Mating between compatible partners allows organisms to create genetic variation within populations and provides them with opportunities for adaptation to environmental change. In contrast, long-term clonal evolution should lead to the accumulation of deleterious mutations within lineages and, ultimately, to extinction (Kondrashov, 1988; Agrawal, 2001; Kaiser and Charlesworth, 2009; Keightley and Eyre-Walker, 2000; McDonald et al., 2016). Some eukaryotic groups have seemingly challenged this theory by evolving for millions of years without observable sexual reproduction. Such organisms have been referred to as ancient asexual scandals, and prominent examples of these evolutionary oddities have included the bdelloid rotifers and the arbuscular mycorrhizal fungi (AMF) (Normark et al., 2003; Judson and Normark, 1996; Sanders, 1999). In rotifers, genome diversity can be generated by reshaping chromosomes through horizontal gene transfers (Flot et al., 2013; Debortoli et al., 2016; Signorovitch et al., 2016). Other asexual mechanisms that increase genetic variability in clonal taxa include transpositions, DNA replication or inter- or intra-chromosomal rearrangements (Faino et al., 2016; Croll, 2014; Seidl and Thomma, 2014). Furthermore, the absence of observable mating does not necessarily indicate that sex is absent as, for example, several organisms long thought to be clonal were eventually found to harbor many genomic signatures of sexual reproduction within their genomes. Evidence of such cryptic sexuality includes the presence of meiosis-specific genes, mating-type loci, sex pheromones, and evidence of inter-strain recombination (Taylor et al., 2015).

AMF are ecologically successful organisms that have long been characterized as having lacked sexual reproduction for hundreds of millions of years (Sanders and Croll, 2010). These fungi form obligate symbioses with the roots of most land plants, and their presence in the soil can improve plant fitness and biodiversity (James et al., 1998; van der Heijden et al., 1998; van der Heijden et al., 2015). Their capacity to colonize early plant lineages such as liverworts and/or hornworts, and the presence of an AMF symbiotic signaling pathway in algal ancestors of land plants, are some indications that AMF-plant associations may have been established when plants colonized land (Bonfante and Selosse, 2010; Delaux et al., 2015). The AMF mycelium is unique among eukaryotes as it harbors hundreds to thousands of nuclei within one continuous cytoplasm, and there is no known life stage with one or two nuclei. Although sexual reproduction is not formally observed in AMF, these fungi contain many genomic regions with homology to those involved in mating in distant relatives (Halary et al., 2011; Riley and Corradi, 2013; Riley et al., 2014), suggesting that they are able to undergo sexual reproduction. The cellular mechanisms that could trigger sexual processes in AMF have however long been elusive.

Using genome analyses, it was recently shown that isolates of the model AMF Rhizophagus irregularis are either homokaryotic – that is co-existing nuclei harbor one putative mating-type (MAT)-locus - or dikaryotic – two populations with different nuclear genotypes coexist in the mycelium, each with a divergent MAT-locus (Ropars et al., 2016). The MAT-locus is the genomic region that gives sexual identity to fungal isolates, and isolates of the AMF genus Rhizophagus harbor a genomic location with significant similarities to the MAT-locus of basidiomycetes (Ropars et al., 2016). AMF dikaryons were proposed to originate from hyphal fusion and nuclear exchange (plasmogamy) between compatible homokaryons (Ropars et al., 2016). In dikaryotic fungi, compatible nuclei fuse (karyogamy) and undergo recombination, but there is no direct evidence yet that these sexual processes occur in AMF dikaryons. In particular, although inter-isolate recombination has been inferred to exist based on analyses of single loci (Riley et al., 2014; Croll and Sanders, 2009; Gandolfi et al., 2003; den Bakker et al., 2010), direct evidence of genetic exchange between nuclei of opposite MAT-loci co-existing within the same mycelium, as is expected for fungal mating processes, has never been reported.

Here, we tested the hypothesis that karyogamy and inter-nuclear recombination occur in the AMF mycelium using a single-nucleus sequencing approach. Sequenced nuclei were isolated from seven isolates of the genus Rhizophagus, including three known Rhizophagus irregularis dikaryons (A4, A5; SL1 (Ropars et al., 2016)), two R. irregularis homokaryons (A1, C2 (Ropars et al., 2016)), and two closely related species (Rhizophagus diaphanus MUCL-43196, Rhizophagus cerebriforme DAOM-227022). Genome data from R. irregularis SL1 (also known as DAOM-240409), R. diaphanus and R. cerebriforme represent new additions to public databases from representatives of the Glomeromycotina, and all sequencing data were assembled using SPAdes (Bankevich et al., 2012) to facilitate comparisons with published genome data (Ropars et al., 2016).

As expected for an AMF dikaryon, the SL1 assembly contains two putative MAT-loci, with a genome architecture identical to that of known R. irregularis dikaryons (Figure 1A). The isolate also harbors the high allele frequency peak at 0.5 characteristic of AMF dikaryons (Table 1, Figure 1B) (Ropars et al., 2016; Corradi and Brachmann, 2017). Despite these similarities, the SL1 assembly is more fragmented than those of all other Rhizophagus relatives (Table 1). Higher fragmentation is also seen in other known dikaryons (Table 1; Ropars et al., 2016), a feature that probably stems from a higher intra-mycelial genetic diversity in these isolates. Importantly, BUSCO, K-mer graphs and transposable elements (TE) analyses all show that all isolates analysed in this study share highly similar gene repertoires, genome sizes and TE counts (Table 2, Supplementary file 1). Thus, the higher assembly fragmentation of SL1 may result from other factors such as, for example, the presence of higher heterokaryosis and/or inter-nuclear recombination. In contrast to SL1, the R. diaphanus and R. cerebriforme assemblies harbor only one MAT-locus and show a conventional homokaryotic allele frequency pattern (Figure 1B).

Figure 1

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To find direct evidence of inter-nuclear recombination in AMF, we obtained and compared partial genome sequences from 86 single nuclei – that is single haploid genotypes – isolated from R. irregularis dikaryons (SL1, n = 15; A4, n = 14; A5, n = 8), R. irregularis homokaryons (A1, n = 12; C2, n = 9), and from the species R. diaphanus (n = 12) and R. cerebriforme (n = 15). Nuclei were isolated and analysed using a combination of fluorescence-activated cell sorting (FACS), whole genome amplification (WGA) (Ropars et al., 2016), and Illumina sequencing. Each nucleus was genotyped by mapping paired-end Illumina reads obtained from WGA-based DNA against their respective genome reference. The combination of PCR-based WGA and Illumina sequencing methods results in variation in average depth position and reference coverage among nuclei and isolates (Table 1). Using basic filtering methodologies (see Material and Methods), we find that nuclear genomes diverge from their respective reference from 0.06% for R. diaphanus, to a maximum of 0.12% for SL1 and A4 (Table 1). Using this method, pairwise nuclear genome comparisons show a high average inter-nuclear similarity ranging from 99.62% in SL1 to a maximum of 99.87% in A1 (Supplementary file 2). When present, variability is scattered across the genome (Supplementary file 3).

It is noteworthy that most attempts to validate SNP scored using basic filtering methods resulted in the identification of many false positives. Specifically, only 4% (6 out of 148 tested) of SNPs identified using the abovementioned methods were confirmed using PCR and Sanger sequencing procedures performed on the original DNA extracts. In order to maximize the detection of true positives in our study, and thus ensure the detection of bone-fide recombinants, we focused our searches for inter-nuclear diversity on the 100 largest scaffolds of each isolate, and scored SNPs only within regions devoid of repeats, manually confirmed to be single copy using reciprocal BLAST procedures, and with frequencies between 0.26–0.74. This conservative approach has likely removed some true positives from our analysis - for example this filtering method resulted in an average 93.8% decrease in total SNP counts (Supplementary file 4) - but we believe that this caveat is largely offset by a much higher SNP validation rate, that is 84%, or 30 out of 36 SNPs tested, were validated using Sanger sequencing procedures.

We selected this stringent methodology to genotype all 86 sequenced nuclei and search for evidence of inter-nucleus recombination in all seven isolates. Using this approach, we find that the total number of SNPs detected is, on average, over 30 times larger in dikaryons compared to homokaryotic relatives, with homokaryons harboring only between 9 (R. diaphanus) to 131 SNPs (R. cerebriforme) along the 100 largest scaffolds (Supplementary file 4). Removing the filtering based on allele frequencies resulted in a slightly higher SNP count, but did not alter the overall results – that is dikaryons are still far more variable than homokaryons (Supplementary file 5). Consistent with previous findings based on much shorter nuclear regions (Ropars et al., 2016), our pairwise sequence comparisons show that nuclei isolated from AMF dikaryons always segregate into two main clades, depending on their genotype (Figure 2), and that each nucleus always harbors one of two co-existing MAT-loci.

Figure 2

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We find that in A4 and A5, nuclei with the same MAT-locus (MAT-1 or MAT-2 for A4; MAT-3 or MAT-6 for A5) always harbor highly similar genotypes. This pattern results in a genetic similarity of single nuclei that is based exclusively on the MAT-locus identity (Figure 2A,B), and we find no evidence that nuclei with opposing MAT-loci cluster together in these isolates. In SL1, nuclei also segregate into two dominant clades, a finding consistent with its dikaryotic state. However, obvious links between the MAT-locus identity of its nuclei and their genotype are not always evident in this isolate (Figure 2C). For example, the nucleus 7 of SL1 harbors the MAT-5 locus, but also shows significant sequence similarity with nuclei harboring the MAT-1 locus (e.g. nucleus 8 and 12). This finding, which we confirmed using Sanger sequencing approaches (see coloured boxes in Figure 2), indicates the presence of inter-nuclear recombination in this isolate, potentially involving the MAT-locus. Other nuclei in SL1 show a similar pattern, as evidenced by the distinct mosaic genotypes of this isolate (Figure 2C). Overall, the genetic make-up of SL1 contrasts with the clear bi-allelic genetic structure of the isolates A4 and A5 (Figure 2A,B), and highlights the higher nuclear genotypic diversity harbored by this isolate.

A detailed look at individual nuclear genotypes highlights evidence of inter-nuclear recombination across the genome (Figure 3, Supplementary file 6). Examples of recombination tracts include those seen in the SL1 nuclei 14, and 15, which all share the MAT-1 locus but can sometimes carry partial genotypes found in co-existing MAT-5 nuclei, and vice versa (Figure 3, Supplementary file 6). This pattern of inter-nuclear genetic exchange occurs among many nuclei in this isolate, regardless of their mating-type, and across all of the largest scaffolds in SL1 (Figure 2, Figure 3, Supplementary file 6). In many cases, recombining genotypes encompass hundreds to thousands of base pairs, (Figure 3, Supplementary file 6). Inter-nuclear exchange between two co-existing genotypes are also seen, although more rarely, in A4 and to a much lesser extent in A5 – for example see scaffold 70 positions 100454 to 100557 (Figure 3, Supplementary file 6) for potential recombination in A4. In this example, a single recombination event between genotypes harbored by the nuclei 22 (MAT-1) and 19 (MAT-2) resulted in a genetic exchange involving at least one thousand base pairs, and similar events are found elsewhere in the genome of A4.

Figure 3

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We performed identical analyses using different, improved assemblies (e.g. ALLPATHS-LG; (Butler et al., 2008); Supplementary file 7) and more stringent variant callers (GATK-HaplotypeCaller (Van der Auwera, 2002), Mutect2 (Cibulskis, 2013); Supplementary file 8, Supplementary file 9), and found that these did not change the overall findings of our study. Specifically, regardless of the methods used to assemble the genome and call variants, evidence for inter-nuclear recombination is always present and more substantial in SL1 than in in A4 and A5. Accordingly, the genetic relationships among co-existing nuclei are also generally unaffected by changes in both assembly and SNP scoring methods (Supplementary files 10–12).

The Meselson effect predicts that the long-term absence of sex and recombination should lead to substantial accumulation of mutations within asexual lineages (Mark Welch and Meselson, 2000; Arkhipova and Meselson, 2005). Many asexual eukaryotes follow this prediction (Weir et al., 2016; Lovell et al., 2017; Neiman et al., 2010) but our data indicate that AMF may not. Specifically, we find that co-existing nuclei are overall very similar in sequence, showing between 0.001% to maximum of 0.38% average genome divergence, depending on the filters applied to score variation, with the vast majority of variation being in a bi-allelic state. The values obtained using basic filtering methods– that is 0.38% average nuclear genome divergence - are consistent with data obtained by others on single nuclei of the model AMF R. irregularis DAOM-197198 (Lin et al., 2014), but we also demonstrate that these are largely inflated by false positives. Future work should take this into consideration when assessing AMF genetic diversity using in-silico methodologies. Our study also shows that low nuclear polymorphism is a hallmark of many AMF species and is not only restricted to this R. irregularis DAOM-197198. It will be now interesting to see if natural AMF populations show a similarly low nuclear polymorphism.

Our analyses of single nuclei data also supports the hypothesis that essentially all fungal species have found means to recombine (Nieuwenhuis and James, 2016). In the case of AMF, our findings build on models previously proposed by Ropars et al. and Corradi and Brachmann (Ropars et al., 2016; Corradi and Brachmann, 2017), as we found evidence that nuclei with opposite MAT-loci can successfully undergo karyogamy in some dikaryotic isolates to generate new nuclear diversity through recombination (Figure 4). This discovery is unaffected by the quality of the assembly, and is supported by different methodologies used to score variation. Interestingly, in SL1, the observed inter-nuclear recombination does not appear to result from a single event – that is each nucleus shows differing recombination patterns - an indication that genetic exchanges among nuclei may have occurred independently at different locations within the same AMF multinucleated mycelium (Figure 4). In contrast, the few recombination events found in A4 involved exchange of genetic material between two co-existing and highly conserved genotypes.

Figure 4

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In fungi, diploid nuclei can undergo recombination either through meiosis, in a sexual cycle, or through aneuploidization, in a parasexual cycle involving nuclear fusion followed by random chromosomal loss. Parasexuality is rare in fungi (Paccola-Meirelles and Azevedo, 1991; Rosada et al., 2010; Seervai et al., 2013; Bennett, 2015), and this process drives recombination via mitotic events (as opposed to meiosis in a sexual cycle). It also requires the stable division of diploid nuclei for many generations to produce recombination through mitosis (Clutterbuck, 1996). To date, flow cytometry data has found that AMF nuclei are haploid (Ropars et al., 2016; Sedzielewska et al., 2011; Hosny et al., 1998) and evidence of widespread diploidy, as evidenced by the mapping of both alternate and reference reads along large portions of genome, could not be found in this study. In contrast, all AMF, including those investigated here, harbor a complete set of meiosis-related genes (Halary et al., 2011) and there is recurring evidence that conspecific isolates can exchange and recombine genetic material, presumably through sex (Riley et al., 2014; Croll and Sanders, 2009; den Bakker et al., 2010; Chen et al., 2018) . For these reasons, we argue that the inter-nuclear recombination we observed in this study is likely driven by meiotic events - that is conventional fungal sexual processes - although it is possible that parasexuality has also been at play in creating some of the observed nuclear diversity.

What causes recombination rates to vary substantially among the dikaryons SL1, A4 and A5 is presently unclear, as all cultures were established around the same time (Ropars et al., 2016; Jansa et al., 2002) and there is no available evidence that they were propagated under different in vitro conditions. Perhaps, in isolates such as A4 and A5 the co-existing genotypes are not permissive for generating recombinants (Idnurm et al., 2015) - for example the isolates have not yet met the right environmental conditions that trigger karyogamy or lack the mating compatibility necessary to undergo frequent meiosis (James et al., 2006; Heitman et al., 2013; Heitman, 2015). In this case, some of the somatic mutations and recombination found in A4 and A5 could result from exposure to mobile viral genotypes or transposable elements, as there is evidence that these are active in AMF genomes (Chen et al., 2018).

In conclusion, our findings demonstrate that arbuscular mycorrhizal fungi can generate genetic diversity via inter-nuclear recombination in the dikaryotic life-stage. This provides a novel understanding of the sexual and genetic potential of these plant symbionts and opens exciting possibilities to enhance their environmental application. In particular, we showed that recombined isolates such as SL1 contain an important source of nuclear diversity, which could lead to genetic enhancement of new AMF strains. Despite evidence of recombination, however, clonality still appears to be the prevalent mode of evolution in lab cultures of these symbionts, including for AMF dikaryons (Figure 4). From a conceptual perspective, future work should now aim to identify the mechanisms that trigger inter-nuclear recombination in AMF isolates, and determine how frequent this process is in natural populations of these widespread plant symbionts. It will also be important to establish whether recombination rates vary depending on soil conditions – for example disturbance - or specific plant hosts (Ritz et al., 2017), and how this process is reflected at the level of the transcriptome. Within this context, future investigations should also determine if specific recombined genomic regions are linked with an isolate’s phenotype (e.g. increased spore production, hyphal density, or mycorrhization rates) or with the fitness of economically important plants and crops.

Single nuclei sequencing and genome reads are available in Genbank under the following bioproject: PRJNA477348. SPAdes assemblies are publicly available on NCBI (R. diaphanus: QZLH00000000, R. cerebrifrome: QZLG00000000, SL1 SPAdes: QZCD00000000, and SL1 ALLPATH-LG: QZCC00000000) along with the core meiosis SL1 genes (RAD21/Rec8: MH974797, MND1: MH974798, DMC1: MH974799, Spo11: MH974800, HOP2: MH974801, MSH4: MH974802, MSH5: MH974803). The whole genome reads are available on NCBI SRA: R. cerebriforme (SRR7418134 and SRR7418135), R. diaphanus (SRR7418169 and SRR7418170), and R. irregularis SL1 (SRR7418171 and SRR7418172). The single nucleus reads are also deposited (R. cerebriforme: SRR7411799 to SRR7411813, R. diaphanus: SRR7410308 to SRR7410319, R. irregularis A1: SRR7416439 to SRR7416450, R. irregularis A4: SRR7416451 to SRR7416464, R. irregularis A5: SRR7416431 to SRR7416438, R. irregularis C2: SRR7416465 to SRR7416473, and R. irregularis SL1: SRR7411814 to SRR7411829).

1. 1. Chen ECH
   2. Mathieu S
   3. Hoffrichter A
   4. Sedzielewska-Toro K
   5. Peart M
   6. Pelin A
   7. Ndikumana S
   8. Ropars J
   9. Dreissig S
   10. Fuchs J
   11. Brachmann A
   12. Corradi N

   (2018) NCBI BioProject

   ID PRJNA477348. Single nuclei sequencing and genome reads from.

   https://www.ncbi.nlm.nih.gov/bioproject/PRJNA477348
2. 1. Chen ECH
   2. Mathieu S
   3. Hoffrichter A
   4. Sedzielewska-Toro K
   5. Peart M
   6. Pelin A
   7. Ndikumana S
   8. Ropars J
   9. Dreissig S
   10. Fuchs J
   11. Brachmann A
   12. Corradi N

   (2018) NCBI Nucleotide

   ID QZLH00000000. SPAdes assemblies from.

   https://www.ncbi.nlm.nih.gov/nuccore/1520279022
3. 1. Chen ECH
   2. Mathieu S
   3. Hoffrichter A
   4. Sedzielewska-Toro K
   5. Peart M
   6. Pelin A
   7. Ndikumana S
   8. Ropars J
   9. Dreissig S
   10. Fuchs J
   11. Brachmann A
   12. Corradi N

   (2018) NCBI Nucleotide

   ID QZLG00000000. SPAdes assemblies from.

   https://www.ncbi.nlm.nih.gov/nuccore/QZLG00000000.1/
4. 1. Chen ECH
   2. Mathieu S
   3. Hoffrichter A
   4. Sedzielewska-Toro K
   5. Peart M
   6. Pelin A
   7. Ndikumana S
   8. Ropars J
   9. Dreissig S
   10. Fuchs J
   11. Brachmann A
   12. Corradi N

   (2018) NCBI Nucleotide

   ID QZCD00000000. SPAdes assemblies from.

   https://www.ncbi.nlm.nih.gov/nuccore/QZCD00000000.1/
5. 1. Chen ECH
   2. Mathieu S
   3. Hoffrichter A
   4. Sedzielewska-Toro K
   5. Peart M
   6. Pelin A
   7. Ndikumana S
   8. Ropars J
   9. Dreissig S
   10. Fuchs J
   11. Brachmann A
   12. Corradi N

   (2018) NCBI Nucleotide

   ID QZCC00000000. SPAdes assemblies from.

   https://www.ncbi.nlm.nih.gov/nuccore/QZCC00000000
6. 1. Chen ECH
   2. Mathieu S
   3. Hoffrichter A
   4. Sedzielewska-Toro K
   5. Peart M
   6. Pelin A
   7. Ndikumana S
   8. Ropars J
   9. Dreissig S
   10. Fuchs J
   11. Brachmann A
   12. Corradi N

   (2018) NCBI Nucleotide

   ID MH974797. Core meiosis SL1 genes from.

   https://www.ncbi.nlm.nih.gov/nuccore/MH974797.1/
7. 1. Chen ECH
   2. Mathieu S
   3. Hoffrichter A
   4. Sedzielewska-Toro K
   5. Peart M
   6. Pelin A
   7. Ndikumana S
   8. Ropars J
   9. Dreissig S
   10. Fuchs J
   11. Brachmann A
   12. Corradi N

   (2018) NCBI Nucleotide

   ID MH974798. Core meiosis SL1 genes from.

   https://www.ncbi.nlm.nih.gov/nuccore/MH974798
8. 1. Chen ECH
   2. Mathieu S
   3. Hoffrichter A
   4. Sedzielewska-Toro K
   5. Peart M
   6. Pelin A
   7. Ndikumana S
   8. Ropars J
   9. Dreissig S
   10. Fuchs J
   11. Brachmann A
   12. Corradi N

   (2018) NCBI Nucleotide

   ID MH974799. Core meiosis SL1 genes from.

   https://www.ncbi.nlm.nih.gov/nuccore/MH974799
9. 1. Chen ECH
   2. Mathieu S
   3. Hoffrichter A
   4. Sedzielewska-Toro K
   5. Peart M
   6. Pelin A
   7. Ndikumana S
   8. Ropars J
   9. Dreissig S
   10. Fuchs J
   11. Brachmann A
   12. Corradi N

   (2018) NCBI Nucleotide

   ID MH974800. Core meiosis SL1 genes from.

   https://www.ncbi.nlm.nih.gov/nuccore/MH974800
10. 1. Chen ECH
    2. Mathieu S
    3. Hoffrichter A
    4. Sedzielewska-Toro K
    5. Peart M
    6. Pelin A
    7. Ndikumana S
    8. Ropars J
    9. Dreissig S
    10. Fuchs J
    11. Brachmann A
    12. Corradi N

    (2018) NCBI Nucleotide

    ID MH974801. Core meiosis SL1 genes from.

    https://www.ncbi.nlm.nih.gov/nuccore/MH974801
11. 1. Chen ECH
    2. Mathieu S
    3. Hoffrichter A
    4. Sedzielewska-Toro K
    5. Peart M
    6. Pelin A
    7. Ndikumana S
    8. Ropars J
    9. Dreissig S
    10. Fuchs J
    11. Brachmann A
    12. Corradi N

    (2018) NCBI Nucleotide

    ID MH974802. Core meiosis SL1 genes from.

    https://www.ncbi.nlm.nih.gov/nuccore/MH974802
12. 1. Chen ECH
    2. Mathieu S
    3. Hoffrichter A
    4. Sedzielewska-Toro K
    5. Peart M
    6. Pelin A
    7. Ndikumana S
    8. Ropars J
    9. Dreissig S
    10. Fuchs J
    11. Brachmann A
    12. Corradi N

    (2018) NCBI Nucleotide

    ID MH974803. Core meiosis SL1 genes from.

    https://www.ncbi.nlm.nih.gov/nuccore/MH974803

1. 1. Ropars J
   2. Toro KS
   3. Noel J
   4. Pelin A
   5. Charron P
   6. Farinelli L
   7. Marton T
   8. Krüger M
   9. Fuchs J
   10. Brachmann A
   11. Corradi N

   (2016) NCBI Nucleotide

   ID LLXH00000000. Rhizophagus irregularis strain A1, whole genome shotgun sequencing project.

   https://www.ncbi.nlm.nih.gov/nuccore/LLXH00000000.1/
2. 1. Ropars J
   2. Toro KS
   3. Noel J
   4. Pelin A
   5. Charron P
   6. Farinelli L
   7. Marton T
   8. Krüger M
   9. Fuchs J
   10. Brachmann A
   11. Corradi N

   (2016) NCBI Nucleotide

   ID LLXI00000000. Rhizophagus irregularis strain A4, whole genome shotgun sequencing project.

   https://www.ncbi.nlm.nih.gov/nuccore/LLXI00000000.1/
3. 1. Ropars J
   2. Toro KS
   3. Noel J
   4. Pelin A
   5. Charron P
   6. Farinelli L
   7. Marton T
   8. Krüger M
   9. Fuchs J
   10. Brachmann A
   11. Corradi N

   (2016) NCBI Nucleotide

   ID LLXJ00000000. Rhizophagus irregularis strain B3, whole genome shotgun sequencing project.

   https://www.ncbi.nlm.nih.gov/nuccore/LLXK00000000.1/
4. 1. Ropars J
   2. Toro KS
   3. Noel J
   4. Pelin A
   5. Charron P
   6. Farinelli L
   7. Marton T
   8. Krüger M
   9. Fuchs J
   10. Brachmann A
   11. Corradi N

   (2016) NCBI Nucleotide

   ID LLXK00000000. Rhizophagus irregularis strain C2, whole genome shotgun sequencing project.

   https://www.ncbi.nlm.nih.gov/nuccore/LLXL00000000.1/
5. 1. Ropars J
   2. Toro KS
   3. Noel J
   4. Pelin A
   5. Charron P
   6. Farinelli L
   7. Marton T
   8. Krüger M
   9. Fuchs J
   10. Brachmann A
   11. Corradi N

   (2016) NCBI Nucleotide

   ID LLXL00000000. Rhizophagus irregularis strain A5, whole genome shotgun sequencing project.

   https://www.ncbi.nlm.nih.gov/nuccore/LLXM00000000.1/

1. 1. Agrawal AF

   (2001) Sexual selection and the maintenance of sexual reproduction

   Nature 411:692–695.

   https://doi.org/10.1038/35079590

   • PubMed
   • Google Scholar
2. 1. Arkhipova I
   2. Meselson M

   (2005) Deleterious transposable elements and the extinction of asexuals

   BioEssays 27:76–85.

   https://doi.org/10.1002/bies.20159

   • PubMed
   • Google Scholar
3. 1. Bankevich A
   2. Nurk S
   3. Antipov D
   4. Gurevich AA
   5. Dvorkin M
   6. Kulikov AS
   7. Lesin VM
   8. Nikolenko SI
   9. Pham S
   10. Prjibelski AD
   11. Pyshkin AV
   12. Sirotkin AV
   13. Vyahhi N
   14. Tesler G
   15. Alekseyev MA
   16. Pevzner PA

   (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing

   Journal of Computational Biology 19:455–477.

   https://doi.org/10.1089/cmb.2012.0021

   • PubMed
   • Google Scholar
4. 1. Bennett RJ

   (2015) The parasexual lifestyle of Candida albicans

   Current Opinion in Microbiology 28:10–17.

   https://doi.org/10.1016/j.mib.2015.06.017

   • PubMed
   • Google Scholar
5. 1. Boetzer M
   2. Henkel CV
   3. Jansen HJ
   4. Butler D
   5. Pirovano W

   (2011) Scaffolding pre-assembled contigs using SSPACE

   Bioinformatics 27:578–579.

   https://doi.org/10.1093/bioinformatics/btq683

   • PubMed
   • Google Scholar
6. 1. Bonfante P
   2. Selosse MA

   (2010) A glimpse into the past of land plants and of their mycorrhizal affairs: from fossils to evo-devo

   New Phytologist 186:267–270.

   https://doi.org/10.1111/j.1469-8137.2010.03196.x

   • PubMed
   • Google Scholar
7. 1. Butler J
   2. MacCallum I
   3. Kleber M
   4. Shlyakhter IA
   5. Belmonte MK
   6. Lander ES
   7. Nusbaum C
   8. Jaffe DB

   (2008) ALLPATHS: de novo assembly of whole-genome shotgun microreads

   Genome Research 18:810–820.

   https://doi.org/10.1101/gr.7337908

   • PubMed
   • Google Scholar
8. 1. Chen ECH
   2. Morin E
   3. Beaudet D
   4. Noel J
   5. Yildirir G
   6. Ndikumana S
   7. Charron P
   8. St-Onge C
   9. Giorgi J
   10. Krüger M
   11. Marton T
   12. Ropars J
   13. Grigoriev IV
   14. Hainaut M
   15. Henrissat B
   16. Roux C
   17. Martin F
   18. Corradi N

   (2018) High intraspecific genome diversity in the model arbuscular mycorrhizal symbiont Rhizophagus irregularis

   New Phytologist 220:1161–1171.

   https://doi.org/10.1111/nph.14989

   • Google Scholar
9. 1. Cibulskis K

   (2013) MuTect - brief summary

   Nature Biotechnology 31:213–219.

   https://doi.org/10.1038/nbt.2514

   • Google Scholar
10. 1. Clutterbuck AJ

    (1996) Parasexual recombination in fungi

    Journal of Genetics 75:281–286.

    https://doi.org/10.1007/BF02966308

    • Google Scholar
11. 1. Corradi N
    2. Brachmann A

    (2017) Fungal mating in the most widespread plant symbionts?

    Trends in Plant Science 22:175–183.

    https://doi.org/10.1016/j.tplants.2016.10.010

    • PubMed
    • Google Scholar
12. 1. Croll D
    2. Sanders IR

    (2009) Recombination in Glomus intraradices, a supposed ancient asexual arbuscular mycorrhizal fungus

    BMC Evolutionary Biology 9:13.

    https://doi.org/10.1186/1471-2148-9-13

    • PubMed
    • Google Scholar
13. 1. Croll D

    (2014) Learn from the fungi: adaptive evolution without sex in fungal pathogens (comment on DOI 10.1002/bies.201300155)

    BioEssays 36:334.

    https://doi.org/10.1002/bies.201400030

    • PubMed
    • Google Scholar
14. 1. Debortoli N
    2. Li X
    3. Eyres I
    4. Fontaneto D
    5. Hespeels B
    6. Tang CQ
    7. Flot JF
    8. Van Doninck K

    (2016) Genetic exchange among bdelloid rotifers is more likely due to horizontal gene transfer than to meiotic sex

    Current Biology 26:723–732.

    https://doi.org/10.1016/j.cub.2016.01.031

    • PubMed
    • Google Scholar
15. 1. Delaux PM
    2. Radhakrishnan GV
    3. Jayaraman D
    4. Cheema J
    5. Malbreil M
    6. Volkening JD
    7. Sekimoto H
    8. Nishiyama T
    9. Melkonian M
    10. Pokorny L
    11. Rothfels CJ
    12. Sederoff HW
    13. Stevenson DW
    14. Surek B
    15. Zhang Y
    16. Sussman MR
    17. Dunand C
    18. Morris RJ
    19. Roux C
    20. Wong GK
    21. Oldroyd GE
    22. Ané JM

    (2015) Algal ancestor of land plants was preadapted for symbiosis

    PNAS 112:13390–13395.

    https://doi.org/10.1073/pnas.1515426112

    • PubMed
    • Google Scholar
16. 1. den Bakker HC
    2. Vankuren NW
    3. Morton JB
    4. Pawlowska TE

    (2010) Clonality and recombination in the life history of an asexual arbuscular mycorrhizal fungus

    Molecular Biology and Evolution 27:2474–2486.

    https://doi.org/10.1093/molbev/msq155

    • PubMed
    • Google Scholar
17. 1. Faino L
    2. Seidl MF
    3. Shi-Kunne X
    4. Pauper M
    5. van den Berg GC
    6. Wittenberg AH
    7. Thomma BP

    (2016) Transposons passively and actively contribute to evolution of the two-speed genome of a fungal pathogen

    Genome Research 26:1091–1100.

    https://doi.org/10.1101/gr.204974.116

    • PubMed
    • Google Scholar
18. 1. Flot JF
    2. Hespeels B
    3. Li X
    4. Noel B
    5. Arkhipova I
    6. Danchin EG
    7. Hejnol A
    8. Henrissat B
    9. Koszul R
    10. Aury JM
    11. Barbe V
    12. Barthélémy RM
    13. Bast J
    14. Bazykin GA
    15. Chabrol O
    16. Couloux A
    17. Da Rocha M
    18. Da Silva C
    19. Gladyshev E
    20. Gouret P
    21. Hallatschek O
    22. Hecox-Lea B
    23. Labadie K
    24. Lejeune B
    25. Piskurek O
    26. Poulain J
    27. Rodriguez F
    28. Ryan JF
    29. Vakhrusheva OA
    30. Wajnberg E
    31. Wirth B
    32. Yushenova I
    33. Kellis M
    34. Kondrashov AS
    35. Mark Welch DB
    36. Pontarotti P
    37. Weissenbach J
    38. Wincker P
    39. Jaillon O
    40. Van Doninck K

    (2013) Genomic evidence for ameiotic evolution in the bdelloid rotifer Adineta vaga

    Nature 500:453–457.

    https://doi.org/10.1038/nature12326

    • PubMed
    • Google Scholar
19. 1. Gandolfi A
    2. Sanders IR
    3. Rossi V
    4. Menozzi P

    (2003) Evidence of recombination in putative ancient asexuals

    Molecular Biology and Evolution 20:754–761.

    https://doi.org/10.1093/molbev/msg081

    • PubMed
    • Google Scholar
20. Preprint

    1. Garrison E
    2. Marth G

    (2012) Haplotype-based variant detection from short-read sequencing

    arXiv.

    https://arxiv.org/abs/1207.3907

    • Google Scholar
21. 1. Halary S
    2. Malik SB
    3. Lildhar L
    4. Slamovits CH
    5. Hijri M
    6. Corradi N

    (2011) Conserved meiotic machinery in Glomus spp., a putatively ancient asexual fungal lineage

    Genome Biology and Evolution 3:950–958.

    https://doi.org/10.1093/gbe/evr089

    • PubMed
    • Google Scholar
22. 1. Heitman J
    2. Sun S
    3. James TY

    (2013) Evolution of fungal sexual reproduction

    Mycologia 105:1–27.

    https://doi.org/10.3852/12-253

    • PubMed
    • Google Scholar
23. 1. Heitman J

    (2015) Evolution of sexual reproduction: a view from the Fungal Kingdom supports an evolutionary epoch with sex before sexes

    Fungal Biology Reviews 29:108–117.

    https://doi.org/10.1016/j.fbr.2015.08.002

    • PubMed
    • Google Scholar
24. 1. Hosny M
    2. Gianinazzi-Pearson V
    3. Dulieu H

    (1998) Nuclear DNA content of 11 fungal species in Glomales

    Genome 41:422–428.

    https://doi.org/10.1139/g98-038

    • Google Scholar
25. 1. Idnurm A
    2. Hood ME
    3. Johannesson H
    4. Giraud T

    (2015) Contrasted patterns in mating-type chromosomes in fungi: hotspots versus coldspots of recombination

    Fungal Biology Reviews 29:220–229.

    https://doi.org/10.1016/j.fbr.2015.06.001

    • PubMed
    • Google Scholar
26. 1. James JD
    2. Smith SE
    3. Read DJ

    (1998)

    Mycorrhizal Symbiosis

    71–72, Plant Growth Regulation, Mycorrhizal Symbiosis, Springer.

    • Google Scholar
27. 1. James TY
    2. Srivilai P
    3. Kües U
    4. Vilgalys R

    (2006) Evolution of the bipolar mating system of the mushroom Coprinellus disseminatus from its tetrapolar ancestors involves loss of mating-type-specific pheromone receptor function

    Genetics 172:1877–1891.

    https://doi.org/10.1534/genetics.105.051128

    • PubMed
    • Google Scholar
28. 1. Jansa J
    2. Mozafar A
    3. Banke S
    4. McDonald BA
    5. Frossard E

    (2002) Intra- and intersporal diversity of ITS rDNA sequences in Glomus intraradices assessed by cloning and sequencing, and by SSCP analysis

    Mycological Research 106:670–681.

    https://doi.org/10.1017/S0953756202006032

    • Google Scholar
29. 1. Judson OP
    2. Normark BB

    (1996) Ancient asexual scandals

    Trends in Ecology & Evolution 11:41–46.

    https://doi.org/10.1016/0169-5347(96)81040-8

    • PubMed
    • Google Scholar
30. 1. Kaiser VB
    2. Charlesworth B

    (2009) The effects of deleterious mutations on evolution in non-recombining genomes

    Trends in Genetics 25:9–12.

    https://doi.org/10.1016/j.tig.2008.10.009

    • PubMed
    • Google Scholar
31. 1. Keightley PD
    2. Eyre-Walker A

    (2000) Deleterious mutations and the evolution of sex

    Science 290:331–333.

    https://doi.org/10.1126/science.290.5490.331

    • PubMed
    • Google Scholar
32. 1. Kondrashov AS

    (1988) Deleterious mutations and the evolution of sexual reproduction

    Nature 336:435–440.

    https://doi.org/10.1038/336435a0

    • PubMed
    • Google Scholar
33. Preprint

    1. Li H

    (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM

    arXiv.

    https://arxiv.org/abs/1303.3997

    • Google Scholar
34. 1. Lin K
    2. Limpens E
    3. Zhang Z
    4. Ivanov S
    5. Saunders DG
    6. Mu D
    7. Pang E
    8. Cao H
    9. Cha H
    10. Lin T
    11. Zhou Q
    12. Shang Y
    13. Li Y
    14. Sharma T
    15. van Velzen R
    16. de Ruijter N
    17. Aanen DK
    18. Win J
    19. Kamoun S
    20. Bisseling T
    21. Geurts R
    22. Huang S

    (2014) Single nucleus genome sequencing reveals high similarity among nuclei of an endomycorrhizal fungus

    PLOS Genetics 10:e1004078.

    https://doi.org/10.1371/journal.pgen.1004078

    • PubMed
    • Google Scholar
35. 1. Lovell JT
    2. Williamson RJ
    3. Wright SI
    4. McKay JK
    5. Sharbel TF

    (2017) Mutation Accumulation in an Asexual Relative of Arabidopsis

    PLOS Genetics 13:e1006550.

    https://doi.org/10.1371/journal.pgen.1006550

    • PubMed
    • Google Scholar
36. 1. Mark Welch D
    2. Meselson M

    (2000) Evidence for the evolution of bdelloid rotifers without sexual reproduction or genetic exchange

    Science 288:1211–1215.

    https://doi.org/10.1126/science.288.5469.1211

    • PubMed
    • Google Scholar
37. 1. McDonald MJ
    2. Rice DP
    3. Desai MM

    (2016) Sex speeds adaptation by altering the dynamics of molecular evolution

    Nature 531:233–236.

    https://doi.org/10.1038/nature17143

    • PubMed
    • Google Scholar
38. 1. Neiman M
    2. Hehman G
    3. Miller JT
    4. Logsdon JM
    5. Taylor DR

    (2010) Accelerated mutation accumulation in asexual lineages of a freshwater snail

    Molecular Biology and Evolution 27:954–963.

    https://doi.org/10.1093/molbev/msp300

    • PubMed
    • Google Scholar
39. 1. Nieuwenhuis BPS
    2. James TY

    (2016) The frequency of sex in fungi

    Philosophical Transactions of the Royal Society B: Biological Sciences 371:20150540.

    https://doi.org/10.1098/rstb.2015.0540

    • Google Scholar
40. 1. Normark BB
    2. Judson OP
    3. Moran NA

    (2003) Genomic signatures of ancient asexual lineages

    Biological Journal of the Linnean Society 79:69–84.

    https://doi.org/10.1046/j.1095-8312.2003.00182.x

    • Google Scholar
41. 1. Paccola-Meirelles LD
    2. Azevedo JL

    (1991) Parasexuality in Beauveria bassiana

    Journal of Invertebrate Pathology 57:172–176.

    https://doi.org/10.1016/0022-2011(91)90113-5

    • Google Scholar
42. 1. Riley R
    2. Charron P
    3. Idnurm A
    4. Farinelli L
    5. Dalpé Y
    6. Martin F
    7. Corradi N

    (2014) Extreme diversification of the mating type-high-mobility group (MATA-HMG) gene family in a plant-associated arbuscular mycorrhizal fungus

    The New Phytologist 201:254–268.

    https://doi.org/10.1111/nph.12462

    • PubMed
    • Google Scholar
43. 1. Riley R
    2. Corradi N

    (2013) Searching for clues of sexual reproduction in the genomes of arbuscular mycorrhizal fungi

    Fungal Ecology 6:44–49.

    https://doi.org/10.1016/j.funeco.2012.01.010

    • Google Scholar
44. 1. Ritz KR
    2. Noor MAF
    3. Singh ND

    (2017) Variation in Recombination Rate: Adaptive or Not?

    Trends in Genetics 33:364–374.

    https://doi.org/10.1016/j.tig.2017.03.003

    • PubMed
    • Google Scholar
45. 1. Ropars J
    2. Toro KS
    3. Noel J
    4. Pelin A
    5. Charron P
    6. Farinelli L
    7. Marton T
    8. Krüger M
    9. Fuchs J
    10. Brachmann A
    11. Corradi N

    (2016) Evidence for the sexual origin of heterokaryosis in arbuscular mycorrhizal fungi

    Nature Microbiology 1:16033.

    https://doi.org/10.1038/nmicrobiol.2016.33

    • PubMed
    • Google Scholar
46. 1. Rosada LJ
    2. Franco CC
    3. Sant'Anna JR
    4. Kaneshima EN
    5. Gonçalves-Vidigal MC
    6. Castro-Prado MA

    (2010) Parasexuality in Race 65 Colletotrichum lindemuthianum isolates

    Journal of Eukaryotic Microbiology 57:383–384.

    https://doi.org/10.1111/j.1550-7408.2010.00486.x

    • PubMed
    • Google Scholar
47. 1. Sanders IR

    (1999) Evolutionary genetics. No sex please, we're fungi

    Nature 399:737–739.

    https://doi.org/10.1038/21544

    • PubMed
    • Google Scholar
48. 1. Sanders IR
    2. Croll D

    (2010) Arbuscular mycorrhiza: the challenge to understand the genetics of the fungal partner

    Annual Review of Genetics 44:271–292.

    https://doi.org/10.1146/annurev-genet-102108-134239

    • PubMed
    • Google Scholar
49. 1. Sedzielewska KA
    2. Fuchs J
    3. Temsch EM
    4. Baronian K
    5. Watzke R
    6. Kunze G

    (2011) Estimation of the Glomus intraradices nuclear DNA content

    New Phytologist 192:794–797.

    https://doi.org/10.1111/j.1469-8137.2011.03937.x

    • PubMed
    • Google Scholar
50. 1. Seervai RN
    2. Jones SK
    3. Hirakawa MP
    4. Porman AM
    5. Bennett RJ

    (2013) Parasexuality and ploidy change in Candida tropicalis

    Eukaryotic Cell 12:1629–1640.

    https://doi.org/10.1128/EC.00128-13

    • PubMed
    • Google Scholar
51. 1. Seidl MF
    2. Thomma BP

    (2014) Sex or no sex: evolutionary adaptation occurs regardless

    BioEssays 36:335–345.

    https://doi.org/10.1002/bies.201300155

    • PubMed
    • Google Scholar
52. 1. Signorovitch A
    2. Hur J
    3. Gladyshev E
    4. Meselson M

    (2016) Evidence for meiotic sex in bdelloid rotifers

    Current Biology 26:R754–R755.

    https://doi.org/10.1016/j.cub.2016.06.051

    • PubMed
    • Google Scholar
53. 1. Taylor JW
    2. Hann-Soden C
    3. Branco S
    4. Sylvain I
    5. Ellison CE

    (2015) Clonal reproduction in fungi

    PNAS 112:8901–8908.

    https://doi.org/10.1073/pnas.1503159112

    • PubMed
    • Google Scholar
54. 1. Van der Auwera GA

    (2002)

    GATK best practices

    Current Protocols in Bioinformatics 11:11.10.1–11.1011.

    • Google Scholar
55. 1. van der Heijden MGA
    2. Klironomos JN
    3. Ursic M
    4. Moutoglis P
    5. Streitwolf-Engel R
    6. Boller T
    7. Wiemken A
    8. Sanders IR

    (1998) Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity

    Nature 396:69–72.

    https://doi.org/10.1038/23932

    • Google Scholar
56. 1. van der Heijden MG
    2. Martin FM
    3. Selosse MA
    4. Sanders IR

    (2015) Mycorrhizal ecology and evolution: the past, the present, and the future

    New Phytologist 205:1406–1423.

    https://doi.org/10.1111/nph.13288

    • PubMed
    • Google Scholar
57. 1. Weir W
    2. Capewell P
    3. Foth B
    4. Clucas C
    5. Pountain A
    6. Steketee P
    7. Veitch N
    8. Koffi M
    9. De Meeûs T
    10. Kaboré J
    11. Camara M
    12. Cooper A
    13. Tait A
    14. Jamonneau V
    15. Bucheton B
    16. Berriman M
    17. MacLeod A

    (2016) Population genomics reveals the origin and asexual evolution of human infective trypanosomes

    eLife 5:e11473.

    https://doi.org/10.7554/eLife.11473

    • PubMed
    • Google Scholar

Author details

1. Eric CH Chen

   Department of Biology, University of Ottawa, Ottawa, Canada

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

2. Stephanie Mathieu

   Department of Biology, University of Ottawa, Ottawa, Canada

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. Anne Hoffrichter

   Institute of Genetics, Faculty of Biology, Ludwig Maximilian University of Munich, Munich, Germany

   Contribution

   Resources, Validation, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

4. Kinga Sedzielewska-Toro

   Institute of Genetics, Faculty of Biology, Ludwig Maximilian University of Munich, Munich, Germany

   Contribution

   Resources, Methodology

   Competing interests

   No competing interests declared

5. Max Peart

   Department of Biology, University of Ottawa, Ottawa, Canada

   Contribution

   Resources, Formal analysis, Validation, Methodology

   Competing interests

   No competing interests declared

6. Adrian Pelin

   Department of Biology, University of Ottawa, Ottawa, Canada

   Contribution

   Data curation, Formal analysis, Validation

   Competing interests

   No competing interests declared

7. Steve Ndikumana

   Department of Biology, University of Ottawa, Ottawa, Canada

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

8. Jeanne Ropars

   Department of Biology, University of Ottawa, Ottawa, Canada

   Present address

   Ecologie Systematique et Evolution, CNRS, Université Paris Sud, AgroParisTech, Université Paris Saclay, Orsay, France

   Contribution

   Conceptualization, Writing—review and editing

   Competing interests

   No competing interests declared

9. Steven Dreissig

   Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany

   Contribution

   Resources, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4766-9698

10. Jorg Fuchs

    Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany

    Contribution

    Resources, Methodology

    Competing interests

    No competing interests declared

11. Andreas Brachmann

    Institute of Genetics, Faculty of Biology, Ludwig Maximilian University of Munich, Munich, Germany

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

12. Nicolas Corradi

    Department of Biology, University of Ottawa, Ottawa, Canada

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    ncorradi@uottawa.ca

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7932-7932

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