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Blackwell Munksgaard 2005

Ecology of Freshwater Fish 2005: 14: 278282

Printed in Singapore All rights reserved

ECOLOGY OF
FRESHWATER FISH

Letter

Ancient and modern mitochondrial haplotypes of

common bream (Abramis brama L.) in Poland
S. Ciesielski1, D. Makowiecki2

Ciesielski S, Makowiecki D. Ancient and modern mitochondrial haplotypes of common bream (Abramis brama L.) in Poland.
Ecology of Freshwater Fish 2005: 14: 278282.
Blackwell
Munksgaard, 2005
Abstract Genetic data on archaeological specimens provide
complementary information for addressing questions on distribution and
migration of shes over long time scales. In this study DNA was extracted
from common bream bones (N 4), dated c. 60001000 bp, and a 172 bp
fragment of mitochondrial cytochrome b gene has been sequenced. The
obtained sequences differed from homologous sequences of breams living
contemporarily (N 4), inhabiting the same geographical areas as
ancient sh. None of ancient mitochondrial haplotype was found in sh
living at present. Our results suggest that sh vicariance could be affected
also by other than glacial retreat historical events, and the mechanisms that
inuenced present distribution of freshwater shes are still unclear.

1
Department of Environmental Biotechnology,
Faculty of Environmental Sciences and Fisheries,
University of Warmia and Mazury in Olsztyn,
Olsztyn, 2Institute of Archaeology and Ethnology,
Polish Academy of Sciences, Poznan, Poland

Key words: Abramis brama L.; ancient DNA;

mitochondrial DNA; phylogeography
Slawomir Ciesielski, Department of Environmental Biotechnology, Faculty of Environmental
Sciences and Fisheries, University of Warmia and
Mazury in Olsztyn, ul. Oczapowskiego 5, 10-718
Olsztyn, Poland; e-mail: slavcm@uwm.edu.pl
Accepted for publication March 31, 2005

Un resumen en espanol se incluye detras del texto principal de este artculo.

Introduction

Climate changes may have an important impact on

distribution of organisms and lead to cycles of range
expansions or contraction (Riddle 1996). Such events
during the Pleistocene and Holocene explain many of
the observed patterns of geographical variation within
ora and fauna (Avise 1994; Hewitt 1996). Analyses
of freshwater shes have revealed molecular traces of
recolonization routes on the North American and
European continent (e.g., Bernatchez & Osinov 1995;
Nesb et al. 1998; Durand et al. 1999; Koskinen et al.
2000). DNA techniques are greatly advancing our
knowledge of the global distribution of genetic
diversity and vicariance of the species (Hewitt
2004), but the details of migration routes could be
provided only by direct analysis of specimens ancestors remnants.
Molecular marker techniques do not only allow for
the study of DNA of contemporary organisms, but also
of some extinct organisms (Cooper & Wayne 1998).
Ancient DNA recovered from archaeological and
palaeontological remains make possible a comparison
of the genetic relationships of extinct organisms with
their contemporary relatives (Hofreiter et al. 2001).
Thus it may be possible to study evolutionary
278

processes and historical events that changed the

distribution of genetic variants, perhaps resulting in
the present distribution of species.
Research on ancient DNA extracted from sh bones
and otoliths has focused on the analysis of mitochondrial DNA (mtDNA) fragments. This is because the
mtDNA genome is a relatively small and well
characterised molecule, is inherited maternally and
evolves faster than nuclear DNA (Kocher et al. 1989).
As each vertebrate cell contains numerous mtDNA
molecules, at least some undegraded mtDNAs often
persist in samples of ancient tissues (Hagelberg et al.
1991). Among the mitochondrial genes as the cytochrome b gene is probably the best studied mtDNA
segment in shes (e.g., Kocher et al. 1989; Briolay
et al. 1998), sequences of this gene is useful in
revealing genetic relationships at intra- and interspecies level.
In this pilot study we examine ancient mtDNA as an
approach to shed light on phylogeography of past and
present mitochondrial DNA haplotypes in common
bream (Abramis brama L., Cyprinidae, Teleostei) in
Poland. The common bream represents a major
component of the sh fauna of Europe, inhabiting
fresh and brackish waters. Due to its sustenance
importance, the remains of common bream have been
doi: 10.1111/j.1600-0633.2005.00097.x

Mitochondrial haplotypes of common bream in Poland

Fig. 1. Map of northern Poland showing the

sampling sites. Dudka, Dabki, and Wolin
were archaeological sites, whereas Bay of
Szczecin, Bukowo Lake, and Niegocin Lake
refer to contemporary sampling sites of
common bream.

uncovered at many archaeological sites (Makowiecki

2000).
We were interested in the following questions: (i)
is it possible to identify common bream mtDNA
haplotypes from archaeological material, (ii) are
modern-type common bream haplotypes present in
archaeological material, and (iii) is this approach
useful for comparing the geographical distribution of
bream mtDNA variants over long periods of time?
Material and methods

The archival material of common bream used in his

work was obtained from three archaeological sites
Wolin, Dabki and Dudka (Fig. 1); samples of three
individuals (one bone from Dabki and Dudka, and two
bones from Wolin) were subjected to DNA analysis.
The contemporary living breams were caught in the
same geographical locations from which archival
samples were obtained (Table 1).
DNA extraction

The method of DNA extraction was performed

following the protocol of Ciesielski et al. (2002). At
rst the bone was soaked in sodium hypochlorite
solution for surface decontamination and then rinsed
several times in ethanol. The bone was powdered with
a ne sand paper, and approximately 0.3 g of a bone
powder was added to 0.5 m ethylenediaminetetraacetic
acid (EDTA) (pH 8.0) in order to decalcify the sample.

Table 1. Description of archival and contemporary samples of common

bream (Abramis brama L.) used in this study.
Catalogue number

Collection location

Sample

Age (years)

Dabki 20
Dabki 01
Dudka 25
Dudka 01
Wolin 11
Wolin 12
Wolin 01a
Wolin 01b

Dabki (Bukowo Lake)

Bukowo Lake
Dudka (Staswiny Lake)
Niegocin Lake
Wolin Island
Wolin Island
Bay of Szczecin
Bay of Szczecin

os cleithrum
Muscle
os operculum
Muscle
os cleithrum
os cleithrum
Muscle
Muscle

6000
1
5500
1
1000
1000
1
1

After 90 h of incubation with EDTA the decalcied

bone powder was transferred into a new tube, and
500 ll of lysis buffer (100 mm Tris-HCl; 10 mm
EDTA; pH 8.0, 0.5 mg of proteinase K, and 0.25 mg
of dithiothreitol) was added. Then the sample was
incubated at 55 C for 3 h. The aqueous phase was
extracted twice with an equal volume of phenol, and
once with chloroform isoamyl alcohol solution
(24:1). The DNA of a sample was concentrated using
Microcon-50 microconcentrators (Millipore Corporation, Billerica, MA, USA). Two consecutive rounds of
concentration gave a nal volume of 50 ll, and the
retante was stored at )20 C.
Modern DNA was extracted using the DNA-direct
kit (Wizard Genomic Purication Kit; Promega,
Madison, WI, USA).
PCR amplification

Primers L: 5-acgcactagtcgacctcc-3 and H: 5-acgtctcggcagatgtgg-3 spanned a 172 bp region of cytochrome b gene from 46 to 218 nucleotide (Ciesielski
et al. 2002).
Double-stranded PCR amplication was performed
in 50 ll reaction volumes containing 2 units of Taq
DNA polymerase (Promega), 5 ll reaction buffer
(500 mm KCl, pH 8.5; Triton X-100), 20 pmol of
each primer (MWG-BIOTECH, Ebersberg, Germany),
2.5 mm MgCl2, 500 of lm dATP, dCTP, dGTP, and
dTTP, and 2 ll of DNA template. DNA was amplied
using a Perkin Elmer 9600 thermal cycler (PE-Applied
Biosystems, Foster City, CA, USA) beginning with
preliminary denaturation at 95 C for 5 min. The
amplication cycle consisted of 94 C for 30 s, 52 C
for 30 s, and 72 C for 45 s, for a total of 30 cycles,
ending with a nal elongation step at 72 C for 3 min.
Initial PCR products of archival samples were reamplied using the same procedure.
The PCR product was separated by electrophoresis
on 1.5% agarose gel, using 1x TBE buffer (0.5 m
EDTA, pH 8.0). Ethidium bromide (0.1 mg/ml) was
added to the gel and the PCR product was visualised
with UV light. Molecular weight marker UX 174/Hinf
I was used.
279

Ciesielski & Makowiecki

DNA sequencing

Before sequencing the PCR product was puried from

oligonucleotides, primers and dimers using Microcon50 spin columns (Millipore Corporation). Sequencing
was performed using a Perkin Elmer ABI 373
automated DNA sequencer and the DyeDeoxy Cycling Sequencing reaction (PE-Applied Biosystems) at
the Institute of Biochemistry and Biophysics in
Warsaw, Poland. The nucleotide sequences were
deposited in GenBank under accession numbers:
AF238082, AY028979, AY028980, AY028981 and
AF486629.
Phylogenetic analysis

Nucleotide composition in the analysed segment was

assessed with MEGA 2.1 (Kumar et al. 2001).
Sequences were aligned with the help of Higgins
and Sharp algorithm using the program CLUSTAL W
(Thompson et al. 1994). Pairwise distance of nucleotide sequences were estimated using the Kimura 2parameter model (Kimura 1980). Genetic relationships
were determined by distance method with MEGA 2.1
program (Kumar et al. 2001) using deduced amino
acids sequences. Distance trees were estimated according to the neighbour-joining method of Saitou & Nei
(1987).
Laboratory precautions

The entire DNA extraction procedure was organised

following strict standard rules to avoid PCR contamination. The preparation of bone sample and DNA
extraction were performed in one laboratory and
stored in a freezer located in another room. Preparation
of buffers and PCR set-up was performed in a separate
laboratory, which was UV illuminated for periods of
time when it was not in use. For each PCR, reaction
blank controls were conducted.
Results

The archaeological sequences (Table 2) agreed with

the sequence of contemporary common bream. All
sequences, including those retrieved from ancient
samples, were complete. For further phylogenetic
analysis we took the middle part of the sequences
without anking segments.
The nucleotide composition of the 117 bp cytochrome b gene segment sequence was G decient
(13,8%), whereas relatively equal frequencies were
observed for the other three nucleotides (A, 25.7%; C,
31.8% and T, 28.7%). Sequence alignment of all eight
specimens was straightforward, no insertions or deletions were observed, and all sequences were translat280

Table 2. Variable sites of 117 bp long region of the mitochondrial

cytochrome b gene (which corresponds to position 46218 of mitochondrial
cytochrome b gene) from eight common bream samples. Identical nucleotides are denoted by dots.
Nucleotide position in mitochondrial cytochrome b gene
Sample

51

61

63

67

69

72

Dudka 01
Dabki 01
Wolin 01a
Wolin 01b
Dudka 25
Dabki 20
Wolin 11
Wolin 12

A
A
A

C
C

able without nonsense codons. Analysis revealed 5

m tDNA haplotypes varying at six positions with the
variable third codon position. One mutation (position
115 of the cytochrome b gene) affected the amino acid
(leucinemethionine). Nucleotide differences were
observed only among the ancient common bream
group, whereas sequences of examined contemporary
living breams were identical (Table 2). The pairwise
nucleotide sequence distance between all haplotypes
ranged from 0.0086 to 0.03516, whereas mean
distance between ancient group (Wol11, Wol12,
Dab20 and Dud25) and the present-day haplotype
(Con01) was 0.0222 (Table 3).
The neighbour-joining tree based on amino acid
sequences revealed two lineages (Fig. 2). A rst clade
included samples: Dabki 01, Dudka 01, Wolin 01a and
Wolin 01b (Con 01 haplotype) and Dudka 25 (Dud 25
haplotype), whereas a second group contained three
ancient samples: Wolin 11, Wolin 12 and Dabki 20
(respectively the Wol 11, Wol 12 and Dab 20
haplotypes). The clustering observed resulted in
leucine or methionine present in 39 amino acid
position in a coded protein.
Discussion

Our results indicated a lack of genetic variation among

analysed contemporary samples, whereas relatively
high divergence among ancient common breams
haplotypes was observed. As ancient sh, lived in
several periods of time, it is difcult to estimate how
this fact inuenced observed genetic divergence. It is
worth noticing that sh remains taken from Wolin
Table 3. Pairwise distance among haplotypes under Kimuras 2-parameter
model.

1.
2.
3.
4.
5.

Con 01
Dab 20
Wol 12
Dud 25
Wol 11

0.03516
0.03502
0.00862
0.00860

0.03516
0.02615
0.02632

0.02609
0.02615

0.01730

Mitochondrial haplotypes of common bream in Poland

Fig. 2. Phylogenetic relationships among mitochondrial DNA

haplotypes of common bream based on amino acids deduced from
117 bp fragment of cytochrome b gene. Genetic distance tree was
estimated according to the neighbour-joining method of Saitou &
Nei (1987). Ancient samples are highlighted with a grey background.

Island (Wol 11, Wol 12; 1000 bp) were genetically

divergent, although they belonged to sh that have
lived at the same time (c. 1000 years bp), contrary to
bream caught in 2001. There was no relationship
between past and present mitochondrial haplotypes of
shes inhabiting the same geographical areas. Each
ancient sh possessed its own mtDNA haplotype, and
unexpectedly none of these were found in contemporary samples.
As some characteristics of cytochrome b gene, such
as saturation effects and the low variation in rst and
second codon position, contribute to problems in
phylogenetic analysis (Meyer 1993), in this study of
reconstruction of genetic relationship we used amino
acid sequences (Adachi & Hasegawa 1996). The
analysis performed revealed two lineages that differed
to each other in one amino acid position. The samples:
Wolin 01a, Wolin 01b, Dabki 01, Dudka 01 (haplotype
Con 01) and Dabki 20 (Dab 20) contained mtDNA
that determined leucine, whereas those from samples
Wolin 11 (Wol11), Wolin 12 (Wol12) and Dabki 20
(Dab 20) coded for methionine at this position. The
pattern observed in phylogenetic partition in analysed
common bream mtDNA is confusing because of the
absence of a clear phylogeographical relationships
between haplotypes. This could result from small
number of studied common bream samples. However
there is a some scheme of relation between analysed
individuals all ancient breams, that inhabited waters
directly connected to the Baltic Sea (Wol 11, Wol 12
and Dab 20 haplotypes) formed one clade (Fig. 2),
whereas sh from Dudka site (Dud 25 haplotype)
joined the group of contemporary sh. It may be
theorized that thousands of years ago a specic group

of common bream stocks inhabited coastline waters of

the Baltic Sea. Later on, because of changing
environmental conditions such as alteration of water
salinity (Muller 2001), this group of sh went extinct.
Some time after that alongside with other changes in
environmental conditions, coastal waters became
inhabited anew by shes, that have immigrated from
the region where sh stocks were genetically more
polymorphic (Hewitt 1999).
In conclusion, it is possible to retrieve and analyse
mitochondrial DNA molecules from archival bones of
common bream. The procedures applied allowed
obtaining information about mitochondrial haplotypes
of sh that lived thousands of years ago. None of
ancient bream mitochondrial haplotypes were found in
contemporary samples, although this observation
could result from small samples size. In order to
compare the geographical distribution of sh mtDNA
variants over long periods of time it is necessary to
analyse larger sample size.
We contemplate that the present distribution of
common bream mitochondrial haplotypes has been
formed no earlier than 1000 years ago and that the
present lack of genetic variation might originate from a
founder effect and restricted gene ow. Although the
glacial history and patterns of postglacial colonization
have played a major role in shaping present-day
genetic differentiation (Hewitt 1996, 1999; Jaarola
et al. 1999) it is also possible that other historical
events in the natural environment might have determined the present genetic variation.
Resumen
1. Descubrimientos recientes han probado que con tecnicas
moleculares no solo es posible estudiar ora y fauna contemporanea sino tambien organismos ya extintos. El acceso a restos
de animales extintos permite extraer y estudiar su ADN y
revelar su posicion taxonomica tanto como sus relaciones con
parientes contemporaneos. Este tipo de informacion puede ser
util para explicar cambios en la distribucion de especies de
peces. Los datos geneticos de especmenes arqueologicos
proveen informacion complementaria para resolver cuestiones
sobre la distribucion y migracion de los peces a traves de
escalas temporales.
2. En este estudio, extraimos el ADN de huesos de Abramis
brama (N 4) datados entre 6000 y 1000 anos AC sobre los
que secuenciamos un fragmento de 172 bp de Citocromo b
mitocondrial. Las secuencias obtenidas dirieron de las secuencias homologas de individuos contemporaneos (N 4)
que habitan la misma area geograca. Ninguno de los
haplotipos mitocondriales antiguos fue encontrado en los
individuos contemporaneos.
3. Sobre estos resultados contemplamos que la distribucion
actual de los haplotipos mitocondriales de A. brama o bien se
ha formado hace menos de mil anos o bien que la falta actual de
variacion genetica puede haberse originado como efecto
fundador o ujo restringido de genes.

281

Ciesielski & Makowiecki

4. Nuestros resultados sugieren que la vicarianza de peces
podra estar afectada por otros eventos historicos distintos de la
retirada glacial. Los mecanismos que inuyen la distribucion
actual de los peces de agua dulce permanecen poco claros.

Acknowledgements
We thank M. Luczynski and F. Volckaert for critical reading
of the manuscript. We are grateful also to J. Filipiak,
Z. Chelkowski, K. Formicki, and A. Wisniewska for their kind
provision of samples. This research was supported by the Polish
Foundation for Sciences under the ARCHEO Nr 5 scheme.

References
Adachi, J. & Hasegawa, M. 1996. Model of amino acid
substitution in proteins encoded by mitochondrial DNA.
Journal of Molecular Evolution 42: 459468.
Avise, J.C. 1994. Molecular markers, natural history and
evolution. London: Chapman and Hall.
Bernatchez, L. & Osinov, A. 1995. Genetic diversity of trout
(genus Salmo) from its most eastern range based on
mitochondrial DNA and nuclear gene variation. Molecular
Ecology 7: 431452.
Briolay, J., Galtier, N., Brito, R.M. & Bouvet, Y. 1998.
Molecular phylogeny of Cyprinidae inferred from cytochrome b DNA sequences. Molecular Phylogenetics and
Evolution 9: 100108.
Ciesielski, S., Brzuzan, P. & Luczynski, M. 2002. Recovery and
analysis of mitochondrial DNA from ancient bones of
common bream, Abramis brama L. Ancient Biomolecules
4: 4346.
Cooper, A. & Wayne, R. 1998. New uses for old DNA. Current
Opinion in Biotechnology 9: 4953
Durand, J.D., Persat, H. & Bouvet, Y. 1999. Phylogeography
and postglacial dispersion of the chub (Leuciscus cephalus)
in Europe. Molecular Ecology 8: 989997.
Hagelberg, E., Bell, L.S., Allen, T., Boyde, A., Jones, S.J. &
Clegg, J.B. 1991. Ancient bone DNA: techniques and
applications. Philosophical Transactions of the Royal Society,
London B 333: 399407.
Hewitt, G.M. 1996. Some genetic consequences of ages, and
their role in divergence and speciation. Biological Journal of
Linnean Society 58: 247276.
Hewitt, G.M. 1999. Post-glacial re-colonization of European
biota. Biological Journal of Linnean Society 68: 87112.
Hewitt, GM. 2004. The structure of biodiversity insights from
molecular phylogeography. Frontiers in Zoology 1: 4.

282

Hofreiter, M., Serre, D., Poinar, H.N., Kuch, M. & Paabo, S.

2001. Ancient DNA. Nature Reviews 2: 353359.
Jaarola, M., Tagelstrom, H. & Fredga, K. 1999. Colonization
history in Fennoscandian rodents. Biological Journal of
Linnean Society 68: 113127.
Kimura, M. 1980. A simple method for estimating evolutionary
rate of base substitution through comparative studies of
nucleotide sequences. Journal of Molecular Evolution 16:
111120.
Kocher, T.D., Thomas, W.K. & Meyer, A. 1989. Dynamics of
mitochondrial DNA evolution in animals: amplication and
sequencing with conserved primers. Proceedings of the
National Academy of Sciences of the United States of
America 86: 61966200.
Koskinen, M.T., Ranta, E., Piironen, J., Veselov, A., Titov, S.,
Haugen, T.O., Nilsson, J., Carlstein, M. & Primmer, C.R.
2000. Genetic lineages and postglacial colonisation of
grayling (Thymallus thymallus, Salmonidae) in Europe, as
revealed by mitochondrial DNA analyses. Molecular Ecology
9: 16091624.
Kumar, S., Tamura, K., Jakobsen, I.B. & Nei, M. 2001.
MEGA2: molecular evolutionary genetics analysis software.
Tempe, AZ: Arizona State University.
Makowiecki, D. 2000. Catalogue of subfossil sh remains from
Poland. Archaeofauna 9: 133149.
Meyer, A. 1993. Evolution of mitochondrial DNA in sh. In:
Hochachka, P.W. & Mommsen, P., eds. Biochemistry and
molecular biology of sh, Vol. 2. New York: Elsevier Press,
pp. 138.
Muller, A. 2001. Late- and postglacial sea-level change and
paleoenvironments in the Oder estuary, Southern Baltic Sea.
Quaternary Research 55: 8696.
Nesb, C.L., Arab, M.O. & Jakobsen, K.S. 1998. Heteroplasmy, length and sequence variation in the mtDNA control
regions of three percid she species (Perca uviatilis,
Acerina cernua, Stizostedion lucioperca). Genetics 148:
19071919.
Riddle, B.R. 1996. The molecular phylogeographic bridge
between deep and shallow history in continental biotas.
Trends in Ecology and Evolution 11: 207211.
Saitou, N. & Nei, M. 1987. The neighbor-joining method: a
new method reconstructing phylogenetic trees. Molecular
Biology and Evolution 4: 406425.
Thompson, J.D., Higgins, D.G. & Gibson, T.J. 1994.
CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weighting,
positions-specic gap penalties and weight matrix choice.
Nucleic Acids Research 22: 46734680.