Journal of Fish Biology (2014) 84, 237–242
doi:10.1111/jfb.12272, available online at wileyonlinelibrary.com
Mechanism of hybridization between bream Abramis
brama and roach Rutilus rutilus in their native range
A. Kuparinen*†||, M. Vinni‡, A. G. F. Teacher§, K. Kähkönen*
and J. Merilä*
*Ecological Genetics Research Unit, Department of Biosciences, P.O. Box 65, FI-00014
University of Helsinki, Finland, ‡Department of Environmental Sciences, P.O. Box 65,
FI-00014 University of Helsinki, Finland and §Department of Biosciences, P.O. Box 65,
FI-00014 University of Helsinki, Finland
(Received 5 December 2012, Accepted 9 October 2013)
Mechanisms of hybridization between bream Abramis brama and roach Rutilus rutilus were studied
within the native range of the species in a lake in southern Finland. Through the genetic analysis of
A. brama, R. rutilus and putative hybrids, hybridization is shown to have occurred between female
A. brama and male R. rutilus. These results match with previous findings from introduced habitats,
suggesting that mating between female A. brama and male R. rutilus is the predominant mechanism
through which the two species hybridize.
© 2014 The Fisheries Society of the British Isles
Key words: Finland; mating; microsatellites; mtDNA; reproduction.
The ability of species to hybridize is a key mechanism determining whether new
species can arise (Mallet, 2007) and is considered to be an important source of new
genetic variation that fuels evolution and adaptation (Dowling & DeMarais, 1993;
Arnold, 1997; Hedrick, 2013). In fishes, hybridization is common (Hubb, 1955), and
hybridization between bream Abramis brama (L. 1758) and roach Rutilus rutilus
(L. 1758) has been particularly well studied and, thus, can even be considered as
a model system for hybridization among northern hemisphere freshwater species
(Hayden et al., 2010; Toscano et al., 2010). Hybridization between A. brama and R.
rutilus is well documented across Europe and the British Isles (Cowx, 1983; Wyatt
et al., 2006) and has been found to be exceptionally commonplace in Ireland, where
the two species have been artificially introduced (Hayden et al., 2010; Toscano et al.,
2010). Studies of the genetic background of hybrids between A. brama and R. rutilus
in four lakes representing a range of environmental conditions across northern and
middle Ireland conducted in the 1990s and 2000s suggest that the primary mechanism
†Author to whom correspondence should be addressed. Tel.: +358 40 731 3120; email:
[email protected]
||Current address: Department of Environmental Sciences, P.O. Box 65, FI-00014 University of Helsinki,
Finland; Environment and Sustainability Institute, University of Exeter, Penryn Campus, Penryn, Cornwall
TR10 9EZ, U.K.
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A . K U PA R I N E N E T A L .
of hybridization is reproduction between male R. rutilus and female A. brama. The
occurrence of backcrosses and F2 hybrids has been found to be low, suggesting
that F1 hybrids experience fitness disadvantages when compared with pure species
(Hayden et al., 2010; Toscano et al., 2010).
Given that the rate of hybridization between A. brama and R. rutilus is exceptionally high in Ireland and considered to be largely linked to the introduction of
these species into a novel environment (Hayden et al., 2010), mechanisms underlying
hybridization in Ireland may differ from those in the native range of the two species.
Analyses of hybrids originating from the native range would therefore provide important new insights into hybridization mechanisms of the species, specifically, whether
mating between male R. rutilus and female A. brama is indeed the predominant
mechanism not only in introduced areas but also within continental and northern
Europe, which constitute the native core range of the species. To study this, hybridization between A. brama and R. rutilus was investigated in southern Finland, which
represents the native range for both these species. Population genetic analyses using
nuclear microsatellites and maternally inherited mtDNA sequence variation were conducted to confirm the identification of hybrids, to resolve the mechanism of hybridization and to disentangle F1 hybrids from possible backcrosses and F2 hybrids.
A total of 53 R. rutilus, 29 A. brama and 26 putative hybrids (based on morphology) were sampled from Iso Ruuhijärvi (61◦ 13 N; 25◦ 3 E). Hybridization
between these two species appears to occur at an exceptionally high rate in this lake,
providing an opportunity for the investigation of hybridization mechanisms in the
wild. The lake is relatively unaffected by human influences and the surrounding area
is dominated by coniferous forest and peatland. The lake is small (13·3 ha), relatively
deep (maximum depth 8 m) and highly humic (335 mg Pt l−1 , Secchi depth 62 cm),
and the concentration of dissolved oxygen may decline below 1 mg l−1 at 6–8 m
depth during the summer months (Olin et al., 2010). Generally, conditions in the
lake are relatively harsh and habitats suitable for fishes are limited to the proximity
of shores. Larvae of phantom midge Chaoborus flavicans, which represent a key
component of the diet for fishes, exist at high densities (500–4000 individuals m−2 )
in the water column deeper than 2 m (Vinni et al., 2004; Estlander et al., 2009). The
natural fish community in the lake consists of R. rutilus, A. brama, their putative
hybrids, perch Perca fluviatilis L. 1758 and pike Esox lucius L. 1758 (Olin et al.,
2010). Low numbers of large specimens of pikeperch Sander lucioperca (L. 1758)
have also been recorded in recent years (Olin et al., 2010).
Fishes were collected by gillnetting (mesh sizes 5–60 mm from knot to knot) from
July to August 2005, and May to June 2010. The lake was divided into two depth
zones: (1) shallows (0–3 m), where nets were set at the bottom, and (2) areas over
3 m deep, where nets were set both at the bottom and on the surface. Fishes were
removed from the nets at 2–12 h intervals. After capture, the fishes were frozen. The
ages of individual fishes were determined using both scales and cleithra, and the age
estimates ranged between 10 and 19 years for A. brama, 2 and 29 years for R. rutilus
and 10 and 22 years for the putative hybrids. Putative hybrids originated from three
cohorts, born in years 1989, 1993 and 1996. Total lengths (LT ) of the fishes ranged
between 19·2 and 47·7 cm for A. brama, 12·6 and 28·4 cm for R. rutilus and 18·0
and 27·0 cm for putative hybrids.
DNA was extracted from the scales using the glass fibre plate DNA isolation
method (Ivanova et al., 2006). Nuclear genetic variation was assessed using 20
© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 84, 237–242
A B R A M I S B R A M A A N D R U T I L U S R U T I L U S H Y B R I D I Z AT I O N
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microsatellite loci: lid1 , lid2 , lid8 , lid11 , rru2 , rru3 , rru4 (Barinova et al., 2004);
ca1 , ca3 (Dimsoski et al., 2000); cypG3 , cypG9 , cypG24 , cypG27 , cypG30 (Baerwald & May, 2004); lco5 (Turner et al. 2004); ppro132 (Bessert & Orti, 2003);
rhco20 (Girard & Angers, 2006); z21908 (Shimoda et al., 1999); mfw1 (Hamilton
& Tyler, 2008); and mfw10 (Crooijmans et al., 1997). PCR was performed in a
total volume of 10 μl: 2 pmol of each primer, 1× Qiagen 2× Master Mix (Qiagen;
www.qiagen.com), 1 μl Q-solution and c. 20 ng of DNA. The PCR programme was
as follows: 95◦ C for 15 min, followed by 34 cycles of 94◦ C for 30 s, 58◦ C for 1 min
30 s and 72◦ C for 1 min, with a final extension at 60◦ C for 10 min. After amplification, 3 μl of PCR-amplicon was run on a 2% agarose (Bioline; www.bioline.com).
The PCR products were diluted 1:100 with MQ water, mixed with Et-ROX 400 standard (GE-Healthcare, Life Sciences; www.gelifesciences.com) and analysed using a
MegaBace 1000 capillary sequencer (GE Healthcare, Life sciences). Genotypes were
scored using Fragment Profiler 1.2 software (GE Healthcare, Life Sciences).
In order to determine the maternal parent of the putative hybrids, a fragment
of the mitochondrial cytochrome b (cytb) gene from each putative hybrid and five
individuals of each parental species were sequenced. The fragment size was 674 bp
in A. brama and hybrids and 452 bp in R. rutilus. The cytb gene fragment was
amplified using the R. rutilus CYTB.F (GenBank accession number Y10440), A.
brama CYTB.F (GenBank accession number Y10441) and Universal CYTB.R
(GenBank accession number Y10440/1/4) primers (Wyatt et al., 2006). PCRs were
performed in a total volume of 20 μl: 5 pmol of each primer, 1× Phire reaction
buffer mix (F-524, Thermo Fisher Scientific; www.thermofisher.com), 200 μM of
each deoxynucleotide triphosphate (dNTP) (F-560 XL, Thermo Fisher Scientific),
0·4 μl of Phire Hot Start DNA Polymerase (Thermo Fisher Scientific) and c. 20 ng
DNA. The cycling profile was as follows: 98◦ C for 30 s, followed by 34 cycles
of 98◦ C for 10 s, 54◦ C for 10 s and 72◦ C for 30 s, with a final extension at 72◦
C for 1 min. After amplification, 2 μl of PCR-amplicon was run on a 2% agarose
(Bioline). The PCR products were purified using EXO-SAP purification (Biolabs;
www.neb.com). The purified PCR products were sequenced in both directions using
the same primers on an ABI 3730xl capillary sequencer at the Institute for Molecular
Medicine, Finland. Sequences were checked and aligned using the programme
Geneious 4.8.4 (Drummond et al., 2006). The similarity of the putative hybrid
sequence to that of the two parental species was checked by visual comparison.
Microsatellite markers were checked for polymorphism, homozygote deficiency
and linkage disequilibrium using GenePop on the Web (Raymond & Rousset, 1995).
Bayesian clustering analysis was performed using Structure analysis (Pritchard et al.,
2000) in order to see how the genotypes of the individual fish clustered. A burn-in
of 10 000 and 10 000 Markov chain Monte Carlo (MCMC) repetitions were used,
with an admixture model, correlated allele frequencies and assuming K = 2 (i.e. the
two pure species). NewHybrids analysis (Anderson & Thompson, 2002) was then
used to estimate the posterior probability that the individuals fit into each of a set
of user-defined hybrid categories: pure A. brama, pure R. rutilus, F1 hybrids, F2
hybrids, A. brama backcross or R. rutilus backcross. A burn-in of 1000 with 1000
MCMC repetitions were used. The microsatellite locus ppro132 was found to be
fixed for a single allele and therefore was removed from the analyses. Locus mfw1
did not amplify in A. brama, but worked well for the putative hybrid and R. rutilus
samples, whereas locus cypg30 did not amplify in R. rutilus, but worked well for the
© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 84, 237–242
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A . K U PA R I N E N E T A L .
1·0
Proportion
0·8
0·6
0·4
0·2
0·0
R. rutilus
Hybrid
A. brama
Fig. 1. Assignment results from the Structure analysis, assuming K = 2 clusters. Each vertical bar represents an
individual. Genetic clusters are indicated by each shade, and the y-axis of each plot shows the proportion
(p) of the genotype for each individual belonging to each cluster ( , assignment probability to Rutilus
rutilus; , assignment probability to Abramis brama). Species of the individual samples are indicated
on the x -axis.
putative hybrid and A. brama. No markers showed consistent homozygote deficiency
in all three groups (R. rutilus, A. brama and hybrid), and there was no consistent
evidence for linkage disequilibrium.
The Structure analysis assigned the 34 R. rutilus samples to the R. rutilus species
(proportion, p = 0·923–0·997) and the 16 A. brama samples to the A. brama species
(p = 0·991–0·996), whereas the 16 putative hybrids were each assigned in part to R.
rutilus (p = 0·389–0·673) and to A. brama (p = 0·327–0·661; Fig. 1). The NewHybrids analysis showed that all putative hybrid individuals were assigned as F1 hybrids
(p = 0·722–1·000). The mtDNA sequencing showed that all five A. brama sequences
were identical to each other, and all five R. rutilus sequences were identical to each
other. The sequence divergence between the two species was 11·5% (52 polymorphic
bases). The putative hybrid sequences were all identical to the A. brama sequences,
indicating that in all cases A. brama was the maternal parent and R. rutilus was the
paternal parent.
The results suggest that hybridization between R. rutilus and A. brama occurs
regularly in the lake examined, as suggested by the fact that the sampled hybrids were
found to belong to three different cohorts. All the hybrids, however, were assigned as
F1 hybrids and mothered by A. brama, and no backcrosses or F2 hybrids were found.
These results comply with the previous observations made in the introduced R. rutilus
and A. brama populations in Ireland, suggesting that (1) the primary hybridization
mechanism between these species is reproduction between female A. brama and male
R. rutilus, and (2) even though these hybrids are known to be viable (Nzau Matondo
et al., 2007), their reproductive success is low (Wyatt et al., 2006; Hayden et al.,
2010; Toscano et al., 2010). The match between present results and those previously
obtained from Irish study sites could be attributable to similarities between the Irish
and Finnish sites; yet the lack of comparable data prevents conclusive discussion
about this.
The findings reported here are in good agreement with the fact that even though
A. brama and R. rutilus are able to hybridize, they still remain separate species
within the habitats where they coexist. In experimental studies, hybridization has
© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 84, 237–242
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been shown to be technically possible between male A. brama and female R. rutilus,
and backcrosses and F2 hybrids were found to be, at least to some extent, viable
(Pitts et al., 1997). Environmental conditions experienced by the species in wild are
therefore likely to disfavour such combinations (Wyatt et al., 2006), for example
through reduced fitness of hybrid offspring or owing to non-overlapping reproductive periods and differences in spawning behaviour between the sexes of the two
species (Hayden et al., 2010). Interestingly, the fact that in this study hybrids were
assigned to three distinct cohorts supports the conclusion by Hayden et al. (2010)
that rate of hybridization varies annually and, thus, is promoted by certain environmental conditions that occur from time to time. Hence, the challenge for future
research remains to identify the environmental factors and processes that promote
hybridization between A. brama and R. rutilus (Hayden et al., 2010; Toscano et al.,
2010). Understanding the role of environmental conditions as drivers for hybridization would not only provide the means to project how future environmental changes
might alter hybridization rates but also provide important insights into the conditions under which processes important for speciation (i.e. premating reproductive
isolation) can break down.
This study was funded by the Academy of Finland (A.K., A.G.F.T. and J.M.) and Bror
Serlachius Foundation (M.V.). We thank Finnish Game and Fisheries Research Institute for
providing equipments for the field work.
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© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, 84, 237–242