Stöck et al., 2011. Simultaneous Mendelian and clonal genome transmission...

An unofficial working translation of a paper of considerable interest.Stöck M., Ustinova J., Betto-Colliard C., Schartl M., Moritz C., Perrin N. Simultaneous Mendelian and clonal genome transmission in a sexually reproducing, all-triploid vertebrate. Proc. R. Soc. B, 2011. doi:10.1098/rspb.2011.1738The Russian name for Bufo baturae — Pushtun toad — is used on the advice of S.N. Litvinchuk and is provisional in nature.Simultaneous Mendelian and Clonal Genome Transmission in a Sexually Reproducing, All-Triploid VertebrateMeiosis in triploid individuals seemingly faces insurmountable difficulties when dividing an odd number of chromosome sets into two. Triploid vertebrates typically circumvent this problem through either asexuality or some form of hybridogenesis, including meiotic hybridogenesis, which involves a reproductive community of varying ploidy levels and differing genome complements. Pushtun toads (Bufo baturae; 3n = 33 chromosomes), however, represent fully triploid sexual reproduction. This hybrid species possesses two copies of a genome bearing a nucleolus organizer region (NOR+) on chromosome 6, and a third genome lacking it (NOR-). Males produce only haploid NOR+ sperm, whereas eggs are diploid and contain one NOR+ and one NOR- chromosome set. Here we perform parentage analysis using codominant microsatellite markers (1) to confirm purely clonal and maternal transmission of the NOR- set, and (2) to demonstrate Mendelian segregation and recombination of NOR+ sets in both sexes. This novel reproductive mode in vertebrates (which we term pre-equational hybrid meiosis) offers an ideal opportunity to study the evolution of non-recombining genomes. Elucidating the mechanisms that permit the simultaneous transmission of two genomes — one Mendelian, the other clonal — may shed light on general processes governing meiosis in vertebrates.1. IntroductionAccording to Mendel's second law [1], alleles at different loci recombine independently of one another during gamete formation. In sexually reproducing species, random transmission of paternal and maternal genomes is achieved through independent segregation of chromosomes during meiosis. Some animal genomes, however, exhibit transmission biases, often depending on their parental origin and typically as a consequence of ancient hybridization [2]. Hybrid lineages of ants, for example, carry two independently evolving genomes transmitted either meiotically [3] or clonally [4,5]. Analogous processes occur in hybridogenetic vertebrates: in diploid hybridogenesis, one genome is transmitted clonally through hybrid lineages, while the other is transmitted sexually from one of the parental species [6]. In meiotic hybridogenesis, both hybridizing genomes may be transmitted sexually, through crosses of diploid and triploid hybrids bearing different genome complements (Fig. 1, [14,16]). Figure 1. Reproductive modes of triploid vertebrates. Shown are the genomes of parents, gametes, and offspring (rows) under various reproductive modes (columns). A, B: genomes of different parental species. Bold colored symbols indicate clonally transmitted copies, while regular black symbols with subscripts denote distinct (recombinant) copies.True parthenogenesis: clonal (no males), exclusively in reptiles [7,8];Sperm-dependent parthenogenesis (i.e., gynogenesis): clonal, embryogenesis requires initiation by allospecific spermatozoa that are not incorporated (rare paternal leakage may transmit a sub-genomic amount of paternal DNA); occurs in teleost fishes and urodele amphibians [9];Kleptogenesis: females acquire, wholly or partially, genomes from their partners through incompletely understood mechanisms, allowing them to purge deleterious alleles from the genome (here BB); described in urodele amphibians [10];Unnamed form of hybridogenesis: clonal diploid eggs are fertilized by sperm of recombining sexually reproducing species, which may be diploid or triploid (as in meiotic hybridogenesis); occurs in anuran amphibians and teleost fishes [11-13];Meiotic hybridogenesis: may occur in triploid males and/or females; found in teleost fishes and anuran amphibians [14,15]; ploidy elevation of diploid offspring, which can produce hybrid diploid gametes, may occur in the next generation (becoming, for example, ABB') in order to restore triploidy (analogous to the preceding form of hybridogenesis);Pre-equational hybrid meiosis: occurs in the Pushtun toad: both sexes are triploid and exhibit Mendelian segregation and recombination in B genomes (equivalent to NOR+ in the present paper), while the A genome (i.e., NOR-) is transmitted clonally from the mother.Bisexual reproduction of pure triploids is impeded by the problem of equal distribution of three chromosome sets during meiosis [17]; see review [18]. Poeciliopsis hybrids, for example, are hybridogenetic in their diploid forms but become gynogenetic as triploids [19,20]. As an alternative to gynogenesis or parthenogenesis (Fig. 1, [7-9,21]), some triploid vertebrates combine clonal and sexual elements in their reproductive mode — for example, kleptogenesis or various forms of hybridogenesis [10,22-24], including meiotic hybridogenesis, which requires a reproductive community of different ploidy levels and genome compositions.In this context, Pushtun toads (Bufo baturae) are unique in being sexually reproducing triploids [25]. This hybrid-origin species inhabits the high mountains of northern Pakistan (above 1,500 m a.s.l.). Its genome (3n = 33; [25,26]) consists of two chromosome sets bearing a nucleolus organizer region (NOR+) on chromosome 6, and one set lacking such a region (NOR-; Figure 2).

Males produce only haploid NOR+ sperm, presumably by eliminating the NOR- set (11 chromosomes) prior to meiosis. In contrast, eggs are diploid (2n = 22) with one NOR+ and one NOR- set. Immature oocytes display 22 lampbrush bivalents [25].Accordingly, it has been proposed that the NOR- set is transmitted purely through the maternal lineage and clonally. Nevertheless, it remains unknown whether the NOR+ set recombines and, if so, by what mechanism. For females, two hypotheses can be advanced (Fig. 2): (a) One NOR+ set is eliminated (either randomly or depending on the parental origin), after which the two remaining sets are auto-duplicated via endomitosis. Meiosis then occurs between pseudo-bivalents [27] and produces one or at most two classes of clonal diploid eggs. The alternative (b) is that the NOR- set is auto-duplicated prior to meiosis, during which the two NOR+ sets recombine normally. Figure 2. Diagram of the reproductive system in triploid Pushtun toads with hypothetical mechanisms (a) and (b) of oogenesis and (c) and (d) of spermatogenesis. Blue NOR- symbol: non-recombining (clonal) chromosome set lacking nucleolus organizers. Red or purple NOR+ symbols: distinct NOR-bearing chromosome sets. Mixed red and purple NOR+ symbols: recombined NOR-bearing sets. Mechanisms confirmed in the present study are enclosed in boxes.For males, analogously, the first hypothesis (Fig. 2, c) is that the entire maternal contribution (NOR+, NOR-) is eliminated, and the paternal NOR+ is subsequently duplicated via pre-meiotic endomitosis. Meiotic pairs (NOR+/NOR+; [25]) would thus represent pseudo-bivalents, implying clonal transmission of one NOR+ set. Spermatocytes would consist of a single multilocus genotype (or at most two, if NOR+ elimination occurred randomly). The alternative (Fig. 2, d) is the elimination of only the NOR- set, with the two NOR+ sets undergoing normal meiosis and recombination.Based on multilocus fingerprinting, Stöck et al. [25] identified several genotypes among offspring of a single pair. However, dominant multilocus markers are not always unambiguously interpretable and thus cannot shed light on the underlying mechanism. In the present study, we performed parentage analysis of offspring groups at 15 codominant microsatellite loci to establish genome-specific transmission and segregation patterns. Our results clearly confirm purely clonal maternal transmission of one chromosome set (NOR-) and demonstrate independent segregation and recombination of the two other sets (NOR+) in both males and females. These shufflings of genetic material should enable efficient purging of the two NOR+ chromosome sets, as in normal sexual reproduction. This is the first example of parallel clonal and meiotic transmission of chromosome sets within a single vertebrate lineage.2. Materials and MethodsAnimals used in controlled experimental crosses were collected at three localities in northern Pakistan (electronic supplementary material, Table S1) during three field periods (June–July 1996, 1997, and 2000). We conducted five breeding experiments with triploid Pushtun toads. In addition, we crossed one female Pushtun toad with both a diploid male Bufo variabilis from Syria (2n = 22, bearing two NOR+ sets) and a tetraploid male Bufo oblongus from Iran (4n = 44, containing two NOR+ and two NOR- sets; [28]). Twenty of 100 offspring were reared in tanks until larvae reached 2–3 cm in length (Gosner stages 30–38, [29]). In total, 85 tadpoles from seven crosses were sampled for genetic analysis. Tadpoles were either karyotyped or their ploidy was determined by flow cytometry; DNA was extracted as described by Stöck et al. [30].We tested series of microsatellite markers from a repetitive-element genomic library of Pushtun toads, some of which had previously been used in other species (electronic supplementary material, Table S2). Alleles were amplified, labeled with GeneMapper v. 3.7 (Applied Biosystems), and named according to their length in base pairs as described [30]. Alleles from NOR+ and NOR- sets, as well as null alleles (0), were identified from inheritance patterns (see §3). Linkage analysis was performed in GENEPOP (https://genepop.curtin.edu.au/, [31,32]) with default parameters, and potential linkage groups were verified by visual inspection. Given the tractable size of the dataset, offspring genotypes were visually inspected for recombination events. The number of recombinations was normalized relative to the number of informative events per pair, and deviations from random segregation were tested for significance (using a chi-square test).3.

Results(a) Inheritance Patterns in the Pushtun ToadIn total, 15 microsatellite primer pairs were amplified in one or more families.Five of these (D103, D105, D5, C224, and C203; electronic supplementary material, Dataset S1) display three alleles per individual, suggesting amplification of products from both the NOR- and the two NOR+ sets. NOR- alleles were readily identified by their constant homomorphism among offspring of a given pair, their identity with the maternal copy, and their difference from the paternal copy in cases where parental copies differed (Table 1 and electronic supplementary material, Table S3 and Dataset S1). Both NOR+ sets, in contrast, reflect inheritance from both parents and Mendelian segregation (electronic supplementary material, Table S3 and Dataset S1). Each heterozygous parent transmitted its two alleles with equal probability (binomial tests).The remaining 10 markers display a maximum of two alleles per individual, with inheritance from both parents and Mendelian segregation, consistent with expectation for meiotic NOR+ sets (electronic supplementary material, Dataset S1).Table 1. Inheritance pattern of NOR- alleles at five loci (rows) in five 3n crosses (columns). Shown is the number of offspring with the maternal NOR- allele / number of informative events. ni indicates non-informativeness; (—) indicates that analysis was not performed. Across all loci and crosses, we tallied 139 cases of maternal inheritance out of 139 informative events. Complete data are presented in electronic supplementary material, Dataset S1. Four linkage groups, each involving two markers, can be defined (Table 2). Out of 199 informative events, we detected a total of 25 recombination events (Table 2), occurring in both sexes (electronic supplementary material, Dataset S1 and Table S3). All other marker pairs were transmitted independently of one another, generating extensive multilocus genotype diversity per family. Notably, the five markers amplifying NOR- products belonged to different linkage groups in the NOR+ genome (Table 2 and electronic supplementary material, Dataset S1), supporting a genome-wide distribution of NOR- markers.(b) Inter-ploidy CrossesLocus D105 was also amplifiable in offspring of a B. baturae female crossed with (i) a diploid B. variabilis male (2n = 22, bearing two NOR+ sets) and (ii) a tetraploid B. oblongus male (4n = 44, including two NOR+ and two NOR- sets). All offspring from insemination by the B. variabilis male were triploid and inherited the maternal NOR- allele at locus D105, while the two NOR+ sets showed inheritance from both parents with Mendelian segregation in both. Offspring from fertilization by B. oblongus were tetraploid and displayed four allelic copies at locus D105, corresponding to two NOR- and two NOR+ sets. One NOR- allele was identical to the maternal copy, while the other was randomly inherited from the two paternal NOR- copies. The two NOR+ sets also showed inheritance from both parents, with Mendelian segregation in both. Consequently, in both crosses, the Pushtun toad mother produced 2n oocytes with a clonally transmitted NOR- and a recombinant NOR+. The B. variabilis male produced sperm with a haploid recombinant NOR+, whereas the B. oblongus male produced diploid spermatozoa with recombinant NOR+ and NOR- sets (see also [25,33]).Table 2. Recombination pattern across four linkage groups. Rec., recombination observed; non-rec., recombination not observed. Values expected under the assumption of independent inheritance are shown in italics. Total indicates the overall number of informative events per pair and per sex. 4. DiscussionOur results demonstrate purely maternal and clonal transmission of all NOR- markers. Offspring inherit only the maternal copies of five markers (D5, D103, D105, C224, and C203) that amplify NOR- alleles. As will be shown, these five markers are located in different linkage groups on the NOR+ genomes (electronic supplementary material, Dataset S1), supporting the inference that the entire NOR- chromosome set undergoes clonal maternal transmission.Moreover, our results provide evidence for NOR+ recombination in both sexes.The 15 markers amplifying NOR+ alleles were distributed across 11 distinct linkage groups (corresponding to the haploid chromosome number of the Pushtun toad), reflecting random and independent segregation in both sexes (electronic supplementary material, Dataset S1). Heterozygous adults always transmit their two alleles with equal probability, indicating random segregation of paternal and maternal NOR+ chromosome sets in both sexes (Fig. 2: pathways (b), (d)). Furthermore, recombination also occurred in both sexes between loci from the same linkage group (Table 2).Taken together, our results exclude hypothesis (a) for oogenesis (which assumed clonal production of NOR+/NOR- oocytes) and hypothesis (c) for spermatogenesis (which assumed clonal production of NOR+ sperm). These results raise important questions concerning analogous mechanisms and evolutionary consequences associated with this unusual reproductive mode.(a) Analogous MechanismsThe NOR- genome of Pushtun toads is eliminated in males but duplicated in females prior to meiosis. Pre-meiotic elimination and/or duplication of genomes have previously been described in hybridogenetic vertebrates, such as the green frog Rana (Pelophylax) esculenta, a hybrid between Rana ridibunda (RR) and Rana lessonae (LL) [33,34]. When cohabiting with R. ridibunda, R. esculenta females (RL) lose the parental genome (R) from the germ line while simultaneously doubling their L genomes via pre-meiotic endomitosis. The subsequent meiosis thus involves fully homozygous pseudo-bivalents (LL) and produces non-recombinant haploid (L) oocytes [35]. Mating with a R. ridibunda male subsequently restores the RL genome. Thus, one chromosome set (R) recombines in the parental species, while the other (L) is transmitted clonally in the hybrids.Both chromosome sets recombine during meiotic hybridogenesis [15,36], which involves reproductive communities of hybrids of varying ploidy and genome composition (RRL, LLR, and RL). Triploid RRL individuals eliminate the L genome from the germ line and produce recombinant haploid R gametes via normal meiosis. Triploid LLR individuals analogously eliminate the R genome prior to meiosis, producing recombinant haploid L gametes. Finally, diploid RL individuals form clonal diploid RL gametes following endomitosis [11]. The combination of these gametes restores the original diploid and triploid genomes [36-38]. Similar mechanisms have been documented in several hybridogenetic teleost fishes [39-44].Thus, pre-meiotic elimination of NOR- in male Pushtun toads, followed by normal diploid meiosis of the two NOR+ sets, bears resemblance to certain processes occurring during meiotic hybridogenesis and other forms of hybridogenetic or kleptogenetic reproduction known in triploid vertebrates (Fig. 1). Furthermore, the duplication of NOR- in female Pushtun toads proceeds via a gametogenetic mechanism (pre-meiotic endomitosis) well known in parthenogenetic and hybridogenetic vertebrates [23,45,46]. Nevertheless, the Pushtun toad system appears unique among vertebrates in that (i) meiotic processes differ between the sexes, and (ii) females simultaneously transmit one genome that is duplicated clonally and another that undergoes normal meiosis. The closest analogous system appears to be found in plants such as heather and dog-rose, in which pollen transmits only the sexually reproduced genome, while eggs additionally transmit a clonally reproduced genome [47-49]. However, in those cases, no pre-meiotic duplication of the clonal genomes occurs. To our knowledge, auto-duplication of one complete chromosome set (NOR-) in the presence of a distinct diploid genome (NOR+/NOR+'), which remains pre-meiotically unchanged and is subsequently transmitted in Mendelian fashion, has not previously been demonstrated (Fig. 1).(b) Evolutionary AspectsThe Pushtun toad shows remarkable intraspecific uniformity of mitochondrial sequences [50], suggesting a unique and recent origin (although none of its potential parental species occur within the species' range, which encompasses the drainage basins of three rivers in northern Pakistan). While triploids can arise directly from crosses between diploid and tetraploid parental species, the complex meiotic processes described here (including whole-genome duplication in females and its elimination in males) could not have emerged immediately at the original hybridization event. Intermediate stages may have involved episodic hybrid interactions among lineages of different ploidy and genome composition, similar to situations currently occurring in northern Kyrgyzstan, where some triploid males arising from natural crosses between 2n Bufo turanensis females and 4n Bufo pewzowi males perform backcrosses with females of either parental species [30]. Such mechanisms, however, must be rare, given the low probability of encountering the complex genetic conditions required to achieve stable hybrid combinations of clonal and Mendelian genomes — an inference recently advanced also for ancient lineages of gynogenetic fishes [51].This reproductive mode also raises questions regarding selective processes and evolutionary fate. Polyploid (3n, 4n) green toad lineages, which have evolved independently multiple times, are clearly associated with harsh habitats [50]. Pushtun toads in particular inhabit conditions of extreme altitude and aridity [52]. One plausible hypothesis is that clonal reproduction of the NOR- genome preserves epistatic components of viability that may be selectively relevant under abiotic, predictably severe environmental conditions [53].

Indeed, asexual lineages are frequently found in marginal habitats with more extreme conditions (cold, dry, high-altitude, and high-UV) than their sexual relatives [54-57]. An important question in this context is whether both genomes are expressed, and if so, whether expression is differential (tissue-specific), as observed in allopolyploid fishes [58] and plants [59].This exclusive mode of NOR- inheritance should also carry adverse evolutionary consequences. First, purely maternal transmission opens opportunities for genomic conflict. We expect, in particular, that feminizing factors will evolve on the maternal NOR-, only to be counterbalanced by masculinizing factors evolving on the NOR+, which is transmitted from both parents. This could lead to sex-specific biases, as observed in hybridogenetic R. esculenta, where the maternally transmitted clonal R genome carries only feminizing factors [60]. Similarly, mutations that are deleterious only in males will not be removed by selection and may accumulate, as occurs in other maternally transmitted mitochondrial [61] or nuclear [62] genomes.Second, the non-recombining NOR- set should gradually accumulate deleterious mutations through a combination of enhanced genetic drift, selective sweeps, background selection, and Muller's ratchet [63-65], as occurs with sex chromosomes (Y or W) and with non-recombining genomes in hemiclonal vertebrates [66]. The Pushtun toad, however, may have arisen too recently for such mutational crises or genomic conflicts over sex determination to be detectable [25,50].Conversely, the Mendelian segregation and recombination found in the NOR+ genome should prevent its evolutionary degeneration, providing the long-term evolutionary potential characteristic of sexually reproducing vertebrates with normal meiosis.Comparison of gene sequences from the NOR- genome of B. baturae with those of its parental species, as well as of tetraploid green toad lineages such as B. oblongus and B. pewzowi, in which NOR- genomes recombine according to cytological [28] and microsatellite (present study) evidence, may help elucidate not only the phylogenetic history of NOR- sets but also the pattern of selection acting on this non-recombining genome. It may also permit investigation of potential conflicts in sex-determination pathways, as well as possible inter-genomic recombinations, particularly those observed in kleptogenetically reproducing Ambystoma salamanders [67].5. ConclusionThe reproductive mode of the Pushtun toad differs from those known in other vertebrates (Fig. 1) not only because meiotic processes differ between the sexes, but also because females exhibit clonal and sexual reproduction simultaneously (the pre-meiotic auto-duplication event affects one chromosome set, while another undergoes normal meiosis). We have designated this process pre-equational hybrid meiosis. Elucidating the mechanisms underlying these features could shed light on general processes governing meiotic regulation in vertebrates. The Pushtun toad also presents an intriguing opportunity to compare the evolutionary forces acting on recombining versus non-recombining genomes within a single organism.References1 Mendel, G. 1866 Versuche uber Pflanzenhybriden [Experiments on plant hybridization]. Verh. Naturforsch. Ver. Briinn 4, 3-47. [In German.]2 Keller, L. 2010 Genetics: biased transmission of genomes according to parents of origin. Curr. Biol.

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