Cytogenetics of the Hybridogenetic Frog Pelophylax grafi and Its Parental Species Pelophylax perezi.

Hybrid taxa from the genus Pelophylax can propagate themselves in a modified way of sexual reproduction called hybridogenesis ensuring the formation of clonal gametes containing the genome of only one parental (host) species. Pelophylax grafi from South-Western Europe is a hybrid composed of P. ridibundus and P. perezi genomes and it lives with a host species P. perezi (P-G system). Yet it is unknown, whether non-Mendelian inheritance is fully maintained in such populations. In this study, we characterize P. perezi and P. grafi somatic karyotypes by using comparative genomic hybridization, genomic in situ hybridization, fluorescent in situ hybridization, and actinomycin D-DAPI. Here, we show the homeology of P. perezi and P. grafi somatic karyotypes to other Pelophylax taxa with 2n = 26 and equal contribution of ridibundus and perezi chromosomes in P. grafi which supports F1 hybrid genome constitution as well as a hemiclonal genome inheritance. We show that ridibundus chromosomes have larger regions of interstitial (TTAGGG)n repeats flanking the nucleolus organizing region on chromosome no. 10 and a high quantity of AT pairs in the centromeric regions. In P. perezi, we found species-specific sequences in metaphase chromosomes and marker structures in lampbrush chromosomes. Pericentromeric RrS1 repeat sequence was present in perezi and ridibundus chromosomes, but the blocks were stronger in ridibundus. Various cytogenetic techniques applied to the P-G system provide genome discrimination between ridibundus and perezi chromosomal sets. They could be used in studies of germ-line cells to explain patterns of clonal gametogenesis in P. grafi and broaden the knowledge about reproductive strategies in hybrid animals.

Genetic and karyotype divergence between parents affect clonality and sterility in hybrids.

Asexual reproduction can be triggered by interspecific hybridization, but its emergence is supposedly rare, relying on exceptional combinations of suitable genomes. To examine how genomic and karyotype divergence between parental lineages affect the incidence of asexual gametogenesis, we experimentally hybridized fishes (Cobitidae) across a broad phylogenetic spectrum, assessed by whole exome data. Gametogenic pathways generally followed a continuum from sexual reproduction in hybrids between closely related evolutionary lineages to sterile or inviable crosses between distant lineages. However, most crosses resulted in a combination of sterile males and asexually reproducing females. Their gametes usually experienced problems in chromosome pairing, but females also produced a certain proportion of oocytes with premeiotically duplicated genomes, enabling their development into clonal eggs. Interspecific hybridization may thus commonly affect cell cycles in a specific way, allowing the formation of unreduced oocytes. The emergence of asexual gametogenesis appears tightly linked to hybrid sterility and constitutes an inherent part of the extended speciation continuum.

Early Development Survival of Pelophylax Water Frog Progeny is Primarily Affected by Paternal Genomic Input.

BACKGROUND: Hybrid taxa exist in nature, but their fitness can vary greatly. Hybrids are usually thought to have lower viability and survival rate than parental species due to the occurrence of two different genomes and divergent evolution in each species. On the other hand, the hybrid vigour of the F1 generation may give hybrids an advantage in mixed populations where they have to live and compete with parental taxa. Post-zygotic selection with endogenous genetic mechanisms may be a significant evolutionary force in hybrid formation. Here we tested principles of post-zygotic reproductive dynamics in mixed populations of Pelophylax water frogs that would help us understand the origin and maintenance of such systems. METHODS: Within experimental crosses, we combined various diploid Pelophylax genotypes resulting in 211 families. Statistical analysis of progeny was used to measure fertilization success, the rate of embryonic/tadpole mortality and the overall survival of the progeny till the time of metamorphosis. Using Generalized Estimating Equations models and variables defined by a mother/father included in mate pairs, we tested which factor best explains the successful embryonal development. RESULTS: The development of Pelophylax offspring significantly varied in survival rate and morphological malformations. These post-zygotic reproductive dynamics were driven by parental combinations of species pairs. The best values in the proportion of developing eggs, embryos, tadpoles and overall survival showed progeny of homospecific P. lessonae crosses. Total survival rates were relatively similar between L-E and R-E population systems but much lower than homospecific crosses in parental taxa. However, once the early stages passed this period, tadpoles mostly of hybrid hemiclonal origin performed even better than pure P. ridibundus progeny. Hybrid × hybrid crosses showed the highest mortality values. Statistical testing revealed that high mortality affected paternal genetic input. CONCLUSIONS: Combined three water frog taxa and both sexes provided patterns of post-zygotic reproduction dynamics of early development in the widespread population systems in Central Europe. The results further showed high survival rates of hybrid F1s created de novo from parental species despite significant divergence between P. ridibundus and P. lessonae DNA. Potential conservation measures of sexual-asexual systems in natural populations are discussed.

The high diversity of gametogenic pathways in amphispermic water frog hybrids from Eastern Ukraine.

Interspecific hybridization can disrupt canonical gametogenic pathways, leading to the emergence of clonal and hemiclonal organisms. Such gametogenic alterations usually include genome endoreplication and/or premeiotic elimination of one of the parental genomes. The hybrid frog Pelophylax esculentus exploits genome endoreplication and genome elimination to produce haploid gametes with chromosomes of only one parental species. To reproduce, hybrids coexist with one of the parental species and form specific population systems. Here, we investigated the mechanism of spermatogenesis in diploid P. esculentus from sympatric populations of P. ridibundus using fluorescent in situ hybridization. We found that the genome composition and ploidy of germ cells, meiotic cells, and spermatids vary among P. esculentus individuals. The spermatogenic patterns observed in various hybrid males suggest the occurrence of at least six diverse germ cell populations, each with a specific premeiotic genome elimination and endoreplication pathway. Besides co-occurring aberrant cells detected during meiosis and gamete aneuploidy, alterations in genome duplication and endoreplication have led to either haploid or diploid sperm production. Diploid P. esculentus males from mixed populations of P. ridibundus rarely follow classical hybridogenesis. Instead, hybrid males simultaneously produce gametes with different genome compositions and ploidy levels. The persistence of the studied mixed populations highly relies on gametes containing a genome of the other parental species, P. lessonae.

Specialization directs habitat selection responses to a top predator in semiaquatic but not aquatic taxa.

Habitat selectivity has become an increasingly acknowledged mechanism shaping the structure of freshwater communities; however, most studies have focused on the effect of predators and competitors, neglecting habitat complexity and specialization. In this study, we examined the habitat selection of semiaquatic (amphibians: Bufonidae; odonates: Libellulidae) and aquatic organisms (true bugs: Notonectidae; diving beetles: Dytiscidae). From each family, we selected one habitat generalist species able to coexist with fish (Bufo bufo, Sympetrum sanguineum, Notonecta glauca, Dytiscus marginalis) and one species specialized in fishless habitats (Bufotes viridis, Sympetrum danae, Notonecta obliqua, Acilius sulcatus). In a mesocosm experiment, we quantified habitat selection decisions in response to the non-consumptive presence of fish (Carassius auratus) and vegetation structure mimicking different successional stages of aquatic habitats (no macrophytes; submerged and floating macrophytes; submerged, floating, and littoral-emergent macrophytes). No congruence between habitat specialists and generalists was observed, but a similar response to fish and vegetation structure defined both semiaquatic and aquatic organisms. While semiaquatic generalists did not distinguish between fish and fishless pools, specialists avoided fish-occupied pools and had a preferred vegetation structure. In aquatic taxa, predator presence affected habitat selection only in combination with vegetation structure, and all species preferred fishless pools with floating and submerged macrophytes. Fish presence triggered avoidance only in the generalist bug N. glauca. Our results highlight the significance of habitat selectivity for structuring freshwater ecosystems and illustrate how habitat selection responses to a top predator are dictated by specialization and life history.

Uniparental Genome Elimination in Australian Carp Gudgeons.

Metazoans usually reproduce sexually, blending the unique identity of parental genomes for the next generation through functional crossing-over and recombination in meiosis. However, some metazoan lineages have evolved reproductive systems where offspring are either full (clonal) or partial (hemiclonal) genetic replicas. In the latter group, the process of uniparental genome elimination selectively eliminates either the maternal or paternal genome from germ cells, and only one parental genome is selected for transmission. Although fairly common in plants, hybridogenesis (i.e., clonal haploidization via chromosome elimination) remains a poorly understood process in animals. Here, we explore the proximal cytogenomic mechanisms of somatic and germ cell chromosomes in sexual and hybrid genotypes of Australian carp gudgeons (Hypseleotris) by tracing the fate of each set during mitosis (in somatic tissues) and meiosis (in gonads). Our comparative study of diploid hybrid and sexual individuals revealed visually functional gonads in male and female hybrid genotypes and generally high karyotype variability, although the number of chromosome arms remains constant. Our results delivered direct evidence for classic hybridogenesis as a reproductive mode in carp gudgeons. Two parental sets with integral structure in the hybrid soma (the F1 constitution) contrasted with uniparental chromosomal inheritance detected in gonads. The inheritance mode happens through premeiotic genome duplication of the parental genome to be transmitted, whereas the second parental genome is likely gradually eliminated already in juvenile individuals. The role of metacentric chromosomes in hybrid evolution is also discussed.

Capture and return of sexual genomes by hybridogenetic frogs provide clonal genome enrichment in a sexual species.

Hybridogenesis is a reproductive tool for sexual parasitism. Hybridogenetic hybrids use gametes from their sexual host for their own reproduction, but sexual species gain no benefit from such matings as their genome is later eliminated. Here, we examine the presence of sexual parasitism in water frogs through crossing experiments and genome-wide data. We specifically focus on the famous Central-European populations where Pelophylax esculentus males (hybrids of P. ridibundus and P. lessonae) live with P. ridibundus. We identified a system where the hybrids commonly produce two types of clonal gametes (hybrid amphispermy). The haploid lessonae genome is clonally inherited from generation to generation and assures the maintenance of hybrids through a process, in which lessonae sperm fertilize P. ridibundus eggs. The haploid ridibundus genome in hybrids received from P. ridibundus a generation ago, is perpetuated as clonal ridibundus sperm and used to fertilize P. ridibundus eggs, yielding female P. ridibundus progeny. These results imply animal reproduction in which hybridogenetic taxa are not only sexual parasites, but also participate in the formation of a sexual taxon in a remarkable way. This occurs through a process by which sexual gametes are being captured, converted to clones, and returned to sexual populations in one generation.

Tracing the maternal origin of the common wall lizard (Podarcis muralis) on the northern range margin in Central Europe.

The maternal origin of isolated populations of the common wall lizard (Podracis muralis) in the Czech Republic, representing the north-eastern range border of the species, was addressed. We compared mitochondrial DNA sequences of the cytochrome b gene of samples from these populations with those from within the continuous range in Slovakia, the northern Balkan region, and those available from previous studies. We recorded five main haplogroups in the studied region, with all available Central European samples belonging to the same haplogroup. The star-like structure of this haplogroup suggests a scenario of relatively recent, post-glacial population expansion, which is further supported by a coalescent-based demographic analysis. The presence of unique haplotypes in two of the three isolated Czech populations together with close phylogenetic relationships to adjacent Slovak populations suggests either autochthonous origin or human-mediated introductions from geographically and genetically closest populations. We therefore support conservation programs for all three isolated Czech populations.

High cryptic diversity of bitterling fish in the southern West Palearctic.

South-east Europe, along with the adjacent region of south-west Asia, is an important biodiversity hotspot with high local endemism largely contributed by contemporary continental lineages that retreated to southern refugia during colder Quaternary periods. We investigated the genetic diversity of the European bitterling fish (Rhodeus amarus) species complex (Cyprinidae) across its range in the western Palearctic, but with a particular emphasis in the region of Balkan, Pontic and Caspian refugia. We genotyped 12 polymorphic microsatellite loci and a partial sequence of mitochondrial gene cytochrome b (CYTB) for a set of 1,038 individuals from 60 populations. We used mtDNA sequences to infer phylogenetic relationships and historical demography, and microsatellite markers to describe fine-scale genetic variability and structure. Our mtDNA analysis revealed six well-supported lineages, with limited local co-occurrence. Two lineages are distributed throughout central and western Europe (lineages "A" and "B"), with two zones of secondary contact. Another two lineages were restricted to the Ponto-Aegean region of Greece (lineages "C" and "D") and the final two lineages were restricted south of the Caucasus mountains (lineage "E" from the Black Sea watershed and lineage "F" from the Caspian watershed). A signal of recent expansion was revealed in the two widespread lineages and the Ponto-Aegean lineage "C". The geographic distribution of clusters detected by nuclear microsatellites corresponded well with mitochondrial lineages and demonstrated finely sub-structured populations. A profound population structure suggested a significant role of genetic drift in differentiation among lineages. Lineage divergence in the Ponto-Aegean and Caspian regions are substantial, supporting the validity of two described endemic species (Rhodeus meridionalis as lineage "D" and Rhodeus colchicus as lineage "E") and invite taxonomic evaluation of the other two southern lineages (Thracean "C" and Caspian "F").

All-male hybrids of a tetrapod Pelophylax esculentus share its origin and genetics of maintenance.

BACKGROUND: Sexual parasites offer unique insights into the reproduction of unisexual and sexual populations. Because unisexuality is almost exclusively linked to the female sex, most studies addressed host-parasite dynamics in populations where sperm-dependent females dominate. Pelophylax water frogs from Central Europe include hybrids of both sexes, collectively named P. esculentus. They live syntopically with their parental species P. lessonae and/or P. ridibundus. Some hybrid lineages consist of all males providing a chance to understand the origin and perpetuation of a host-parasite (egg-dependent) system compared to sperm-dependent parthenogenesis. METHODS: We focused on P. ridibundus-P. esculentus populations where P. ridibundus of both sexes lives together with only diploid P. esculentus males. Based on 17 microsatellite markers and six allozyme loci, we analyzed (i) the variability of individual genomes, (ii) the reproductive mode(s) of all-male hybrids, and (iii) the genealogical relationships between the hybrid and parental genomes. RESULTS: Our microsatellite data revealed that P. esculentus males bear Mendelian-inherited ridibundus genomes while the lessonae genome represents a single clone. Our data indicate that this clone did not recently originate from adjacent P. lessonae populations, suggesting an older in situ or ex situ origin. CONCLUSIONS: Our results confirm that also males can perpetuate over many generations as the unisexual lineage and successfully compete with P. ridibundus males for eggs provided by P. ridibundus females. Natural persistence of such sex-specific hybrid populations allows to studying the similarities and differences between male and female reproductive parasitism in many biological settings.

Genomic islands of differentiation in two songbird species reveal candidate genes for hybrid female sterility.

Hybrid sterility is a common first step in the evolution of postzygotic reproductive isolation. According to Haldane's Rule, it affects predominantly the heterogametic sex. While the genetic basis of hybrid male sterility in organisms with heterogametic males has been studied for decades, the genetic basis of hybrid female sterility in organisms with heterogametic females has received much less attention. We investigated the genetic basis of reproductive isolation in two closely related avian species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (L. luscinia), that hybridize in a secondary contact zone and produce viable hybrid progeny. In accordance with Haldane's Rule, hybrid females are sterile, while hybrid males are fertile, allowing gene flow to occur between the species. Using transcriptomic data from multiple individuals of both nightingale species, we identified genomic islands of high differentiation (FST ) and of high divergence (Dxy ), and we analysed gene content and patterns of molecular evolution within these islands. Interestingly, we found that these islands were enriched for genes related to female meiosis and metabolism. The islands of high differentiation and divergence were also characterized by higher levels of linkage disequilibrium than the rest of the genome in both species indicating that they might be situated in genomic regions of low recombination. This study provides one of the first insights into genetic basis of hybrid female sterility in organisms with heterogametic females.

Morphologically indistinguishable hybrid Carassius female with 156 chromosomes: A threat for the threatened crucian carp, C. carassius, L.

The crucian carp Carassius carassius (Linnaeus, 1758), is native to many European freshwaters. Despite its wide distribution, the crucian carp is declining in both the number and sizes of populations across much of its range. Here we studied 30 individuals of a putative pure population from Helsinki, Finland. Despite clear external morphological features of C. carassius, an individual was of a higher ploidy level than the others. We therefore applied a set of molecular genetic (S7 nuclear and cytochrome b mitochondrial genes) and cytogenetic tools (sequential fluorescent 4', 6-diamidino-2-phenylindole [DAPI], Chromomycin A3 [CMA3], C-banding and in situ hybridization [FISH] with both 5S and 28S ribosomal DNA probes) to determine its origin. While all examined characteristics of a diploid representative male (CCAHe2Fi) clearly corresponded to those of C. carassius, a triploid individual (CCAHe1Fi) was more complex. Phylogenetic analysis revealed that the nuclear genome of CCAHe1Fi contained three haploid sets: two C. gibelio and one C. carassius. However the mitochondrial DNA was that of C. gibelio, demonstrating its hybrid origin. The FISH revealed three strong (more intensive) 5S rDNA loci, confirming the triploid status, and an additional 24 weak (less intensive) signals were observed in the chromosome complement of CCAHe1Fi. On the other hand, only two strong and 16 weak 5S rDNA signals were visible on the chromosomes of the CCAHe2Fi male. 28S rDNA FISH revealed four strong signals in both CCAHe1Fi and CCAHe2Fi individuals. CMA3 staining revealed four to six CMA3-positive bands of CCAHe1Fi, while that of diploids contained only two to four. The fact that a polyploid hybrid Carassius female with a strong invasive potential may share morphological characters typical for endangered C. carassius highlights a need to combine genetic investigations of Carassius cryptic diversity with conservation measures of C. carassius in Europe.

Hybrid asexuality as a primary postzygotic barrier between nascent species: On the interconnection between asexuality, hybridization and speciation.

Although sexual reproduction is ubiquitous throughout nature, the molecular machinery behind it has been repeatedly disrupted during evolution, leading to the emergence of asexual lineages in all eukaryotic phyla. Despite intensive research, little is known about what causes the switch from sexual reproduction to asexuality. Interspecific hybridization is one of the candidate explanations, but the reasons for the apparent association between hybridization and asexuality remain unclear. In this study, we combined cross-breeding experiments with population genetic and phylogenomic approaches to reveal the history of speciation and asexuality evolution in European spined loaches (Cobitis). Contemporary species readily hybridize in hybrid zones, but produce infertile males and fertile but clonally reproducing females that cannot mediate introgressions. However, our analysis of exome data indicates that intensive gene flow between species has occurred in the past. Crossings among species with various genetic distances showed that, while distantly related species produced asexual females and sterile males, closely related species produce sexually reproducing hybrids of both sexes. Our results suggest that hybridization leads to sexual hybrids at the initial stages of speciation, but as the species diverge further, the gradual accumulation of reproductive incompatibilities between species could distort their gametogenesis towards asexuality. Interestingly, comparative analysis of published data revealed that hybrid asexuality generally evolves at lower genetic divergences than hybrid sterility or inviability. Given that hybrid asexuality effectively restricts gene flow, it may establish a primary reproductive barrier earlier during diversification than other "classical" forms of postzygotic incompatibilities. Hybrid asexuality may thus indirectly contribute to the speciation process.

Is premeiotic genome elimination an exclusive mechanism for hemiclonal reproduction in hybrid males of the genus Pelophylax?

BACKGROUND: The ability to eliminate a parental genome from a eukaryotic germ cell is a phenomenon observed mostly in hybrid organisms displaying an alternative propagation to sexual reproduction. For most taxa, the underlying cellular pathways and timing of the elimination process is only poorly understood. In the water frog hybrid Pelophylax esculentus (parental taxa are P. ridibundus and P. lessonae) the only described mechanism assumes that one parental genome is excluded from the germline during metamorphosis and prior to meiosis, while only second genome enters meiosis after endoreduplication. Our study of hybrids from a P. ridibundus-P. esculentus-male populations known for its production of more types of gametes shows that hybridogenetic mechanism of genome elimination is not uniform. RESULTS: Using comparative genomic hybridization (CGH) on mitotic and meiotic cell stages, we identified at least two pathways of meiotic mechanisms. One type of Pelophylax esculentus males provides supporting evidence of a premeiotic elimination of one parental genome. In several other males we record the presence of both parental genomes in the late phases of meiotic prophase I (diplotene) and metaphase I. CONCLUSION: Some P. esculentus males have no genome elimination from the germ line prior to meiosis. Considering previous cytological and experimental evidence for a formation of both ridibundus and lessonae sperm within a single P. esculentus individual, we propose a hypothesis that genome elimination from the germline can either be postponed to the meiotic stages or absent altogether in these hybrids.

Asexual Reproduction Does Not Apparently Increase the Rate of Chromosomal Evolution: Karyotype Stability in Diploid and Triploid Clonal Hybrid Fish (Cobitis, Cypriniformes, Teleostei).

Interspecific hybridization, polyploidization and transitions from sexuality to asexuality considerably affect organismal genomes. Especially the last mentioned process has been assumed to play a significant role in the initiation of chromosomal rearrangements, causing increased rates of karyotype evolution. We used cytogenetic analysis and molecular dating of cladogenetic events to compare the rate of changes of chromosome morphology and karyotype in asexually and sexually reproducing counterparts in European spined loach fish (Cobitis). We studied metaphases of three sexually reproducing species and their diploid and polyploid hybrid clones of different age of origin. The material includes artificial F1 hybrid strains, representatives of lineage originated in Holocene epoch, and also individuals of an oldest known age to date (roughly 0.37 MYA). Thereafter we applied GISH technique as a marker to differentiate parental chromosomal sets in hybrids. Although the sexual species accumulated remarkable chromosomal rearrangements after their speciation, we observed no differences in chromosome numbers and/or morphology among karyotypes of asexual hybrids. These hybrids possess chromosome sets originating from respective parental species with no cytogenetically detectable recombinations, suggesting their integrity even in a long term. The switch to asexual reproduction thus did not provoke any significant acceleration of the rate of chromosomal evolution in Cobitis. Asexual animals described in other case studies reproduce ameiotically, while Cobitis hybrids described here produce eggs likely through modified meiosis. Therefore, our findings indicate that the effect of asexuality on the rate of chromosomal change may be context-dependent rather than universal and related to particular type of asexual reproduction.

Genetic diversity and distribution patterns of diploid and polyploid hybrid water frog populations (Pelophylax esculentus complex) across Europe.

Polyploidization is a rare yet sometimes successful way for animals to rapidly create geno- and phenotypes that may colonize new habitats and quickly adapt to environmental changes. In this study, we use water frogs of the Pelophylax esculentus complex, comprising two species (Pelophylax lessonae, genotype LL; Pelophylax ridibundus, RR) and various diploid (LR) and triploid (LLR, LRR) hybrid forms, summarized as P. esculentus, as a model for studying recent hybridization and polyploidization in the context of speciation. Specifically, we compared the geographic distribution and genetic diversity of diploid and triploid hybrids across Europe to understand their origin, maintenance and potential role in hybrid speciation. We found that different hybrid and parental genotypes are not evenly distributed across Europe. Rather, their genetic diversity is structured by latitude and longitude and the presence/absence of parental species but not of triploids. Highest genetic diversity was observed in central and eastern Europe, the lowest in the northwestern parts of Europe. This gradient can be explained by the decrease in genetic diversity during postglacial expansion from southeastern glacial refuge areas. Genealogical relationships calculated on the basis of microsatellite data clearly indicate that hybrids are of multiple origin and include a huge variety of parental genomes. Water frogs in mixed-ploidy populations without any parental species (i.e. all-hybrid populations) can be viewed as evolutionary units that may be on their way towards hybrid speciation. Maintenance of such all-hybrid populations requires a continuous exchange of genomes between diploids and triploids, but scenarios for alternative evolutionary trajectories are discussed.

Distinguishing between incomplete lineage sorting and genomic introgressions: complete fixation of allospecific mitochondrial DNA in a sexually reproducing fish (Cobitis; Teleostei), despite clonal reproduction of hybrids.

Distinguishing between hybrid introgression and incomplete lineage sorting causing incongruence among gene trees in that they exhibit topological differences requires application of statistical approaches that are based on biologically relevant models. Such study is especially challenging in hybrid systems, where usual vectors mediating interspecific gene transfers--hybrids with Mendelian heredity--are absent or unknown. Here we study a complex of hybridizing species, which are known to produce clonal hybrids, to discover how one of the species, Cobitis tanaitica, has achieved a pattern of mito-nuclear mosaic genome over the whole geographic range. We appplied three distinct methods, including the method using solely the information on gene tree topologies, and found that the contrasting mito-nuclear signal might not have resulted from the retention of ancestral polymorphism. Instead, we found two signs of hybridization events related to C. tanaitica; one concerning nuclear gene flow and the other suggested mitochondrial capture. Interestingly, clonal inheritance (gynogenesis) of contemporary hybrids prevents genomic introgressions and non-clonal hybrids are either absent or too rare to be detected among European Cobitis. Our analyses therefore suggest that introgressive hybridizations are rather old episodes, mediated by previously existing hybrids whose inheritance was not entirely clonal. Cobitis complex thus supports the view that the type of resulting hybrids depends on a level of genomic divergence between sexual species.

Comparative phylogeography of three trematomid fishes reveals contrasting genetic structure patterns in benthic and pelagic species.

Population genetics patterns of marine fish in general and of Southern Ocean fish in particular range from virtual panmixia over ocean-wide scale to deeply fragmented populations. However the causes underlying these different patterns are not properly understood. In this paper, we tested the hypotheses that population connectivity is positively related to a combination of life history traits, namely duration of pelagic larval period and the tendency towards pelagic life style in the adulthood. To do so, we analysed the variability of six microsatellite and one mitochondrial marker (cytochrome b) in three Southern Ocean fish species (Trematomus newnesi, Trematomus hansoni and Trematomus bernacchii). They share a recent common ancestor but notably differ in their duration of pelagic larval period as well as pelagic versus benthic lifestyle. We sampled over a range of more than 5000 km for all three species and used a number of population genetics tools to investigate past and contemporary levels of connectivity. All species experienced population fluctuations, but coalescent simulations suggested that contemporary populations are in migration-drift equilibrium. Although global F(ST) values were rather low, a significant population structure separated the High-Antarctic from the Peninsular regions in all species. The level of genetic differentiation was much lower in the pelagic versus benthic species. Present data suggest that past and present genetic structuring in the Southern Ocean are indeed related with the ecological traits of Antarctic fish, however the relative importance of individual factors remains unclear.

Dynamic formation of asexual diploid and polyploid lineages: multilocus analysis of Cobitis reveals the mechanisms maintaining the diversity of clones.

Given the hybrid genomic constitutions and increased ploidy of many asexual animals, the identification of processes governing the origin and maintenance of clonal diversity provides useful information about the evolutionary consequences of interspecific hybridization, asexuality and polyploidy. In order to understand the processes driving observed diversity of biotypes and clones in the Cobitis taenia hybrid complex, we performed fine-scale genetic analysis of Central European hybrid zone between two sexual species using microsatellite genotyping and mtDNA sequencing. We found that the hybrid zone is populated by an assemblage of clonally (gynogenetically) reproducing di-, tri- and tetraploid hybrid lineages and that successful clones, which are able of spatial expansion, recruit from two ploidy levels, i.e. diploid and triploid. We further compared the distribution of observed estimates of clonal ages to theoretical distributions simulated under various assumptions and showed that new clones are most likely continuously recruited from ancestral populations. This suggests that the clonal diversity is maintained by dynamic equilibrium between origination and extinction of clonal lineages. On the other hand, an interclonal selection is implied by nonrandom spatial distribution of individual clones with respect to the coexisting sexual species. Importantly, there was no evidence for sexually reproducing hybrids or clonally reproducing non-hybrid forms. Together with previous successful laboratory synthesis of clonal Cobitis hybrids, our data thus provide the most compelling evidence that 1) the origin of asexuality is causally linked to interspecific hybridization; 2) successful establishment of clones is not restricted to one specific ploidy level and 3) the initiation of clonality and polyploidy may be dynamic and continuous in asexual complexes.

Synthesis of clonality and polyploidy in vertebrate animals by hybridization between two sexual species.

Because most clonal vertebrates have hybrid genomic constitutions, tight linkages are assumed among hybridization, clonality, and polyploidy. However, predictions about how these processes mechanistically relate during the switch from sexual to clonal reproduction have not been validated. Therefore, we performed a crossing experiment to test the hypothesis that interspecific hybridization per se initiated clonal diploid and triploid spined loaches (Cobitis) and their gynogenetic reproduction. We reared two F1 families resulting from the crossing of 14 pairs of two sexual species, and found their diploid hybrid constitution and a 1:1 sex ratio. While males were infertile, females produced unreduced nonrecombinant eggs (100%). Synthetic triploid females and males (96.3%) resulted in each of nine backcrossed families from eggs of synthesized diploid F1s fertilized by haploid sperm from sexual males. Five individuals (3.7%) from one backcross family were genetically identical to the somatic cells of the mother and originated via gynogenesis; the sperm of the sexual male only triggered clonal development of the egg. Our reconstruction of the evolutionary route from sexuality to clonality and polyploidy in these fish shows that clonality and gynogenesis may have been directly triggered by interspecific hybridization and that polyploidy is a consequence, not a cause, of clonality.