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PUBLISHED BY THE ACADEMY OF SCIENCES OF ALBANIA

AJNTSTHE ALBANIAN JOURNAL OF NATURAL

AND TECHNICAL SCIENCES

2004 (2)X (16)

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LOW GENETIC VARIABILITY IN THE SUMMER KORAN(SALMO LETNICA AESTIVALIS STEFANOVIC) OF LAKE

OHRID

Iain WILSONExternal Lecturer, Agricultural University of Tirana (AUT) at Kamza,

Tirana, AlbaniaContact address: Rr. ‘Sadik Petrela’, No. 88, P.O. Box 8291, Tirana, Albania

Tel/Fax: +355 4 377558; e-mail: ifwilson@netscape.net

ABSTRACT

Three populations of the summer koran (Salmo letnica aestivalis) and a sampleof hatchery-reared winter koran (S. l. typicus), two forms of a salmonidendemic to Lake Ohrid, were analysed for variation at genetic enzyme loci(allozymes). Samples were run alongside one another and alongside oneindividual Atlantic salmon (S. salar) to enable comparison of allele mobilitiesrelative to those present in a well-studied species. In all, 31 loci were resolved.In this, the first genetic, study of different populations of the koran, one locusonly (SOD-2*) was found to be variable. Overall, levels of geneticheterozygosity and polymorphism were exceptionally low for a salmonid. Theallozyme pattern at GPI-1*, -2* and -3* was the same as in S. salar.Keywords: allozymes, Salmo letnica, genetic differentiation

1. INTRODUCTION

Lake Ohrid is unique among the large freshwater bodies of the world. It is oneof the Dassaretes Lakes (lakes Ohrid and Prespa, and the now dried out LakeMaliq), which lie in the Balkans and which are acknowledged to be the oldestlakes in Europe, between two and four million years old [22]. Of these lakes,Lake Ohrid, shared between Albania and the Former Yugoslav Republic ofMacedonia (FYROM), is unusually deep (286m), as a consequence of which ithas been never been glaciated, with the implication that its fauna and florahave a direct ancestry that go back perhaps four million years. Given its uniquetaxa (some 60% of its forms of fish species are endemic) the lake provides a

N0.16.2004. The Albanian Journal of Natural & Technical Sciences

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unique opportunity to investigate relict life forms, which include ancientpopulations of salmonids.The koran Trutta letnica was first described by Karaman [11], and four differentforms, or subspecies, are recognised: i) Salmo letnica typicus Stefanovic, awinter form; ii) S. l. lumi Poljakov et al., a river form; iii) S. balcanicusKaraman, recognised by Stankovic [22] as S. l. balcanicus, a second winterform; iv) S. l. aestivalis Stefanovic, a summer form. The last of these forms isnot mentioned in Rakaj [18], while according to FishBase [9] it was preoccupiedby S. ischchan aestivalis Fortunatov, which is now S. aphelios, but these twonames refer to a trout endemic to Lake Sevan (Caucasus).The summer and winter forms of koran are distinguishable by both theirmorphology and ecology [22]. The summer form grows to a smaller size andhas fainter dark spots on the dorsal surface than the other forms. The winterforms spawn near the east and north-west shores of Lake Ohrid, while thesummer form spawns only in the sublittoral. A river form spawns in the laketributaries during December and January. There are also sterile individuals ofOhrid trout, studied by Karaman, but which were later shown to be S. l. typicuscaught outside their breeding season [22]. Karaman later regarded S.balcanicus as a separate species and not as a form of S. letnica, which henamed S. obtusirostris letnica, and which he thought was related to S.obtusirostris of the River Keta a tributary of the Montenegrin part of LakeShkodra, which is shared with Albania [reviewed in 22].The taxonomy of the salmonids of Lake Ohrid is further complicated by thepresence of the endemic belushka (belvica in Slavic; Acantholingua ohridana(Salmothymus ohridanus), classified with Salmothymus obtusirostris Berg,first designated Salar obtusirostris Heckel, in a genus Berg created for thesetwo Dinaric trout [21]), which spawns in December and June and which ismore or less profundal [22]. The belushka is smaller than the koran, moresilvery in appearance and has larger eyes in proportion to body size, butnevertheless even experienced fishermen can confuse it with the summer koran(pers. obs.). The consensus from DNA analysis of belushka [14, 16, 17, 21] isthat this species is a member of the genus Salmo; the last of these authors alsoplace Salmothymus obtusirostris in that same genus.Up to now the only published genetic studies on the koran are those of Apostolidiset al. [1, 2, 3], on a single sample collected during the summer. The presentpaper undertook for the first time allozyme analysis of different populations oftwo of the four different forms of koran, S. l. aestivalis and S. l. typicus.Three wild samples of summer koran and a hatchery-reared sample of S. l.typicus were screened for genetic variation along with a sample of S. salar,which was used for allelic comparison. The hatchery on the Albanian side ofthe border collects eggs of the form typicus each year (C & M Shegani, pers.

Iain WILSON

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comm.), in an effort to prevent a reduction in genetic variation that can arisefrom sustained use and re-use of genetic material over more than one generation[e.g. 24].Allozyme markers have already been identified that can be used to distinguishbetween populations of brown trout at the level of region. For example, allelesat LDH-5* and mMEP-1* have been found to discriminate between trout fromAtlantic and Danubian drainages [5]. Moreover, allozymes can be used toseparate local populations. For example, alleles at LDH-5* can distinguishbetween populations of trout inhabiting Lough Melvin, Ireland [8]. Thus theuse of allozymes should provide interesting results from an analysis of salmonidsseparated by millions of years, such as S. trutta, S. salar, S. letnica, and S.ohridanus, and perhaps between the different forms of koran, while at thesame time provide a basis for comparisons with DNA techniques.This paper presents a first step in applying molecular genetics techniques tounravel the complexities of western Balkan salmonid taxonomy, while providingthe beginnings of genetic research of the ecological relationships among theancient populations of salmonids of Lake Ohrid.

2. MATERIALS AND METHODS

2.1 Sample sitesSamples of Salmo letnica aestivalis were obtained from fishermen duringMay, June and July 2002 from three locations approximately one kilometre offthe western shore of Lake Ohrid (Piskopat, Hudenisht, Memelisht, each 5kmapart from the next in a north-south line; see Figure 1), each sample beingcaught within an area of 1km2. A sample of winter koran (S. l. typicus) wasobtained from the hatchery near Lin, while an individual farmed S. salar wasbought from a market in Tirana.Samples were photographed, labelled and stored at -200C until analysed forallozyme variation, when tissues (muscle, liver and eye) were dissected andanalysed.

2.2 ElectrophoresisPartially frozen tissue samples (approximately 250 mg) were mechanicallyhomogenised in 0.1 mL of distilled water, centrifuged at 13,000g for five minutesand the supernatant subjected to horizontal electrophoresis using 11 per centstarch gels (Sigma-Aldrich). Gels were run above a cooling plate in whichwater circulated at 50C.Samples were screened for enzyme variation at 31 loci, reported in Table 1aalong with their tertiary structure and the three different buffer systems andtissues used. Loci are named according to Shaklee et al. [20]. Alleles at each

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locus in S. letnica were named according to their mobility relative to the allelepresent at the same locus in S. salar.

2.3 Statistical analysisAllele frequencies were estimated from the genotype frequencies calculatedfrom observation of the allozyme phenotypes on each gel. Because of thesmall number of expected genotypes, samples were tested for deviation fromHardy-Weinberg expectation by using Fisher’s exact test. Analysis of genefrequencies was performed by using BIOSYS-1 [23], from which overall levelsof heterozygosity ( H ) and polymorphism ( P ) were estimated. Cluster analysis(Unweighted Pair Group Method with Arithmetic Averaging, UPGMA) wasperformed using Nei’s unbiased genetic distance [13].

Iain WILSON

Fig. 1. Map of Europe showing location of Lake Ohrid, shared betweenFYROM and Albania, and the four sampling locations, marked with a dot.

Albania

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3. RESULTS

All of the 31 loci screened were well resolved. Only one locus (SOD-2*) wasfound to be variable in samples of koran (Table 1b), the other 30 loci investigatedbeing monomorphic. The individual Atlantic salmon was monomorphic at allthe 31 loci that were screened.

The common allele in S. l. aestivalis was different from that in S. salar at onethird (ten from 31) of the loci screened (Table 1b). The common allele in koranmigrated farther than the allele present in the salmon at mAAT-1*, FH-1,2*,LDH-2*, mMEP-1*, sMEP-2*, PGDH* and SOD-2*, and migrated a shorterdistance at MDH-2* and PGM-1*, while the allozyme pattern at the threeGPI loci was identical in the two species.

Low genetic variability in the summer koran (Salmo letnica aesti........

Table 1. a) E.C. number and subunit structure of, and buffer andtissues used to screen, the 31 enzyme loci in samples of S. letnica and S.

salar; b) alleles recorded in the two species.

a) Locus† E.C. No. Structure Buffer‡ Tissue# b) Allele Koran Salmon

mAAT-1* 2.6.1.1 Dimer RW M *200 *100 mAAT-2* " RW M *100 *100 sAAT-1,2* " RW M *100 *100 sAAT-3* " RW E *100 *100 sAAT-4* " RW L *100 *100 FH-1,2* 4.2.1.2 Tetramer TC L,M,E *110 *100 G3PDH-1* 1.1.1.8 Dimer TC M *100 *100 GPI-1,2* 5.3.1.9 Dimer CAM M *100 *100 GPI-3* " CAM L,M *100 *100 LDH-1* 1.1.1.27 Tetramer RW M *100 *100 LDH-2* " RW M *200 *100 LDH-3* " RW M *100 *100 LDH-4* " RW L *100 *100 LDH-5* " RW E *100 *100 MDH-1* 1.1.1.37 Dimer CAM L,E *100 *100 MDH-2* " CAM E *85 *100 MDH-3,4* " CAM M *100 *100 mMEP-1* 1.1.1.40 Tetramer CAM M *110 *100 mMEP-2* " CAM M *100 *100 sMEP-2* " CAM M *110 *100 MPI-1* 5.3.1.8 Monomer TC M *100 *100 MPI-2* " TC L,E,M *100 *100 PGDH* 1.1.1.44 Dimer TC M *145 *100 PGM-1* 5.4.2.2 Monomer CAM L,M *90 *100 PGM-2* " CAM M,L *100 *100 SOD-1* 1.15.1.1 Tetramer RW H *100 *100 SOD-2* Dimer RW L *110;*55 *100

‡: RW, modified discontinuous buffer [19] described in [12]; CAM, continuous buffer [6]adjusted to pH 6.8; TC [25]. #: L, liver; M, muscle; E, eye.

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Allelic frequencies at SOD-2* in the different populations of koran are reportedin Table 2, along with the levels of polymorphism and heterozygosity. Therewas no significant deviation from expected Hardy-Weinberg frequency in anyof the samples. The frequency of SOD-2*55 in the three samples of summerkoran ranged between 0.42 and 0.48, while in the hatchery-reared sample thefrequency was slightly lower, at 0.31.

There was a mean genetic distance between the koran and salmon of 0.38,while that among the samples of koran was very slight (0.001 between thehatchery-reared sample and the other, summer, samples). Figure 2 shows thetree obtained using the UPGMA method, in which the koran samples clusteredtogether, but in which they were very distinct from the Atlantic salmon.

Iain WILSON

Fig. 2. UPGMA tree based upon Nei’s unbiased genetic distance [13]among the four samples of koran and the sample of Atlantic salmon.

Table 2. Frequencies of alleles at SOD-2* among the hatchery sampleand three different wild samples of Salmo letnica aestivalis from Lake

Ohrid.

Locus Allele Frequency Piskopat Hudenisht Memelisht Hatchery

N 17 22 18 26 SOD-2* *100 0.56 0.52 0.58 0.69

*55 0.44 0.48 0.42 0.31 H 0.017 0.019 0.009 0.012 P 0.032 0.032 0.032 0.032

.40 .33 .27 .20 .13 .07 .00

.40 .33 .27 .20 .13 .07 .00

Genetic distance

S. salar

Memelisht

Piskopat

Hudenisht

Hatchery

Genetic distance

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4. DISCUSSION

This study reports for the first time molecular genetic data on more than onesample of the koran (Salmo letnica), and is the first study to make a geneticcomparison between two of its different forms: the summer koran S. l. aestivalisand a hatchery-reared population of the winter form S. l. typicus. For alleliccomparison, samples were run alongside an individual S. salar for which thereare substantial genetic data reported in the literature [reviewed in 27].Despite the high level of endemism present in Lake Ohrid, there has beenpublished only one allozyme study of any of its species [1]. In that study, SOD-2*, LDH-2* and AAT-4* were found to be variable in a single sample of koranthat was compared with trout from around Europe. Neither LDH-2* nor AAT-4* were found to be variable in the present study. There may be a number ofreasons for this difference, including differences in electrophoretic conditionsbetween the two laboratories and the possibility that the sample in the earlierstudy might have included a mixture of different forms of the koran that haveyet to be compared genetically.Most of the alleles resolved in the present study correspond with those reportedpreviously for S. trutta and S. salar [e.g. 10], though S. letnica and S. salarwere indistinguishable here at MPI*, GPI-1,2* and GPI-3*.Given the great age of Lake Ohrid one would expect evolutionary differencesto have accumulated, such as in allozyme frequencies, between forms thatexhibit such marked ecological and reproductive behavioural differences.Though the allele frequency difference at SOD-2* between the summer formand the hatchery-reared winter form of koran is slight it is nevertheless interestingand requires further investigation to see whether it is consistent. Such adifference may reflect an effect of sampling or of changes brought about underthe conditions in the hatchery, or it may reflect a real genetic difference betweenthe two forms of koran.Apostolidis et. al [1] reported a frequency for SOD-2*100 of 0.625, a figurethat lies within the range of those reported here for the two different forms ofkoran. It would be interesting to investigate in these forms regions of DNAthat evolve more quickly than allozymes, such as microsatellites, which mayprovide markers that could distinguish between them.Osinov [15] investigated the genetics of four different ecological forms of browntrout (Salmo ischchan) inhabiting Lake Sevan that, like the koran, are separatedmorphologically and reproductively: the different forms of Sevan trout breed indifferent parts of the lake system, some in the rivers and others near the lakeshore, but little genetic differentiation was found among the different forms.This holds true for the koran of Lake Ohrid, as far as the sampling in thepresent study has found.

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The most striking finding in the present study is the very low level ofpolymorphism ( P =0.032) detected for the koran. Atlantic salmon is known tobe much less variable at allozyme loci than brown trout. For example, levels ofpolymorphism in diverse populations of S. salar, from Scotland, Spain andIreland, and in a farmed strain of Norwegian origin, have been found to bebetween 0.097 and 0.167 [26], while for S. trutta, levels are commonly foundof between 0.06 and 0.38 [e.g. 10]. These levels are from two to twelve timeshigher than those estimated here for the koran. On the other hand, for LakeSevan trout levels of polymorphism of between 0.000 and 0.034 have beenreported [15], with only one locus, sAAT-1,2*, found to be variable out of the29 loci investigated.Such low levels of polymorphism may reflect either the presence of naturalselection reducing genetic variability or that the populations have experienceda recent bottleneck. In contrast to this finding, A. Duguid (pers. comm.) hasfound in koran unique alleles, as well as DNA sequences characteristic ofpopulations inhabiting various drainages of the native area of the Europeanbrown trout. The difference between the results obtained for the differenttechniques requires further investigation.The genetic distance found between S. salar and S. letnica is similar to thevalues obtained for differences between S. salar and S. trutta [e.g. 7, 10].However, a triangular comparison among S. salar, S. trutta and S. letnica hasyet to be undertaken.Up until the 1960s, when hydroelectric dams were constructed along the RiverDrin, which connects Lake Ohrid with the Adriatic Sea along a long riversystem that includes many tributaries, including drainage from Lake Shkodra,this huge freshwater system had been interconnected. Within this system thereare many different forms of brown trout [22, 18]. It has been noted [4] that thewestern Balkans is home to the most phenotypic diversity recorded for thebrown trout. Further work on these different forms needs to be carried outbefore their phylogenetic relationships can be determined, and the present papermarks a beginning towards that end.Finally, given the uniqueness of the fauna of Lake Ohrid, it is essential that itsendemic life forms, which include the koran, are conserved and managed withcontemporary practices.

ACKNOWLEDGEMENTS

This work was carried out in the recently reconstructed genetics laboratory inthe Department of Animal Production at AUT. It has been equipped mostlythrough funding from TEMPUS. I particularly wish to thank Dr Anila Hodaand Professor Vladimir Spaho for collaboration, initiated by Dr Joerg Lohman

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of GTZ. Professor Costas Triantaphyllidis kindly donated some reagents andcommented on the manuscript, while Dr John Taggart kindly providedphotographs from his past work on allozymes in S. trutta. Samples of S. letnicawere obtained from Defrim Lloga, and from Çelnika and Mihallaq Shegani ofthe hatchery at Lin. Project support was provided by the Know How Fund ofthe British Embassy, Tirana, GTZ-AUT, Naum Gegprifti, Alba Dakoli-Wilsonand the Soros Foundation.

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