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Mating combinations and descent in a mixed Rana lessonae (genotype LL ) – Rana esculenta (genotype RL ) assemblage (females are represented darker than males). Prior to meiosis, R. esculenta excludes the lessonae hemigenome, and thus, its gametes only contain the R genome. Since the L genome is of paternal origin, RL × LL matings lead to daughters only. Only one out of the four mating combinations gives LL tadpoles (homotypic LL pairing). The cross indicates that the other homotypic matings ( RL × RL ) usually produce non- or little-viable RR female offspring. Both heterotypic combinations result in new hybrid individuals
Source publication
Hybridization is a widespread phenomenon in many vertebrate groups. Prezygotic isolating mechanisms, probably caused by selection against hybrids with reduced fitness, reduce the likelihood of such events. Although hybrid-reduced fitness relatively to parental species is common, hybridization can also be beneficial, and hybrids sometimes outperform...
Contexts in source publication
Context 1
... hybridization involves high costs (i.e. when hybrid fitness is lower than that of parental taxa), natural selection should promote reproductive isolation by favouring pre- mating mechanisms, the so-called reinforcement process (Loftus-Hills and Littlejohn 1971; Noor 1995; Saetre et al. 1997; Pfennig and Simovitch 2002). Whereas reinforcement was first considered a rare phenomenon, recent studies suggest that it may have been involved in numerous cases of species isolation (Coyne and Orr 1989; Noor 1997). Gerhardt et al. (1994) attributed the apparent rarity of reinforcement processes to the fact that nearly all studies have focused on courtship displays or signals of males rather than on the way by which females select their mates. If signals diverge significantly and are perceptually distinguishable at the time when sympatry is achieved, there is no logical necessity for further divergence in male signals (Waage 1979). Conversely, selection could mainly operate to sharpen the selectivity of females because they usually have more to loose from a mating mistake than males. When fitness values of hybrid genotypes equal or exceed those of parental species, other mechanisms could promote gene flow between species (Rosenfield and Kodric-Brown 2003). In animals, the causes of hybridization are to be first approached at the level of mating behaviour (Grant and Grant 1997). Indeed, several behavioural mechanisms such as female mate choice or male – male competition may facilitate interbreeding (Randler 2002): (i) The “ scarcity of conspecifics ” hypothesis known as Hubbs ’ (1955) principle states that hybridization occurs when the availability of conspecific individuals is limited by such ecological factors as resource depletion, disturbance or habitat fragmentation. Females of the rarer species initially reject males of the more common species, but the longer they search for males of their own species the less discriminating they become. As a consequence, they may mate with males of the commoner species as a result of flexibility in mate preferences (Grant and Grant 1992). In this context, hybrid offspring are produced by females of the rare species and males of the common species (Wirtz 1999). (ii) Sexual selection could also promote heterospecific mating through male – male competition. Indeed, competition among males can contribute to hybridization processes since competitive interactions are indicative of male quality. For instance, in the hybrid zone between the white-collared manakin, Manacus candei , and the golden-collared manakin, Manacus vitellinus , sexual selection through asymmetries in male aggression behaviour plays an important role in the dynamics of hybridization (McDonald et al. 2001). Moreover, in a study of hybridization between several species of the genus Cyprinodon , Rosenfield and Kodric-Brown (2003) showed that hybrid males dominate the males of both parental species under certain conditions because of their extreme aggressiveness. Competitive interactions among males may thus promote hybridization. In the present study, we investigate pair formation in the European waterfrogs of the Rana lessonae – Rana esculenta hybrid system. The hybridization that results from such breaking of homotypic mating is one of the key conditions of the hybridogenesis model (Schultz 1969; Tunner 1974). While this model is widely accepted for explaining the composition of waterfrog assemblages (Graf and Polls Pelaz 1989), the mechanisms explaining pair formation remain poorly understood. Nevertheless, this system is an excellent model system to test hypotheses on hybridization dynamics because of the high variability of assemblage composition among breeding sites and the high density of males that could impede mate choice by females and promote male – male competition at each chorus. The edible frog R. esculenta (genotype RL ) is an interspecific hybrid produced through mating between the pool frog R. lessonae (genotype LL ) and the lake frog Rana ridibunda (genotype RR ). Mixed R. lessonae – R. esculenta populations represent the most widespread system in the waterfrog complex (L – E system). Until recently, R. ridibunda has been absent from these systems recolonised after the last ice age. These two taxa may easily be confused because morphological identification in the field requires experi- ence and should be confirmed by bioacoustic analysis or genetic analysis. Gamete formation in the RL hybrid obeys the hybridogenesis model (Schultz 1969; Uzzel and Berger 1975). Prior to meiosis, one of the parental genomes ( L ) is discarded while the other ( R ) is replicated and clonally transmitted to eggs or sperm. Therefore, the hybrid can only produce viable offspring through sexual parasitism on the parental species. R. esculenta thus regains the lost L genome by mating with R. lessonae , its sexual host. The hybrid coexists with the parental species, and thus, males of both taxa gather at the same breeding ponds where they form choruses that reach up to 400 males. Observations conducted at night revealed a strong competition between males. In such mixed populations, four different mating combinations are possible (Fig. 1). Homotypic matings between LL males and females lead to LL tadpoles. Homotypic RL pairings produce RR offspring, usually with null or low survival, probably because of the homozygosity of deleterious mutations accumulated owing to the lack of recombination ( R genome clonally transmitted). Only when RL parents belong to different hemiclones do the tadpoles develop normally (Hotz et al. 1992; Vorburger 2001). All known R. esculenta lineages originate from matings between female R. ridibunda and male R. lessonae . Therefore, clonal R genomes contain an X chromosome, and all resulting offspring of successful matings between hybrids are females. Heterotypic LL × RL matings result in new R. esculenta individuals, but the outcomes differ according to mate gender. Mating between LL male and RL female produced three times more tadpoles (1:1 sex ratio) than the reverse combination ( RL male × LL female, all-daughter progeny; Berger 1977; Joly and Moyen unpublished data). Moreover, Berger et al. (1988) showed that sex ratios of the two combinations are not the same because males and females R. esculenta produce only gametes with the female genome (see Fig. 1). Thus, in such mixed populations, there is a conflict over the best mating strategy between R. lessonae and R. esculenta . Whereas preference for the parental species is the only way to produce viable offspring in hybrid RL frogs, such a mating is expected to be avoided by parental LL frogs as the resulting RL hybrids will exclude their parental L genome in the next generation. On the other hand, if a wrong mating induces relatively low cost in males, it could imply a total fitness failure in LL females. Because hybrid individuals are often numerous in mixed assemblages, one expects that heterospecific mating is promoted by behavioural mechanisms. Pairing rules might have been adaptively shaped by natural selection, and heterospecific mate choice in RL females suggests a breaking of genetic correlation between male trait and female preference for male trait. However, such assertion is a postulate which is a by-product of the hybridogenesis model. This model indeed supposes that the mating frequency of hybrid females with parental males is higher than expected on the basis of homotypic mating or random mating. Until now, only female mate choice has been investigated to explain such unbalanced mating frequency. However, because these choice experiments have not been conducted within the usual behavioural context of sexual selection (noisy background of the chorus, possibility of scramble competition for mate capturing), the extrapolation of these results to the outcome of natural pairing remains hypothetical. Avoiding the temptation of an adaptationist logic, our study is the first to repeat extensive observations within natural choruses and to propose and explore two alternative hypotheses to that of adaptive female mate choice. We hypothesised that (i) pairs form according to the proportion of males and females taxa in each taxon at the chorus (the proportion of heterotypic matings is confounded by the scarcity of hybrid males), and (ii) the proportion of heterotypic mating may be explained by interspecific variation in mating speed. We examined natural choruses of the R. lessonae – R. esculenta assemblage. An experimental design was used to estimate the pairing success of ...
Context 2
... hybridization involves high costs (i.e. when hybrid fitness is lower than that of parental taxa), natural selection should promote reproductive isolation by favouring pre- mating mechanisms, the so-called reinforcement process (Loftus-Hills and Littlejohn 1971; Noor 1995; Saetre et al. 1997; Pfennig and Simovitch 2002). Whereas reinforcement was first considered a rare phenomenon, recent studies suggest that it may have been involved in numerous cases of species isolation (Coyne and Orr 1989; Noor 1997). Gerhardt et al. (1994) attributed the apparent rarity of reinforcement processes to the fact that nearly all studies have focused on courtship displays or signals of males rather than on the way by which females select their mates. If signals diverge significantly and are perceptually distinguishable at the time when sympatry is achieved, there is no logical necessity for further divergence in male signals (Waage 1979). Conversely, selection could mainly operate to sharpen the selectivity of females because they usually have more to loose from a mating mistake than males. When fitness values of hybrid genotypes equal or exceed those of parental species, other mechanisms could promote gene flow between species (Rosenfield and Kodric-Brown 2003). In animals, the causes of hybridization are to be first approached at the level of mating behaviour (Grant and Grant 1997). Indeed, several behavioural mechanisms such as female mate choice or male – male competition may facilitate interbreeding (Randler 2002): (i) The “ scarcity of conspecifics ” hypothesis known as Hubbs ’ (1955) principle states that hybridization occurs when the availability of conspecific individuals is limited by such ecological factors as resource depletion, disturbance or habitat fragmentation. Females of the rarer species initially reject males of the more common species, but the longer they search for males of their own species the less discriminating they become. As a consequence, they may mate with males of the commoner species as a result of flexibility in mate preferences (Grant and Grant 1992). In this context, hybrid offspring are produced by females of the rare species and males of the common species (Wirtz 1999). (ii) Sexual selection could also promote heterospecific mating through male – male competition. Indeed, competition among males can contribute to hybridization processes since competitive interactions are indicative of male quality. For instance, in the hybrid zone between the white-collared manakin, Manacus candei , and the golden-collared manakin, Manacus vitellinus , sexual selection through asymmetries in male aggression behaviour plays an important role in the dynamics of hybridization (McDonald et al. 2001). Moreover, in a study of hybridization between several species of the genus Cyprinodon , Rosenfield and Kodric-Brown (2003) showed that hybrid males dominate the males of both parental species under certain conditions because of their extreme aggressiveness. Competitive interactions among males may thus promote hybridization. In the present study, we investigate pair formation in the European waterfrogs of the Rana lessonae – Rana esculenta hybrid system. The hybridization that results from such breaking of homotypic mating is one of the key conditions of the hybridogenesis model (Schultz 1969; Tunner 1974). While this model is widely accepted for explaining the composition of waterfrog assemblages (Graf and Polls Pelaz 1989), the mechanisms explaining pair formation remain poorly understood. Nevertheless, this system is an excellent model system to test hypotheses on hybridization dynamics because of the high variability of assemblage composition among breeding sites and the high density of males that could impede mate choice by females and promote male – male competition at each chorus. The edible frog R. esculenta (genotype RL ) is an interspecific hybrid produced through mating between the pool frog R. lessonae (genotype LL ) and the lake frog Rana ridibunda (genotype RR ). Mixed R. lessonae – R. esculenta populations represent the most widespread system in the waterfrog complex (L – E system). Until recently, R. ridibunda has been absent from these systems recolonised after the last ice age. These two taxa may easily be confused because morphological identification in the field requires experi- ence and should be confirmed by bioacoustic analysis or genetic analysis. Gamete formation in the RL hybrid obeys the hybridogenesis model (Schultz 1969; Uzzel and Berger 1975). Prior to meiosis, one of the parental genomes ( L ) is discarded while the other ( R ) is replicated and clonally transmitted to eggs or sperm. Therefore, the hybrid can only produce viable offspring through sexual parasitism on the parental species. R. esculenta thus regains the lost L genome by mating with R. lessonae , its sexual host. The hybrid coexists with the parental species, and thus, males of both taxa gather at the same breeding ponds where they form choruses that reach up to 400 males. Observations conducted at night revealed a strong competition between males. In such mixed populations, four different mating combinations are possible (Fig. 1). Homotypic matings between LL males and females lead to LL tadpoles. Homotypic RL pairings produce RR offspring, usually with null or low survival, probably because of the homozygosity of deleterious mutations accumulated owing to the lack of recombination ( R genome clonally transmitted). Only when RL parents belong to different hemiclones do the tadpoles develop normally (Hotz et al. 1992; Vorburger 2001). All known R. esculenta lineages originate from matings between female R. ridibunda and male R. lessonae . Therefore, clonal R genomes contain an X chromosome, and all resulting offspring of successful matings between hybrids are females. Heterotypic LL × RL matings result in new R. esculenta individuals, but the outcomes differ according to mate gender. Mating between LL male and RL female produced three times more tadpoles (1:1 sex ratio) than the reverse combination ( RL male × LL female, all-daughter progeny; Berger 1977; Joly and Moyen unpublished data). Moreover, Berger et al. (1988) showed that sex ratios of the two combinations are not the same because males and females R. esculenta produce only gametes with the female genome (see Fig. 1). Thus, in such mixed populations, there is a conflict over the best mating strategy between R. lessonae and R. esculenta . Whereas preference for the parental species is the only way to produce viable offspring in hybrid RL frogs, such a mating is expected to be avoided by parental LL frogs as the resulting RL hybrids will exclude their parental L genome in the next generation. On the other hand, if a wrong mating induces relatively low cost in males, it could imply a total fitness failure in LL females. Because hybrid individuals are often numerous in mixed assemblages, one expects that heterospecific mating is promoted by behavioural mechanisms. Pairing rules might have been adaptively shaped by natural selection, and heterospecific mate choice in RL females suggests a breaking of genetic correlation between male trait and female preference for male trait. However, such assertion is a postulate which is a by-product of the hybridogenesis model. This model indeed supposes that the mating frequency of hybrid females with parental males is higher than expected on the basis of homotypic mating or random mating. Until now, only female mate choice has been investigated to explain such unbalanced mating frequency. However, because these choice experiments have not been conducted within the usual behavioural context of sexual selection (noisy background of the chorus, possibility of scramble competition for mate capturing), the extrapolation of these results to the outcome of natural pairing remains hypothetical. Avoiding the temptation of an adaptationist logic, our study is the first to repeat extensive observations within natural choruses and to propose and explore two alternative hypotheses to that of adaptive female mate choice. We hypothesised that (i) pairs form according to the proportion of males and females taxa in each taxon at the chorus (the proportion of heterotypic matings is confounded by the scarcity of hybrid males), and (ii) the proportion of heterotypic mating may be explained by interspecific variation in mating speed. We examined natural choruses of the R. lessonae – R. esculenta assemblage. An experimental design was used to estimate the pairing success of ...
Context 3
... on allozyme identification of 679 males sampled from six choruses, the LL – RL ratio in the studied pond was 64 – 36%. The mating pattern did not match this ratio since RL males were found in less than 15% of the observed breeding pairs, both with LL and RL females ( Fig. 2). All the other pairs involved LL males that were amplexed with LL , RL and RR females (55, 44 and 1%, respectively). The female R. ridibunda probably came from R. esculenta pairings because no introduction of R. ridibunda was known in the reserve (Fig. 1). We refined this general pattern by taking into account variation among choruses. For this purpose, we performed a log-linear analysis to investigate the relationship between mating frequency and availability of each taxon within each chorus (Table 1 and Fig. 3). Chorus × taxon × mating status and chorus × mating status interactions were removed as non-significant, whereas chorus × taxon and taxon × mating status interactions proved significant. This shows, first, that the proportions of each taxon differed among choruses and, second, that the mating probability of a male was linked to its taxon. With respect to the composition of each chorus, LL males were always over-represented in the amplexed pairs, whereas RL males were under-represented. Taking into account the proportion of LL and RL males in the chorus, the probability for an LL male to mate was 3.7 (CI 95% 2.15 – 6.38) times greater than that of an RL male. In spite of the low number of pairs collected on each chorus, a chorus by chorus analysis emphasised in three choruses a counter-selection of RL males compared to LL males since pair composition was bias towards LL males: chorus 1 ( χ 2 test, χ 2 =6.105, df =1, P =0.013), chorus 3 (Exact Fisher test, P =0.035) and chorus 4 ( χ 2 =4.892, df =1, P =0.027). The same pattern was observed on the other three choruses, but the difference between the two taxa was not statistically significant: chorus 2 (Exact Fisher test, P =0.733), chorus 5 (Exact Fisher test, P =0.559) and chorus 6 ( χ 2 =2.195, df =1, P ...
Citations
... Reproductive interference occurs when interspecific interactions during mating or mate acquisition negatively affect fitness (Gröning and Hochkirch 2008). The most common cause of reproductive interference is imperfect species recognition (Gröning and Hochkirch 2008), which is most likely to arise in mating systems ruled by malemale competition and sexual coercion where males are under strong selection not only for superior competitive abilities but also for indiscriminateness and a prompt reaction to mating opportunities (Andersson 1994;Clutton-Brock and Parker 1995;Lengagne et al. 2006). When two species with such a mating system co-occur, indiscriminate males may also harass heterospecific females and mate with them coercively (Kyogoku and Nishida 2012;Noriyuki et al. 2012) thus acting as Bsatyrs^ (Ribeiro and Spielman 1986). ...
... In species exhibiting explosive breeding (sensu Wells 1977), the reproductive season is short, males largely outnumber females over large parts of the breeding season, and intense male-male competition overrules direct female choice (Davies and Halliday 1979;Engeler and Reyer 2001;Hettyey et al. , 2009a. Males are selected for large body size (Davies and Halliday 1979;Liao and Lu 2012;Rausch et al. 2014), swiftness in pairing (high mating speed; Lengagne et al. 2006), and indiscriminateness (Wells 1977;Engeler and Reyer 2001;Hettyey et al. 2005). ...
... Despite their potential importance for breeding success of individuals and for the coexistence of species, behavioral mechanisms of male-male competition in anuran amphibians and their relationship with physical traits of males have been studied rarely in the heterospecific context. In hybridogenetic waterfrogs, where heterospecifics compete for females, Pelophylax lessonae males are faster in achieving amplexus with females than are P. esculentus males (Lengagne et al. 2006), whereas P. esculentus males are more aggressive, call more often, and orient more frequently toward females (Bergen et al. 1997). These differences in male behavior, presumably along with mating preferences of females (Abt and Reyer 1993;Engeler and Reyer 2001), result in higher mating success of males of the parental species, P. lessonae. ...
Reproductive interference can seriously affect the reproductive success of involved individuals and may lead to local exclusion of the competitively inferior species. The components of male competitiveness in direct bodily fights for females have been studied rarely in a heterospecific context. In explosively breeding anuran amphibians, males are often coercive and indiscriminate, which frequently leads to interactions among heterospecific males. We experimentally assessed the competitiveness of male Rana dalmatina (RD) and R. temporaria (RT), two species with overlapping breeding both in time and space. The mating speed of the RD males decreased over the course of the breeding season, whereas RT males retained their swiftness. In addition, the RD males were rarely able to replace the RT males from amplexus with RD females, while takeovers frequently occurred among the RD males. Further, larger RD males were more successful in maintaining amplexus and in achieving takeovers when facing smaller conspecifics. Our results suggest that male body size is important for intraspecific competition among the RD males, but the RT males outcompete the RD males of all sizes, perhaps due to their larger body size. Hence, RT males are likely to be effective satyrs of RD females, because they may lower the reproductive success of the latter. Such interspecific differences in the competitiveness of males may represent an important mechanism that contributes to the arousal of asymmetric reproductive interference between species.
Significance statement
Reproductive interference—interactions between species during mating with negative consequences on the fitness of involved individuals—is widespread in nature. Even though differences in the competitiveness of males of interfering species may have severe consequences, competitiveness of males has rarely been studied in an interspecific scenario in vertebrates. Males of anuran amphibians are often indiscriminate and coercive and engage in bodily fights for the possession of both conspecific and heterospecific females, which may often compromise the reproductive success of involved males and females. Here, we demonstrate that interspecific differences among ranid males can be large in the swiftness of pair formation, in the ability to defend the mating position and to perform takeovers. These differences are likely to lower the reproductive performance of the competitively inferior species, potentially leading to its local extinction.
... Second, an increase in male density may favor scramble competition (Wells 1977;Arak 1983), leading to an increase in female harassment. This may create sexual conflicts, in which competing males impose sexual coercion and thus restrict female opportunity for mate choice (Bergen et al. 1997;Lengagne et al. 2006;Lengagne and Joly 2010). ...
Mate preference was long assumed to be static and open-ended. However, a growing body of evidence actually suggests that the predominant factor in mate choice fluctuates due to the context dependence of the factors involved in sexual selection. In particular , fluctuation in the local environment, the density of individuals, and the presence of heterospecifics can all alter the strength, form, or direction of sexual selection by shaping the fitness costs and benefits of the traits that mediate male–female interactions. Amphibians constitute useful biological models to examine context dependence in sexual selection as the availability of suitable breeding resources may significantly impact the cost–benefit trade-off between mating strategies. Using an experimental approach, we examined how the risk of larval competition in breeding ponds affects pond occupancy, male–male spacing behavior, and spawning decisions in an anuran, the yellow-bellied toad (Bombina variegata). Then, we examined how female mate choice is affected by male body condition and how the risk of larval competition might influence females when choosing a mate. Our results indicate that male pond preference is strongly affected by the presence of conspecific tadpoles. Males preferentially selected ponds free of tadpoles, and spawning mainly occurred in these same ponds. This preference influenced male spacing behavior by enhancing the proximity of competing males when the availability of good-quality sites was experimentally manipulated, which had further significant consequences on females' choice of mate. The article also discusses the evolutionary causes and consequences of such context-dependent mate choice in amphibians and beyond.
... Alternatively, male-male competition may be asymmetrical; males outcompete heterospecifics in only one of the two species. Thus, everything else being equal, competitive males are expected to hybridize whereas males from the other species should mate only with conspecific females (Lengagne et al. 2006). Here, we assessed the relative importance of mate recognition and quality signals and the effects of male-male competition on mate choice and mating patterns among three genetically divergent lineages of the amphipod Gammarus fossarum. ...
Mate preference for conspecifics does not necessarily lead to assortative mating in cases where mating outcomes also depend on preferences based on mate quality and on individual competitiveness. We tested how such traits affected mate choice among genetically divergent lineages (called molecular operational taxonomic units; MOTU) of the amphipod Gammarus fossarum. We presented males with two females, including one from its own MOTU. Females also potentially differed in body size, and therefore fecundity, and in time before reproduction, two traits previously recognized as important in male mate choice. Males generally preferred females from their own MOTU when females originated from highly divergent MOTUs (17 % genetic divergence), but not when they were more closely related (3.5 % genetic divergence). Contrary to expectations, they did not prefer larger females, but they consistently paired with the female closest to reproduction. A second experiment involving duos of males of different MOTUs in competition for a female also revealed that males consistently won the competition over pairing with females of their own MOTU. Overall, these results reveal a strong influence of genetic divergence on mate recognition and reproductive isolation between sympatric MOTUs. However, male preference for females that are close to being available for reproduction also potentially results in hybridization among closely related MOTUs. We examine these results in the light of field mating patterns observed in a previous study of G. fossarum and discuss the importance of considering competitiveness and preferences for mate quality signals when studying evolutionary consequences of secondary contact between divergent lineages.
... In contrast, genetic fixation of mate preferences in a particular genotype should be impossible in diploid-triploid all-hybrid populations because here the to-be preferred mating partners alternate each generation: diploid LR females producing diploid eggs should choose triploid LLR or LRR males; the resulting triploid daughters should choose diploid LR males, etc. For females from L-E and E-E systems, these predictions from theoretical models (Hellriegel and Reyer 2000;Som et al. 2000;Som and Reyer 2006) have, indeed, been supported both in natural populations (Blankenhorn 1974;Günther and Plötner 1990;Lengagne et al. 2006Lengagne et al. , 2008Lengagne and Joly 2010;GAT, unpublished data) and in experimental settings (Abt and Reyer 1993;Reyer et al. 1999;Roesli and Reyer 2000;Engeler and Reyer 2001;Rondinelli 2006). For R-E systems, we do not know of any detailed study on mate choice and mating behavior; therefore, we will not refer to R-E systems for the rest of this paper. ...
... Hybrids take an intermediate position between the two parental species for many characteristics, including morphology and male vocalization (e.g., Kierzkowski et al. 2011;Hoffmann and Reyer 2013), and they occupy a broader spectrum of biotic and abiotic habitat conditions than the parental species (Plenet et al. 2000;Pagano et al. 2001;Holenweg Peter et al. 2002). In terms of mating behavior, P. esculentus males seem to share the tendency to be stationary at territories with P. ridibundus (Weidenberg 1999) but are also active in intercepting and clasping females like P. lessonae (Lengagne et al. 2006(Lengagne et al. , 2008. In all-hybrid E-E systems, the intermediate position of P. esculentus has been investigated on an even finer scale, namely, with respect to a genome dosage effect. ...
Mating success depends not only on genetic and phenotypic characteristics of males and females but also on their spatial position relative to other individuals, which influences the chances for interactions. Hence, any behavior that affects proximity to other individuals can potentially translate into fitness gain or loss. Here, we investigate the effects of genotype on male movement and distance to nearest neighbor (DNN) in three populations of the edible frog Pelophylax esculentus, a natural hybrid between its parental species Pelophylax ridibundus (genotype RR) and Pelophylax lessonae (LL). The system is particularly suited for such an analysis because the fitness differences between mating with a certain genotype are particularly strong. Moreover, which genotype should be preferred differs among populations where diploid hybrids (LR) live in sympatry with P. lessonae (L–E system) and those where diploid hybrids occur in all-hybrid populations together with triploids (LLR and/or LRR) (E–E system). Hence, we expected differences among genotypes in movement patterns and spatial arrangement within the breeding pond. We did, indeed, find such differences. They were predominantly due to density differences between populations, followed by size and condition differences between males. Most relevant for our question was a difference in DNN: in the E–E system, distances between all three hybrid types were equal, whereas in the L–E system LR hybrids tended to stay closer to LL than to other LR. The results are discussed in relation to previous mate choice experiments and theoretical models about mating preferences in the two systems.
... All these 82 ancient esculentus lineages harbored lessonae mtDNA, as expected from a long history of coexistence. For size and behaviorrelated reasons, L9E crosses often take place between esculentus females and lessonae males (Berger 1970), but reverse crosses also occur at some lower rate (Lengagne et al. 2006). Assuming lessonae females to contribute as few as 10 % of the crosses, the ridibundus mtDNA is expected to decline at a 10 % rate per generation, reaching frequencies below 5 % within 30 generations. ...
The water-frog L–E system, widespread in Western Europe, comprises the pool frog Pelophylax lessonae and the hybridogenetic edible frog P. esculentus, which originated from hybridization between pool frogs and marsh frogs (P. ridibundus). In P. esculentus, the lessonae (L) genome is eliminated during meiosis and has to be gained anew each generation from a P. lessonae partner, while the ridibundus (R′) genome is transmitted clonally. It therefore accumulates deleterious mutations, so that R′R′ offspring from P.
esculentus×P.
esculentus crosses are normally unviable. This system is now threatened by invasive P. ridibundus (RR) imported from Eastern Europe and the Balkans. We investigated the genetic interactions between invasive marsh frogs and native water frogs in a Swiss wetland area, and used genetic data collected in the field to validate several components of a recently postulated mechanism of species replacement. We identified neo-ridibundus individuals derived from crosses between invasive ridibundus and native esculentus, as well as newly arisen hybridogenetic esculentus lineages stemming from crosses between invasive ridibundus (RR) and native lessonae (LL). As their ridibundus genomes are likely to carry less deleterious mutations, such lineages are expected to produce viable ridibundus offspring, contributing to species replacement. However, such crosses with invasive ridibundus only occurred at a limited scale; moreover, RR×LL crosses did not induce any introgression from the ridibundus to the lessonae genome. We did not find any ridibundus stemming from crosses between ancient esculentus lineages. Despite several decades of presence on the site, introduced ridibundus individuals only represent 15 % of sampled frogs, and their spatial distribution seems shaped by specific ecological requirements rather than history of colonization. We therefore expect the three taxa to coexist stably in this area.
... The combination of the relative abundance and heterosis should out-compete P. lessonae in L-E complexes. The widespread distribution of L-E complexes, although with different percentages of hybrids, reveals that there are mechanisms which contribute to the stability of such complexes [37][38][39][40]. Of these mechanisms, sexual selection seems to be one of the most important. ...
Background
Some species of water frogs originated from hybridization between different species. Such hybrid populations have a particular reproduction system called hybridogenesis. In this paper we consider the two species Pelophylax ridibundus and Pelophylax lessonae, and their hybrids Pelophylax esculentus. P. lessonae and P. esculentus form stable complexes (L-E complexes) in which P. esculentus are hemiclonal. In L-E complexes all the transmitted genomes by P. esculentus carry deleterious mutations which are lethal in homozygosity.
Results
We analyze, by means of an individual based computational model, L-E complexes. The results of simulations based on the model show that, by eliminating deleterious mutations, L-E complexes collapse. In addition, simulations show that particular female preferences can contribute to the diffusion of deleterious mutations among all P. esculentus frogs. Finally, simulations show how L-E complexes react to the introduction of translocated P. ridibundus.
Conclusions
The conclusions are the following: (i) deleterious mutations (combined with sexual preferences) strongly contribute to the stability of L-E complexes; (ii) female sexual choice can contribute to the diffusion of deleterious mutations; and (iii) the introduction of P. ridibundus can destabilize L-E complexes.
... Population size A lack of available conspecific mates is considered one of the most common causes of hybridization in birds and other taxa (e.g., Hubbs 1955;Mayr 1963;Short 1969;Wirtz 1999), although formal tests of this hypothesis are few (but see, e.g., Grant and Grant 1997;Nuechterlein and Buitron 1998;Randler 2002Randler , 2006aGee 2003;Lengagne et al. 2006;Gerlai 2007). Mate availability might be unaffected by population size; however, it may be lower in smaller populations due to lower densities (e.g., Gibbs and Faaborg 1990), greater proportions of individuals at the range limit, greater sex ratio bias (e.g., Dale 2001) or higher risks of inbreeding (Keller and Waller 2002). ...
Hybridization is important in the evolution of many animal groups; however, broad scale patterns of natural hybridization are still poorly understood. Using phylogenetic comparative analyses, we tested for relationships between demographic, ecological, phenotypic and phylogenetic variables and the incidence of natural hybridization among 45 species of North American wood warbler. Since 1980, hybrids have been documented in 24 species (53 %). We detected negative relationships between the incidence of hybridization and both breeding range size and phylogenetic distance, and positive relationships between the incidence of hybridization and (1) song similarity, (2) the extent of breeding sympatry of species pairs and (3) the number of additional heterospecific warbler species that co-occur during breeding. Neither population size nor breeding habitat quality (as measured by threats to survival or reproduction in breeding areas) explained variation in the incidence of hybridization. Our results suggest a potential role of limited breeding habitat in wood warbler hybridization events and a net positive effect of breeding sympatry. They also support the hypothesis that mating signal similarity facilitates hybridization events and are consistent with an increase in reproductive isolation with increasing genetic divergence. Our results also suggest the presence of phylogenetic signal in wood warbler hybridization. By investigating natural hybridization at the taxonomic level of family, we have identified several previously undocumented patterns of natural hybridization. This study demonstrates the utility of examining the combined effects of ecology, demography, phenotype and phylogeny when studying variation in the expression of natural hybridization among taxa.
... Besides female choice and, although less commonly in anurans, male choice, there may also be a difference in male breeding behaviour, which can influence the probability of amplexus formation. Field studies suggest that P. lessonae males do not defend territories, are more mobile and attract a female more readily than P. esculentus and P. ridibundus males ( Lengagne et al., 2006;Lengagne & Joly, 2010). Rare matings between hybrids and P. ridibundus can also be influenced by undiscovered ecological differences operating in studied populations, such as unequal species densities, species-specific spatial distribution or shifted reproductive seasons. ...
Besides several exceptions, asexual metazoans are usually viewed as ephemeral sinks for genomes, which become 'frozen' in clonal lineages after their emergence from ancestral sexual species. Here, we investigated whether and at what rate the asexuals are able to introgress their genomes back into the parental sexual population, thus more or less importantly affecting the gene pools of sexual species. We focused on hybridogenetic hybrids of western Palaearctic water frogs (Pelophylax esculentus), which originate through hybridization between P. ridibundus and P. lessonae, but transmit only clonal ridibundus genome into their gametes. Although usually mating with P. lessonae, P. esculentus may upon mating with P. ridibundus or another hybrid produce sexually reproducing P. ridibundus offspring with the introgressed ex-clonal genome. We compared the rate of nuclear amplified fragment length polymorphism (AFLP) and mitochondrial introgression in two types of populations, that is, those where P. ridibundus occurs in isolation and those where it lives with the hybridogens. Although significant differentiation (Φpt) between sexual and clonal ridibundus genomes suggested limited gene flow between sexuals and hybridogens, a non-negligible (~5%) proportion of P. ridibundus bore introgressed mtDNA and AFLP markers. Whereas transfer of mtDNA was exclusively unidirectional, introgression of nuclear markers was bidirectional. The proportion of introgressed P. ridibundus was highest in syntopic populations with P. esculentus, proving an ongoing and site-specific interspecific genetic transfer mediated by hybridogenetic hybrids. It turns out that asexual hybrids are not just a sink for genes of sexual species, but may significantly influence the genetic architecture of their sexual counterparts.
© 2014 The Authors. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.
... Previous studies have provided extensive data on the genetic and inheritance patterns in populations with different ploidies, but empirical data on phenotypic manifestations in triploid versus diploid water frogs, or in recombining versus hybridogenetically reproducing hybrids are restricted to cell planimetry and body morphology [23,[67][68][69][70][71]. Where vocalization and other behaviors were investigated and found to vary within hybrid lineages [72][73][74], these studies were mostly restricted to mixed populations of diploid P. esculentus and one or both of its parental species. The same is true for studies using male advertisement calls for distinguishing between water frog species and populations [12a, 36b]. ...
Zusammenfassung Der Teichfrosch (Pelophylax esculentus, Genotypen LR, LLR oder LRR) ist ein natürlicher Hybrid zwischen dem Kleinen Wasserfrosch (P. lessonae, Genotyp LL) und dem Seefrosch (P. ridibundus, Genotyp RR). In der rein diploiden Form (LR) pflanzt sich der Hybrid via Hybridogenese fort, bei welcher ein Teil des elterlichen Genoms (entweder der L- oder der R-spezifische Teil) während der Gametogenese eliminiert und der verbleibende Teil klonal auf haploide Keimzellen übertragen wird. Rekombination zwischen dem L- und R-spezifischen Teil des Genoms ist in der Regel nicht möglich, weshalb es durch die wiederholte klonale Weitergabe innerhalb des Genoms zu einer Ansammlung schädlicher Mutationen kommt. Bei der Verpaarung zweiter Hybriden aus derselben Population sind deren Nachkommen durch die Kombination zweier klonaler Genome daher nicht überlebensfähig. Um lebensfähige Nachkommen zu zeugen, müssen sich die Hybriden mit der jeweiligen Elternart rückkreuzen, deren Teil des Hybridgenoms während der Gametogenese verloren ging. Aus dieser fortplanzungsbedingten Abhängigkeit heraus entwickelten sich verschiedene Formen eines gemischten Populationssystems aus Hybriden und Elternarten. In einigen Populationen produziert P. esculentus sowohl haploide (L oder R) als auch diploide Keimzellen (LR-Gameten, welche normalerweise nur von Individuen des Genotyps LR gebildet werden). Aus der Verschmelzung von diploiden und haploiden Keimzellen entstehen triploide Hybriden der Genotypen LLR und LRR, welche ihrerseits bei der Gametogenese jenen Genomteil ausschliessen, der nur in einzelner Ausführung vorliegt, und aus dem doppelt vorhandenen Genomteil haploide Gameten bilden (so produziert der Genotyp LLR haploide L-Gameten und der Genotyp LRR haploide R-Gameten). Dadurch entstehen über die Generationen hinweg abwechselnd diploide und triploide Hybride, welche sich unabhängig von der Anwesenheit der Elternarten erfolgreich miteinander fortpflanzen. Die reproduktive Unabhängigkeit dieser sogenannten gemischt-ploiden Systeme wird dadurch ermöglicht, dass die in den triploiden Fröschen doppelt vorhandenen Genomteile rekombiniert werden können, wodurch verhindert wird, dass sich in den Genomen zu viele schädliche Mutationen ansammeln. Sowohl die gemischten Systeme aus Hybriden und Elternarten als auch gemischt-ploide Systeme wurden in den letzten Jahrzehnten unter verschiedenen Aspekten und in unterschiedlichen geographischen Regionen untersucht. Jedoch fehlte bislang aufgrund der ungleichmässigen Verbreitung gemischt-ploider Populationssysteme in Europa ein umfassender und vergleichender populationsgenetischer Überblick im grösseren geographischen Masstab. Ferner sind die populationsgenetischen und phänotypischen Unterschiede zwischen Hybriden aus Elternarten-Populationen und gemischt-ploiden Populationen bisher noch weitgehend unbekannt, obwohl zwischen den unterschiedlichen Systemen, als auch zwischen geographischen Regionen, potentiell unterschiedliche Selektionsbedingungen herrschen. Die Zielsetzungen meiner Arbeit waren daher: a) gemischt-ploide Populationen aus unterschiedlichen Gegenden Europas genealogisch zu untersuchen und herauszufinden, ob deren ungleichmässige Verteilung auf eine unabhängige Entstehungsgeschichte zurückzuführen ist, b) zwei augenscheinlich unterschiedliche gemischt-ploide Fortpflanzungssysteme auf Unterschiede in der Keimzellenproduktion zu untersuchen, c) die bioakustischen Eigenschaften männlicher Paarungsrufe einer Anzahl gemischt-ploider Populationen unterschiedlicher geographischer Herkunft zu analysieren und mit den Eigenschaften der Paarungsrufe aus gemischten Hybrid-Elternarten-Populationen zu vergleichen, und d) die räumlichen Bewegungsmuster und das Distanzverhalten zwischen Männchen verschiedener Genotypen aus gemischt-ploiden und gemischten Hybrid- Elternart-Populationen zu untersuchen und potentielle Unterschiede auf Zusammenhänge mit phänotypischen Eigenschaften der Männchen sowie mit Eigenschaften der untersuchten Teiche zu testen. In Kapitel eins untersuchte ich durch Analysen von Mikrosatelliten-DNA und mitochondrialer DNA populationsgenetische Parameter für mehr als 2000 Gewebeproben, welche aus 72 Lokalitäten in Nord-, Mittel- und Osteuropa stammten. Die Ergebnisse dieser Studie zeigten, dass die auf der Mikrosatelliten- Analyse basierende genetische Diversität von der geographischen Lage, dem Vorhandensein der Elternarten P. lessonae und P. ridibundus sowie dem Populationstyp beeinflusst wird. Während sich die meisten gemischt-ploiden Populationen aus Mitteleuropa und dem östlichen Mitteleuropa genetisch nicht sehr unterscheiden, zeigen einige Populationen aus der Ukraine ein deutlich anderes genetisches Profil. Dieses Ergebnis wird durch den Fund ungewöhnlicher mitochondrialer DNA-Typen in Individuen jener Populationen bestätigt und legt die von den gemischt-ploiden Populationen Nord- und Mitteleuropas unabhängige Entstehung jener östlichen Populationen nahe. In der Diskussion interpretiere ich diese Ergebnisse mit Bezug auf nach- und zwischeneiszeitliche Kolonisationsszenarien in Europa. Kapitel zwei präsentiert eine Studie, welche in Zusammenarbeit mit Nicolas Pruvost durchgeführt wurde und in welcher wir Kreuzungsexperimente und Analysen von Mikrosatelliten-DNA benutzten, um fünf Populationen von unterschiedlicher Populationsstruktur zu vergleichen. Dafür untersuchten wir mit Hilfe von Indizes für Heterozygotie und genetische Differenzierung die Interaktionen zwischen verschiedenen Genotypen (LL, LLR, LR, LRR and RR). Die Ergebnisse dieser Studie erlaubten uns verschiedene Fortpflanzungssysteme zu definieren und zu unterscheiden, sowie ein evolutionäres Szenario für das Auftreten und die Aufrechterhaltung eines alternativen Systems gemischt-ploider Populationen in Mitteleuropa vorzuschlagen. In Kapitel drei befasste ich mich mit den bioakustischen Eigenschaften männlicher Paarungsrufe innerhalb und zwischen gemischt-ploider sowie gemischten Populationen aus Hybriden und den Elternarten P. lessonae und P. ridibundus. Aus der Analyse von Feldaufnahmen der Rufe leitete ich fünf Rufparameter ab, welche alle einen Dosiseffekt des jeweiligen Genoms L oder R zeigten, d.h. sie nahmen mit steigendem L:R-Verhältnis der Genotypen in der Reihenfolge LL-LLR-LR-LRR-RR entweder zu oder ab. Zwei der fünf Rufparameter unterschieden sich zudem zwischen Populationssystemen. Die Effektgrössen nahmen in der Reihenfolge Genotyp-Populationssystem –geographische Lage der Population ab. Die Rufe diploider Hybriden (LR) variierten zwischen den Populationssystemen in Abhängigkeit davon, ob die Hybriden zur erfolgreichen Fortpflanzung eine der beiden Elternarten benötigen, oder nicht. In Kapitel vier überprüfte ich innerhalb dreier Teiche (zwei mit gemischt- ploiden Populationen, einer mit einer Population aus LR-Hybriden und P. lessonae), ob sich das räumliche Mobilitätsmuster und Distanzverhalten der Männchen während der Paarungszeit zwischen Genotypen unterscheidet. Darüber hinaus testete ich die räumlichen Parameter auf Zusammenhänge mit der Körpergrösse und Kondition der Männchen, sowie mit der beobachteten Häufigkeit, in der die einzelnen Männchen im Amplexus mit Weibchen beobachtet wurden. Die Ergebnisse zeigten, dass weder Genotyp noch Kondition das räumliche Bewegungsmuster beeinflussen und deuteten darauf hin, dass vorhandene Unterschiede zwischen den Teichen wahrscheinlich auf Unterschiede in der Populationsdichte zurückzuführen sind. Die Verteilung der Genotypen der Männchen im Amplexus entsprach für gemischt-ploide Populationen der tatsächlichen Verteilung der Genotypen der Männchen im Teich. Bei den Amplexus-Männchen der gemischten Hybrid-P. lessonae-Population waren P. lessonae-Männchen leicht überproportional vertreten. In Kapitel fünf präsentiere ich eine kollaborative Studie mit Anke Stöhr über Ranavirus-Infektionen in wilden Wasserfrosch-Populationen. Die Studie kombiniert die Fallstudie eines Ranavirus-Ausbruchs unter in Gehegen gehaltenen Wasserfröschen mit der Beschreibung eines neuen Ranavirus und dessen phylogenetischer Klassifizierung. Die Kapitel eins bis vier meiner Dissertation ermöglichen ein tieferes Verständnis der Diversität, Verbreitung sowie der genetischen und phänotypischen Differenzierung von P. esculentus-Populationen. Als Schlussfolgerung daraus argumentiere ich, dass es keinen „Allerwelts“-Hybriden gibt, sondern dass Teichfrosch-Populationen in Europa so divers sind, dass sie als signifikante Evolutionseinheiten denselben Respekt und dieselbe Aufmerksamkeit verdienen wie „reine“ Arten. Summary The edible frog (Pelophylax esculentus, genotypes LR, LLR or LRR) is a natural hybrid between the pool frog (P. lessonae, genotype LL) and the marsh frog (P. ridibundus, genotype RR). Diploid hybrids (LR) reproduce by hybridogenesis, where one part of the hybrid’s parental genome (either the L or the R chromosome set) is excluded during gametogenesis and the other part is clonally transmitted into haploid gametes. Recombination between the L and R genome within the hybrid is usually not possible. Therefore, repeated clonal transmission of one part of the genome leads to the accumulation of deleterious mutations which normally renders offspring from inter-hybrid crossings within the same population unviable. In order to produce viable offspring, the hybrid is thus forced to mate with the parental species whose part of the genome was excluded. This reproductive dependence has led to several forms of mixed hybrid-parental population systems. In some populations, P. esculentus can produce both haploid (L or R) and diploid gametes, LR gametes usually coming only from LR individuals. The fusion of diploid with haploid gametes results in triploid hybrids of the genotypes LLR and LRR, which exclude the single copy genome and produce only haploid gametes of the other genome (LLR produce L, LRR produce R gametes). Thus, in a perpetuating way, diploid and triploid hybrids are generated and can successfully reproduce with each other, independent of the presence of the parental species. The reproductive independence of these so-called mixed-ploidy systems is due to the fact that triploids recombine the part of their genome which is present in a double copy and thus prevent the accumulation of deleterious alleles in the genetic pool of the population. Both the mixed hybrid-parental and the mixed-ploidy systems have been studied over the last decades in several aspects and geographic regions, but due to the patchy geographic distribution of mixed-ploidy systems in Europe, a comprehensive and comparative population genetic overview across a larger area has been lacking. Furthermore, population genetic and phenotypic differences between hybrid P. esculentus from mixed-parental and mixed-ploidy systems are vastly unknown, despite potentially different selection regimes between population types and geographic regions. The objectives of my thesis were thus: a) to compare mixed-ploidy populations from different European areas in a genealogical approach and to find out whether these patchily distributed populations are of independent origin, b) to examine the gamete production patterns between two supposedly different mixed-ploidy breeding systems, c) to study bioacoustic characteristics of male advertisement calls across a number of geographically distant mixed-ploidy populations and compare them with hybrids from mixed hybrid-parental systems, and d) to examine the spatial movement and spacing behavior of male frogs within and between mixed-ploidy and mixed hybrid-parental systems and relate potential differences to male genotype, male morphology and pond characteristics. In chapter one, I used microsatellite DNA and mitochondrial DNA analysis to obtain population genetic parameters for more than 2000 samples from 72 localities across Northern, Central and Eastern Europe. The results from this study showed that genetic diversity among populations based on microsatellites is structured by geographic latitude and longitude, the presence of parental genotypes (P. lessonae and P. ridibundus) and population type. Most mixed-ploidy populations from Central and East-Central Europe did not genetically differ substantially, but some populations from Ukraine showed a distinctively different genetic profile. This was confirmed by the novel finding of unusual types of mitochondrial DNA in specimens from there. My findings suggest an independent origin of polyploid water frogs from this area, which I discuss with reference to postglacial re-colonization scenarios in Europe. Chapter two presents a collaborative study with Nicolas Pruvost, where we used microsatellite DNA analyses and crossing experiments to compare five populations of different population structures. Indices of heterozygosity and genetic differentiation were used to depict the genetic interactions between the different genotypes (LL, LLR, LR, LRR and RR). The results from this study allowed us to define and differentiate between different breeding systems and propose an evolutionary scenario for the occurrence and maintenance of an alternative mixed- ploidy population type in Central Europe. In chapter three I studied the bioacoustic properties of male advertisement calls within and between mixed-ploidy and mixed hybrid-parental populations. From field recordings I derived five call parameters which all showed a genomic dosage effect, i.e. they either decreased or increased with the L/R ratio among genotypes in the order LL-LLR-LR-LRR-RR. Two of the five call parameters were also affected by the population system. Effect sizes decreased from genotype through population system to geographic location of the population. Calls of diploid (LR) hybrids varied between population systems, depending on whether they belonged to a system that required a sexual host for successful reproduction, or not. In chapter four I tested within three ponds whether male spatial movement and spacing behavior during the breeding season differs between genotypes. Furthermore, I related spatial parameters to male body size and condition, and to the observed frequency of amplexus by individual males. As a result, I found that neither genotype nor size nor condition affected spatial movement patterns and that differences are most likely to be explained by population density. The frequency of amplexus events among genotypes corresponded to the observed male genotype distribution in the two mixed-ploidy ponds, and slightly favored P. lessonae males in the mixed hybrid-parental population in the other pond. Chapter five represents a collaborative study with Anke Stöhr on ranavirus infection in wild populations of European water frogs. The study combines a case study of a ranavirus outbreak among captive water frogs with the description of a novel ranavirus and its phylogenetic classification. In conclusion, chapters one to four of my thesis allow a better understanding of the diversity, distribution and differentiation of P. esculentus populations in terms of genetic and phenotypic characteristics. I argue that there is no such thing as a “common” water frog hybrid, but rather that hybrid populations are so diverse that they represent evolutionary significant units which deserve the same respect and attention as other “true” anuran species.
... Previous studies have provided extensive data on the genetic and inheritance patterns in populations with different ploidies, but empirical data on phenotypic manifestations in triploid versus diploid water frogs, or in recombining versus hybridogenetically reproducing hybrids are restricted to cell planimetry and body morphology [23,[67][68][69][70][71]. Where vocalization and other behaviors were investigated and found to vary within hybrid lineages [72][73][74], these studies were mostly restricted to mixed populations of diploid P. esculentus and one or both of its parental species. The same is true for studies using male advertisement calls for distinguishing between water frog species and populations [12,36]. ...
In anurans, differences in male mating calls have intensively been studied with respect to taxonomic classification, phylogeographic comparisons among different populations and sexual selection. Although overall successful, there is often much unexplained variation in these studies. Potential causes for such variation include differences among genotypes and breeding systems, as well as differences between populations. We investigated how these three factors affect call properties in male water frogs of Pelophylax lessonae (genotype LL), P. ridibundus (RR) and their interspecific hybrid P. esculentus which comes in diploid (LR) and triploid types (LLR, LRR).
We investigated five call parameters that all showed a genomic dosage effect, i.e. they either decreased or increased with the L/R ratio in the order LL-LLR-LR-LRR-RR. Not all parameters differentiated equally well between the five genotypes, but combined they provided a good separation. Two of the five call parameters were also affected by the breeding system. Calls of diploid LR males varied, depending on whether these males mated with one or both of the parental species (diploid systems) or triploid hybrids (mixed ploidy systems). With the exception of the northernmost mixed-ploidy population, call differences were not related to the geographic location of the population and they were not correlated with genetic distances in the R and L genomes.
We found an influence of all three tested factors on call parameters, with the effect size decreasing from genotype through breeding system to geographic location of the population. Overall, results were in line with predictions from a dosage effect in L/R ratios, but in three call parameters all three hybrid types were more similar to one or the other parental species. Also calls of diploid hybrids varied between breeding systems in agreement with the sexual host required for successful reproduction. The lack of hybrid call differences in a mixed-ploidy population at the northern edge of the water frog distribution is likely to be associated with genetic particularities, including a) low genetic variability and/or b) a local loss of genes coding for genotype-dependent call differentiation under conditions where female discrimination between diploid and triploid males is not beneficial.
