Aniolas Sruoga*,**, Isaak Rashal***, Dalius Butkauskas*, and Linas Loþys*
* Institute of Ecology, Vilnius University, Akademijos 2, LT-08412, Vilnius, LITHUANIA;
E-mail: [email protected]
** Vytautas Magnus University, K. Donelaicio g. 58, LT-44248 Kaunas, LITHUANIA
*** Institute of Biology, University of Latvia, Miera iela 3, Salaspils, LV-2169, LATVIA;
E-mail: [email protected]
Contributed by Isaak Rashal
A total of 56 perch individuals attributed to three different populations—the Baltic Sea (Lithuanian costal zone), the Curonian Lagoon (Lithuania) and the Gulf of Rîga (Latvia)—were investigated.
The amplified mtDNA fragment consisting of 378 bases includes the trnT gene and the 5‘-fragment of the control region consisting of 260 bases. Ten variable positions were identified and 15 haplotypes of the European perch of mitochondrial DNA (mtDNA) D-loop were estab-lished. Three new haplotypes C4, F8 and G2 characteristic of the perch caught in Lithuanian terri-torial waters and two haplotypes L1 and L2 characteristic of the perch from the Gulf of Rîga were determined. Five rarer haplotypes were found in the samples of the Lithuanian perch and four in the samples of the Latvian perch, which characterise the scope of reproductive isolation among the populations. The similarity in the distribution of the most frequently found haplotypes A and F in the Lithuanian samples investigated illustrate the similarity of the perch populations and sub-stantiate the assumption of possible seasonal migration of the perch between the Curonian La-goon and the coastal zone of the Baltic Sea.
Key words: perch, mtDNA, haplotypes.
However, till now there has been no DNA marker data available concerning the genetic diversity of the perch in the inland waters of Lithuania and Latvia or in the coastal waters of the Baltic Sea. The only survey known in the re-gion is the analysis of perch populations in Kaunas city res-ervoir, in the Curonian Lagoon and in the Baltic Sea coastal zone by means of isoenzymes as biochemical markers (Paulauskas and Loþys, 2001). The aim of the present work was to determine polymorphism of sequences of mtDNA D-loop and to evaluate the genetic diversity of the popula-tions of perch representing different areas of the Baltic Sea.
MATERIAL AND METHODS
A total of 56 European perches (Perca fluviatilis) caught in the years 2004–2006 were used for the investigation. Alto-gether 15 individuals were caught in the Baltic Sea, 18 indi-viduals in the Curonian Lagoon and 23 in the Gulf of Rîga.
Among them, in 2004, nine individuals were caught in the Baltic Sea near Monciðkës and six individuals caught in the Baltic Sea near Bûtingë, both Lithuania. In 2005, ten indi-viduals were caught in the Curonian Lagoon (Lithuania) near Kiaulës nugara and eight individuals in the same la-goon near Ventës ragas, and in 2006, 23 European perches in the Gulf of Rîga near the mouth of the River Daugava (Latvia).
Genomic DNA was isolated from the samples of muscle tis-sues fixed in ethanol by means of the salt extraction method (Aljanabi and Martinez, 1997).
Amplification of mtDNA fragments was carried out using Mastercycler Gradien amplifier manufactured by the Ep-pendorf Company. For amplification of mitochondrial DNA sequences the following primers were used: HV2:
TTCCCCGGTCTTGTAAACC; and CSB-D: GGAAC-CAAATGCCAGGAA.
PCR was carried out by denaturation at 96oC temperature for 5 minutes, then 30 cycles at 96 oC for one minute, at 54oC (HV2 – CSB-D) for one minute, followed by an elon-gation step at 72oC for 2 minutes and finishing with a final elongation step at the 72oC for 5 minutes. The PGR prod-uct was checked in 1.5% agarose gel and purified by CIAP and ExoI nucleases: for 15 minutes at 37oC and then for 15 minutes at 85 oC.
Sequencing was carried out at the Sequencing Centre of the Institute of Biotechnology (Vilnius, Lithuania). DNA se-quences were determined by an ABI Prism 377 automatic sequencer. Sequencing results were compared with the PFY14724 sequence using the BLAST database. The com-puter programme CLC Free Workbench version 0.91 was used for the sequence analysis and for the construction of phylogenetic tree.
RESULTS
A total of 56 perches attributed to three different
popula-of Rîga were investigated. The amplified mtDNA fragment consisting of 378 bases includes the trnT gene and the 5‘-fragment of the control region consisting of 260 bases. Ten variable positions were identified and 15 haplotypes of the European perch of mtDNA D-loop were established (Table 1). Three new haplotypes, we designated as C4, F8, and G2, characteristic of the perch caught in Lithuanian territorial waters and two haplotypes, designated as L1 and L2, char-acteristic of the Latvian perch were determined (Table 1).
The distribution of haplotypes in the studied populations is presented on Figure 1. The most frequent F haplotype was discovered in samples of perch caught both in the Curonian Lagoon and the Baltic Sea near Ðventoji. Haplotype A was found to be the second by frequency in the populations of the Lithuanian perch, and haplotypes C and E are most of-ten found in the populations of perch of the Gulf of Rîga.
Haplotypes C1 and A7 were found in all investigated popu-lations. Five rarer haplotypes were found in the samples of the Lithuanian perch and four in the samples of the Latvian perch, which characterises the scope of reproductive isola-tion among the populaisola-tions of these perches. Haplotype E was found with minimum frequency on the coastal zone of the Baltic Sea near Ðventoji only in the samples of 2004 and 2005.
T a b l e 1 HAPLOTYPES OF mtDNR trnT gene AND CONTROL REGION FRAG-MENT DETERMINED IN EUROPEAN PERCH Perca fluviatilis SAM-PLES CAUGHT IN THE TERRITORIAL WATERS OF LITHUANIA AND LATVIA (data are compared with the mtDNA sequence PFY14724, haplotype A presented in the Gene Bank)
Haplo-types
Position N
98–99 103 129 130 135 136 137 139 264 299
A - A G C A A T T T C 6
C4, G2, F8, L1 and L2 – newly identified haplotypes.
N, number of individuals.
T, A, and G, substitution of nucleotides.
- – deletion of nucleotides.
DISCUSSION
Nesb¸ et al. (1998) and Refseth et al. (1998) determined in total 35 haplotypes grouped into eight closely related groups by the nature of base substitutions. Based on this data, several zones, separated from the phylogenetic point of view, were postulated, with prevailing different groups of mtDNA haplotypes (Nesb¸ et al., 1999). In all perch populations of Western Europe, haplotypes of group F dominated, with the exception of those in river basins
(oldest in the term of origin), in which haplotypes of group M (M, M1) prevailed. In samples of perch of the continental part of the mainland, in the basins of the Nemunas, Dnieper rivers, as well as in the remote Lake Baikal, the Angara River basin, haplotypes of group C (C, C1–C3) prevailed.
This indicated separation of perch in the basins of the rivers stretching across this territory to a separate Euro-Asian phylo- geographical region.
Haplotype E was found with minimum frequency on the coastal zone of the Baltic Sea near Ðventoji only in the sam-ples of 2004 and 2005 which could be related to the anadro-mous behaviour of those perch, i.e., a periodical change in the environment when fish feeding in more saline waters of the coastal zone of the Baltic Sea swim to spawn to the fresh waters of the river mouths. A repeated discovery of haplotype E in the samples of the perch caught in the coastal strip near Monciðkës not far from the settlement of Ðventoji in different years can be related to a larger part of the individuals keeping near the spawning site located in the approaches of the mouth of the Ðventoji River, and rarer variants of haplotypes (haplotype E), inherited with steady, though small frequency, reflect the Hardy-Weinberg bal-ance of the population.
Newly identified haplotypes C4 and F8 were found in the samples of the Baltic Sea. Also, haplotype G2 found in the samples from the Curonian Lagoon are characteristic of the Lithuanian population of the perch and are related to the group of haplotypes E–F (Fig. 2). The presence of an inser-tion of ten nucleotides characteristic of the newly identified haplotypes L1 and L2 could be accounted for by a close phylogenetic link of these haplotypes. However, on the ba-sis of grouping of these samples in the phylogenetic tree, according to the nature of single base substitutions,
haplo-Fig. 1. Frequencies of mtDNR haplotypes in the investigated populations of European perch in the Gulf of Rîga (a), the Baltic Sea near Ðventoji (b) and the Curonian Lagoon (c)
Fig. 2. The Neighbour joining phylogenetic tree representing the origin of haplotypes.
type L1 is closest to the group of haplotypes F1 and F2, which is related to the group of haplotypes F fully dominat-ing in Western Europe, and haplotype L2 is closest to the group of haplotypes A typical of the western and northern parts of Scandinavia (Norway).
A combination of haplotypes A and F is characteristic of the samples of the Matsalu perch population investigated in the geographical range of the Baltic Sea (territorial waters of Estonia) (Table 2), which geographically is one of the clos-est to the sample of the Latvian perch invclos-estigated by us but, however, which differs greatly from the Emjajogi and Peipsi populations (the Gulf of Finland, territorial waters of Estonia) in which the haplotypes of groups A and C suggest influence of the Northern and Eastern phylo-geographical regions on the formation of the perch population of the Gulf of Finland.
The phylogenetic link between the Estonian and Latvian perch populations is confirmed by the most frequently pres-ence of haplotypes of groups A and F in the Matsalu popu-lation of the Gulf of Rîga, but the Latvian perch popupopu-lation is noted for a rather high frequency of haplotypes C, which is related to the Euro-Asian phylo-geographical region ex-tending in the East. The frequency of haplotypes of group C is three times lower in the populations of the Lithuanian perch in the samples of both the Curonian Lagoon and the coastal zone of the Baltic Sea, hence, the total spectrum of
Europe, although the “eastern” haplotypes of group C en-tirely dominate in the perch in the basins of the Vilija and Nemunas rivers in other than Lithuanian territory.
The lowest diversity of haplotypes (six) was observed in the perch population of the Curonian Lagoon. Similarly, perch populations of the Gulf of Bothnia in the territorial waters of Sweden, non-migrating perch populations had three or four different haplotypes, the lowest diversity of haplo-types, while perch with an anadromous behaviour had six different haplotypes (the size of samples fluctuated from 17 to 20 individuals). In brackish coastal waters of the Baltic Sea near Ðventoji and in the Gulf of Rîga, where the perch generally have anadromous behaviour, a greater diversity of haplotypes—eight or nine—was found. The similarity in the distribution of the most frequently found haplotypes A and F in the samples investigated illustrates the similarity of the populations of the Lithuanian perch, supporting the idea of possible seasonal migration of the perch between the Curo-nian Lagoon and the coastal zone of the Baltic Sea: adult in-dividuals migrate in summer to the brackish Baltic Sea probably due to the beneficial effect of water salinity on the perch growth, whereas in autumn they return to the fresh-water Lagoon, stay there in winter and spawn the following spring (Loþys, 2004). In this case, the lower diversity of haplotypes in summer population of the perch of the Curo-nian Lagoon suggests seasonal migrations of part of the
T a b l e 2 SAMPLE COLLECTION SITES AND DISTRIBUTION OF HAPLOTYPES IN POPULATIONS OF THE EUROPEAN PERCH
Population Region Number of
individuals studied
Water basin Haplotypes (N*)
Curonian Lagoon** Lithuanian territorial waters 15 Baltic Sea, mouth of the Nemunas A(2), A7(1), C(1), C1(1), F(9), G2(1) Baltic Sea** Lithuanian territorial waters 18 Baltic Sea near Ðventoji A(2), A2(1), C(1), C4(1), E(2) F(9),
F1(1), F8(1) Gulf of Rîga** Latvian territorial waters 23 Baltic Sea, the Gulf of Rîga near the
Daugava mouth
A(2), A7(3), C(6), C1(2), F(5), F2(1), F7(2), L1(1), L2(1)
L. Krylovo*** Russia, inland waters 5 Basins of the Vilija/Nemunas Rivers C(5)
L. Hencza*** Inland waters of Poland 5 The Nemunas basin C(4), C1(1)
L. Dubrovskoje*** Inland waters of Russia 10 The Dnieper basin C(10)
Gulf of Matsalu*** Inland waters of Estonia 10 Baltic Sea A8(3), F(4), F1(1), F7(1), E2(1) Emjajogi R.** * Inland waters of Estonia 5 Baltic Sea, the Gulf of Finland A(1), A7(1), A8(1), C(2), J1(1) L. Peipsi *** Inland waters of Estonia 9 Baltic Sea, the Gulf of Finland A(1), A7(1), A8(1),C(2), J1(1)
L. Vourasjarve *** Inland waters of Norway 10 Karasjakka/Tana A(4), A2(6)
The Angara River*** Inland waters of Russia 10 Baikal Lake, the Angara basin C(10)
L. Rybinsk*** Inland waters of Russia 14 The Volga basin A(1), C(7), C2(1), C3(2), G1(1), J(2) The gulf of Bothnia*** Territorial waters of Sweden 20 Baltic Sea A7(2), C(14), F1(4)
Anadromous A*** Territorial waters of Sweden 20 Baltic Sea A(2), A7(4), C(8), F(3), F1(2), G(1) Anadromous B*** Territorial waters of Sweden 17 Baltic Sea A(4), C(6), C1(1), F(2), F1(2), G(2) L. Ängersjön*** Territorial waters of Sweden 19 Lake near the Baltic Sea A5(1), C(13), C1(4), F1(1)
L. Race*** Inland waters of Slovenia 10 Drava/Danube C(1), M1(9)
* in brackets the number of individuals of the particular haplotype is given
** data from this research
*** data from Nesb¸ et al., 1999
Further work on the structure of populations in relation to seasonal migration to brackish waters requires the use of microsatellite markers (Brunner et al., 1998; Englbrecht et al., 2002). Several specific microsatellite primers have al-ready been developed for species of yellow perch, which is taxonomically close to the European perch (Leclerc et al., 2000), thereby aiding to overcome the stage of creating spe-cific primers for the species, and allowing investigators to make use of the available information in continuing investi-gations into the interspecific genetic diversity and popula-tion structure of the European perch.
ACKNOWLEDGEMENTS
We express our gratitude to the Mutual Funds for the Scien-tific Co-operation of the joint project of Taiwan, the Repub-lics of Latvia and Lithuania “Application of genetic and mi-cro-chemical markers as implements for diadromous and endangered commercial fish species populations manage-ment” for its financial assistance and the provided possibil-ity to carry out this project.
REFERENCES
Aljanabi, M. S., Martinez, I. (1997) Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleid Acids Re-search, 25 (22), 1772–1773.
Bernatchez, W. (1998) Comparative phylogeography of Nearctic and Palaearctic fishes. Mol. Ecol., 7, 431–452.
Brunner, P. C, Douglas, M. R., Bernatchez, L. (1998) Microsatellite and mi-tochondrial DNA assessment of population structure and stocking effects
in Arctic charr Salvelinus alpinus from central Alpine lakes. Mol. Ecol., 7, 209–223.
Englbrecht, C. C., Schlliewen, U., Tautz, D. (2002) The impact of stocking on the genetic integrity of Arctic charr (Salvelinus) populations from the Alpine region. Mol. Ecol., 11, 1017–1027.
Leclerc, D., Wirth, T., Bernatchez, L. (2000) Isolation and characterization of microsatellite loci in the yellow perch (Perca flavescens) and cross-species amplification within the family Percidae. Mol. Ecol., 9, 993–1011.
Loþys, L. (2004) The growth of pikeperch (Sander lucioperca L.) and perch (Perca fluviatilis L.) under different water temperature and salinity condi-tions in the Curonian Lagoon and Lithuanian coastal waters of the Baltic Sea. Hydrobiologia, 514 (1–3), 105–113.
Mardsen, J. E., Kassler, T., Philip, D. (1995) Allozyme confirmation that North American yellow perch (Perca flavescens) and Eurasian yellow perch (Perca fluviatilis) are separate species. Copeia, 4, 977–981.
Nesb¸, C. L., Maghagen, C., Jakobsen, K. S. (1998) Genetic differentiation among stationary and anadromous perch (Perca fluviatilis) in the Baltic sea. Hereditas, 129, 241–249.
Nesb¸, C. L., Fossheim, T., V¸llestad, L. A., Jakobsen, K. S. (1999) Genetic divergence and phylogeographic relationships among European perch (Perca fluviatilis) populations reflect glacial refugia and postglacial colo-nisation. Mol. Ecol., 8, 1387–1404.
Paulauskas, A., Loþys, L. (2001). Isoenzyme analysis of percids (Stizostedion lucioperca, Perca fluviatilis, Gymnocephalus cernua) in Lithuanian costal zone of the Baltic sea, the Curonian lagoon and Kaunas water reservoir. Acta Zool. Lituanica, 11 (1), 29–38.
Refseth, U. H., Nesb¸, C. L., V¸llestad, L. A., Fjeld, E., Stacy, J. E., Jakobsen, K. S. (1998) Genetic evidence for different migration routes of freshwaterfish into Norway revealed by analysis of current perch (Perca fluviatilis) populations in Scandinavia. Mol. Ecol., 7, 1015–1027.
mtDNS HAPLOTIPU DAÞÂDÎBA KURÐU LÎÈA, BALTIJAS JÛRAS PIEKRASTES ZONAS UN RÎGAS LÎÈA ASARU (Perca fluviatilis) POPULÂCIJÂS
Îpatòiem no trim asaru populâcijâm tika pçtîts mtDNS haplotipu sadalîjums. Trîs jauni haplotipi atrasti starp Lietuvas paraugiem, divi – Received 20 November 2006