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We found in two water frogs captured in Cyprus, the distribution area of P. cypriensis, nuclear alleles and mitochondrial haplotypes that are characteristic of Anatolian water frogs, Pelophylax cf. bedriagae. Mitochondrial and nuclear DNA data strongly indicate that the ancestors of these two frogs did not arrive on Cyprus via a land bridge or overseas dispersal but were unintentionally introduced by humans, most probably together with carp fingerlings. Both individuals exhibited recombined nuclear genomes characteristic of backcross hybrids. Further investigations are needed to estimate the amount of introgression of allochthonous alleles into the gene pool of P. cypriensis.

Water frogs inhabiting Cyprus represent a distinct evolutionary species of Messinian origin that is formally described in this paper. The systematic status of Cypriot frogs is evidenced by specific characters in their mitochondrial (mt) and nuclear (nu) DNA sequences, and the fact that they form a well supported monophyletic clade in both mtDNA and nuDNA phylogenies. While genetic data revealed clear and reproducible differences between this new taxon and all other western Palearctic water frog species including Pelophylax bedriagae in the Levant and two Anatolian water frogs lineages (P. cf. bedriagae -1 and P. cf. bedriagae -2), there is no diagnostic morphological or morphometric character that allows a clear discrimination between Cyprus frogs and frogs from the adjacent mainland. If several morphometric indices are combined as predictor variables in a discriminant analysis, however, both females and males of Cypriot water frogs are correctly distinguished from the other eastern Mediterranean lineages. While phylogenies based on concatenated sequences of two mitochondrial genes (ND2 + ND3) suggest a sister group relationship of Cypriot and Anatolian water frog lineages, our nuclear data hypothesize a sister group relationship between Cypriot frogs (sp. n.) and Crete frogs (P. cretensis), thus speaking for the same isolation time of both island populations (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

RrS1-like sequences of water frogs (genus Pelophylax) display varied genomic organization, whereas the centromeric hybridization pattern reveals species-specific differences. Using fluorescent in situ hybridization, Pelophylax cf. bedriagae, Pelophylax kurtmuelleri, and Pelophylax ridibundus showed a hybridization signal at centromeres of chromosomes 1-5, but in P. kurtmuelleri the medium-small chromosome labeled was 10 rather than 8. Pelophylax cretensis had almost 16 of 26 centromeres labeled, as did Pelophylax lessonae from Poland when its chromosomes are hybridized with a homologous probe. When StuI-digested genomic DNA was hybridized with RrS1 probe, hybridization ladders for P. ridibundus from Poland have evenly spaced steps (about 100 bp) of uniform intensity from about 200 bp upward. Steps in hybridization ladders from circum-Aegean taxa vary in intensity: larger, odd-numbered steps are often fainter. A strong double band (800/900 bp) in Anatolian P. cf. bedriagae, emphasized by a weak 700 bp band, distinguishes them from P. kurtmuelleri from the Peloponnisos, in which the 900 bp band is almost absent. The ladder in P. cretensis lacks odd-numbered steps. A and B repeats, observed originally within the RrS1 satellite of P. ridibundus, occur also in the circum-Aegean frogs and in P. lessonae, Pelophylax epeiroticus, Pelophylax saharicus, and Pelophylax shqipericus. It is plausible that AB dimers or ABB trimers rather than A or B monomers correspond to functional/evolutionary units. The presence of regions similar to yeast CDEs and mammalian CENP-B boxes suggests a role for RrS1 sequences in centromere organization.

AIM: Our aims were to assess the phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs and to estimate divergence times using different geological scenarios. We related divergence times to past geological events and discuss the relevance of our data for the systematics of eastern Mediterranean water frogs. LOCATION: The eastern Mediterranean region. METHODS: Genetic diversity and divergence were calculated using sequences of two protein-coding mitochondrial (mt) genes: ND2 (1038 bp, 119 sequences) and ND3 (340 bp, 612 sequences). Divergence times were estimated in a Bayesian framework under four geological scenarios representing alternative possible geological histories for the eastern Mediterranean. We then compared the different scenarios using Bayes factors and additional geological data. RESULTS: Extensive genetic diversity in mtDNA divides eastern Mediterranean water frogs into six main haplogroups (MHG). Three MHGs were identified on the Anatolian mainland; the most widespread MHG with the highest diversity is distributed from western Anatolia to the northern shore of the Caspian Sea, including the type locality of Pelophylax ridibundus. The other two Anatolian MHGs are restricted to south-eastern Turkey, occupying localities west and east of the Amanos mountain range. One of the remaining three MHGs is restricted to Cyprus; a second to the Levant; the third was found in the distribution area of European lake frogs (P. ridibundus group), including the Balkans. MAIN CONCLUSIONS: Based on geological evidence and estimates of genetic divergence we hypothesize that the water frogs of Cyprus have been isolated from the Anatolian mainland populations since the end of the Messinian salinity crisis (MSC), i.e. since c. 5.5-5.3 Ma, while our divergence time estimates indicate that the isolation of Crete from the mainland populations (Peloponnese, Anatolia) most likely pre-dates the MSC. The observed rates of divergence imply a time window of c. 1.6-1.1 million years for diversification of the largest Anatolian MHG; divergence between the two other Anatolian MHGs may have begun about 3.0 Ma, apparently as a result of uplift of the Amanos Mountains. Our mtDNA data suggest that the Anatolian water frogs and frogs from Cyprus represent several undescribed species.

Genotypic variation at six of 67 microsatellite loci we developed from Rana ridibunda, containing di- or trinucleotide simplesequence repeats, confirms the value of microsatellites as an evolutionary genetic tool for studying western Palearctic water frogs, a model system characterized by clonal reproduction in natural hybrid lineages. R. ridibunda, Rana lessonae. and their hemiclonal hybrid Rana esculenta, which transmits only its ridibunda genome to gametes, are highly variable in central Poland and northern Switzerland: with one exception (a locus that is part of a coding region), each locus was polymorphic in each taxon when scorable, and the number of alleles was high (in Swiss lessonae-esculenta populations, 1-6 per locus, total 13, for ridibunda genomes; 1-9 per locus, total 23, for lessonae genomes; 3 shared by both genomes). Estimated repeat numbers ranged from 3 to 14 for trinucleotide- and from 6 to 27 for dinucleotide-repeats. Nonamplifying (null) alleles, problematic because they underestimate genotypic variability if undetected, occur in ridibunda genomes of Swiss R. esculenta at one locus: apparent lessonae null alleles at another locus were subsequently resolved for several segregating alleles evidenced by bands of much lower intensity. Two loci had multiple ridibunda alleles in Swiss R. esculenta and thus discriminate between hemiclones. with a resolution superior to that of allozymes. Adding these two loci to our protein electrophoretic data (7 loci) more than doubled the number of hemiclones detected, showed that several allozyme-defined hemiclones are composites of multiple microsatellite hemiclones, and revealed clonal diversity in populations that are uniclonal by allozyme data. The extent to which microsatellite-defined hemiclones result from primary hybridizations or from subsequent mutations in ridibunda genomes of R. esculenta lineages remains to be determined. A sample of newly metamorphosed R. ridibunda from hybrid × hybrid matings collected at a Swiss population in the fall had a significant heterozygote excess at the two loci that varied in ridibunda genomes; this shows that most, probably all, of them originated from inter- rather than intrahemiclonal matings, in accord with the idea that the usual hybrid × hybrid inviability in lessonae-esculenta systems is caused by deleterious recessive alleles on the clonally inherited ridibunda genomes. Three of four microsatellite loci tested amplified in most species of western Palearctic water frogs.

Rana esculenta progeny from experimental crosses (using R. esculenta from natural lineages and F1 Ram ridihztncia x Rnrlo lessonarr), reared in artificial ponds and in cages immersed in natural breeding ponds, showed three different types of malformations reflecting developmental disturbances, each affecting few individuals overall. Giant tadpoles arrested development at the foot-paddle stage, independent from a size at metamorphosisitime to metamorphosis trade-off that may be under selection. The fused nostrils/eyeless syndrome probably reflects a disturbed gastrulation. The observed polymely, affecting the hind limbs, may result from infectious agents in pond water used to inoculate the tanks. Although known parentage and distribution among rearing tanks for each of the three malformations exclude a simple genetic basis and a direct influence of particular human-caused factors, it is not possible to further discriminate among genetic, biotically-induced, and non-biotically induced environmental causes of the developmental disturbances and their interactions. Direct experimental approaches will be needed to disentangle these possibilities and to assess their relative contributions to the observed decline of amphibian populations.

Complete mitochondrial genomes (mtDNA) of five individuals representing two haplotypes of Bombina bombina and three of Bombina variegata were compared using restriction site maps. Phylogenetic analyses reveal three ancient mitochondrial lineages: (1) two very similar haplotypes A and B of B. bombina; (2) almost identical haplotypes D and E of B. variegata; and (3) haplotype C of B. variegata. Haplotype C is as different from haplotypes D/E as from A/B. These data are strikingly discordant with relationships based on morphology and allozymes. Haplotypes C and D/E represent a pre-Pleistocene mitochondrial divergence within B. variegata, nearly coincident with speciation between B. variegata and B. bombina. Geographical partitioning of the two divergent B. variegata mitochondrial lineages indicates repeated localization of the lineages in separate glacial refugia during the Pleistocene. That nuclear genes do not show a similar divergence, but rather indicate relatively free genetic exchange between populations with divergent mtDNAs, suggests that males dispersed much more widely than females during expansions from glacial refugia. Comparison of Bombina mtDNA maps with a restriction site map of Xenopus laevis mtDNA revealed 16 homologous sites; 12 of these may be nearly invariant across primitive anuran mtDNAs. Two distinct regions of heteroplasmy, representing two regions with variable numbers of sequence repeats [length variable (LV) regions], were characterized. Comparison with the Xenopus map places LV1, present in all five haplotypes, near the 5′-end of the control region, and LV2 present only in B. variegata, near the 3′-end. Although phylogenetic analyses did not group the two major B. variegata lineages together, presence of LV2 in both lineages supports placement of both within B. variegata.

A family of interspersed repetitive elements, RlBamHI, with sequence similarity to the transposase of hAT DNA transposons, occurs in genomes of eight western Palaearctic water frog taxa and the brown frog Rana dalmatina, but was not detected in Xenopus laevis or Salamandra salamandra. RlBamHI elements are not tandemly arrayed, are dispersed across all chromosomes although not uniformly distributed, and based on dot-blot hybridizations may constitute as much as approximately 10% of the genomes of Rana lessonae, Rana ridibunda, and Rana perezi, but only approximately 1% of that of Rana saharica. Eleven nucleotide sequences of a 572 bp fragment from the nine taxa are very similar (pairwise differences 0.4–8.1% for nucleotides, 0.6–18.2% for amino acids), and all share a single open reading frame across the whole RlBamHI fragment. The reading frame is maintained despite several indels, most of which are multiples of 3 bp, but a pair of which in one species alters and, after 13 codons, restores the reading frame. It is possible that the reading frame is selectively maintained, suggesting recent or even present transposition capacity. The amino acid sequences encoded by RlBamHI elements, but not the nucleotide sequences themselves, reveal the similarity of RlBamHI to members of the widespread hAT superfamily of DNA transposons. Amino acid sequence comparisons permitted no convincing phylogenetic placement of RlBamHI among 32 representative hAT transposons across organisms, probably reflecting both the genetic divergence of RlBamHI elements and the paucity of aligned residues available for analysis. Among DNA transposons, a genomic fraction of 10% is extraordinarily high. Phylogenetic analyses of Rana RlBamHI sequences match poorly with independent genetic and molecular phylogenies; the elements compared probably are not orthologous, which renders their sequences inadequate for reconstructing organismal phylogenetic histories.

Three potent broad-spectrum antimicrobial peptides (HFIAP-1, -2, and -3) isolated from intestinal tissues of Myxine glutinosa (Atlantic hagfish) are identified as ancient members of the cathelicidin family of antimicrobial peptides, hitherto known only from mammals. In situ hybridization reveals that HFIAPs are produced in nests of myeloid cells within the loose connective tissue of the gut wall, a tissue reminiscent of both gut-associated lymphoid tissue (GALT) and vertebrate spleen. We suggest that this tissue organization provides local defense of the hagfish gastrointestinal tract via innate immunity and possibly served as the architectural plan upon which the adaptive immune system evolved.

Using five restriction enzymes, geographical variation of mitochondrial DNA (mtDNA) in Bombina bombina and B. variegata was studied in samples from 20 locations. Each restriction enzyme produced a species-specific fragment pattern. B. bombina haplotypes A and B were closely related to each other. In contrast, haplotypes A and B of B. variegata formed two distinct lineages. A very distinctive haplotype (C) was found in the Carpathian Mountains, whereas two other haplotypes, D and E (differing by a single AvaI site), were present in western Europe and the Balkans, respectively. Populations polymorphic for haplotypes D and E occurred in the central Balkans where the haplotypes could replace each other clinally. mtDNA sequence divergence between B. bombina and B. variegata was estimated as 6.0-8.1% and 4.7-5.2% between type C and types D/E of B. variegata. The latter divergence is contrary to allozyme and morphological data that place the western and Carpathian B. v. variegata together (Nei's D = 0.07) and separate them from the Balkan subspecies B. v. scabra (Nei's D = 0.18). Broad interspecific correlation among morphology, allozymes and mtDNA types in European fire-bellied toads argues that, despite continuous hybridization (interrupted perhaps during Pleistocene glacial maxima), little or no mtDNA introgression between the species has occurred outside the narrow hybrid zones that separate these parapatric species.

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.

AIM: Our aims were to assess the phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs and to estimate divergence times using different geological scenarios. We related divergence times to past geological events and discuss the relevance of our data for the systematics of eastern Mediterranean water frogs. LOCATION: The eastern Mediterranean region. METHODS: Genetic diversity and divergence were calculated using sequences of two protein-coding mitochondrial (mt) genes: ND2 (1038 bp, 119 sequences) and ND3 (340 bp, 612 sequences). Divergence times were estimated in a Bayesian framework under four geological scenarios representing alternative possible geological histories for the eastern Mediterranean. We then compared the different scenarios using Bayes factors and additional geological data. RESULTS: Extensive genetic diversity in mtDNA divides eastern Mediterranean water frogs into six main haplogroups (MHG). Three MHGs were identified on the Anatolian mainland; the most widespread MHG with the highest diversity is distributed from western Anatolia to the northern shore of the Caspian Sea, including the type locality of Pelophylax ridibundus. The other two Anatolian MHGs are restricted to south-eastern Turkey, occupying localities west and east of the Amanos mountain range. One of the remaining three MHGs is restricted to Cyprus; a second to the Levant; the third was found in the distribution area of European lake frogs (P. ridibundus group), including the Balkans. MAIN CONCLUSIONS: Based on geological evidence and estimates of genetic divergence we hypothesize that the water frogs of Cyprus have been isolated from the Anatolian mainland populations since the end of the Messinian salinity crisis (MSC), i.e. since c. 5.5-5.3 Ma, while our divergence time estimates indicate that the isolation of Crete from the mainland populations (Peloponnese, Anatolia) most likely pre-dates the MSC. The observed rates of divergence imply a time window of c. 1.6-1.1 million years for diversification of the largest Anatolian MHG; divergence between the two other Anatolian MHGs may have begun about 3.0 Ma, apparently as a result of uplift of the Amanos Mountains. Our mtDNA data suggest that the Anatolian water frogs and frogs from Cyprus represent several undescribed species.

Water frogs [genus Pelophylax (Rana)] that occur around the eastern Mediterranean Sea provide an opportunity to study early stages of speciation. The geography of the eastern Mediterranean region has changed dramatically since the Middle Miocene as a result of motions of adjoining lithospheric plates and regional-scale vertical crustal motions (uplift and subsidence). For several hundred thousand years between 6 and 5 million years ago (Mya), the Mediterranean basin was isolated from the Atlantic Ocean, and became desiccated (the Messinian Salinity Crisis; MSC). Geological data suggest that the endemic water frog lineage on Cyprus was isolated by the flooding of the Mediterranean basin by salt water at the end of the MSC, circa 5.5–5.3 Mya. This suggests a rate of uncorrected genetic divergence of approximately 1.1% per million years (My). Divergence time estimates based on this rate are in good agreement with the chronology of events in the history of crustal deformation and landscape development in the eastern Mediterranean region.

A 5′ truncated non-LTR CR1-like retrotransposon, named RanaCR1, was identified in the serum albumin intron-1 (SAI-1) of at least seven species of western Palearctic water frogs (WPWF). Based on sequence similarity of the carboxy-terminal region (CTR) of ORF2 and/or the highly conserved 3′ untranslated region (3′ UTR), RanaCR1-like elements occur also in the genome of Xenopus tropicalis and Rana temporaria. Unlike other CR1 elements, RanaCR1 contains a CA microsatellite in its 3′ UTR. The low nucleotide diversity of the 3′ UTR compared to the CTR and to SAI-1 suggests that this region still plays a role in WPWF, either as a structure-stabilizing element, or within a species-specific transcriptional network. Length variation of water frog SAI-1 sequences is caused by deletions that extend in some cases beyond the 5′ or 3′ ends of RanaCR1, probably a result of selection for structural and functional stability of the primary transcript. The impact of RanaCR1 on SAI-1 evolution is also indicated by the significant negative correlation between the length of both SAI-1 and RanaCR1 and the percentage GC content of RanaCR1. Both SAI-1 and RanaCR1 sequences support the sister group relationship of R. perezi and R. saharica, which are placed in the phylogenetic tree at a basal position, the sister clade to other water frog taxa. It also supports the monophyly of the R. lessonae group; of Anatolian water frogs (R. cf. bedriagae), which are not conspecific with R. bedriagae, and of the European ridibunda group. Within the ridibunda clade, Greek frogs are clearly separated, supporting the hypothesis that Balkan water frogs represent a distinct species. Frogs from Atyrau (Kazakhstan), the type locality of R. ridibunda, were heterozygous for a ridibunda and a cf. bedriagae specific allele.

The complete mitochondrial genomes of two basal anurans, Bombina bombina and B. variegata (Anura; Bombinatoridae), were sequenced. The gene order of their mitochondrial DNA (mtDNA) is identical to that of canonical vertebrate mtDNA. In contrast, we show that there are structural differences in regulatory regions and protein coding genes between the mtDNA of these two closely related species. Corrected sequence divergence between the mtDNA of B. bombina and B. variegata amounts to 8.7% (2.3% divergence in amino acids). Comparisons with two East Asian congeners show that the control region contains two repeat regions, LV1 and LV2, present in all species except for B. bombina, in which LV2 has been secondarily lost. The rRNAs and tRNAs are characterized by low nucleotide divergence. The protein coding genes are considerably more disparate, although functional constraint is high but variable among genes, as evidenced by dN/dS ratios. A mtDNA phylogeny established the distribution of autapomorphic nonsynonomous substitutions in the mitogenomes of B. bombina and B. variegata. Nine of 98 nonsynonomous substitutions led to radical amino acid replacements that may alter mitochondrial protein function. Most radical substitutions were found in ND2, ND4, or ND5, encoding mitochondrial subunits of complex I of the electron transport system. The extensive divergence between the mitogenomes of B. bombina and B. variegata is discussed in terms of its possible role in impeding gene flow in natural hybrid zones between these two species.

European water frog hybrids Rana esculenta (R. ridibunda×R. lessonae) reproduce hemiclonally, by hybridogenesis: in the germ line they exclude the genome of one parental species and produce haploid gametes with an unrecombined genome of the other parental species. In the widespread L-E population system, both sexes of hybrids (E) coexist with R. lessonae (L). They exclude the lessonae genome and produce ridibunda gametes. In the R-E system, hybrid males coexist with R. ridibunda (R); they exclude either their ridibunda or their lessonae genome and produce sperm with a lessonae or with a ridibunda genome or a mixture of both kinds of sperm. We examined 13 male offspring, 12 of which were from crosses between L-E system and R-E system frogs. All were somatically hybrid. With one exception, they excluded the lessonae genome in the germ line and subsequently endoreduplicated the ridibunda genome. Spermatogonial metaphases contained a haploid or a diploid number of ridibunda chromosomes, identified through in situ hybridization to a satellite DNA marker, and by spermatocyte I metaphases containing a haploid number of ridibunda bivalents. The exception, an F1 hybrid between L-E system R. lessonae and R-E system R. ridibunda, was not hybridogenetic, showed no genome exclusion, and evidenced a disturbed gametogenesis resulting from the combination of two heterospecific genomes. None of the hybridogenetic hybrids showed any cell lines excluding the ridibunda genome, the pattern most frequent in hybrids of the R-E system, unique to that system, and essential for its persistence. A particular combination of R-E system lessonae and R-E system ridibunda genomes seems necessary to induce the R-E system type of hemiclonal gametogenesis.

The fire-bellied toads Bombina bombina and Bombina variegata, interbreed in a long, narrow zone maintained by a balance between selection and dispersal. Hybridization takes place between local, genetically differentiated groups. To quantify divergence between these groups and reconstruct their history and demography, we analysed nucleotide variation at the mitochondrial cytochrome b gene (1096 bp) in 364 individuals from 156 sites representing the entire range of both species. Three distinct clades with high sequence divergence (K2P = 8-11%) were distinguished. One clade grouped B. bombina haplotypes; the two other clades grouped B. variegata haplotypes. One B. variegata clade included only Carpathian individuals; the other represented B. variegata from the southwestern parts of its distribution: Southern and Western Europe (Balkano-Western lineage), Apennines, and the Rhodope Mountains. Differentiation between the Carpathian and Balkano-Western lineages, K2P approximately 8%, approached interspecific divergence. Deep divergence among European Bombina lineages suggests their preglacial origin, and implies long and largely independent evolutionary histories of the species. Multiple glacial refugia were identified in the lowlands adjoining the Black Sea, in the Carpathians, in the Balkans, and in the Apennines. The results of the nested clade and demographic analyses suggest drastic reductions of population sizes during the last glacial period, and significant demographic growth related to postglacial colonization. Inferred history, supported by fossil evidence, demonstrates that Bombina ranges underwent repeated contractions and expansions. Geographical concordance between morphology, allozymes, and mtDNA shows that previous episodes of interspecific hybridization have left no detectable mtDNA introgression. Either the admixed populations went extinct, or selection against hybrids hindered mtDNA gene flow in ancient hybrid zones.

Abundant natural interspecies hybrids between the European water frog Rana ridibunda and at least three other taxa reproduce hemiclonally, by hybridogenesis: the non-ridi- bunda genome is excluded in the germ line before meiosis, and the unrecombined ridibunda ge- nome is transmitted to haploid gametes. In contrast, natural hybrids between Rana ridibunda and either of two Balkan species (Rana shqiperica and Rana epeirotica) do not show such genome exclusion. This plausibly results from failure of Balkan Rana ridibunda genomes to "induce" such exclusion in the germ line of hybrids, from "resistance" of Rana shqiperica and Rana epeirotica genomes to such exclusion in hybrids with an "inducing" Rana ridibunda genome, or both. We tested the second hypothesis by examining lampbrush chromosome patterns in oocytes of hybrids that in the soma contain one "inducing" ridibunda genome and one genome of either of the two Balkan species. Several lampbrush chromosome markers (e.g., presence and location of certain giant loops and conspicuousness and width of centromeres) discriminate sets of Rana ridibunda chromosomes from those of Rana shqiperica and Rana epeirotica. Based on such markers, nine diploid female hybrids between Rana ridibunda or Rana esculenta from natural hybridogenetic lineages (Rana ridibunda ◊ Rana lessonae, making ridibunda gametes) from central Poland and either Rana shqiperica or Rana epeirotica each contained both parental genomes in primary oo- cytes; the bivalents showed reduced numbers of chiasmata compared with parental species. It follows that none of these hybrids was hybridogenetic. This conclusion is confirmed, for two hy- brids between Rana epeirotica and either Rana ridibunda or Rana esculenta, by protein electro- phoretic comparison of somatic tissues with primary oocytes, all of which evidenced allelic markers of both parental species. Because Rana ridibunda genomes that are known to induce germ line genome exclusion when combined in hybrids with Rana lessonae genomes were used, these data provide the first compelling evidence for resistance of Rana shqiperica as well as Rana epeirotica genomes to such exclusion. J. Exp. Zool. 279:163-176, 1997. © 1997 Wiley-Liss, Inc.

Among progeny of a hybrid (Rana shqiperica × R. lessonae) × R lessonae, 14 of 22 loci form four linkage groups (LGs): (1) mitochondrial aspartate aminotransferase, carbonate dehydratase-2, esterase 4, peptidase D; (2) mannosephosphate isomerase, lactate dehydrogenase-B, sex, hexokinase-1, peptidase B; (3) albumin, fructose-biphosphatase-1, guanine deaminase; (4) mitochondrial superoxide dismutase, cytosolic malic enzyme, xanthine oxidase. Fructose-biphosphate aldolase-2 and cytosolic aspartate aminotransferase possibly form a fifth LG. Mitochondrial aconitate hydratase, α-glucosidase, glyceraldehyde-3+hosphate dehydrogenase, phosphogluconate dehydrogenase, and phosphoglucomutase-2 are unlinked to other loci. All testable linkages (among eight loci of LGs 1, 2, 3, and 4) are shared with eastern Palearctic water frogs. Including published data, 44 protein loci can be assigned to 10 of the 13 chromosomes in Holarctic Rana. Of testable pairs among 18 protein loci, agreement between Palearctic and Nearctic Rana is complete (125 unlinked, 14 linked pairs among 14 loci of five syntenies), and Holarctic Rana and Xenopus laevis are highly concordant (125 shared nonlinkages, 13 shared linkages, three differences). Several Rana syntenies occur in mammals and fish. Many syntenies apparently have persisted for 60–140 × 106 years (frogs), some even for 350–400 × 106 years (mammals and teleosts).