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A molecular approach to detect

hybridisation between crucian carp
(Carassius carassius) and non-
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Gary Carvalho

Freshwater Biology

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Freshwater Biology (2005) 50, 403–417 doi:10.1111/j.1365-2427.2004.01330.x

A molecular approach to detect hybridisation between

crucian carp (Carassius carassius) and non-indigenous
carp species (Carassius spp. and Cyprinus carpio)
B . H Ä N F L I N G , * P . B O L T O N , † M . H A R L E Y * A N D G . R . C A R V A L H O *
*Molecular Ecology and Fisheries Genetics Laboratory, Department of Biological Sciences, University of Hull, Hull, U.K.
†
Environment Agency Kingsmeadow House, Reading, Berkshire, U.K.

SUMMARY
1. Releases of non-native fish into the wild is an increasing problem posing considerable
ecological and genetic threats through direct competition and hybridisation.
2. We employed six microsatellite markers to identify first generation hybrids and
backcrosses between native crucian carp (Carassius carassius) and introduced goldfish
(C. auratus) and common carp (Cyprinus carpio) in the U.K. We also investigated the genetic
characteristics of the taxonomically controversial gibel carp (Carassius spp.) from sites
across Europe.
3. Natural hybridisation between goldfish and crucian carp occurs frequently, although
hybrids between all other species pairs were observed. Only 62% of British crucian carp
populations (n ¼ 21) consisted exclusively of pure crucian carp. In some populations
hybrids were so frequent, that no pure crucian carp were caught, indicating a high
competitive ability of hybrids.
4. Most hybrids belonged to the F1 generation but backcrossing was evident at a low
frequency in goldfish · crucian carp hybrids and goldfish · common carp hybrids.
Furthermore, some local populations had high frequencies of backcrosses, raising the
opportunity for introgression.
5. Gibel carp from Germany and Italy belonged to two triploid clonal lineages that were
genetically closely related to goldfish, whereas all individuals identified from British
populations proved to be crucian carp · goldfish hybrids.
6. Our study suggests that the release of closely related exotic cyprinids not only poses a
threat to the genetic integrity and associated local adaptations of native species, but may
also contribute to shifts in community structure through competitive interactions.

Keywords: alien invasion, Carassius auratus, cyprinidae, introgression, microsatellites

the popularity of ornamental ponds and aquaria and

Introduction
Problems arising from the accidental or deliberate
releases of non-native fish species into the wild have
increased markedly in recent decades due mainly to
Correspondence: B. Hänfling, Molecular Ecology and Fisheries
Genetics Laboratory, Department of Biological Sciences,
University of Hull, Hull, HU6 7RX, U.K.
E-mail: b.haenfling@hull.ac.uk

2005 Blackwell Publishing Ltd
demand from recreational fisheries (Cambray, 2003;
Simon & Townsend, 2003; Hickley & Chare, 2004;
Semmens et al., 2004). Releases of exotic species can
pose considerable ecological threats not only through
direct competition, but also by hybridisation with
native species (Epifanio & Nielsen, 2000; Allendorf
et al., 2001). Hybridisation is of particular concern if
hybrids are more vigorous than native species, or if
backcrossing leads to introgression into the gene pool
403
404 B. Hänfling et al.
of native species (Arnold & Hodges, 1995; Rhymer &
Simberloff, 1996). The crucian carp (Carassius carassius
L.) is considered a native species of the British Isles,
Central and Eastern Europe (Wheeler, 1977, 2000). In
contrast, a closely related cyprinid, originally native to
East Asia, the goldfish (Carassius auratus L.), has been
introduced into most of Europe by human activities.
The brown variety of the goldfish bears a morpholo-
gical resemblance to the crucian carp, and this has
previously hampered conservation efforts for crucian
carp (Wheeler, 1998). The common carp (Cyprinus
carpio L.) is native to Eastern Europe and parts of
Central Europe but was introduced to British waters
probably during the 15th century (Hoole et al., 2001).
It is known that artificially produced first and second
generation hybrids between all three species are
viable but the degree to which hybridisation occurs
in natural populations is unclear (Aduma-Bossman,
1971). Efforts to clarify the situation are impeded by
difficulties in identification of pure-bred Carassius
carassius and hybrids by external morphological
investigation and by some unresolved taxonomic
problems in the genus Carassius (Wheeler, 2000). Of
particular note is the species in Europe commonly
known as the ‘gibel’ or ‘Prussian carp’; despite
numerous reports, we still do not know whether this
is a species in its own right (C. gibelio; Kottelat, 1997), a
subspecies of the goldfish (C. auratus gibelio; Bloch,
1783), a morphotype of the goldfish (C. auratus;
Hensel, 1970; Lusková et al., 2004) or whether it is
indeed a hybrid between the goldfish and another
related species. The origin of this taxon is thought to
be eastern Russia, possibly the Amur basin, but it has
colonised Europe successively during the 20th cen-
tury. The Western margins of this invasion are not
clear. Although Banarescu & Bogutskaya (2003)
assume an absence of gibel carp in the U.K.,
populations resembling gibel carp have been reported
by U.K. Environment Agency officers and anglers
(G. Howes and C. Williams, pers. comm.) and the
British Museum of Natural History contains gibel carp
specimens collected in the U.K. In addition to
taxonomic ambiguities, the gibel carp varies in ploidy
level and mode of reproduction (Zhou, Wang & Gui,
2000a). Until the 1990s, invasive populations in
Europe seem to have consisted exclusively of triploid
females which reproduced gynogenetically, whereas
more recent studies have found a high proportion of
both diploid males and females in Czech waters,
indicating sexual reproduction (Lusková et al., 2004).
Such findings raise the additional concern that these
fish might hybridise with native crucian carp. Overall,
there is now a strong, but as yet unsubstantiated,
belief that the crucian carp is under increasing threat
from hybridisation, competition and disease caused
by non-native species. This is supported by many
reports of dramatic declines or local extirpations in
previously healthy crucian carp populations, a prob-
lem that is thought to be Europe-wide (Wheeler, 1998;
K. Wesley, R. Klupp, D. de Charleroy, pers. comm.).
Increased focus on the extent and nature of such
hybridisation in the wild is not only vital to assess
associated environmental risks, but also can provide
insightful opportunities to explore the ecological and
evolutionary consequences of hybridisation and col-
onisation.
Genetic methods provide powerful tools with
which to identify cryptic species or species with
few readily identifiable phenotypic characteristics.
Furthermore, nuclear genetic markers in particular
allow species hybrid identification because they can
identify the contributions of both the father and
mother to the hybrid genome. Providing sufficient
loci are used, it is also possible to differentiate
subsequent generations of hybrids as well as back-
crosses (Boecklen & Howard, 1997). Microsatellites
are particularly suitable for hybridisation studies
because of their genome-wide distribution and asso-
ciated potential for detecting genome-wide processes
such as introgression. Moreover, assuming a step-
wise mutation model of microsatellite evolution,
inferences can be made concerning the genealogical
origin of alleles, so increasing the power of the
approach by identifying diagnostic allele size classes
(instead of diagnostic alleles).
Here, we (i) identify microsatellite markers to
identify crucian carp (CA), goldfish (AU), common
carp (CY), and their hybrids respectively, (ii) identify
British populations of true crucian carp and (iii)
describe the genetic characteristics of gibel carp. Such
data will facilitate the protection of native crucian
carp by providing robust guidance for managing
movements of Carassius spp. and their hybrids, as well
as assessing the impact of such releases into the wild.
Furthermore individuals genetically identified as pure
species can be used to develop a morphology-based
key to differentiate between, crucian carp, goldfish
and gibel carp.

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417

Methods
Sampling
Samples were taken from each of the three species
involved (C. carassius, C. auratus and C. carpio) and
from individuals identified as hybrids or gibel carp on
morphological grounds (Table 1). The initial morpho-
logical identification was tentative and carried out
using simple external observation by U.K. Environ-
ment Agency officers as applied in the field. The
sampling scheme was carried out between January
and July 2003 and had three components: (i) individ-
uals identified as pure species to document species-
specific genetic characteristics as baseline data; (ii)
individuals of British Carassius populations, to con-
firm their status as pure species or hybrids; (iii)
individuals from British, German and Italian popula-
tions identified tentatively as gibel carp. Furthermore,
we included pure species and hybrids from Germany
and New Zealand as outgroups to test the applicabil-
ity of the genetic markers in a larger geographical
context. The outgroup samples were collected be-
tween July 1997 and September 2002. Such a design
does of course depend upon the initial unequivocal
identification of individuals as ‘pure species’. How-
ever, the use of analysis methods that are independent
of the assumption of such status do allow for the
subsequent assessment of the efficacy of an a priori
morphological identification. Where possible, general
background information was gathered for each site
including stocking history and presence or absence of
all three studied species. Accordingly, sample sites
were classified into three categories (Table 1): retailer
(commercial trader or breeder), semi-wild (regular
management through stocking and fish removal) and
wild (no or little stocking and fishing pressure). Tissue
samples (fin-clippings) were stored in 98% ethanol for
DNA analysis. Total genomic DNA was extracted
using a modified salting-out protocol based on
Bruford et al. (1992). DNA-pellets were re-suspended
in TE buffer and a dilution containing 10–50 ng lL)1
DNA was prepared from this stock solution.
Microsatellite analysis
Microsatellites are DNA sequences comprising a sim-
ple repeat motif of one to six bases (e.g. AT or CGT) that
is repeated in tandem multiple times. Alleles at a given
locus differ in the number of repeat units and thus in

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417
Hybridisation between native and introduced carp 405
overall size. Assuming that stepwise mutation is the
predominant force in microsatellite evolution (Gold-
stein & Schlötterer, 1999) one would expect that lineage
sorting processes will lead to some differences in allele
size ranges among species and ultimately to non-
overlapping allele ranges at some loci. Such diagnostic
allele size ranges facilitate species and hybrid identifi-
cation because newly found alleles can be assigned to a
certain species with a high probability. The aim of the
study was therefore to identify loci which showed these
diagnostic properties for the target species.
One confounding factor in the application of micro-
satellite markers across species is that primers devel-
oped to amplify microsatellite loci are often species-
specific or restricted to closely related species. For the
present study it was necessary to identify primer pairs
that could be used across all taxa under investigation.
The first step was therefore to test a set of published
microsatellite primers (goldfish: Zheng et al., 1995;
common carp: Crooijmans et al., 1997) for cross-ampli-
fication in all three species. PCR reactions were carried
out in 10 lL volumes using a standard protocol: 10–
50 ng DNA, 10x NH4 reaction buffer (Bioline, London,
U.K.), 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 units of Taq
polymerase (Bioline), 200 nM of each primer. Polym-
erase chain reaction (PCR) was conducted on a Hybaid
Omnigene thermal cycler using the following thermal
profile: Initial denaturation for 2 min at 94 C followed
by 40 cycles of 15 s at 92 C, 20 s at annealing
temperature (TA) and 30 s at 72 C, with a final
extension step of 10 min at 72 C. All primers were
first tested for amplification on a set of 12 individuals
(four of each species AU-NA/1–4; CY-FFF1–4; CA-SC/
1–4; Table 1) under generic conditions (TA ¼ 48).
Following separation of reaction products on agarose
gels, primers yielding consistent amplification of
products in the expected size range (100–400 bp) for
at least two species were selected for further optimi-
sation. Amplification of a larger number of individuals
from the pure species (AU-NA/1–4, AU-F/1–5, CA-
FFF/1–4 CA-CFF/1–8, CY-HI/1–7, CY-SC/1–5, CY-
RCF/1–9; Table 1) were carried out and repeated
under a range of annealing temperatures in order to
eliminate unspecific products and to guarantee repro-
ducible results. Fragments were separated on 6%
polyacrylamide gels on an ALFexpressTM automatic
sequencer (Amersham-Pharmacia Biotech, Uppsala,
Sweden). After suitable loci were identified, all indi-
viduals were genotyped at the target loci using three to
406 B. Hänfling et al.
Table 1 Sampling locations

Location Country Code Status AU CA CY GI CA · AU AU · CY CA · CY

Pure species
Yorkshire U.K. NA R 4 (4) – – – – – –
Yorkshire U.K. F R 5 (5) – – – – – –
Reading U.K. FFF R – 4 (4) – – – – –
Nottinghamshire U.K. CFF R – 10 (10) – – – – –
Kent U.K. RCF R – – 9 (9) – –
Leicestershire U.K. HI SW – – 7 (7) – – – –
Hampshire U.K. SC SW – – 5 (5) – – – –
British Carassius populations
Greenwich U.K. CCS W – 30 (30) – –
High Wycombe U.K. TL W – 20 (20) – –
Epping U.K. BW W X 31 (30) X (1)
Bedfordshire U.K. CAC W – 9 (9) X
Corby U.K. CBL W – 30 (30) X
Essex U.K. HC W – 30 (30) X
Redditch U.K. CFP SW 4 (4)
Wiltshire U.K. CP W 6 (6)
Devon U.K. EA SW 2 (2)
Dorset U.K. SFS W 3 (3)
Squareshill U.K. SP W 7 (7)
St Helens U.K. MN W – 16 (16) – – – – –
Cambridgeshire U.K. WAC W – 20 (20) X
Pelham U.K. HR W 6 (6)
Doncaster U.K. LLF SW X 1 (5) 4
Altricham U.K. A W X 1 (1) 2 (2)
Newquay U.K. PGB W X X 2 (2)
Midlands U.K. BMC W 1 X (9) 8
Buntingford U.K. GPB SW 17 (17) X X 3 (3) 11 (11)
Wiltshire U.K. MFF SW 4 X 9 (13)
Cheshire U.K. MHF W 10 3 (13)
Kent U.K. CM W X 2 (2) X 19 (19) 2 (2)
Hertfordshire U.K. MG W – 3 (3) 4 (10) 8 (2)
Kent U.K. RCF R X – X (10) 10
European gibel carp
Elbe, Germany G 201 W X 4 (4)
Lake San Pietro I SPI W 9 (9)
Lake Laceno I LI W 9 (9)
Pond Krappmann G KG SW X X 9 (8) (1)
Bieberbach G BG W X 1 (1)
Pond Popp G PG SW X 9 (9)
Outgroup samples
Kruegersee G 2014 N 4 (4) X
Danube G 301 W X 1 (1) X
Elbe G 201 W X 1 (1) X
South Island NZ SNZ W X X 1 (1)
North Island NZ NNZ W 1 (1) 1 (1) 3 (3)
R 32 248 28 63 14 15 35

Country: G, Germany; I, Italy; NZ, New Zealand; U.K., United Kingdom. Code – used in further analyses. Status of populations:
R, fish retailer; SW, semi-wild; W, wild (see text for definitions). Values are number of individual sampled for each taxon
according to initial morphological classification (post hoc classification based on the genetic analysis is indicated in brackets).
AU, goldfish; CA, crucian carp; CY, common carp; GI, gibel carp.
Additionally presence (X) or absence (–) of unsampled species is indicated; if no information was available field is left blank.

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417


four internal size standards. Additionally, the same
three individuals (one from each species) were run on
all gels to facilitate cross-referencing among gels.
The first step of the analysis was to determine the
locus-specific range in allele sizes for each species
using the individuals classified as pure species in
baseline data. Subsequently, the alleles of every indi-
vidual genotype at each locus were assigned according
to their origin (Goldfish: AU; crucian carp: CA,
common carp: CY). Alleles which were shared
between species were classified as typical for one
species or another according to their frequencies. Each
individual was assigned to a certain genotypic cate-
gory based on the relative distribution of alleles of each
species among the multilocus genotypes (assignment
by inspection). Considering the number of loci under
investigation (Boecklen & Howard, 1997) the assign-
ment was limited to four genotypic categories con-
sidering the classification of Nason & Ellstrand (1993).
(i) Genotypes containing 100% species-specific alleles
of one species (AU, CA, CY), typical for pure species.
(ii) Genotypes containing alleles of each parental
species at each locus (e.g. HAU)CA), typical for F1
hybrids. (iii) Genotypes containing both loci with
alleles of one species and loci with alleles of both
parental species (e.g. IAU/AU)CA), typical for backcros-
ses. (iv) Genotypes containing at least one locus
homozygote for one species and at least one locus
homozygous for another species (e.g. SAU/CA), typical
for the F2 generation. Furthermore, a hybrid index
based on allele frequencies from the pure species as
classified with the ‘assignment by the inspection
method’ was calculated according to (Campton &
Utter, 1985). The power to infer the true genealogical
classes of pure species (P), F1 or F2 hybrids (F1, F2) or
backcrosses (BP) from genotypic categories depends
on the number of loci under investigation. The
‘assignment by inspection method’ is only 100%
reliable if an unlimited number of loci is used
(Epifanio & Philipp, 1997). We therefore applied the
method of Anderson & Thompson (2002) to calculate
posterior probabilities that each individual belongs to
either of the hybrid categories and mean posterior
probability for a certain hybrid category in the
population. Such an approach assumes that only two
species hybridise. Thus, for analyses the dataset was
recombined into three sets consisting of two species
each and their respective hybrids. After initial tests,
the value for the Markov chain sampling was set on

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417
Hybridisation between native and introduced carp 407
50 000 for BurnIn and 100 000 for successive sweeps
(Anderson & Thompson, 2002). The analysis was
repeated 10 times for each species pair and probabil-
ities averaged among runs. The usefulness of multi-
variate methods based on multilocus genotypes for
visual identification of hybrids was demonstrated by
(Young et al., 2001). Here we conducted a Factorial
Correspondence Analysis (FCA) using the programme
GENETIX version 4.01 (Belkhir et al., 2000) in order to
visualise the genetic separation between pure species
and hybrids. This analysis shows the genetic similarity
of individuals and is based on the presence or absence
of alleles in each multilocus genotype.
Results
Identification of species-specific markers
Twenty-five unlabelled primer pairs developed to
amplify di-nucleotide repeat microsatellites in goldfish
[GF1, GF11, GF17, GF22 and GF29; (Zheng et al., 1995)]
and carp [MFW1–20 (Crooijmans et al., 1997)] were
tested as diagnostic markers for the three species and
their hybrids. Ten loci showed amplification products
of an appropriate size range (100–300 bp) in more than
one species and were therefore potentially useful in
hybrid identification. Optimal conditions across all
species could be established for six loci which were
deemed appropriate as diagnostic markers (Table 2)
Four loci displayed alleles with a clearly distinct size
range for each species and (GF1, GF17, GF29, MFW2).
One of these loci did however not produce amplifica-
tion products for common carp. Two loci (MFW7 and
MFW17) showed overlapping range sizes among the
three species but most of the alleles were diagnostic for
one of the three species (Table 2; Appendix). However,
three out of 33 alleles were shared between crucian carp
and common carp at locus MFW7. At locus MFW17 five
out of 25 alleles were shared between goldfish and
common carp, and two alleles were shared between
crucian carp and common carp. The identification of
species specific alleles was further facilitated by the fact
that a shift in allele sizes between odd and even sizes
occurred among species in three of the loci (GF29,
MFW2, and MFW7). In most cases outgroup individ-
uals from Germany and New Zealand fell within the
allele size range of individuals determined from U.K.
populations, indicating the usefulness of the diagnostic
markers identified here over a larger geographic area.
408 B. Hänfling et al.
Table 2 Size range for goldfish (AU), crucian carp (CA), common carp (CY) and clonal lineages of gibel carp (GI)
Allele size ranges
Locus AU CA CY
GF1 300–312 298 296
GF17 184–212 182 – 186–220
GF29 189–207 210–228 243–283
MFW2 157 160 169–271
MFW7 157–191 178–196 188–276
MFW17 216–256 242–250 234–280
Diagnostic properties between species pairs (R, diagnostic allele size range; S, one-base pair shift between allele sizes; A, diagnostic
alleles, in brackets is the percentage of alleles which are completely diagnostic, the remaining alleles show marked frequency
differences) and amplification conditions at the six investigated microsatellite loci.
Only one common carp from Germany had a shorter
allele (174) than the shortest allele found in a British
population (188) at locus MFW7.
Genotyping
Overall, 435 individuals were genotyped at six
microsatellite loci. Alleles were numbered according
to their length in basepairs (bp), and individual
genotypes recorded for each locus (available from
authors upon request). Most individuals showed one
or two alleles per locus suggesting a regular diploid
genome. In some individuals of populations BMC and
LLF (Table 1), and in all but one individual from the
European samples of gibel carp, three alleles were
found for one or more loci indicating polyploidy; a
result which was highly repeatable. Furthermore, 40
European gibel carp from six populations belonged to
just two multilocus genotypes. One of these geno-
types, which possibly represent two clonal lineages,
was found exclusively in Germany whereas the
second one was restricted to Italy; no clonal gibel
carp were found in the U.K. Based on the specific
allele ranges of each species and using the ‘assign-
ment by inspection method’ most individuals could
be attributed to the genotypic categories of pure
species or F1 hybrids, including hybrids between all
species pairs. Allele frequencies of pure species and
hybrids are displayed in the Appendix. Most of the
alleles found in the putative clonal lineages of Euro-
pean gibel carp were typical goldfish alleles or fell
into allele size range of goldfish. Twenty-two percent
of gibel carp alleles were specific to gibel carp
(Appendix). A small number of individuals of
AU · CA (six) and AU · CY (three) hybrids fell into
the genotypic categories of backcrosses being homo-
Diagnostic properties
GI AU · CA AU · CY CA · CY TA (C)
300–308 R R R 55
R – – 52
193–207 R, S R R, S 55
157 R, S R R, S 58
175–199 A, S A, S A (90) 58
222–232 A A (80) A (92) 50
zygote for one species for at least one locus and
heterozygote for two species for at least one locus
(Table 3). In concordance, hybrid indices for each of
the three species pairs were distributed in three
distinct groups, the two parental species occupying
positions at the far left and right of the distribution
and F1 hybrids occupying an intermediate position
(Fig. 1). Only a few individuals showed hybrid
indices which fell between the three main hybrid
index classes. These were mainly the individuals
identified as backcrosses by other methods. However,
the representatives of crucian carp from Germany also
showed a hybrid index which was intermediate to
pure crucian carp and F1 hybrids. These results were
confirmed by analyses which did not require any a
priori assignment of pure species. Firstly, the multi-
variate analysis (FCA) showed that multilocus geno-
types can clearly be assigned into six major groups
representing the three species and their F1 hybrids
(Fig. 2). Secondly, posterior probabilities of individual
assignment to either category showed that most
individuals belonged to either pure species or F1
hybrids with a probability of 0.99 < P < 1.00. The
analysis of the hybrid group AU/CA showed that two
individuals had a probability 0.99 < P < 1.00 to be a
second generation hybrid. Accordingly, fish BW-31
represents either a F2 hybrid (P ¼ 0.33) or a BCCA
(P ¼ 0.64;); fish BMC-1 represents either a F2 hybrid
(P ¼ 0.24) or a BCAU (P ¼ 0.76). Five individuals from
this group had a probability of 0.02 < P < 0.69 to be a
second generation hybrid. In Group AU/CY, three
individuals had a small probability (0.02 < P < 0.06)
to be second generation hybrids. No possible second
generation hybrid could be detected in group CA/CY.
The estimated overall frequencies for second genera-
tion backcrosses are <0.01 in every case (Table 4).

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417
 Hybridisation between native and introduced carp 409
Table 3 Initial morphological assignment of individuals (AU, goldfish; CA, crucian carp; CY, common carp; GI, gibel carp)

Initial
morphological Genotypic Genological
Location n assignment category Max A class

NA, F 9 AU AU 2 P
FFF, CFF, CCS, TL, CAC, CBL, 201 CA CA 2 P
HC, CFP, CP, EA, SFS, SP, MN,
WAC, HR, 2014
HI, SC, 301, 201 23 CY CY 2 P
BW 30 CA CA 2 P
1 CA ICA/AU)CA 2 BCCA (0.64), F2 (0.33)
LLF 4 CA-CY HAU)CY 3 BCAU*
1 AU-CY HAU)CY 2 F1
A 1 CA CA 2 P
2 AU-CA HAU)CA 2 F1
PGB 2 AU-CY HAU)CY 2 F1
BMC 1 AU IAU/AU)CA 2 AU (0.69), BCAU (0.29)
1 AU-CY IAU/AU)CA 2 BCAU (0.76), F2 (0.24)
4 AU-CY HAU)CA 2 F1
2 AU-CY HAU)CA 3 BCCA*
1 AU-CY ICA/AU)CA 2 F1 (0.93), BCCA (0.06), F2 (0.03)
GPB 17 AU AU 2 P
3 GI HAU)CA 2 F1
11 CA-CY HCA)CY 2 F1
MFF 1 AU IAU/AU)CA 2 F1 (0.48), BCAU (0.38), F2 (0.13)
3 AU HAU)CA 2 F1
9 AU-CA HAU)CA 2 F1
MHF 13 AU-CA HAU)CA 2 F1
CM 2 CA CA 2 P
19 GI HAU)CA 2 F1
2 AU-CY HAU)CY 2 F1
MG 10 CY CY 2 P
2 CA-CY HCA)CY 2 F1
3 CA CA 2 P
RCF 2 CA-CY IAU/AU)CY 2 F1 (0.95) BCAU (0.04) F2 (0.01)
8 CA-CY HAU)CY 2 F1
NZS 1 AU-CY ICY/AU)CY 2 CY (0.90), F1 (0.03), BCCY (0.07)
NZN 3 AU-CY HAU)CY 2 F1
1 AU AU 2 P
1 CY CY 2 P
201 4 GI AU 3 Clonal
KG 8 GI AU 3 Clonal
1 GI IAU/AU)CA 2 F1 (0.95), BCAU (0.04)
PG 9 GI AU 3 Clonal
BG 1 GI AU 3 Clonal
SPI 9 GI AU 3 Clonal
LI 9 GI AU 3 Clonal

Assignment of individuals into genotypic categories based on diagnostic alleles (AU, CA, CY ¼ pure species; HXY ¼ F1; IX/X)Y ¼
backcross with species X); Max A, maximum number of alleles found at an individual locus and most probable genealogical class
inferred from a Bayesian approach (Anderson & Thompson, 2002) and in some cases (*) from ploidy-level. (P, pure species; F1, first
generation hybrid; F2, second generation; F1 · F1, hybrid; BCX, backcross to species X).
Discordance between morphological and genetic assignment is indicated in bold.

exclusively of genetically pure crucian carp whereas

Population analysis
Overall, the samples sourced from 13 wild or semi-wild
populations and two fish farm populations consisted

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417
eight samples from natural populations contained
crucian carp hybrids. Seven of these contained crucian
carp · goldfish hybrids and three samples contained
410 B. Hänfling et al.
160 amplification without null-alleles was successful for
140
Pure AU Pure CA six microsatellite loci previously characterised for
goldfish (GF1, GF17, GF29) and common carp (MFW2,
Frequency

MFW7, MFW17), respectively. Four of these loci

AU-MFF/12; GI-KG/2
60
showed clearly distinct allele size ranges among all
40 species and the two remaining loci were completely

HY-BMC/1
AU-BMC/5

CA-BW/31
HY-BMC/8
diagnostic for the separation of the two Carassius
20
species and partly diagnostic for the separation
0 between Carassius and Cyprinus (Table 2). Thus, the
0.0 0.2 0.4 0.6 0.8 1.0 assignment of species-specific alleles could be made
14 with high confidence. In such a situation the ‘assign-
Pure AU Pure CY
12 ment by inspection method’ proved particularly
10 powerful for identifying genotypic categories even
Frequency

8 from geographical distant locations, because all avail-

HY-RCF/6

able information, including allele size differences and

6
HY-RCF/9

one basepair shifts in allele sizes, can be exploited.

HY-SNZ/1

4
This method can be expected, however, to be less
2 reliable for characterising AU · CY and CA · CY
0 hybrids, because at some loci allele size ranges
0.0 0.2 0.4 0.6 0.8 1.0
140
overlapped and some alleles were shared between
Pure CA Pure CY these species pairs. For such hybrid categories, other
120 methods are deemed more appropriate, such as those
Frequency

that are not dependent upon diagnostic alleles (e.g.

100
hybrid indices) or those which do not even require a
priori assignment of pure species (e.g. FCA or a
40 probability model, Anderson & Thompson, 2002).
20 Note however, that these methods, although more
objective, are more sensitive to sampling bias such as
0
0.0 0.2 0.4 0.6 0.8 1.0 the inclusion of individuals from different popula-
tions. Under such circumstances, the calculation of
Hybrid index
hybrid indices in particular can lead to a potential
Fig. 1 Histogram of hybrid indices for the three possible pairs of misidentification of pure individuals or F1 hybrids as
species. Filled bars represent individuals from British popula- backcrosses as the ambiguous position of outgroup
tions, open bars represent outgroup individuals from German samples in Fig. 1 showed.
and New Zealand populations. Positions of potential backcros-
ses are marked by individual codes.
The power to infer the true genealogical classes
from multilocus genotypic categories depends on the
number of loci analysed (Boecklen & Howard, 1997).
crucian carp · common carp hybrids (Tables 1 and 2). The probability that an individual is heterozygote for
Furthermore, three British samples and the two New at least one locus declines from the F1 hybrid
Zealand samples contained goldfish · common carp generation to subsequent generations and is depen-
hybrids. dent on the number of loci used. For example, using
five diagnostic markers, the probability that a first
generation backcross (F1 · parent species, BC-1) has
Discussion alleles of only one species at all loci (and may
therefore be misdiagnosed as a pure species) is 3%
Molecular identification of species hybrids
whereas it is 24% for BC-2, 51% for BC-3, 72% for BC-
Microsatellite markers are indeed useful for the 4 and 85% for BC-5 (Boecklen & Howard, 1997).
identification of species and hybrids of the genus Based on these probabilities alone it can therefore be
Carassius and Cyprinus. Reproducible cross-species assumed that the present dataset allows a confident

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417
 Hybridisation between native and introduced carp 411

Fig. 2 Factorial Correspondence Analysis

of the multilocus genotypes of all ana-
lysed individuals. The different symbols
indicate the initial morphological classifi-
cation of individuals ( , crucian carp; ,
common carp; d, goldfish; h, crucian
carp · common carp hybrid; , crucian
carp · goldfish hybrid; , gold-
fish · common carp hybrid; , gibel
carp). Note that, because of overlapping
data points, not all individuals are visible
in this plot. Individuals which are poten-
tial backcrosses as identified from other
analyses are marked with a cross.

Table 4 Mean posterior probabilities for each hybrid category possibility of backcrossing appears to be high. Four of
according to (Anderson & Thompson, 2002) eight hybrid individuals had an increased probability
AU · CY AU · CA CA · CY to be backcrosses, as further supported by the pres-
ence of two individuals of this population that were
Pure A 0.309 0.085 0.712
polyploid. Polyploidy can arise through hybridisation
Pure B 0.410 0.727 0.112
F1 0.272 0.176 0.123 in the second generation if chromosomal incompati-
F2 0.003 0.003 0.002 bilities prevent segregation of homologous chromo-
BC A 0.003 0.005 0.001 somes during meiosis, leading to diploid gametes.
BC B 0.003 0.003 0.002
These gametes would consequently have alleles of
both parents at each locus (Vrijenhoek, 1994).
identification of first and second generation hybrid
categories.
Status of British Carassius populations
The data demonstrated that hybridisation occurred
among all three species. Most hybrid individuals Molecular evidence indicated clearly that hybridisa-
fitted the expected multilocus genotype of the F1 tion between native crucian carp and introduced
generation (each locus has paternal and maternal goldfish and common carp frequently occurs. Around
alleles). Although the morphological classification 38% of investigated crucian carp populations con-
into pure species or hybrids was in fact correct in tained hybrids with goldfish or common carp, respect-
most cases, some individuals initially identified as ively. Hybrids between common carp and both
pure species were genetically identified as hybrids crucian carp and goldfish are produced artificially
(Table 3; Fig. 2). Moreover, it proved difficult to for commercial proposes. However, all of the hybrid
allocate individuals into precise hybrid combinations samples taken from the wild are believed to have been
based on morphological grounds. This is true especi- produced naturally rather than been stocked after
ally for the differentiation between AU · CY and being artificially propagated. There were no known
CA · CY, both of which, in contrast to AU · CA, U.K. fish farms that produced goldfish/crucian carp
possess barbules. As indicated by the probability hybrids prior to sampling (Live Fish Movement Data-
analysis, the overall proportion of backcrosses is base – Environment Agency U.K./Centre for Environ-
clearly very low and restricted to three populations. ment, Fisheries and Aquaculture Science). Although
However, within one of these populations (BMC) the background information about species distribution in

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417
412 B. Hänfling et al.
each site is not comprehensive, we consider this
sufficient to show general trends. AU · CA hybrids
were more common than the other hybrid crosses.
Furthermore, hybrids were found in all populations in
which crucian carp and goldfish occurred in sympatry.
In contrast, crucian carp · common carp hybrids were
found in only three populations, whereas co-habitation
of both species was indicated for at least ten popula-
tions. These differences in hybridisation rates might be
linked to reproductive strategies or genetic factors. The
low frequency of AU · CY hybrids (three populations)
might reflect the relatively low co-habitation of the
parental species in the investigated habitats (four
populations). A high frequency of natural hybridisa-
tion between these two species was reported from New
Zealand were both species where introduced during
the 20th century (Pullan & Smith, 1987).
Implications for conservation
The high rates of hybridisation raise two major
concerns. Firstly, hybrid vigour of the F1 generation
might lead to competition between pure species and
hybrids (Arnold & Hodges, 1995). Such impacts might
be particularly severe in habitats characterised by small
still waters, with relatively small population sizes,
typical of crucian carp. Where crucian carp · goldfish
hybrids were caught in the wild (at sites A, BMC, CM,
GPB, MFF and MHF) the original crucian populations
were stated to either be in decline or none at all were
caught during sampling, supporting the view that
crucian carp are out-competed by hybrid fish (K.
Wesley, R. Klupp, D. de Charleroy, pers comm.). For
example, in a sample of 30 fish taken by the U.K.
Environment Agency at site A in 2003, mainly pure
species (14 crucian carp and 14 goldfish) were found
together with two AU · CA hybrids; a subsequent
sample of 47 fish taken in 2004 at the same site recorded
45 AU · CA hybrids and two goldfish (P. Bolton,
unpubl. data).
Secondly, backcrossing between F1 hybrids and
pure species might lead to introgression. The fre-
quency and distribution of backcrosses indicated that
F1 hybrid fertility is either very low or further
generation hybrids are less viable or strongly selected
against. Such data suggest the operation of an efficient
postzygotic mechanism for maintaining species integ-
rity. Interestingly, reproductive isolation appears to
be highest between crucian carp and common carp
and is therefore well correlated with overlap in the
species ranges. All three species occur sympatrically
in the eastern range of their distribution, which is also
thought to be the geographical centre for the radiation
of cyprinid fishes, but the geographical ranges of
crucian and common carp overlap almost completely.
Such a pattern is consistent with the hypothesis that
reinforcement contributed to reproductive isolation
between crucian and common carp and has also been
observed in hybridising Galaxid fishes of New Zea-
land (Esa, Waters & Wallis, 2001; Waters, Esa &
Wallis, 2001). Because of the low frequency of back-
crossing it appears to be unlikely that hybridisation
would pose a major threat for native crucian carp
through immediate outbreeding depression, which
could be the case if reproductive isolation is low as in
the case of genetically distinct salmon populations
(McGinnity et al., 2003). However, even at a low level,
backcrossing raises the opportunity for introgression
if selection is relaxed (Arnold, 1997). The finding of
several goldfish/crucian carp hybrids with well
developed ovaries (CM population) provides sup-
porting evidence of the reproductive potential of these
fish, raising concerns about potential introgressive
hybridisation. Furthermore, the results also indicate
that backcrossing could be high locally, as evident in
population BMC. Thus, hybrid fitness might differ
among populations depending on local conditions. In
certain environments hybrids might have equal fit-
ness to the parental species or even a selective
advantage, and therefore evolve into a highly com-
petitive form. In this case hybridisation could have
strong evolutionary consequences (Barton, 2001).
Simulation studies have shown that introgression
can lead to extinction of parental lineages even under
strong negative selection of the F1 if further genera-
tions have only a slight selective advantage (Epifanio
& Philipp, 2000). Introgressive hybridisation was also
proposed as a source for the invasive potential of
plant populations (Milne & Abbott, 2000; Hänfling &
Kollmann, 2002; Abbott et al., 2003; Bleeker, 2003)
adding to the potential negative impact of human-
mediated hybridisation. However, it should also be
emphasised that natural hybridisation is now increas-
ingly seen as an important evolutionary factor to
initiate speciation and adaptive radiations (Alves,
Coelho & CollaresPereira, 1997; Dowling & Secor,
1997; Seehausen, 2004). Although the long-term con-
sequences of introgressive hybridisation for fitness

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417

and survival of crucian carp population are currently
speculative, the demonstration of its existence here,
although geographically variable, represents a
marked threat to the genetic identity of native
C. carassius.
Such threats from non-native populations are par-
ticularly relevant because of additional risks posed by
habitat deterioration, where the habitats typical of C.
carassius, small stillwaters, have declined dramatically
through agricultural and urban development. The
number of ponds in Great Britain, for example, has
fallen from 470 000 to 243 000 (Countryside Survey
2000; http://www.cs2000.org.uk). As current collec-
tions were made, two ponds have been drained and
had their crucian stocks removed. Therefore active
conservation measures should be considered to pro-
tect endemic crucian carp populations. Allendorf et al.
(2001) defined six major categories of hybrid popula-
tions that require the application of different conser-
vation policies. According to our data, most British
Carassius populations fall into category 4 (human
mediated hybridisation without introgression). Re-
moval of goldfish and hybrids from these populations
is likely to be beneficial and habitat improvement
could further help decrease rates of hybridisation
(Allendorf et al., 2001). Some populations, however,
such as BMC fit category 5 (human mediated hybrid-
isation with widespread introgression) and are of little
conservation value (Allendorf et al., 2001). Further-
more the introduction of goldfish and hybrids into
habitats containing crucian carp populations should
be avoided at all costs. This is of particular concern
because the morphological resemblance of brown
goldfish and crucian carp has previously led to
unintentional release of goldfish (Wheeler, 1998,
2000).
Genetic characteristics of gibel carp
The molecular analysis of phenotypically identified
gibel carp included 63 individuals from four German,
two Italian and three British populations (Table 1).
The genetic analyses showed that the samples fell into
two categories of genotypes. (i) Polyploid individuals
exclusively with goldfish alleles and some private
alleles. All but one Continental individual fell into this
category which comprised, furthermore, only two
multilocus genotypes, suggesting the existence of two
clonal lineages representing German and Italian sam-

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417
Hybridisation between native and introduced carp 413
ples respectively. These characteristics are consistent
with the description of gibel carp as a clonally-
reproducing subspecies of goldfish, Carassius auratus
gibelio (Zhou, Wang & Gui, 2001) and with the
suggestion, based on chromosome studies on Asian
Carassius, that gibel carp have a triploid genome
(Zhou & Gui, 2002). (ii) Diploid F1 generation hybrids
between crucian carp and goldfish. All individuals
from the three British gibel carp populations belong to
this second category. The diversity of multilocus
genotypes detected suggests that clonal reproduction
in these individuals is unlikely. Furthermore, based
on genetic data British ‘gibel carp’ could not be
distinguished from individuals subjected to the ana-
lysis as F1 hybrids. These results suggest that, in U.K.
waters, diploid sexually reproducing goldfish and
their hybrids are present, but clonal gibel carp are
absent or rare. Interestingly, one individual from the
German gibel carp populations was also identified as
a goldfish · crucian carp hybrid. The multilocus
genotype of this individual, however, excludes the
possibility that one of the two clonal lineages could be
one of the parental species. Such an observation
suggests that there are either more clonal lineages
present from which some switch back to sexual
reproduction, or that genetically independent lineages
of diploid goldfish are also present in these popula-
tions. Such apparent diversity is of particular interest
because during the last 20 years an increasing number
of diploid male and female gibel carp have been
reported among gynogenetically reproducing indi-
viduals across Europe (Lusková et al., 2004). Their
increasing incidence may be indicative of a change in
reproductive mode from complete parthenogenesis
towards occasional sexual reproduction, as suggested
by Zhou, Wang & Gui (2000b). One would expect that
even occasional sexual reproduction would result in a
high number of clonal lineages. In contrast, our results
raise the question whether sexually and gynogeneti-
cally reproducing lineages are in fact of independent
origin, and whether diploid lineages represent a new
independent invasion. Additional samples encom-
passing a wider geographic range are required to
examine such an assertion. Notwithstanding, our
results demonstrate that what is referred to as ‘gibel
carp’ in fact represents an assemblage of lineages of
different origins (various clonal goldfish lineages as
well as hybrids between goldfish and crucian carp)
which might have contributed to the taxonomic
414 B. Hänfling et al.
confusion in the past. We suggest that in future
genetic data should be used to identify monophyletic
lineages of Carassius populations prior to their formal
description. Our data provide such information for a
necessary taxonomic revision of the genus Carassisus.
Conclusion
Collectively, our data demonstrate that microsatel-
lites, when used judiciously with appropriate sample
identification, can yield informative assessments of
species identity and hybridisation in wild carp
species. The release of closely related exotic cypri-
nids, a group characterised by their propensity for
hybridisation (Schwartz, 1981), not only poses a
threat to the genetic integrity and associated local
adaptations of pure species (Allendorf et al., 2001;
Costedoat et al., in press), but importantly may also
contribute to shifts in community structure through
competitive interactions. There is clearly a need to
further document the geographic patterns of molecu-
lar and morphological variation in wild carp, not
only to determine the extent and nature of such
impacts, but also to reveal fundamental information
on local variation in breeding systems and associated
genotypic diversity. Additionally, any program of
conservation measures must also include preserva-
tion of habitats typical of respective pure species,
such as, for example, small, fragmented ponds, often
associated with C. carassius.
Acknowledgments
Assistance during sampling was provided by Pier
Giorgio Bianco, Robert Klupp, Kai Kuhlen, Chris
Longsdon, Graeme Pierson, Manfred Popp, John
Talbot, Ian Welby, Keith Wesley and many U.K.
Environment Agency officers. Peter Smith provided
outgroup samples from New Zealand. Helen Wilcock
and Ole Seehausen provided useful comments on the
manuscript. Eric Anderson kindly made a modified
version of his programme NewHybrids available. The
study was funded by Environment Agency R & D
grant W2–077.
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Appendix
Allele frequencies of six microsatellite loci in common carp (CY) crucian carp (CA), goldfish (AU), clonal gibel carp (GI) and their
respective hybrids

GI-clones AU · CA AU · CY CA · CY
Locus Allele CY (n¼35) CA (n¼237) AU (n¼27) (n¼40) (n¼61) (n¼25) (n¼13)

GF1 296 1.00 0 0 0 0 0.52 0.50

298 0 1.00 0 0 0.49 0 0.50
300–312 0 0 1.00 1.00 0.51 0.48 0
Ho 0 0 0.48 0.45 0.95 0.96 1
A 7 1 1 4 3 4 4 2
GF17 182 – 1.00 0 0 0.49 0 1.00
184–212 – 0 1.00 0.78 0.51 1.00 0
220 – 0 0 0.23 0 0 0
Ho – 0 0.52 1 0.98 0.08 0
A 9 0 1 4 4 5 3 1
GF29 189–207 0 0 1.00 1.00 0.52 0.48 0
210–228 0 1.00 0 0 0.48 0 0.50
243–283 1.00 0 0 0 0 0.52 0.50
Ho 0.77 0.39 0.59 1.00 1.00 1.00 1.00
A 27 11 6 5 3 7 10 9
MFW2 157 0 0 1.00 1.00 0.50 0.48 0
160 0 1.00 0 0 0.50 0 0.50
169–271 1.00 0 0 0 0 0.52 0.50
Ho 0.66 0 0 0 0.97 1.00 1.00
A 25 15 1 1 1 2 10 9
MFW7 157 0 0 0.19 0 0.06 0.10 0
159 0 0 0 0 0.01 0 0
169 0 0 0.13 0 0.10 0 0
171 0 0 0 0 0 0.04 0
174 0.01 0 0 0 0 0.02 0
175 0 0 0.61 0.23 0.34 0.40 0
177 0 0 0.06 0 0 0 0
178 0 0.03 0 0 0 0 0
180 0 0.01 0 0.28 0 0 0
182 0 0.01 0 0 0 0 0
184 0 0.05 0 0 0.01 0 0
188 0.23 0.01 0 0 0.02 0.12 0.04
191 0 0 0.02 0.23 0 0 0
192 0.09 0.87 0 0 0.47 0 0.62
194 0.09 0 0 0 0 0.06 0.04
196 0.20 0.03 0 0 0 0.02 0.08

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417

 Hybridisation between native and introduced carp 417
Appendix (Continued)

GI-clones AU · CA AU · CY CA · CY
Locus Allele CY (n¼35) CA (n¼237) AU (n¼27) (n¼40) (n¼61) (n¼25) (n¼13)

198 0.07 0 0 0 0 0.14 0

199 0 0 0 0.28 0 0 0
200–276 0.31 0 0 0 0 0.10 0.23
Ho 0.83 0.16 0.52 1.00 1.00 0.92 0.77
A 33 16 7 5 4 7 12 8
MFW17 214 0 0 0 0 0.02 0 0
216 0 0 0.02 0 0.01 0 0
222 0 0 0 0.23 0 0 0
224 0 0 0 0.28 0 0 0
228 0 0 0.13 0.28 0.16 0.02 0
230 0 0 0 0 0 0 0
232 0 0 0 0.23 0 0 0
234 0.01 0 0 0 0 0 0
240 0.01 0 0.15 0 0.01 0 0
242 0.33 0.60 0 0 0.02 0.18 0.42
244 0.09 0 0.09 0 0.03 0.08 0.04
246 0.07 0.20 0 0 0.46 0.06 0.12
250 0 0.19 0 0 0 0 0
252 0 0 0.07 0 0 0.08 0
254 0 0 0.07 0 0.01 0 0
256 0.01 0 0.46 0 0.27 0.36 0.08
258 0.04 0 0 0 0 0 0
262–280 0.43 0 0 0 0 0.22 0.35
Ho 0.69 0.31 0.67 1.00 0.95 1.00 1.00
A 25 12 4 7 4 9 11 8

Shaded areas represent allele frequencies > 0.

Ho, observed heterozygosity; A, number of alleles.

2005 Blackwell Publishing Ltd, Freshwater Biology, 50, 403–417