3.4.2. Diversity of common carp (Cyprinus carpio L.) genetic ... - IGB

KOHLMANN, K., GROSS, R. 1 , MURAKAEVA, A.
3.4.2. Diversity of common carp (Cyprinus carpio L.)
genetic resources
Diversität genetischer Ressourcen des Karpfens (Cyprinus carpio L.)
Key words: common carp, Cyprinus carpio, diversity, genetic resources
Abstract
The common carp is among the most important species in freshwater fish
culture. It has a long history of domestication, and numerous strains and
breeds have been developed from the wild ancestor. The most
comprehensive study on the population structure and genetic relationships
of these breeds, as well as between them and wild carp, has been performed
by our research team. Our study comprises of 22 European, six Central
Asian, and four East/South-East Asian, wild and domesticated populations
which were analysed for three classes of genetic markers (allozymes,
microsatellites, and PCR-RFLP of mitochondrial DNA). Results showed that
allozyme variability was much lower than microsatellite variability. Wild
caught populations differed from domesticated stocks by a higher variability,
independent of their geographical origin. All three classes of genetic markers
grouped the populations into two highly divergent clusters: Europe/Central
Asia and East/South-East Asia. Thus, our genetic data support the two
subspecies C. c. carpio (Europe) and C. c. haematopterus (East Asia) formerly
distinguished on the basis of morphological and morphometric differences.
The close relationship of European and Central Asian carp is also reflected
in the fact that all but one of the European populations we examined were
fixed for the same mtDNA composite haplotype that also dominated in
Central Asian but was completely missing in East/South-East Asian
populations. Moreover, this result also indicates a single origin of European
carp in Central Asia. The exception among the European populations was
the wild caught carp from river Danube near Straubing, Germany, for which
mtDNA and allozyme data suggest not only mixture but also hybridisation
with Asian carp. Our results emphasize the great importance of wild
populations and their genetic purity for the conservation of common carp
genetic resources. Out of the two wild/feral populations known in Germany,
the one from river Rhine near Riedstadt-Erfelden is more valuable than the
one from river Danube since the former does not show any sign of
mixing/hybridisation with Asian carp so far.
1 Department of Fish Farming, Institute of Animal Sciences, Estonian Agricultural University,
Tartu
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Zusammenfassung
Der Karpfen gehört zu den weltweit bedeutendsten Arten der
Süßwasserfischzucht. Er weist eine lange Geschichte der Domestikation auf,
und zahlreiche Linien und Zuchtformen wurden von seiner Wildform
abgeleitet. Die bisher umfassendste Studie zur Populationsstruktur und den
verwandtschaftlichen Beziehungen zwischen diesen Zuchtformen, aber auch
zwischen ihnen und Wildkarpfen, wurde in unserer Arbeitsgruppe erstellt: sie
schließt zur Zeit 22 europäische, sechs mittelasiatische und vier ost-/südost-
asiatische Wild- und Zuchtpopulationen ein, die mittels dreier genetischer
Markersysteme (Allozyme, Mikrosatelliten, PCR-RFLP mitochondrialer
DNA) charakterisiert wurden. Dabei zeigte sich, dass die Variabilität der
Allozym-Loci wesentlich geringer war als die der Mikrosatelliten-Loci.
Wildlebende Populationen unterschieden sich von den Zuchtbeständen –
unabhängig von der geografischen Herkunft – durch eine höhere Variabilität.
Alle drei Klassen genetischer Marker gruppierten die Populationen in zwei
hochdivergente Cluster: Europa/Mittelasien und Ost-/Südostasien. Damit
unterstützen unsere genetischen Daten die bereits anhand morphologischmorphometrischer
Unterschiede beschriebenen beiden Unterarten C. c. carpio
(Europa) bzw. C. c. haematopterus (Ostasien). Die enge Verwandtschaft
europäischer und mittelasiatischer Karpfen äußert sich auch darin, dass mit
einer Ausnahme alle untersuchten europäischen Populationen für den
gleichen mtDNA-Komposithaplotyp fixiert waren, der auch in Mittelasien
dominierte, in Ost- und Südostasien aber völlig fehlte. Dies deutet darüber
hinaus auf einen singulären Ursprung der europäischen Karpfen in
Mittelasien hin. Die Ausnahme unter den europäischen Populationen ist die
wildlebende aus der Donau bei Straubing, bei der die mtDNA- und Allozym-
Daten nicht nur auf eine Vermischung, sondern auch auf eine bereits
erfolgte Hybridisierung mit asiatischen Karpfen hinweisen. Unsere
Ergebnisse unterstreichen, dass für den Erhalt genetischer Ressourcen des
Karpfens den Wildpopulationen und ihrer Reinerhaltung eine entscheidende
Bedeutung zukommt. Von den beiden aus Deutschland bekannten
wildlebenden Populationen besitzen dabei die Karpfen aus dem Rhein bei
Riedstadt-Erfelden einen höheren Wert als die aus der Donau, da bei ihnen
noch keine Anzeichen für eine Vermischung/Hybridisierung mit asiatischen
Karpfen festgestellt werden konnten.
3.4.2.1 Introduction
The common carp is among the most important species in freshwater fish
culture. Its annual production amounts to more than 3.2 million t worldwide
(FAO Fishery Statistics 2002). It is one of the few fish species that can be
considered as a domestic animal (Steffens 1980). European pond carp
originate from wild carp from the river Danube. Rearing of wild carp caught
in rivers was already described by Aristoteles. Modern pond farming,
however, started to develop only in medieval times since 16 th century. Asia
(China) was a second center of domestication. There, carp rearing in ponds
started earlier (2,500 to 3,000 years ago) but true domestication began later
and proceeded slower. As a result Chinese pond carp remained more similar
to their wild ancestors than European pond carp (Kirpitchnikov 1999).

The wild common carp is characterized by an elongated torpedo-shaped
body completely covered by scales. In Europe as well as in Asia, numerous
breeds, local races, and lines have been derived from it, mainly for human
nutrition. An exception from utilization as food fish, is the Japanese Koi
carp that is reared as an ornamental fish in garden ponds and tanks.
Kirpitchnikov (1967) distinguished four subspecies of carp based on
morphological and morphometric differences (Figure 1).
Fig. 1: Distribution of common carp subspecies as proposed by Kirpitchnikov (1967):
(a) European-Transcaucasian carp, C. carpio carpio; (b) Central Asian carp, C. c. aralensis;
(c) East Asian carp, C. c. haematopterus; (d) South-East Asian carp, C. c. viridiviolaceus.
Later he revised his view and recognized only two valid subspecies: C. c.
carpio in Europe, Caucasus and Central Asia and C. c. haematopterus in East
Asia (Kirpitchnikov 1999), similar to the opinion of Balon (1995). In
contrast, Baruš et al. (2002) hypothesized that a third subspecies, C. c.
viridiviolaceus, still might exist in South-East Asia.
In order to clarify the intraspecific systematics of common carp molecular
biological studies are needed. Biochemical-genetic markers (allozymes and
proteins) have been examined since the end of the 1970s, followed by
molecular-genetic markers (nuclear and mitochondrial DNA) since the end
of 1990s. However, most of these studies were restricted to regional levels.
The few studies comparing carp populations from different geographical
regions could identify at least two highly divergent groups – Europe and
East Asia (Brody et al. 1979; Paaver 1983; Kohlmann and Kersten 1999;
Gross et al. 2002). Our present study is the first one worldwide to include
wild and domesticated populations from the whole natural distribution range
of common carp that were simultaneously analysed for three classes of
genetic markers (allozymes, microsatellites and mitochondrial DNA).
3.4.2.2 Material and Methods
A total of 32 carp populations of differing genetic status had been collected
(Table 1). Domesticated pond carp dominated among the 22 European
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populations. With only two exceptions (EU5d and EU19d) all of these pond
carp were of the mirror carp type. Most European carp were collected in
Germany. Some of these populations were designated as wild/feral (wf)
since their exact genetic status is not known. Six true wild populations
originated from Central Asia (Uzbekistan). East and South-East Asia were
represented by two wild and two domesticated populations.
Table 1: List of common carp populations and number of individuals analysed for each
genetic marker. Genetic status: D = domesticated; W = wild; W/F = wild/feral. *captive stock.
Population Geographic region/ Country of Genetic Number of individuals analysed for
code Population name origin status Allozymes Microsatellites mtDNA
Europe
EU1wf r. Danube (Maier) Germany W/F* 50 30 8
EU2wf r. Danube (Straubing) Germany W/F 30 28 27
EU3wf r. Tisza Hungary W/F 9 9
EU4wf r. Rhine Germany W/F 30 30 29
EU5d Scaly carp Germany D 50 30 29
EU6d Fiedler Germany D 50 30 23
EU7d Kauppa Germany D 50 29
EU8d Seckendorff Germany D 50
EU9d Wiesinger Germany D 30
EU10d Scheuermann Germany D 25
EU11d Hertlein Germany D 50
EU12d Glinzig Germany D 25 25
EU13d Kreba Germany D 25
EU14d Petkampsberg Germany D 50
EU15d Petershain Germany D 28 30
EU16d Dor-70 Israel D 20
EU17d Ropscha Russia D 40
EU18d Badajoz Spain D 20 18
EU19d Tata Hungary D 18 19
EU20d Pohorelice Czech Republic D 30 18
EU21d Zator Poland D 30 17
EU22wf Lake Velence Hungary W/F 11
Central Asia
CA1w Lake Tuzkan Uzbekistan W 50 30 29
CA2w r. Syr-Darya Uzbekistan W 19 28 28
CA3w Syr-Darya channels Uzbekistan W 25 28
CA4w Lake Arnasaiskie Uzbekistan W 25 28 27
CA5w Lake Aidar Uzbekistan W 50 29
CA6w r. Kli Uzbekistan W 30 28
East and South-East Asia
EA1w r. Amur Russia W* 50 30 22
EA2w r. Red Vietnam W 50 30 26
EA3d Wuhan China D 20 20
EA4d Koi Japan D 50 30 30
The methods of allozyme (16 loci), microsatellite (four loci) and PCR-RFLP
(mitochondrial ND-3/4 and ND-5/6 genes) analyses, including statistics,
have been described in detail by Kohlmann and Kersten (1999), Gross et al.
(2002), Murakaeva et al. (2003) and Kohlmann et al. (2003). Due to logistical
reasons not all of the populations could be analysed by all three classes of
genetic markers. Nevertheless, for most individuals complete data sets were
available.

3.4.2.3 Results
Genetic variability within populations
As could be expected, microsatellites displayed a much higher variability than
allozymes for all parameters measured. The average number of alleles per
locus was only 1.06 to 1.81 at allozymes compared to 2.50 to 14.25 at
microsatellites. The percentage of polymorphic loci ranged from 6.2% to
43.8% at allozymes whereas all of the four microsatellites analysed were
found to be polymorphic. The observed heterozygosity was also much
higher at microsatellite loci (0.492 to 0.909) than at allozyme loci (0.006 to
0.181).
The genetic variability of carp populations did not show a clear geographical
pattern. At allozymes the East/South-East Asian populations displayed a
higher variability than the European and Central Asian populations that
showed a similar level of variability. In contrast, at microsatellites the Central
Asian populations were the most variable ones, followed by the European
and East/South-East Asian populations that showed a similar variability level
for these genetic markers. However, these comparisons might be biased, due
to unequal proportions of domesticated to wild populations in the three
geographical regions. A clear variability pattern became evident if
populations were grouped according to their genetic status independent of
geographical origin: domesticated stocks generally suffered from a reduced
variability in comparison to wild caught populations. At allozyme loci, this
difference was statistically significant in Europe but only tendentious in the
other two regions. At microsatellites, the differences were more pronounced:
the average number of alleles per locus was only approximately half as high
in domesticated stocks (4.44) as in wild populations (8.22). However,
observed heterozygosity was similar in both groups (0.734 in domesticated
stocks vs. 0.799 in wild populations).
The distribution of the 10 composite haplotypes resulting from the
restriction digestion of the mitochondrial ND-3/4 and ND-5/6 genes with
10 restriction enzymes each revealed an interesting pattern (Table 2). All but
one of the European populations were fixed for the same composite
haplotype H1 that also dominated in Central Asia, but was completely
missing in East/South-East Asia. The only exceptions were two out of 27
carp individuals caught in the river Danube near Straubing, Germany
(EU2wf). These fish expressed a haplotype differing by only two nucleotide
substitutions from haplotype H3 for which wild carp from the river Amur,
Russia (EA1w) were fixed. Ornamental Koi carp was another population
fixed for a specific haplotype (H7). The most variable populations were the
Central Asian wild carp (CA1w to CA6w), followed by Chinese domesticated
carp (EA3d) and Vietnamese wild carp (EA2w). The fixation of river Amur
wild carp and their reduced variability at allozyme and microsatellite loci in
comparison to other wild carp populations (Kohlmann et al. 2003) can be
considered as an effect of captive breeding: a probably low number of
founder individuals (bottleneck effect) and/or low effective population sizes
during culture in captivity. In contrast, the high variability of Chinese
domesticated carp, also at microsatellite loci (Kohlmann et al. in press),
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reflects the extensive management of this population: a high number of
spawners can reproduce naturally in large ponds; only fry for transfer into
small intensively managed ponds or marketable carp are caught.
Table 2: Distribution of mtDNA composite haplotypes derived after digestion of ND-3/4 and
ND-5/6 coding regions by 10 restriction enzymes. *based on two nucleotide substitutions
designated as H3a subsequently.
Population
Composite haplotype
code H1 H1b H3 H5 H6 H7 H9 H11 H12 H13
EU1wf 8
EU2wf 25 2*
EU3wf 9
EU4wf 29
EU5d 29
EU6d 23
EU15d 30
EU18d 18
EU19d 19
EU20d 18
EU21d 17
CA1w 16 6 6 1
CA2w 15 7 6
CA3w 28
CA4w 12 4 9 2
CA5w 15 6 8
CA6w 11 8 9
EA1w 22
EA2w 15 11
EA3d 7 12 1
EA4d 30
Genetic diversity among populations
In order to estimate genetic differentiation between populations and
geographical regions, the fixation index FST has been used. Based on
allozyme loci significant differences between populations were mainly found
if they originated from different geographical regions. Non-significant
differentiation was observed for several comparisons within regions, in
particular between German pond carp or between Uzbek wild carp
(Kohlmann et al. 2003). Average population differentiation was highest in
East/South-East Asia (FST = 0.29), followed by Europe (FST = 0.10) and
Central Asia (FST = 0.008). Based on microsatellite loci the average
population differentiation increased for East/South-East Asia (FST = 0.343)
and Europe (FST = 0.138) whereas differentiation of Central Asian
populations decreased (FST = 0.002) (Kohlmann et al. in press). However,
the ranking of the three regions did not change.
Allozyme as well as microsatellite polymorphisms clustered the carp
populations into two highly divergent groups (Figures 2 and 3):
Europe/Central Asia and East/South-East Asia with the bootstrap value
being higher for microsatellite data (91%) than for allozyme data (73%). The
Central Asian wild carp formed a distinct subgroup within the
European/Central Asian cluster in both cases.

Fig. 2: UPGMA clustering of common carp populations based on allozyme polymorphisms at
16 loci and Nei´s (1972) standard genetic distances. Bootstrap values were calculated by
PHYLIP version 3.573c software (Felsenstein 1995).
Fig. 3: UPGMA clustering of common carp populations based on microsatellite variability at
four loci and DA distances (Nei et al. 1983) among pairs of populations. Bootstrap values
were calculated by DISPAN software (Ota 1993).
The mtDNA polymorphisms revealed a similar pattern: the composite
haplotypes (Figure 4) as well as the populations themselves (Figure 5)
formed the same two major groups (Europe/Central Asia and East/South-
East Asia). Again, a separate subgroup consisted of the Central Asian wild
carp.
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Fig. 4: Unrooted network of common carp mtDNA composite haplotypes at the ND-3/4 and
ND-5/6 genes. Bootstrap values were obtained from the maximum likelihood tree.
Fig. 5: UPGMA clustering of common carp populations based on the estimated number of
net nucleotide substitutions between all pairs of populations (dA) at the mitochondrial ND-3/4
and ND-5/6 genes.
3.4.2.4 Discussion
A reduced genetic variability of captive stocks in comparison to wild caught
populations as observed in the present study of common carp was also
found in other fish species such as Atlantic salmon (Staahl 1983; Verspoor
1988), masu salmon (Nakajima et al. 1986), rainbow trout (Paaver 1986), and

own trout (Vuorinen 1984). The loss of genetic variability can be explained
by genetic drift due to small population sizes and/or by inbreeding due to
high selection intensities (e.g. in ornamental Koi carp). Bottleneck effects at
the beginning or during the culture of populations as well as genetic
adaptations to captive and local environmental conditions could be
additional contributing factors.
All three classes of genetic markers grouped populations into two highly
divergent clusters: Europe/Central Asia and East/South-East Asia. Thus,
our present genetic data support the two subspecies C. c. carpio (Europe) and
C. c. haematopterus (East Asia) formerly distinguished on the basis of
morphological and morphometric differences. Preliminary results of mtDNA
sequencing indicate a divergence time between both groups of approximately
500,000 years. In contrast, the subspecies status of C. c. aralensis for Central
Asian carp does not seem to be justified. However, the high degree of
genetic differentiation in East/South-East Asia suggests that further
evolutionary significant units might exist in this region. In this respect, the
morphological and colour varieties of common carp from Northern Vietnam
described by Tran Dinh-Trong (1967) are of special interest. Detailed genetic
studies are needed in order to confirm or reject the existence of the
supposed third subspecies C. c. viridiviolaceus.
The close relationship of European and Central Asian carp is also reflected
by the fact that all but one of the European populations examined were fixed
for the same mtDNA composite haplotype that also dominated in Central
Asia but was completely missing in East/South-East Asia. Moreover, this
also indicates a single origin of European carp in Central Asia (postglacial
immigration of wild carp from Central Asia to Europe into the Danube basin
and further distribution and domestication by humans). The exception
among the European populations was the wild/feral one from the river
Danube near Straubing, where two out of 27 individuals possessed a
composite haplotype very similar to river Amur wild carp from Asia.
Moreover, allozyme data of this population suggest not only mixing but also
hybridisation with Asian carp (Kohlmann et al. 2003).
Our results emphasize that wild populations and the preservation of their
genetic purity are of utmost importance for the conservation of common
carp genetic resources. However, wild carp are already extinct or endangered
in many areas of their natural distribution range, partly due to losses of
habitats, but mainly because of displacement by domesticated pond carp
which are preferred for farming due to their faster growth. Out of the two
wild/feral populations known in Germany, the one from the river Rhine
near Riedstadt-Erfelden is more valuable than the one from the river
Danube since the former does not so far show any sign of
mixing/hybridisation with Asian carp.
Acknowledgement
We thank P. Kersten for technical assistance in the field and laboratory. We
are also grateful to numerous carp farmers from Germany and abroad who
provided the tissue samples. Special thanks go to F. Geldhauser (Germany),
M. Flajšhans and O. Linhart (Czech Republic), M. Luczynski (Poland) and L.
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Varadi (Hungary). The study was partly financed by scholarships to R. Gross
(Forschungsverbund Berlin e.V.) and A. Murakaeva (DAAD – German
Academic Exchange Service) and by grants from Landesfischereiverband
Bayern e.V. to K. Kohlmann and from the Estonian Science Foundation
(no. 4826) to R. Gross.
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