GENETIC VARIABILITY OF THE POLISH BROWN HARE (LEPUS EUROPAEUS) BASED ON PCR-RFLP MTDNA ANALYSIS (PRELIMINARY RESULTS)

Tomasz Strzała¹, Costas Stamatis², Barbara Kosowska³, Magdalena Moska¹, Bożena Marszałek-Kruk³, Zissis Mamuris^2
1 Department of Genetics, Wrocław University of Environmental and Life Sciences, Poland
² University of Thessaly, Department of Biochemistry and Biotechnology, Larissa, Greece
³ Department of Genetics and Animal Breeding, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

We have made an analysis of 136 brown hare individuals (Lepus europaeus Pallas, 1778) from 13 locations situated in Poland. The analysis was based on the data from PCR-RFLP of three mitochondrial DNA fragments. For digestion, we used five enzymes (revealed as diagnostic for Lepus by Stamatis et al.): MboI HinfI, MseI for Cytochrome-b (Cyt-b)/Control Region (CR), HhaI, for Cytochrome Oxidase I (COI) and MseI for 16S rRNA. As a result of the analysis, we found 17 haplotypes created by joining all digestion profiles from all enzymes. The data analysis revealed high haplotype diversity (12 from 17 haplotypes were unique, they were found only in one location) and high nucleotide diversity. The most variable region was south-eastern Poland (the samples from the area around the city of Zamość).

Key words: Lepus europaeus, RFLP, genetic diversity, mtDNA.

INTRODUCTION

Brown hare (Lepus europaeus Pallas, 1778) is one of the most widespread species of the Leporidae family in Europe. In spite of its big spreading, this species is now endangered. In Europe, we can observe permanent reduction, and, in some years, a sharp drop of its abundance. In the fifties of the 20th century, approximately 50 hares were observed per 1 km² [9,10] in Poland. Nowadays, this number is much smaller. According to the data from the Department of Environment and Central Administration of the Polish Hunting Society in 2002, the estimated number of all Polish brown hares were about 460 000 individuals. When we compare this data with the same statistics from 1990/1991 we can observe that this population decreased by half in 11 years. In the early seventies, about 700 000 hares per year were hunted. Since the beginning of the nineties it was just 505 000 per year [4].

The biggest decrease of the brown hare population number was observed in the middle of the eighties of the 20th century [12]. Nowadays, hare shooting  is abandoned in many hunting districts due to the very small hare population.

According to many authors, the reasons for the decrease in the hares population are as follows: European Brown Hare Syndrome (EBHS) [3], the superabundant number of predators (foxes, birds), monoculture crops, balks and tree clumps removal [2], worsening of the climate conditions, mechanization and chemicalization of  agriculture, and the increasing traffic and poaching [2].

Genetic research, which analyzes diversity among and within local brown hare populations, is very important part of the current analysis relating to brown hares. Genetic research will provide knowledge which is necessary to preserve local endemism and brown hare genetic biodiversity. Our research will analyze genetic diversity of the brown hares from several regions of Poland.

MATERIAL AND METHODS

We have analyzed the total number of 136 brown hare individuals from 13 locations (Fig. 1).

Fig. 1. Sample collecting sites of all individuals

Samples (muscle or liver fragments) were collected by hunters during the two hunting seasons – 2005 and 2006. Tissues were stored in 90% alcohol or were frozen until DNA was isolated. The isolation was carried out with the use of the Sherlock AX kit (A&A Biotechnology) according to the manual. Samples differentiation was proceeded by the RFLP analysis of the three mtDNA fragments: cytochrome b/control region (cyt-b/CR), cytochrome oxidase I (COI), and 16s rRNA. Primers and PCR conditions were analogical with Mamuris et al. [5]. Amplified fragments were digested with the following endonucleases: MboI, HinfI, MseI for cyt-b/CR fragment, HhaI for COI and MseI for 16s rRNA. All enzymes were qualified as diagnostic enzymes for Lepus by Stamatis et al. (in printing). The digestion results were verified by electrophoresis on 6 % polyacrylamide gels (PAGE). Then the digestion profiles were created for every enzyme. The connected profiles created haplotypes – specific for each individual. Haplotype diversity (Nei, 1978) and nucleotide diversity (Nei and Tajima, 1981) were calculated with REAP 4.0 [6]. Diversity within and among populations was calculated with the hierarchical variance analysis (AMOVA, [1]), implemented in Arlequin 2.0 [13]. A significance level of the F statistics and variance components was obtained for 10000 randomizations.

RESULTS

As a results of our analysis, we have got 17 haplotypes created by connecting all digestion profiles (Table 1).

Table 1. Haplotypes found within analyzed populations connected with sampling areas and a number of each haplotype

Haplotypes*		Samples localization
		Pl_Zam	Pl_Tor	Pl_Suw	Pl_Rze	Pl_Op	Pl_Nos	Pl_Lub	Pl_Kon	Pl_Kie	Pl_Kal	Pl_Cie	Pl_Osw	Pl_Sw
1	BCBAB	1
2	BCBBA	7	9			1	2	7	2	5	13	9	2
3	BIBBA								1		1
4	CCBBA	3	3	1	1		4	5	4	1	7	1	7	1
5	CCBBC		5
6	CCNBA	1
7	CHBBA	2	1					8				7	2
8	CHBBC		1
9	CRBBA							1
10	CSBBA	1
11	FBAAB	2
12	IBAAB	1										1
13	ITBAB							1
14	TCBBA							1
15	UBAAA												1
16	UBMAA						1
17	VBMAA	1
Number of individuals		19	19	1	1	1	7	23	7	6	21	18	12	1

* Above haplotypes were created with following enzymes: Cytochrome-b (Cyt b)/Control Region (CR): MboI, HinfI, MseI; Cytochrome oxidase I (COI): HhaI; 16s rRNA: MseI

The most popular haplotypes were numbered: 2, 4 and 7. Together those haplotypes were found in 115 out of 136 analyzed hares, which is almost 85% of the whole analyzed population.

12 haplotypes were found unique, and were spotted only in one population. The most divergent region of Poland was the neighbourhood of the city of Zamość, where we found 9 haplotypes out of which 5 were unique. The less divergent region was the city of Kalisz and its neighbourhood, we found only 3 haplotypes there within 21 individuals.

Table 2. Sample size (N), the number of total haplotypes (T) and of population-specific haplotypes (P) found within each population, sample and percentages of the number of total haplotypes/number of individuals (V), the number of population-specific haplotypes/number of individuals (W), haplotype diversity (h) and nucleotide diversity (π)

Samples locatization:	N	T	P	V	W	h	π
Zamosc (Pl_Zam)	19	9	4	47.37%	21.05%	0.85	0.063
Toruń (Pl_Tor)	19	5	2	26.32%	10.53%	0.71	0.015
Suwałki (Pl_Suw)	1	1	-	-	-	0.00	0.000
Rzeszów (Pl_Rze)	1	1	-	-	-	0.00	0.000
Opole (Pl_Op)	1	1	-	-	-	0.00	0.000
Nowy Sacz (Pl_Nos)	7	3	1	42.86%	14.29%	0.67	0.034
Lublin (Pl_Lub)	23	6	3	26.09%	13.04%	0.77	0.027
Konin (Pl_Kon)	7	3	-	42.86%	-	0.67	0.011
Kielce (Pl_Kie)	6	2	-	33.33%	-	0.33	0.005
Kalisz (Pl_Kal)	21	3	1	14.29%	4.76%	0.53	0.008
Ciechanów (Pl_Cie)	18	4	-	22.22%	-	0.63	0.028
Oswięcim (Pl_Osw)	12	4	1	33.33%	8.33%	0.65	0.021
Swiebodzin (Pl_Sw)	1	1	-	-	-	0.00	0.000
Average/Total for the whole country	136	3	12	32.07%	12.00%	0.45	0.016

Data from Table 2 suggest that the analyzed populations have high haplotype and nucleotide diversity. The most divergent region was again south-eastern Poland – Zamość and its neighbourhood. Haplotype diversity in that region was almost twice as a high and nucleotide diversity was three times higher than an average for the whole country.

Table 3. Results of the hierarchical analysis of molecular variance (AMOVA)

Source of variation	Degrees of freedom	Sum of squares	Variance components	% Total variance
Among populations	12	43.289	0.11860 Va	4.65
Within populations	123	298.917	2.43022 Vb	95.35
Total	135	342.206	2.54882
Fst= 0.04653		P = 0.09

As the main source of the genetic diversity, we found differentiation within population (95%). Diversification among populations was rather low, only 5%.

DISCUSSION

Our results show that the Polish brown hare population reveals high genetic diversity within the analyzed mtDNA fragments. That diversity changes with a geographic location from where samples were collected. The further we go to the south-eastern part of Poland the higher diversity we find. The main source of diversity is differentiation within population, which is high for all populations. It suggests that south-eastern part of Poland has regular genes flow from Eastern Europe, and probably indicates some migration route.

Haplotype diversity estimated by Stamatis et al. [11], as a result of the RFLP analysis of the same mtDNA fragments, was 60% for haplotypes compounded with digestion profiles from 20 endonucleases. In our results, this value was lower and amounted to 45% for only 5 diagnostic enzymes. This suggests that the Polish brown hare population is one of the most divergent populations in Europe.

An analogical situation can be found when we compare other diversity factors describing the Polish and European populations. The number of population-specific (unique) haplotypes (W) in Poland was 12% which is nearly twice as high as a European average (7%). Even bigger disproportion between the Polish end European populations can be found when we compare diversity expressed as a fraction of the number of haplotypes to the number of individuals in each population (V). An average for Poland is 32% – for Europe it is much less – 12%.

Diversity among and within populations is much different for our results and those from other parts of Europe [11]. In our research, we found out that diversity within populations was about 5%. Stamatis showed that average diversity within populations for the European hares is 50%. Diversity among populations in Poland is the main source of diversity. Its level is very high – 95%. Stamatis estimates diversity among population to be much lower, only 46%. That disproportion is probably a result of the different data amount used for the analysis in our research and Stamatis. Stamatis used twice as much loci as we did, and in our opinion, it is the main reason for such differences. At this point, we would like to add that the presented results are just a preliminary study. As the final result, RFLP will be verified by sequencing.

CONCLUSIONS

In conclusion, we can confirm that our results place the Polish brown hare population as one of the most divergent in Europe. The source of this diversity is not well positioned (among or within populations) and we need more data to perform this. Our future results will enable us to create a more precise study of the genetic diversity within the Polish brown hare population, to refer those results to the whole European population, and maybe to define some brown hare migration paths in Europe.

ACKNOWLEDGEMENTS

The research was financed by the project: "Second scholarship program for University of Environmental and Life Sciences PhD students". The project was co-founded by European Union.

The research was co-financed by the Project "Hare research program" realized by the University of Environmental and Life Sciences, agreement No. 15/4-W/2007.

REFERENCES

1. Excoffier L., Smouse P., Quattro J., 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics, 131, 479–491.

2. Edwards P.J., Fletcher M.R., Bernyc P., 2000. Review of the factors affecting the decline of the European brown hare, Lepus europaeus (Pallas, 1778) and the use of wildlife incident data to evaluate the significance of parquet. Agriculture, Ecosystems and Environment 79, 95–103.

3. Frolich K., Meyer H.H., Pielowski Z., 1996. European brown hare syndrome in free-ranging hares in Poland. Journal of Wildlife Diseases, 32(2), 280-285

4. Hartl G.B., Markowski J., Świątecki A., 1992. Genetic diversity in the Polish brown hare Lepus europaeus Pallas, 1778: Implications for conservation and managment. Acta Theriologica 37(1-2), 15-25.

5. Mamuris Z., Sfougaris A.I., Stamatis C., 2001. Genetic structure of Greek brown hare (Lepus europaeus) populations as revealed by mtDBNA RFLP-PCR analysis: implications for conserving genetic diversity. Biological Conservation 101, 187-196.

6. McElroy D., Moran P., Bermingham E., Kornfield J., 1991. The Restriction Enzyme Analysis Package, Version 4.0. University of Maine, Orono, ME.

7. Nei M., 1987. Molecular Evolutionary Genetics. Columbia University Press, New York.

8. Nei M., Tajima F., 1981. DNA polymorphism detectable by restriction endonucleases. Genetics, 97, 145–163.

9. Pielowski Z., 1976. On the present state and perspectives of the European hare breeding in Poland. PWRiL, Warszawa, 25-27.

10. Pielowski Z., 1979. Brown hare. Nature and hunting monography. PWRiL, Warszawa, 1-54

11. Stamatis C., Suchentrunk F., Giacometti M., Haerer G., Davidovic M., Vapa L., Vukovic M., Tvrtkovic N., Sert H., Mamuris Z. Phylogeography of the brown hare, Lepus europaeus, in Europe: a legacy of southeastern Mediterranean refugia? [in press].

12. Sikorski J., 1987. Hunting economy expansion directions. Łow. Pol. 11, 4-7.

13. Schneider S., Roessli D., Excoffier L., 2000. ARLEQUIN, Version 2.0 A Software for Population Genetic Data Analysis. Genetics and Biometry Laboratory, University of Geneva, Geneva, Switzerland.

 

Accepted for print: 8.02.2008

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Tomasz Strzała
Department of Genetics, Wrocław University of Environmental and Life Sciences, Poland
Kożuchowska 7
51-631 Wrocław
Poland
email: arrow00@poczta.fm

Costas Stamatis
University of Thessaly,
Department of Biochemistry and Biotechnology, Larissa, Greece
26 Ploutonos & Aiolou Str. 41221, Larissa, Greece

Barbara Kosowska
Department of Genetics and Animal Breeding,
Wrocław University of Environmental and Life Sciences, Poland
Kożuchowska 7, 51-631 Wrocław, Poland
email: basia@gen.ar.wroc.pl

Magdalena Moska
Department of Genetics, Wrocław University of Environmental and Life Sciences, Poland
Kożuchowska 7
51-631 Wrocław
Poland
Phone +48 71 320 5921
email: magdalena.moska@up.wroc.pl

Bożena Marszałek-Kruk
Department of Genetics and Animal Breeding,
Wrocław University of Environmental and Life Sciences, Poland
Kożuchowska 7, 51-631 Wrocław, Poland

Zissis Mamuris
University of Thessaly,
Department of Biochemistry and Biotechnology, Larissa, Greece
26 Ploutonos & Aiolou Str. 41221, Larissa, Greece

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Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

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