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Aquaculture 295 (2009) 30–37

Contents lists available at ScienceDirect

Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Suitability of European green frogs for intensive culture: Comparison between

different phenotypes of the esculenta hybridogenetic complex
André Neveu ⁎
INRA, Agrocampus Rennes, UMR 985, F-35042 Rennes Cedex, France

a r t i c l e i n f o a b s t r a c t

Article history: European countries are probably the greatest consumers of frogs' legs, although this is occurring in a context
Received 11 August 2008 of amphibian decline. Imports of frogs' legs have been increasing over the last few decades in relation to the
Received in revised form 19 June 2009 development of deep-frozen products. This demand has stimulated farming with neotropical species for
Accepted 20 June 2009
international trade. Today, in spite of some trials on European species, no production is effective. From the
early 1990s, small-scale experimental rearing trials have been undertaken in Brittany (France) with a local
Keywords:
Raniculture
green frog belonging to the esculenta complex (Rana ridibunda) which accepts granulated feed. This complex
Phenotype comparisons comprises two Mendelian species R. lessonae (LL) and R. ridibunda (RR) as well as their hybridogenetic
Survival rates hybrid Rana esculenta, which can be either diploid (RL) or triploid (RLL, RRL).
Growth The purpose of this study is to analyse the ability of each taxon to be reared. During the two months
Rana esculenta complex following metamorphosis, froglets were trained to eat pellets. The surviving individuals were reared for one
year under regulated conditions to estimate the survival rate, growth and production of each frog type.
The results show low survival rates (14.8 to 26.2%) for LL and RLL, higher rates (39–46.4%) for F1 and F2 from
wild RR and RLL, with the highest rates (63.0 to 77.4%) for RL, RRL and all RR, using data from several years of
rearing. After three years under rearing conditions, the adult survival rate is highest for rearing strain RR-
Rivan 92* (53.7%). The growth rate varies greatly according to phenotype, and only frogs with an RR
phenotype reach marketable size. In the wild, LL exhibits some difficulty in reaching this limit. As a result,
production increases from 1.5–8.7 kg/m2 for individuals with an L hemiclone to 22.9–35.7 kg/m2 for each
cohort of phenotype RR from at least two generations under rearing conditions.
In relation to hybridogenesis processes, hybrids with diploid or triploid genomes do not seem to offer any
advantage for production purposes. However, R. lessonae individuals from intensive rearing can produce
fertile eggs and tadpoles, which could enable production for restocking to preserve wild diversity or build up
new populations in suitable habitats.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction consisted of specimens of the Rana (Pelophylax) ridibunda group

European countries are currently among the greatest consumer of
frogs' legs, accounting for 8000–10,000 T/year, with France repre-
senting around 45% of this consumption (Hardouin, 1994; Neveu,
1985, 2004a). The French market comprises two types of product:
fresh frogs' legs (for restaurants) and frozen or deep-frozen frogs' legs
(for major distribution outlets). In Europe, the decline in frog
populations increased after the Second World War leading to recourse
to imports during the 1960s, which was further reinforced by the
development of deep-frozen products. For France, imports of frozen
frogs' legs rose considerably over the least 30 years, and currently
account for 3000–4000 T/year, coming mainly from the Far East and
consisted of local species. During the same period, imports of live frogs
(for fresh frogs' legs) remained stable at 600–800 T/year, and
⁎ Tel.: +33 2 23 48 54 46; fax: +33 2 23 48 54 40.
E-mail address: Andre.Neveu@rennes.inra.fr.
from Mediterranean countries (Turkey, Egypt, etc.).
For a long time, imports were composed of wild specimens,
harvested in various aquatic systems according to traditional methods.
Humans were historically and quantitatively perhaps the most
important predators of frogs. While amphibians are declining more
rapidly than either birds or mammals in many areas, there is no
agreement about the causes of this decline which seem to vary
according to the country concerned (Stuart et al., 2004; Reading,
2007).
Whereas the majority of imports are still collected in the wild, a
substantial demand over the last decade has stimulated research on
intensive farming with various neotropical species. At present, the
American bullfrog (Rana catesbeiana) appears to be the most
commercially important species produced in various systems based
on the concept proposed by Lima and Agostinho (1992). Data
concerning European species are very scarce, but there is nevertheless
some continuing interest in local frog farming using Rana (Pelophylax)
perezi in Spain (Real et al., 2005; Alvarez and Real, 2006) or Rana

0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2009.06.027
 Author's personal copy
A. Neveu / Aquaculture 295 (2009) 30–37
temporaria in England (Miles et al., 2004). One of the major difficulties
of frog farming is that anurans require moving prey to elicit feeding
behaviour. To be economic, cultivated frogs would have to be fed on
artificial foods rather than live prey. Hence, the most current practice
in bullfrog cultures involves the use of pellets mixed with live
domestic fly larvae (to stimulate pellet consumption). Nevertheless, it
would not appear possible to completely suppress the addition of
larvae in the case of R. perezi (Real et al., 2005), and there is currently
no intensive production system applied in Europe.
In Western France, rearing trials began during the 1980s using local
green frogs belonging to the Rana (Pelophylax) esculenta complex. This
complex is composed of two Mendelian species [R. (Pelophylax) lessonae
and R. (Pelophylax) ridibunda] and their natural hybridogenetic hybrid
(R. esculenta), which can be either diploid or triploid. Among these
species, R. lessonae is the smallest, R. ridibunda is the largest, while R.
esculenta is intermediate. R. lessonae lives in small water bodies (pools,
ponds, marshes, etc.), R. ridibunda inhabits large water systems (lakes,
rivers, etc.) and R. esculenta can be found in all waters (Berger and
Rybacki, 1997). In the wild, pure populations of R. lessonae and R.
ridibunda are very scarce in the studied area, while the most common
situation consists of mixed populations associating a parental species
(lessonae) with the hybrid (Régnier and Neveu, 1986). Generally
speaking, pure R. esculenta populations are not fertile, but the crossing
with one or the other parental species is fertile. In the latter case, the F1 of
R. esculenta × R. lessonae pair gives R. esculenta and the F1 of
R. esculenta ×R. ridibunda gives R. ridibunda. The hemiclone R. lessonae
is eliminated prior to meiosis (hybridogenesis) and the remaining
R. ridibunda genome is transmitted clonally to eggs and sperm cells
(review in Graf and Polls Pelaz, 1989; Berger, 2008).
In the West of France, the growth curves of green frogs under wild
conditions are related to the taxon concerned (Neveu, 1991, 1996).
After some years of rearing trials, we only succeeded in the 1990s to
get R. ridibunda to accept floating pellets. After a strong mass
selection, a small experimental rearing setup is currently operational
with adults that can eat any pellets (Neveu, 2004b, 2005 and
unpublished data). This is the first case of intensive culture of frogs
living in temperate zone of the Eurasian continent.
In view of the genetic complexity of European green frogs, we
considered it interesting to compare the rearing potential of the
different components of the R. esculenta complex. The purpose of
the present study is to compare the survival rates, growth and the
average life span of each taxon by comparing R. ridibunda frogs from
intensive rearing and frogs under wild conditions. We attempt
to estimate the impact of these characteristics at the level of in-
tensive rearing, i.e. with a very large number of individuals (30,216
froglets).
2. Materials and methods
In the following, species are designated by LL for R. lessonae and RR
for R. ridibunda, with RL for the diploids and RLL or RRL for the
triploids (males only) of R. esculenta, using a subscript w for wild
adults and r for reared adults.
The adult frogs resulted either from natural biotopes or from an
intensive rearing of R. ridibunda (code: RR-Rivan 92*). [This strain
is protected by a know-how license of the French National Institute
of Agronomic Research. Today, it is the only species which is
permitted by Law for rearing in France and not exportable in a
living state for ethical and ecological reasons]. This intensive
rearing takes place at Rennes (48°6′52″N, 1°40′45″W) in Brittany
(Western France). The wild adults were captured just before
pairing, at two sites: site A is located in marshes near Redon
(47°39′08″N, 2°05′01″W) with a LL–RL population, while site B is
located in ponds near Rennes with a RR–RLL population, and is the
same area used for collecting frogs to set up the intensive rearing.
The distance between the two sites is approximately 70 km. A
31
survey along a north–south axis (140 km) showed that LL–RL
populations are the most common in Brittany, with LL as the most
abundant in marshes and RL dominant in ponds, often containing
numerous RLL triploids. Some patches from pools in meadows
show pure hybrid populations, with RLL triploids accounting for up
to 95% (Régnier and Neveu, 1986). RR is found only in the central
part of the region (around Rennes) in pure populations as well as in
RR–RL and RR–RLL populations (Neveu, 1996). According to some
studies, this species seems to be more common than expected in
France (Pagano et al., 2001, 2003).
Determination of frog age was performed by osteochronology. The
characteristics of these populations have been established in previous
studies. At site A, the lifespan is short and limited to around two years,
with only some rare individuals older than 3 years (Régnier and Neveu,
1986; Neveu, 1991, 1992). At site B, after a period of colonisation of
new ponds by RL–RLL, the population evolved progressively into a new
RR–RLL system (Neveu,1996). Growth rates are similar for both taxa, but
RR females appear to have a longer life (up to 7–8 years) and hence their
weight might attain 150 g (unpublished data).
The first data on the intensive rearing of RR showed that froglets
could eat pellets while attaining good survival (up to 92%), and that
adults of trade size are produced within one year. However, intensive
rearing without winter resting seemed to have reduced the annual
clutch number per female and also decreased egg fertility (Neveu,
2004b, 2005).
The taxonomic identification of individuals was based on classic
morphological characters as proposed by Berger (1966), an approach
still used regularly by many authors (Ragghianti et al., 2007; Plötner
et al., 2008). The most significant of these characters include colour,
spot pattern and callus morphology, but according to Fog (1994), the
femur spot pattern is the most constant difference. These criteria were
supplemented in some samples of individuals by classic analysis of
proteins (LDH, albumin), as has been applied by numerous authors
since Tunner (1973). The possibility that a cryptic species of
R. ridibunda was introduced cannot be ruled out. Due to lack of
relevant marker for genotyping, we will consider all individuals with a
R. ridibunda phenotype as RR strictly speaking, which is in agreement
with most studies conducted in Western Europe.
In our study, triploids were detected by the size of their
erythrocytes at age of 14 months (10 erythrocytes measured for
each blood smear) according to Ogielska-Nowak (1978). This method
is the simplest and quickest for the analysis of large samples. There is a
good agreement between the chromosome counts and erythrocyte
size (Uzzel et al., 1975), and hence this criterion is widely used (Hotz
et al., 1997; Christiansen et al., 2005; Plötner et al., 2008). The
detection of triploids was confirmed by gene–dose effects in the
results of electrophoresis (LDH) and during some amplification
performed for molecular studies (Plénet, pers. comm.). At site A, we
determined erythrocyte areas of 268 µ2 (σ = 24.0, n = 154) for
diploids and 415 µ2 (σ = 21.2, n = 16) for triploids, while, at site B
we obtained 265 µ2 (σ = 26.8, n = 30) for diploids and 387 µ2
(σ = 28.6, n = 90) for triploids (t = 20.5, P b 0.0005; t = 23.5,
P b 0.0005, respectively). During a 25-year monitoring study, we
found 9% triploids on average in hybrids at site A, and from 50% (1989)
to 100% (from 1993 to present) at site B (hybrid males only, no female
detected). At seven different sites, only 1% of a sample of 692
individuals was difficult to classify (no cases at site A or B).
Since our main purpose was to test the rearing characteristics
(survival and growth) of the various phenotypes, only froglets that
survived on pelleted feed during the first two months of their life were
kept for experimentation. Pairs producing less than around 100
froglets were not taken into consideration. This was the case with
many usually infertile hybrid pairs (RL × RL). Even if some of these
pairs produced RR froglets, they rarely accepted pelleted feed.
Similarly, for some individuals, the survival during the first two
months was highly variable from one pair to another.
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32 A. Neveu / Aquaculture 295 (2009) 30–37

After 12 months of rearing with pellets, the frogs (19,163 individuals,
14 months old) were counted and determined. Measurements were
performed for the mean individual size (TL = snout–vent length to
the nearest mm) and the harvested biomass (live weight to the
nearest 0.1 g). We converted length into biomass using the relation-
ship established on 70 individuals (14 months old) of both sexes:
W = 5.01 10− 5TL3.26, with W expressed in g of fresh wet weight and TL
in mm. Only adults could be sexed, the males being distinguished by
the presence of vocal sacs and thumb pads.
Two-month-old froglets were reared in different fibreglass tanks (at
the same height from bottom) with maximum densities equivalent to
300–400 m− 2 (preliminary tests with RR showed no density effect at
this level; total rearing area availability: 12 m2 each year) and housed in
a room at 20–22 °C (ventilated to homogenize the temperature; inter-
tank difference: ≈1 °C) with a 12 h–12 h photoperiod. Water level (tap
water) in the tanks was about 1 cm and renewed approximately twice
daily. In all cases, if red legs disease was detected in a given tank, the
whole sample was destroyed for prophylactic purposes. A complete
disinfection of each tank was carried out every 2–3 months, with a tank
change for each frog batch. The fish pellets were distributed twice daily
ad libitum. The floating pellets were 2 mm in diameter and contained
34% crude protein. According to Somsueb and Boonyaratpalin (2001),
such content appears sufficient for R. rugulosa.
Data were analysed using parametric and non-parametric tests
according to Siegel and Castellan (1998) and Tomassone et al. (1993).
We used the following abbreviations: t for Student test (tc, corrected
for unequal variance), r for Pearson correlation coefficient, F for Fisher
test, Rs for Spearman correlation coefficient, χ2 for Chi-square test
and U for Wilcoxon–Mann–Whitney test.
The results include all the data acquired over eight years, subject to
the availability of certain taxa. During this period, the cultured strain
(RR-Rivan 92*) failed to show any correlation between annual survival
and growth (Rs = 0.02, Rs = 0.14, respectively), or between survival
rate and growth with density at harvest (Rs = 0.24, Rs = 0.48,
respectively). Hence the rearing conditions could be considered as
standardized and the results of all trials were grouped according to the
composition of pairs. Biometrical comparisons confirm that frog size
shows a normal distribution.
3. Results
3.1. Survival at 14 months under rearing conditions
After one year of growth under intensive rearing conditions, the
survival rate differed between taxa (Table 1). In most cases, only one
phenotype was harvested for each pair, and sometimes two, so these
would represent potential individuals for rearing.
For F1 of the first group with wild parents, the data showed that LL
had the lowest survival rate, being lower than either RLL (χ2 = 13.0,
P b 0.005) or RR (C5 and 6) (χ2 = 74.5, P b 0.005). The survival rate of
RLL was lower than RR (C5 and 6) (χ2 = 91.7, P b 0.005), while the
survival of RL was higher (C2 and 3) (χ2 = 37.6, P b 0.005). For the
same pair, the survival of RLL was lower than RR (χ2 = 316.7,
P b 0.005). For the second generation under rearing conditions, the
triploid RLL displayed a better survival (χ2 = 30.9, P b 0.005) than for
the first generation, but this was not the case for the corresponding RR
(C6). The survival rate of RL (C2) from site A was higher than RL (C3)
of mixed origin (χ2 = 4.5, P b 0.05).
The mean survival of RR-Rivan 92* (C8) was better than F1 + F2
(χ2 = 173.0, P b 0.005). It is difficult to draw any conclusions for the RR
produced by the pair C3, due to the insufficient number of frogs in the
sample.
Back-crossing between an intensively-reared female and a RR wild
male increased the survival of F1 (χ2 = 40.1, P b 0.005). By contrast,
back-crossing between an intensively-reared male and a RR wild
female did not change the survival of F1 (χ2 = 0.17). The first hybrid
lineage yielded a better survival rate than the second (χ2 = 32.0,
P b 0.005), while the self-crossing of this lineage gave the same
survival at F2. Back-crossing between an intensive rearing female and
a RLL wild male yielded two genotypes as observed with the wild pair
(C4), with the survival rate of descendants increasing even more for
RR (χ2 = 91.8, P b 0.005) than for RLL (χ2 = 4.4, P = 0.03).
A rare triploid male RRL from site B was crossed with an
intensively-reared female (C12). The resulting lineage was made up
solely of triploid males with a good survival, which was less than with
a wild RR male (C9) (χ2 = 9.6, P b 0.005) but better than from crossing
with a RLL male (C13, χ2 = 55.2, P b 0.005).

Table 1
Comparison between survival rates and mean sizes of phenotypes from green frog esculenta complex after 14 months of life under intensive rearing conditions with granulated feed.

Cross no. Pairs Origin n Descendants N0 Nf Survival NTL TL (σ)

♀×♂ (site) Phenotypes F (%) (mm)
A. Wild pairs
C1 LL × LL A 5 LL 1 793 118 14.8 118 42.8 (3.5)
C2 LL × RL A 1 RL 1 311 242 77.8 32 43.2 (4.5)
C3 RR × RL B.A 1 RL + 1 408 289 69.1 35 37.4 (4.3)
RR 1 10 6 60.0 – –
C4 RR × RLL B 3 RLL+ 1 1078 231 21.4 89 41.8 (2.5)
RR 1 288 113 39.2 74 62.1 (5.3)
C5 RR × RR B 4 RR 1 765 299 39.0 126 58.6 (3.7)
C6 RR(F1C5) × RR(F1C5) B 3 RR 2 882 345 39.1 84 59.3 (4.2)
C7 RL × RLL (F1C4) A.B 3 RLL 2 97 45 46.4 54 46.6 (2.2)

B. Rearing pairs
C8 RR × RR B 73 RR 6–9 21,541 14,652 67.9 3087 63.3 (3.8)

C. Hybrid pairs
C9 (RR)r × (RR)w B 4 RR 1 1030 798 77.4 128 60.8 (5.0)
C10 (RR)w × (RR)r B 6 RR 1 1938 1309 67.5 201 61.5 (5.0)
C11 RR(F1C9) × RR(F1C9) B 2 RR 2 494 379 76.7 60 67.7 (5.0)
C12 (RR)r × (RRL)w B 1 RRL 1 92 58 63.0 58 45.0 (6.1)
C13 (RR)r × (RLL)w B 1 RLL+ 1 210 59 28.0 20 43.7 (2.9)
RR 1 279 220 78.8 107 66.5 (7.0)

Cross no.: pair reference; w: wild; r: rearing; pairs: with phenotype of parents and, eventually, whether they come from a first-generation living under rearing conditions; origin:
harvest site of pairs; n: tank number; descendants: phenotypes surviving after 14 months and generation level; N0: number of froglets at the beginning of the study period, 2 months
old; Nf: number of adults surviving after 12 months; survival: Nf/N0 ratio in percent; NTL: number of adults measured; TL: mean total length in mm, 14 months old; σ: standard
deviation.
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A. Neveu / Aquaculture 295 (2009) 30–37
Table 2
Comparison between mean sizes (TL: total length in mm, σ: standard deviation) of
green frog (esculenta complex) attained after two summers of life at wild sites where
experimental pairs were caught.
Sites Phenotypes n TL (σ) t P b 0.0005), and were more variable (F = 4.18, P b 0.005). The RL
(mm)
A. Redon LL 66 48.2 (3.1) 1.72 (P b 0.05)
RL 50 49.2 (3.1)
RLL 5 59.4 (6.5)
B. Rennes RL 10 59.5 (2.8)
RLL 114 58.7 (3.1) 4.71 (P b 0.0005)
RR 80 61.0 (2.2)
Site A: males and females; site B: males only; t: Student test for each site; n: number of
frogs.
3.2. Size at 14 months
3.2.1. Size under wild conditions
The 14-month-old reared frogs corresponded to 1+ frogs (two
summers old) caught during field studies in September (Régnier and
Neveu, 1986; Neveu, 1992). There were some differences in mean size
according to the sites and the species composition (Table 2). At site A,
the mean size of RL was larger than LL (t = 1.7, P b 0.05), while, at site
B, the mean size of RR (males) was larger than RLL (males) (t = 4.7,
P b 0.0005). The mean size of RLL at site B was larger than RL at site
A (t = 18.5, P b 0.0005). This difference appears related to the biotope
qualities because, at site A, the rare triploids RLL (n = 5, TL = 59.4,
σ = 6.5) do not differ from RL (t = 1.3, P = 0.10) and, at site B, the
rare diploids RL (n = 10, TL = 59.5, σ = 2.8) do not differ from RLL
(t = 0.7, P = 0.24).
3.2.2. Size comparison between wild and intensive rearing conditions
At 14 months, there was no significant difference in size between
males and females of wild RL and LL (Régnier, 1983). The average size of
RR-Rivan 92* remained unchanged over the study period (1998–2005),
with measurements showing no significant trend (Rs = 0.09) or
difference between male (TL = 61.1 mm, σ = 2.2, n = 80) and female
(TL = 60.7 mm, σ = 2.5, n = 46) (t = 0.72). This data set can be used as a
reference in comparison with the other phenotypes (Table 1).
At the same age, the F1 mean sizes of reared LL, RL(C2) and RL(C3)
were smaller than the corresponding wild individuals (Table 2)
(t = 10.8, tc = 6.6, t = 14.7, respectively, all with P b 0.0005). The wild
RLL individuals from site B (Table 2) were larger than the corresponding
reared F1 individuals (tc = 43.0, P b 0.0005), and were more hetero-
geneous (F = 1.53, P b 0.02). In comparison with the reared F2, they were
also larger (tc = 29.1, P b 0.0005) and more heterogeneous (F = 1.98,
P b 0.005). However, the second generation showed an increase in mean
size compared to the first (t = 11.6, P b 0.0005).
At site B, the mean size of wild RR at the same age (Table 2) was
larger than F1 under rearing conditions (tc = 5.0, P b 0.0005) but more
homogeneous (F = 2.82, P b 0.005), the same being true with F2
(tc = 3.2, P b 0.02; F = 3.64, P b 0.005). However, the individuals were
smaller than RR-Rivan 92* (tc = 5.4, P b 0.0005) and less variable
(F = 2.98, P b 0.005).
3.2.3. Size comparison between phenotypes under rearing conditions
In the first generation, the LL frogs were larger than the RLL frogs
(tc = 2.4, P = 0.01) and more variable in size (F = 1.96, P b 0.005). The
first-generation LL frogs were smaller in size than the second-generation
RLL (tc = 8.6, P b 0.0005), but more heterogeneous (F = 2.53, P b 0.005).
For the pair from site A, LL frogs had the same size as RL (tc = 0.5), but
were less variable (F = 1.65, P b 0.005). On the contrary, the LL frogs were
larger when RL was of mixed origin (female from site B, male from site
A) (C3) (t = 7.6, P b 0.0005). There was also a difference in the mean size
of both RL types (t = 5.4, P b 0.0005).
33
For the first generation in captivity, RL diploid hybrids from the
site-A pair were larger than RLL triploid hybrids from the site-B pair
(t = 1.7, P b 0.05) as well as more variable (F = 3.24, P b 0.005). They
were both smaller than the second-generation RLL (tc = 4.4,
from the pair of mixed origins were smaller than RLL (C4 and 7)
(tc = 5.7, tc = 11.7, respectively, all P b 0.0005), and also more variable
(F = 2.95, F = 3.82, respectively, all P b 0.005). The RLL triploids were
always smaller than RR and, in the case of the same pair, they were
much smaller than their RR brothers (tc = 30.2, P b 0.0005) but more
homogenous (F = 4.5, P b 0.005). These latter were more heteroge-
neous than the RR from a RR × RR pair (F = 2.05, P b 0.005) and larger
(tc = 5.0, P b 0.0005).
Although the RR individuals from homogeneous RR pairs had the
same size in F1 and F2 (t = 1.27), they were smaller than the RR-Rivan
92* (t = 13.6, t = 9.5, respectively, all P b 0.0005).
3.2.4. Influence of back-crossing
The reared female × wild male pair (C9) produced smaller
individuals than the rearing pairs (C8) (tc = 5.6, P b 0.0005) and
more heterogeneous (F = 1.73, P b 0.005). The reverse combination in
the parental pair (C10) produced smaller individuals than the rearing
pairs (tc = 5.0, P b 0.0005), but always more heterogeneous (F = 1.73,
P b 0.005). The crossing between previous hybrids led to an increase in
the mean size of F2 (t = 8.8, t = 8.4, respectively, all P b 0.0005), which
became even greater for individuals from the rearing pairs (tc = 6.7,
P b 0.0005), with the individuals remaining more heterogeneous
(F = 1.73, P b 0.005). It should be noted that, initially, the reared
individuals were larger at the age of 2 months compared with
previous hybrids (TL = 32.5 mm, σ = 3.4, n = 117; TL = 30.3 mm,
σ = 3.5, n = 96, respectively) (t = 4.6, P b 0.0005). For RR-Rivan 92,
there was a correlation between the weight (in g) at 14 months (Wf)
and the weight at 2 months (W0): Wf = 19.90W00.43 (r2 = 0.80, n = 12
samples, P b 0.005).
The reared RR female × wild RLL male pair gave rise to larger
individuals than the wild pair (t = 3.0, P b 0.005 for RLL; t = 4.5,
P b 0.0005 for RR) and more heterogeneous in RR (F = 1.74, P b 0.01).
The reared RR female × wild RRL male pair yielded smaller individuals
than the rearing pairs (tc=22.6, P b 0.0005) and more variable (F = 2.6,
P b 0.005). The mean size was greater than for F1 RLL (tc=3.8, P b 0.0005)
and smaller than F2 RLL (tc=1.8, P b 0.05), while the individuals were
more heterogeneous (F = 5.6, F = 7.7, respectively, all P b 0.005).
4. Growth and survival of phenotypes during study period
Certain groups were monitored over several years, with some
individuals able to live a few years in intensive breeding and others
rapidly showing a significant mortality (Fig. 1). The LL showed the
fastest decline in density, with some individuals living for three years
and reproducing during the second summer. The RL diploid hybrids
showed a moderate mortality rate during the first two years, but then
the mortality increased with individuals living no older than LL. Both
triploid hybrids (RLL and RRL) had a better survival than diploids at
3 years (around 40%). However, the rate of mortality increased more
rapidly at the beginning of growth for RLL. Some individuals were able
to live for five years. The intensively-reared RR-Rivan 92* showed the
best survival and the longest lifespan (at present, some individuals are
16 years old). At the beginning of their life, both RRL and RR-Rivan 92*
displayed very similar survival curves.
In the same way, the different frogs showed differences between
their growth curves (Fig. 2). After several years of rearing, RR-Rivan
92* had a higher growth curve compared with wild RR at the same age
(Fig. 2A). In the wild, the growth of RLw diploid hybrids was greater
than for LLw at the same site. On the other hand, RLr2 and RLr3 (F1
from C2 and C3, respectively) showed a slowing down of growth
during rearing, comparable to LLr (Fig. 2B). The RLLw wild triploids
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34 A. Neveu / Aquaculture 295 (2009) 30–37

assuming 1000 froglets (2 months old) per square metre at the

beginning of the study period. After 12 months of growth, the
production potential varies over a wide range, from 1.5 to 35.5 kg/
m2 according to phenotype (Table 3). The production potential with
RR-Rivan 92* is 16 times that of LL. Back-crossing with wild individuals
seems to increase (×1.4) the production after two generations. In
cases where two phenotypes are produced by the same pair (C4), RR
accounts for 67.6% of the production with only 24.6% of the total
density. Under the same conditions, assuming RR is the only
phenotype produced by RR × RLL, then the production would be
13.8 kg/m2, i.e. very close to the results with wild RR × RR. In the

Fig. 1. Survival of phenotypes from green frog esculenta complex during the first five
years of their lives under intensive rearing conditions (LLr: R lessonae; RRr: R. ridibunda;
RLr2, RLr3: R. esculenta diploid, F1 from C2 and C3 in Table 1 respectively; RLLr, RRLr:
R. esculenta triploid).

showed a greater growth compared to individuals under rearing

conditions (Fig. 2C).
During the cold period, from mid October to mid March
(5 months), the wild frogs cease their feeding activity. We can
compare the growth curves by considering only the months with
feeding activity in the wild. The results show that RRw, RLw and RLLw
had a very similar growth curve to RRr, but this was not the case for
LLw (Fig. 3). According to this approach, frogs of the same size could
have very different ages (e.g. at a mean size of 80.6 mm, RRw were
60 months old, while at 81.2 mm, RRr were only 35 months old). The
differences in growth between wild and rearing conditions were
clearly more marked in hybrids RL and RLL than in LL.
The best-fitted logarithmic models for each growth curve yield
correlation coefficients that are all significant below the 5% threshold
(Fig. 4), which is not the case for the K value estimated from Van
Bertalanffy curves in relation to winter inactivity of wild individuals.
We can thus distinguish two groups among the growth curves: G1
refers to frog types associated with the best growth (RRr and RRw, as
well as the wild hybrids RLw and RLLw), while G2 refers to those
displaying the lowest growth (LLw, LLr and all hybrids from rearing).

5. Aquaculture potential of taxa

Taken as a whole, the data set suggests that only RR have a good
potential for intensive rearing purposes (Table 3). After 14 months
under rearing conditions, the mean individual size for all the RR group
was 61.9 mm (σ = 3.02, n = 7 groups), as against 42.8 mm (σ = 3.15,
n = 6 groups) for others with at least one L (with a significant
difference between sizes: U = 0, P b 0.001). The survival rate for RR
groups was low (39.0–39.2%) in F1 or F2 generations (Table 1: C4, 5, 6),
but after 6–9 generations, it increased to 67.9% (C8), while back- Fig. 2. Growth of phenotypes from green frog esculenta complex during the first five
crossing with wild individuals led to good survivals of between 67.5 years of their lives under intensive rearing conditions. Comparison between frogs under
rearing conditions and frogs in the wild at the same age (TL: total length in mm).
and 76.7% (C9, 10, 11). A. Ridibunda group (RRr: reared, RRw: wild). B. lessonae and diploid esculenta group
The production potential can be simulated using survival data from (LLr: reared; RLr2, RLr3: reared, F1 from C2 and C3 in Table 1, respectively; LLw, RLw:
Table 1 and individual mean weights corresponding to mean sizes, wild). Triploid esculenta group (RLLr, RRLr: reared; RLLw: wild).
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A. Neveu / Aquaculture 295 (2009) 30–37 35

Table 3
Simulation of potential production (P in kg m− 2) for each green frog type (esculenta
complex), assuming an initial density of 1000 froglets m− 2 (2 months old) and after
12 months under intensive rearing conditions (same caption as in Table 1; subscript:
parental origin; W̄: mean individual weight in g; market size of around 30 g each).

References Descendants W̄ P Market

Phenotypes F (g) (kg/m2) size

1 (LL)w 1 10.44 1.54 −

2 (RL)w 1 10.85 8.44 −
3 (RL)w 1 6.73 4.65 −
4 (RLL)w 1 8.30 1.63 −
(RR)w 1 35.17 3.41 +
5 (RR)w 1 29.11 11.35 +
6 (RR)w 2 30.20 11.80 +
7 (RLL)w 2 18.80 8.72 −
8 (RR)r 6–9 37.69 25.55 +
9 (RR)r w 1 32.82 25.41 +
10 (RR)r w 1 34.07 22.99 +
11 (RR)r w 2 46.60 35.75 +
12 (RRL)r w 1 12.30 7.75 −
13 (RLL)r w 1 11.16 1.35 −
(RR)r w 1 43.87 19.70 +

After 14 months of life, only RR-type individuals show an average

Fig. 3. Growth of phenotypes from green frog esculenta complex after the first winter
and according to months of effective feeding activity. Comparison between frogs under
rearing conditions (curves with points) and frogs in the wild (curves without points)
(TL: total length in mm; GET: Growth Effective Time, i.e. number of months with
effective feeding activity, assuming winter inactivity during five months for wild
individuals).
preceding pair, the substitution of the RR wild female with a RR reared
female (C13) gives RR, accounting for 93.5% of the production with
78.7% of the total density. When RR is the only type produced, then the
production would be 34.52 kg/m2, which is very close to the result for
the mixed pair C11.
Fig. 4. Logarithmic models fitted to growth curves of phenotypes from green frog es-
culenta complex. G1: fast-growing group with RRw, RLw and RLLw wild frogs, as well as
the rearing strain RRr; G2: slow-growing group with several rearing frogs (LLr, RLr1,
RLr2, RLLr and RRLr) and only one wild type LLw. The phenotypes are arranged in order
of decreasing size at 12 months (TL: total length in mm).
weight above 30 g, which can be regarded as the minimum size for the
French market.
6. Discussion
According to Dubois and Ohler (1994), the esculenta complex
should be incorporated into the subgenus Rana (Pelophylax). For
practical reasons, and in agreement with virtually all authors over the
last few years, we have kept the name Rana. In addition, we prefer to
retain the vernacular term “green frogs”, which is the older
established usage in English based on colour names (like brown
frogs) and not habitat names (like water frogs or tree frogs) (see
discussion in Dubois and Ohler, 1994).
Previous results have shown that certain morphological indexes
(Dp/Ch, T/Ch) could be appropriately used to separate local species
with efficiency close to 90% (Régnier, 1983; Régnier and Neveu, 1986).
However, Fog (1994) considered that characters based on coloration
led to less overlap than morphological characters, and claimed that
using the femur spot pattern increased efficiency. We fully agree with
this view, which was supported by the results of electrophoresis (test
yielded 1 misclassified non-mature adult among 70 frogs from a
mixed LL–RL–RLL).
Triploids are frequent today in Brittany, with the result that, at site
B, all hydrids are RLL triploid males (with a colour very similar to LL)
mixed with RR diploids (Neveu, 1996). This assumes a production of
LL diploid male gametes similar to that observed in a population near
Paris (Graf and Polls Pelaz, 1989). For the rare RRL triploid male, this
assumes a production of RL diploid male gametes (Brychta and
Tunner, 1994; Rybacki, 1994). A particular case is exhibited by RR × RL,
giving rise to RL (with some RR) progeny, which may be explained by
an L transmission by the male (Rybacki, 1994; Binkert et al., 1982;
Christiansen et al., 2005; Ragghianti et al., 2007).
The intensive rearing strain can be used as control group to
assess stability under experimental conditions. During the study
period, we find correlations of survival and growth with the year for
this strain, as well as correlations between survival, growth and
density at 14 months. In addition, we not only observe growth
differences between tanks during the same year (for frog size
comparisons, all values of t b 1.06), but also a high stability in mean
frog size. For example, the mean size in 1998 was TL = 63.0 mm,
with σ = 3.3 between tanks (n = 20 tanks) and CV = 0.05. In 1999,
we obtained TL = 65.7 mm, σ = 4.5 and CV = 0.06 (n = 17 tanks),
while, in 2004, TL = 62.8, σ = 4.0 and CV = 0.06 (n = 9 tanks). All
these results show that experimental conditions remained stable
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36 A. Neveu / Aquaculture 295 (2009) 30–37
during the experimental period and that differences are strongly
correlated with taxonomic status.
Under rearing conditions, the survivals of the different frog types
are very different after 14 months of life. Three groups can be
distinguished: the first, with the lowest survival rates between 14.8
and 26.2%, corresponds to the descendants of wild pairs LL or RLL. The
second group, with intermediate survival rates between 39.0 and
46.4%, is made up of F1 and F2 of wild RR and F2 RLL. The third group
shows the best survival rates, between 63.0 and 77.4%, consisting of
the RL and RRL hybrids as well as all RR individuals with several years
under rearing conditions. After the first year of life, there are
increasing differences between frogs. At 3 years old, the triploid
hybrids show a good survival of between 35 and 38%, while RR-Rivan
92* yields the best result with 55.7%. On the other hand, the other
phenotypes display a very low survival of only 4.7 to 8.3%. After
61 months under rearing conditions, survivals are represented only by
the RR-Rivan 92* type, corresponding to 52.9% of the initial number
remaining alive. There are no literature data providing comparisons
with these results. Otherwise, the present data can be compared with
some results obtained by osteochronology on wild frogs (Neveu, 1991
and unpublished data). At site A, the study of 763 lessonae yields a
mean survival rate of 7% after 4 summers of life and 4% for esculenta
(n = 266) (with a sex ratio very close to 1 for both taxa). Other results
from the mixed RR-RLL population at site B under semi-natural
conditions (enclosures without net) indicate survival rates ranging
from 8 to 42.8% at 14 months of life (Neveu, 1996). For the same
population, the monitoring of 960 wild adults shows that 4% of RLL
(males) are 4 years old, as against 6.7% for RR males and 27.4% for RR
females (unpublished data). In other words, rearing conditions increase
the lifespan of ridibunda, but do not affect the others in comparison to
the wild.
The growth of individuals is also highly variable according to
phenotype. The first group shows a mean size of 37.4 to 46.2 mm after
14 months, being characterized by the presence of at least one L in
each type and with triploids larger than diploids (t = 6.0, P b 0.0005).
The second group has a mean size of 58.6 to 67.7 mm, and is made up
exclusively of ridibunda, which could reach a marketable size.
Moreover, the growth performances of the first group are less
satisfactory under rearing conditions than in the wild. This difference
could be linked to a reduced food intake during captivity (author's
personal observation). For the second group, the growth rate increases
over several rearing years. However, the back-crossing of the oldest
strain in rearing (RR-Rivan 92*) with new wild frogs (from the same
site of origin) seems to increase performances. This would appear to
be an effect of inbreeding, a common problem in fish aquaculture
(Ledberg, 1990; Smith, 1994). However, it is surprising that the wild
hemiclone increases survival, which appears to contradict previous
results at the level of F1 or F2 from wild pairs, suggesting a lack of
outbreeding at the level of pellet acceptance. More appropriate trials
of quantitative genetics are necessary to study these limited
preliminary results and the heritability of performances.
All these results allow us to test the suitability of using green frog
types for raniculture. For 14-month-old frogs, the potential production
increases from 1.5 to 8.7 kg/m2 in the case of taxa containing the
lessonae hemiclone to 22.9–35.7 kg/m2 for ridibunda after at least two
generations under rearing conditions. For the first generations derived
from wild ridibunda pairs reared on pellets, we obtain an intermediate
production level of 11.3–13.7 kg/m2. Only ridibunda allows us to
exceed the market size, i.e. around 25–30 g in France. In the case of
green frogs, the natural triploids do not seem to offer any advantage
for aquaculture, while in fish, the presence of triploids generally has no
influence on the growth of males in contrast to the females (Breton et al.,
1996). In the present study, another difference is that the triploids are
fertile males derived from natural hybrids. Similarly, hybridizing with
lessonae [under the influence of hybridogenesis] provides no advantage
for frog rearing, since the L hemiclone decreases the growth rate.
However, some improvements could be obtained, possibly by hybridiza-
tion between ridibunda of different origins, as well as with other cryptic
species (R. bedriagae, etc.).
It is difficult to make a comparison with the farming of neotropical
species since the climatic conditions are very different and the higher
temperatures increase the growth rate. However, Lutz and Avery (1999)
showed that R. catesbeiana can reach 50 g after four months outdoors in
a temperate climate, but at very low density (50/m2). Lima et al. (2003)
report that this species attains 100 g in 100 days (in South American
cultures under low densities: 4 kg/m2). In an experimental design in
Thailand, Somsueb and Boonyaratpalin (2001) used Thai frogs
(R. rugulosa) which reached 50–90 g in three months. As far back as
1992, Lima and Agostinho proposed an exemplary bullfrog
(R. catesbeiana) culture in Brazil with a potential production of around
45 kg/m2. This culture produced several generations each year, with
adult growth in 75 days, a market size of around 100 g and a high
survival rate (90%). For R. ridibunda, a limited trial at 25–28 °C showed
an accelerated growth with mature adults in six months (i.e. at the
market size). Therefore, it seems possible to have two generations in a
year, which presupposes a controlled reproduction throughout the year.
However, this is not currently the case for temperate species, since they
require a winter rest to obtain a good fertility.
The rearing of phenotypes with at least part of their genome
containing lessonae could yield mature adults producing fertile eggs,
which would enable production for restocking. In wild populations, the
lessonae species guarantees fertility in hybrid mixed populations.
Similarly, esculenta hybrid females could be produced with higher
fertility than lessonae females of the same total length, and then paired
with lessonae males to allow the building up of new populations in
suitable habitats.
Acknowledgments
I thank the Ecology and Ecotoxicology Experimental Station of INRA-
Rennes for its technical support. I am especially grateful to P.M. Lucas for
his patient technical assistance and efficiency in managing frog rearing.
All experiments were conducted under a Certificate of Approval
no. 04744 accorded by the French government for experimentation
with animals. The English style was edited by M.S.N. Carpenter. I am
grateful to anonymous reviewers for helpful comments on the
manuscript.
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