The Coevolution of Fungal Mitochondrial Introns and Their Homing Endonucleases (GIY-YIG and LAGLIDADG)

Liu W., Cai Y., Zhang Q., Chen L., Shu F., Ma X., Bian Y.

The complete mitochondrial genome of Morchella importuna, the famous edible and medicinal mushroom, was assembled as a 272,238 bp single circular dsDNA. As the largest mitogenome among fungi, it exhibits several distinct characteristics. The mitogenome of M. importuna encoded 14 core conserved mitochondrial protein-coding genes and 151 mitochondrial non-conserved open reading frames (ncORFs) were predicted, of which 61 were annotated as homing endonuclease genes, and 108 were confirmed to be expressed during the vegetative growth stages of M. importuna. In addition, 34 introns were identified in seven core genes (cob, cox1, cox2, cox3, nad1, nad4 and nad5) and two rRNA genes (rrnS and rrnL) with a length from 383 bp to 7453 bp, and eight large introns with a length range of 2340 bp to 7453 bp contained multiple intronic mtORFs. Moreover, 34 group I (IA, IB, IC1, IC2, ID and derived group I introns) and four group II intron domains were identified for the 34 introns, including five hybrid ones. Furthermore, the M. importuna mitogenome showed the presence of about 18.7% mitogenomic interspersed repeats. These and the aforementioned ncORFs and introns, contributed to the enlarged size of the mitogenome.

Kortsinoglou A., Korovesi A.G., Theelen B., Hagen F., Boekhout T., Kouvelis V.N.

Abstract Cryptococcus spp. are fungal species belonging to Tremellomycetes, Agaricomycotina, Basidiomycota, and several members are responsible for cryptococcosis, one of the most ubiquitous human mycoses. Affecting mainly immune suppressed patients, but also immune competent ones, the members of this genus present a high level of genetic diversity. In this study, two mitochondrial intergenic regions, i.e. nad1-cob and cob-rps3, were tested for the intra- or interspecies discrimination and identification of strains and species of the genus Cryptococcus. Phylogenetic trees were constructed based on individual and concatenated sequences from representative pathogenic strains of the C. neoformans/C. gattii complex, representing serotypes and AFLP genotypes of all newly introduced species of this complex. Using both intergenic regions, as well as the concatenated dataset, the strains clustered in accordance with the new taxonomy. These results suggest that identification of Cryptococcus strains is possible by employing these mitochondrial intergenic regions using PCR amplification as a quick and effective method to elucidate genotypic and taxonomic differences. Thus, these regions may be applicable to a broad range of clinical studies, leading to a rapid recognition of the clinical profiles of patients.

Wu B., Hao W.

Mitochondrial recombination in yeast is well recognized, yet the underlying genetic mechanisms are not well understood. Recent progress has suggested that mobile introns in mitochondrial genomes (mitogenomes) can facilitate the recombination of their corresponding intron-containing genes through a mechanism known as intron homing. As many mitochondrial genes lack introns, there is a critical need to determine the extent of recombination and underlying mechanism of intron-lacking genes. This study leverages yeast mitogenomes to address these questions. In Saccharomyces cerevisiae, the 3'-end sequences of at least three intron-lacking mitochondrial genes exhibit elevated nucleotide diversity and recombination hotspots. Each of these 3'-end sequences is immediately adjacent to or even fused as overlapping genes with a stand-alone endonuclease. Our findings suggest that SAEs are responsible for recombination and elevated diversity of adjacent intron-lacking genes. SAEs were also evident to drive recombination of intron-lacking genes in Lachancea kluyveri, a yeast species that diverged from S. cerevisiae more than 100 million years ago. These results suggest SAEs as a common driver in recombination of intron-lacking genes during mitogenome evolution. We postulate that the linkage between intron-lacking gene and its adjacent endonuclease gene is the result of co-evolution.

Lee H., Ke H., Lin C.I., Lee T.J., Chung C., Tsai I.J.

AbstractComparative genomics of fungal mitochondrial genomes (mitogenomes) have revealed a remarkable pattern of rearrangement between and within major phyla owing to horizontal gene transfer and recombination. The role of recombination was exemplified at a finer evolutionary time scale in basidiomycetes group of fungi as they display a diversity of mitochondrial DNA inheritance patterns. Here, we assembled mitogenomes of six species from the Hymenochaetales order of basidiomycetes and examined 59 mitogenomes from 2 genetic lineages of Phellinus noxius. Gene order is largely collinear, while intergene regions are major determinants of mitogenome size variation. Substantial sequence divergence was found in shared introns consistent with high horizontal gene transfer frequency observed in yeasts, but we also identified a rare case where an intron was retained in five species since speciation. In contrast to the hyperdiversity observed in nuclear genomes of Phellinus noxius, mitogenomes’ intraspecific polymorphisms at protein-coding sequences are extremely low. Phylogeny network based on introns revealed turnover as well as exchange of introns between two lineages. Strikingly, some strains harbor a mosaic origin of introns from both lineages. Analysis of intergenic sequence indicated substantial differences between and within lineages, and an expansion may be ongoing as a result of exchange between distal intergenes. These findings suggest that the evolution in mitochondrial DNAs is usually lineage specific but chimeric mitotypes are frequently observed, thus capturing the possible evolutionary processes shaping mitogenomes in a basidiomycete. The large mitogenome sizes reported in various basidiomycetes appear to be a result of interspecific reshuffling of intergenes.

Kolesnikova A.I., Putintseva Y.A., Simonov E.P., Biriukov V.V., Oreshkova N.V., Pavlov I.N., Sharov V.V., Kuzmin D.A., Anderson J.B., Krutovsky K.V.

Species in the genus Armillaria (fungi, basidiomycota) are well-known as saprophytes and pathogens on plants. Many of them cause white-rot root disease in diverse woody plants worldwide. Mitochondrial genomes (mitogenomes) are widely used in evolutionary and population studies, but despite the importance and wide distribution of Armillaria, the complete mitogenomes have not previously been reported for this genus. Meanwhile, the well-supported phylogeny of Armillaria species provides an excellent framework in which to study variation in mitogenomes and how they have evolved over time. Here we completely sequenced, assembled, and annotated the circular mitogenomes of four species: A. borealis, A. gallica, A. sinapina, and A. solidipes (116,443, 98,896, 103,563, and 122,167 bp, respectively). The variation in mitogenome size can be explained by variable numbers of mobile genetic elements, introns, and plasmid-related sequences. Most Armillaria introns contained open reading frames (ORFs) that are related to homing endonucleases of the LAGLIDADG and GIY-YIG families. Insertions of mobile elements were also evident as fragments of plasmid-related sequences in Armillaria mitogenomes. We also found several truncated gene duplications in all four mitogenomes. Our study showed that fungal mitogenomes have a high degree of variation in size, gene content, and genomic organization even among closely related species of Armillara. We suggest that mobile genetic elements invading introns and intergenic sequences in the Armillaria mitogenomes have played a significant role in shaping their genome structure. The mitogenome changes we describe here are consistent with widely accepted phylogenetic relationships among the four species.

Pogoda C.S., Keepers K.G., Nadiadi A.Y., Bailey D.W., Lendemer J.C., Tripp E.A., Kane N.C.

Reductions in genome size and complexity are a hallmark of obligate symbioses. The mitochondrial genome displays clear examples of these reductions, with the ancestral alpha-proteobacterial genome size and gene number having been reduced by orders of magnitude in most descendent modern mitochondrial genomes. Here, we examine patterns of mitochondrial evolution specifically looking at intron size, number, and position across 58 species from 21 genera of lichenized Ascomycete fungi, representing a broad range of fungal diversity and niches. Our results show that the cox1gene always contained the highest number of introns out of all the mitochondrial protein-coding genes, that high intron sequence similarity (>90%) can be maintained between different genera, and that lichens have undergone at least two instances of complete, genome-wide intron loss consistent with evidence for genome streamlining via loss of parasitic, noncoding DNA, in Phlyctis boliviensisand Graphis lineola. Notably, however, lichenized fungi have not only undergone intron loss but in some instances have expanded considerably in size due to intron proliferation (e.g., Alectoria fallacina and Parmotrema neotropicum), even between closely related sister species (e.g., Cladonia). These results shed light on the highly dynamic mitochondrial evolution that is occurring in lichens and suggest that these obligate symbiotic organisms are in some cases undergoing recent, broad-scale genome streamlining via loss of protein-coding genes as well as noncoding, parasitic DNA elements.

Wai A., Shen C., Carta A., Dansen A., Crous P.W., Hausner G.

In the mitochondrial genomes of the filamentous Ascomycota, aside from the usual 'core' set of genes, one can encounter genes encoding for ribosomal protein S3 (rps3), N-acetyltransferase, and in a few instances aminotransferases. Based on a survey using sequence data from various databases, it was observed that these genes can be located within introns or exist as freestanding genes in intergenic regions. Furthermore, they can also be absent from fungal mitochondrial genomes. The rps3 gene is highly conserved among fungal mitochondrial genomes although examples were noted where the mtDNA version of this gene has been translocated into the nuclear genome. The N-acetyltransferase gene was less frequently encountered and may be a more recent import from the nuclear genome. Both genes serve as examples of genetic elements that appear to be capable of 'cycling' or mobilizing between introns and intergenic regions and possible between the nuclear and mitochondrial genomes. This 'cycling' mechanism is currently not understood but may involve recombination events and/or movement via RNA intermediates.

Parenteau J., Maignon L., Berthoumieux M., Catala M., Gagnon V., Abou Elela S.

Introns are ubiquitous features of all eukaryotic cells. Introns need to be removed from nascent messenger RNA through the process of splicing to produce functional proteins. Here we show that the physical presence of introns in the genome promotes cell survival under starvation conditions. A systematic deletion set of all known introns in budding yeast genes indicates that, in most cases, cells with an intron deletion are impaired when nutrients are depleted. This effect of introns on growth is not linked to the expression of the host gene, and was reproduced even when translation of the host mRNA was blocked. Transcriptomic and genetic analyses indicate that introns promote resistance to starvation by enhancing the repression of ribosomal protein genes that are downstream of the nutrient-sensing TORC1 and PKA pathways. Our results reveal functions of introns that may help to explain their evolutionary preservation in genes, and uncover regulatory mechanisms of cell adaptations to starvation.Transcriptomic and genetic analyses of a deletion set of all known introns in genes of the budding yeast Saccharomyces cerevisiae indicate that introns promote resistance to starvation.

Brankovics B., Kulik T., Sawicki J., Bilska K., Zhang H., de Hoog G.S., van der Lee T.A., Waalwijk C., van Diepeningen A.D.

There is a gradual shift from representing a species’ genome by a single reference genome sequence to a pan-genome representation. Pan-genomes are the abstract representations of the genomes of all the strains that are present in the population or species. In this study, we employed a pan-genomic approach to analyze the intraspecific mitochondrial genome diversity of Fusarium graminearum. We present an improved reference mitochondrial genome for F. graminearum with an intron-exon annotation that was verified using RNA-seq data. Each of the 24 studied isolates had a distinct mitochondrial sequence. Length variation in the F. graminearum mitogenome was found to be largely due to variation of intron regions (99.98%). The “intronless” mitogenome length was found to be quite stable and could be informative when comparing species. The coding regions showed high conservation, while the variability of intergenic regions was highest. However, the most important variable parts are the intron regions, because they contain approximately half of the variable sites, make up more than half of the mitogenome, and show presence/absence variation. Furthermore, our analyses show that the mitogenome of F. graminearum is recombining, as was previously shown in F. oxysporum, indicating that mitogenome recombination is a common phenomenon in Fusarium. The majority of mitochondrial introns in F. graminearum belongs to group I introns, which are associated with homing endonuclease genes (HEGs). Mitochondrial introns containing HE genes may spread within populations through homing, where the endonuclease recognizes and cleaves the recognition site in the target gene. After cleavage of the “host” gene, it is replaced by the gene copy containing the intron with HEG. We propose to use introns unique to a population for tracking the spread of the given population, because introns can spread through vertical inheritance, recombination as well as via horizontal transfer. We demonstrate how pooled sequencing of strains can be used for mining mitogenome data. The usage of pooled sequencing offers a scalable solution for population analysis and for species level comparisons studies. This study may serve as a basis for future mitochondrial genome variability studies and representations.

Zubaer A., Wai A., Hausner G.

Endoconidiophora resinifera (=Ceratocystis resinifera) is a blue-stain fungus that occurs on conifers. The data showed that the Endoconidiophora resinifera mitochondrial genome is one of the largest mitochondrial genomes (>220 kb) so far reported among members of the Ascomycota. An exceptional large number of introns (81) were noted and differences among the four strains were restricted to minor variations in intron numbers and a few indels and single nucleotide polymorphisms. The major differences among the four strains examined are due to size polymorphisms generated by the absence or presence of mitochondrial introns. Also, these mitochondrial genomes encode the largest cytochrome oxidase subunit 1 gene (47.5 kb) reported so far among the fungi. The large size for this gene again can be attributed to the large number of intron insertions. This study reports the first mitochondrial genome for the genus Endoconidiophora, previously members of this genus were assigned to Ceratocystis. The latter genus has recently undergone extensive taxonomic revisions and the mitochondrial genome might provide loci that could be applied as molecular markers assisting in the identification of taxa within this group of economically important fungi. The large mitochondrial genome also may provide some insight on mechanisms that can lead to mitochondrial genome expansion.

Ahrendt S.R., Quandt C.A., Ciobanu D., Clum A., Salamov A., Andreopoulos B., Cheng J., Woyke T., Pelin A., Henrissat B., Reynolds N.K., Benny G.L., Smith M.E., James T.Y., Grigoriev I.V.

Environmental DNA surveys reveal that most fungal diversity represents uncultured species. We sequenced the genomes of eight uncultured species across the fungal tree of life using a new single-cell genomics pipeline. We show that, despite a large variation in genome and gene space recovery from each single amplified genome (SAG), ≥90% can be recovered by combining multiple SAGs. SAGs provide robust placement for early-diverging lineages and infer a diploid ancestor of fungi. Early-diverging fungi share metabolic deficiencies and show unique gene expansions correlated with parasitism and unculturability. Single-cell genomics holds great promise in exploring fungal diversity, life cycles and metabolic potential. Single-cell genome sequencing of eight uncultured fungal species provides insights into the phylogenetic placement of early-diverging lineages, highlights metabolic deficiencies and identifies gene expansions correlated with parasitism and unculturability.

Korovesi A.G., Ntertilis M., Kouvelis V.N.

The nuclear ribosomal protein S3 (Rps3) is implicated in the assembly of the ribosomal small subunit. Fungi and plants present a gene copy in their mitochondrial (mt) genomes. An analysis of 303 complete fungal mt genomes showed that, when rps3 is found, it is either a free-standing gene or an anchored gene within the omega intron of the rnl gene. Early divergent fungi, Basidiomycota and all yeasts but the CTG group belong to the first case, and Pezizomycotina to the second. Its position, size and genetic code employed are conserved within species of the same Order. Size variability is attributed to different number of repeats. These repeats consist of AT-rich sequences. MtRps3 proteins lack the KH domain, necessary for binding to rRNA, in their N-terminal region. Their C-terminal region is conserved in all Domains of life. Phylogenetic analysis showed that nuclear and mtRps3 proteins are descendants of archaeal and a-proteobacterial homologues, respectively. Thus, fungal mt-rps3 gene is an ancient gene which evolved within the endosymbiotic model and presents different evolutionary routes: (a) coming from a-proteobacteria, it was relocated to another region of the mt genome, (b) via its insertion to the omega intron, it was transferred to the nucleus and/or got lost, and (c) it was re-routed to the mt genome again. Today, Basidiomycota and Saccharomycetales seem to follow the first evolutionary route and almost all Pezizomycotina support the second scenario with their exceptions being the result of the third scenario, i.e., the gene's re-entry to the mt genome.

Deng Y., Hsiang T., Li S., Lin L., Wang Q., Chen Q., Xie B., Ming R.

Mitochondrial DNA (mtDNA) is a core non-nuclear genetic material found in all eukaryotic organisms, the size of which varies extensively in the eumycota, even within species. In this study, mitochondrial genomes of six isolates of Annulohypoxylon stygium (Lév.) were assembled from raw reads from PacBio and Illumina sequencing. The diversity of genomic structures, conserved genes, intergenic regions and introns were analyzed and compared. Genome sizes ranged from 132 kb to 147 kb and contained the same sets of conserved protein-coding, tRNA and rRNA genes and shared the same gene arrangements and orientation. In addition, most intergenic regions were homogeneous and had similar sizes except for the region between cytochrome b (cob) and cytochrome c oxidase I (cox1) genes which ranged from 2,998 bp to 8,039 bp among the six isolates. Sixty-five intron insertion sites and 99 different introns were detected in these genomes. Each genome contained 45 or more introns, which varied in distribution and content. Introns from homologous insertion sites also showed high diversity in size, type and content. Comparison of introns at the same loci showed some complex introns, such as twintrons and ORF-less introns. There were 44 short fragment insertions detected within introns, intergenic regions, or as introns, some of them located at conserved domain regions of homing endonuclease genes. Insertions of short fragments such as small inverted repeats might affect or hinder the movement of introns, and these allowed for intron accumulation in the mitochondrial genomes analyzed, and enlarged their size. This study showed that the evolution of fungal mitochondrial introns is complex, and the results suggest short fragment insertions as a potential factor leading to larger mitochondrial genomes in A. stygium.

Novikova O., Belfort M.

The duality of group II introns, capable of carrying out both self-splicing and retromobility reactions, is hypothesized to have played a profound role in the evolution of eukaryotes. These introns likely provided the framework for the emergence of eukaryotic retroelements, spliceosomal introns and other key components of the spliceosome. Group II introns are found in all three domains of life and are therefore considered to be exceptionally successful mobile genetic elements. Initially identified in organellar genomes, group II introns are found in bacteria, chloroplasts, and mitochondria of plants and fungi, but not in nuclear genomes. Although there is no doubt that prokaryotic and organellar group II introns are evolutionary related, there are remarkable differences in survival strategies between them. Furthermore, an evolutionary relationship of group II introns to eukaryotic retroelements, including telomeres, and spliceosomes is unmistakable.

Zhao C., Pyle A.M.

While the major architectural features and active-site components of group II introns have been known for almost a decade, information on the individual stages of splicing has been lacking. Recent advances in crystallography and cryo-electron microscopy (cryo-EM) have provided major new insights into the structure of intact lariat introns. Conformational changes that mediate the steps of splicing and retrotransposition are being elucidated, revealing the dynamic, highly coordinated motions that are required for group II intron activity. Finally, these ribozymes can now be viewed in their larger, more natural context as components of holoenzymes that include encoded maturase proteins. These studies expand our understanding of group II intron structural diversity and evolution, while setting the stage for rigorous mechanistic analysis of RNA splicing machines.

de Assis P.M., Rodrigues-Oliveira I.H., Batista da Silva I., Pasa R., Menegidio F.B., Kavalco K.F.

Li M., Sun X., Liu Y., Qin S., Li M., He X.

Pleosporales , the largest order in Dothideomycetes, has a broad host range and inhabits host plants as epiphytes, endophytes, parasites and saprophytes. Trematosphaeriaceae is a monophyletic family in Pleosporales, composed of species of deviated ecological background and morphological traits. In this study, we described a new fungal taxon under Trematosphaeriaceae, based on root endophytic fungi recovered from the desert plant Gymnocarpos przewalskii in Gansu Province, China. The taxon is characterised by simple, aseptate conidia and pycnidia in unusually small sizes. Multilocus phylogenetic analysis, based on ITS, LSU, SSU and TEF sequences and a morphology study indicated that the taxon represented a new genus within the Trematosphaeriaceae and was named Nigromargarita tarda. Intriguingly, an intron of 355 bp in length located at site 453 on the ribosomal SSU gene was detected in one strain of N. tarda. Sequence analysis and phylogenetic analysis indicated that the intron belongs to an intron position class (Pcl) restricted to Pleosporales. Phylogeny affiliated distribution of this Pcl was confined at the genus or lower level, suggesting a horizontal transmission pattern of this Pcl. This study established a new genus in Trematosphaeriaceae and depicted the spread features of a less-documented Pcl amongst Pleosporales families with high resolution, which promotes our understanding of the origin and transmission mechanism of such mobile genetic elements.

Xie S., Ma X., Wu H., Zang R., Li H., Liu M., Li Q., Ma Q., Guo Y., Zhang M.

In this study, the complete mitogenomes of three Diaporthe species (Diaporthe eres ZM79-3, D. phaseolorum ZM33-4 and Diaporthe sp. ZM41-5) were sequenced, assembled and compared with the other three previously sequenced Diaporthe mitogenomes (D. caulivora VNIIKR SE Dcaul3, D. longicolla MSPL 10-6 and D. sojae VNIIKR SE Dps12). The six Diaporthe mitogenomes were found to be circular DNA molecules, with lengths ranging from 53,646 bp to 108,865 bp. The mitogenomes of the six Diaporthe species mainly comprised the same set of 15 core protein-coding genes (PCGs), two rRNAs, and a certain number of tRNAs and unidentified open reading frames (ORFs). The PCG length, AT skew and GC skew showed large variability among the 15 PCGs in the six mitogenomes. The nad1 gene had the least K2P genetic distance of the 15 core PCGs among the 13 Diaporthales species, indicating that this gene was highly conserved. The Ka/Ks values for all 15 core PCGs were < 1, suggesting that these genes were all subject to purifying selection. Comparative mitogenome analysis showed that introns contributed the most to the size variation of Diaporthe mitogenomes. Frequent intron loss/gain events were detected to have occurred in the cox1 gene during the evolution of the Diaporthales mitogenomes. Although the mitogenomes of 13 species from Diaporthales had undergone large-scale gene rearrangements, six mitogenomes of Diaporthe species had identical gene arrangements. Phylogenetic analysis based on combined mitochondrial gene datasets showed that the six Diaporthe species formed well-supported topologies. To our knowledge, this study is the first report on the mitogenomes of D. phaseolorum ZM33-4 and Diaporthe sp. ZM41-5, as well as the first comparison of mitogenomes among Diaporthe species. Our findings will further promote investigations of the genetics, evolution and phylogeny of the Diaporthe species.

Tao G., Ahrendt S., Miyauchi S., Zhu X., Peng H., Labutti K., Clum A., Hayes R., Chain P.S., Grigoriev I.V., Bonito G., Martin F.M.

Morchella species have considerable significance in terrestrial ecosystems, exhibiting a range of ecological lifestyles along the saprotrophism-to-symbiosis continuum. However, the mitochondrial genomes of these ascomycetous fungi have not been thoroughly studied, thereby impeding a comprehensive understanding of their genetic makeup and ecological role. In this study, we analysed the mitogenomes of 30 Morchellaceae species, including yellow, black, blushing and false morels. These mitogenomes are either circular or linear DNA molecules with lengths ranging from 217 to 565 kbp and GC content ranging from 38% to 48%. Fifteen core protein-coding genes, 28–37 tRNA genes and 3–8 rRNA genes were identified in these Morchellaceae mitogenomes. The gene order demonstrated a high level of conservation, with the cox1 gene consistently positioned adjacent to the rnS gene and cob gene flanked by apt genes. Some exceptions were observed, such as the rearrangement of atp6 and rps3 in Morchella importuna and the reversed order of atp6 and atp8 in certain morel mitogenomes. However, the arrangement of the tRNA genes remains conserved. We additionally investigated the distribution and phylogeny of homing endonuclease genes (HEGs) of the LAGLIDADG (LAGs) and GIY-YIG (GIYs) families. A total of 925 LAG and GIY sequences were detected, with individual species containing 19–48HEGs. These HEGs were primarily located in the cox1, cob, cox2 and nad5 introns and their presence and distribution displayed significant diversity amongst morel species. These elements significantly contribute to shaping their mitogenome diversity. Overall, this study provides novel insights into the phylogeny and evolution of the Morchellaceae.

Trinca V., Silva S.R., Almeida J.V., Miranda V.F., Costa-Macedo J.V., Carnaval T.K., Araújo D.B., Prosdocimi F., Varani A.M.

Mimosa tenuiflora, popularly known as “Jurema-Preta”, is a perennial tree or shrub native to the tropical regions of the Americas, particularly among Afro-Brazilian and Indigenous Brazilian communities. Known for producing N,N-Dimethyltryptamine, a psychedelic compound with profound psychological effects, Jurema-Preta has been studied for its therapeutic potential in mental health. This study offers a comprehensive analysis of the plastid (ptDNA) and mitochondrion (mtDNA) genomes of M. tenuiflora. The 165,639 bp ptDNA sequence features the classical quadripartite structure with 130 protein-coding genes. Comparative genomics among Mimosa species shows high sequence identity in protein-coding genes, with variation in the rpoC1, clpP, ndhA, and ycf1 genes. The ptDNA junctions display distinct features, such as the deletion of the rpl22 gene, and specific simple sequence repeats highlight genetic variation and unique motifs as valuable genetic markers for population studies. Phylogenetic analysis places M. tenuiflora in the Caesalpinioideae, closely related to M. pigra and M. pudica. The 617,839 bp mtDNA sequence exhibits a complex structure with multiple genomic arrangements due to large repeats, encoding 107 protein-coding genes, including the ptDNA petG and psaA genes, and non-retroviral RNA mitoviruses sequences. Comparative analysis across Fabaceae species reveals limited conservation, emphasizing the dynamic nature of plant mitochondrial genomes. The genomic characterization of M. tenuiflora enhances understanding of its evolutionary dynamics, providing insights for population studies and potential applications in ethnopharmacology and conservation.

Schmoll M.

The genus Trichoderma comprises more than 400 species and fungi from diverse ecological niches as well as model fungi for different phenomena, such as plant cell wall degradation, biocontrol, or light response. The widespread uses that arise from these characteristics initiated applications in industry and agriculture, but also development of a plethora of molecular tools for genome manipulation and strain improvement with Trichoderma. Analysis of evolution with high-quality genome sequences revealed the acquisition of an important share of enzymes by horizontal gene transfer (HGT). The “core genome” of Trichoderma was narrowed down to 7000 genes and repeat-induced point mutation (RIP) was discovered as one mechanism for evolutionary genome modification. Genome sequencing studies further revealed numerous mitochondrial genomes and the presence of a surprising diversity of mycoviruses in Trichoderma genomes. Moreover, discoveries on the relevance of long non-coding RNA (lncRNA) and small RNAs as well as selected transcriptome and proteome studies are discussed. Besides new insights from comparative analysis of genomes, genome mining yielded new enzymes, pathways, and progress in the investigation of secondary metabolism of diverse species of the genus.

van Westerhoven A.C., Dijkstra J., Aznar Palop J.L., Wissink K., Bell J., Kema G.H., Seidl M.F.

ABSTRACT Mitochondria are present in almost all eukaryotic lineages. The mitochondrial genomes (mitogenomes) evolve separately from nuclear genomes, and they can therefore provide relevant insights into the evolution of their host species. Fusarium oxysporum is a major fungal plant pathogen that is assumed to reproduce clonally. However, horizontal chromosome transfer between strains can occur through heterokaryon formation, and recently, signs of sexual recombination have been observed. Similarly, signs of recombination in F. oxysporum mitogenomes challenged the prevailing assumption of clonal reproduction in this species. Here, we construct, to our knowledge, the first fungal pan-mitogenome graph of nearly 500 F . oxysporum mitogenome assemblies to uncover the variation and evolution. In general, the gene order of fungal mitogenomes is not well conserved, yet the mitogenome of F. oxysporum and related species are highly colinear. We observed two strikingly contrasting regions in the F. oxysporum pan-mitogenome, comprising a highly conserved core mitogenome and a long variable region (6–16 kb in size), of which we identified three distinct types. The pan-mitogenome graph reveals that only five intron insertions occurred in the core mitogenome and that the long variable regions drive the difference between mitogenomes. Moreover, we observed that their evolution is neither concurrent with the core mitogenome nor with the nuclear genome. Our large-scale analysis of long variable regions uncovers frequent recombination between mitogenomes, even between strains that belong to different taxonomic clades. This challenges the common assumption of incompatibility between genetically diverse F. oxysporum strains and provides new insights into the evolution of this fungal species. IMPORTANCE Insights into plant pathogen evolution is essential for the understanding and management of disease. Fusarium oxysporum is a major fungal pathogen that can infect many economically important crops. Pathogenicity can be transferred between strains by the horizontal transfer of pathogenicity chromosomes. The fungus has been thought to evolve clonally, yet recent evidence suggests active sexual recombination between related isolates, which could at least partially explain the horizontal transfer of pathogenicity chromosomes. By constructing a pan-genome graph of nearly 500 mitochondrial genomes, we describe the genetic variation of mitochondria in unprecedented detail and demonstrate frequent mitochondrial recombination. Importantly, recombination can occur between genetically diverse isolates from distinct taxonomic clades and thus can shed light on genetic exchange between fungal strains.

van Ingen-Buijs V.A., van Westerhoven A.C., Skiadas P., Zuijdgeest X.C., Haridas S., Daum C., Duffy K., Guo J., Hundley H., LaButti K., Lipzen A., Pangilinan J., Riley R., Wang J., Yan M., et. al.

Phyllosticta citricarpa is an important citrus-pathogen and a quarantine organism in the European Union. Its recently described relative, P. paracitricarpa, is very closely related and not listed as a quarantine organism. P. paracitricarpa is very difficult to distinguish from P. citricarpa, since its morphological features overlap and the barcoding gene sequences that were originally used to delimit them as distinct species have a low number of species-specific polymorphisms that have subsequently been shown to overlap between the two clades. Therefore, we performed extensive genomic analyses to determine whether the genetic variation between P. citricarpa and P. paracitricarpa strains should be considered to represent infraspecific variation within P. citricarpa, or whether it is indicative of distinct species. Using a phylogenomic analysis with 3,000 single copy ortholog genes and whole-genome comparisons, we determined that the variation between P. citricarpa and P. paracitricarpa can be considered as infraspecies variation within P. citricarpa. We also determined the level of variation in mitochondrial assemblies of several Phyllosticta species and concluded there are only minimal differences between the assemblies of P. citricarpa and P. paracitricarpa. Thus, using several orthogonal approaches, we here demonstrate that variation within the nuclear and mitochondrial genomes of other Phyllosticta species is larger than variation between genomes obtained from P. citricarpa and P. paracitricarpa strains. Thus, P. citricarpa and P. paracitricarpa should be considered as conspecific.

Yu Y., Liu T., Wang Y., Liu L., He X., Li J., Martin F.M., Peng W., Tan H.

Pleurotus pulmonarius, commonly known as the mini oyster mushroom, is highly esteemed for its crisp texture and umami flavor. Limited genetic diversity among P. pulmonarius cultivars raises concerns regarding its sustainable industrial production. To delve into the maternal genetic diversity of the principal P. pulmonarius cultivars, 36 cultivars and five wild isolates were subjected to de novo sequencing and assembly to generate high-quality mitogenome sequences. The P. pulmonarius mitogenomes had lengths ranging from 69,096 to 72,905 base pairs. The mitogenome sizes of P. pulmonarius and those of other mushroom species in the Pleurotus genus showed a significant positive correlation with the counts of LAGLIDAG and GIY-YIG homing endonucleases encoded by intronic open reading frames. A comparison of gene arrangements revealed an inversion of a fragment containing atp9-nad3-nad2 between P. pulmonarius and P. ostreatus. The mitogenomes of P. pulmonarius were clustered into three distinct clades, two of which were crowded with commercial cultivars. Clade I, all of which possess an inserted dpo gene, shared a maternal origin linked to an ancestral cultivar from Taiwan. Primers were designed to target the dpo gene, potentially safeguarding intellectual property rights. The wild isolates in Clade III exhibited more divergent mitogenomes, rendering them valuable for breeding.

Yang M., Xie Y., Zhu L., Li X., Xu W.

Mukhopadhyay J., Hausner G.

Group I and II introns are large catalytic RNAs (ribozymes) that are frequently encountered in fungal mitochondrial genomes. The discovery of respiratory mutants linked to intron splicing defects demonstrated that for the efficient removal of organellar introns there appears to be a requirement of protein splicing factors. These splicing factors can be intron-encoded proteins with maturase activities that usually promote the splicing of the introns that encode them ( cis-acting) and/or nuclear-encoded factors that can promote the splicing of a range of different introns ( trans-acting). Compared to plants organellar introns, fungal mitochondrial intron splicing is still poorly explored, especially in terms of the synergy of nuclear factors with intron-encoded maturases that has direct impact on splicing through their association with intron RNA. In addition, nuclear-encoded accessory factors might drive the splicing impetus through translational activation, mitoribosome assembly, and phosphorylation-mediated RNA turnover. This review explores protein-assisted splicing of introns by nuclear and mitochondrial-encoded maturases as a means of mitonuclear interplay that could respond to environmental and developmental factors promoting phenotypic adaptation and potentially speciation. It also highlights key evolutionary events that have led to changes in structure and ATP-dependence to accommodate the dual functionality of nuclear and organellar splicing factors.

Mendoza H., Lamb E.A., Thomas J., Tavares D.G., Schroeder L.A., Müller C., Agrawal N., Schirawski J., Perlin M.H.

IntroductionModern understanding of the concept of genetic diversity must include the study of both nuclear and organellar DNA, which differ greatly in terms of their structure, organization, gene content and distribution. This study comprises an analysis of the genetic diversity of the smut fungus Sporisorium reilianum f. sp. zeae from a mitochondrial perspective.MethodsWhole-genome sequencing data was generated from biological samples of S. reilianum collected from different geographical regions. Multiple sequence alignment and gene synteny analysis were performed to further characterize genetic diversity in the context of mitogenomic polymorphisms.ResultsMitochondria of strains collected in China contained unique sequences. The largest unique sequence stretch encompassed a portion of cox1, a mitochondrial gene encoding one of the subunits that make up complex IV of the mitochondrial electron transport chain. This unique sequence had high percent identity to the mitogenome of the related species Sporisorium scitamineum and Ustilago bromivora.DiscussionThe results of this study hint at potential horizontal gene transfer or mitochondrial genome recombination events during the evolutionary history of basidiomycetes. Additionally, the distinct polymorphic region detected in the Chinese mitogenome provides the ideal foundation to develop a diagnostic method to discern between mitotypes and enhance knowledge on the genetic diversity of this organism.

Li H., Liang T., Liu Y., Wang P., Wang S., Zhao M., Zhang Y.

Limited exploration in fungal mitochondrial genetics has uncovered diverse inheritance modes. The mitochondrial genomes are inherited uniparentally in the majority of sexual eukaryotes, our discovery of persistent mitochondrial heterogeneity within the natural population of the basidiomycete fungus Thelephora ganbajun represents a significant advance in understanding mitochondrial inheritance and evolution in eukaryotes. Here, we present a comprehensive analysis by sequencing and assembling the complete mitogenomes of 40 samples exhibiting diverse cox1 heterogeneity patterns from various geographical origins. Additionally, we identified heterogeneous variants in the nad5 gene, which, similar to cox1, displayed variability across multiple copies. Notably, our study reveals a distinct prevalence of introns and homing endonucleases in these heterogeneous genes. Furthermore, we detected potential instances of horizontal gene transfer involving homing endonucleases. Population genomic analyses underscore regional variations in mitochondrial genome composition among natural samples exhibiting heterogeneity. Thus, polymorphisms in heterogeneous genes, introns, and homing endonucleases significantly influence mitochondrial structure, structural variation, and evolutionary dynamics in this species. This study contributes valuable insights into mitochondrial genome architecture, population dynamics, and the evolutionary implications of mitochondrial heterogeneity in sexual eukaryotes.

van Westerhoven A., Dijkstra J., Aznar Palop L., Wissink K., Bell J., Kema G., Seidl M.F.

AbstractMitochondria are present in almost all eukaryotic lineages. The mitochondrial genomes (mitogenomes) evolve separately from nuclear genomes, and they can therefore provide relevant insights into the evolution of their host species.Fusarium oxysporumis a major fungal plant pathogen that is assumed to reproduce clonally. However, horizontal chromosome transfer between strains can occur through heterokaryon formation, and recently signs of sexual recombination have been observed. Similarly, signs of recombination inF. oxysporummitogenomes challenged the prevailing assumption of clonal reproduction in this species. Here, we construct, to our knowledge, the first fungal pan-mitogenome graph of nearly 500F. oxysporummitogenome assemblies to uncover the variation and evolution. In general, the gene order of fungal mitogenomes is not well conserved, yet the mitogenome ofF. oxysporumand related species are highly co-linear. We observed two strikingly contrasting regions in theFusarium oxysporumpan-mitogenome, comprising a highly conserved core mitogenome and a long variable region (6-16 kb in size), of which we identified three distinct types. The pan-mitogenome graph reveals that only five intron insertions occurred in the core mitogenome and that the long variable regions drive the difference between mitogenomes. Moreover, we observed that their evolution is neither concurrent with the core mitogenome nor with the nuclear genome. Our large-scale analysis of long variable regions uncovers frequent recombination between mitogenomes, even between strains that belong to different taxonomic clades. This challenges the common assumption of incompatibility between genetically diverseF. oxysporumstrains and provides new insights into the evolution of this fungal species.Importance statementInsights into plant pathogen evolution is essential for the understanding and management of disease.Fusarium oxysporumis a major fungal pathogen that can infect many economically important crops. Pathogenicity can be transferred between strains by the horizontal transfer of pathogenicity chromosomes. The fungus has been thought to evolve clonally, yet recent evidence suggests active sexual recombination between related isolates, which could at least partially explain the horizontal transfer of pathogenicity chromosomes. By constructing a pan-genome graph of nearly 500 mitochondrial genomes, we describe the genetic variation of mitochondria in unprecedented detail and demonstrate frequent mitochondrial recombination. Importantly, recombination can occur between genetically diverse isolates from distinct taxonomic clades and thus can shed light on genetic exchange between fungal strains.

Song X., Geng Y., Xu C., Li J., Guo Y., Shi Y., Ma Q., Li Q., Zhang M.

AbstractIn the present study, three mitogenomes from the Bipolaris genus (Bipolaris maydis, B. zeicola, and B. oryzae) were assembled and compared with the other two reported Bipolaris mitogenomes (B. oryzae and B. sorokiniana). The five mitogenomes were all circular DNA molecules, with lengths ranging from 106,403 bp to 135,790 bp. The mitogenomes of the five Bipolaris species mainly comprised the same set of 13 core protein-coding genes (PCGs), two rRNAs, and a certain number of tRNAs and unidentified open reading frames (ORFs). The PCG length, AT skew and GC skew showed large variability among the 13 PCGs in the five mitogenomes. Across the 13 core PCGs tested, nad6 had the least genetic distance among the 16 Pleosporales species we investigated, indicating that this gene was highly conserved. In addition, the Ka/Ks values for all 12 core PCGs (excluding rps3) were < 1, suggesting that these genes were subject to purifying selection. Comparative mitogenomic analyses indicate that introns were the main factor contributing to the size variation of Bipolaris mitogenomes. The introns of the cox1 gene experienced frequent gain/loss events in Pleosporales species. The gene arrangement and collinearity in the mitogenomes of the five Bipolaris species were almost highly conserved within the genus. Phylogenetic analysis based on combined mitochondrial gene datasets showed that the five Bipolaris species formed well-supported topologies. This study is the first report on the mitogenomes of B. maydis and B. zeicola, as well as the first comparison of mitogenomes among Bipolaris species. The findings of this study will further advance investigations into the population genetics, evolution, and genomics of Bipolaris species.