Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, that is, 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, that is, meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin.     

Unisexual hybrids break through an evolutionary dead end by two-way backcrossing

Unisexual vertebrates (i.e., those produced through clonal or hemiclonal reproduction) are typically incapable of purging deleterious mutations, and, as a result, are considered short-lived in evolutionary terms. In hemiclonal reproduction (hybridogenesis), one parental genome is eliminated during oogenesis, producing haploid eggs containing the genome of a single parent. Hemiclonal hybrids are usually produced by backcrossing hemiclonal hybrids with males of the paternal species. When hemiclonal hybrids from a genus of greenlings (Hexagrammos) are crossed with males of the maternal species, the progeny are phenotypically similar to the maternal species and produce recombinant gametes by regular meiosis. The present study was conducted to determine if the hemiclonal genome is returned to the gene pool of the maternal species in the wild. Using a specific cytogenetic marker to discriminate between such progeny and the maternal species, we observed that Hexagrammos hybrids mated with maternal and paternal ancestors at the same frequency. This two-way backcrossing in which clonal genomes are returned to the gene pool where they can undergo recombination plays an important role in increasing the genetic variability of the hemiclonal genome and reducing the extinction risk. In this way, hybrid lineages may have survived longer than predicted through occasional recombinant generation.     

Non-hybrid offspring from matings between hemiclonal hybrid waterfrogs suggest occasional recombination between clonal genomes

The hemiclonal waterfrog Rana esculenta , a hybrid between R. ridibunda and R. lessonae , eliminates the lessonae genome from the germline and clonally transmits the ridibunda genome (hybridogenesis). Such genomes are prone to accumulate deleterious mutations, which may explain why offspring from matings between hybrids are typically inviable. Here I present field data from a population for which experimental crossings showed that some R. esculenta pairs produce viable R. ridibunda offspring. I demonstrate: (1) that R. ridibunda metamorphs are also produced and survive under natural conditions; (2) that their genotypes are consistent with combinations of clonal ridibunda genomes found in hybrids; and (3) that all R. ridibunda are female. These females possibly recombine the clonal genomes they inherited and, upon mating with syntopic R. lessonae , produce new hemiclones with novel combinations of alleles. Hence, occasional recombination between otherwise clonal ridibunda genomes seems plausible and may provide an escape from the evolutionary dead end they were proposed to be trapped in.     

Homoploid hybrid speciation and genome evolution via chromosome sorting

Genomes of numerous diploid plant and animal species possess traces of interspecific crosses, and many researches consider them as support for homoploid hybrid speciation (HHS), a process by which a new reproductively isolated species arises through hybridization and combination of parts of the parental genomes, but without an increase in ploidy. However, convincing evidence for a creative role of hybridization in the origin of reproductive isolation between hybrid and parental forms is extremely limited. Here, through studying Agrodiaetus butterflies, we provide proof of a previously unknown mode of HHS based on the formation of post-zygotic reproductive isolation via hybridization of chromosomally divergent parental species and subsequent fixation of a novel combination of chromosome fusions/fissions in hybrid descendants. We show that meiotic segregation, operating in the hybrid lineage, resulted in the formation of a new diploid genome, drastically rearranged in terms of chromosome number. We also demonstrate that during the heterozygous stage of the hybrid species formation, recombination was limited between rearranged chromosomes of different parental origin, representing evidence that the reproductive isolation was a direct consequence of hybridization.     

Heterozygous fitness effects of clonally transmitted genomes in waterfrogs

The European waterfrog Rana esculenta (RL‐genotype) is a natural hybrid between R. ridibunda (RR) and R. lessonae (LL) and reproduces by hybridogenesis, i.e. it eliminates the L‐genome from the germline and produces gametes only containing the clonally transmitted R‐genome. Because of the lack of recombination, R‐genomes are prone to accumulate spontaneous deleterious mutations. The homozygous effects of such mutations become evident in matings between hybrids: their offspring possess two clonal R‐genomes and are generally inviable. However, the evolutionary fate of R. esculenta mainly depends on the heterozygous effects of mutations on the R‐genome. These effects may be hidden in the hybrid R. esculenta because it has been shown to benefit from spontaneous heterosis. To uncouple clonal inheritance from hybridity, I crossed R. esculenta with R. ridibunda to produce nonhybrid offspring with one clonal and one sexual R‐genome, and compared their survival and larval performance with normal, sexually produced R. ridibunda tadpoles. Because environmental stress can enhance the negative effects of mutation accumulation, I measured the performance at high and low food levels. There was no indication that tadpoles with a clonal genome performed worse at either food level, suggesting that at least in the larval stage, R. esculenta benefits from heterosis without incurring any costs because of heterozygous effects of deleterious mutations on the clonally transmitted R‐genome.     

Mutation accumulation and fitness effects in hybridogenetic populations: a comparison to sexual and asexual systems

Background  

Female only unisexual vertebrates that reproduce by hybridogenesis show an unusual genetic composition. They are of hybrid origin but show no recombination between the genomes of their parental species. Instead, the paternal genome is discarded from the germline prior to meiosis, and gametes (eggs only) contain solely unrecombined maternal genomes. Hence hybridogens only transmit maternally inherited mutations. Hybridity is restored each generation by backcrossing with males of the sexual parental species whose genome was eliminated. In contrast, recombining sexual species propagate an intermixed pool of mutations derived from the maternal and paternal parts of the genome. If mutation rates are lower in female gametes than males, it raises the possibility for lower mutation accumulation in a hybridogenetic population, and consequently, higher population fitness than its sexual counterpart.     

Identification of hemiclonal reproduction in three species of Hexagrammos marine reef fishes

Natural hybrids between the boreal species Hexagrammos octogrammus and two temperate species Hexagrammos agrammus and Hexagrammos otakii were observed frequently in southern Hokkaido, Japan. Previous studies revealed that H. octogrammus is a maternal ancestor of both hybrids; the hybrids are all fertile females and they frequently breed with paternal species. Although such rampant hybridization occurs, species boundaries have been maintained in the hybrid zone. Possible explanations for the absence of introgressions, despite the frequent backcrossing, might include clonal reproduction: parthenogenesis, gynogenesis and hybridogenesis. The natural hybrids produced haploid eggs that contained only the H. octogrammus genome (maternal ancestor) with discarded paternal genome and generated F₁‐hybrid type offspring by fertilization with the haploid sperm of H. agrammus or H. otakii (paternal ancestor). This reproductive mode was found in an artificial backcross hybrid between the natural hybrid and a male of the paternal ancestor. These findings indicate that the natural hybrids adopt hybridogenesis with high possibility and produce successive generations through hybridogenesis by backcrossing with the paternal ancestor. These hybrids of Hexagrammos represent the first hybridogenetic system found from marine fishes that widely inhabit the North Pacific Ocean. In contrast with other hybridogenetic systems, these Hexagrammos hybrids coexist with all three ancestral species in the hybrid zone. The coexistence mechanism is also discussed.     

Introgression in the genus Salmo via allotriploids

Hybridization between sympatric species is not uncommon in the wild. Wild allotriploids (individuals with two chromosome sets from a species + one chromosome set from another species) are generally the result of a backcross between interspecific hybrids that produce unreduced gametes and one of the parental species. In animals, allotriploids are commonly sterile, except for some vertebrate species complexes in which allotriploids reproduce by parthenogenesis, gynogenesis and/or hybridogenesis, producing generally clonal or hemiclonal gametes; nuclear DNA introgression between hybridizing species is considered to be extremely rare. Employing species-specific molecular markers, we show genetic introgression between the chromosomally well-differentiated salmonids Atlantic salmon (2n = 58) and brown trout (2n = 80) through spontaneous bisexual reproduction of allotriploids leading to salmon-like offspring bearing some brown trout genes. Although introgression between these Salmo species can occur via allotriploids, we hypothesize that extinction of parental species can be discarded based on very low survival of allotriploid offspring.     

Reproductive isolation in a nascent species pair is associated with aneuploidy in hybrid offspring

Speciation may occur when the genomes of two populations accumulate genetic incompatibilities and/or chromosomal rearrangements that prevent inter-breeding in nature. Chromosome stability is critical for survival and faithful transmission of the genome, and hybridization can compromise this. However, the role of chromosomal stability on hybrid incompatibilities has rarely been tested in recently diverged populations. Here, we test for chromosomal instability in hybrids between nascent species, the ‘dwarf’ and ‘normal’ lake whitefish (Coregonus clupeaformis). We examined chromosomes in pure embryos, and healthy and malformed backcross embryos. While pure individuals displayed chromosome numbers corresponding to the expected diploid number (2n = 80), healthy backcrosses showed evidence of mitotic instability through an increased variance of chromosome numbers within an individual. In malformed backcrosses, extensive aneuploidy corresponding to multiples of the haploid number (1n = 40, 2n = 80, 3n = 120) was found, suggesting meiotic breakdown in their F₁ parent. However, no detectable chromosome rearrangements between parental forms were identified. Genomic instability through aneuploidy thus appears to contribute to reproductive isolation between dwarf and normal lake whitefish, despite their very recent divergence (approx. 15–20 000 generations). Our data suggest that genetic incompatibilities may accumulate early during speciation and limit hybridization between nascent species.     

The origin and evolution of a unisexual hybrid: Poecilia formosa

Clonal reproduction in vertebrates can always be traced back to hybridization events as all known unisexual vertebrates are hybrids between recognized species or genetically defined races. Interestingly, clonal vertebrates often also rely on interspecific matings for their reproduction because gynogenesis (sperm-dependent parthenogenesis) and hybridogenesis are common modes of propagation. While in most cases these hybridization events leave no hereditary traces in the offspring, occasionally the genome exclusion mechanism fails and either small parts of male genetic material remain inside the oocyte in the form of microchromosomes, or fusion of the sperm nucleus with the oocyte nucleus leads to polyploid individuals. In this review, we highlight the important role of hybridization for the origin and evolution of a unisexual hybrid: the Amazon molly, Poecilia formosa.     

Did backcrossing contribute to the origin of hybrid edible bananas?

Background

Bananas and plantains (Musa spp.) provide a staple food for many millions of people living in the humid tropics. The cultivated varieties (cultivars) are seedless parthenocarpic clones of which the origin remains unclear. Many are believed to be diploid and polyploid hybrids involving the A genome diploid M. acuminata and the B genome M. balbisiana, with the hybrid genomes consisting of a simple combination of the parental ones. Thus the genomic constitution of the diploids has been classified as AB, and that of the triploids as AAB or ABB. However, the morphology of many accessions is biased towards either the A or B phenotype and does not conform to predictions based on these genomic formulae.

Scope

On the basis of published cytotypes (mitochondrial and chloroplast genomes), we speculate here that the hybrid banana genomes are unbalanced with respect to the parental ones, and/or that inter-genome translocation chromosomes are relatively common. We hypothesize that the evolution under domestication of cultivated banana hybrids is more likely to have passed through an intermediate hybrid, which was then involved in a variety of backcrossing events. We present experimental data supporting our hypothesis and we propose a set of experimental approaches to test it, thereby indicating other possibilities for explaining some of the unbalanced genome expressions. Progress in this area would not only throw more light on the origin of one of the most important crops, but provide data of general relevance for the evolution under domestication of many other important clonal crops. At the same time, a complex origin of the cultivated banana hybrids would imply a reconsideration of current breeding strategies.     

Genome relationships in the genus Dasypyrum (Gramineae)

To elucidate the genome relationships in the genus Dasypyrum and the ancestry of tetraploid D. breviaristatum, two cytotypes of D. breviaristatum and D. villosum were reciprocally crossed with one another. Chromosome pairing at the first metaphase of meiosis and fertility were examined in the F1 hybrids and the parental plants. The mean pairing configuration and mean arm pairing frequency in D. villosum-D. breviaristatum (2x) hybrids were 11.12I + 1.44II per cell and 0.107, respectively, and they were almost completely sterile. In D. breviaristatum (4x)-D. breviaristatum (2x) hybrid, up to seven trivalents were formed, and the mean pairing configuration was 3.38I + 3.20II + 3.74III + 0.005IV per cell. The mean arm pairing frequency and relative affinity calculated in that F1 hybrid were 0.915 and 0.641, respectively. Seven bivalents and seven univalents were characteristically formed in D. villosum-D. breviaristatum (4x) hybrids. Based on the present results, we clearly concluded that the genome of diploid D. breviaristatum is distantly related to the genome V of D. villosum, and that these two species have different basic genomes. We, therefore, proposed the symbol Vb for the haploid genome of diploid cytotype of D. breviaristatum. Moreover, we concluded that tetraploid D. breviaristatum is an autotetraploid with doubled sets of the genomes homologous with that of diploid D. breviaristatum, and we proposed the genome constitution VbVb for the haploid genome set of tetraploid cytotype of D. breviaristatum. Furthermore, from the chromosome pairing in the F1 hybrids involving Moroccan and Greek accessions, it was suggested that complicated rearrangements of chromosome structure have occurred in tetraploid D. breviaristatum in its natural populations across the entire distribution area.     

Cytonuclear evidence for hybridogenetic reproduction in natural populations of the Australian carp gudgeon (Hypseleotris: Eleotridae)

Although most vertebrates reproduce sexually, a small number of fishes, amphibians and reptiles are known in which reproduction is asexual, i.e. without meiotic recombination. In fishes, these so-called unisexual lineages usually comprise only females and utilize co-occurring males of a related sexual species to reproduce via gynogenesis or hybridogenesis. Here, we examine patterns of microsatellite and mitochondrial DNA (mtDNA) variation in a widespread group of freshwater fishes (carp gudgeons; Hypseleotris spp.) to investigate a long-standing proposal that this group includes unisexual forms. We show that the mtDNA genome of most carp gudgeons in tributaries of the Goulburn River belongs to one of two deeply divided clades (～10% cyt b divergence) and that nuclear variation divides the same individuals into four distinct groups. Group 1 exhibits the genotypic proportions of a random mating population and has a 1:1 sex ratio. Two other groups are extremely sex-biased (98% male, 96% female), exhibit excess heterozygosity at most loci and share at least one allele per locus with group 1. We propose that these two groups represent 'unisexual' hybridogenetic lineages and that both utilize co-occurring group 1 as sexual host. Interestingly, the fourth distinct group appears to represent hybrid offspring of the two putative hybridogenetic lineages. The propagation of clonal haploid genomes by both males and females and the ability of these clones to unite and form sexually mature diploid hybrid offspring may represent a novel mechanism that contributes to the dynamics of coexistence between hybridogenetic lineages and their sexual hosts.     

Individual variability and inheritance of the number and size of the nucleolar organizer regions in chromosomes of domestic swine

Individual variability of the number and size of silver-staining nucleolar organiser regions (NOR) was studied in 76 pigs of Semirechenskaya, Big White and Duroc breeds. The variability of the NOR number is exclusively caused by the presence of the 8NOR- allele (the chromosomes of the 8th homologous pair lacking the silver-staining NOR). Concentration of the 8NOR- allele is about 0.7 in the average for all three breeds. All 7 offsprings from the homozygous parents male 8NOR- /8NOR- and female 8NOR+/8NOR+ were heterozygotes 8NOR- /8NOR+. These data, together with good agreement between found and expected (according to the Hardy-Weinberg law) frequencies of homo- and heterozygotes suggest the codominant character of the NOR number inheritance. This allows using the NOR analysis to control the parentage announced. The study of inheritance of the size of NOR did not prove unequivocally its codominant character.     

Genetic dissection of clonally inherited genomes of poeciliopsis. I. Linkage analysis and preliminary assessment of deleterious gene loads

Theoretical considerations suggest that a high load of deleterious mutations should accumulate in asexual genomes. An ideal system for testing this hypothesis occurs in the hybrid all-female fish Poeciliopsis monacha-lucida. The hybrid genotype is retained between generations by an oogenetic process that transmits only a nonrecombinant haploid monacha genome to their ova. The hybrid genotype is re-established in nature by fertilization of these monacha eggs with sperm from a sexual species, P. lucida. The unique reproductive mechanism of these hybrids allows the genetic dissection of the clonal monacha genome by forced matings with males of P. monacha. The resultant F₁ hybrids and their backcross progeny were examined to determine the amount and kinds of genetic changes that might have occurred in two clonal monacha genomes.—Using six allozyme markers, four similar linkage groups were identified in each clonal genome. Segregation and assortment at these loci revealed no apparent differences between monacha genomes from sexually and clonally reproducing species. Mortality of F₁ and backcross progeny revealed differences between the two clonal genomes, suggesting that deleterious genes may accumulate in genomes sheltered from recombination.     

Genomic compatibility between two phyllotine rodent species evaluated through their hybrids

In order to investigate the genomic compatibility between allopatric rodent species, Phyllotis darwini and Phyllotis magister, we have studied several cytogenetic and reproductive features of their laboratory hybrids. Of thirty-one pairings between species, only five were successful, producing eleven newborns. Like parents, hybrids had 38 metacentric chromosomes, except for the subtelocentric Y chromosome inherited from P. magister. There was almost total C and G band correspondence between homeologous autosomes. However, parental sex chromosomes had different morphology, C and G bands. Ag-NOR bands appeared as small telomeric Ag+ regions, distributed in four chromosomal pairs of darwini, three of magister and four homeologous chromosomes of the hybrids. The three forms had similar indexes of NOR activity per cell, in spite of the variability in NOR expression which was always detected. Usually, only one member of parental homologous chromosomes showed AgNOR+; nevertheless, both homeologous chromosomes were active in many hybrid cells. The frequencies of cells that expressed their ribosomal genes in the two homologous or homeologous NOR chromosomes were similar in parental and hybrid cells. These results strongly suggest that ribosomal genes of both parental genomes would function codominantly in the hybrids. The gonad histological and morphometric analyses showed that hybrids conformed to Haldane's rule, since females were fertile and males were infertile. Our results indicate that P. darwini and P. magister genomes can function in relative harmony and compatibility when they are placed together in their laboratory generated hybrids, suggesting that these species have few genetic differences, probably because they have recently diverged.     

Gametogenesis of intergroup hybrids of hemiclonal frogs

European water frog hybrids Rana esculenta (R. ridibundaxR. 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.     

Pervasive genetic associations between traits causing reproductive isolation in Heliconius butterflies

Ecological speciation proceeds through the accumulation of divergent traits that contribute to reproductive isolation, but in the face of gene flow traits that characterize incipient species may become disassociated through recombination. Heliconius butterflies are well known for bright mimetic warning patterns that are also used in mate recognition and cause both pre- and post-mating isolation between divergent taxa. Sympatric sister taxa representing the final stages of speciation, such as Heliconius cydno and Heliconius melpomene, also differ in ecology and hybrid fertility. We examine mate preference and sterility among offspring of crosses between these species and demonstrate the clustering of Mendelian colour pattern loci and behavioural loci that contribute to reproductive isolation. In particular, male preference for red patterns is associated with the locus responsible for the red forewing band. Two further colour pattern loci are associated, respectively, with female mating outcome and hybrid sterility. This genetic architecture in which ‘speciation genes’ are clustered in the genome can facilitate two controversial models of speciation, namely divergence in the face of gene flow and hybrid speciation.     

Simultaneous production of triploid and haploid eggs by triploid Squalius alburnoides (Teleostei: Cyprinidae)

The hybrid minnow Squalius alburnoides comprises diploid and polyploid forms with altered modes of reproduction. In the present paper, we report a cross where a triploid female generated both large, triploid and small, haploid eggs simultaneously, which were fertilized with S. pyrenaicus sperm. Although the large eggs were rarer (15%), they originated offspring with higher survivorship, so that tetraploids were dominant among the surviving siblings. The cross yielded apparently all female progeny. Inheritance patterns were inferred using four microsatellite markers and NORs (Nucleolus Organizer Regions) phenotypes, and suggested that haploid eggs were probably produced by an atypical hybridogenesis, in which the elimination of the unmatched genome permitted random segregation and recombination between the homospecific genomes, while the triploid eggs were clonal. The present results suggest that the occurrence of triploid unreduced eggs may be a new route for the natural tetraploidization in the complex.