The mitochondrial cytochrome oxidase II gene in Bacillus stick insects: ancestry of hybrids, androgenesis, and phylogenetic relationships

Sequencing of a cytochrome oxidase II (COII) gene fragment in Bacillus taxa provided evidence that the bisexual B. rossius is the maternal ancestor of the hybridogenetic B. rossius-grandii strains and revealed the same ancestry for both parthenogenetic hybrids: the diploid B. whitei (B. rossius/grandii grandii) and the triploid B. lynceorum (B. rossius/grandii grandii/atticus). Present data clearly demonstrate that all Bacillus unisexuals arose through asymmetrical hybridization events and realized a paraphyletic derivation from the B. rossius redtenbacheri subspecies. The invention of B. rossius mitochondrial DNA haplotypes in specimens with B. grandii grandii nuclear genomes revealed the occurrence of androgenesis in nature. Natural androgens represent a peculiar escape from hybridity and can help maintain the hybridogenetic system through the production of the fathering taxon via hybrid females. Results from the COII gene support the phyletic relationships among taxa suggested by previous taxonomical approaches, but also indicate a departure of B. grandii subspecies from the established taxonomy. Assuming the existence of a molecular clock, the evaluated substitution rate brings the splitting between B. rossius and B. grandii/B. atticus back to 22.79 +/- 2.65 myr before present, while the origin of hybrids appears to be much more recent (1.06 +/- 0.53 myr).     

Genome exclusion and gametic DAPI-DNA content in the hybridogenetic Bacillus rossius-grandii benazzii complex (Insecta Phasmatodea).

Among Sicilian stick insects, two hybridogenetic complexes have been discovered: Bacillus rossius-grandii benazzii and B. rossius-grandii grandii, which also produce androgenetic offspring. The egg maturation of the former is analyzed here through DAPI fluorometry, which, besides the assessment of the meiotic stages, also allows their DNA measurements and the analysis of sperm-head evolution into male pronuclei in these polyspermic eggs. Hybridogenetic eggs undergo an extrasynthesis of chromosomes, because two groups of n autobivalents (4C each) are segregated at metaphase 1st; the two groups must correspond to the pure parental species haplosets. Then the grandii chromosomes degenerate (1st polar body), while the rossius chromosomes divide further to produce two groups of n autodiads (2C each); one of them degenerates (2nd polar body), and the other is ready to perform syngamy (female pronucleus). Meanwhile, several B. grandii sperm evolve into male pronuclei by doubling their DNA (from 1C to 2C content) and assuming an interphase nucleus appearance. If regular mixis occurs, the F1 hybrid constitution is restored but, if it fails, a fusion between two sperms may occur, originating fully paternal descendants (natural androgenesis). The genome exclusion mechanism of stick-insect hybridogens appears to be more primitive than those observed in the already known hybridogenetic complexes of Poeciliopsis and Rana esculenta. Unfertilized eggs of hybridogens are capable of self-activation, but the cytology of the related clonally reproducing B. whitei indicates that its parthenogenetic mechanism stems from the hybridization event (hybrid theory) rather than from tychoparthenogenetic potentialities (spontaneous theory).     

Unisexuality and Molecular Drive: Bag320 Sequence Diversity in <Emphasis Type="Italic">Bacillus</Emphasis> Taxa (Insecta Phasmatodea)

Satellite DNA variability follows a pattern of concerted evolution through homogenization of new variants by genomic turnover mechanisms and variant fixation by chromosome redistribution into new combinations with the sexual process. Bacillus taxa share the same Bag320 satellite family and their reproduction ranges from strict bisexuality (B. grandii) to automictic (B. atticus) and apomictic (B. whitei = rossius/ grandii; B. lynceorum = rossius/grandii/atticus) unisexuality. Thelytokous reproduction clearly allows uncoupling of homogenization from fixation. Both trends and absolute values of satellite variability were analyzed in all Bacillus taxa but B. rossius, on 906 sequenced monomers at all level of comparisons: intraspecimen, intrapopulation, interpopulation, intersubspecies, and interspecies. For unisexuals, allozymic and mitochondrial clones were also taken into account. Different reproductive modes (sexual/parthenogenetic) appear to explain observed variability trends, supporting Dover's hypothesis of sexuality acting as a driving force in the fixation of sequence variants, but the present analyses also highlight current spreading of new variants in B. grandii maretimi specimens and point to a biased sequence inheritance at the time of hybrid onset in the apomictic hybrids B. whitei and B. lynceorum. Evidence of biased gene conversion events suggests that, given enough time, sequence homogenization can take place in a unisexual such as B. lynceorum. On the contrary, the absolute values of sequence diversity in each taxon are linked to the species' range, time of divergence, and repeat copy number and, possibly, to transposon features. Satellite dynamics appears therefore to be the outcome of both general molecular processes and specific organismal traits.     

Satellite sequence turnover in parthenogenetic systems: the apomictic triploid hybrid Bacillus lynceorum (Insecta, Phasmatodea)

In the genus Bacillus (Insecta, Phasmatodea) the Bag320 satellite DNA family is present in the bisexual B. grandii and in the related automictic nonhybrid B. atticus; it is lacking in the other bisexual taxon of the genus, B. rossius. This family of highly repeated sequences was analyzed for 11 populations of the apomictic triploid hybrid B. lynceorum. In the neighbor-joining dendrogram, B. lynceorum nucleotide sequences distribute, regardless of geographical origin, among two clusters, one also including all clones of the three B. atticus races, and the other including sequences of the B. grandii grandii subspecies. Thus, B. lynceorum is a trihybrid taxon: as the molecular approach definitively demonstrates, it embodies one haploid complement each of both B. grandii grandii and B. atticus, which must be added to that of B. rossius. The contribution of the latter species has already been assessed on karyological and allozymic grounds. A statistical analysis performed on p-distances shows that for the parental taxa, nucleotide substitution values are of comparable magnitudes at the population level but differ at the subspecific level, being higher for the bisexual taxon. In the apomictic hybrid, atticus- and grandii grandii- like sequences coexist with significantly different p-distance values. For three clones, the nucleotide compositions at the diagnostic loci suggest that gene conversion can occur between atticus- and grandii grandii-like monomers. On the whole, this supports bisexuality as a driving force in variant fixation and suggests that in Bacillus, different gametogenetic processes and different origins of the unisexuals are mirrored in genomic turnover rates of satellite DNA.      

Chromosomal evidence of hemiclonal and all-paternal offspring production in Bacillus rossius-grandii benazzii (Insecta Phasmatodea)

The stick insects Bacillus rossius-grandii benazzii and B. rossius-grandii grandii naturally reproduce by hybridogenesis and androgenesis. The hybrid karyotype of the former (2n=35, XX female; 34, X0 male) clearly sums up a B. rossius haploset (r) with n=18 and a B. grandii benazzii one (gb) with n=17. The two sets keep the parental features for C-heterochromatin amount (much larger in the gb complement) and satellites/NORs (nuclear organizer regions) (more numerous and variably located in the r set); hybridogenetically produced males always show severely impaired gametogenesis and are therefore sterile, whereas hybridogenetic females are fertile. Reproductive, karyological and cytogenetical properties of the hybridogenetic system have been exploited to obtain the chromosomal evidence of whole haploset exchanges. In progenies obtained by crossing B. rossiusgrandii benazzii females to B. rossius males with either standard or repatterned (with Robertsonian fusions) karyotypes, there has always been complete agreement between electrophoretically genotyped and karyologically analyzed hybridogenetic offspring: the unassorted maternal r haploset (r ^m) is transmitted and the gb ^m haploset replaced by that of the fathering male (r ^p), thus evidencing the hemiclonal reproduction and the new r ^m-r ^p chromosomal constitution. New karyotype traits of the offspring relate to chromosome number (2n=36, female; 35, male), C-heterochromatin pattern (the heterochromatin-rich gb haploset completely disappears) and satellite/NOR features (corresponding to r ^m plus r ^p locations). The same crosses also produce genetically and chromosomally all-paternal descendants (androgenetics), of both sexes and fully fertile, with an r ^p r ^p structure. These androgenetic progeny show segregation for alleles and chromosomes at which fathering males are heterozygous: it was therefore possible to demonstrate that androgenetics can derive from syngamy of two sperm nuclei, of the several present in the polyspermic hybridogenetic egg. The production of androgenetics from field fertilized females of B. rossius-grandii benazzii, B. rossius-grandii grandii and parthenogenetic Bacillus whitei (=B. rossius/grandii grandii) suggests the occurrence of unsuspected relationships between hybrids and their parental species, so that the hybrids cannot be simply considered as sexual parasites. Furthermore, there is a suggestion of evolution of parthenogenetic clonal species from selection of initially hybridogenetic strains. The ability to produce uniparental progeny naturally from the spermatic genome may open a new field of investigation on genomic imprinting.     

HYBRIDOGENESIS AND ANDROGENESIS IN THE STICK-INSECT BACILLUS ROSSIUS-GRANDII BENAZZII (INSECTA,PHASMATODEA)

In northwestern Sicily interspecific hybrid females between Bacillus rossius and B. grandii benazzii (Insecta, Phasmatodea) are sympatric with facultatively parthenogenetic demes of the former and bisexual populations of the latter. Preliminary observations suggested that hybrid females are maintained by hybridogenetic reproduction, not by current F₁ hybrid production nor through parthenogenesis. Being hybridogens, a complex of hemiclonal lineages, we informally refer to them as B. rossius-grandii benazzii, according to Schultz's proposal. In this study B. rossius-g. benazzii females were crossed with males of B. g. benazzii, B. g. grandii, B. g. maretimi, and B. rossius. Allozyme analysis of the progeny showed that the great majority of them were actually produced by hybridogenesis with a hemiclonal inheritance of the maternal B. rossius genotype (Br^m) and actual syngamy with a sperm from the fathering male, so that Br^m-gb^p, Br^m-gg^p, Br^m-gm^p, and Br^m-r^p offspring were obtained in the respective crosses. All-paternal progeny (androgenetics) were also produced (Bgb^pgb^p, Bgm^pgm^p, Br^pr^p) and two gynogenetic descendants were observed. Cytological investigations on virgin eggs that failed to hatch revealed in most of them a haploid-diploid blocked blastoderm; this rudimentary parthenogenesis appears to be an important prerequisite for further evolution of this hybridogen. Reproductive modes of descendants were also analyzed; although Br^m-g^p hybrids are still able to reproduce by hybridogenesis, a progressive disruption of the hybridogenetic-androgenetic system takes place in synthetic B. rossius (Br^m-r^p, Br^pr^p) and abundant thelytokous parthenogenetic offspring are obtained from females of androgenetic origin. The evolutionary role of these hybridogens appears to be linked to their shift towards parthenogenesis; this has apparently occurred in the southeastern Sicilian hybrid B. whitei (=B. rossius/g. grandii), which exhibits both hybridogenesis and parthenogenesis.     

Linkage between sexual and asexual lineages: genome evolution in Bacillus stick insects

The sexually reproducing stick insects Bacillus rossius and B. grandii are sharply differentiated in terms of allozyme gene alleles; B. atticus is a polyclonal automictic parthenogen sister to B. grandii grandii. Although well differentiated for coding genes, these hybridize to produce diploid (B. whitei=rossius/grandii) or triploid (B. lynceorum=rossius/grandii/atticus) clonal forms which reproduce apomictically. Allozyme analyses of unisexual Bacillus clearly establish their relationships from bisexual ancestor species as does the existence in all of them of several clones (especially in B. atticus) whose egg maturation allows regular recombination to occur. Bacillus taxa share the Bag320 satellite DNA family within different reproductive frameworks, allowing satellite variant homogenization to be uncoupled from fixation. The nested analysis of monomers reveals different patterns of sequence diversity: sexual reproduction includes both homogenization and variant fixation, whereas the slowing of molecular turnover processes and the absence of syngamy in the parthenogens realizes a similar range of sequence diversity at the level of the individual and supra‐individual, but with no fixation. On the other hand, the actual values of sequence diversity appear mostly linked to species traits – range size, copy number of repeats, number of hybrid crosses – and possibly transposon activity, rather than to the reproductive mode. In addition, the mitochondrial genome reveals a comparable level of cox2 sequence variability in sexual and parthenogenetic taxa, thus adding to clonal variability. From Bacillus and other stick insect complexes, an overall picture of genomic diversification of parthenogens is therefore beginning to emerge. To define those animals that reproduce by non‐canonical sexual modes (i.e. parthenogenesis, hybridogenesis), but make use of egg and meiotic mechanisms, the term meta‐sexual is proposed. © 2003 The Linnean Society of London. Biological Journal of the Linnean Society, 2003, 79 , 137–150.     

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.     

Genome elimination in diploid and triploid Rana esculenta males: cytological evidence from DNA flow cytometry

Cytological aspects of hemiclonal (meroclonal) inheritance in diploid and triploid males of the hybridogenetic frog Rana esculenta (Rana ridibunda x Rana lessonae) have been studied by DNA flow cytometry. The fact that the R. ridibunda genome contains 16% more DNA than the R. lessonae genome provides the ability to discern cells containing genomes of any species from the water-frog complex under study. Data are presented showing that elimination of the R. ridibunda genome occurs in hybridogenetic males from certain populations. In triploid males, the cytogenetic mechanism of hemiclonal inheritance is simpler than in diploids: after the elimination of a genome (always the genome in the minority in the triploid set; "homogenizing elimination"), no compensatory duplication of the remaining genetic material is necessary, as it is in diploids. The process of elimination can be visualized in triploid males by using DNA flow cytometry to identify cells in the special phase of the spermatogonial cell cycle that we termed the E phase.     

The Bag320 satellite DNA family in Bacillus stick insects (Phasmatodea): different rates of molecular evolution of highly repetitive DNA in bisexual and parthenogenic taxa

The Bag320 satellite DNA (satDNA) family was studied in seven populations of the stick insects Bacillus atticus (parthenogenetic, unisexual) and Bacillus grandii (bisexual). It was characterized as widespread in all zymoraces of B. atticus and in all subspecies of B. grandii. The copy number of this satellite is higher in the bisexual B. grandii (15%-20% of the genome) than in the parthenogenetic B. atticus (2%-5% of the genome). The nucleotide sequences of 12 Bag320 clones from B. atticus and 17 from B. grandii differed at 13 characteristic positions by fixed nucleotide substitutions. Thus, nucleotide sequences from both species cluster conspecifically in phylogenetic dendrograms. The nucleotide sequences derived from B. grandii grandii could be clearly discriminated from those of B. grandii benazzii and B. grandii maretimi on the basis of 25 variable sites, although all taxa come from Sicily. In contrast, the Bag320 sequences from B. atticus could not be discriminated accordingly, although they derive from geographically quite distant populations of its three zymoraces (the Italian and Greek B. atticus atticus, the Greek and Turkish B. atticus carius, and the Cyprian B. atticus cyprius). The different rate of evolutionary turnover of the Bag320 satDNA in both species can be related to their different modes of reproduction. This indicates that meiosis and chromosome segregation affect processes in satDNA diversification.      

Effect of gamma-irradiation on oogenesis of Drosophila mutants defective for reparation and meiotic recombination

Effects of mutations rad201, mei-9, and mei-41 on cell sensitivity to gamma-radiation in Drosophila oogenesis were studied. Females of the control (Oregon R) and mutant strains were irradiated at a dose of 15 Gy. For 9 days after the irradiation, the number of eggs in consecutive day batches, the frequency of dominant lethals (DLs) among the eggs, and the cytologically recorded distribution of oocytes for stages of their development, and the frequency of egg chamber degeneration in female ovaries were estimated. As a result of joint analysis of the data, different oogenesis stages were characterized with regard to the frequency of two radiation-induced events: appearance of DLs in oocytes and degeneration of egg chambers due to apoptosis of nurse cells. It was shown that the mutations affect these parameters only at particular stages of early oogenesis, at which previtellogenetic growth of egg follicles and meiotic recombination in oocytes occur. Mutation rad201G1 increased the frequency of DLs and egg chamber degeneration, mei-41D5 affected only the DL frequency, and mei-9a, in addition to enhancing the chamber degeneration frequency, promoted radiation "rescue" of some oocytes from the DL induction.     

Maintenance of sister chromatid attachment in mouse eggs through maturation-promoting factor activity

Mammalian eggs naturally arrest at metaphase of the second meiotic division, until sperm triggers a series of Ca(2+) spikes that result in activation of the anaphase-promoting complex/cyclosome (APC/C). APC/C activation at metaphase targets destruction-box containing substrates, such as cyclin B1 and securin, for degradation, and as such eggs complete the second meiotic division. Cyclin B1 degradation reduces maturation (M-phase)-promoting factor (MPF) activity and securin degradation allows sister chromatid separation. Here we examined the second meiotic division in mouse eggs following expression of a cyclin B1 construct with an N-terminal 90 amino acid deletion (Delta 90 cyclin B1) that was visualized by coupling to EGFP. This cyclin construct was not an APC/C substrate, and so following fertilization, sperm were incapable of stimulating Delta 90 cyclin B1 degradation. In these eggs, chromatin remained condensed and no pronuclei formed. As a consequence of the lack of pronucleus formation, sperm-triggered Ca(2+) spiking continued indefinitely, consistent with a current model in which the sperm-activating factor is localized to the nucleus. Because Ca(2+) spiking was not inhibited by Delta 90 cyclin B1, the degradation timing of securin, visualized by coupling it to EGFP, was unaffected. However, despite rapid securin degradation, sister chromatids remained attached. This was a direct consequence of MPF activity because separation was induced following application of the MPF inhibitor roscovitine. Similar observations regarding the ability of MPF to prevent sister chromatid separation have recently been made in Xenopus egg extracts and in HeLa cells. The results presented here show this mechanism can also occur in intact mammalian eggs and further that this mechanism appears conserved among vertebrates. We present a model in which metaphase II arrest is maintained primarily by MPF levels only.     

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.     

Genome reduction in a hemiclonal frog Rana esculenta from radioactively contaminated areas

A decrease in genome size was found in the hemiclonal hybridogenetic frog Rana esculenta (R. ridibunda x R. lessonae) from areas of radioactive contamination that resulted from the Chernobyl fallout. This genome reduction was of up to 4% and correlated with the background level of gamma-radiation (linear regression corresponded on average to -0.4% per doubling of radiation level). No change in genome size was observed in the coexisting parental species R. lessonae. There was no correlation between genome size and body mass in R. esculenta froglets, which have metamorphosed in the year of the study. The hemiclonal forms may become a suitable object for study on biological significance of individual DNA sequences (and of genome size as a whole) because mutant animals with deletions in a specified genome can arise after a low radiation dose. The proneness to genetic damage makes such forms also a prospective bioindicator of radioactive (and possibly other mutagenic) pollution with the effects of genetic damage conveniently and rapidly monitored by DNA flow cytometry.     

Effects of Gamma-Radiation in Oogenesis of Drosophila Mutants Defective for Repair and Meiotic Recombination

Effects of mutations rad201, mei-9, and mei-41on cell sensitivity to gamma-radiation in Drosophilaoogenesis were studied. Females of the control (Oregon R) and mutant strains were irradiated at a dose of 15 Gy. For 9 days after the irradiation, the number of eggs in consecutive day batches, the frequency of dominant lethals (DLs) among the eggs, and the cytologically recorded distribution of oocytes for stages of their development, and the frequency of egg chamber degeneration in female ovaries were estimated. As a result of joint analysis of the data, different oogenesis stages were characterized with regard to the frequency of two radiation-induced events: appearance of DLs in oocytes and degeneration of egg chambers due to apoptosis of nurse cells. It was shown that the mutations affect these parameters only at particular stages of early oogenesis, at which previtellogenetic growth of egg follicles and meiotic recombination in oocytes occur. Mutation rad201 ^G1increased the frequency of DLs and egg chamber degeneration, mei-41 ^D5affected only the DL frequency, and mei-9 ^a, in addition to enhancing the chamber degeneration frequency, promoted radiation rescue of some oocytes from the DL induction.     

Egg activation and timing of sperm acceptance by an egg in honeybees (Apis mellifera L.)

Summary. Mechanical stresses by a narrow glass capillary were applied to unfertilized eggs of honeybees to determine whether the removal of meiotic blocks of the eggs could be caused by simple mechanical stimuli. The treated eggs developed into the anaphase of the first meiotic division at 15 min after treatment, whereas the untreated eggs remained arrested at the metaphase of the first meiotic division. The results of histological examination of the common oviduct showed that its inner widths were sufficiently narrow to cause the distortion of eggs passing through it. The distorted eggs could be fertilized and develop into diploid embryos if they were exposed to the semen immediately (within 30 sec) after egg distortion. However, this would not happen if the distorted eggs were exposed to semen later (30 min). The eggs exposed to the semen but not given mechanical stimuli could initiate the embryonic development with diploid chromosomes. The interval between mechanical distortion and sperm acceptance by eggs in vitro is compatible with that of natural oviposition of fertilized eggs by honeybee queens. These results suggest that egg activation by mechanical stresses in the common oviduct is valid for the natural oviposition in honeybees.     

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.     

Simultaneous formation of haploid, diploid and triploid eggs in diploid–triploid mosaic loaches

In the loach Misgurnus anguillicaudatus , very few diploid–triploid mosaic individuals, which are generated by accidental incorporation of the sperm nucleus into diploid eggs produced by clonal diploid loach, occur in nature. Ploidy examination of gynogenetic progeny induced by activation with ultraviolet-irradiated goldfish sperm indicated that diploid–triploid mosaic females laid haploid, diploid and triploid eggs, simultaneously. In addition, triploid eggs exhibited larger egg sizes. Microsatellite genotyping of diploid–triploid mosaics revealed that triploid genotypes of mosaic mothers possessed two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from a mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of the three alleles of the mosaic mother was transmitted to haploid eggs. These results suggested that diploid germ cells, which had a clonal genome, were differentiated into clonal diploid eggs, and triploid and haploid eggs were produced from triploid germ cells in the same ovary of mosaic individuals.     

Ancient versus reticulate origin of a hemiclonal lineage

Crossing experiments revealed that a diploid hybridogenetic fish (genus Poeciliopsis) from the Río Mocorito (Sinaloa, Mexico) is trihybrid. Its haploid maternal genome is inherited clonally (i.e., hemiclonally), and it expresses a mixture of morphological traits found in the closely related species P. monacha and P. viriosa. Its haploid paternal genome is replaced in each generation by mating with males of a more distantly related sexual species, P. lucida. However, expression of mixed (monacha X viriosa) traits by this hemiclone is also consistent with retention of shared ancestral polymorphisms. If true, this hemiclonal lineage would be one of the few examples of an ancient asexual taxon. We used mitochondrial DNA and allozymes to test whether the maternal progenitor of the Mocorito hybridogen was a recent P. monacha X P. viriosa hybrid or a remnant of their most recent common ancestor. Our results clearly link the hemiclonal genome to contemporary P. monacha and therefore support the hypothesis of a recent origin. Additionally, our findings suggest that this unisexual fish may serve as a vehicle for introgression between two allopatric sexual species.