Hansjürg Hotz

GEOLOGICALLY DATED SEA BARRIERS CALIBRATE A PROTEIN CLOCK FOR AEGEAN WATER FROGS

Reliable estimates of phylogenetic relationships and divergence times are a crucial requirement for many evolutionary studies, but are usually difficult because fossils are scarce and their interpretation is often uncertain. Frogs are fresh water animals that generally are unable to cross salt water barriers (their skin is readily permeable to both salt and water). The geologically determined ages of salt water barriers that isolate related frog populations thus provide an independent measure of the minimum date of genetic divergence between pairs of such populations. For the genetically well‐studied western Palearctic water frogs (Rana esculenta group), the Aegean region provides an ideal area for determining the relationship between genetic divergence and time of spatial isolation, using a nested set of geologically determined isolation times (12,000 yr, 200,000 yr, 1.8 Myr, 2–3 Myr, and 5.2 Myr).

COMPETITION AMONG TADPOLES OF COEXISTING HEMICLONES OF HYBRIDOGENETIC RANA ESCULENTA: SUPPORT FOR THE FROZEN NICHE VARIATION MODEL

Vertebrate animals reproducing without genetic recombination typically are hybrids, which have large ranges, are locally abundant, and live in disturbed or harsh habitats. This holds for the hemiclonal hybridogenetic frog Rana esculenta: it is widespread in Europe and commonly is found in disturbed habitats such as gravel pits. We hypothesize that its widespread occurrence may either be the result of natural selection for a single hemiclone acting as a broadly adapted “general‐purpose” genotype, or of interclonal selection, which maintains multiple hemiclones that each are relatively narrowly adapted and perform differently across environments, that is, the Frozen Niche Variation model. We tested these competing hypotheses using 1000‐L outdoor artificial ponds to rear tadpoles of the parental species (Rana lessonae [LL] and Rana ridibunda [RR]) alone, and each of three hemiclones of Rana esculenta (GUT1, GUT2, GUT3) alone, and in mixed hemiclonal populations from hatching to metamorphosis. Tadpoles of three coexisting hemiclones from a single natural population (near Gütighausen, Switzerland) were reared in both two‐ and three‐way mixtures in equal total numbers at high and low density. For each species and hemiclone, the proportion of tadpoles metamorphosing decreased as the density of tadpoles increased, with the three hemiclones spanning the range of values exhibited by the two parental species. LL and GUT1 tadpoles produced the highest proportion of metamorphs, whereas tadpoles of RR produced the fewest metamorphs at both densities. GUT1 tadpoles also produced the largest metamorphs at low density, GUT2 and GUT3 tadpoles produced smaller metamorphs than did GUT1 tadpoles at the low density, but the three hemiclones did not differ from each other at high density. The parental species (LL and RR) were intermediate in metamorphic size to the hemiclones at low density, but all genotypes converged on a similar size at high density. Length of the larval period also was affected by density, but its effect was dependent on genotype. GUT1 tadpoles had the shortest larval period at the low density, but larval period was longer and not different between GUT1, GUT3, and LL at high density. RR tadpoles had the longest larval period at both densities. The most dramatic results were that three genotypes (GUT1, GUT2, and RR) maintained rank order and increased days to metamorphosis from low to high density, whereas two genotypes (GUT3 and LL) changed rank order and decreased days to metamorphosis from low to high density. Mixtures of hemiclones in two‐ and three‐way combinations facilitated the proportion of tadpoles metamorphosing for GUT1 and GUT2 at both densities, but only at the low density for GUT3 tadpoles. Results from this experiment are incompatible with the General‐Purpose Genotype model as a global explanation of hybrid abundance in these frogs. Alternatively, the Frozen Niche Variation prediction of general performance superiority of clonal mixtures relative to single clone populations is strongly supported. The data confirm that fitness advantages of hemiclones change, depending on the environment, such that in temporally and spatially heterogeneous habitats like ponds, frequency‐dependent selection among hemiclones may promote coexistence in hemiclonal assemblages. Yet, differential dispersal or colonization ability and historical factors affecting hemiclone distribution may also be important in shaping patterns of clonal coexistence.

OVERWINTER SURVIVAL OF RANA LESSONAE AND ITS HEMICLONAL ASSOCIATE RANA ESCULENTA

In central Europe, the hybridogenetic water frog Rana esculenta is a sexual parasite that only persists in the presence of the parental species, R. lessonae, with which it must mate in order to reproduce. R. esculenta is a superior larval competitor, and adult females are about three times more fecund than R. lessonae. This suggests that, in the absence of some balancing advantage to R. lessonae, R. esculenta should drive the parental species locally extinct, closely followed by itself. We measured annual survival rates over six years at two widely separated ponds using modern capture–mark–recapture methods to test whether differences in adult survival might contribute to the persistence of the water frog system. We marked 856 adult frogs and recovered 182 at least once. The data did not deviate significantly from the assumptions of the Cormack-Jolly-Seber model at either pond. There were no detectable differences in recapture probabilities between taxa, but females were, overall, less likely to be recaptured. Survival was higher for R. esculenta in all but one year. Therefore, some mechanism other than adult survival must be invoked to explain the persistence of this unusual breeding system. There was considerable year-to-year variation in survival, ranging from 6% over the winter of 1996–1997, to 98% over the winter of 1995–1996. Although the two ponds are separated by 35 km, pond identity did not contribute to the minimum models. A composite measure of winter severity constructed from a principal components analysis of weather data during the course of the study had a very close relationship with survival (R. lessonae, r = 0.905; R. esculenta, r = 0.889). Survival was lowest in winters with low minimum temperatures coupled with high and variable maximum temperatures. Corresponding Editor: B. Sinervo.

Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid frogRana esculenta and its parental species

Hybrid water frogsRana esculenta reproduce by hybridogenesis: one parental genome (ofRana lessonae) is excluded in the germ line, the other (ofRana ridibunda) is clonally transmitted to haploid gametes. The two parental species differ in that the amount of centromeric heterochromatin revealed by differential staining is much higher inRana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed inRana ridibunda genomes by the restriction endonucleaseStul, which generates a major repetitive sequence fragment of 300 and a minor one of 200 bp. This AT-rich (68%) satellite family is located at the centromeres of the five largest chromosomes (1–5) and of a medium to small heterobrachial one (8 or 9); it thus constitutes only part of the centromeric heterochromatin that characterizes allRana ridibunda chromosomes. RrS1 represents about 2.5% of the genome ofRana ridibunda; it may represent as little as 0.2% of the genome ofRana lessonae, and cannot be detected inXenopus laevis frogs orSalamandra salamandra andTriturus carnifex salamanders. Segments of the satellite sequence are similar to sequences of yeast centromeric DNA element CDEIII and of the mammalian CENP-B box. A role for RrS1 and other centromeric satellite DNAs in the germ line genome exclusion of the hybridogenetic frog hybrids, although suggested, has not yet been demonstrated.

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

SummaryHybridogenetic 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 Poznań, 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.

Differential Morphology and Jumping Performance of Newly Metamorphosed Frogs of the Hybridogenetic Rana esculenta Complex

Closely related clonal and sexual populations may coexist in spite of the theorized lower potential for the evolution of clonal genotypes. Water frogs of the Rana esculenta complex have hemiclonal inheritance but most populations coexist with one of the recombinant parental species. We examine whether hemiclonal lineages may counterbalance their limitations of genetic variability by the adoption of one or more non-exclusive mechanisms: the general-purpose genotype or the frozen niche-variation model. Three coexisting hemiclones of the hybrid R. esculenta (GUT1, GUT2, GUT3) and both parental species (syntopic R. lessonae and allopatric R. ridibunda) were raised at two larval densities to examine morphological traits affecting jumping performance at the time of metamorphosis and size-independent jumping ability tested at three temperatures. Hind leg length and body mass at metamorphosis, traits that explain most of the variance in jumping performance, differed across hemiclones of R. esculenta. Metamorphs of hemiclone GUT1 had the longest hindlimbs and were bigger than metamorphs of the other hemiclones at low larval density but not at high density. Size adjusted jumping performance exhibited a significant genotype by larval density interaction. Metamorphs of GUT1 showed maximal jumping performance when raised at low larval density but at high density metamorphs of GUT2 were the best jumpers. In addition, within particular traits, differences were found between hemiclones across densities. These results appear to be consistent with both frozen niche-variation model and the general-purpose genotype model. Comparison with parental species revealed syntopic R. lessonae was smaller than hemiclones at metamorphosis but conversely exhib- ited better size-adjusted jumping performance when raised at low larval density. Temperature affected size- adjusted jumping performance only for frogs raised at low larval density but not for those raised at high larval densities. There was no significant temperature by hemiclone interaction.

Bioaccumulation of organochlorine pesticides in frogs of the Rana esculenta complex in central Italy

Concentrations of commonly used organochlorine pesticides (OCPs) were determined in tissues of 23 adult and 24 larval water frogs of two coexisting species (Rana lessonae and the hemiclonal hybrid R. esculenta) and in the water of their breeding pond in an agricultural zone in Umbria, central Italy, where increased occurrence of infectious diseases and distinctly oversized tadpoles were recently observed. The concentrations of OCP in tissues of both species were lower than those in the water of their breeding pond, except for DDT, which was more concentrated in adult frogs than in pond water (bioaccumulation factor 7 for R. lessonae, 15 for R. esculenta). Total OCP concentration and adult body weight were positively correlated for both species, which is consistent with bioaccumulation. In accord, adults contained higher OCP concentrations than tadpoles. Oversized tadpoles had higher OCP concentrations than normal tadpoles. Mean OCP concentrations in individual organs were about an order of magnitude higher than those in whole-frog homogenates. They were highest in brain, higher in ventral than in dorsal skin, and moderately high in ovaries; transmission of bioaccumulation loads to the next generation is therefore possible. The observed OCP concentrations appear too low to directly cause mortality in water frogs, but effects of cumulative exposure to low-level pollutants and their synergistic interactions with the effects of other natural and anthropogenic environmental stressors are unknown.

DIFFERENTIAL PERFORMANCE AMONG LDH-B GENOTYPES IN RANA LESSONAE TADPOLES

Abstract 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.

Masked Damage: Mutational Load in Hemiclonal Water Frogs

Hemiclonal hybrids of Western Palearctic water frogs of the Rana esculenta complex transmit only one parental genome to their offspring without recombination (hybridogenesis). Such genomes are thus prone to accumulate deleterious mutations. The frog complex is unique among hybridogens in that hemiclonal hybrids occur in both sexes. This provides the opportunity of using experimental crosses to produce offspring possessing two clonal genomes of various origins and thereby study their homozygous and heterozygous effects on fitness. Here we review work that made use of this possibility to assess the evolutionary consequences of clonal inheritance in water frogs. Overall, these studies indicate that clonally transmitted genomes bear a substantial load of fixed deleterious mutations, yet these mutations appear to have minor effects on fitness in the heterozygous state. We also point out potential mechanisms for episodic recombination by which otherwise clonal genomes may be purged of deleterious alleles, and we present evidence for such episodic recombination to occur in natural populations of hybridogenetic frogs. Finally, we provide an outlook on work in progress that exploits the peculiarities of this system to obtain relevant estimates of the frequency of segregating lethal mutations in sexual populations of water frogs.

Balancing a Cline by Influx of Migrants: A Genetic Transition in Water Frogs of Eastern Greece

Variation patterns of allozymes and of ND3 haplotypes of mitochondrial DNA reveal a zone of genetic transition among western Palearctic water frogs extending across northeastern Greece and European Turkey. At the western end of the zone, allozymes characteristic of Central European frogs known as Pelophylax ridibundus predominate, whereas at the eastern end, alleles characteristic of western Anatolian water frogs (P. cf. bedriagae) prevail. The ND3 haplotypes reveal 2 major clades, 1 characteristic of Anatolian frogs, the other of European; the European clade itself has distinct eastern and western subclades. Both the 2 major clades and the 2 subclades overlap within the transition zone. Using Bayesian model selection methods, allozyme data suggest considerable immigration into the Nestos River area from eastern and western populations. In contrast, the ND3 data suggest that migration rates are so high among all locations that they form a single panmictic unit; the best model for allozymes is second best for mitochondrial DNA (mtDNA). Nuclear markers (allozymes), which have roughly 4 times as deep a coalescent history as mtDNA data and thus may reflect patterns over a longer time, indicate that eastern and western refugial populations have expanded since deglaciation (in the last 10,000 years) and have met near the Nestos River, whereas the mtDNA with its smaller effective population size has already lost the signal of partitioning into refugia.