Available online at www.sciencedirect.com
ScienceDirect Genome diversity in arbuscular mycorrhizal fungi J Peter W Young Arbuscular mycorrhizal fungi (Glomeromycota) are the most widespread and important symbionts of plants. They cannot be cultured without plants, are apparently asexual, and have multiple nuclei in a common cytoplasm. There is evidence for genetic variation among nuclei, and for segregation of this variation during growth, but these findings remain contentious. Recently, two papers have reported whole genome sequences for a strain of Rhizophagus irregularis; both suggest that genetic variation among nuclei is low. Genome assembly is very incomplete, though, so significant nuclear diversity cannot be excluded. While the diversity of nuclear genomes remains unresolved, multiple complete mitochondrial genomes are now available; there is virtually no variation within isolates, but significant variation between them. Address Department of Biology, University of York, York YO10 5DD, UK Corresponding author: Young, J Peter W ([email protected]
Current Opinion in Plant Biology 2015, 26:113–119 This review comes from a themed issue on Biotic interactions Edited by Uta Paszkowski and D Barry Scott
http://dx.doi.org/10.1016/j.pbi.2015.06.005 1369-5266/# 2015 Elsevier Ltd. All rights reserved.
Introduction Mycorrhizas are symbioses between fungi and plant roots, and many different fungi are involved in such associations. They are so widespread and important that it has been claimed that ‘mycorrhizas, not roots, are the chief organs of nutrient uptake by land plants’ . By far the most prevalent, and possibly the oldest, are the arbuscular mycorrhizas, which are found in more than 80% of plant families . They are also among the least well understood, due to the intractable nature of the fungal partner. All known arbuscular mycorrhizal (AM) fungi are in the phylum Glomeromycota, and all known Glomeromycota are AM fungi, with the exception of Geosiphon, which forms a symbiosis with cyanobacteria . The Glomeromycota are probably the sister group of the Mucoromycotina [3–5,6] which, interestingly, form mycorrhiza-like associations with basal plant lineages , although the last common ancestor of these two fungal groups probably lived well before plants colonised land . AM fungi cannot be cultured without their plant partner. They have www.sciencedirect.com
no observed sexual cycle, and multiple nuclei share a common cytoplasm, even in the spores. The combination of phylogenetic isolation and life-cycle peculiarities suggests that AM fungi might have an unusual genomic architecture, but their experimental intractability has been frustrating. After considerable effort, we now have some genome-scale sequence data, and this sheds a little light on one species and leads the way towards the exploration of genomic diversity across the whole phylum. This review is largely concerned with the evidence for genomic diversity within a mycelial network. It begins with a consideration of the ‘pre-genomic’ observations that coloured our expectations and speculations about genome organisation in the AM fungi, and then the relevant aspects of the genome papers are summarised and interpreted. After considering what we know about nuclear genomes, we review the evidence concerning mitochondrial genomes, which are currently better characterised. Besides the nucleus and mitochondrion, AM fungi have a third ‘genome compartment’ that is beginning to attract increasing attention, though we can only give it the briefest of mentions here. The majority of AM fungi that have been examined have at least one, and sometimes more than one, bacterial endosymbiont living within the mycelium [9,10,11], though Rhizophagus, the AM fungal genus with the best-studied genome (see Box 1 for information on names), is unusual in not having bacterial endosymbionts. Two very different groups of bacteria are involved, and in both cases the association appears to be ancient [9,10,11]. Indeed, one type seems to be so well integrated with the fungus that it has a much reduced genome and has adopted many fungal genes [12,13]. There is genetic variation among the bacteria, and it is likely that this may have functional consequences, but this needs more investigation.
Hints and speculations about the genome diversity of AM fungi What we know of the life cycle of AM fungi suggests that there might be genomic diversity even among the nuclei in a single isolate. Unlike most organisms, there is no stage in the cycle at which the organism develops from a single nucleus. When a spore forms, multiple nuclei move into it from the hypha , so the spore carries a sample of the population of nuclei that are mingled in the shared cytoplasm. With no single-nucleus stage to reset diversity to zero, we can expect that differences will build up among the nuclei that share a hyphal network, and this diversity will be perpetuated through the spores. In effect, the nuclei behave like a population rather than an individual. Current Opinion in Plant Biology 2015, 26:113–119
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Box 1 The names of the AM fungi. In the past few years, while genomic information has been accumulating for AM fungi, the names of the species have been changed, making the literature hard to follow. In this review, the current names are given, but older names will be found in many of the articles that are cited. This box provides an explanation and a translation table (Table 1). Originally, the taxonomy of AM fungi was based on morphological differences, largely in the spores. Since the spores are tiny (<1 mm) and have only a limited repertoire of distinguishable characteristics, the classification was coarse-grained. Those fungi without distinguishing spore features were lumped together in the genus Glomus. With the advent of molecular sequence data, it became evident that this was a heterogeneous group of unrelated organisms, and some of the more egregious misfits were given new names . More recently, this process took a further major step, resulting in a set of genera that correspond to unambiguous clades in the phylogeny based on ribosomal RNA genes . The job is far from complete, as there are no molecular data associated with many of the older species descriptions, and no cultures available for the increasing number of putative new taxa that are known only from sequences obtained from roots or soil. However, the fungi discussed in this review are among the best characterised, so we can hope that their nomenclature will change less in future. Ironically, the best-studied AM fungus of all has undergone the most convoluted sequence of renamings. In 2004, efforts began to sequence the genome of Glomus intraradices DAOM197198 . The task eventually took a decade , by which time the organism was called Rhizophagus irregularis. First, a comparison of molecular sequences showed that DAOM197198 was closer to G. irregulare than to G. intraradices [40,41]. Accordingly, the strain became G. irregulare, even though it lacked the irregular spores that were originally considered diagnostic of this species. Then the bloated genus Glomus was carved up into a number of new genera [38,42]. The name Glomus had to stay with the type species, G. macrocarpum, while G. irregulare became Rhizophagus irregularis. The name Rhizophagus was resurrected from a very old description of a putative root pathogen , but it has recently been argued that the modern AM fungi do not match this description, so a new name is needed . The name Rhizoglomus is proposed, so DAOM197198 would become Rhizoglomus irregulare. While it is likely that this name will be adopted in future, it is not used in any of the literature cited here so, for consistency, the names used by Schu¨ßler and Walker  are adopted throughout this review. Interested readers can consult a commentary with a simplified phylogeny that explains this classification . While a considerable range of AM fungi are available for research (see Table 1), it is notable that genomic studies have been concentrated on very few of them. Many of the others are included here only because they have featured in surveys of endosymbiotic bacteria [9,10], but their genomes should also be accessible.
The level of diversity of this intracytoplasmic population will depend on a number of processes (Figure 1). New variants are generated by point mutation, deletion or rearrangement. Another way to top up the genetic diversity in a population is by immigration from other populations, and there is a well-documented process by which this happens in AM fungi. When the hyphae of two separate mycelia meet, a cytoplasmic bridge called an anastomosis can be formed between them. Anastomoses between hyphae from different spores have been Current Opinion in Plant Biology 2015, 26:113–119
observed in Acaulospora, Claroideoglomus, Glomus, and Rhizophagus, but not Gigaspora or Scutellospora , and nuclei can pass from one fungus to the other [15,16], so this may be a widespread, but not universal, mechanism for bringing together genetically different nuclei. Recombination between nuclei would also generate novel genotypes, but there is as yet no evidence that this occurs in AM fungi. These processes that increase nuclear diversity will be balanced by losses resulting from random drift and perhaps due to selection against certain genotypes. Neutral genetic drift is a predictable consequence of bottlenecks in the population size, such as the entry into a spore. The number of independent nuclei that enter the spore is the crucial factor here, and the evidence for Claroideoglomus etunicatum is that a spore is populated by a stream of separate nuclei rather than by replication of one or few founder nuclei , so the bottleneck may not be very severe. It is not entirely clear how selection operates in a population of nuclei that share a common cytoplasm, and hence share the phenotypic traits that arise from gene expression. One can imagine that there will be an advantage to nuclei that replicate faster, and they could achieve this through massive genomic deletions, which might not be lethal if they are compensated by other nuclei in a shared cytoplasm. This raises the theoretical possibility that nuclei become radically different in genomic content and mutually interdependent . The variation in such a system would be locked in, because a mix of nuclei would be required for normal functioning. Even if the degree of interdependence were not as extreme as this, any mutual compensation between the nuclei would provide a frequency-dependent effect that would stabilise the diversity and reduce the rate of loss through drift.
Evidence for variation among genomes within an isolate There is substantial observational and experimental evidence for genetic diversity within AM strains, and for changes in diversity. The early evidence concerned ribosomal RNA operons. These have multiple sequences within an isolate but, because they are usually found in multiple copies per nucleus, the variation might be within, rather than between, nuclei . In situ hybridisation suggested, though not conclusively, that there were differences in ribosomal RNA spacer sequences (ITS) between nuclei of Racocetra castanea, but some nuclei carried both variants . On the other hand, a study of C. etunicatum found that microdissected single nuclei each carried three ITS variants, with no sign of variation between nuclei . The same studies also documented variation within an isolate in protein-coding loci. Kuhn et al.  found 15 sequence variants in the BiP gene (encoding an Hsp70-family chaperone) of a single isolate of Rhizophagus irregularis, and argued that these represented allelic variation among nuclei. This argument falls www.sciencedirect.com
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drift Current Opinion in Plant Biology
Processes that cause gains and losses of nuclear diversity. (a) Gain of nuclei through anastomosis with a different mycelium. (b) Gain through mutation. (c) Loss through selection, in this case by the plant host. (d) Loss by random drift, in this case at the bottleneck during spore formation.
if each nucleus has multiple BiP loci. Pawlowska and Taylor  found that 13 variants of the PLS1 gene (POL1-like, but probably a pseudogene ) were all inherited by all spores that they examined in C. etunicatum, and argued that every nucleus must have all the variants because such stability would be very unlikely if each variant were carried by an independently inherited nucleus. This argument loses force if the inheritance is not entirely independent, which could be the case if each nucleus carried several (but not all) variant loci, or if nuclei were mutually interdependent; anastomosis could also stem the loss of variation. Hijri and Sanders  estimated that each nucleus of C. etunicatum carried just one copy of PLS1, but they did not explain the stable inheritance of so many variants. A series of experimental studies from the laboratory of Ian Sanders have documented shifts in gene frequencies when AM fungi are subcultured, and also changes in phenotype. For example, in two studies [23,24], cultures were propagated from lines that had previously been made heterokaryotic by anastomosis between genetically distinct lines . Some of the resulting fungal lines, which had differences in AFLP (amplified fragment length polymorphism) patterns and Bg112 allele frequencies, had strikingly different effects on host plant gene expression and growth. In a more recent paper, multiple genetically distinct lineages of R. irregularis showed changes in AFLP pattern when shifted from carrot roots to potato roots, with some consistency in the set of loci affected . Sequence-based allele frequencies of the Bg112 marker also changed significantly, though not in a consistent way. The fact that nuclear-encoded sequences can change in frequency is consistent with genetically diverse nuclei, but hard to explain if all nuclei carry the same alleles. Spore production changed significantly, indicating that phenotype is also altered during culture. www.sciencedirect.com
Evidence from the first published nuclear genomes Two independent papers, published within weeks of each other, presented genome sequences of the same R. irregularis isolate [6,26]. Despite different methods, their conclusions were remarkably similar. Tisserant et al.  sequenced genomic DNA of R. irregularis DAOM-197198 from multinucleate hyphae growing from a carrot root culture. They estimated the genome size as 153 Mb, although the assembly was only 101 Mb because roughly a third of the genome consists of repeated transposable elements. This assembly is believed to include almost all the protein-coding genes (23,561 high-confidence gene models), as there is good concordance with the previously published transcriptome data . However, the assembly is highly fragmented because of the repeated sequences, so it comprises 12,421 separate scaffolds, with half the assembly being in scaffolds under 15.16 kb in length. With an average of less than two genes per scaffold, many genes have very little genomic context, which limits our ability to distinguish between orthologous (allelic) and paralogous (duplicated) genes. If some loci had alleles that were substantially divergent, these would probably assemble in separate contigs. Tisserant et al.  did find that 5.6% of contigs were >90% identical to other contigs, but concluded that these mostly involved repetitive sequences. Among reads that were sufficiently similar to be assembled together, the level of single nucleotide polymorphisms (SNPs) was 0.43 per kb. This is low, but not negligible. The same analysis gave a value of just 0.06 SNP per kb for Tuber melanosporum, a truly homokaryotic ascomycete, but 0.78 SNP per kb for Laccaria bicolor, a dikaryotic basidiomycete. Hence, the polymorphism within the R. irregularis assembly is closer Current Opinion in Plant Biology 2015, 26:113–119
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to that of a dikaryon, which has two genomes sampled from the population, supporting the view that the isolate has a population of different nuclei, though with rather a low level of polymorphism. By contrast, the Rhizoctonia solani isolate AG8, which is known to be a heterokaryon with genetically diverse nuclei, had 15.9 SNPs per kb . While is must be recognised that this was not calculated by exactly the same method, it is clear that this is an order of magnitude more variation than seen in DAOM-197198. Genomic variation within the isolate DAOM-197198 seems to be substantially lower than across the species, since RNA-Seq reads from another R. irregularis isolate, C2, had 2.6 SNP per kb when compared to the DAOM-197198 assembly. Lin et al.  sequenced the same R. irregularis isolate as Tisserant et al. , though a separate subculture that they designate DAOM197198w. Their study addressed the question of genomic diversity more directly, because they sequenced four individual nuclei using whole genome amplification, as well as two separate multinuclear samples. The overall assembly was 141 Mb, comparable to that of Tisserant et al. , but the individual nuclear assemblies were smaller, 71–115 Mb. This does not, however, indicate that the individual nuclei have large deletions, since all assemblies are incomplete. In fact, the two papers draw strikingly similar conclusions on genomic diversity. Lin et al.  estimate that more than 99.7% of the aligned sequence is identical between nuclei, and overall polymorphism, 0.1 SNP per kb, was even lower than the corresponding estimate by Tisserant et al., though this could reflect differences in the sequencing and analysis as well as in the biological material. A comparison of the two assemblies gives 8 SNP per kb, a much larger value, which suggests that these two cultures of the same original isolate may have diverged, though technical differences in the assemblies might also affect this result . Lin et al.  also looked specifically at the loci that had featured in earlier controversies over variation between and within nuclei. They found three distinct BiP loci, with no nucleotide polymorphism within any of them. One of the nuclei (N6) had all three loci; the others had just one or two, but this may reflect incomplete sequencing. The lack of variation clearly differs from the earlier report of 15 BiP alleles in another isolate of R. irregulare . Lin et al.  found that the Bg112 microsatellite sequence also came in three distinct forms, again with no polymorphism detected within each. Three nuclei had two variants, one (N31) had all three, again demonstrating that these were three loci, not three allelic variants. The assumption that all nuclei have all three loci is clearly incompatible with the observation that the relative abundance of Bg112 variants changes with shifts in host plant . In the case of the ribosomal ITS and LSU sequences, Lin et al.  confirmed the common observation that these were polymorphic, and added the Current Opinion in Plant Biology 2015, 26:113–119
information that most of the SNPs varied within a single nucleus, but the relative abundance of alleles appeared to differ between nuclei. These sequences occur in tandem arrays in the genome, and the existence of polymorphism suggests that the process of homogenisation by recombination (concerted evolution) is less effective in this organism than in many others.
Reconciling the evidence The two genome papers [6,26] assert that there is little genomic variation among the nuclei of R. irregularis DAOM197198, except perhaps at the rRNA locus. On the other hand, it is clear that mycelium containing genetically different nuclei can be created by anastomosis between different parental lines [15,16], and there is persistent evidence that AM fungal isolates do have internal genetic variation that can segregate, and that subcultures can have different phenotypic properties [23,24,25]. How can we reconcile these apparently incompatible views of the genomic structure of AM fungi? The first point to make is that the sequenced isolate, R. irregularis DAOM197198, has been in culture for decades. If there is segregation of variant nuclei, it may have lost diversity and become clonal. By contrast, most of the studies of nuclear diversity have involved other isolates, which may be very different. Indeed, many studies [20–22,30] concern Claroideoglomus, which has been diverging from Rhizophagus for almost 500 million years , so may differ in fundamental ways. The studies of diversity in R. irregularis have not used DAOM197198 but other, potentially less ‘domesticated’, isolates, and in some cases have deliberately enhanced variation through anastomosis [23,25,31]. Only three distinct sequences were found at the BiP locus in the DAOM197198 genome , but many more are reported for other R. irregularis strains . One recent study compared new genome sequence data for DAOM197198 and other isolates of R. irregularis , and reported a much smaller ‘pangenome’ (unique sequence length) for this isolate, suggesting lower genomic complexity, but the very low sequence coverage of the DAOM197198 genome in this study (just 86 Mb, less than onefold) may have affected this result. This study also reported very high sequence variation for some putatively single-copy loci, but this finding has been challenged by Ropars and Corradi , who failed to confirm, using Illumina sequencing, the high SNP count reported for DAOM197198 by Boon et al. . Although the 454 pyrosequencing used by Boon et al. can potentially generate more errors, these are usually missed or extra bases rather than SNPs, and the higher stringency of the filtering that Ropars and Corradi applied to the sequence data may have been the main contributor to the lower level of polymorphism that they reported. It would be illuminating to apply the same analyses, with a range of stringencies, to both sets of sequences. www.sciencedirect.com
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The difficulties of working with AM fungi mean that the evidence, on either side, is not as crisp as we would like. Genetic changes during culture have been demonstrated using AFLP, which provides only an indirect proxy for genetic markers, or rRNA loci that are repetitive and highly variable, or relative frequencies of other sequence variants that might not be allelic. On the other hand, the genome sequence data were assembled using tools designed for genetically uniform individuals, and the assembly is so fragmentary that alleles might be confused with paralogs. The observation that different variants of BiP and Bg112 can be found in the same nucleus  is strong evidence that these are duplicated loci rather than alleles, but there is no evidence that every nucleus has all the loci, and changes in relative abundance  could be accommodated if this were not the case.
inheritance of nuclei in AM fungi resembles that of mitochondria in more conventional organisms, and the processes that could generate and maintain diversity of mitochondrial genomes are the same as those already discussed for nuclei (Figure 1). The first complete mtDNA sequence was of R. irregularis FACE494 . It is a typical fungal mtDNA, circular and 70606 bp in length, and there was no evidence of sequence polymorphism. Five additional complete mtDNA sequences from the same species revealed substantial differences in size and sequence between isolates, but confirmed the absence of polymorphism within each isolate . The differences between isolates were largely generated by the activity of three classes of mobile element. Four further Rhizophagus mtDNAs show similar features [35–37].
Mitochondrial genomes and their diversity We currently have much clearer data on mitochondrial genomes (mtDNA) of AM fungi than on their nuclear genomes. It is worth considering what we have learned about the diversity of mtDNA, not only because it is important in its own right, but for the light that it might shed on the issues surrounding nuclear genomics. The
Mitochondrial sequences have been determined for two Gigaspora isolates belonging to different species [4,5]. They are both large (>90 kb) and, unlike R. irregularis, have genes encoded on both strands, as well as unusual trans-spliced introns. Again, there is no evidence of polymorphism within the isolates.
Table 1 Genera and species of Glomeromycota used in studies covered in this review, showing the name formerly used and the recent name, arranged in the four orders of the phylum (based on Refs. [38,42]) Former name
Glomerales Glomus intraradices, G. intraradice Glomus clarum Glomus manihotis Glomus diaphanum Glomus caledonium Glomus mosseae Glomus geosporum Glomus claroideum Glomus etunicatum Glomus cerebriforme Glomus formosanum
Rhizophagus irregularis Rhizophagus clarus Rhizophagus manihotis Rhizophagus diaphanus Funneliformis caledonium Funneliformis mosseae Funneliformis geosporum Claroideoglomus claroideum Claroideoglomus etunicatum Uncertain Uncertain
[3,6,9,16,19,21,23,24,25,26,27,31,32,34–37] [9,15]       [8,9,14,15,20–22,30]  
Diversisporales Acaulospora Kuklospora colombiana Glomus versiforme Scutellospora calospora Scutellospora castanea Scutellospora persica Scutellospora verrucosa Scutellospora gilmorei Scutellospora pellucida Scutellospora heterogama Scutellospora erythropus Gigaspora
Acaulospora Acaulospora colombiana Diversispora epigaea Scutellospora calospora Racocetra castanea Racocetra persica Racocetra verrucosa Uncertain (Cetraspora?) Uncertain (Cetraspora?) Uncertain (Dentiscutata?) uncertain (Dentiscutata?) Gigaspora
    [10,19]       [4,5,9,10,11,15]
Archaeosporales Ambispora Geosiphon piriformis
Ambispora Geosiphon piriformis
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Set against all these reports of homoplasmy (genetic uniformity) of mtDNA in AM fungi is the observation that mitochondria (like nuclei) passed through anastomoses between distinct isolates of R. irregularis, generating spores that carried two distinct mtDNA genotypes, although this heteroplasmy was transient . It remains to be determined whether AM fungi have mechanisms that purge mitochondrial diversity (or, indeed, nuclear diversity) faster than random drift alone.
Schu¨ßler A, Schwarzott D, Walker C: A new fungal phylum, the Glomeromycota: phylogeny and evolution. Mycol Res 2001, 105:1413-1421.
Lee J, Young JPW: The mitochondrial genome sequence of the arbuscular mycorrhizal fungus Glomus intraradices isolate 494 and implications for the phylogenetic placement of Glomus. New Phytol 2009, 183:200-211.
Nadimi M, Beaudet D, Forget L, Hijri M, Lang BF: Group I intronmediated trans-splicing in mitochondria of Gigaspora rosea and a robust phylogenetic affiliation of arbuscular mycorrhizal fungi with Mortierellales. Mol Biol Evol 2012, 29:2199-2210.
Pelin A, Pombert JF, Salvioli A, Bonen L, Bonfante P, Corradi N: The mitochondrial genome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals two unsuspected trans-splicing events of group I introns. New Phytol 2012, 194:836-845.
Conclusions This is a pivotal moment for our understanding of genomic diversity and adaptation in AM fungi, which is of considerable theoretical interest but also vital if we are to make the best use of this symbiosis. We need much better assemblies of nuclear genomes, and we need them from a much wider range of AM fungi. Most studies so far have used just a handful of species (see Table 1). Fortunately, a range of AM fungi are included within the plans for the ‘1000 Fungal Genomes’ project of the Joint Genome Institute (http://genome.jgi.doe.gov/pages/fungi-1000-projects.jsf). We need studies of genomic change when AM fungi are cultured on different hosts and in different conditions, and these need to use much more robust genetic markers. We need more studies of the genetic mixing that results from anastomosis, and of the subsequent fate of the nuclear heterogeneity that this creates. The fragmentary evidence pulls in different directions, but our increasing experience and improving technologies should enable us to move forward rapidly. We have shown that we can obtain large amounts of high-quality sequence data from a range of AM fungi, either using multiple spores or, with whole genome amplification, even from single nuclei. This should allow genome sequencing of the wide range of species available as cultures on plants. We can expect to see high-throughput sequencing increasingly deployed to illuminate experimental studies as well as to map genomic diversity across species, genera and families of AM fungi. There are also advances in transcriptomics, proteomics and microscopy, which lie outside the scope of this review, that will need to be integrated with the genomic perspective. The next few years promise to answer many of our current questions, though no doubt they will also raise new ones.
Acknowledgement The author’s research on arbuscular mycorrhizal fungi is supported by the Biotechnology and Biological Sciences Research Council grant BB/ H014373/1.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Smith SE, Read DJ: Mycorrhizal Symbiosis. Academic Press; 2008.
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Lin K, Limpens E, Zhang Z, Ivanov S, Saunders DGO, Mu D, Pang E, Cao H, Cha H, Lin T et al.: Single nucleus genome sequencing reveals high similarity among nuclei of an endomycorrhizal fungus. PLoS Genet 2014, 10:e1004078. The genome sequence of Rhizophagus irregularis DAOM197198 is presented, including sequences obtained by whole genome amplification from single nuclei that show little sequence variation between nuclei. 7.
Bidartondo MI, Read DJ, Trappe JM, Merckx V, Ligrone R, Duckett JG: The dawn of symbiosis between plants and fungi. Biol Lett 2011, 7:574-577.
VanKuren NW, Bakker den HC, Morton JB, Pawlowska TE: Ribosomal RNA gene diversity, effective population size, and evolutionary longevity in asexual Glomeromycota. Evolution 2013, 67:207-224. A careful analysis of rRNA gene variation within and between isolates of Claroideoglomus. 9.
Naumann M, Schu¨ßler A, Bonfante P: The obligate endobacteria of arbuscular mycorrhizal fungi are ancient heritable components related to the Mollicutes. ISME J 2010, 4:862-871.
10. Mondo SJ, Toomer KH, Morton JB, Lekberg Y, Pawlowska TE: Evolutionary stability in a 400-million-year-old heritable facultative mutualism. Evolution 2012, 66:2564-2576. 11. Desiro` A, Salvioli A, Ngonkeu EL, Mondo SJ, Epis S, Faccio A, Kaech A, Pawlowska TE, Bonfante P: Detection of a novel intracellular microbiome hosted in arbuscular mycorrhizal fungi. ISME J 2014, 8:257-270. The genetic diversity of two types of endosymbiotic bacteria found in the spores of Gigaspora. 12. Naito M, Morton JB, Pawlowska TE: Minimal genomes of mycoplasma-related endobacteria are plastic and contain host-derived genes for sustained life within Glomeromycota. Proc Natl Acad Sci U S A 2015 http://dx.doi.org/10.1073/pnas. 1501676112. 13. Torres-Corte´s G, Ghignone S, Bonfante P, Schu¨ßler A: Mosaic genome of endobacteria in arbuscular mycorrhizal fungi: transkingdom gene transfer in an ancient mycoplasma-fungus association. Proc Natl Acad Sci U S A 2015 http://dx.doi.org/ 10.1073/pnas.1501540112. 14. Jany JL, Pawlowska TE: Multinucleate spores contribute to evolutionary longevity of asexual Glomeromycota. Am Nat 2010, 175:424-435. 15. de Novais CB, Sbrana C, Ju´nior OJS, Siqueira JO, Giovannetti M: Vegetative compatibility and anastomosis formation within and among individual germlings of tropical isolates of arbuscular mycorrhizal fungi (Glomeromycota). Mycorrhiza 2013, 23:325-331. Anastomosis between genetically distinct hyphae is demonstrated for representatives of a range of different AM fungal genera, showing that the creation of nuclear heterogeneity by this means is potentially widespread. 16. Croll D, Giovannetti M, Koch AM, Sbrana C, Ehinger M, Lammers PJ, Sanders IR: Nonself vegetative fusion and genetic exchange in the arbuscular mycorrhizal fungus Glomus intraradices. New Phytol 2009, 181:924-937. 17. Young JPW: Kissing cousins: mycorrhizal fungi get together. New Phytol 2009, 181:751-753. www.sciencedirect.com
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18. Sanders IR, Clapp JP, Wiemken A: The genetic diversity of arbuscular mycorrhizal fungi in natural ecosystems — a key to understanding the ecology and functioning of the mycorrhizal symbiosis. New Phytol 1996, 133:123-134. 19. Kuhn G, Hijri M, Sanders IR: Evidence for the evolution of multiple genomes in arbuscular mycorrhizal fungi. Nature 2001, 414:745-748. 20. Pawlowska TE, Taylor JW: Organization of genetic variation in individuals of arbuscular mycorrhizal fungi. Nature 2004, 427:733-737. 21. Boon E, Zimmerman E, Lang BF, Hijri M: Intra-isolate genome variation in arbuscular mycorrhizal fungi persists in the transcriptome. J Evol Biol 2010, 23:1519-1527. 22. Hijri M, Sanders IR: Low gene copy number shows that arbuscular mycorrhizal fungi inherit genetically different nuclei. Nature 2005, 433:160-163. 23. Angelard C, Colard A, Niculita-Hirzel H, Croll D, Sanders IR: Segregation in a mycorrhizal fungus alters rice growth and symbiosis-specific gene transcription. Curr Biol 2010, 20:1216-1221. 24. Colard A, Angelard C, Sanders IR: Genetic exchange in an arbuscular mycorrhizal fungus results in increased rice growth and altered mycorrhiza-specific gene transcription. Appl Environ Microbiol 2011, 77:6510-6515. 25. Angelard C, Tanner CJ, Fontanillas P, Niculita-Hirzel H, Masclaux F, Sanders IR: Rapid genotypic change and plasticity in arbuscular mycorrhizal fungi is caused by a host shift and enhanced by segregation. ISME J 2014, 8:284-294. A recent example of nuclear diversity and segregation, in this case reflecting a change in host plant. 26. Tisserant E, Malbreil M, Kuo A, Kohler A, Symeonidi A, Balestrini R, Charron P, Duensing N, Frei dit Frey N, Gianinazzi-Pearson V et al.: Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis. Proc Natl Acad Sci U S A 2013, 110:20117-20122. The genome sequence of Rhizophagus irregularis DAOM197198 is presented, based on a major collaborative project, and the content of the genome is discussed in detail. 27. Tisserant E, Kohler A, Dozolme Seddas P, Balestrini R, Benabdellah K, Colard A, Croll D, Da Silva C, Gomez SK, Koul R et al.: The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont. New Phytol 2012, 193:755-769. 28. Hane JK, Anderson JP, Williams AH, Sperschneider J, Singh KB: Genome sequencing and comparative genomics of the broad host-range pathogen Rhizoctonia solani AG8. PLoS Genet 2014, 10:e1004281. 29. Limpens E, Geurts R: Plant-driven genome selection of arbuscular mycorrhizal fungi. Mol Plant Pathol 2014, 15:531-534. A thoughtful commentary on the possible causes and consequences of variation among nuclei. 30. Boon E, Zimmerman E, St-Arnaud M, Hijri M: Allelic differences within and among sister spores of the arbuscular mycorrhizal fungus Glomus etunicatum suggest segregation at sporulation. PLoS ONE 2013, 8:e83301.
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