1. Genetics and Genomics
  2. Microbiology and Infectious Disease
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Uniparental nuclear inheritance following bisexual mating in fungi

  1. Vikas Yadav
  2. Sheng Sun
  3. Joseph Heitman  Is a corresponding author
  1. Department of Molecular Genetics and Microbiology, Duke University Medical Center, United States
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Cite this article as: eLife 2021;10:e66234 doi: 10.7554/eLife.66234

Abstract

Some remarkable animal species require an opposite-sex partner for their sexual development but discard the partner’s genome before gamete formation, generating hemi-clonal progeny in a process called hybridogenesis. Here, we discovered a similar phenomenon, termed pseudosexual reproduction, in a basidiomycete human fungal pathogen, Cryptococcus neoformans, where exclusive uniparental inheritance of nuclear genetic material was observed during bisexual reproduction. Analysis of strains expressing fluorescent reporter proteins revealed instances where only one of the parental nuclei was present in the terminal sporulating basidium. Whole-genome sequencing revealed that the nuclear genome of the progeny was identical with one or the other parental genome. Pseudosexual reproduction was also detected in natural isolate crosses where it resulted in mainly MATα progeny, a bias observed in Cryptococcus ecological distribution as well. The mitochondria in these progeny were inherited from the MATa parent, resulting in nuclear-mitochondrial genome exchange. The meiotic recombinase Dmc1 was found to be critical for pseudosexual reproduction. These findings reveal a novel, and potentially ecologically significant, mode of eukaryotic microbial reproduction that shares features with hybridogenesis in animals.

eLife digest

Sexual reproduction enables organisms to recombine their genes to generate progeny that have higher levels of evolutionary fitness. This process requires reproductive cells – like the sperm and egg – to fuse together and mix their two genomes, resulting in offspring that are genetically distinct from their parents.

In a disease-causing fungus called Cryptococcus neoformans, sexual reproduction occurs when two compatible mating types (MATa and MATα) merge together to form long branched filaments called hyphae. Cells in the hyphae contain two nuclei – one from each parent – which fuse in specialized cells at the end of the branches called basidia. The fused nucleus is then divided into four daughter nuclei, which generate spores that can develop into new organisms. In nature, the mating types of C. neoformans exhibit a peculiar distribution where MATα represents 95% or more of the population. However, it is not clear how this fungus successfully reproduces with such an unusually skewed distribution of mating types.

To investigate this further, Yadav et al. tracked the reproductive cycle of C. neoformans applying genetic techniques, fluorescence microscopy, and whole-genome sequencing. This revealed that during hyphal branching some cells lose the nucleus of one of the two mating types. As a result, the nuclei of the generated spores only contain genetic information from one parent.

Yadav et al. named this process pseudosexual reproduction as it defies the central benefit of sex, which is to produce offspring with a new combination of genetic information. Further experiments showed that this unconventional mode of reproduction can be conducted by fungi isolated from both environmental samples and clinical patient samples. This suggests that pseudosexual reproduction is a widespread and conserved process that may provide significant evolutionary benefits.

C. neoformans represents a flexible and adaptable model organism to explore the impact and evolutionary advantages of sex. Further studies of the unique reproductive strategies employed by this fungus may improve the understanding of similar processes in other eukaryotes, including animals and plants. This research may also have important implications for understanding and controlling the growth of other disease-causing microbes.

Introduction

Most multicellular organisms in nature undergo (bi)sexual reproduction involving two partners of the opposite sex to produce progeny. In most cases, following the fusion of the two haploid gametes, the diploid zygote receives one copy of the genetic material from each parent. To produce these haploid gametes, a diploid germ cell of the organism undergoes meiosis, which involves recombination between the two parental genomes, generating recombinant product. Recombination confers benefits by bringing together beneficial mutations and segregating away deleterious ones (Dimijian, 2005; Meirmans, 2009). In contrast, some organisms undergo variant forms of sexual reproduction, including parthenogenesis, gynogenesis, androgenesis, and hybridogenesis, and in doing so, produce clonal or hemi-clonal progeny (Avise, 2015; Neaves and Baumann, 2011).

In parthenogenesis, a female produces clonal progeny from its eggs without any contribution from a male partner (Avise, 2015; Horandl, 2009). Gynogenesis and androgenesis occur when the fusion of an egg with a sperm induces cell division to produce clonal female or male zygotes, respectively (Lehtonen et al., 2013). During hybridogenesis, an egg from one species fuses with the sperm from another species to generate a hybrid diploid zygote (Lavanchy and Schwander, 2019). However, one of the parental genomes is excluded during development, in a process termed genome exclusion that occurs before gametogenesis. The remaining parental genome undergoes replication followed by meiosis to produce an egg or a sperm. The sperm or egg then fuses with an opposite-sex gamete to generate a hemiclonal progeny. Because only one parent contributes genetic material to the progeny, but both parents are physically required, this phenomenon has been termed sexual parasitism (Lehtonen et al., 2013; Umphrey, 2006). While most of the reported cases of hybridogenesis are from female populations, recent reports suggest that it may also occur in male populations of some species (Doležálková et al., 2016; Schwander and Oldroyd, 2016). Currently, hybridogenesis has only been observed in the animal kingdom in some species of frogs, fishes, and snakes. Plants also exhibit parthenogenesis (aka apomixis), along with gynogenesis and androgenesis (Lehtonen et al., 2013; Mirzaghaderi and Hörandl, 2016).

Unlike animals, most fungi do not have sex chromosomes; instead, cell-type identity is defined by the mating-type (MAT) locus (Heitman, 2015; Heitman et al., 2013). While many fungi are heterothallic, with opposite mating types in different individuals, and undergo sexual reproduction involving two partners of compatible mating types, other fungi are homothallic, with opposite mating types residing within the same organism, and can undergo sexual production during solo culture in the absence of a mating partner. One class of homothallic fungi undergoes unisexual reproduction, during which cells of a single mating type undergo sexual reproduction to produce clonal progeny, similar to parthenogenesis (Heitman, 2015; Lee et al., 2010). Gynogenesis and hybridogenesis have not been identified in the fungal kingdom thus far.

Cryptococcus neoformans is a basidiomycete human fungal pathogen that exists as either one of two mating types, MATa or MATα (Sun et al., 2019a). During sexual reproduction, two haploid yeast cells of opposite mating types interact and undergo cell-cell fusion (Kwon-Chung, 1975; Kwon-Chung, 1976; Sun et al., 2019b). The resulting dikaryotic zygote then undergoes a morphological transition and develops into hyphae whose termini mature to form basidia. In the basidium, the two parental nuclei fuse (karyogamy), and the resulting diploid nucleus undergoes meiosis to produce four daughter nuclei (Idnurm, 2010; Kwon-Chung, 1976; Sun et al., 2019b; Zhao et al., 2019). These four haploid nuclei repeatedly divide via mitosis and bud from the surface of the basidium to produce four long spore chains. Interestingly, in addition to this canonical heterothallic sexual reproduction, a closely related species, C. deneoformans can undergo unisexual reproduction (Lin et al., 2005; Roth et al., 2018; Sun et al., 2014).

In a previous study, we generated a genome-shuffled strain of C. neoformans, VYD135α, by using the CRISPR-Cas9 system targeting centromeric transposons in the lab strain H99α. This led to multiple centromere-mediated chromosome arm exchanges in strain VYD135α when compared to the parental strain H99α, without any detectable changes in gene content between the two genomes (Yadav et al., 2020). In addition, strain VYD135α exhibits severe sporulation defects when mated with strain KN99a (which is congenic with strain H99α but has the opposite mating type), likely due to the extensive chromosomal rearrangements introduced into the VYD135α strain. In this study, we show that the genome-shuffled strain VYD135α can in fact produce spores in crosses with MATa C. neoformans strains after prolonged incubation. Analysis of these spores reveals that the products from each individual basidium contain genetic material derived from only one of the two parents. Whole-genome sequencing of the progeny revealed an absence of recombination between the two parental genomes. The mitochondria in these progeny were found to always be inherited from the MATa parent, consistent with known mitochondrial uniparental inheritance (UPI) patterns in C. neoformans (Sun et al., 2020a). Using strains with differentially fluorescently labeled nuclei, we discovered that in a few hyphal branches as well as in basidia, only one of the two parental nuclei was present and produced spores, leading to uniparental nuclear inheritance. We also observed the occurrence of such uniparental nuclear inheritance in wild-type and natural isolate crosses. Furthermore, we found that the meiotic recombinase Dmc1 plays a central role during this unusual mode of reproduction of C. neoformans. Overall, this mode of sexual reproduction of C. neoformans exhibits striking parallels with hybridogenesis in animals.

Results

Chromosomal translocation strain exhibits unusual sexual reproduction

Previously, we generated a strain (VYD135α) with eight centromere-mediated chromosome translocations compared to the wild-type parental isolate H99α (Yadav et al., 2020). Co-incubation of the wild-type strain KN99a with the genome-shuffled strain VYD135α resulted in hyphal development and basidia production, but no spores were observed during a standard 2-week incubation. However, when sporulation was assessed at later time points in the VYD135α×KN99a cross, we observed a limited number of sporulating basidia (16/1201=1.3%) after 5 weeks compared to a much greater level of sporulation in the wild-type H99α×KN99a cross (524/599=88%) (Figure 1A–D). None of these strains exhibited any filamentation on their own even after 5 weeks of incubation, indicating that the sporulation events were not a result of unisexual reproduction (Figure 1A–B). To analyze this delayed sporulation process in detail, spores from individual basidia were dissected and germinated to yield viable F1 progeny. As expected, genotyping of the mating-type locus in the H99α×KN99a progeny revealed that both MATa and MATα progeny were produced from each basidium (Figure 1E and G, Table 1). In contrast, the same analysis for VYD135α×KN99a revealed that all germinating progeny from each individual basidium possessed either only the MATα or the MATa allele (Figure 1E and G, Table 1). Polymerase chain reaction (PCR) assays also revealed that the mitochondria in all of these progeny were inherited from the MATa parent, in accord with known UPI (Figure 1F–G). These results suggest the inheritance of only one of the parental nuclei in the VYD135α×KN99a F1 progeny. The presence of mitochondria from only the MATa parent in MATα progeny further confirmed that these progeny were the products of fusion between the parent strains and were not the products of unisexual reproduction.

Chromosome shuffled strain exhibits unusual sexual reproduction.

(A, B) Images of cultures for the individual strains H99α, KN99a, and VYD135α, showing no self-filamentation on mating medium. Magnification=10×. (C, D) Light microscopy images showing robust sporulation in the H99α×KN99a cross, whereas the VYD135α×KN99a cross exhibited robust hyphal development but infrequent sporulation events. The inset images in colored boxes show examples of basidia observed in each of the crosses. Scale bar, 100 µm. (E, F) A scheme showing the MATα (H99α and VYD135α) and MATa (KN99a) alleles at the STE20 (E) and COX1 (F) loci. Primers used for PCR analysis are marked by blue triangles. (G) Gel images showing PCR amplification of STE20 and COX1 alleles in the progeny obtained from four different basidia for both H99α×KN99a and VYD135α×KN99a crosses. PCR analysis for the parental strains is also shown, and key bands for DNA marker are labeled. PCR, polymerase chain reaction.

Table 1
Genotype analysis of basidia-specific spores germinated from H99α×KN99a and VYD135α×KN99a crosses.
Basidia #H99α×KN99a crossVYD135α×KN99a cross
Spores germinated/ dissected% GerminatedMATMitoSpores germinated/ dissected% GerminatedMATMito
15/14364α+1aa12/2450All αa
214/141007α+7aa6/1060All αa
312/14862α+7a+3aa15/15100All aa
410/14714α+6aa22/2781All aa
57/13546a+1aa3/1225All αa
613/14936α+7aa25/2793All αa
711/14796α+5aa4/4100All αa
814/1410012α+2aa10/1377All αa
910/14714α+6aa13/1587All αa
1014/141007α+7aa31/6151All αa
1114/1410010α+4aa10/10100All aa
1212/14868α+4aa4/580All aa
134/1136All aa24/2886All aa
1413/131008α+5aa16/2857All aa
1514/141007α+7aa11/11100All aa
1614/141006α+8aa10/2245All αa
  1. Mito refers to Mitochondria.

Fluorescence microscopy reveals uniparental nuclear inheritance after mating

Next, we tested whether the uniparental inheritance detected at the MAT locus also applied to the entire nuclear genome. To address this, we established a fluorescence-based assay in which the nuclei of strains H99α and VYD135α were labeled with GFP-H4, whereas the KN99a nucleus was marked with mCherry-H4. In a wild-type cross (H99α×KN99a), the nuclei in the hyphae as well as in the spores were yellow to orange because both nuclei were in a common cytoplasm and thus incorporated both the GFP-tagged and the mCherry-tagged histone H4 proteins (Figure 2—figure supplement 1A and B). We hypothesized that in the cases of uniparental nuclear inheritance, only one of the nuclei would reach the terminal basidium and would thus harbor only one fluorescent nuclear color signal (Figure 2—figure supplement 1A).

After establishing this fluorescent tagging system using the wild-type strains H99α×KN99a, shuffled-strain VYD135α×KN99a crosses with fluorescently labeled strains were examined. In the wild-type cross, most of the basidia formed robust spore chains with both fluorescent colors observed in them, while a small population (~1%) of basidia exhibited spore chains with only one color, representing uniparental nuclear inheritance (Figure 2A and Figure 2—figure supplement 2A). In contrast, the majority of the basidium population in the shuffled-strain VYD135α×KN99a cross did not exhibit sporulation, and the two parental nuclei appeared fused but undivided (Figure 2B and Figure 2—figure supplement 2B). A few basidia (~1%) bore spore chains with only one fluorescent color, marking uniparental nuclear inheritance events. While the basidia with uniparental nuclear inheritance in the H99α×KN99a cross were a small fraction (~1%) of sporulating basidia, the uniparental basidia accounted for all of the sporulating basidia in the VYD135α×KN99a cross. Taken together, these results show that the uniparental nuclear inheritance leads to the generation of clonal progeny but requires mating, the cell-cell fusion between parents of two opposite mating types. Thus, this process defies the main purpose of sexual reproduction, which is to produce recombinant progeny from two parents. Based on these observations, we define the process of uniparental nuclear inheritance during sporulation in C. neoformans as pseudosexual reproduction (and it is referred to as such hereafter). The progeny obtained via this process will be referred to as the uniparental progeny because they inherit a nuclear genome derived from only one of the two parents.

Figure 2 with 2 supplements see all
Fluorescence microscopy reveals uniparental nuclear inheritance in the wild-type crosses.

(A) Crosses of GFP-H4 tagged H99α and mCherry-H4 tagged KN99a revealed the presence of both fluorescent markers in most spore chains along with uniparental nuclear inheritance in rare cases (~1%). In these few sporulating basidia, only one of the fluorescent signals was observed in the spore chains, reflecting the presence of only one parental nucleus in these basidia. (B) Crosses involving GFP-H4 tagged VYD135α and mCherry-H4 tagged KN99a revealed the presence of spore chains with only one fluorescent color. In the majority of basidia that have both parental nuclei, marked by both GFP and mCherry signals, spore chains are not produced, consistent with a failure of meiosis in these basidia. Scale bar, 10 µm.

Pseudosexual reproduction also occurs in natural isolates

After establishing the pseudosexual reproduction of lab strains, we sought to determine whether such events also occur with natural isolates. For this purpose, we selected two wild-type natural isolates, Bt63a and IUM96-2828a (referred to as IUM96a hereafter) (Desjardins et al., 2017; Keller et al., 2003; Litvintseva et al., 2003). IUM96a belongs to the same lineage as H99α/KN99a (VNI) and exhibits approximately 0.1% genome divergence from the H99α reference genome. Bt63a belongs to a different lineage of the C. neoformans species (VNBI) and exhibits ~0.5% genetic divergence from the H99α/KN99a genome. Both the Bt63a and the IUM96a genomes exhibit one reciprocal chromosome translocation with H99α, and as a result, share a total of 10 chromosome-level changes with the genome-shuffled strain VYD135α (Figure 3A). None of these strains are self-filamentous even after prolonged incubation on mating media but both cross efficiently with H99α and VYD135α (Figure 3—figure supplement 1A).

Figure 3 with 7 supplements see all
VYD135α progeny exhibit strict uniparental nuclear inheritance and lack the signature of meiotic recombination.

(A) Chromosome maps for H99α/ΚN99a, VYD135α, Bt63a, and IUM96a showing the karyotype variation. The genome of the wild-type strain H99α served as the reference. Black arrowheads represent chromosome translocations between VYD135α and H99α whereas red arrowheads mark chromosomes with a translocation between H99α and Bt63a or IUM96a. (B) Whole-genome sequencing, followed by SNP identification, of H99α×Bt63a progeny revealed evidence of meiotic recombination in all of the progeny. The left panel shows SNPs with respect to the Bt63a genome whereas the right panel depicts SNPs against the H99α genome. H99α and Bt63a Illumina sequencing data served as controls for SNP calling. (C) SNP analysis of VYD135α ×Bt63a progeny revealed no contribution of the Bt63a parental genome in the progeny as evidenced by the presence of SNPs only against Bt63a (left panel) but not against the VYD135α genome (right panel). The presence of a few SNPs observed in VYD135α, as well as all VYD135α×Bt63a progeny, are within nucleotide repeat regions. GF stands for germination frequency and P stands for progeny. (D) SNP analysis of H99α×Bt63a and VYD135α×Bt63a progeny using mitochondrial DNA as the reference revealed that mitochondrial DNA is inherited from Bt63a in all of the progeny. Progeny obtained from VYD135α×Bt63a basidium 18 also revealed recombination between the two parental mitochondrial genomes as marked by the absence or presence of two SNPs when mapped against VYD135α and Bt63a mitochondrial genomes, respectively. The green bar in each panel depicts the locus used for PCR analysis of the mitochondrial genotype in the progeny. PCR, polymerase chain reaction; SNP, single nucleotide polymorphism.

The H99α×Bt63a strains crossed rapidly (within a week) producing robust sporulation from most of the basidia observed. The VYD135α×Bt63a cross underwent a low frequency of sporulation (12 spore-producing basidia/840 basidia=1.4%) in 2–3 weeks (Figure 3—figure supplement 1B). Dissection of spores from the H99α×Bt63a cross revealed a low germination frequency (average of 25%) with two of the basidia showing no spore germination at all (Supplementary file 1a). This result is consistent with previous results, and the low germination frequency could be explained by the genetic divergence between the two strains (Morrow et al., 2012). Genotyping of germinated spores from the H99α×Bt63a cross revealed both MATa and MATα progeny from individual basidia, with almost 75% of the meiotic events generating progeny that were heterozygous for the MAT locus (Figure 3—figure supplement 1C and Supplementary file 1a). For the VYD135α×Bt63a cross, spores from 15/20 basidia germinated and displayed a higher germination frequency than the H99α×Bt63a cross (Supplementary file 1a). Interestingly, all germinated progeny harbored only the MATα mating type, whereas the mitochondria were in all cases inherited from the MATa parent (Figure 3—figure supplement 1C). These results suggest that pseudosexual reproduction also occurs with Bt63a and accounts for the high germination frequency of progeny from the VYD135α×Bt63a cross. The occurrence of pseudosexual reproduction was also identified using the fluorescence-based assay with crosses between the GFP-H4 tagged VDY135α and mCherry-H4 tagged Bt63a strains (Figure 3—figure supplement 2).

Crosses with strain IUM96a also revealed a low level of sporulation (19/842=2.3%) with VYD135α but a high sporulation frequency with H99α (91%) (Figure 3—figure supplement 1D). Analysis of progeny from crosses involving IUM96a revealed a similar pattern to what was observed with crosses involving KN99a. The progeny from H99α×IUM96a exhibited variable basidium-specific germination frequencies and inherited both MATa and MATα in each basidium, whereas VYD135α×IUM96a progeny from each basidium inherited exclusively either MATa or MATα (Figure 3—figure supplement 1E, and Supplementary file 1b). Interestingly, we observed co-incident uniparental MAT inheritance and a high germination frequency in progeny of basidia 7, 8, and 9 from the H99α×IUM96a cross as well (Figure 3—figure supplement 1E, and Supplementary file 1b). Taken together, these results suggest that this unusual mode of sexual reproduction occurs with multiple natural isolates. We further propose that pseudosexual reproduction occurs in nature in parallel with canonical sexual reproduction.

Uniparental progeny completely lack signs of nuclear recombination between the two parents

As mentioned previously, H99α (as well as the H99α-derived strain VYD135α) and Bt63a have approximately 0.5% genetic divergence. The occurrence of pseudosexual reproduction in the VYD135α×Bt63a cross allowed us to test if the two parental genomes recombine with each other during development. We subjected progeny from crosses VYD135α×Bt63a and H99α×Bt63a to whole-genome sequencing. As expected, for the H99α×Bt63a cross, both parents contributed to the nuclear composition of their progeny, and there was clear evidence of meiotic recombination as determined by variant analysis (Figure 3B). However, when the VYD135α×Bt63a progeny were similarly analyzed, the nuclear genome in each of the progeny was found to be inherited exclusively from only the VYD135α parent (Figure 3C and Figure 3—figure supplement 3), and the progeny exhibited sequence differences across the entire Bt63a genome. In contrast, the mitochondrial genome was inherited exclusively from the Bt63a parent (Figure 3D and Figure 3—figure supplement 4), in accord with the PCR assay results discussed above. In addition, the whole-genome sequencing data also revealed that while most of the H99α×Bt63a progeny exhibited aneuploidy, the genome-shuffled strain VYD135α×Bt63a progeny were euploid (Figure 3—figure supplement 5A and B), and based on flow cytometry analysis, these uniparental progeny were haploid (Figure 3—figure supplement 5C).

The progeny from crosses involving IUM96a as the MATa partner were also sequenced. Similar to the Bt63a analysis, the H99α×IUM96a progeny exhibited signs of meiotic recombination, whereas the VYD135α ×IUM96a progeny did not (Figure 3—figure supplement 6). Congruent with the mating-type analysis, the progeny in each of the basidia exclusively inherited nuclear genetic material from only one of the two parents. Furthermore, the H99α×IUM96a progeny were found to be aneuploid for some chromosomes, while the VYD135α×IUM96a progeny were completely euploid (Figure 3—figure supplement 7). We also sequenced four progeny from basidium 7 from the H99α×IUM96a cross, which were suspected to be uniparental progeny based on mating-type PCRs. This analysis showed that all four progeny harbored only H99α nuclear DNA and had no contribution from the IUM96a nuclear genome, further supporting the conclusion that pseudosexual reproduction occurs in wild-type crosses (Figure 3—figure supplement 6A). Similar to other progeny, the mitochondria in these progeny were inherited from the MATa parent (Figure 3—figure supplement 1E, and Supplementary file 1b). Combined, these results affirm the occurrence of a novel mode of sexual reproduction in C. neoformans, which is initiated by the fusion of two strains of opposite mating types, but whose progeny inherit DNA exclusively from one parent.

Pseudosexual reproduction stems from nuclear loss via hyphal branches

Fluorescence microscopy revealed that only one of the two parental nuclei undergoes meiosis and produces spores in approximately 1% of the total basidia population. Based on this finding, we hypothesized that the basidia with only one parental nucleus might arise due to nuclear segregation events during hyphal branching. To gain further insight into this process, the nuclear distribution pattern along the sporulating hyphae was studied. As expected, imaging of long hyphae in the wild-type cross revealed the presence of pairs of nuclei with both fluorescent markers along the length of the majority of hyphae (Figure 4A). In contrast, tracking of hyphae from basidia with spore chains in the genome-shuffled strain VYD135α×KN99a cross revealed hyphal branches with only one parental nucleus, which were preceded by a hyphum with both parental nuclei (Figure 4B, Figure 4—figure supplement 1A and B). Unfortunately, a majority of the hyphae (>30 independent hyphae) we tracked were embedded into the agar, and most of these could not be tracked to the point of branching. For some others, we were able to image the hyphal branching point where two nuclei separate from each other but were then either broken or did not have mature basidia on them (Figure 4—figure supplement 1B). In total, we observed seven events of nuclear loss at hyphal branching in independent experiments and were able to track two of them to observe sporulation or basidia formation at the tip. We also observed long hyphae with only one parental nucleus in the VYD135α×Bt63a cross as well, suggesting the mechanism might be similar between strains.

Figure 4 with 1 supplement see all
Pan-hyphal microscopy reveals the loss of one parental nucleus during pseudosexual reproduction.

Spore-producing long hyphae were visualized in both (A) wild-type H99α×KN99a and (B) VYD135α×KN99a crosses to study the dynamics of nuclei in hyphae. Both nuclei were present across the hyphal length in the wild-type and resulted in the production of recombinant spores. On the other hand, one of the nuclei was lost during hyphal branching in the VYD135α×KN99a cross and resulted in uniparental nuclear inheritance in the spores that were produced. The arrow in (B) marks the hyphal branching point after which only one of the parental nuclei is present (also see Figure 4—figure supplement 1A). The images were captured as independent sections and assembled to obtain the final presented image. Scale bar, 10 µm.

These results suggest that hyphal branching may facilitate the separation of one parental nucleus from the main hyphae harboring both parental nuclei. While this is the most plausible explanation based on our results, we cannot rule out other possible mechanisms, such as a role for clamp cells, leading to nuclear separation during hyphal growth. As a result, one of the parental genomes is excluded at a step before diploidization and meiosis, similar to the process of genome exclusion observed in hybridogenesis. We hypothesize that nuclear segregation can be followed by endoreplication occurring in these hyphal branches or in the basidium to produce a diploid nucleus that then ultimately undergoes meiosis and produces uniparental progeny, which will be explored in future studies.

Meiotic recombinase Dmc1 is important for pseudosexual reproduction

Because the genomes of the uniparental progeny did not show evidence of meiotic recombination between the two parents, we tested whether pseudosexual reproduction involves meiosis. In addition, we sought to test our hypothesis that pseudosexual reproduction involves endoreplication that is followed by meiosis. We therefore tested whether Dmc1, a key component of the meiotic machinery, is required for pseudosexual reproduction. The meiotic recombinase gene DMC1 was deleted in congenic strains H99α, VYD135α, and KN99a, and the resulting mutants were subjected to crossing. A previous report documented that dmc1Δ bilateral crosses (both the parents are mutant for DMC1) display significantly reduced, but not completely abolished, sporulation in Cryptococcus (Lin et al., 2005). We observed a similar phenotype with the H99α dmc1Δ×KN99a dmc1Δ cross. While most of the basidia were devoid of spore chains, a small percentage (21/760=2.7%) of the population bypassed the requirement for Dmc1 and produced spores (Figure 5A and Figure 5—figure supplement 1A). When dissected, the germination frequency for these spores was found to be very low (~22% on average) with spores from many basidia not germinating at all (Supplementary file 1c). Furthermore, MAT-specific PCRs revealed that some of the progeny were aneuploid or diploid. For VYD135α dmc1Δ×KN99a dmc1Δ, many fewer basidia (~0.1%) produced spore chains as compared to ~1% sporulation in VYD135α×KN99a (Figure 5A,B and Figure 5—figure supplement 1B). dmc1 mutant unilateral crosses (one of the two parents is mutant and the other one is wild-type) sporulated at a frequency of 0.4% suggesting that only one of the parental strains was producing spores (Figure 5B). When a few sporulating basidia from the VYD135α dmc1Δ×KN99a dmc1Δ bilateral cross were dissected, two different populations of basidia emerged, one with no spore germination, and the other with a high spore germination frequency and uniparental MAT inheritance (Supplementary file 1c). We hypothesized that the basidia with a high spore germination frequency represent those that have escaped the normal requirement for Dmc1. Overall, the DMC1 deletion led to a 20-fold reduction in viable sporulation in the VYD135α×KN99a cross, observed as a ten fold decrease from the number of sporulation events in the bilateral cross and a further two fold reduction in the number of basidia producing viable spores.

Figure 5 with 2 supplements see all
Meiotic recombinase Dmc1 is required for pseudosexual reproduction.

(A) Light microscopy images showing the impact of dmc1 mutation on sexual and pseudosexual reproduction in C. neoformans. Scale bar, 100 µm. (B) A graph showing quantification (n=3) of sporulation events in multiple crosses with dmc1Δ mutants. At least 3000 basidia were counted in each experiment.

To further support these findings, DMC1 was deleted in mCherry-H4 tagged KN99a and crossed with GFP-H4 tagged VYD135α. We hypothesized that GFP-H4 tagged VYD135α would produce spore chains in this cross because it harbors DMC1, whereas mCherry-H4 tagged KN99a dmc1Δ would fail to do so. Indeed, all 11 observed basidia with only the GFP-H4 fluorescence signal were found to produce spores, but only 2 out of 19 mCherry-H4 containing basidia exhibited sporulation (Figure 5—figure supplement 2). These results combined with the spore dissection findings show that Dmc1 is critical for pseudosexual reproduction. While these results provide concrete evidence for meiosis as a part of pseudosexual reproduction, they also suggest the occurrence of a preceding endoreplication event. However, further studies will need to be conducted to validate and confirm endoreplication or alternate mechanisms to achieve the ploidy necessary for a classical meiosis event.

Discussion

Hybridogenesis and parthenogenesis are mechanisms that allow some organisms to overcome some hurdles of sexual reproduction and produce hemiclonal or clonal progeny (Avise, 2015; Horandl, 2009; Lavanchy and Schwander, 2019). However, harmful mutations are not filtered in these processes, making them disadvantageous during evolution and thus restricting the occurrence of these processes to a limited number of animal species (Lavanchy and Schwander, 2019). In this study, we discovered and characterized the occurrence of a phenomenon in fungi that resembles hybridogenesis and termed it pseudosexual reproduction (Figure 6—figure supplement 1). Fungi are known to exhibit asexual, (bi)sexual, unisexual, and parasexual reproduction, and can switch between these reproductive modes depending on environmental conditions (Heitman, 2015; Heitman et al., 2013). The discovery of pseudosexual reproduction further diversifies known reproductive modes in fungi, suggesting the presence of sexual parasitism in this kingdom.

Hybridogenesis in animals occurs between two different species. The result of hybridogenesis is the production of gametes that are clones of one of the parents, which then fuse with an opposite-sex gamete of the second species, generating hemiclonal offspring. In our study, we observed a similar phenomenon where only one parent contributes to spores, the counterpart of mammalian gametes. However, we observed this phenomenon occurring between different strains of the same species, C. neoformans. It is important to note that these strains vary significantly from each other in terms of genetic divergence and in one case by chromosome rearrangements to the extent that they could be considered different species. This suggests that hybridogenesis in animals and pseudosexual reproduction in fungi are similar to each other. Hybridogenesis requires the exclusion of one of the parents, which is followed by endoreplication of the other parent’s genome and meiosis. The whole-genome sequence of the progeny in our study revealed the complete absence of one parent’s genome, suggesting manifestations of genome exclusion during hyphal growth. The mechanism by which the retained parental genome increases its ploidy before meiosis remains to be further investigated in C. neoformans. Endoreplication is known to occur in the sister species C. deneoformans during unisexual reproduction, and we think that this is the most likely route via which ploidy is increased during pseudosexual reproduction.

The mechanism and time of genome exclusion during hybridogenesis in animals are not entirely understood, except for a few insights from diploid fishes of the genus Poeciliopsis and water frogs, Pelophylax esculentus. Studies using Poeciliopsis fishes showed that haploid paternal genome exclusion takes place during the onset of meiosis via the formation of a unipolar spindle, and thus, only the haploid set of maternal chromosomes is retained (Cimino, 1972a; Cimino, 1972b). On the other hand, studies involving P. esculentus revealed that genome exclusion occurs during mitotic division, before meiosis, which is followed by endoreplication of the other parental genome (Heppich et al., 1982; Tunner and Heppich-Tunner, 1991; Tunner and Heppich, 1981). A recent study, however, proposed that genome exclusion in P. esculentus could also take place during early meiotic phases (Doležálková et al., 2016). Using fluorescence microscopy, we examined the steps of nuclear exclusion in C. neoformans and found that it occurs during mitotic hyphal growth and not during meiosis. We also observed that genome exclusion could happen with either of the two parents in C. neoformans, similar to what has also been reported for water frogs. However, for most other species, genome exclusion was found to occur with the male genome only, leaving behind the female genome for meiosis (Cimino, 1972a; Holsbeek and Jooris, 2010; Lavanchy and Schwander, 2019; Umphrey, 2006; Uzzell et al., 1976; Vinogradov et al., 1991). Multiple studies have shown the formation of meiotic synaptonemal complexes during hybridogenesis, clearly establishing the presence of meiosis during this process (Dedukh et al., 2019; Dedukh et al., 2020; Nabais et al., 2012). Our results showed that the meiotic recombinase Dmc1 is required for pseudosexual reproduction, suggesting the presence of meiosis, whereas there is no direct evidence for the role of a meiotic recombinase in hybridogenetic animals. Taken together, these results indicate that the mechanism might be at least partially conserved across distantly related species. Future studies will shed more light on this, and if established, the amenability of C. neoformans to genetic manipulation will aid in deciphering some of the unanswered questions related to hybridogenesis in animals.

The occurrence of pseudosexual reproduction might also have significant implications for C. neoformans biology. Most (>95%) of Cryptococcus natural isolates belong to only one mating type, α (Zhao et al., 2019). While the reason behind this distribution is unknown, one explanation could be the presence of unisexual reproduction in the sister species C. deneoformans and C. gattii (Fraser et al., 2005; Lin et al., 2005; Phadke et al., 2014). The presence of pseudosexual reproduction in C. neoformans might help explain the mating-type distribution pattern for this species. In this report, one of the MATa natural isolates, Bt63a, did not contribute to pseudosexual reproduction and the other isolate, IUM96a, produced uniparental progeny in only one basidium, while the rest of the basidia produced MATα progeny. We hypothesized that MATa isolates may be defective in this process due to either a variation in their genomes or some other as yet undefined sporulation factor. As a result, pseudosexual reproduction could lead to the generation of predominantly α progeny in nature, reducing the MATa population and thus favoring the expansion of the α mating-type population. However, it is still possible that the preferential inheritance of the nuclear genome from one of the two parents is decided by genetic elements located in regions other than MAT, and whether the uniparental nuclear inheritance is mating-type specific remains to be elucidated. Furthermore, the occurrence of pseudosexual reproduction in other pathogenic species such as C. deneoformans and non-pathogenic species such as C. amylolentus will be investigated in future studies. Attempts to identify the occurrence of pseudosexual reproduction between species where hybrids are known to occur, C. neoformans and C. deneoformans hybrids, will also be made. These studies will help establish the scope of pseudosexual reproduction in Cryptococcus species and could be extended to other basidiomycetes.

We propose that pseudosexual reproduction can occur between any two opposite mating-type strains as long as each of them is capable of undergoing cell-cell fusion and at least one of them can sporulate. We speculate that pseudosexual reproduction might play a key role in C. neoformans survival during unfavorable conditions. In conditions where two mating partners are fully compatible, pseudosexual reproduction will be mostly hidden and might not be important (Figure 6, top panel). However, when the two mating partners are partially incompatible or completely incompatible due to high genetic divergence or karyotypic variation, pseudosexual reproduction will be important (Figure 6, left, right, and bottom panels). For example, most of the basidia in H99α and Bt63a cross largely produce aneuploid and/or inviable progeny leading to unsuccessful sexual reproduction. However, a small yet significant proportion of the basidia generate clonal progeny that are viable and fit via pseudosexual reproduction. We hypothesized that these progeny will have a better chance of survival and find a suitable mating partner in the environment whereas, the unfit recombinant progeny might fail to do so. In nature, this might allow a new genotype/karyotype to not only survive but also expand and will prove advantageous. If a new genotype/karyotype had only the option of undergoing sexual reproduction, it might not survive, restricting the evolution of a new strain. Overall, this mode of pseudosexual reproduction might act as an escape path from genomic incompatibilities between two related isolates and allow them to produce spores for dispersal.

Figure 6 with 1 supplement see all
Model for the role of pseudosexual reproduction in C. neoformans ecology.

Scenarios showing possible roles for pseudosexual reproduction under various hypothetical mating conditions. Except for one condition where the two parents are completely compatible with each other, pseudosexual reproduction could play a significant role in survival and dissemination despite its occurrence at a low frequency.

One of the key differences between pseudosexual reproduction and unisexual reproduction observed in the Cryptococcus species complex is the inheritance of mitochondrial DNA. While both unisexual and pseudosexual reproduction result in clonal progeny with respect to the nuclear genome, the mitochondria in pseudosexual reproduction are almost exclusively inherited from the MATa parent (Figure 6—figure supplement 1). This results in the exchange of mitochondrial DNA in the progeny that inherit the MATα nuclear genome, resembling the nuclear-mitochondrial exchange observed during cytoduction in Saccharomyces cerevisiae. During cytoduction, mutants defective in nuclear fusion produce haploid progeny with nuclear genome from one parent, but a mixture of both parents cytoplasm resulting in the inheritance of one parental mitochondrial genome with the other parent’s nuclear genome (Conde and Fink, 1976; Lancashire and Mattoon, 1979; Zakharov and Yarovoy, 1977). This process was used to study mitochondrial genetics with respect to the transfer of drug-resistance genes and other mitochondrial mutations. Similar to cytoduction, pseudosexual reproduction could be employed to study mitochondrial genetics, such as functional analysis of mitochondrial encoded drug resistance, and cytoplasmic inheritance of factors such as prions in C. neoformans.

The fungal kingdom is one of the more diverse kingdoms with approximately 3 million species (Sun et al., 2020b). The finding of hybridogenesis-like pseudosexual reproduction hints toward unexplored biology in this kingdom that might provide crucial clues for understanding the evolution of sex. Fungi have also been the basis of studies focused on understanding the evolution of meiosis, and the presence of genome reduction, as well as the parasexual cycle in fungi, have led to the proposal that meiosis evolved from mitosis (Hurst and Nurse, 1991; Wilkins and Holliday, 2009). Pseudosexual reproduction may be a part of an evolutionary process wherein genome exclusion followed by endoreplication and meiosis was an ancestral form of reproduction that preceded the evolution of sexual reproduction. Evidence supporting such a hypothesis can be observed in organisms undergoing facultative sex or facultative parthenogenesis (Booth et al., 2012; Fields et al., 2015; Hodač et al., 2019; Hojsgaard and Horandl, 2015). The presence of these organisms also suggests that a combination of both sexual and clonal modes of reproduction might prove to be evolutionarily advantageous.

Materials and methods

Strains and media

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C. neoformans wild-type strains H99α and KN99a served as the wild-type isogenic parental lineages for the experiments (Nielsen et al., 2003; Perfect et al., 1993), in addition to MATa strains Bt63a and IUM96-2828a (Keller et al., 2003; Litvintseva et al., 2003). Strains were grown in YPD media for all experiments at 30°C unless stated otherwise. G418 and/or NAT were added at a final concentration of 200 and 100 µg/ml, respectively, for the selection of transformants. MS media was used for all the mating assays, which were performed as described previously (Sun et al., 2019b). Basidia-specific spore dissections were performed after 2–5 weeks of mating, and the spore germination frequency was scored after 5 days of dissection. All strains and primers used in this study are listed in Supplementary file 1d and Supplementary file 1e, respectively.

Genotyping for mating-type locus and mitochondria

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Mating type (MAT) and mitochondrial genotyping for all the progeny were conducted using PCR assays. Genomic DNA was prepared using the MasterPure Yeast DNA Purification Kit from Lucigen. To determine the MAT, the STE20 allele present within the MAT locus was detected because it differs in length between the two different mating types. Primers specific to both MATa and MATα (JOHE50979-50982 in Supplementary file 1e) were mixed in the same PCR mix, and the identification was made based on the length of the amplicon (Figure 1E–G). For the mitochondrial genotyping, the COX1 allele present in the mitochondrial DNA was probed to distinguish between H99α/VYD135α and KN99a/IUM96a. For the differentiation between Bt63a and H99α/VYD135α, the COB1 allele was used because COX1 in Bt63a is identical to H99α/VYD135α. The difference for both COX1 and COB1 is the presence or absence of an intron and results in significantly different size products between MATα and MATa parents (Figure 1 and Figure 3—figure supplement 1). The primers used for these assays (JOHE51004-51007) are mentioned in Supplementary file 1e.

Genomic DNA isolation for sequencing

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Genomic DNA for whole-genome sequencing was prepared using the CTAB-based lysis method, as described previously (Yadav et al., 2020). Briefly, 50 ml of an overnight culture was pelleted, frozen at −80°C, and subjected to lyophilization. The lyophilized cell pellet was broken into a fine powder, mixed with lysis buffer, and the mix was incubated at 65°C for an hour with intermittent shaking. The mix was then cooled on ice, and the supernatant was transferred into a fresh tube, and an equal volume of chloroform (~15 ml) was added and mixed. The mix was centrifuged at 3200 rpm for 10 min, and the supernatant was transferred to a fresh tube. An equal volume of isopropanol (~18–20 ml) was added into the supernatant and mixed gently. This mix was incubated at −20°C for an hour and centrifuged at 3200 rpm for 10 min. The supernatant was discarded, and the DNA pellet was washed with 70% ethanol. The pellet was air-dried and dissolved in 1 ml of RNase containing 1× TE buffer and incubated at 37°C for 45 min. The DNA was again chloroform purified and precipitated using isopropanol, followed by ethanol washing, air drying, and finally dissolved in 200 µl 1× TE buffer. The DNA quality was estimated with NanoDrop, whereas DNA quantity was estimated with Qubit.

Whole-genome Illumina sequencing, ploidy, and SNP analysis

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Illumina sequencing of the strains was performed at the Duke sequencing facility core (https://genome.duke.edu/), using Novaseq 6000 as 150 paired-end sequencing. The Illumina reads, thus obtained, were mapped to the respective genome assembly (H99α, VYD135α, Bt63a, or IUM96a) using Geneious (RRID:SCR_010519) default mapper to estimate ploidy. The resulting BAM file was converted to a. tdf file, which was then visualized through IGV to estimate the ploidy based on read coverage for each chromosome.

For SNP calling and score for recombination in the progeny, Illumina sequencing data for each progeny was mapped to parental strain genome assemblies individually using the Geneious default mapper with three iterations. The mapped BAM files were used to perform variant calling using Geneious with 0.8 variant frequency parameter and at least 90× coverage for each variant. The variants thus called were exported as VCF files and imported into IGV for visualization purposes. H99α, Bt63a, IUM96a, and VYD135α Illumina reads were used as controls for SNP calling analysis.

PacBio/Nanopore genome assembly and synteny comparison

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To obtain high-molecular-weight DNA for Bt63a genome PacBio and IUM96a genome Nanopore sequencing, DNA was prepared as described above. The size estimation of DNA was carried out by electrophoresis of DNA samples using PFGE. For this purpose, the PFGE was carried out at 6 V/cm at a switching frequency of 1–6 s for 16 hr at 14°C. Samples with most of the DNA ≥100 kb or larger were selected for sequencing. For PacBio sequencing, the DNA sample was submitted to the Duke sequencing facility core. Nanopore sequencing was performed in our lab using a MinION device on an R9.4.1 flow cell. After sequencing, reads were assembled to obtain a Bt63a genome assembly via Canu (RRID:SCR_015880) using PacBio reads >2 kb followed by five rounds of pilon polishing (RRID:SCR_014731). For IUM96a, one round of nanopolish was also performed before pilon polishing. Once completed, the chromosomes were numbered based on their synteny with the H99α genome. For chromosomes involved in translocation (Chr 3 and Chr 11), the chromosome numbering was defined by the presence of the respective syntenic centromere from H99. Centromere locations were mapped based on BLASTn analysis with H99α centromere flanking genes.

Synteny comparisons between the genomes were performed with SyMAP v4.2 using default parameters (Soderlund et al., 2011) (http://www.agcol.arizona.edu/software/symap/). The comparison block maps were exported as .svg files and were then processed using Adobe Illustrator (RRID:SCR_010279) and Adobe Photoshop (RRID:SCR_014199) for representation purposes. The H99α genome was used as the reference for comparison purposes for plotting VYD135α, Bt63a, and IUM96a genomes. The centromere and telomere locations were manually added during the figure processing.

Fluorescent tagging and microscopy

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GFP and mCherry tagging of histone H4 were performed by integrating respective constructs at the safe haven locus (Arras et al., 2015). GFP-H4 tagging was done using the previously described construct, pVY3 (Yadav and Sanyal, 2018). For mCherry-H4 tagging, the GFP-containing fragment in pVY3 was excised using SacI and BamHI and was replaced with mCherry sequence PCR amplified from the plasmid pLKB25 (Kozubowski and Heitman, 2010). The constructs were then linearized using XmnI and transformed into desired strains using CRISPR transformation, as described previously (Fan and Lin, 2018). The transformants were screened by PCR, and correct integrants were obtained and verified using fluorescent microscopy.

To observe the fluorescence signals in the hyphae and basidia, a 2- to 3-week-old mating patch was cut out of the plate and directly inverted onto a coverslip in a glass-bottom dish. The dish was then used to observe filaments under a DeltaVision microscope available at the Duke University Light Microscopy Core Facility (https://microscopy.duke.edu/dv). The images were captured at 60× magnification with 2×2 bin size and z-sections of either 1 or 0.4 µm each. GFP and mCherry signals were captured using the GFP and mCherry filters in the Live-Cell filter set. The images were processed using Fiji-ImageJ (https://imagej.net/Fiji) (RRID:SCR_002285) and exported as tiff files as individual maximum projected images. The final figure was then assembled using Adobe Photoshop software for quality purposes.

Sporulation frequency counting

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To visualize hyphal growth and sporulation defects during mating assays, the mating plates were directly observed under a Nikon Eclipse E400 microscope. Hyphal growth and basidia images were captured using the top-mounted Nikon DXM1200F camera on the microscope. The images were processed using Fiji-ImageJ and assembled in Adobe Photoshop software.

For crosses involving wild-type H99α, VYD135α, KN99a, Bt63a, and IUM96a, approximately 1000 total basidia were counted after 4 weeks of mating, and the sporulation frequency was calculated. For crosses involving VYD135 dmc1Δ strain, three mating spots were setup independently. From each mating spot periphery, six images were captured after 3–4 weeks of mating. Basidia (both sporulating and non-sporulating) in each of these spots were counted manually after some processing of images using ImageJ. The sporulation frequency was determined by dividing the sporulating basidia by the total number of basidia for each spot. Each mating spot was considered as an independent experiment and at least 3000 basidia were counted from each mating spot.

Flow cytometry

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Flow cytometry analysis was performed as described previously (Fu and Heitman, 2017). Cells were grown on YPD medium for 2 days at 30°C, harvested, and washed with 1× phosphate-buffered saline buffer followed by fixation in 70% ethanol at 4°C overnight. Next, cells were washed once with 1 ml of NS buffer (10 mM Tris-HCl, pH=7.2, 250 mM sucrose, 1 mM EDTA, pH=8.0, 1 mM MgCl2, 0.1 mM CaCl2, 0.1 mM ZnCl2, 0.4 mM phenylmethylsulfonyl fluoride, and 7 mM β-mercaptoethanol), and finally resuspended in 180 μl NS buffer containing 20 μl 10 mg/ml RNase and 5 μl 0.5 mg/ml propidium iodide (PI) at 37°C for 3–4 hr. Then, 50 μl stained cells were diluted in 2 ml of 50 mM Tris-HCl, pH=8.0, transferred to FACS compatible tube, and submitted for analysis at the Duke Cancer Institute Flow Cytometry Shared Resource. For each sample, 10,000 cells were analyzed on the FL1 channel on the Becton-Dickinson FACScan. Wild-type H99α and previously generated AI187 were used as haploid and diploid controls, respectively, in these experiments. Data analysis was performed using the FlowJo software (RRID:SCR_008520).

Data availability

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

The following data sets were generated
    1. Yadav V
    2. Sun S
    3. Heitman J
    (2020) NCBI BioProject
    ID PRJNA682203. Uniparental reproduction in Cryptococcus neoformans.
The following previously published data sets were used
    1. Broad Institute
    (2012) NCBI Sequence Read Archive
    ID SRR642222. Illumina whole genome shotgun sequencing of genomic DNA paired-end library 'Pond-151755' containing sample 'Cryptococcus neoformans H99'.
    1. Broad Institute
    (2012) NCBI Sequence Read Archive
    ID SRR647805. Illumina whole genome shotgun sequencing of genomic DNA paired-end library 'Pond-151755' containing sample 'Cryptococcus neoformans H99'.

References

  1. Book
    1. Horandl E
    (2009) Geographical parthenogenesis: Opportunities for asexuality
    In: Schön I, Martens K, Dijk P, editors. Lost Sex: The Evolutionary Biology of Parthenogenesis. Springer. pp. 161–186.
    https://doi.org/10.1007/978-90-481-2770-2_8
    1. Kwon-Chung KJ
    (1975)
    A new genus, Filobasidiella, the perfect state of Cryptococcus neoformans
    Mycologia 67:1197–1200.
    1. Kwon-Chung KJ
    (1976)
    Morphogenesis of Filobasidiella neoformans, the sexual state of Cryptococcus neoformans
    Mycologia 68:821–833.
  2. Book
    1. Meirmans S
    (2009)
    The evolution of the problem of sex
    In: Schön I, Martens K, Dijk P, editors. Lost Sex: The Evolutionary Biology of Parthenogenesis. Springer Netherlands: Dordrecht. pp. 21–46.
    1. Schwander T
    2. Oldroyd BP
    (2016) Androgenesis: Where males hijack eggs to clone themselves
    Philosophical Transactions of the Royal Society B: Biological Sciences 371:20150534.
    https://doi.org/10.1098/rstb.2015.0534
    1. Uzzell T
    2. Günther R
    3. Berger L
    (1976)
    Rana Ridibunda and Rana esculenta: A Leaky Hybridogenetic System (Amphibia Salientia)
    PNAS 128:147–171.

Decision letter

  1. Luis F Larrondo
    Reviewing Editor; Pontificia Universidad Católica de Chile, Chile
  2. Patricia J Wittkopp
    Senior Editor; University of Michigan, United States
  3. Christina Hull
    Reviewer; University of Wisconsin-Madison, School of Medicine and Public Health, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Hybridogenesis is an unusual form of reproduction described in some animal species, that requires an opposite-sex partner although the partner's genome is discarded before gamete formation, producing hemi-clonal progeny. In this work, the authors characterize in the human fungal pathogen Cryptococcus neoformans, an unusual form of reproduction that exhibits striking parallels with hybridogenesis, and that is termed pseudosexual reproduction. The discovery of pseudosexual reproduction not only further expands the described reproductive modes in fungi, but it could help addressing yet unresolved mechanistic aspects of hybridogenesis in animals. This work will be of interest to the fungal community, and also to a broader audience of biologists interested in the evolution of sexual reproduction and its consequences for species survival.

Decision letter after peer review:

Thank you for submitting your article "Uniparental nuclear inheritance during bisexual mating in fungi" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Patricia Wittkopp as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Christina Hull (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) It is not clear whether the observed process can be properly classified as hybridogenesis or not. While in some parts of the text the authors are very careful to refer to a process "similar" or "akin" to hybridogenesis, in others they plainly call it hybridogenesis. Yet, it appears that there are several reasons why this should not be referred to as hybridogenesis, among others: (i) no hybridization and (ii) some of the hallmarks that occur in hybridogenesis (e.g. endoreplication) are not shown or further discussed. Presenting this as a possible new form of sexual reproduction would be reasonable, but the authors would need to definitively show that (1) this is not unisexual reproduction as seen in C. deneoformans (easy to do – grow the strains in monoculture and evaluate for spore production) and (2) provide compelling data showing that mt inheritance is uniparental.

2) The reviewers suggest a series of points that would help improving the manuscript, making it accessible to a larger audience. In particular several of the clarifications revolve around one core aspect: is this hybridogenesis, or is it a new mechanism of sexual development for C. neoformans that more closely resembles hybridogenesis than other known forms of sexual development? To explicitly compare and contrast the features of both would be helpful.

3) It is important to clarify what is supported by direct evidence and what is not (i.e endoreplication?

4) Only a minimal set of experiments are suggested, particularly related to rule out unisexual reproduction, and to clearly support the uniparental inheritance of mt, while also providing clearer information on datasets (i.e germination rate H99alpha x IUM96a).

Reviewer #1 (Recommendations for the authors):

1) The authors state that "this process of uniparental sexual reproduction of C. neoformans exhibits striking parallels with hybridogenesis in animals". Yet, they don't explicitly comment what are the features/issues that would preclude them from claiming that the process is fully comparable to hybridogenesis.

Moreover, later on (page 10, line 19) they appear to indicate that the characterized phenomenon is, actually, hybridogenesis: "The discovery of hybridogenesis further diversifies known reproductive modes in fungi, suggesting the presence of sexual parasitism in this kingdom".

Thus, it is unclear throughout the text whether all the requirements to define this process as bona fide hybridogenesis are met or not.

2) An important aspect of hybridogenesis that is not explicitly discussed in the manuscript either, relates to the fact that hybridogenesis is normally defined as "an unusual form of reproduction that is found in hybrids between different species" (i.e PMID: 30721675). Yet, in the present manuscript the authors obtain their data in crosses between isolates that are part of the same species (albeit with enough genetic differences to clearly differentiate their nuclei, but below the threshold of species differences). This is not acknowledged or discussed in the text.

Moreover, the authors have recently published fine work in the context of hybrids between C. neoformans and C. deneoformans, and how species boundary (and conventional sexual reproduction) are stablished (PMID: 33465111). Indeed, it is quite relevant that such hybrids can be regularly identified from both environmental and clinical samples, in some cases representing a large percentage of the isolates (~20% of the isolates identified in Europe, with even higher prevalence in some EU countries -i.e. Portugal 31%- PMID: 32511709). Detecting hybridogenesis in such context would be a major thing, and would help putting the work into perspective. Indeed, it appears that hybridogenesis tends to occur between a hybrid and another species (related to the hybrid) which genome is then discarded. Thus, the reports of hybridogenesis in several animal species suggest that its origin may imply diverse hybridization events involving intergeneric paternal ancestors. None of these complexities and peculiarities are commented or discussed. Albeit hybridogenesis is not as "popular" as other unusual reproduction modes (i.e. Parthenogenesis), its existence in fungi would be a relevant finding, which would be better appreciated by a general audience if the basics of hybridogenesis are clearly stated, and a checklist of its basics is completed, based on the findings in C. neoformans.

3) It would be valuable that the authors would extend their discussions to other members of the Cryptococcus neoformans species complex. Indeed, the current evidence indicates that C. neoformans exhibits bisexual reproduction, whereas in C. deneoformans displays both bisexual and unisexual mating. Therefore, it would be interesting to see whether hybridogenesis appears as a peculiarity of C. neoformans, or as general property of the species complex (and particularly in the context of hybrids, see previous point). Likewise, the same question could be extended to the six members of the Cryptococcus gattii species complex. Nevertheless, as conducting those experiments would involve massive amount of work, the authors could expand the discussion of the topic (somehow covered already in page 11). In this context the authors could explicitly discuss whether they expect hybridogenesis to be a phenomenon that was acquired in C. neoformans, or a property that was lost in other members of the Cryptococcus species complex.

4) Page 6, Line 33: "Nuclear segregation can be followed by endoreplication occurring in these hyphal branches or in the basidia to produce a diploid nucleus that then ultimately undergoes meiosis and produces uniparental progeny"

It is not clear if with this phrase the authors are stating a possibility or a fact. What is the evidence that endoreplication is occurring?

Related to this, it is interesting that endoreplication has been observed as a one of the mechanisms of unisexual mating in C. deneoformans.

5) Page 8, Line 33: "Congruent with the mating-type analysis, the progeny exclusively inherited nuclear genetic material from only one of the two parents".

Please specify which one.

6) Page 10, Line 7: "These results combined with spore dissection data show that Dmc1 is critical for uniparental sporulation". Authors could enrich the phrase by explicitly comparing the requirement of meiotic recombinase-based mechanisms in animal hybridogenesis.

Page 11, Line 23: "As a result, hybridogenesis would result in the generation of predominantly progeny in nature reducing the MATa population and thus favoring the expansion of the mating-type population" While this is an interesting idea, it is also hard to ignore the fact that the hybridogenesis process described by the authors is rare (approx. 1 %) compared to classic sexual reproduction. Therefore, it would contribute to a minor fraction of the reproductive events occurring in nature, unless some environmental conditions facilitate the former mode over the latter.

Reviewer #3 (Recommendations for the authors):

Thank you to the authors for the opportunity to review their excellent and interesting work! I have several recommendations for the authors to consider.

1) This is an exciting discovery that could be of broad interest, but it is difficult to keep up with what the authors are trying to communicate in this manuscript. Specifically, the terminology to refer to the various forms of sexual development and spore production (i.e. uniparental sporulation, uniparental nuclear inheritance, bisexual reproduction, unisexual sporulation, etc.) are not well defined and used inconsistently. The challenge is confounded by the (necessary) inclusion in the mix of "uniparental mitochondrial inheritance." The authors could improve the manuscript substantially by carefully defining and consistently using intentional labels for the numerous, relevant processes (including "mating," which is best used when referring to only the fusion process and not all of sexual development). It will help readers understand their beautiful data.

2) A second point of possible confusion is the use of "Germination Rate" to refer to the proportion of spores that can germinate into viable progeny. In the case of spores, it is probably more accurate to refer to this as "Germination Frequency" (high vs. low). Germination Rate is perhaps better used to indicate the efficiency with which any given spore differentiates into a yeast over time (slow vs. fast).

3) The manuscript would also benefit from careful editing so that all figures are referenced in the text, figure legends and the text provide the same information, and the prose all makes sense. Some specific problems are indicated below.

4) With respect to the mitochondrial inheritance data, Cryptococcus researchers likely know how one determines the parental source of the mitochondria in F1 progeny, but others may not. It is also challenging because all of the progeny (with only one exception) harbor "a"-derived mitochondria. It is a somewhat annoying but necessary question: How do you know your primers can discern between "a" and α-derived mitochondria? What is the basis for this discrimination? What if the primers were mislabeled – would you know? More information and context in the manuscript would help resolve any doubts.

5) After Figure 2, there is a leap and a disconnect. The authors show clear evidence of one nuclear loss event at a hyphal branch, supporting their model. Then they indicate that endoreplication and meiosis can occur but do not provide any data for this. Then the manuscript addresses the presence or absence of meiotic recombination and the role of DMC1 in uniparental nuclear inheritance. It is not clear how this transition is made – particularly if one is outside the Cryptococcus field and does not know about the events that occur during unisexual reproduction in C. deneoformans. The absence of meiotic recombination in uniparental nuclear inheritance would make sense, but then why investigate a meiotic recombinase subsequent to that? Providing context for the DMC1 experiments would be extremely useful, as would including an interpretation of the findings in the Discussion.

6) Food for thought: What if loss of one parent nucleus is just a mistake? What if, at some frequency, in all forms of Cryptococcus reproduction clamp cells mess up and a nucleus gets lost (maybe more at branchpoints) but it "just happens" in ~1% of all filaments. Is that really hybridosis or akin to hybridosis? It seems a little risky to liken a very rare event that occurs during sexual development to a form of animal development that is the primary mechanism of reproduction within certain animal species. Is it then reasonable to posit that understanding the seemingly very rare uniparental nuclear inheritance process in a fungus will inform sexual evolution in larger eukaryotes? On the other hand, if the C. neoformans response to losing one of the parental nuclei is endoreplication (as the authors suggest but do not show), that seems to be more of a potential parallel.

How likely is this very low frequency event to contribute to fitness if it is occurring coincident with bisexual reproduction, as suggested by the authors? Perhaps it contributes to fitness, but perhaps not. Can such a rare event be reasonably compared to sexual parasitism?

Page 3, line 1: "Most organisms in nature undergo sexual reproduction between two partners of the opposite sex to produce progeny." Is it most? There are a lot of microbes in the world that don't undergo sexual reproduction as described here.

Page 5, line 17: At that point, how do you know it's sexual?

Page 9, lines 26-30: "Dmc1 mutant unilateral crosses sporulated at a frequency of

0.4% suggesting that only one of the parental strains was producing spores (Figure 4B). When a few sporulating basidia from multiple mating spots were dissected, two different populations of basidia emerged, one with no spore germination, and the other with a high spore germination rate and uniparental DNA inheritance (Table 2)." Something here seems amiss – why are there two populations of spores? Perhaps re-phrasing could help clarify.

Page 9, line 24: Is the presence of both MAT alleles indicative of aneuploidy? Couldn't it also be diploidy? Also, need to change "much fewer" to "many fewer."

Is bisexual reproduction normal in nature?

There is no specific reference to Figure S2B in the manuscript.

Figure 2 Legend refers to unisexual reproduction – should be uniparental? This happens several times.

Labels for which consolidation and/or definitions would be helpful:

From Title and Abstract:

Uniparental nuclear inheritance

Bisexual mating

Bisexual reproduction

Uniparental sporulation

Uniparental reproduction

From Introduction:

Sexual reproduction

Unisexual reproduction

Heterothallic sexual reproduction

Mitochondrial Uniparental Inheritance

Uniparental inheritance of nuclei

Partner-stimulated uniparental sexual reproduction

From Results:

Unusual sexual reproduction

Uniparental mitochondrial genome inheritance

Unisexual reproduction

Uniparental inheritance

Uniparental sporulation

Uniparental reproduction

Biparental sporulation

Uniparental meiosis and sporulation

Uniparental MAT inheritance

this unusual mode of unisexual reproduction occurs in nature in parallel with normal bisexual reproduction

Bilateral crosses

Unilateral crosses

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Uniparental nuclear inheritance following bisexual mating in fungi" for further consideration by eLife. Your revised article has been evaluated by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Patricia Wittkopp (Senior Editor).

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

I am confident that incorporating the different suggestions can be easily done and should not take much time.

Reviewer #1 (Recommendations for the authors):

The revised manuscript has included key recommendations previously pointed by reviewers. In particular, the authors have been more conservative in classifying the observed phenomenon as pseudosexual reproduction, which shares several features of hybridogenesis, albeit it may be too soon to classify it unequivocally us such. They also have provided additional data solidifying their conclusions about mitochondria inheritance. These findings, described for the first time in the fungal kingdom, may provide important insights for understanding the evolution of sex. Indeed, this pseudosexual reproduction (particularly related to genome exclusion followed by endoreplication and meiosis) could correspond to an ancestral form of reproduction that preceded the evolution of sexual reproduction. While the fine mechanisms allowing pseudosexual reproduction still remain undefined (i.e. endoreplication is suspected to occur, although no experiments actually confirm or deny its occurrence), the authors acknowledge the current limitations of the study.

Reviewer #3 (Recommendations for the authors):

In the resubmitted manuscript "Uniparental nuclear inheritance following bisexual mating in fungi," the authors offer a thorough and thoughtful response to reviewer criticisms and extensive revisions to the paper. Through their efforts, the manuscript is substantially improved – offering a more accurate presentation of the data, more clarity in the data presentation, and consideration of points that will make the findings more accessible to a broad audience of researchers.

The authors were particularly responsive to the criticism that the term hybridogenesis might not apply to their findings. They modified the manuscript accordingly by providing pivotal evidence to rule out unisexual reproduction, engaging in a richer consideration of alternative hypotheses, and clarifying/unifying their use of language to refer to sexual processes. As a result, the data are more clear, and the arguments are more compelling.

Points for consideration:

Figure 4: It is still unclear how many times the authors determined that nuclear loss occurred at a branchpoint during hyphal growth in pseudosexual reproduction. I recognize that capturing these events is difficult, so a reference to the images shown as being representative of what is seen in Figure 4 and a general accounting of the number of times this pattern has been observed would be useful (in text and/or legend).

Figure 6: In terms of understanding the mechanism(s) by which pseudosexual reproduction could occur and influence fitness, Figure 6A could be eliminated (and referred to in supplemental). Figure 6B could be retained as simply Figure 6 and be referred to in the Discussion.

Throughout text:

Clarity of the manuscript could be improved even further by using the term "mating" to refer only to the fusion event between parents (the first step of sexual development) and not to the entire process of development.

Reviewer #4 (Recommendations for the authors):

The authors have responded satisfactorily to the comments from the previous review.

I have some additional suggestions for the authors:

1. The title might be improved to indicate that the uniparental nuclear genome inheritance enables nuclear-mitochondrial genome swapping, which the authors both demonstrate and is a key piece of the mechanistic argument.

2. The authors may want to examine more cases of the observed phenomenon before concluding that a particular mating type is the favored result of the pseudo sexual process. In other words, it may not be mating type but rather nuclear genotype more generally that determines which nucleus is lost during dikaryotic development.

3. The authors may want to compare their observations with cytoduction in S. cerevisiae.

https://doi.org/10.7554/eLife.66234.sa1

Author response

Essential Revisions (for the authors):

1) It is not clear whether the observed process can be properly classified as hybridogenesis or not. While in some parts of the text the authors are very careful to refer to a process "similar" or "akin" to hybridogenesis, in others they plainly call it hybridogenesis. Yet, it appears that there are several reasons why this should not be referred to as hybridogenesis, among others: i) no hybridization and ii) some of the hallmarks that occur in hybridogenesis (e.g. endoreplication) are not shown or further discussed. Presenting this as a possible new form of sexual reproduction would be reasonable, but the authors would need to definitively show that 1) this is not unisexual reproduction as seen in C. deneoformans (easy to do – grow the strains in monoculture and evaluate for spore production) and 2) provide compelling data showing that mt inheritance is uniparental.

We thank the editor and reviewers for their insightful and critical comments. While some of the crosses analyzed in our study could be considered as hybridization given the significant structural chromosomal rearrangements between the two isolates (e.g. VYD135 vs. KN99a), we agree that the novel phenomenon of the production of clonal progeny with a uniparental nuclear inheritance could also occur between strains that are genetically closely related. Thus, we have decided to refrain from calling it hybridogenesis. Instead, we now define it as a novel form of reproduction and term it “pseudosexual reproduction”, reflecting the finding that although the observed process starts with bisexual mating, the two parental nuclei do not recombine and the progeny are clonal with the nuclear genome that is identical to one of the two parents.

Following the reviewers’ suggestions, we included compelling additional data definitively demonstrating mitochondrial uniparental inheritance during pseudosexual reproduction by both PCR as well as whole-genome sequencing (Figures 1, 4, Figure 3—figure supplement 1, and Figure 3—figure supplement 4).

We also included images for monocultures of the strains used in our crosses under sex-inducing conditions, confirming that none of them is capable of selfing and thus excluding the possibility that the observed process is unisexual reproduction (Figures 1 and Figure 3—figure supplement 1).

Moreover, our microscopic observation of fluorescently labelled strains of opposite mating type undergoing sexual reproduction provides additional evidence that hyphae containing both parental nuclei experience nuclear mis-segration at branch points, resulting in more distal hyphal compartments and basidia containing only one or the other parental nucleus.

Taken together, these findings exclude unisexual reproduction as an explanation for our observations of an unusual sexual cycle involving two parents but giving rise to clonal progeny with nuclear genomes related to only one parental nuclear genotype.

We revised our manuscript accordingly and hope that the editor and reviewers find these revisions satisfactory.

2) The reviewers suggest a series of points that would help improving the manuscript, making it accessible to a larger audience. In particular several of the clarifications revolve around one core aspect: is this hybridogenesis, or is it a new mechanism of sexual development for C. neoformans that more closely resembles hybridogenesis than other known forms of sexual development? To explicitly compare and contrast the features of both would be helpful.

This is a great suggestion! We revised our manuscript thoroughly and included additional discussion. As mentioned in our previous response, we now consider the phenomenon that we discovered represents a novel form of sexual reproduction that we have termed pseudosexual reproduction, which does share similarities with hybridogenesis. We have also revised the discussion to provide a detailed comparison of the two processes, pseudosexual reproduction and hybridogenesis.

3) It is important to clarify what is supported by direct evidence and what is not (i.e endoreplication?

We revised the manuscript accordingly to explicitly state the results and hypothesis. While our experiments are consistent with and suggestive of endoreplication, we do not have direct evidence to state this unequivocally. We revised the text to clarify that the presence of endoreplication currently is only a hypothesis. While we do think endoreplication is likely occurring during pseudosexual reproduction, given that our molecular studies provide evidence of meiosis resulting in the production of four spore chains, other mechanisms are possible and assessing these requires further investigation, which is beyond the scope of the current study.

4) Only a minimal set of experiments are suggested, particularly related to rule out unisexual reproduction, and to clearly support the uniparental inheritance of mt, while also providing clearer information on datasets (i.e germination rate H99alpha x IUM96a).

We thank the editor and reviewers for the opportunity to revise and resubmit the manuscript. As mentioned above, we have provided additional data to rule out unisexual reproduction and definitively supporting uniparental mitochondrial inheritance (Figures 1, 4, Figure 3—figure supplement 1, and Figure 3—figure supplement 4). We also revised the discussion to include more details as suggested by the reviewers, for example with respect to germination rates.

Reviewer #1 (Recommendations for the authors):

1) The authors state that "this process of uniparental sexual reproduction of C. neoformans exhibits striking parallels with hybridogenesis in animals". Yet, they don't explicitly comment what are the features/issues that would preclude them from claiming that the process is fully comparable to hybridogenesis.

Moreover, later on (page 10, line 19) they appear to indicate that the characterized phenomenon is, actually, hybridogenesis: "The discovery of hybridogenesis further diversifies known reproductive modes in fungi, suggesting the presence of sexual parasitism in this kingdom".

Thus, it is unclear throughout the text whether all the requirements to define this process as bona fide hybridogenesis are met or not.

We appreciate the reviewer’s comment. As mentioned in responses to previous comments from the other reviewers and editor, we now consider the mode of reproduction discovered in our study as a novel form of sexual reproduction and term it as pseudosexual reproduction, which resembles hybridogenesis. We have revised the manuscript accordingly and included a detailed comparison between pseudosexual reproduction and hybridogenesis. We hope the reviewer finds our revision now satisfactory.

2) An important aspect of hybridogenesis that is not explicitly discussed in the manuscript either, relates to the fact that hybridogenesis is normally defined as "an unusual form of reproduction that is found in hybrids between different species" (i.e PMID: 30721675). Yet, in the present manuscript the authors obtain their data in crosses between isolates that are part of the same species (albeit with enough genetic differences to clearly differentiate their nuclei, but below the threshold of species differences). This is not acknowledged or discussed in the text.

Moreover, the authors have recently published fine work in the context of hybrids between C. neoformans and C. deneoformans, and how species boundary (and conventional sexual reproduction) are stablished (PMID: 33465111). Indeed, it is quite relevant that such hybrids can be regularly identified from both environmental and clinical samples, in some cases representing a large percentage of the isolates (~20% of the isolates identified in Europe, with even higher prevalence in some EU countries -i.e. Portugal 31%- PMID: 32511709). Detecting hybridogenesis in such context would be a major thing, and would help putting the work into perspective. Indeed, it appears that hybridogenesis tends to occur between a hybrid and another species (related to the hybrid) which genome is then discarded. Thus, the reports of hybridogenesis in several animal species suggest that its origin may imply diverse hybridization events involving intergeneric paternal ancestors. None of these complexities and peculiarities are commented or discussed. Albeit hybridogenesis is not as "popular" as other unusual reproduction modes (i.e. Parthenogenesis), its existence in fungi would be a relevant finding, which would be better appreciated by a general audience if the basics of hybridogenesis are clearly stated, and a checklist of its basics is completed, based on the findings in C. neoformans.

We thank the reviewer for a great suggestion! We have revised our manuscript accordingly. Specifically, we made it clear that the described phenomenon is not yet reported in hybrids of Cryptococcus neoformans and Cryptococcus deneoformans and represents a novel form of reproduction that resembles hybridogenesis. We also provided a detailed comparison between pseudosexual reproduction and hybridogenesis as suggested by the reviewer. We are currently expanding this work to study whether pseudosexual reproduction occurs during sexual reproduction of other Cryptococcus species, as well as during hybridization between different species.

3) It would be valuable that the authors would extend their discussions to other members of the Cryptococcus neoformans species complex. Indeed, the current evidence indicates that C. neoformans exhibits bisexual reproduction, whereas in C. deneoformans displays both bisexual and unisexual mating. Therefore, it would be interesting to see whether hybridogenesis appears as a peculiarity of C. neoformans, or as general property of the species complex (and particularly in the context of hybrids, see previous point). Likewise, the same question could be extended to the six members of the Cryptococcus gattii species complex. Nevertheless, as conducting those experiments would involve massive amount of work, the authors could expand the discussion of the topic (somehow covered already in page 11). In this context the authors could explicitly discuss whether they expect hybridogenesis to be a phenomenon that was acquired in C. neoformans, or a property that was lost in other members of the Cryptococcus species complex.

We are currently studying the occurrence of this new mode of reproduction in other species of Cryptococcus. We aim to conduct those experiments and answer some of the questions raised here in the next study. While we did not include any data from these experiments in this study, we added relevant discussion in the revised manuscript. We also included a model figure (Figure 6A) to differentiate between unisexual and pseudosexual reproduction along with a more thorough comparison in the revised manuscript.

4) Page 6, Line 33: "Nuclear segregation can be followed by endoreplication occurring in these hyphal branches or in the basidia to produce a diploid nucleus that then ultimately undergoes meiosis and produces uniparental progeny"

It is not clear if with this phrase the authors are stating a possibility or a fact. What is the evidence that endoreplication is occurring?

Related to this, it is interesting that endoreplication has been observed as a one of the mechanisms of unisexual mating in C. deneoformans.

While we suspect the occurrence of endoreplication after nuclear segregation, we do not have concrete evidence to support this. Thus, we revised the manuscript suggesting endoreplication as a possible mechanism, which requires further study. As the reviewer mentioned, the occurrence of endoreplication in C. deneoformans during unisexual reproduction raises the possibility that C. neoformans might similarly undergo endoreplication during pseudosexual reproduction. We included this possibility in the revised manuscript.

5) Page 8, Line 33: "Congruent with the mating-type analysis, the progeny exclusively inherited nuclear genetic material from only one of the two parents".

Please specify which one.

The referred statement is about VYD135α x IUM96a cross where we observed inheritance of MATα nuclei in progeny from all of the basidia, with exception of one in which all of the progeny inherited the nuclear genome from the MATa parent. Given that, none of the parental strains is capable of selfing; our data suggest pseudosexual reproduction can involve either of the two parental nuclei to produce uniparental progeny. We revised the text to make this point clearer.

6) Page 10, Line 7: "These results combined with spore dissection data show that Dmc1 is critical for uniparental sporulation". Authors could enrich the phrase by explicitly comparing the requirement of meiotic recombinase-based mechanisms in animal hybridogenesis.

This is a great suggestion! We conducted a thorough search in the literature on studies of hybridogenesis. While we could not find any study suggesting the requirement for a meiotic recombinase, we did find previous studies showing synaptonemal complex formation as evidence of meiosis during hybridogenesis. We added discussion of these studies in the revised manuscript.

Page 11, Line 23: "As a result, hybridogenesis would result in the generation of predominantly progeny in nature reducing the MATa population and thus favoring the expansion of the mating-type population" While this is an interesting idea, it is also hard to ignore the fact that the hybridogenesis process described by the authors is rare (approx. 1 %) compared to classic sexual reproduction. Therefore, it would contribute to a minor fraction of the reproductive events occurring in nature, unless some environmental conditions facilitate the former mode over the latter.

We agree with the reviewer that 1% of sporulation events under normal conditions might not be considered to be of significance. However, we discuss a few hypothetical scenarios where even a 1% successful sporulation rate could be of great advantage as compared to sexual reproduction events (Figure 6B). Unfortunately, Cryptococcus mating occurs under highly specific conditions restricting testing of variable environmental conditions. At the same time, we cannot rule out that there might be some yet-to-be-identified natural conditions that facilitate pseudosexual reproduction and might even promote it.

Reviewer #3 (Recommendations for the authors):

Thank you to the authors for the opportunity to review their excellent and interesting work! I have several recommendations for the authors to consider.

1) This is an exciting discovery that could be of broad interest, but it is difficult to keep up with what the authors are trying to communicate in this manuscript. Specifically, the terminology to refer to the various forms of sexual development and spore production (i.e. uniparental sporulation, uniparental nuclear inheritance, bisexual reproduction, unisexual sporulation, etc.) are not well defined and used inconsistently. The challenge is confounded by the (necessary) inclusion in the mix of "uniparental mitochondrial inheritance." The authors could improve the manuscript substantially by carefully defining and consistently using intentional labels for the numerous, relevant processes (including "mating," which is best used when referring to only the fusion process and not all of sexual development). It will help readers understand their beautiful data.

We thank the reviewer for their very useful comment. As recommended, we have removed multiple terms from the manuscript and defined the ones that we have retained. We also revised our manuscript thoroughly to make sure that we use the same term for a defined process consistently, thus avoiding multiple terms. We hope the reviewer will find these revisions satisfactory.

2) A second point of possible confusion is the use of "Germination Rate" to refer to the proportion of spores that can germinate into viable progeny. In the case of spores, it is probably more accurate to refer to this as "Germination Frequency" (high vs. low). Germination Rate is perhaps better used to indicate the efficiency with which any given spore differentiates into a yeast over time (slow vs. fast).

We modified the text as suggested by the reviewer.

3) The manuscript would also benefit from careful editing so that all figures are referenced in the text, figure legends and the text provide the same information, and the prose all makes sense. Some specific problems are indicated below.

We revised the manuscript to include all necessary details and referred to all of the data presented.

4) With respect to the mitochondrial inheritance data, Cryptococcus researchers likely know how one determines the parental source of the mitochondria in F1 progeny, but others may not. It is also challenging because all of the progeny (with only one exception) harbor "a"-derived mitochondria. It is a somewhat annoying but necessary question: How do you know your primers can discern between "a" and α-derived mitochondria? What is the basis for this discrimination? What if the primers were mislabeled – would you know? More information and context in the manuscript would help resolve any doubts.

We included additional and clearer details on genotyping of mitochondrial DNA in the revised manuscript. We have provided the schemes of alleles that were employed to discern MATα mitochondria versus MATa mitochondria (Figures 1 and Figure 3—figure supplement 1). The maps also include the locations of primers to indicate the length of amplicons. Furthermore, we included images of the PCR assay to show the controls and results from our genotyping data.

5) After Figure 2, there is a leap and a disconnect. The authors show clear evidence of one nuclear loss event at a hyphal branch, supporting their model. Then they indicate that endoreplication and meiosis can occur but do not provide any data for this. Then the manuscript addresses the presence or absence of meiotic recombination and the role of DMC1 in uniparental nuclear inheritance. It is not clear how this transition is made – particularly if one is outside the Cryptococcus field and does not know about the events that occur during unisexual reproduction in C. deneoformans. The absence of meiotic recombination in uniparental nuclear inheritance would make sense, but then why investigate a meiotic recombinase subsequent to that? Providing context for the DMC1 experiments would be extremely useful, as would including an interpretation of the findings in the Discussion.

We revised the manuscript to alter the flow of information in the Results section. We now first show results suggesting the occurrence of uniparental nuclear inheritance in lab strains. Then we show the occurrence of the same phenomenon in the natural isolates along with the evidence that two parental genomes do not mix with each other in the generation of the uniparental progeny. Then, we delve into the mechanism that shows that the two nuclei separate during hyphal branching. In the end, we show that the meiotic recombinase DMC1 is required for the generation of uniparental progeny, providing evidence that meiosis is still a key part of the uniparental nuclear inheritance. This also suggests that the remaining nucleus undergoes endoreplication or another process of genome duplication. We included additional text to provide a better context for the Dmc1 experiments and revised the discussion accordingly.

6) Food for thought: What if loss of one parent nucleus is just a mistake? What if, at some frequency, in all forms of Cryptococcus reproduction clamp cells mess up and a nucleus gets lost (maybe more at branchpoints) but it "just happens" in ~1% of all filaments. Is that really hybridosis or akin to hybridosis? It seems a little risky to liken a very rare event that occurs during sexual development to a form of animal development that is the primary mechanism of reproduction within certain animal species. Is it then reasonable to posit that understanding the seemingly very rare uniparental nuclear inheritance process in a fungus will inform sexual evolution in larger eukaryotes? On the other hand, if the C. neoformans response to losing one of the parental nuclei is endoreplication (as the authors suggest but do not show), that seems to be more of a potential parallel.

We appreciate the reviewer’s very insightful comment. Our initial hypothesis included the possibility that this process is occurring due to some errors. However, a 1% error rate is probably too high to be explained by nuclear segregation errors or clamp cells. Therefore, we think the phenomenon observed cannot be entirely explained by these errors. If multiple errors are contributing to the phenomenon, it is important to understand the process and the causal factors. While we accept the reviewer’s point of view, we think that this is a novel reproductive process and could have significant implications for Cryptococcus biology.

We agree that this may not be hybridogenesis as such and we have revised the manuscript to suggest that this is a novel form of reproduction in Cryptococcus neoformans. However, the two processes share many similarities and we cannot rule out the possibility that studying one will help in understanding the other. Future studies will provide more clarity on the connection between these two phenomena.

We agree with the reviewer that endoreplication might be a response of C. neoformans to loss of one of the nuclei. If so, we are not sure we could call this process a result of errors alone. Also, what might seem like an error to us could be a survival strategy for C. neoformans. In other words, pseudosexual reproduction may be a well-programmed mechanism that enables C. neoformans to survive under unfavorable conditions.

We included some of these hypothetical scenarios where uniparental nuclear inheritance might be more beneficial than sexual reproduction (Figure 6B). Thus, understanding this process might provide important insights into the evolution of this human fungal pathogen.

How likely is this very low frequency event to contribute to fitness if it is occurring coincident with bisexual reproduction, as suggested by the authors? Perhaps it contributes to fitness, but perhaps not. Can such a rare event be reasonably compared to sexual parasitism?

Albeit at low frequency, pseudosexual reproduction can be beneficial in certain conditions when the two parents can initiate mating, but are not able to complete classic sexual reproduction due to genetic incompatibilities such as sequence divergence, chromosomal structural variation, or a combination of the two. We have revised the manuscript to include a discussion of multiple possible scenarios (Figure 6B and discussion). While it cannot be compared explicitly to sexual parasitism, their outcomes are similar.

Page 3, line 1: "Most organisms in nature undergo sexual reproduction between two partners of the opposite sex to produce progeny." Is it most? There are a lot of microbes in the world that don't undergo sexual reproduction as described here.

We modified the sentence as suggested by the reviewer.

Page 5, line 17: At that point, how do you know it's sexual?

We modified the text in the revised manuscript to make this point clearer.

Page 9, lines 26-30: "Dmc1 mutant unilateral crosses sporulated at a frequency of

0.4% suggesting that only one of the parental strains was producing spores (Figure 4B). When a few sporulating basidia from multiple mating spots were dissected, two different populations of basidia emerged, one with no spore germination, and the other with a high spore germination rate and uniparental DNA inheritance (Table 2)." Something here seems amiss – why are there two populations of spores? Perhaps re-phrasing could help clarify.

We revised the text to make this clearer. The two populations most likely arise because some basidia can bypass the requirement for Dmc1 while most others cannot.

Page 9, line 24: Is the presence of both MAT alleles indicative of aneuploidy? Couldn't it also be diploidy? Also, need to change "much fewer" to "many fewer."

We thank the reviewer for bringing this to our attention. The presence of both MAT alleles could be due to diploidy as well. We modified the text as suggested by the reviewer.

Is bisexual reproduction normal in nature?

We think the reviewer may be referring to the presence of bisexual reproduction in Cryptococcus neoformans with this point. While there is no direct evidence for bisexual reproduction in C. neoformans in nature, several lines of evidence suggest that this is likely occurring, at least in certain areas. First, many natural isolates are capable of undergoing sexual reproduction in the lab. Second, while there is an overall bias toward the α mating type among natural isolates, in certain geographic regions the distribution of the two mating types in the natural population is more balanced, suggesting the opportunity for sexual reproduction is present in nature. Third, population genomics studies of the strains isolated from regions with balanced mating types show clear signatures of recombination at the population level. Fourth, naturally occuring inter-species AD hybrids are diploid and most often harbor MAT alleles of opposite mating type. Finally, sexual reproduction has been shown to occur on plants, or on pigeon guano media, two common natural habitats of Cryptococcus.

There is no specific reference to Figure S2B in the manuscript.

We previously referred to this figure as “Figure S2” (now Figure 2—figure supplement 1) in the second Results section, which was meant to refer to both panels A and B. We modified the text to specifically refer to these as “Figure 3—figure supplement 1A and B” in the revised draft.

Figure 2 Legend refers to unisexual reproduction – should be uniparental? This happens several times.

We have extensively revised the manuscript to avoid any confusion arising due to different terminology. We have entirely refrained from using the term “unisexual” for the process described in this study.

Labels for which consolidation and/or definitions would be helpful:

From Title and Abstract:

Uniparental nuclear inheritance

Bisexual mating

Bisexual reproduction

Uniparental sporulation

Uniparental reproduction

From Introduction:

Sexual reproduction

Unisexual reproduction

Heterothallic sexual reproduction

Mitochondrial Uniparental Inheritance

Uniparental inheritance of nuclei

Partner-stimulated uniparental sexual reproduction

From Results:

Unusual sexual reproduction

Uniparental mitochondrial genome inheritance

Unisexual reproduction

Uniparental inheritance

Uniparental sporulation

Uniparental reproduction

Biparental sporulation

Uniparental meiosis and sporulation

Uniparental MAT inheritance

this unusual mode of unisexual reproduction occurs in nature in parallel with normal bisexual reproduction

Bilateral crosses

Unilateral crosses

We thank the reviewer for bringing this to our attention. We now revised the manuscript, reduced the terms used throughout the manuscript, and defined the ones that are being used. We really appreciate the reviewer’s effort in helping us to simplify the manuscript and clarify the text.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #1 (Recommendations for the authors):

The revised manuscript has included key recommendations previously pointed by reviewers. In particular, the authors have been more conservative in classifying the observed phenomenon as pseudosexual reproduction, which shares several features of hybridogenesis, albeit it may be too soon to classify it unequivocally us such. They also have provided additional data solidifying their conclusions about mitochondria inheritance. These findings, described for the first time in the fungal kingdom, may provide important insights for understanding the evolution of sex. Indeed, this pseudosexual reproduction (particularly related to genome exclusion followed by endoreplication and meiosis) could correspond to an ancestral form of reproduction that preceded the evolution of sexual reproduction. While the fine mechanisms allowing pseudosexual reproduction still remain undefined (i.e. endoreplication is suspected to occur, although no experiments actually confirm or deny its occurrence), the authors acknowledge the current limitations of the study.

We are once again thankful for the insightful and critical review that helped us significantly improve the clarity and presentation of the manuscript.

Reviewer #3 (Recommendations for the authors):

In the resubmitted manuscript "Uniparental nuclear inheritance following bisexual mating in fungi," the authors offer a thorough and thoughtful response to reviewer criticisms and extensive revisions to the paper. Through their efforts, the manuscript is substantially improved – offering a more accurate presentation of the data, more clarity in the data presentation, and consideration of points that will make the findings more accessible to a broad audience of researchers.

The authors were particularly responsive to the criticism that the term hybridogenesis might not apply to their findings. They modified the manuscript accordingly by providing pivotal evidence to rule out unisexual reproduction, engaging in a richer consideration of alternative hypotheses, and clarifying/unifying their use of language to refer to sexual processes. As a result, the data are more clear, and the arguments are more compelling.

We would like to extend our sincere thanks to the reviewers for their insights and thoughtful suggestions. Their comments helped us in significantly improving the manuscript and making it both clearer and, we think, more compelling.

Points for consideration:

Figure 4: It is still unclear how many times the authors determined that nuclear loss occurred at a branchpoint during hyphal growth in pseudosexual reproduction. I recognize that capturing these events is difficult, so a reference to the images shown as being representative of what is seen in Figure 4 and a general accounting of the number of times this pattern has been observed would be useful (in text and/or legend).

We have now revised the text to include specific details and modified the paragraph accordingly. The paragraph now reads:

“…Unfortunately, a majority of the hyphae (>30 independent hyphae) we tracked were embedded into the agar, and most of these could not be tracked to the point of branching. For some others, we were able to image the hyphal branching point where two nuclei separate from each other but were then either broken or did not have mature basidia on them (Figure 4—figure supplement 1B). In total, we observed seven events of nuclear loss at hyphal branching in independent experiments and were able to track two of them to observe sporulation or basidia formation at the tip. We also observed long hyphae with only one parental nucleus in the VYD135α x Bt63a cross as well, suggesting the mechanism might be similar between strains.”

Figure 6: In terms of understanding the mechanism(s) by which pseudosexual reproduction could occur and influence fitness, Figure 6A could be eliminated (and referred to in supplemental). Figure 6B could be retained as simply Figure 6 and be referred to in the Discussion.

We modified the figure as suggested by the reviewer. The original Figure 6A is now included as Figure 6—figure supplement 1 and the original figure 6B is now main Figure 6.

Throughout text:

Clarity of the manuscript could be improved even further by using the term "mating" to refer only to the fusion event between parents (the first step of sexual development) and not to the entire process of development.

This is a great suggestion. We modified the text to only use the term “mating” for cell-cell fusion. Everywhere else, the term has been modified according to the reviewer’s suggestions provided in the marked PDF file.

Reviewer #4 (Recommendations for the authors):

The authors have responded satisfactorily to the comments from the previous review.

I have some additional suggestions for the authors:

1. The title might be improved to indicate that the uniparental nuclear genome inheritance enables nuclear-mitochondrial genome swapping, which the authors both demonstrate and is a key piece of the mechanistic argument.

We thank the reviewer for highlighting this point. While it is indeed the case that pseudosexual reproduction enables nuclear-mitochondrial genome exchange in the MATα progeny, it does not do so in the MATa progeny. Thus, we added a sentence in the abstract to highlight this point, but have kept the same title in the revised manuscript. The modified abstract now reads as follows:

“…Pseudosexual reproduction was also detected in natural isolate crosses where it resulted in mainly MATα progeny, a bias observed in Cryptococcus ecological distribution as well. The mitochondria in these progeny were inherited from the MATa parent, resulting in nuclear-mitochondrial genome exchange….”

2. The authors may want to examine more cases of the observed phenomenon before concluding that a particular mating type is the favored result of the pseudo sexual process. In other words, it may not be mating type but rather nuclear genotype more generally that determines which nucleus is lost during dikaryotic development.

We have modified the text to clarify that this is a possible hypothesis and not a conclusion. The text now reads:

“…We hypothesize that MATa isolates may be defective in this process due to either a variation in their genomes or some other as yet undefined sporulation factor. As a result, pseudosexual reproduction could lead to the generation of predominantly α progeny in nature, reducing the MATa population and thus favoring the expansion of the α mating-type population. However, it is still possible that the preferential inheritance of the nuclear genome from one of the two parents is decided by genetic elements located in regions other than MAT, and whether the uniparental nuclear inheritance is mating-type specific remains to be elucidated….”

3. The authors may want to compare their observations with cytoduction in S. cerevisiae.

We thank the reviewer for this excellent suggestion. We added a paragraph in the discussion about cytoduction that reads as follows:

“One of the key differences between pseudosexual reproduction and unisexual reproduction observed in the Cryptococcus species complex is the inheritance of mitochondrial DNA. While both unisexual and pseudosexual reproduction result in clonal progeny with respect to the nuclear genome, the mitochondria in pseudosexual reproduction are almost exclusively inherited from the MATa parent (Figure 6—figure supplement 1). This results in the exchange of mitochondrial DNA in the progeny that inherit the MATα nuclear genome, resembling the nuclear-mitochondrial exchange observed during cytoduction in Saccharomyces cerevisiae. During cytoduction, mutants defective in nuclear fusion produce haploid progeny with nuclear genome from one parent, but a mixture of both parents cytoplasm resulting in the inheritance of one parental mitochondrial genome with the other parent’s nuclear genome (Conde and Fink, 1976; Lancashire and Mattoon, 1979; Zakharov and Yarovoy, 1977). This process was used to study mitochondrial genetics with respect to the transfer of drug-resistance genes and other mitochondrial mutations. Similar to cytoduction, pseudosexual reproduction could be employed to study mitochondrial genetics, such as functional analysis of mitochondrial encoded drug resistance, and cytoplasmic inheritance of factors such as prions in C. neoformans.”

https://doi.org/10.7554/eLife.66234.sa2

Article and author information

Author details

  1. Vikas Yadav

    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2650-9035
  2. Sheng Sun

    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States
    Contribution
    Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2895-1153
  3. Joseph Heitman

    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    heitm001@duke.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6369-5995

Funding

National Institute of Allergy and Infectious Diseases (AI50113-16)

  • Joseph Heitman

National Institute of Allergy and Infectious Diseases (AI39115-24)

  • Joseph Heitman

National Institute of Allergy and Infectious Diseases (AI33654-04)

  • Joseph Heitman

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors thank Shelby Priest and Arti Dumbrepatil for the critical reading of this manuscript. This study was supported by NIH/NIAID R01 award AI39115-24, R01 grant AI50113-16 awarded to JH, and R01 grant AI33654-04 awarded to JH, David Tobin, and Paul Magwene. JH is also Co-Director and Fellow of the CIFAR program Fungal Kingdom: Threats and Opportunities.

Senior Editor

  1. Patricia J Wittkopp, University of Michigan, United States

Reviewing Editor

  1. Luis F Larrondo, Pontificia Universidad Católica de Chile, Chile

Reviewer

  1. Christina Hull, University of Wisconsin-Madison, School of Medicine and Public Health, United States

Publication history

  1. Preprint posted: December 3, 2020 (view preprint)
  2. Received: January 4, 2021
  3. Accepted: July 27, 2021
  4. Accepted Manuscript published: August 2, 2021 (version 1)
  5. Version of Record published: September 2, 2021 (version 2)

Copyright

© 2021, Yadav et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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