Mitochondrial comparative genomics and phylogenetic signal assessment of mtDNA among arbuscular mycorrhizal fungi.

Mitochondrial (mt) genes, such as cytochrome C oxidase genes (cox), have been widely used for barcoding in many groups of organisms, although this approach has been less powerful in the fungal kingdom due to the rapid evolution of their mt genomes. The use of mt genes in phylogenetic studies of Dikarya has been met with success, while early diverging fungal lineages remain less studied, particularly the arbuscular mycorrhizal fungi (AMF). Advances in next-generation sequencing have substantially increased the number of publically available mtDNA sequences for the Glomeromycota. As a result, comparison of mtDNA across key AMF taxa can now be applied to assess the phylogenetic signal of individual mt coding genes, as well as concatenated subsets of coding genes. Here we show comparative analyses of publically available mt genomes of Glomeromycota, augmented with two mtDNA genomes that were newly sequenced for this study (Rhizophagus irregularis DAOM240159 and Glomus aggregatum DAOM240163), resulting in 16 complete mtDNA datasets. R. irregularis isolate DAOM240159 and G. aggregatum isolate DAOM240163 showed mt genomes measuring 72,293bp and 69,505bp with G+C contents of 37.1% and 37.3%, respectively. We assessed the phylogenies inferred from single mt genes and complete sets of coding genes, which are referred to as "supergenes" (16 concatenated coding genes), using Shimodaira-Hasegawa tests, in order to identify genes that best described AMF phylogeny. We found that rnl, nad5, cox1, and nad2 genes, as well as concatenated subset of these genes, provided phylogenies that were similar to the supergene set. This mitochondrial genomic analysis was also combined with principal coordinate and partitioning analyses, which helped to unravel certain evolutionary relationships in the Rhizophagus genus and for G. aggregatum within the Glomeromycota. We showed evidence to support the position of G. aggregatum within the R. irregularis 'species complex'.

Independent mitochondrial and nuclear exchanges arising in Rhizophagus irregularis crossed-isolates support the presence of a mitochondrial segregation mechanism.

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) are members of the phylum Glomeromycota, an early divergent fungal lineage that forms symbiotic associations with the large majority of land plants. These organisms are asexual obligate biotrophs, meaning that they cannot complete their life cycle in the absence of a suitable host. These fungi can exchange genetic information through hyphal fusions (i.e. anastomosis) with genetically compatible isolates belonging to the same species. The occurrence of transient mitochondrial length-heteroplasmy through anastomosis between geographically distant Rhizophagus irregularis isolates was previously demonstrated in single spores resulting from crossing experiments. However, (1) the persistence of this phenomenon in monosporal culture lines from crossed parental isolates, (2) its correlation with nuclear exchanges and (3) the potential mechanisms responsible for mitochondrial inheritance are still unknown. Using the AMF model organism R. irregularis, we tested whether the presence of a heteroplasmic state in progeny spores was linked to the occurrence of nuclear exchanges and whether the previously observed heteroplasmic state persisted in monosporal in vitro crossed-culture lines. We also investigated the presence of a putative mitochondrial segregation apparatus in Glomeromycota by identifying proteins similar to those found in other fungal groups. RESULTS: We observed the occurrence of biparental inheritance both for mitochondrial and nuclear markers tested in single spores obtained from crossed-isolates. However, only one parental mitochondrial DNA and nuclear genotype were recovered in each monosporal crossed-cultures, with an overrepresentation of certain mitochondrial haplotypes. These results strongly support the presence of a nuclear-independent mitochondrial segregation mechanism in R. irregularis. Furthermore, a nearly complete set of genes was identified with putative orthology to those found in other fungi and known to be associated with the mitochondrial segregation in Saccharomyces cerevisiae and filamentous fungi. CONCLUSIONS: Our findings suggest that mitochondrial segregation might take place either during spore formation or colony development and that it might be independent of the nuclear segregation machinery. We present the basic building blocks for a better understanding of the mitochondrial inheritance process and segregation in these important symbiotic fungi. The comprehension of these processes is of great importance since it has been shown that different segregated lines of the same isolate can have variable effects on the host plant.

Intraisolate mitochondrial genetic polymorphism and gene variants coexpression in arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are multinucleated and coenocytic organisms, in which the extent of the intraisolate nuclear genetic variation has been a source of debate. Conversely, their mitochondrial genomes (mtDNAs) have appeared to be homogeneous within isolates in all next generation sequencing (NGS)-based studies. Although several lines of evidence have challenged mtDNA homogeneity in AMF, extensive survey to investigate intraisolate allelic diversity has not previously been undertaken. In this study, we used a conventional polymerase chain reaction -based approach on selected mitochondrial regions with a high-fidelity DNA polymerase, followed by cloning and Sanger sequencing. Two isolates of Rhizophagus irregularis were used, one cultivated in vitro for several generations (DAOM-197198) and the other recently isolated from the field (DAOM-242422). At different loci in both isolates, we found intraisolate allelic variation within the mtDNA and in a single copy nuclear marker, which highlighted the presence of several nonsynonymous mutations in protein coding genes. We confirmed that some of this variation persisted in the transcriptome, giving rise to at least four distinct nad4 transcripts in DAOM-197198. We also detected the presence of numerous mitochondrial DNA copies within nuclear genomes (numts), providing insights to understand this important evolutionary process in AMF. Our study reveals that genetic variation in Glomeromycota is higher than what had been previously assumed and also suggests that it could have been grossly underestimated in most NGS-based AMF studies, both in mitochondrial and nuclear genomes, due to the presence of low-level mutations.

Mating type gene homologues and putative sex pheromone-sensing pathway in arbuscular mycorrhizal fungi, a presumably asexual plant root symbiont.

The fungal kingdom displays a fascinating diversity of sex-determination systems. Recent advances in genomics provide insights into the molecular mechanisms of sex, mating type determination, and evolution of sexual reproduction in many fungal species in both ancient and modern phylogenetic lineages. All major fungal groups have evolved sexual differentiation and recombination pathways. However, sexuality is unknown in arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota, an ecologically vital group of obligate plant root symbionts. AMF are commonly considered an ancient asexual lineage dating back to the Ordovician, approximately 460 M years ago. In this study, we used genomic and transcriptomic surveys of several AMF species to demonstrate the presence of conserved putative sex pheromone-sensing mitogen-activated protein (MAP) kinases, comparable to those described in Ascomycota and Basidiomycota. We also find genes for high mobility group (HMG) transcription factors, homologous to SexM and SexP genes in the Mucorales. The SexM genes show a remarkable sequence diversity among multiple copies in the genome, while only a single SexP sequence was detected in some isolates of Rhizophagus irregularis. In the Mucorales and Microsporidia, the sexM gene is flanked by genes for a triosephosphate transporter (TPT) and a RNA helicase, but we find no evidence for synteny in the vicinity of the Sex locus in AMF. Nonetheless, our results, together with previous observations on meiotic machinery, suggest that AMF could undergo a complete sexual reproduction cycle.