtDNA (Bernatchez & Dodson, 1994;Reist et al., 1998) studies where muksun clustered among forms belonging to theC. lavaretus group but did not form a sister clade to them.

Identification of muksun and Siberian whitefish was impossible by any genetictechniques employed. Individuals identified morphologically as C. muksun andC. lavaretus pidschian are genetically very similar. Relatively easy morphologicalrecognition of muksun is possible by its higher gill raker counts and wider snout(Berg, 1948). However, these traits, especially gill raker number are distinctiveonly when muksun is compared with sympatric low gill-rakered Siberianwhitefish. Some North European whitefish forms of the C. lavaretus lavaretuscomplex (i.e., conspecific to Siberian whitefish) have counts overlapping withmuksun (e.g. Berg, 1948). At present it is difficult to decide whether the muksunis a distinct species, or just one of the numerous ecotypes of the polymorphicspecies C. lavaretus. Conspecificity and monophyletic origin of muksun popu-lations from different parts of the nominal species’ range should be confirmedfurther. If muksun and Siberian whitefish do represent separate species, theirlow genetic differentiation may indicate that gene flow between them has ceasedrecently.

Interspecific v. intraspecific variationFrom Table V we calculated that, on average, mtDNA haplotypes differed at

3·16 sites within species v. 18·87 sites between species. Muksun and Siberianwhitefishes that share some haplotypes were combined here in a single OTU(operational taxonomic unit). Besides these whitefishes, only peled P5 and leastcisco CS1 had just one non-shared site followed by CS1 v. Siberian whitefishP502 (eight sites), Arctic cisco AU1 v. Baikal omul O5 (nine sites). In all otherpairs the differences were even greater. Judging from this, misidentification ofcoregonid species is very unlikely with the markers used. The above-mentionedexceptions represent cases of recently diverged forms that would requireemployment of additional highly variable genetic markers.

Introgressive hybridization and identification of coregonid speciesIn several cases allozyme and mtDNA diagnoses did not correspond with the

initial morphological ones (Table IV). The least cisco haplotypes CS1 and CS35appeared in muksun specimens 230M34 and 229M33, respectively. Broadwhitefish haplotype N17 was found in the individual identified as a Siberianwhitefish 468P11. Composite genotype P3 was found not only in eight Siberianwhitefish and nine muksun specimens but also in one specimen each identifiedinitially as least cisco, broad whitefish, and Arctic cisco. The Arctic ciscospecimen 453AU71 carried several allozyme alleles typical for whitefishes.

As these examples illustrate, the presence of haplotypes typical for anotherspecies might be caused by mis-identification of species in the field, or byintrogression among species. These two possible causes are not necessarilyindependent, as mis-identification is more likely among individuals withintermediate morphology, which, in turn, reflects probable hybrid origin.

68 . . .

Hybridization is reported frequently among coregonid species (Berg, 1948;Svardson, 1998) and certified by genetic studies (Luczynski et al., 1992;Reist et al., 1992; Bickham et al., 1997). Vertebrate mtDNA has maternalinheritance, and when used alone reflects only partly interspecific gene flow.Employment of different types of genetic markers (especially nuclear ones) isdesired and helpful in inferring both possible parents (Carr et al., 1986; Catheyet al., 1998) and, potentially, generation of the hybrid (F1, F2, different types ofbackcrosses etc.).

CONCLUSIONS

Allozyme loci and RFLP analysis of PCR-amplified ND-1 fragments ofmitochondrial DNA proved to be reliable for the discrimination of coregonidspecies. Creatine kinase zymograms, and restriction digestions with enzymes RsaI and Dpn II discriminated among all the studied Palearctic Coregonus speciesexcept for the muksun–Siberian whitefish pair. The reliability of identificationincreased with more genetic markers employed. Application of at least two typesof markers (e.g. maternally inherited mtDNA and nuclear genes like isozymeloci) is especially helpful in the identification of hybrids. Non-typical mtDNAhaplotypes for a particular species might have originated from another species asa result of past or recent introgression. Taking hybridization into account is ofgreat importance not only theoretically for understanding evolutionary path-ways in fish (Altukhov, 1982; Smith, 1992) but also practically for geneticidentification of coregonids (Bickham et al., 1997).

The genetic techniques used here are proposed not as replacements oftraditional morphological keys, but to assist taxonomists to create new, morepowerful methods of identification. Morphological keys should be based oncarefully selected characters that appear in all typical representatives of par-ticular species. Genetic methods can help to select such representatives that werenot subjected to interspecific crossing. Deviated or intermediate forms such ashybrids can also be revealed by molecular genetic markers, morphologicallyre-examined and included into such key tables as separate items.

Unlike most morphological methods, it is possible to use genetic techniqueswhen whole fish are unavailable. Small pieces of frozen tissue or even tissuepreserved at room temperature in DMSO/sodium chloride solution can be usedsuccessfully for identification. This feature is important when populations andspecies are overexploited and fisheries management requires monitoring ofgenetic resources. Commercial catches, where often samples available are onlyheadless fillets, salted or otherwise preserved fish products, may be subjected togenetic analysis for species composition and proportion of different species in thetotal product.

Further development of powerful new genetic methods of identification willensure differentiation not only among species but perhaps also among localpopulations or stocks.

The authors thank sponsors, organizers and participants of the expedition ‘ TundraEcology–94 ’, the crew of ‘ Akademik Fedorov ’ research vessel, and to two helicoptercrews for their invaluable help during field work; and V. V. Smirnov and L. Yu.Yampolsky for assistance in obtaining and transporting Baikal omul samples. The

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laboratory treatment of samples was supported by the Russian Foundation for BasicResearch, projects #96-04-49174 and #95-04-12614a, and by the Program for Support ofthe Scientific Schools, grant #96-15-97862. This paper represents contribution #00 of theCenter for Biosystematics and Biodiversity, Texas A&M University.

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