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Amphibia-Reptilia 41 (2020): 361-371 brill.com/amre
Body size variation in hybrids among populations of European water
frogs (Pelophylax esculentus complex) with different breeding
systems
Adam Hermaniuk1,∗, Magdalena Czajkowska2, Anetta Borkowska2, Jan R.E. Taylor1
Abstract. In some populations, hybrids reproduce with a parental species by eliminating the genome of this species from their
own germline and produce gametes that only contain the genome of the other parental species (sexual host). This mode of
reproduction, known as hybridogenesis, leads to a conflict of interest between the two parties because the sexual host should
avoid mating with the hybrid to prevent a reduction in reproductive success, whereas the hybrid depends on such matings for
survival. We investigated European water frogs (Pelophylax esculentus complex), including hybrids (P. esculentus, genotype
LR) and two sexual host species (P. lessonae,LLandP. ridibundus, RR). We hypothesized that to maximize fitness, hybrid
males should be morphologically more similar to the sexual host that is preferred by females for successful reproduction. To
test this hypothesis, we compared hybrid males in two different population types, L-E (hybrids coexist with LL) and L-E-R
(hybrids coexist with both LL and RR). The latter was described in terms of genome composition, sex ratio, and mate choice
preferences; the sex ratio of hybrids was significantly male-biased. We found that LR males from the L-E-R populations were
significantly larger than those from the L-E, which makes them more similar to P. ridibundus, the largest species within the P.
esculentus complex. We suggest that a larger body size of hybrid males may provide a reproductive advantage in the L-E-R
population type, where the most common type of pair caught in the breeding season was LR males ×RR females.
Keywords: body size, hybridogenesis, hybrids, L-E-R population type, male-biased sex ratio, mate choice.
Introduction
Complexes of species composed of hybrids and
their parental species have been described in
fish, amphibians, and reptiles (Avise, 2008).
To maintain their own lineage, hybrids may
present an unusual reproductive mode known
as hybridogenesis which involves the selective
transmission of one of the parental genomes,
while the other one is renewed by mating
with the other parental species used as sexual
host (Schultz, 1969; Lavanchy and Schwander,
2019). Such systems are interesting for evo-
lutionary ecology because of the various im-
pacts of hybridogenesis on the population dy-
namics and genetics of the species involved.
1 - Department of Evolutionary and Physiological Ecol-
ogy, Faculty of Biology, University of Białystok,
Ciołkowskiego 1J, 15-245 Białystok, Poland
2 - Department of Zoology and Genetics, Faculty of Biol-
ogy, University of Białystok, Ciołkowskiego 1J, 15-245
Białystok, Poland
∗Corresponding author; e-mail: adamher@uwb.edu.pl
The continuance of hemiclonal lineages re-
quires that hybrids must “trick” a sexual host
to reproduce successfully. On the other hand,
host species should resist mating with the hy-
brids because of the exclusion of their genome
in the next generation (Dries, 2003). Because
mismating induces relatively low costs in males
in terms of sperm production, females should
be the more selective sex because they invest
more in eggs (Emlen and Oring, 1977; Clutton-
Brock and Vincent, 1991). Furthermore, fe-
males of host species potentially face a con-
flict between species recognition (identifica-
tion of conspecifics) and mate-quality recog-
nition (identification of high-quality males) if
high-quality conspecific males resemble het-
erospecific males (Sherman, Reeve and Pfen-
ning, 1997). In that case, the preferences of fe-
males for extreme traits may increase the prob-
ability of mistakingly mating with heterospe-
cific males, especially when the two species dif-
fer only slightly in mate recognition character-
istics (Pfennig, 2000; Randler, 2002). For both
©Koninklijke Brill NV, Leiden, 2020. DOI:10.1163/15685381-bja10005
362 A. Hermaniuk et al.
reasons, mate preference of sexual host females
may shape the behaviour and morphology of hy-
brid males.
One of the most extensively investigated hy-
bridogenetic species complexes, European wa-
ter frogs (Pelophylax esculentus complex), con-
sist of the parental species Pelophylax lessonae
(Camerano, 1882, pool frog, LL genotype)
and Pelophylax ridibundus (Pallas, 1771, marsh
frog, RR), and their hybrid Pelophylax esculen-
tus (Linnaeus, 1758, edible frog, LR). Hybrids
reproduce by back-crossing with the parental
species (heterotypic matings), the genome of
which is eliminated (for review, see Graf and
Polls Pelaz, 1989). Three major breeding sys-
tems have been described in detail in Europe
in various populations: the L-E, R-E, and E-E
systems (initial letters refer to species Latin
names) (Rybacki and Berger, 2001; Plötner,
2005). In the most widespread L-E system, hy-
brids coexist with P. lessonae, exclude the L
genome from the germline, and clonally trans-
mit the R genome to gametes. In the mir-
ror image of the L-E system, the R-E sys-
tem, hybrids mate with P. ridibundus, exclude
the R genome, and produce mainly L gametes
(Uzzell, Günther and Berger, 1977; Rybacki
and Berger, 2001; Doležálková-Kaštánková et
al., 2018). Mating among hybrids, at least in
the L-E system, results in non-hybrid offspring
(LL or RR) that typically die before sexual
maturity presumably because of the homozy-
gosity of deleterious mutations accumulated as
a result of the lack of recombination in the
clonally transmitted genomes (Vorburger, 2001;
Guex, Hotz and Semlitsch, 2002). In the third
breeding system E-E (also known as all-hybrid
populations) diploid hybrids (LR) coexist with
triploids with different combinations of parental
genomes (LLR and LRR). The persistence of
this type of population is made possible by ge-
netic recombination of homospecific genomes
in triploids (see Christiansen and Reyer (2009)
for details). In some areas, many variants of the
systems described above have been found, in-
cluding different combinations of the parental
species with diploids and triploid hybrids (Ry-
backi and Berger, 2001). An intriguing popu-
lation is the L-E-R type, very rare in western
Europe, where all members of the P. esculen-
tus complex exist together. Although the L-E-
R type is widely distributed in eastern Europe
(Lada, Borkin and Vinogradov, 1995; Borkin et
al., 2002) and was occasionally found in cen-
tral Europe (Mikulíˇ
cek et al., 2015; Herczeg et
al., 2017), little is known about how hybrids are
perpetuated in these populations.
In the majority of breeding systems in Eu-
rope, an asymmetry in the hybrid sex ratio has
been found (Rybacki and Berger, 2001). Water
frogs exhibit X-Y sex determination (Schempp
and Schmid, 1981; Christiansen, 2009). It has
been observed that most primary hybridizations
take place between P. lessonae males and P.
ridibundus females (Berger, Uzzell and Hotz,
1988). As a consequence, the male Y factor
becomes almost exclusively assigned to the L
genome (fig. 1), which may cause sex asym-
metry in the next generations of hemiclon-
ally reproducing hybrids (Graf and Polls Pelaz,
1989). Hybrids in some L-E populations ex-
hibit a female-biased sex ratio (Berger, Uzzell
and Hotz, 1988). In R-E systems, almost half
Figure 1. Primary hybridization between Pelophylax
lessonae male and P. ridibundus female. For behavioural
reasons, this type of mating prevails in natural populations.
Consequently, the hybrid L genome can have an X or a Y
factor, while their R genome only have an X factor. Y de-
notes a male-determining factor, and X denotes a female-
determining factor. Males are black, females are white.
Body size variation in hybrid water frogs 363
of the examined populations exhibit a sex ra-
tio skewed in favour of males (Rybacki and
Berger, 2001). In some regions, the R-E system
consists of males and females of P. ridibundus
and only males of P. esculentus (Doležálková-
Kaštánková et al., 2018). Nothing is known
about the hybrid sex ratio in the L-E-R popu-
lation type.
Because mating among diploid hybrids re-
sults in nonviable offspring, strong selection
against LR ×LR matings should occur. In that
case, P. esculentus in the L-E and R-E systems
should prefer parental species over their own
to achieve reproductive success. Furthermore,
compared to males, sexual host females should
be more choosy in terms of mating with hy-
brids because of the higher cost of gamete pro-
duction (Emlen and Oring, 1977; Clutton-Brock
and Vincent, 1991). The experiments on mate
choice in the L-E system confirmed that females
(both LL and LR) showed the predicted pref-
erence for LL males, whereas males (LL and
LR) did not discriminate between LL and LR fe-
males (Abt and Reyer, 1993; Engeler and Reyer,
2000; Roesli and Reyer, 2000). Another exper-
iment showed that LL and LR females reduce
clutch size when amplexed by undesired hybrid
males (Reyer, Frei and Som, 1999). It must be
emphasized that frogs within the P. esculentus
complex differ greatly in body size: P. lessonae
is the smallest, P. ridibundus is larger by half,
and the hybrid P. esculentus is intermediate be-
tween the two other species (Berger, 2008).
Therefore, in order to maximize mating suc-
cess P. esculentus males from different popula-
tion types should be morphologically more sim-
ilar to the sexual host males that are preferred
by females for successful reproduction. To test
this hypothesis, we examined the body mass
and body size of P. esculentus in two different
population types, L-E and L-E-R, in the Biebrza
River Valley (north-eastern Poland). Since there
are no published data examining the reproduc-
tive mode of hybrids in L-E-R, we examined
naturally formed pairs in amplexus during the
breeding season to determine sexual host and
female mating preference in this type of popu-
lation.
Material and methods
Study area and data collection
The study area, Biebrza National Park, is unique in Europe
for its marshes and peatlands, as well as for its highly diver-
sified fauna. Adult water frogs were collected in 2010-2016
from eight sites (table 1, supplementary fig. S1). Frogs were
caught during or shortly after the breeding season (May to
July), usually at night by hand and rarely during the day with
a net. Water bodies were searched extensively by wading
and from a raft. To reduce non-random sampling of males
and females, frogs were collected beyond the most active
period of chorusing, when males do not bunch together. Two
sites (Krasnoborki and Jagłowo) were sampled only once;
the other sites were sampled two or three times. Since taxa
and sex frequency within sites did not differ between years,
particular samples were pooled and analysed together. The
only exception was Osowiec, due to a detected population
composition change between 2010-2011 and 2016 (see re-
sults). For that reason, the earlier and later samples were
analysed separately (table 1). After being caught, all frogs
were transported to the field station in Gugny and were kept
for one day in plastic tanks (40 cm ×20 cm ×20 cm)
filled with plants and shallow water. Animals were sexed
by the presence or absence of the nuptial pads on thumbs
as well as vocal sac openings and weighed to the nearest
0.01 g. For each animal, we also measured the body length
(from snout to vent; SVL), tibia length (T), first toe length
(digitus primus; DP), and metatarsal tubercle length (cal-
lus internus; CI) with an electronic calliper (error =0.01
mm). For taxonomical identification, we used the morpho-
logical indices DP/CI and T/CI, the shape of the metatarsal
tubercle, male nuptial colouration, and colour of the vo-
cal sacs (Plötner, 2005; Berger, 2008; Kierzkowski et al.,
2011). Photographs of the unique patterns of back spots in
each captured frog allowed their identification so that frogs
would not be recounted. After the biometrical measure-
ments, we made blood smears from a cut fingertip on mi-
croscopic slides and finally returned to the original place of
collection. All fingertips were fixed in 75% ethanol and in-
dividually stored in labelled plastic tubes. Ploidy was deter-
mined using the erythrocyte size and densitometric method
of DNA content measurement in erythrocyte nuclei (Ogiel-
ska, Kierzkowski and Rybacki, 2004; Hermaniuk et al.,
2013). All frogs from 2016 were genotyped using a mi-
crosatellites (see below). Additionally, to examine the accu-
racy of previously used methods in correctly identify water
frogs, 20 randomly chosen animals from 2010-2013 were
genotyped.
In 2016, at one of the sites (Osowiec), mate choice
was examined to explain the skewed sex ratio in hybrids
observed in the L-E-R population type. For this reason, we
determined the taxonomic affiliation of the frogs caught
in amplexus during the breeding season. Unlike the other
samples (table 1), pairs in amplexus were collected during
the most active period of chorusing.
364 A. Hermaniuk et al.
Tab le 1 . Numbers of frogs (males and females) of the Pelophylax esculentus complex at eight sites in the Biebrza River Valley;
LL, Pelophylax lessonae; RR, P. ridibundus;LR,P. esculentus (the hybrid of the two species). The sites are listed according to
the population type found there: L-E and L-E-R (the letters refer to species’ Latin names: lessonae,esculentus,ridibundus).
Sex ratios of hybrids are also shown (significant deviations from the 1:1 ratio are in bold). N, number of individuals.
Population Site Biotope Year of sampling Genotype N ♂N♀Hybrid sex ratio
type (♂:♀)
L-E Jagłowo (Ja) artificial 2012 LL 16 10
pond LR 11 5 2.2:1 (P=0.134)
Krasnoborki (Kr) gravel pit 2012 LL 34 14
LR 7 0 males only
Tajno (Ta) lake 2013 LL 7 1
LR 13 3 4.3:1 (P=0.012)
Wizna (Wi) oxbow 2011-2012 LL 30 19
LR 25 13 1.9:1 (P=0.052)
Zajki (Za) artificial 2010 LL 9 4
pond LR 11 10 1.1:1 (P=0.827)
L-E-R Dobarz (Db) artificial 2013 LL 66 14
pond LR 24 3 8.0:1 (P< 0.0001)
RR 2 4
Dolistowo (Dl) artificial 2011-2013 LL 4 7
pond LR 42 3 14: 1 (P< 0.0001)
RR 1 5
Osowiec (Os) moat 2010-2011 LL 11 3
LR 62 6 10:1 (P< 0.0001)
RR 6 12
Osowiec (Os)amoat 2016 LR 36 3 12:1 (P< 0.0001)
RR 3 16
aThe population structure in Osowiec was studied for the second time in 2016 to examine mate choice. Pelophylax lessonae
was not found in 2016.
Microsatellite analysis
We chose and organized autosomal and unlinked mi-
crosatellite markers into one multiplex set that maximized
the number of loci suitable for simultaneous amplification
and analysis, with no allele overlap between loci labelled
with the same dye. This set consisted of four loci (supple-
mentary table S1). Two (Cala27 and RlCA18) are ampli-
fied only in P. lessonae (Garner et al., 2000); the other two
(Res22 and Rrid169A) are amplified only in P. ridibundus
(Zeisset, Rowe and Beebee, 2000; Hotz et al., 2001). These
four markers are among the most polymorphic loci tested
so far and precisely identify ridibundus and lessonae geno-
types (Czernicka, 2013). The forward primer of each pair
was fluorescently labelled. Multiplex PCRs were performed
with the GeneAmp PCR System 9700 (Applied Biosystems)
in a 5-μl reaction volume containing 2 μl of isolated ge-
nomic DNA, 1.7 μl Qiagen Multiplex PCR Master Mix
(1×), 0.3 μl mix of primers (0.2 μM of each primer), and
1μl RNase-free water. The PCR products were mixed with
10 μl ultragrade formamide and 0.2 μl GeneScan 500-LIZ
size standard (Applied Biosystems), denatured at 95°C for
5 min, rapidly cooled, and detected using an ABI 3130 Ge-
netic Analyser (Applied Biosystems). The allele fragment
lengths were estimated using the Auto Bin feature in the
GeneMapper 4.0 software (Applied Biosystems). Individu-
als that amplified only alleles of Cala27 and R1CA18 were
identified as P. lessonae; individuals that amplified alleles
of Res22 and Rrid169A loci only were identified as P. rid i -
bundus. Diploid hybrids (LR) amplify one allele from each
of the four loci. LLR triploids amplify one allele in both
loci specified by the P. ridibundus genotype and two alleles
in at least one locus specified by the P. lessonae genotype
(Czernicka, 2013).
All procedures concerning animals adhered to princi-
ples of animal care and to specific national laws (approvals:
DOPpn-4102-858/43327/10/RS, DOPpn-4102-150/10939/
12/RS, WPN.6401.173.2016.MN from the Ministry of En-
vironment and Regional Directorate for Environmental Pro-
tection; permits: 32/2009, 20/2012 from the local ethical
committee in Białystok).
Statistics
To compare morphology of hybrids between population
types, we used only adult males. Females were excluded
from the analysis because of the difficulty in determining
their sexual maturity. This analysis was performed on the
material from 2010-2013, which included 195 P. esculentus
males. Body mass, SVL, T, and DP were compared between
population types by means of a nested ANOVA model
that included the population type as a fixed factor and
the site nested in the population type as a random factor.
To check whether the hybrid sex ratios observed in the
studied populations differed significantly from equality, the
Body size variation in hybrid water frogs 365
binomial test was applied. All tests were performed using
SAS, ver. 9.3 (SAS Institute, Cary, NC, USA). ANOVAs
were performed using the MIXED procedure based on the
restricted maximum likelihood method (REML).
Results
Genome composition and sex ratio within
population types
Of the 517 adult individuals caught in the eight
localities sampled in the Biebrza River Val-
ley in 2010-2013, 249 were identified as P.
lessonae (48.2%), 238 as P. esculentus (46.0%),
and 30 as P. ridibundus (5.8%). Pelophylax
lessonae and P. esculentus were present at all
sites. Pelophylax ridibundus was found only in
Dolistowo, Dobarz, and Osowiec. Based on the
species composition, we classified five popula-
tions composed of P. lessonae and P. esculentus
only as L-E population type and three having all
three species as L-E-R type (table 1). The struc-
ture at Osowiec changed between 2010-2011
and 2016. Among the 90 frogs examined in
2016, we did not find any P. lessonae; we can-
not entirely exclude, however, that they were
not there. Only diploid frogs were found in
samples from 2010-2016. Microsatellite analy-
sis showed that all 20 randomly chosen frogs
from 2010-2013 were correctly identified using
biometrical and cytogenetic methods.
Of the 242 frogs caught in the five localities
within the L-E population type, 59.5% were P.
lessonae and 40.5% were P. esculentus. Con-
sidering the study sites separately, P. lessonae
was the dominant taxon at three out of the five
sites (Jagłowo, Krasnoborki, and Wizna). Pelo-
phylax esculentus prevailed in two sites (Zajki
and Tajno). The hybrid sex ratio differed signif-
icantly from 1:1 at two sites (Krasnoborki and
Tajno). In the other sites with the L-E popula-
tion type, we did not find statistically significant
asymmetry in the hybrid sex ratio (table 1).
Considering all three locations with the L-
E-R population type (Dolistowo, Osowiec, and
Dobarz, 275 individuals together), P. esculentus
was the dominant taxon, and individuals of this
species represented 50.9% of all frogs caught.
Pelophylax lessonae represented 38.2% and P.
ridibundus constituted 10.9%. Dolistowo and
Osowiec were similar to each other in taxon
composition. At both sites, P. esculentus was the
dominant taxon, with a lower frequency of P.
lessonae and P. ridibundus. At Dobarz, in con-
trast, P. lessonae was the most abundant taxon,
considerably outnumbering P. esculentus.Atall
sites with the L-E-R population type, a highly
significant male-biased asymmetry was found
in hybrids, varying from 1:8 to 1:14 (table 1).
Mate choice at Osowiec in 2016 was examined
in 16 naturally formed amplectant pairs. Only
RR female ×LR male (12 pairs, 75%) and
LR female ×LR male (4 pairs, 25%) were ob-
served.
Morphology of hybrid males
P. esculentus males were heavier and larger in
L-E-R than in the L-E population type. Means
of body mass, SVL, and lengths of tibia and
the first toe in L-E-R males of P. esculentus
significantly exceeded those in the L-E popula-
tions (table 2, fig. 2). Despite the fact that the
population structure in Osowiec had changed
between 2010-2011 and 2016 (table 1), there
were no differences in body mass, SVL, T, and
DP in hybrid males from that locality between
these years (ANOVA, F1, 96 =0.15, P=0.696;
F1,96 =0.69, P=0.407; F1,96 =0.52, P=
0.472; F1,96 =2.19, P=0.142, respectively).
Tab le 2 . Mean values (±SE) of snout-vent length (SVL),
body mass, length of tibia (T) and length of the first toe of
the hind leg (DP) of Pelophylax esculentus males in L-E
and L-E-R population types in the Biebrza River Valley.
All mean values are adjusted from ANOVA (see Statistics
for details). Pvalues of the statistically significant effects
between population types are shown in bold. N, number of
individuals.
Pop. N SVL Body mass T DP
type (mm) (g) (mm) (mm)
L-E 67 65.0 ±1.2 26.5 ±1.5 30.1 ±0.6 8.2 ±0.2
L-E-R 128 71.5 ±1.4 35.0 ±1.6 32.9 ±0.7 9.0 ±0.2
F1,6– 12.40 14.28 10.23 12.45
P–0.012 0.009 0.019 0.012
366 A. Hermaniuk et al.
Figure 2. Comparison of body size and mass of Pelophylax esculentus males in L-E (circles) and L-E-R (squares) population
types at different sites. See table 1 for abbreviations of the site names and supplementary fig. S1 for their location. The sites
are shown in the geographic order, downstream along the river. Mean values (±SE) of a) snout-vent length (SVL), b) body
mass, c) tibia length (T), d) length of the first toe of the hind leg (DP).
Discussion
Genome composition and hybrid sex ratio
within population types
Our research revealed two types of popula-
tions, L-E and L-E-R, in different parts of the
Biebrza River Valley. The L-E type, found in
five populations in the Biebrza River Valley, is
widespread from western France to the Volga
River in Russia and usually occurs in small,
shallow water bodies (Plötner, 2005). This is
also the most widespread system in Poland (Ry-
backi and Berger, 2001). In the L-E system,
matings between LR females and LL males pro-
duce hybrid offspring in an equal sex ratio,
while matings between LR males and LL fe-
males produce daughters only (Graf and Polls
Pelaz, 1989) (fig. 3). We did not observe a
prevalence of P. esculentus females among hy-
brids at any of the studied sites. Given that
at three sites, the hybrid sex ratio did not dif-
fer from 1:1, we infer that the most common
type of pair within the L-E population type
that produced hybrids in the study area was
LR female ×LL male (fig. 3). Such a situa-
tion is common in L-E populations (Blanken-
horn, 1977). For example, the most common
types of naturally formed pairs in the L-E sys-
tem in western Poland were LL female ×LL
male (46%) and LR female ×LL male (41.3%)
(Skierska, 2011). Nevertheless, in some popu-
lations, mating between LR male ×LL fe-
male seem to be more frequent, as evidenced by
the female-biased asymmetry in hybrids found
in several L-E populations in central Europe
(Berger, Uzzell and Hotz, 1988).
The L-E-R population type, which we found
in the study area, is known mainly from cen-
tral and eastern Europe, but the knowledge
about hybrid persistence in these populations is
still insufficient (Lada, Borkin and Vinogradov,
Body size variation in hybrid water frogs 367
Figure 3. Possible combinations of mating between Pelophylax lessonae (LL) and Pelophylax esculentus (LR) in L-E
population type. In this type of population, both sexes of P. esculentus exclude the L genome prior to meiosis and clonally
transmit the R genome to gametes. Both combinations result in new hybrids but crosses between LR males and LL females
produce daughters only, which causes female-biased asymmetry in hybrid offspring. The description of symbols is the same
as in fig. 1.
Figure 4. Mating of P. esculentus (LR) males with P. ridibundus (RR) females is the most frequent mating type in the L-E-R
population type in the Biebrza River Valley. Two possible ways of hybridogenesis in P. esculentus males are shown. If the R
genome is excluded, the mating produces hybrid males only (left side). If the L genome is excluded, the mating produces P.
ridibundus females only (right side). Symbols are the same as in fig. 1.
1995; Rybacki and Berger, 2001; Borkin et al.,
2002; Borkin et al., 2004; Mikulíˇ
cek et al.,
2015; Herczeg et al., 2017). Regardless of the
differences in taxa contribution, at all three sites
we found significant male-biased asymmetry
in the hybrid sex ratio (table 1). Our obser-
vation that most naturally formed pairs at Os-
owiec consisted of RR females and LR males,
together with the highly male skewed sex ra-
tio, may suggest that male hybrids here exclude
the R genome in gametogenesis and clonally
transmit an L genome with a Y factor. Mat-
ings of RR females and LR males would thus
produce only LR males (fig. 4) similar to the
R-E system pattern that has been described in
detail in the upper Oder River, Czech Repub-
lic (Doležálková-Kaštánková et al., 2018). Al-
though the population structure changed in Os-
owiec in 2016, our conclusion was still sup-
ported by the highly male-biased sex ratio ob-
served in hybrids. Nevertheless, it must be
pointed out that further genetic investigation
(i.e. crossing experiments or extended popula-
tion genetics) is needed to determine the repro-
ductive mode of hybrids in this population type.
Similar mating preference to that in Biebrza
368 A. Hermaniuk et al.
was observed in the R-E populations of the
Barycz Valley (Skierska, 2011). The most com-
mon types of pairs observed in the breeding
season (54 pairs in amplexus were examined)
were also RR female ×LR male, despite the
fact that P. ridibundus males considerably out-
numbered the hybrid males. In this popula-
tion, a small fraction of P. lessonae was ob-
served (2.4% of the whole sample) and hybrid
males significantly outnumbered hybrid females
(Skierska, 2011). Considering the results of our
study, we cannot exclude the possibility that
some matings between RR females ×LR males
produce P. ridibundus females (fig. 4). Genetic
evidence suggests that P. ridibundus may also
originate from crosses between two hybrids,
which should skew the sex ratio in favour of fe-
males (Hotz, Beerli and Spolsky, 1992; Guex,
Hotz and Semlitsch, 2002; Mikulíˇ
cek et al.,
2014). In all L-E-R populations in the study
area, RR females were at least twice as com-
mon as RR males, which may reflect this phe-
nomenon.
Among the 607 individuals examined be-
tween 2010 and 2016, we did not record
any polyploids. In contrast, two decades ago,
Schröer (1996) observed a high proportion of
triploids in this area. Of the 39 frogs caught
by Schröer at three sites, two of which (Wizna
and Zajki) were examined in this study, 56.4%
were classified as triploids. It is difficult to ex-
plain such a high frequency of polyploids in this
area in the past, given the rarity or absence of
triploids in eastern Poland, as well as in adja-
cent Republic of Belarus and in the Kaliningrad
region (Borkin et al., 1986; Rybacki and Berger,
2001; Litvinchuk et al., 2015). Given the large
number of frogs examined in our study, the re-
sults of Schröer (1996) should be treated with
caution.
Morphology of hybrid males
We found that male hybrids in L-E-R population
type were larger than male hybrids in L-E type,
therefore, they were more similar to P. ridibun-
dus – the largest species in the P. esculentus
complex. This may reflect female (RR) mat-
ing preference in the L-E-R population type de-
scribed herein and in Barycz Valley (Skierska,
2011). Furthermore, the smaller size of male
hybrids in L-E populations may reflect mating
preferences of LL and LR females – both prefer
smaller P. lessonae males (Abt and Reyer, 1993;
Roesli and Reyer, 2000). Our results are parallel
to those of Hoffmann and Reyer’s (2013), who
found that call parameters – an important trait
in amphibians that serves as a signal of male
quality and is used for species recognition –
varied in LR hybrids between population types
in agreement with the sexual host required for
successful reproduction. The authors indicated
greater similarity of LR calls to P. ridibundus
from the L-E-R populations compared to those
from the E-E, where RR males were absent. The
larger body size of LR males in L-E-R (with low
frequency of LL males) or R-E populations type
may provide an additional reproductive bene-
fit, because they show, as RR males, territorial
behaviour (Lengagne, Plenet and Joly, 2008).
Larger hybrids presumably defend their territo-
ries more efficiently, which could be more at-
tractive for RR females. Such reproductive be-
haviour cannot be effective in the L-E popula-
tion type because P. lessonae males do not de-
fend territories; they are more mobile and attract
females more readily than P. esculentus males
(Lengagne, Plenet and Joly, 2008).
Diploid hybrids in general clonally transmit
only their unrecombined genome to progeny
(figs. 3, 4). Therefore, to affect body size of hy-
brid males the selection forces would generally
require inter-clonal selection that was proposed
by Vrijenhoek and Parker (2009). Alterna-
tive mechanisms, not based on selection, could
also explain our observations. Due to clonally
transmitted genomes, LR hybrids may accu-
mulate deleterious mutations through “Muller’s
ratchet”, even if there is a possibility of episodic
recombination of clonal genomes and/or occa-
sional introgression between parental genomes
(Vorburger et al., 2009; Mikulíˇ
cek et al., 2014).
If such deleterious mutations affect the genes
Body size variation in hybrid water frogs 369
responsible for a male trait, it may cause hy-
brid males to resemble the parental species phe-
notype more. This is because clonal genomes
are sheltered by sexual host genomes that pre-
vent their occurrence in the homozygous state
and reduce the predisposition to a certain dis-
order by their epistatic interactions (Vorburger
et al., 2009). Counterintuitive, such deleterious
mutations could be beneficial for hybrids by in-
creasing their chances of mating with females
of the sexual host (Bove, Milazzo and Barbuti,
2014).
Another explanation of the larger body size
of male hybrids in the L-E-R population type
may refer to potential episodes of spontaneous
heterosis (Hotz et al., 1999). In the L-E-R
type, especially with a high frequency of P.
lessonae males (e.g., in Dobarz in our study
area), the probability of primary hybridization
increases considerably. The combination of dif-
ferent parental genomes in newly generated F1
hybrids can lead to increased levels of somatic
heterozygosity and therefore to production of
extreme phenotypes. It has been shown that
these F1hybrid offspring have higher survival,
higher early growth rates, and earlier metamor-
phosis compared with either parental species
(Hotz et al., 1999). Thus, in the L-E-R popula-
tion type, high frequency of primary hybridiza-
tion could even strengthen the effect of sexual
selection since spontaneous heterosis may in-
crease body size of hybrid males as well. The
largest LR males observed in Dobarz (see Re-
sults) may support our assumption.
Conclusions
Two types of population, L-E and L-E-R, were
described in the study area. We found sig-
nificant male-biased asymmetry in the hybrid
sex ratio in the L-E-R population type, which
might be the consequence of predominant mat-
ing between LR males and RR females. In the
present study, we demonstrated for the first
time that diploid hybrid P. esculentus males
from the L-E-R populations were consider-
ably larger than those from the L-E popula-
tions. We suggest that this could be the ef-
fect of selection pressure associated with re-
quirements of sexual hosts female for success-
ful reproduction. Another explanation of hy-
brid body size variation might be the effect of
epistatic interaction between differently inher-
ited genomes affecting the male traits used by
females to species recognition. The larger body
size of hybrid males could also be the effect of
higher somatic heterozygosity that may occur
in L-E-R breeding as a result of primary hy-
bridization between sexual host species. How-
ever, this area of research requires further ge-
netic study.
Acknowledgements. We thank S. Gadomska, J. Perkowska,
K. Butwiłowska, Ł. Ołdakowski, and R. Kossakowski for
their assistance with field and laboratory work. We also
thank P. Kierzkowski and M. Ogielska for their support and
advice during the densitometric analysis of DNA content.
We are grateful to Thomas Uzzell and three anonymous
reviewers for valuable comments on an earlier draft of the
manuscript. Natalia Ochman kindly improved the English.
This article has received financial support from the Polish
Ministry of Science and Higher Education under a subsidy
to maintain the research potential of the Faculty of Biology
and Chemistry, University of Białystok.
Supplementary material. Supplementary material is avail-
able online at:
https://doi.org/10.6084/m9.figshare.11902011
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Submitted: July 10, 2019. Final revision received:
February 24, 2020. Accepted: February 25, 2020.
Associate Editor: Ariel Rodríguez.
