Uniparental sexual reproduction following cell-cell fusion of opposite 1 mating-type partners

Some animal species require an opposite-sex partner for their sexual development but discard the partner genome before gamete formation, generating hemi-clonal progeny in a process called hybridogenesis. In this study, we discovered hybridogenesis-like reproduction in a basidiomycete fungus, Cryptococcus neoformans. C. neoformans has two mating types, MATa and MAT, which fuse to produce a dikaryotic zygote that completes a sexual cycle producing recombinant meiotic progeny. Here, we discovered exclusive uniparental inheritance of nuclear genetic material in a fraction of the F1 progeny produced during bisexual reproduction of two opposite mating-type partners. By analyzing strains expressing fluorescent reporter proteins, we observed that dikaryotic hyphae were produced, but only one parental nuclei was found in the terminal basidium where sporulation occurs. Whole-genome sequencing revealed the nuclear genome of the progeny was identical with one or the other parental genome, whereas the mitochondrial genome was always inherited from the MATa parent. Uniparental sporulation was also observed in natural isolate crosses occurring in concert with biparental sporulation. The meiotic recombinase Dmc1 was found to be critical for uniparental reproduction. These findings reveal an unusual mode of eukaryotic microbial unisexual reproduction that shares features with hybridogenesis in animals.

Most organisms in nature undergo sexual reproduction between two partners of the 2 opposite sex to produce progeny. In most cases, the diploid zygote receives one copy of the 3 genetic material from each parent following fusion of the two haploid gametes. To produce 4 these haploid gametes, the diploid cell of the organism undergoes meiosis, which involves common cytoplasm and thus incorporate both the GFP-and the mCherry-tagged histone H4 1 proteins ( Figure S2). We hypothesized that in the cases of uniparental sporulation, only one 2 of the nuclei would reach the terminal basidium and thus would harbor only one fluorescent 3 nuclear color signal ( Figure S2A). 4 After establishing this fluorescent tagging system using wild-type strains, H99α x 5 KN99a and shuffle-strain VYD135α x KN99a crosses with fluorescently labeled strains were 6 examined. In the wild-type cross, most of the basidia formed robust spore chains with both 7 fluorescent colors observed in them while a small population (~1%) of basidia exhibited 8 spore chains with only one color, representing uniparental reproduction ( Figure 1A and S3A). 9 On the other hand, the majority of the basidia population in the shuffle-strain VYD135α x 1 0 KN99a cross did not exhibit sporulation, and the two parental nuclei appeared fused but  Fluorescence microscopy revealed that only one of the two parental nuclei is present 1 9 in a small proportion of the basidia, which results in uniparental meiosis and sporulation.

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Based on this finding, we hypothesized that the basidia with only one parental nucleus might 2 1 arise due to nuclear segregation events during hyphal branching. To gain further insight into 2 2 this process, the nuclear distribution pattern along the sporulating hyphae was studied. As 2 3 expected, imaging of long hyphae in the wild-type cross revealed the presence of pairs of 2 4 nuclei with both fluorescent markers along the length of the majority of hyphae ( Figure 2A).

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In contrast, tracking of hyphae from basidia with spore chains in the genome-shuffle strain 2 6 VYD135α x KN99a cross revealed hyphal branches with only one parental nucleus, which 2 7 were preceded by a hypha with both parental nuclei ( Figure 2B).

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These results suggest that hyphal branching may facilitate the separation of one 2 9 parental nucleus from the main hyphae harboring both parental nuclei. As a result, one of the 3 0 parental genomes is excluded at a step before meiosis and the generation of F1 progeny, 3 1 similar to the process of genome exclusion observed in hybridogenesis. Nuclear segregation 3 2 can be followed by endoreplication occuring in these hyphal branches or in the basidia to 3 3 7 produce a diploid nucleus that then ultimately undergoes meiosis and produces uniparental 1 progeny.

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Uniparental reproduction also occurs in natural isolates 4 After establishing the uniparental sporulation in lab strains, we attempted to determine 5 whether such events also occur with natural isolates. For this purpose, we selected two wild-6 type natural isolates, Bt63a and IUM96-2828a (referred to as IUM96a hereafter) (Keller et 7 al. 2003, Litvintseva et al. 2003, Desjardins et al. 2017. IUM96a belongs to the same lineage VYD135α ( Figure 3A).

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During mating, the H99α x Bt63a cross rapidly (within a week) produced robust  (Table S1). This result is consistent with previous results and the low germination rate   (Table S1). Interestingly, all germinated progeny harbored only the MATα mating-type 2 6 whereas the mitochondria were inherited from the MATa parent. These results suggest 2 7 uniparental sporulation also occurs with Bt63a and accounts for high germination rates of 2 8 progeny from the VYD135α x Bt63a cross. The occurrence of uniparental sporulation was 2 9 also identified using the fluorescence-based assay with crosses between the GFP-H4 tagged 3 0 VDY135α and mCherry-H4 tagged Bt63a strains ( Figure S4).

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Mating assays with strain IUM96a also revealed a low level of sporulation 3 2 (19/842=2.2%) with VYD135α but a high sporulation rate with H99α (91%) (Figure S1E-F).  (Table S2). Interestingly, we also 4 observed co-incident uniparental MAT inheritance and with a high germination rates in 5 basidia 7, 8, and 9 from the H99α x IUM96a cross as well (Table S2). Taken together, these 6 results suggest that this unusual mode of sexual reproduction occurs with multiple natural 7 isolates. We further propose that this unusual mode of unisexual reproduction occurs in 8 nature in parallel with normal bisexual reproduction. Uniparental progeny completely lack signs of recombination between the two parents 1 1 As mentioned previously, H99α (as well as the H99α-derived strain VYD135α) and Bt63a, as well as the H99α x Bt63a, progeny to whole-genome sequencing. As expected, for analysis ( Figure 3B). When the VYD135α x Bt63a progeny were similarly analyzed, the 1 9 nuclear genome in each progeny was found to be inherited exclusively from only the 2 0 VYD135α parent ( Figure 3C and S5), and the progeny exhibited sequence differences across 2 1 the entire Bt63a genome. In contrast, the mitochondrial genome was inherited exclusively 2 2 from the Bt63a parent, in accord with the PCR assay results discussed above. Additionally, 2 3 the whole-genome sequencing data also revealed that while most of the H99α x Bt63a F1 2 4 progeny exhibited aneuploidy, the genome-shuffle strain VYD135α x Bt63a progeny were 2 5 euploid ( Figure S6A-B), and based on flow cytometry analysis these uniparental F1 progeny were haploid ( Figure S6C).

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The F1 progeny from crosses involving IUM96a as the MATa partner were also 2 8 sequenced. Similar to the Bt63a analysis, the H99α x IUM96a F1 progeny exhibited signs of 2 9 meiotic recombination, whereas the VYD135α x IUM96a F1 progeny did not ( Figure S7).

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Congruent with the mating-type analysis, the progeny exclusively inherited nuclear genetic 3 1 material from only one of the two parents. Furthermore, the H99α x IUM96a progeny were 3 2 found to be aneuploid for some chromosomes while the progeny of VYD135α x IUM96a 1 were completely euploid ( Figure S8). We also sequenced four progeny from basidium 7 from 2 the H99α x IUM96a cross, which were suspected to be uniparental progeny based on mating-3 type PCRs. This analysis showed that all four progeny harbored only H99α nuclear DNA and 4 had no contribution from the IUM96a genome further confirming the conclusion that 5 uniparental reproduction occurs in wild-type crosses ( Figure S7A). Combined, these results 6 suggest the occurrence of a novel mode of sexual reproduction in C. neoformans, where two 7 parents participate but the two parental genomes do not recombine with each other. bypassed the requirement for Dmc1 and produced spores ( Figure 4A and S9A). When 2 0 dissected, the germination rate for these spores was found to be very low with spores from 2 1 many basidia not germinating at all (Table 2). Furthermore, MAT-specific PCRs revealed that 2 2 some of the progeny were aneuploid. For VYD135α dmc1Δ x KN99a dmc1Δ, much fewer 2 3 basidia (~0.1%) produced spore chains as compared to ~1% sporulation in VYD135α x 2 4 KN99a ( Figure 4A, B and S9B). Dmc1 mutant unilateral crosses sporulated at a frequency of 2 5 0.4% suggesting that only one of the parental strains was producing spores ( Figure 4B).

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When a few sporulating basidia from multiple mating spots were dissected, two different 2 7 populations of basidia emerged, one with no spore germination, and the other with a high 2 8 spore germination rate and uniparental DNA inheritance (Table 2). Combined together, the 2 9 DMC1 deletion led to a 20-fold reduction in viable sporulation, observed as a 10-fold  To further support these conclusions, DMC1 was deleted in mCherry-H4 tagged 1 KN99a and crossed with GFP-H4 tagged VYD135α. We hypothesized that GFP-H4 tagged 2 VYD135α would produce spore chains in this cross because it harbors DMC1 whereas 3 mCherry-H4 tagged KN99a, lacking DMC1, would fail to do so. Indeed, all 11 observed 4 basidia with only the GFP-H4 fluorescence signal were found to produce spores but only 2 5 out of 19 mCherry-H4 containing basidia exhibited sporulation ( Figure S10). These results 6 combined with spore dissection data show that Dmc1 is critical for uniparental sporulation. presence of sexual parasitism in this kingdom.

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Hybridogenesis requires the exclusion of one of the parents, which is followed by 2 1 endoreplication of the other parent's genome and meiosis to produce hemiclonal progeny. A 2 2 similar process was found to occur in a human fungal pathogen, C. neoformans, in this study.

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The whole-genome sequence of the progeny revealed the complete absence of one parent's 2 4 genome, suggesting manifestations of genome exclusion during hyphal growth. The Tunner and Heppich-Tunner 1991). A recent study, however, proposed that genome 1 exclusion in P. esculentus could also take place during early meiotic phases (Dolezalkova et 2 al. 2016). Using fluorescence microscopy, we examined the steps of nuclear exclusion in C. 3 neoformans and found that it occurs during mitotic hyphal growth and not during meiosis. 4 We also observed that genome exclusion could happen with either of the two parents in C. 5 neoformans, similar to what has also been reported for water frogs (Uzzell et al. 1976, 6 Vinogradov et al. 1991, Holsbeek and Jooris 2009. Taken together, these results indicate that 7 the mechanism might be at least partially conserved between these two distantly related 8 organisms. The amenability of C. neoformans to genetic manipulation will aid in deciphering 9 some of the unanswered questions related to hybridogenesis in animals.

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The occurrence of hybridogenesis might also have significant implications for C. presence of a hybridogenetic mechanism in C. neoformans might help explain the mating-1 6 type distribution pattern for this species specifically. In this report, one of the MATa natural 1 7 isolates, Bt63a, did not contribute to uniparental sporulation and the other isolate, IUM96a, MATα progeny. We hypothesize that MATa isolates may be defective in this process due to population. Furthermore, we propose that hybridogenesis can occur between any two 2 4 opposite mating-type strains as long as each one of them is capable of undergoing cell-cell 2 5 fusion and at least one of them can sporulate. This mode of reproduction might act as an 2 6 escape path from genomic incompatibilities between two related, yet karyotypically-2 7 incompatible isolates and allow them to produce spore progeny for dispersal and infection.

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The fungal kingdom is one of the more diverse kingdoms with approximately 3 2 9 million species. The finding of hybridogenesis hints towards unexplored biology in this organisms also suggests that a combination of both sexual and clonal modes of reproduction 7 might prove to be evolutionarily advantageous. Basidia-specific spore dissections were performed after two-five weeks of mating, and the 1 9 spore germination rate was scored after five days of dissection. Strains and primers used in 2 0 this study are listed in Table S3 and S4, respectively. See Supplementary methods for details.

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The sequence data generated in this study were submitted to NCBI with the BioProject 2 2 accession number PRJNA682203.