Genetic Differentiation of Polish Sea Trout, Salmo Trutta M

Not to be cited without prior reference. to the authors

International Council for the CM 1998/K:4 Exploration of the Sea

Genetic differentiation of Polish sea trout, Salmo trutta m. trutta, populations based on RFLP analysis of PeR-amplified mtDNA segments

E. Wlodarczyk & R. Wenne

Sea Fisheries Institute, ul. Kollataja I, 81-332 Gdynia, Poland *Fax: (+48 58) 620-28-31 E-mail: [email protected]

ABSTRACT

Genetic differentiation among the populations of sea trout, Salrna trutta m. trutta, spawning in five Polish rivers has been investigated by RFLP ofPCR-amplified NADH-dehydrogenase 1 and 5/6 segments ofmtDNA. Total of 16 composite haplotypes were identified and the number of haplotypes per population ranged from 6 to 8. Three haplotypes were shared by all five populations and had similar frequencies ranging from 0.100 to 0.450. One haplotype was observed in 4 populations, two haplotypes in 3 populations and one haplotype was shared by 2 populations. Nine Tare.haplotypes were found in 4 populations, at frequencies ranging from 0.025 to 0.075. The highest number of rare haplotypes, three, was observed in the samples from the rivers Slupia and Pars((ta.. No rare haplotypes were detected in the river Wieprza. All populations were fixed at one morph for the ND-I segment digested with Hin!1.

Key words: mtDNA, population genetics, RFLP, Salrna trutta m. trutta, sea trout.

INTRODUCTION

The sea trout ( Salrna trutta m. trutta) is an anadromous salmonid species of high commercial value, widely distributed in Europe. Its freshwater counterpart, the brown trout, has been extensively researched, and a number of studies have shown a strong population subdivision within the species (reviewed by Ferguson, 1989). A pronounced genetic differentiation has been identified on a large geographical scale (Bernatchez et al., 1992; Giuffra et aI., 1994; Garcia Marin and Pia, 1996) and within smaller geographical ranges (Ferguson and Taggart, 1991; McVeigh et al., 1995; Hansen and Loeschcke, 1996; Apostolidis et al., 1997). Due to environmental degradation and extensive stocking of hatchery-raised trout into the wild it is likely that the genetic integrity of some natural gene pools might be endangered (Largiader and Scholl, 1996; Skaala et aI., 1996). Therefore, it is imperative to collect information on the genetics of indigenous trout populations in order to follow the dynamics of their population structure. Population genetics data will also become indispensable when managerial decisions have to be made about continous restocking, enhancement or reintroduction of trout under the constraint of retaining the genetic diversity of the species.

MATERIALS AND METHODS

Sample collection Between September and December 1996, tail fin clippings were collected from the sea trout spawners entering five Polish rivers: Vistula, Slupia, Wieprza, Pars"ta and Rega (Fig. 1). At four sites fish were caught in traps positioned at a fixed distance from the river mouth (3-40 km) while the samples from the river Vistula consisted of fish caught by commercial fishermen, possibly very close to the,river mouth. Forty individuals were sampled at each site. Fin clippings were preserved in 95% ethanol and kept at+40 C aftercollection.

Baltic Sea

Figure 1. Map of northern Poland. Marked are the rivers from which samples of sea trout were collected; the sample from Drw"ca R. is still being analyzed, therefore it is not included in this paper. The black bar in the bottom center indicates a dam on the river Vistula.

MtDNA analysis Total genomic DNA was extracted with the use of "Genomic DNA Prep Plus" kit (A&A Biotechnology, Gdaiisk). The ND-l and ND-5/6 segments ofmtDNA were amplified following the PCR protocol of Hansen and Loeschcke (1996). The amplified ND-l segment was screened for polymorphism with the restriction endonucleases Alu I, Ava'II, Hae ill, HinfI, Hpa II, andthe ND-5/6 segment with A va II, Hae ill, HinfI, Taq I and Xba 1. For each sample 3-8 III of the peR product were digested with 1-2 units of the appropriate restriction enzyme followed by electrophoresis on 8% (29: I) polyacrylamide or 2% agarose gels. Polyacrylamide gels were silver stained and dried. The DNA fragments in agarose were visualized with ethidium bromide in UV light and recorded on Polaroid film. The molecular weight standard, a 100 bp ladder (Gibco BRL), was used to size the restriction fragments (Table 1). Each restriction morph was assigned a capital 2 letter. Composite haplotypes were designated by a sequence of letters, i.e. one letter per each restriction endonuclease used for screening the ND-I and ND-5/6 segments (see Table 2 for sequence details).

Table 1. Approximate fragment sizes of restriction morphs for ND-I and ND-5/6 regions of .mtDNA. Fragments smaller than 100 bp'not shown.

·ND·l Restriction Alu I Ava II Hae III Hpa II endoilUclease ------Restriction morph A B A B A B C A B

Fragment sizes 700 700 950 700 700 700 800 (bp) '. I,~ '. 490 750 540 540 550 550 390 390 425 425 415 480 290 320 320 290 290 290 460 460 235 235 225 225 220 220 265 185 160 200 200 200 185 185 180 180 150 150 185 130 130 100 100 105

ND·S/6 Restriction Ava II Hae III HinfIII TaqI XbaI endonuclease ------A ------Restriction morph A B C A B C D A B C D B C D A B

Fragment sizes 1900 1900 950 950 950 950 1200 800 2600 (bp) 1000 830 830 1050 1050 1050 670 670 670 670 2350 950 800 800 475 475 475 650 650 250 900 650 650 460 630 600 600 630 630 320 320 320 315 325 325 325 325 250 250 200 275 275 275 275 315 315 315 I 125 125 175 175 175 175 240 240 240 240 300 135 135 135 185 185 185 185 290 290 290 290 120 120 120 120 150 150 175 175 175 175 140 140 140 120 120 120 120

RESULTS

The results of the genetic analysis are compiled in Table 2. Total of 16 composite haplotypes were identified and the number of haplotypes per population ranged from 6 to 8. Three haplotypes were shared by all five populations and had similar frequencies, ranging from 0.100 to 0.450. One haplotype was shared by 4 populations, two haplotypes by 3 populations and one haplotype was observed in 2 populations. Nine rare haplotypes were found in 4 populations at frequencies ranging from 0.025 to 0.075. The highest number of rare haplotypes, namely three, was observed in the samples from the rivers Slupia and Pars«ta. No rare haplotypes were detected in the river Wieprza. All populations were fixed at one morph for the ND-I segment digested with HinfI (not shown in Table 2). 3 Table 2. Frequencies of composite haplotypes in sea trout samples from five Polish rivers, based on the RFLP of the ND-l and ND-5/6 regions of mtDNA. Haplotypes are denoted by capitaUetters in the following order: (ND-I) Alu I, Ava II, Hae m, Hpa II; (ND-5/6) Ava II, Hae m, HinfI, Taq I, XbaI. In parenthesis, the number of individuals displaying a given haplotype; n=40. River populations are arranged according to their geographical proximity in the eastward direction.

Population AABA BAAA ABAB AABA AABA AACA AABA ABAB BAAA ABAA ABAB AABA ABAB ABAB ABAB AABA AABBB CAAAA BBDCA CABBA ADCDB CAAAA BBDCA AABBB AABBB AABBB BBBCA ACCDB BCDCA BBBBA BBDCA AAAAB I 2 3 4 5 6 7 8 . 9 10 11 12 13 14 15 16 .

Rega 0.325 0.275 0.175 0.125 0.025 0 0 0 0.025 0 0 0 0.050 0 0 0 (13) (II) (7) (5) (1) (1) (2)

Pars~ta 0.450 0.225 0.150 0.050 0.025 0 0 0 0 0 0 0 0 0.025 0.050 0.025 (18) (9) (6) (2) (1) (1) (2) (1)

Wieprza 0.325·. 0.325 0.100 0.100 Om5 Om5 0 0 0 0 0 0 0 0 0 0 (13) (13) (4) (4) (3) (3) . Slupia 0.350 0.325 0 0.100 0 I· 0.025 0 0 0.025 0.025 Om5 Om5 0 0 0 0 (14) (13) (4) (1) (1) ( 1) (3) (3) .

Wisla 0.325 0.250 0.150 0.125 0 0.050 0.075 0.025 0 0 0 0 0 0 0 0 (13) (10) (6) (5) (2) (3) (1)

r - -,~-; DISCUSSION

Previous. genetic studies of Polish sea trout consisted of sequence analysis of the PCR-amplified mtDNA control region and comparative allozyme analysis of four coastal river populations (Bematchez .et al:; 1992; Luczynski et aI., 1997). According to Bernatchez et al. (1992), sea trout from the Polish rivers Slupia and Vistula belong to the Atlantic grouping. The allozyme study of sea trout from the rivers Vistula, Rega, Slupia and Pars«ta revealed very short genetic distances (Nei's D) among the sampled populations, ranging from 0.000 to 0.001 (Luczynski et aI., 1997) . . 'The RFLP analysis of mtDNA segments employed in this study also revealed low genetic variability among the sea trout populations from the rivers Vistula, Rega, Slupia, Pars«ta and Wieprza (see Table ;2). From 57.5 to 67.5% of 40 spawners sampled from each river displayed either haplotype 1 or 2. The frequencies of haplotype 1 were identical or almost identical in the samples from the rivers Vistula, Rega, Slupia and Wieprza. The closest similarity between the frequeneies of tlje most common haplotypes 1 and 2 were observed in the neighboring Wieprza and Slupiarivers; Nine rare haplotypes, observed in the Vistula, Rega, Pars«ta and Slupia I populations, will be very useful during statistical analysis which is planned after completing the data set (a sample from Drw«ca River was being still analyzed at the time of the paper preparation). The finding that all five Polish sea trout populations were fixed at one morph for the ND-l segment digested with Hinflis consistent with the same observations for Danish wild and hatchery strains (Hansen and Loeschcke, 1996; Hansen et aI., 1997) and Turkish wild populations (Togan, pers. comm.). . The lack of pronounced genotypic variation among geographically close freshwater trout populations is not unusual, particularly in the areas where intense stocking with non-indigenous or with local but mixed stocks has been conducted (Hansen et al.,1995; Garcia et aI., 1991), or in localities accesible from the sea {Hansen and Loeschcke, 1996). Only landlocked pristine populations seem to have retained a certain degree of genetic 'uniqueness due to the lack of gene flow and large genetic drift in such, usually small, populations (Hindar et aI., 1991). The five Polish rivers from which sea trout were sampled in this study have been intensely stocked with smolts and alevins (Bartel and Debowski, 1996). In the years 1972-1991, under the management of the Commission for Stocking and Salmon Management from 2 to over 7 million of alevins and from 170 thousand to 190 thousand smolts were being released yearly into most of the rivers inhabited by sea trout (Bartel, 1993a), The stocking material was obtained through the artificial spawning of fish ascending the presumably natal rivers. Some mixing of stocking material between rivers had occurred when insufficient amounts of indigenous eggs and sperm had to be supplementd with smolts from other coastal rivers in order to maintain the Vistula sea trout population (Bartel, 1993b). However, the presently applied stocking practices of the Commission do not allow stock mixing, The complete data set will be compared with the results of other RFLP studies on PCR-· amplified ND-l and ND-5/6 mtDNA segments for trout populations within the Baltic Sea, after conducting an inter-laboratory comparative analysis of restriction morph sizes.

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