Genetic compatibility between sexual and clonal genomes in local populations of the hybridogeneticRana esculenta complex

Summary Hybridogenetic species possess a hybrid genome: half is clonally inherited (hemiclonal reproduction) while the other half is obtained each generation by sexual reproduction with a parental species. We addressed the question of whether different hemiclones of the hybridogenetic water frogRana esculenta are locally adapted for genetic compatibility with their sexual parental hostRana lessonae. We artificially crossedR. esculenta females of three hemiclones (GUT1, GUT2 and GUT3) from a pond near Gütighausen, Switzerland and one hemiclone (HEL1) from near Hellberg, Switzerland each toR. lessonae males from both populations. We also created primary hybrids by crossing the sameR. lessonae males from both populations toR. ridibunda females from Pozna, Poland (POZ). Tadpoles were then reared in the laboratory at two food levels to assess their performance related to early larval growth rate, body size at metamorphosis and length of the larval period. Tadpoles from hemiclones GUT1, GUT3 and POZ had higher growth rates than those from hemiclones GUT2 and HEL1 at the low food level, but at the high food level all growth rates were higher and diverged significantly between hemiclones GUT2 and HEL1. Tadpoles from the intrapopulational crosses GUT2 × GUT and HEL1 × HEL were larger at metamorphosis than those from the interpopulational crosses GUT2 × HEL and HEL1 × GUT. A high food level increased the size at metamorphosis in all tadpoles. A high food level also decreased the days to metamorphosis and tadpoles from GUT1, GUT3 and POZ had the shortest larval period whereas those from GUT2 and HEL1 had the longest. These results indicate that the differential compatibility of clonal genomes may play an important role in hybridogenetic species successfully using locally adapted sexual genomes of parental species and that interclonal selection is likely important in determining the distribution of hemiclones among local populations.     

PERFORMANCE OF TADPOLES FROM THE HYBRIDOGENETIC RANA ESCULENTA COMPLEX: INTERACTIONS WITH POND DRYING AND INTERSPECIFIC COMPETITION

The performance of three genotypes (LL, LR, RR) of tadpoles resulting from the hybrid mating system of Rana lessonae (phenotype L, genotype LL) and Rana esculenta (phenotype E, genotype LR) was determined in artificial ponds. The effects of interspecific competition and pond drying on growth, development, and survival of tadpoles were used to measure the performance of genotypes and the relative fitness of offspring. Among the three genotypes, tadpoles from the homogametic mating RR had the lowest survival, growth, and development under all environmental conditions. Body size of the LL and LR genotype tadpoles at metamorphosis was reduced by competition and pond drying. Days to metamorphosis were also higher for the LL and LR genotype tadpoles in competition ponds. The proportion of individuals metamorphosing of each genotype was differentially lowered by competition and pond drying. The LL genotype produced more metamorphs than the LR genotype in the constant water level ponds, but the LR genotype produced more in drying ponds. In competition ponds, the LR genotype produced more metamorphs than the LL genotype, but the LL genotype produced more metamorphs in ponds without competition. The RR genotype produced no metamorphs in any of the experimental environments. Increased performance of LR offspring from the heterogametic mating, in harsh conditions, and reduced performance of RR offspring from the homogametic mating, even under favorable conditions, relative to the parental genotype (LL) suggests that the population dynamics of this hybridogenetic system is strongly dependent on mate choice in mixed populations and the subsequent pond environment females select for oviposition and larval development.     

FIXATION OF DELETERIOUS MUTATIONS IN CLONAL LINEAGES: EVIDENCE FROM HYBRIDOGENETIC FROGS

Abstract The hemiclonal waterfrog Rana esculenta (RL genotype), a bisexual hybrid between R. ridibunda (RR) and R. lessonae (LL), eliminates the L genome from its germline and clonally transmits the R genome (hybridogenesis). Matings between hybrids produce R. ridibunda offspring, but they generally die at an early larval stage. Mortality may be due to fixed recessive deleterious mutations in the clonally inherited R genomes that were either acquired through the advance of Muller's ratchet or else frozen in these genomes at hemiclone formation. From this hypothesis results a straightforward prediction: Matings between different hemiclones, that is, between R. esculenta possessing different R genomes of independent origin, should produce viable R. ridibunda offspring because it is unlikely that different clonal lineages have become fixed for the same mutations. I tested this prediction by comparing survival and larval performance of tadpoles from within‐ and between‐population crossings using R. esculenta from Seseglio (Se) in southern, Alpnach (Al) in central, and Elliker Auen (El) in northern Switzerland, respectively. Se is isolated from the other populations by the Alps. Enzyme electrophoresis revealed that parents from Se belonged to a single hemiclone that was different from all hemiclones found north of the Alps. Parents from Al also belonged to one hemiclone, but parents from El belonged to three hemiclones, one of which was indistinguishable from the one in Al. Rana esculenta from Se produced inviable tadpoles when crossed with other hybrids of their own population, but when crossed with R. esculenta from Al and El, tadpoles successfully completed metamorphosis, supporting the hypothesis I tested. Within‐population crosses from Al were also inviable, but some within‐population crosses from El, where three hemiclones were present, produced viable offspring. Only part of the crosses between Al and El were viable, but there was no consistent relationship between hemiclone combination and tadpole survival. When backcrossed with the parental species R. ridibunda, hybrids from all source populations produced viable offspring. Performance of these tadpoles with a sexual and a clonal genome was comparable to that of normal, sexually produced R. ridibunda tadpoles. Thus, in the heterozygous state, the deleterious mutations on the clonal R genomes did not appear to reduce tadpole fitness.     

ASYMMETRIC COMPETITION IN MIXED POPULATIONS OF TADPOLES OF THE HYBRIDOGENETIC: RANA ESCULENTA COMPLEX

Hybridogenetic Rana esculenta tadpoles display tolerance to extreme environmental conditions and fit criteria of the “general-purpose” genotype. A trade-off between generality and competitive ability is assumed to occur in asexual species, but the evidence remains unclear. The purpose of my experiment was to test the competitive ability of hemiclonal hybrid Rana esculenta tadpoles relative to the parental species Rana lessonae. Mixed and single genotype populations of R. esculenta and R. lessonae tadpoles were reared at three densities in artificial ponds. Survival of R. esculenta was higher than for R. lessonae tadpoles, but did not differ among densities. Body size at metamorphosis was the same between genotypes, but decreased with increasing density. Larval period was not affected by density, but R. esculenta tended to metamorphose earlier than R. lessonae. Percentage of individuals metamorphosing was higher for R. esculenta at both medium and high densities, but the same as R. lessonae at the low density. The difference in survival, body size, and larval period between tadpoles reared in single and mixed genotype populations was unaffected by genotype or density. The difference in the percentage of metamorphs, however, was strongly affected. The percentage of hybrids metamorphosing was 9% above the responses of single genotype populations at the highest density. Conversely, the percentage of R. lessonae metamorphosing was 12% below the responses of single genotype populations at the same density. Hybrid success in this experiment further supports the criterion of a “general-purpose” genotype without assumptions of reduced competitive ability.     

Deleterious alleles and differential viability in progeny of natural hemiclonal frogs

Abstract.-Spontaneous deleterious mutations are expected to accumulate through Muller's ratchet in clonally reproducing organisms and may lead to their extinction. We study deleterious mutations and their effects in a system of European frogs. Rana esculenta (RL), natural hybrids R. ridibunda (RR) X R. lessonae (LL), reproduce hemiclonally; both sexes exclude the L genome in the germ line and produce unrecombined R gametes; hybridity is restored each generation by matings of RL with coexisting LL. Different allozyme-defined hybrid hemiclones (R genome haplotypes) are thought to have originated independently from primary hybridizations RR x LL. Natural matings between two hybrids usually lead to inviable RR tadpoles. This inviability is thought to result from unmasked deleterious alleles on the clonally transmitted R genomes. Most simply it reflects homozygosity for recessive deleterious alleles at particular loci; alternatively (consistent with absence of RR adults in multiclonal populations) it may reflect hemiclone-specific sets of incompletely recessive deleterious mutations that cumulatively cause inviability when two such genomes are combined. If inviability results from the former, progeny of two hybrids of different hemiclones, whether allopatric or coexisting, should be viable, because it is improbable that their R genomes share recessive deleterious alleles at the same set of loci; if inviability results from the latter, progeny of hybrids of different hemiclones should be inviable, especially when hybrid lineages are old. We tested these hypotheses in artificial crosses, using frogs from three regions: hemiclonal hybrids outside R. ridibunda's range from northern Switzerland (two abundant coexisting allozyme-defined hemiclones; estimated lineage age < or = 5,000 generations) and from Sicily, Italy (one hemiclone; estimated age > or = 25,000 generations) and R. ridibunda from Poland. We generated RR progeny, which we reared under benign conditions in the laboratory, by crossing (1) two hybrids from the same region (H x H local); (2) two hybrids from different regions (H X H foreign); (3) hybrids and R. ridibunda (H X R); and (4) two R. ridibunda (R X R). Survival to metamorphosis was similar and high for R x R, H X H foreign, and H X R, whereas all tadpoles of H X H local died before metamorphosis. This supports the hypothesis that homozygosity for recessive deleterious mutations at particular loci causes inviability. Crosses within and between the two coexisting hemiclones from Switzerland were, however, equally inviable. This result may reflect episodic sexual recombination in RR progeny from exceptional successful interclonal hybrid X hybrid matings, followed by matings of such RR with LL. This process would both slow down or halt Muller's ratchet and disrupt genetic independence of coexisting hemiclones, so that the same remaining deleterious R alleles could exist in different allozyme-defined hemiclones. Whereas all data are consistent with the prediction of Muller's ratchet operating on clonally transmitted R genomes of natural hybrid lineages, they are insufficient to demonstrate such operation, because deleterious recessives that mutated after clone formation and those that preexisted in the R. ridibunda source populations that formed the hemiclonal lineages are not distinguished. The possibility of episodic sexual recombination must be carefully taken into account when studying Muller's ratchet in natural populations of this Rana system.     

ADAPTIVE GENETIC VARIATION IN GROWTH AND DEVELOPMENT OF TADPOLES OF THE HYBRIDOGENETIC RANA ESCULENTA COMPLEX

The distribution and proportion of the sexual species Rana lessonae to the hemiclonal hybrid R. esculenta among natural habitats suggests that these anurans may differ in adaptive abilities. I used a half-sib design to partition phenotypic and quantitative genetic variation in tadpole responses at two food levels into causal variance components. Rana lessonae displays strong phenotypic variation across food levels. Growth rate is strictly determined by environmental factors and includes weak maternal effects. Larval period and body size at metamorphosis both contain moderate levels of additive genetic variance. The sire x food interactions and the lack of environmental correlations indicate that adaptive phenotypic plasticity is present in both of these traits. In contrast, R. esculenta displays less phenotypic variation across food levels, especially for larval period. Variation in body size at metamorphosis is underlain by genetic variation as shown by high levels of additive genetic variance, yet growth rate and larval period are not. Significant environmental correlations between larval period at high food level and growth, larval period, and body size at low food, indicate phenotypic plasticity is absent. A positive phenotypic correlation between body size at metamorphosis and larval period for R. lessonae at both food levels suggests a trade-off between growing large and metamorphosing quickly to escape predation or pond drying. The lack of a similar correlation for R. esculenta at the high food level suggests it may be less constrained. Different levels of adaptive genetic variation among larval traits suggest that the sexual species and the hybridogenetic hemiclone differ in their abilities to cope with temporally and spatially heterogeneous environments.     

Growth and development of larval green frogs (Rana clamitans) exposed to multiple doses of an insecticide

Our objective was to determine how green frogs (Rana clamitans) are affected by multiple exposures to a sublethal level of the carbamate insecticide, carbaryl, in outdoor ponds. Tadpoles were added to 1,000-l ponds at a low or high density which were exposed to carbaryl 0, 1, 2, or 3 times. Length of the larval period, mass, developmental stage, tadpole survival, and proportion metamorphosed were used to determine treatment effects. The frequency of dosing affected the proportion of green frogs that reached metamorphosis and the developmental stage of tadpoles. Generally, exposure to carbaryl increased rates of metamorphosis and development. The effect of the frequency of carbaryl exposure on development varied with the density treatment; the majority of metamorphs and the most developed tadpoles came from high-density ponds exposed to carbaryl 3 times. This interaction suggests that exposure to carbaryl later in the larval period stimulated metamorphosis, directly or indirectly, under high-density conditions. Our study indicates that exposure to a contaminant can lead to early initiation of metamorphosis and that natural biotic factors can mediate the effects of a contaminant in the environment.     

Brain plasticity over the metamorphic boundary: carry-over effect of larval environment on froglet brain development

Brain development shows high plasticity in response to environmental heterogeneity. However, it is unknown how environmental variation during development may affect brain architecture across life history switch points in species with complex life cycles. Previously, we showed that predation and competition affect brain development in common frog (Rana temporaria) tadpoles. Here, we studied whether larval environment had carry-over effects in brains of metamorphs. Tadpoles grown at high density had large optic tecta at metamorphosis, whereas tadpoles grown under predation risk had small diencephala. We found that larval density had a carry-over effect on froglet optic tectum size, whereas the effect of larval predation risk had vanished by metamorphosis. We discuss the possibility that the observed changes may be adaptive, reflecting the needs of an organism in given environmental and developmental contexts.     

Quantitative genetic analysis of larval life history traits in two alpine populations of Rana temporaria

We estimated genetic and maternal variance components of larval life history characters in alpine populations of Rana temporaria (the common frog) using a full-sib/half-sib breeding design. We studied trait plasticity by raising tadpoles at 14 or 20°C in the laboratory. Larval period and metamorphic mass were greater at 14°C. Larval period did not differ between populations, but high elevation metamorphs were larger than low elevation metamorphs. Significant additive variation for larval period was detected in the low altitude population. No significant additive variation was detected for mass at metamorphosis (MM), which instead displayed significant maternal effects. Plasticity in metamorphic mass of froglets was greater in the high altitude population. The plastic response of larval period to temperature did not differ between the populations. Evolution of metamorphic mass is likely constrained by lack of additive genetic variation. In contrast, significant heritability for larval period suggests this trait may evolve in response to environmental change. These results differ from other studies on R. temporaria, suggesting that populations of this broadly distributed species present substantial geographic variation in the genetic architecture and plasticity of tadpole life history traits.     

Influence of density and salinity on larval development of salt‐adapted and salt‐naïve frog populations

Environmental change and habitat fragmentation will affect population densities for many species. For those species that have locally adapted to persist in changed or stressful habitats, it is uncertain how density dependence will affect adaptive responses. Anurans (frogs and toads) are typically freshwater organisms, but some coastal populations of green treefrogs (Hyla cinerea) have adapted to brackish, coastal wetlands. Tadpoles from coastal populations metamorphose sooner and demonstrate faster growth rates than inland populations when reared solitarily. Although saltwater exposure has adaptively reduced the duration of the larval period for coastal populations, increases in densities during larval development typically increase time to metamorphosis and reduce rates of growth and survival. We test how combined stressors of density and salinity affect larval development between salt‐adapted (“coastal”) and nonsalt‐adapted (“inland”) populations by measuring various developmental and metamorphic phenotypes. We found that increased tadpole density strongly affected coastal and inland tadpole populations similarly. In high‐density treatments, both coastal and inland populations had reduced growth rates, greater exponential decay of growth, a smaller size at metamorphosis, took longer to reach metamorphosis, and had lower survivorship at metamorphosis. Salinity only exaggerated the effects of density on the time to reach metamorphosis and exponential decay of growth. Location of origin affected length at metamorphosis, with coastal tadpoles metamorphosing slightly longer than inland tadpoles across densities and salinities. These findings confirm that density has a strong and central influence on larval development even across divergent populations and habitat types and may mitigate the expression (and therefore detection) of locally adapted phenotypes.     

Feeding behaviour, food consumption, and growth efficiency of hemiclonal and parental tadpoles of the Rana esculenta complex

1. Clonally reproducing species are often assumed to lack sufficient genetic variability to evolve specific local adaptations to cope with environmental perturbation and competition from sexual species. Yet, many asexuals are extremely successful judged by abundance and wide range, suggesting high competitive abilities in resource exploitation.
2. In this study, food use and its effects on larval growth in a water frog system consisting of the two parental sexual species, Rana lessonae (Camerano 1882) and Rana ridibunda (Pallas 1771), and three different coexisting hemiclones of their hybrid, Rana esculenta (Linnaeus 1758) were investigated.
3. R. esculenta tadpoles spent 18·6% more time feeding than did tadpoles of either parental species, but feeding time was not affected by interspecific mixture.
4. R. esculenta tadpoles consumed 50·8% more food over the whole test period than did tadpoles of the two parental species.
5. R. esculenta tadpoles exhibited higher growth rates than did tadpoles of either parental species.
6. R. lessonae tadpoles had the highest and R . ridibunda tadpoles the lowest growth efficiencies with the R. esculenta tadpoles ranging between the two parentals.
7. The results obtained indicate that hemiclonal hybridogenetic R . esculenta tadpoles display significant phenotypic variation among coexisting hemiclones as well as out-perform tadpoles of the parental sexual species R. lessonae and R . ridibunda. The primary mechanism for success of the hybrid tadpoles is probably behavioural, through increased feeding time and food consumption, and not physiological via growth efficiency.     

Ecological constraints on amphibian metamorphosis: interactions of temperature and larval density with responses to changing food level

Phenotypic plasticity is adaptive for an organism inhabiting a variable environment if the optimal phenotype of a trait that affects fitness varies with environmental conditions, and if the organism can perceive environmental conditions and respond appropriately. Wilbur and Collins have proposed that amphibian larvae might respond adaptively to changes in their resource environment. If conditions for growth in the aquatic environment deteriorate, then a tadpole should metamorphose earlier and smaller than a tadpole under constant high growth conditions. Several experiments on a variety of species have tested this prediction, but only one demonstrated such a response. That experiment involved Couch's spadefoot toads (Scaphiopus couchii) and employed a gradual decrease in food level, whereas the others all used an abrupt switch from high to low food. The purpose of the present experiment was to examine the response of S. couchii to an abrupt change in food level, and to determine if the response depended on the level of two other factors, density and temperature, that also affect larval development. The average effects of the abrupt change in food level were similar to those seen in studies on other species: age at metamorphosis was primarily determined by the early food regime, and size at metamorphosis was determined by food level late in the larval period, suggesting that the effect of decreased food depends on how the food change is done. However, the response to even an abrupt food change depended on interactions with other environmental factors. At high temperature, high initial food, and low density, development was very rapid and tadpoles switched from high to low food metamorphosed at about the same time and size as those at constant high food. In contrast, under high temperature and high initial food conditions, but at high density, tadpoles switched to low food metamorphosed somewhat earlier and smaller, on average, than tadpoles kept at high food. At low temperature, the direction of response depended on density: tadpoles metamorphosed much smaller and slightly, but significantly, earlier at low density, but smaller and later at high density. The developmental response to increased food also varied with temperature. Larvae at high temperature metamorphosed earlier and larger than those at constant low food. At low temperature, larvae metamorphosed larger, but at nearly the same time as their counterparts at constant low food. The combination of high density and constant low food prevented any tadpoles from metamorphosing at high temperature, and allowed relatively few metamorphs at low temperature. Under conditions which impose either very rapid or retarded development, the opportunity to respond to altered food level may be limited. Interactions among environmental factors, therefore, may constrain responses to changing conditions, and may even prevent completion of development. Received: 3 February 1997 / Accepted: 2 October 1997     

Influence of density and predators on metamorphic size in Rhinella schneideri tadpoles raised in mesocosm conditions

Amphibians exhibit extreme plasticity in the timing of metamorphosis, and several species respond to water availability, accelerating metamorphosis when their ponds dry. We analyzed the plasticity of the developmental response to water volume in Rhinella schneideri tadpoles. We raised tadpoles in mesocosm. Covariation between body size at metamorphosis and timing of development was positive. Nevertheless, the first approximately 53% of the metamorphoses finishing the cycle required between 34 and 56 days, and the covariation between body size at metamorphosis and timing of development was negative. For these tadpoles, the larval density and the presence of predators did not significantly affect their mass to metamorphosis. Nevertheless, predators affected time to metamorphosis. For the remainder of the tadpoles that reached metamorphosis at > 56 days, the relationship between body size at metamorphosis and timing of development was positive. For these tadpoles, larval density was important for mass at metamorphosis and presence of predators was also important for time to metamorphosis. Two dominant features were observed: (i) approximately 53% of metamorphs had morphological features similar to individuals developing in desiccating ponds, and (ii) the other individuals had morphological characteristics comparable to metamorphs developing in an unchanging environment.     

Reproductive features of homospecific hybridogenetically-derived stick insects suggest how unisexuals can evolve

Hybridogenetic reproduction has been demonstrated in both vertebrate and invertebrate unisexual hybrids. Its most peculiar feature is the transmission to the progeny of one invariant genome (hemiclone) through the egg and the replacement of the other by host fathering males. Bacillus hybridogens are the only known example of hemiclonal invertebrates; their comparison to Poeciliopsis and Rana systems helps in understanding peculiar and shared features of vertebrate and insect hybridogenesis. In P. monacha-lucida, the experimental production of non-hybrid progeny through the reunion of the maternal hemiclone with a homospecific paternal genome provided by males of the maternal ancestor leads to inviable or severely impaired sterile specimens, whereas in Rana esculenta viable offspring are the rule. The comparable synthetic B. rossius progeny (Rr) embodying the maternal R hemiclone and a paternal r haploset, appear perfectly viable and fertile, clearly demonstrating compatibility between the two homospecific genomes, and also supporting a lack of deterioration of the R hemiclone. This condition can be ascribed to the recent origin of the hemiclones, and also to the absence of lethal recessives, owing to their most likely derivation from an automictic doubling in the parthenogenetic mechanisms of the maternal ancestor. However, the hybridogenetic system breaks down in the gamete production of the majority of Rr females, since normal allele segregation also occurs in their progeny. These reproductive modes suggest a likely evolutionary dynamic for newly originated hybridogens: to achieve stability, an interruption of reproductive interactions with the maternal ancestor seems necessary. In stick insects, this constraint appears to be fulfilled in both areas of sympatry. The microevolutionary pathway suggested by the ecological scenario also supports the possibility that a shift of hemiclonal stick insect strains to clonality has occurred.     

Differential performance among LDH-B genotypes in Rana lessonae tadpoles

The European pool frog, Rana lessonae, is widely polymorphic for two common alleles (b,e) at the lactate dehydrogenase-B (LDH-B) locus. We compared fitness-related larval life-history traits among LDH-B genotypes, which originated from segregation in heterozygous parents, in an artificial pond experiment where tadpoles of R. lessonae from a Swiss population were raised together with tadpoles of the hemiclonal hybrid R. esculenta at two densities. In R. lessonae, LDH-B e/e homozygotes at each density had a higher proportion of metamorphs among survivors, reached metamorphosis earlier, and were heavier at metamorphosis than b/b homozygotes; b/e heterozygotes had intermediate values. That e/e individuals were superior to b/b in both time to and mass at metamorphosis is surprising because these two life-history traits are thought to reflect a performance trade-off; e/e genotypes apparently compensated for shorter time to metamorphosis by a higher growth rate. The two alleles showed the same performance ranking when combined in hybrids with a R. ridibunda allele: When R. esculenta from Swiss populations reared in the same ponds had received the e allele rather than the b allele from their R. lessonae parent, they reached metamorphosis earlier, but did not differ in mass at metamorphosis. The degree of linkage disequilibrium in the source population of the eight R. lessonae used as parents of the R. lessonae tadpoles is unknown, so we cannot exclude the possibility that the performance differences are caused by some anonymous tightly linked gene, rather than the LDH-B locus, that constitutes the genomically localized target of natural selection. A causal involvement of LDH-B is plausible, nevertheless, because this enzyme takes part in the central energy-metabolizing processes and has been reported to underlie fitness differences in other animals; also, differential performance of LDH-B genotypes has been observed in R. lessonae larvae from another population. The present results suggest strong directional selection for allele e; the sum of available data, including an independent laboratory experiment, suggests that partial environment-dependent overdominance combined with balancing selection favoring e/e homozygotes under some and b/b homozygotes under other conditions may be partially responsible for the broad maintenance of the LDH-B polymorphism in R. lessonae.     

Interacting effects of predation risk and food availability on larval anuran behaviour and development

Age and size at metamorphosis are two important fitness components in species with complex life cycles. In anurans, metamorphic traits show remarkable phenotypic plasticity, especially in response to changes in growth conditions. It is also possible that the perception of risk directly determines changes in larval period and the size of metamorphs. This study examines how the perception of predation risk affects the timing of and size at metamorphosis in common frogs (Rana temporaria). I raised tadpoles at two risk levels (fish-conditioned water or unconditioned water) crossed with the availability or lack of food at night (all tadpoles had food available in the day). Tadpoles reacted to chemical cues from predatory fish by decreasing activity. A novel behavioural result was a predation×food interaction effect on refuge use, which also accounted for most of the predator main effect: predation risk only caused increased refuge use in the night-starved treatment. Despite these behavioural modifications, the perception of predation risk did not affect growth rate and mass at metamorphosis in a simple way: the effects of food regime on growth and size at metamorphosis were dependent on the level of predation risk as revealed by significant predation×food interaction effects. Tadpoles who had food withheld at night metamorphosed at the smallest size, suggesting a negative relationship between size at metamorphosis and refuge use. Tadpoles raised in fish-conditioned water had longer larval periods than those in unconditioned water, but these differences were significant only if food was available at night. These results conflict with the hypotheses that tadpoles should reduce their larval period or growth rates (and hence metamorphose at a smaller size) as the risk of predation increases. In contrast to predation risk, food availability strongly affected the length of the larval period: night-starved tadpoles metamorphosed relatively early with or without fish stimulus. Thus, early metamorphosis resulted from periods of low food availability, but not from a heightened ”perceived risk” of predation. This example counters the hypothesis of acceleration of the developmental rate (which shortens the time to metamorphosis) as a mechanism to escape a risky environment. Received: 18 August 1999 / Accepted: 10 January 2000     

Experimental analysis of the evolutionary potential of hybridization in leopard frogs (Anura: Ranidae)

Artificial crossing using Rana blairi and R. sphenocephala frogs produced conspecific, interspecific and F₁ backcross hybrid genotypes. Although hybrid males used in the crosses were sterile, crosses using hybrid females produced viable larvae. The larval performance of resultant parental and hybrid genotypes was measured in experimental ponds at two densities. Density significantly affected survival, body mass at metamorphosis, larval period length and metamorphosis for all genotypes. Survival was the same among genotypes, but decreased with increasing density. Body mass at metamorphosis was the same among genotypes, but decreased with increasing density. Larval period increased with increasing density. Among genotypes, larvae from the conspecific R. sphenocephala cross had the shortest larval period while larvae from the conspecific R. blairi cross had the longest larval period. All hybrid genotypes had larval periods longer than R. sphenocephala, but shorter than R. blairi. The percentage of individuals metamorphosing was highest for R. sphenocephala ponds and lowest for R. blairi ponds across densities. Ponds with hybrid larvae produced a greater proportion of metamorphs than those with R. blairi larvae, but a smaller proportion than R. sphenocephala ponds. Equivalent or increased relative larval performance of hybrid genotypes under the conditions of our experiment suggests that hybrid genotypes may possess similar or higher fitnesses relative to their progenitors in some environments. Reduced fertility of adult hybrid males is a powerful selective force against natural hybridization. Nevertheless, because of the successful reproduction by female hybrids, natural hybridization has the potential to serve as a mechanism for the introgression of novel genetic variation that can benefit both R. blairi and R. sphenocephala in fluctuating and unpredictable larval environments. Experimental determination of the fitness of parental and hybrid genotypes is crucial for a comprehensive understanding of the effects of hybridization on organismal evolution.     

Hemiclone diversity in the hybridogenetic frog Rana esculenta outside the area of clone formation: the view from protein electrophoresis

European water frog hybrids Rana esculenta reproduce hemiclonally, by hybridogenesis: In the germ line they exclude the genome of the parental species Rana lessonae and produce haploid, unrecombined gametes with a genome of the parental species Rana ridibunda . These hybrids coexist with and depend as sexual parasites on the host parental species R. lessonae (the L-E population system); matings with R. lessonae restore somatic hybridity in each generation of R. esculenta . We investigated 15 L-E system populations in northern Switzerland, which is outside R. ridibunda 's native range. Frequency of hybrids in samples varied from 8% in marsh ponds to 100% in gravel pits and forest ponds. Clonal diversity (variation among R. ridibunda genomes of hybrids), detected by six protein electrophoretic marker loci, revealed a total of eight hemiclones and locally ranged from uniclonal populations in southern parts of the survey region to six coexisting hemiclones in the north. All alleles distinguishing hemiclones occur commonly in the nearest native R. ridibunda populations of east-central Europe; the most probable source of clonal diversity in our samples is multiple clone formation by primary hybridizations in the sympatry area of R. ridibunda and R. lessonae and subsequent dispersal of hemiclonal lineages. A positive correlation between amount of clonal diversity and hybrid frequency, predicted by the Frozen Niche Variation (FNV) model (each hemiclone is characterized by a relatively narrow niche, coexistence is possible through niche partitioning), was not found; this contrasts with hemiclonally reproducing fish hybrids ( Poeciliopsis ). Historical factors, such as availability of different colonizing hemiclones may be strong enough to override the signal from operation of the FNV.     

Sequential predator effects across three life stages of the African tree frog, <Emphasis Type="Italic">Hyperolius spinigularis</Emphasis>

While theoretical studies of the timing of key switch points in complex life cycles such as hatching and metamorphosis have stressed the importance of considering multiple stages, most empirical work has focused on a single life stage. However, the relationship between the fitness components of different life stages may be complex. Ontogenetic switch points such as hatching and metamorphosis do not represent new beginnings—carryover effects across stages can arise when environmental effects on the density and/or traits of early ontogenetic stages subsequently alter mortality or growth in later stages. In this study, I examine the effects of egg- and larval-stage predators on larval performance, size at metamorphosis, and post-metamorphic predation in the African tree frog Hyperolius spinigularis. I monitored the density and survival of arboreal H. spinigularis clutches in the field to estimate how much egg-stage predation reduced the input of tadpoles into the pond. I then conducted experiments to determine: (1) how reductions in initial larval density due to egg predators affect larval survival and mass and age at metamorphosis in the presence and absence of aquatic larval predators, dragonfly larvae, and (2) how differences in mass or age at metamorphosis arising from predation in the embryonic and larval environments affect encounters with post-metamorphic predators, fishing spiders. Reduction in larval densities due to egg predation tended to increase per capita larval survival, decrease larval duration and increase mass at metamorphosis. Larval predators decreased larval survival and had density-dependent effects on larval duration and mass at metamorphosis. The combined effects of embryonic and larval-stage predators increased mass at metamorphosis of survivors by 91%. Larger mass at metamorphosis may have immediate fitness benefits, as larger metamorphs had higher survival in encounters with fishing spiders. Thus, the effects of predators early in ontogeny can alter predation risk even two life stages later.     

Populational Changes of the Enteric Protozoans Opalina spp. and Nyctotherus cordiformis During the Ontogeny of Anuran Tadpoles

ABSTRACT. We studied ontogenetic population changes of Opalina and Nyctotherus cordiformis in eight species of tadpoles from 10 sites in east-central Mississippi. Most tadpoles acquired Opalina early in development, while the acquisition of N. cordiformis was variable. Developmental stage, species and collection site explained significant amounts of the variation in Opalina density of tadpoles ( F = 11.6; df = 27, 235; P < 0.0001) and metamorphs ( F = 7.31; df = 24, 84; P < 0.0001). Relationships between Opalina density and host stage showed either (1) a gradual decrease or (2) a gradual increase throughout host ontogeny. Opalina densities declined during metamorphosis. Density variations of N. cordiformis were explained by host species of tadpoles ( F = 9.30; df = 7, 142; P < 0.0001) and by host species and stage of metamorphs ( F = 5.85; df = 8, 62; P < 0.0001).
The length of larval period, habitat duration and generation time of the protozoans are suggested as major modifiers of the protozoan densities. Hosts with long larval periods show a decreasing population density and hosts with short developmental periods show a pattern of increasing density. Neither pattern was detected in tadpoles from temporary sites. Metamorphic declines in protozoan density, but not necessarily the loss of protozoans, reflect metamorphic alterations of the gut common to all hosts.