Mitogenomics and mitochondrial gene phylogeny decipher the evolution of Saccharomycotina yeasts

Hao W.

Analysis of genome variation provides insights into mechanisms in genome evolution. This is increasingly appreciated with the rapid growth of genomic data. Mitochondrial genomes (mitogenomes) are well known to vary substantially in many genomic aspects, such as genome size, sequence context, nucleotide base composition and substitution rate. Such substantial variation makes mitogenomes an excellent model system to study the mechanisms dictating mitogenome variation. Recent sequencing efforts have not only covered a rich number of yeast species but also generated genomes from abundant strains within the same species. The rich yeast genomic data have enabled detailed investigation from genome variation into molecular mechanisms in genome evolution. This mini-review highlights some recent progresses in yeast mitogenome studies.

Theelen B., Christinaki A.C., Dawson T.L., Boekhout T., Kouvelis V.N.

ABSTRACT Malassezia furfur is a yeast species belonging to Malasseziomycetes, Ustilaginomycotina and Basidiomycota that is found on healthy warm-blooded animal skin, but also involved in various skin disorders like seborrheic dermatitis/dandruff and pityriasis versicolor. Moreover, Malassezia are associated with bloodstream infections, Crohn's disease and pancreatic carcinoma. Recent advances in Malassezia genomics and genetics have focused on the nuclear genome. In this work, we present the M. furfur mitochondrial (mt) genetic heterogenicity with full analysis of 14 novel and six available M. furfur mt genomes. The mitogenome analysis reveals a mt gene content typical for fungi, including identification of variable mt regions suitable for intra-species discrimination. Three of them, namely the trnK–atp6 and cox3–nad3 intergenic regions and intron 2 of the cob gene, were selected for primer design to identify strain differences. Malassezia furfur strains belonging to known genetic variable clusters, based on AFLP and nuclear loci, were assessed for their mt variation using PCR amplification and sequencing. The results suggest that these mt regions are excellent molecular markers for the typing of M. furfur strains and may provide added value to nuclear regions when assessing evolutionary relationships at the intraspecies level.

Mukhopadhyay J., Hausner G.

Introns are ubiquitous in eukaryotic genomes and have long been considered as ‘junk RNA’ but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed.

Ramsey J., Rasche H., Maughmer C., Criscione A., Mijalis E., Liu M., Hu J.C., Young R., Gill J.J.

In the modern genomic era, scientists without extensive bioinformatic training need to apply high-power computational analyses to critical tasks like phage genome annotation. At the Center for Phage Technology (CPT), we developed a suite of phage-oriented tools housed in open, user-friendly web-based interfaces. A Galaxy platform conducts computationally intensive analyses and Apollo, a collaborative genome annotation editor, visualizes the results of these analyses. The collection includes open source applications such as the BLAST+ suite, InterProScan, and several gene callers, as well as unique tools developed at the CPT that allow maximum user flexibility. We describe in detail programs for finding Shine-Dalgarno sequences, resources used for confident identification of lysis genes such as spanins, and methods used for identifying interrupted genes that contain frameshifts or introns. At the CPT, genome annotation is separated into two robust segments that are facilitated through the automated execution of many tools chained together in an operation called a workflow. First, the structural annotation workflow results in gene and other feature calls. This is followed by a functional annotation workflow that combines sequence comparisons and conserved domain searching, which is contextualized to allow integrated evidence assessment in functional prediction. Finally, we describe a workflow used for comparative genomics. Using this multi-purpose platform enables researchers to easily and accurately annotate an entire phage genome. The portal can be accessed at https://cpt.tamu.edu/galaxy-pub with accompanying user training material.

Nguyen D.T., Wu B., Xiao S., Hao W.

AbstractGenome-wide nucleotide composition varies widely among species. Despite extensive research, the source of genome-wide nucleotide composition diversity remains elusive. Yeast mitochondrial genomes (mitogenomes) are highly A + T rich, and they provide a unique opportunity to study the evolution of AT-biased landscape. In this study, we sequenced ten complete mitogenomes of the Saccharomycodes ludwigii yeast with 8% G + C content, the lowest genome-wide %(G + C) in all published genomes to date. The S. ludwigii mitogenomes have high densities of short tandem repeats but severely underrepresented mononucleotide repeats. Comparative population genomics of these record-setting A + T-rich genomes shows dynamic indel mutations and strong mutation bias toward A/T. Indel mutations play a greater role in genomic variation among very closely related strains than nucleotide substitutions. Indels have resulted in presence–absence polymorphism of tRNAArg (ACG) among S. ludwigii mitogenomes. Interestingly, these mitogenomes have undergone recombination, a genetic process that can increase G + C content by GC-biased gene conversion. Finally, the expected equilibrium G + C content under mutation pressure alone is higher than observed G + C content, suggesting existence of mechanisms other than AT-biased mutation operating to increase A/T. Together, our findings shed new lights on mechanisms driving extremely AT-rich genomes.

Hill G.E.

DNA barcoding based on mitochondrial (mt) nucleotide sequences is an enigma. Neutral models of mt evolution predict DNA barcoding cannot work for recently diverged taxa, and yet, mt DNA barcoding accurately delimits species for many bilaterian animals. Meanwhile, mt DNA barcoding often fails for plants and fungi. I propose that because mt gene products must cofunction with nuclear gene products, the evolution of mt genomes is best understood with full consideration of the two environments that impose selective pressure on mt genes: the external environment and the internal genomic environment. Moreover, it is critical to fully consider the potential for adaptive evolution of not just protein products of mt genes but also of mt transfer RNAs and mt ribosomal RNAs. The tight linkage of genes on mt genomes that do not engage in recombination could facilitate selective sweeps whenever there is positive selection on any element in the mt genome, leading to the purging of mt genetic diversity within a population and to the rapid fixation of novel mt DNA sequences. Accordingly, the most important factor determining whether or not mt DNA sequences diagnose species boundaries may be the extent to which the mt chromosomes engage in recombination.

Megarioti A.H., Kouvelis V.N.

Abstract Fungal mitochondrial (mt) genomes exhibit great diversity in size which is partially attributed to their variable intergenic regions and most importantly to the inclusion of introns within their genes. These introns belong to group I or II, and both of them are self-splicing. The majority of them carry genes encoding homing endonucleases, either LAGLIDADG or GIY-YIG. In this study, it was found that these intronic homing endonucleases genes (HEGs) may originate from mt free-standing open reading frames which can be found nowadays in species belonging to Early Diverging Fungi as “living fossils.” A total of 487 introns carrying HEGs which were located in the publicly available mt genomes of representative species belonging to orders from all fungal phyla was analyzed. Their distribution in the mt genes, their insertion target sequence, and the phylogenetic analyses of the HEGs showed that these introns along with their HEGs form a composite structure in which both selfish elements coevolved. The invasion of the ancestral free-standing HEGs in the introns occurred through a perpetual mechanism, called in this study as “aenaon” hypothesis. It is based on recombination, transpositions, and horizontal gene transfer events throughout evolution. HEGs phylogenetically clustered primarily according to their intron hosts and secondarily to the mt genes carrying the introns and their HEGs. The evolutionary models created revealed an “intron-early” evolution which was enriched by “intron-late” events through many different independent recombinational events which resulted from both vertical and horizontal gene transfers.

Cinget B., Bélanger R.R.

Group I catalytic introns are widespread in bacterial, archaeal, viral, organellar, and some eukaryotic genomes, where they are reported to provide regulatory functions. The group I introns are currently divided into five types (A-E), which are themselves distributed into several subtypes, with the exception of group I type D intron (GI-D). GI-D introns belong to the rarest group with only 17 described to date, including only one with a putative role reported in fungi, where it would interfere with an adaptive response in the cytochrome b (COB) gene to quinone outside inhibitor (QoI) fungicide resistance. Using homology search methods taking into account both conserved sequences and RNA secondary structures, we analysed the mitochondrial genomes or COB genes of 169 fungal species, including some frequently under QoI selection pressure. These analyses have led to the identification of 216 novel GI-D introns, and the definition of three distinct subtypes, one of which being linked with a functional activity. We have further uncovered a homing site for this GI-D intron type, which helps refine the accepted model of quinone outside inhibitor resistance, whereby mobility of the intron across fungal mitochondrial genomes, would influence a fungus ability to develop resistance to QoIs.

De Chiara M., Friedrich A., Barré B., Breitenbach M., Schacherer J., Liti G.

Mitochondria are essential organelles partially regulated by their own genomes. The mitochondrial genome maintenance and inheritance differ from the nuclear genome, potentially uncoupling their evolutionary trajectories. Here, we analysed mitochondrial sequences obtained from the 1011 Saccharomyces cerevisiae strain collection and identified pronounced differences with their nuclear genome counterparts. In contrast with pre-whole genome duplication fungal species, S. cerevisiae mitochondrial genomes show higher genetic diversity compared to the nuclear genomes. Strikingly, mitochondrial genomes appear to be highly admixed, resulting in a complex interconnected phylogeny with a weak grouping of isolates, whereas interspecies introgressions are very rare. Complete genome assemblies revealed that structural rearrangements are nearly absent with rare inversions detected. We tracked intron variation in COX1 and COB to infer gain and loss events throughout the species evolutionary history. Mitochondrial genome copy number is connected with the nuclear genome and linearly scale up with ploidy. We observed rare cases of naturally occurring mitochondrial DNA loss, petite, with a subset of them that do not suffer the expected growth defect in fermentable rich media. Overall, our results illustrate how differences in the biology of two genomes coexisting in the same cells can lead to discordant evolutionary histories.

Pote S.T., Sonawane M.S., Rahi P., Shah S.R., Shouche Y.S., Patole M., Thakar M.R., Sharma R.

Species of genus Candida are part of the common microbiota of humans; however, some of the Candida species are known opportunistic pathogens. Formation of biofilms, resistance to antifungal drugs, and increase in asymptomatic infections demands more studies on isolation, identification and characterization of Candida from clinical samples.The present manuscript deals with assessment of authentic yeast identification by three methods viz., DNA sequencing of 28S rRNA gene, protein profiles using MALDI-TOF MS, and colony coloration on chromogenic media. Antifungal susceptibility and in vitro cell invasion assays were performed to further characterize these isolates.Comparison of three methods showed that DNA sequence analysis correctly identified more than 99.4% of the isolates up to species level as compared to 89% by MALDI-TOF MS. In this study, we isolated a total of 176 yeasts from clinical samples and preliminary morphological characters indicated that these yeast isolates belong to the genus Candida. The species distribution of isolates was as follows: 75 isolates of Candida albicans (42.61%), 50 of C. tropicalis (28.40%), 22 of C. glabrata (12.5%), 14 of C. parapsilosis (7.95%) and 4 of Clavispora lusitaniae (2.27%). Other species like Cyberlindnera fabianii, Issatchenkia orientalis, Kluyveromyces marxianus, Kodamaea ohmeri, Lodderomyces sp., and Trichosporon asahii were less than 2%. Antifungal susceptibility assay performed with 157 isolates showed that most of the isolates were resistant to the four azoles viz., clotrimazole, fluconazole, itraconazole, and ketoconazole, and the frequency of resistance was more in non-albicans Candida isolates. The susceptibility to azole drugs ranged from 7% to 48%, while 75% of the tested yeasts were susceptible to nystatin. Moreover, 88 isolates were also tested for their capacity to invade human cells using HeLa cells. In vitro invasion assay showed that most of the C. albicans isolates showed epithelial cell invasion as compared to isolates belonging to C. glabrata, C. parapsilosis and C. tropicalis.The identification of yeasts of clinical origin by sequencing of 28S rRNA gene performed better than MALDI-TOF MS. The present study reiterates the world scenario wherein there is a shift from Candida strains to emerging opportunistic pathogens which were earlier regarded as environmental strains. The present study enlightens the current understanding of identification methods for clinical yeast isolates, increased antifungal drug resistance, epithelial cell invasion as a virulence factor, and diversity of yeasts in Indian clinical samples.

Schoch C.L., Ciufo S., Domrachev M., Hotton C.L., Kannan S., Khovanskaya R., Leipe D., Mcveigh R., O’Neill K., Robbertse B., Sharma S., Soussov V., Sullivan J.P., Sun L., Turner S., et. al.

AbstractThe National Center for Biotechnology Information (NCBI) Taxonomy includes organism names and classifications for every sequence in the nucleotide and protein sequence databases of the International Nucleotide Sequence Database Collaboration. Since the last review of this resource in 2012, it has undergone several improvements. Most notable is the shift from a single SQL database to a series of linked databases tied to a framework of data called NameBank. This means that relations among data elements can be adjusted in more detail, resulting in expanded annotation of synonyms, the ability to flag names with specific nomenclatural properties, enhanced tracking of publications tied to names and improved annotation of scientific authorities and types. Additionally, practices utilized by NCBI Taxonomy curators specific to major taxonomic groups are described, terms peculiar to NCBI Taxonomy are explained, external resources are acknowledged and updates to tools and other resources are documented.Database URL: https://www.ncbi.nlm.nih.gov/taxonomy

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.

LaBella A.L., Opulente D.A., Steenwyk J.L., Hittinger C.T., Rokas A.

Variation in synonymous codon usage is abundant across multiple levels of organization: between codons of an amino acid, between genes in a genome, and between genomes of different species. It is now well understood that variation in synonymous codon usage is influenced by mutational bias coupled with both natural selection for translational efficiency and genetic drift, but how these processes shape patterns of codon usage bias across entire lineages remains unexplored. To address this question, we used a rich genomic data set of 327 species that covers nearly one third of the known biodiversity of the budding yeast subphylum Saccharomycotina. We found that, while genome-wide relative synonymous codon usage (RSCU) for all codons was highly correlated with the GC content of the third codon position (GC3), the usage of codons for the amino acids proline, arginine, and glycine was inconsistent with the neutral expectation where mutational bias coupled with genetic drift drive codon usage. Examination between genes’ effective numbers of codons and their GC3 contents in individual genomes revealed that nearly a quarter of genes (381,174/1,683,203; 23%), as well as most genomes (308/327; 94%), significantly deviate from the neutral expectation. Finally, by evaluating the imprint of translational selection on codon usage, measured as the degree to which genes’ adaptiveness to the tRNA pool were correlated with selective pressure, we show that translational selection is widespread in budding yeast genomes (264/327; 81%). These results suggest that the contribution of translational selection and drift to patterns of synonymous codon usage across budding yeasts varies across codons, genes, and genomes; whereas drift is the primary driver of global codon usage across the subphylum, the codon bias of large numbers of genes in the majority of genomes is influenced by translational selection.

Nielsen J.

For thousands of years, the yeast Saccharomyces cerevisiae (S. cerevisiae) has served as a cell factory for the production of bread, beer, and wine. In more recent years, this yeast has also served as a cell factory for producing many different fuels, chemicals, food ingredients, and pharmaceuticals. S. cerevisiae, however, has also served as a very important model organism for studying eukaryal biology, and even today many new discoveries, important for the treatment of human diseases, are made using this yeast as a model organism. Here a brief review of the use of S. cerevisiae as a model organism for studying eukaryal biology, its use as a cell factory, and how advances in systems biology underpin developments in both these areas, is provided.

Chan P.P., Lowe T.M.

Transfer RNAs are the largest, most complex non-coding RNA family, universal to all living organisms. tRNAscan-SE has been the de facto tool for predicting tRNA genes in whole genomes. The newly developed version 2.0 has incorporated advanced methodologies with improved probabilistic search software and a suite of new gene models, enabling better functional classification of predicted genes. This chapter describes the use of the UNIX command-driven and online web versions, illustrating different search modes and options.

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.

Shen X., Cao X., Huang X., Zhuo L., Yang H., Fan L., Hou C.

Shiraia-related species are well-known bambusicolous fungi in Dothideomycetes class, with high value in traditional medicine for producing hypocrellin, as an anticipated photosensitiser. The complete mitogenomes of hypocrellin-producing Pseudoshiraia conidialis strains were analysed in the present study, with functional gene variations through comparative genomics and transcriptomics. Five strains (ZZZ816, CNUCC1353PR, JAP103846, CNUCC C72, CNUCC C151) were sequenced, which indicated similar genome characteristics. Two of them possess an extra atp6 gene, and the associated variable fragment “HSP1-HSP2-atp6_2” correlates closely with hypocrellin production capacity. Therefore, these five strains were divided into three groups: ZZZ816 and CNUCC1353PR possessing high production efficiency, CNUCC C72 and JAP103846 with low yield and CNUCC C151 as a transition type. The gene expression changes were screened under various conditions. ZZZ816-related species showed significant changes in mitochondrial genes, especially HSP1, HSP2 and atp6_2, linked closely to hypocrellin synthesis and stress response; rps3 expression also consistently correlated with hypocrellin production. JAP103846 group showed a stable expression pattern divergently, except for rps3 suppression by blue light. These findings would provide new insights into secondary metabolite regulation and ROS resistance. Above all, this study conducted the comprehensive analysis of Shiraia-like fungi mitogenomes and functional gene expression, which can update the understanding of fungal evolution and potential for improved hypocrellin production.

Liu C., Li W., Zheng L., Dao M., Chen H., Han L.

The genus Strobilomyces, representing a diverse and widespread group of ectomycorrhizal mushroom-forming fungi, plays a crucial ecological and economical role. However, until now, a comprehensive description of its mitochondrial genome (mitogenome) has been lacking. In our current study, we have successfully assembled and analysed the mitogenomes of five Strobilomyces species. These mitogenomes span a range from 35,618 base pairs (bp) to 42,088 bp, exhibiting a higher nucleotide abundance of AT compared to GC. All five mitogenomes harbour 14 conserved protein-coding genes (PCGs), two ribosomal RNAs (rRNAs) and 24 transfer RNAs (tRNAs). Notably, the overall ratio of Ka/Ks for all PCGs was found to be less than 1.0, indicating that these genes have undergone purifying selection during evolution. Intriguingly, the mitogenomic comparison revealed two instances of gene re-arrangement, which were directly linked to the geographical distribution of the Strobilomyces species. The concatenated mitochondrial PCGs (mtPCGs) and nuclear ribosomal DNA (nrDNA) phylogenies displayed a robust congruent topology at the family level. Specifically, the Strobilomyces species clustered together and formed sister relationship with other Boletaceae species in the mtPCGs tree. In contrast, the Strobilomyces species grouped at the base of the nrDNA tree when concerning Boletaceae. This study represents the first report on the mitogenomes of the Strobilomyces genus, providing valuable insights into fungal evolution within Boletales.

Ludwig C.D., Maughan P.J., Jellen E.N., Davis T.M.

AbstractHigh-quality nuclear, chloroplast, and preliminary mitochondrial genomes have been assembled and annotated for the B-genome diploid (BB: 2n = 2x = 18) figleaf goosefoot (Chenopodium ficifolium). The primary objective was to advance a simplified model system for genetic characterization and improvement of allotetraploid (AABB: 2n = 4x = 36) quinoa (Chenopodium quinoa), a nutritionally valuable, halophytic orphan crop. In addition to its diploidy and favorably small genome size, theC. ficifoliummodel provides a shorter generational period and smaller overall plant size as compared toC. quinoa, while displaying relevant agronomic trait variations amenable to gene-trait association studies. TheC. ficifolium‘Portsmouth’ nuclear genome was sequenced using PacBio HiFi Long Read technology and assembled using Hifiasm. After manual adjustments, the final ChenoFicP_1.0 assembly consisted of nine pseudochromosomes spanning 711.5 Mbp, while 22,617 genes were identified and annotated. BUSCO analyses indicated a nuclear genome completeness of 97.5%, and a proteome and transcriptome completeness of 98.4 percent. The chloroplast genome assembly detected two equally represented structural haplotypes differing in the orientation of the Short Single Copy region relative to the Long Single Copy region. Phylogenetic and parentage analyses pointed to an unspecified AA diploid species and away fromC. ficifoliumas the likely maternal chloroplast and mitochondrial genome donor(s) during the initial tetraploidization event in theC. quinoalineage. Using the new ChenoFicP_1.0 reference genome, a GWAS was performed on a previously studiedC. ficifoliumF2 population to further define region(s) implicated in the control of three key agronomic traits: days to flowering, plant height, and branch number. This analysis localized control of all three traits to a 7 Mb interval on pseudochromosome Cf4. This region contains approximately 770 genes, including theFTL1locus, thus confirming and extending our prior, single-marker analysis showing association of these three traits with anFTL1amplicon length polymorphism. The use of these data to further developC. ficifoliumas a model species for genetics and breeding of quinoa serves to expand knowledge and germplasm resources for quinoa improvement.

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.

Anikin M., Henry M.F., Hodorova V., Houbaviy H.B., Nosek J., Pestov D.G., Markov D.

Respiration in eukaryotes depends on mitochondrial protein synthesis, which is performed by organelle-specific ribosomes translating organelle-encoded mRNAs. Although RNA maturation and stability are central events controlling mitochondrial gene expression, many of the molecular details in this pathway remain elusive. These includecis-andtrans-regulatory factors that generate and protect the 3′ ends. Here, we mapped the 3′ ends of mitochondrial mRNAs of yeasts classified into multiple families of the subphylum Saccharomycotina. We found that the processing of mitochondrial 15S rRNA and mRNAs involves species-specific sequence elements, which we term 3′-end RNA processing elements (3′-RPEs). InSaccharomyces cerevisiae,the 3′-RPE has long been recognized as a conserved dodecamer sequence, which recent studies have shown specifically interacts with the nuclear genome-encoded pentatricopeptide repeat protein Rmd9. We also demonstrate that, analogous to Rmd9 inS. cerevisiae, two Rmd9 orthologs from theDebaryomycetaceaefamily interact with their respective 3′-RPEs found in mRNAs and 15S rRNA. Thus, Rmd9-dependent processing of mitochondrial RNA precursors may be a common mechanism among the families of the Saccharomycotina subphylum. Surprisingly, we observed that 3′-RPEs often occur upstream of stop codons in complex I subunit mRNAs from yeasts of the CUG-Ser1 clade. We examined two of these mature mRNAs and found that their stop codons are indeed removed. Thus, translation of these stop-codon-less transcripts would require a noncanonical termination mechanism. Our findings highlight Rmd9 as a key evolutionarily conserved factor in both mitochondrial mRNA metabolism and mitoribosome biogenesis in a variety of yeasts.

Terbot J.W., Soni V., Versoza C.J., Pfeifer S.P., Jensen J.D.

ABSTRACTThe nocturnal aye-aye,Daubentonia madagascariensis, is one of the most elusive lemurs on the island of Madagascar. The timing of its activity and arboreal lifestyle has generally made it difficult to obtain accurate assessments of population size using traditional census methods. Therefore, alternative estimates provided by population genetic inference are essential for yielding much needed information for conservation measures and for enabling ecological and evolutionary studies of this species. Here, we utilize genomic data from 17 unrelated individuals — including 5 newly sequenced, high-coverage genomes — to estimate this history. Essential to this estimation are recently published annotations of the aye-aye genome which allow for variation at putatively neutral genomic regions to be included in the estimation procedures, and regions subject to selective constraints, or in linkage to such sites, to be excluded owing to the biasing effects of selection on demographic inference. By comparing a variety of demographic estimation tools to develop a well-supported model of population history, we find strong support for the species to consist of two demes, separating northern Madagascar from the rest of the island. Additionally, we find that the aye-aye has experienced two severe reductions in population size. The first occurred rapidly, approximately 3,000 to 5,000 years ago, and likely corresponded with the arrival of humans to Madagascar. The second occurred over the past few decades and is likely related to substantial habitat loss, suggesting that the species is still undergoing population decline and remains at great risk for extinction.

Viot C.R.

AbstractThe textile use of cotton fibers from sister speciesGossypium arboreumL. (Tree Cotton) andG. herbaceumL. (Levant Cotton) began, respectively, in Asia in the eighth millennium BP and in Africa in the third millennium BP and probably earlier. Archaeological data show that cotton cultivation spread in the Antiquity over most tropical and subtropical regions suitable for agriculture in Afroeurasia. The geographical expansions of the two Old World cotton species appear very contrasted, in terms of their chronologies, in terms of the areas conquered, and as for their speed of diffusion.G. arboreumspread over humid or sub-humid regions of southern, western and eastern Asia atca. 0.8 to 1.7 km/yr, a rate close to that estimated for wheat from the Fertile Crescent to northwestern Europe during Antiquity, whileG. herbaceumspread over arid to sub-arid regions of Africa and western and central Asia, much later and more than five times faster thanG. arboreum. The very high speed of diffusion ofG. herbaceumcan be explained by the context and by the agricultural adaptations of this species but also leads to revisit the history of its domestication and beginning of cultivation.

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.

Ebdon S., Laetsch D.R., Vila R., Baird S.J., Lohse K.

AbstractMany closely related species continue to hybridise after millions of generations of divergence. However, the extent to which current patterning in hybrid zones connects back to the speciation process remains unclear: does evidence for current multilocus barriers support the hypothesis of speciation due to multilocus divergence? We analyse whole-genome sequencing data to investigate the speciation history of the scarce swallowtailsIphiclides podaliriusandI. feisthamelii, which abut at a narrow (∼25 km) contact zone north of the Pyrenees. We first quantify the heterogeneity of effective migration rate under a model of isolation with migration, using genomes sampled across the range to identify long-term barriers to gene flow. Secondly, we investigate the recent ancestry of individuals from the hybrid zone using genome polarisation and estimate the coupling coefficient under a model of a multilocus barrier. We infer a low rate of long-term gene flow fromI. feisthameliiintoI. podaliriusthe direction of which matches the admixture across the hybrid zone and complete reproductive isolation across≈33% of the genome. Our contrast of recent and long-term gene flow shows that regions of low recent hybridisation are indeed enriched for long-term barriers which maintain divergence between these hybridising sister species. This paves the way for future analysis of the evolution of reproductive isolation along the speciation continuum.Author summaryEfforts to understand how new species evolve typically approach the problem through either: 1) investigating patterns of genetic exchange across ’hybrid zones’ — where closely related species interbreed — or 2) modelling the demographic history of closely related species using geneological trees. Both approaches are capable of quantifying variation in genetic exchange, or ’gene flow’, along the genome to identify regions of reproductive isolation; yet they leverage genetic signatures across vastly different timescales. The former exploits very recent signatures, while the latter averages long-term signatures over the history of divergence. Hence, we can compare and contrast each approach to understand how patterns of gene flow change across the speciation continuum. Here we use this strategy to capture the speciation dynamics of a pair of hybridising papilionid butterflies. Our results show that not only are these species continuing to produce hybrids after more than a million years since the onset of divergence, but there is a significant degree of concordance between patterns of gene flow observed along the genome across time scales.

Tran Lu Y A., Ruault S., Daguin-Thiebaut C., Le Port A., Ballenghien M., Castel J., Gagnaire P., Bierne N., Arnaud-Haond S., Poitrimol C., Thiebaut E., Lallier F., Broquet T., Jollivet D., Bonhomme F., et. al.

AbstractHow the interplay of biotic and abiotic factors shapes current genetic diversity at the community level remains an open question, particularly in the deep sea. Comparative phylogeography of multiple species can reveal the influence of past climatic events, geographic barriers, and species life history traits on spatial patterns of genetic structure across lineages.Here, we conducted a comparative population genomic study on seven hydrothermal vent species co-distributed in the Back-arc Basins of the Southwest Pacific region. Using ddRAD-seq, we compared distribution range-wide genomic diversity patterns across species and discovered a shared zone of phylogeographic break. Although species exhibit congruent patterns of spatial structure, they also show variation in the degree of divergence among lineages across the suture zone. Additionally, two species have a sympatric contact zone in the Woodlark Basin.Demogenetic inference revealed shared histories of lineage divergence and secondary contact. Low levels of asymmetric gene flow probably occurred in most species between the Woodlark and North Fiji basins, but the exact location of contact zones varied from species to species. Results show that gene flow is heterogeneous across the genome, indicating possible partial reproductive isolation between lineages and early speciation.Our comparative study sheds light on the factors that shape genetic variation at the community level, and our findings enrich our understanding of deep-sea biogeography patterns. Emphasizing the pivotal role of historical and contemporary factors, our research underscores the necessity for a holistic approach, and in particular filling in the gaps regarding life history traits of deep-sea species (generation time, development type, duration of the larval phase).

Lemke O., Heineike B.M., Viknander S., Cohen N., Steenwyk J.L., Spranger L., Li F., Agostini F., Lee C.T., Aulakh S.K., Nielsen J., Rokas A., Berman J., Zelezniak A., Gossmann T.I., et. al.

AbstractThe functions of cells and proteins depend on their biochemical microenvironment. To understand how biochemical constraints shaped protein structural evolution, we coupled the extensive genetic and metabolic data from theSaccharomycotinasubphylum with the capability of AlphaFold2 to systematically predict protein structures from sequence. Determining how 11,269 enzyme structures catalysing 361 different metabolic reactions evolved over 400 million years alongside their molecular functions, we report that metabolism has shaped the structural evolution of enzymes at different levels: the organism’s overall metabolism; the topological organisation of the metabolic network; and each enzyme’s molecular properties. For example, structural evolution depends on each enzyme’s reaction mechanism, on the variability rather than the amount of metabolic flux, and on biosynthetic cost. Evolutionary cost-optimization is stronger on highly abundant enzymes and acts differently on different structural domains, with the exception of small-molecule binding sites, which are prioritised over other structural domains and lack cost-optimisation. Finally, while enzyme surfaces are less constrained, surface residues can also be exposed to positive selection for the co-evolution of protein-protein interaction sites. Accessing AlphaFold’s power to predict protein structures systematically and across species barriers, facilitating the integration of protein structures with functional genomics, we were thus able to map biological constraints which shape protein structural evolution at scale and over long timelines.

Varassas S.P., Amillis S., Pappas K.M., Kouvelis V.N.

Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this study, the mitochondrial DNA polymerase γ (Mip1) of the entomopathogenic fungus Metarhizium brunneum is characterized and analyzed with disruption experiments and its in silico interactions with key proteins implicated in mt gene transcription, i.e., mt RNA polymerase Rpo41 and mt transcription factor Mtf1. Disruption of mip1 gene and its partial expression influences cell growth, morphology, germination and stress tolerance. A putative in silico model of Mip1-Rpo41-Mtf1, which is known to be needed for the initiation of replication, was proposed and helped to identify potential amino acid residues of Mip1 that interact with the Rpo41-Mtf1 complex. Moreover, the reduced expression of mip1 indicates that Mip1 is not required for efficient transcription but only for replication. Functional differences between the M. brunneum Mip1 and its counterparts from Saccharomyces cerevisiae and higher eukaryotes are discussed.

Sawasawa W., van Huysduynen A.H., Gresham S., Sungani H., Rusuwa B., De Boeck G., Turner G.F., Ngochera M., Svardal H.

AbstractFish is an important source of animal protein for many people living around Lake Malawi. The evaluation of population structure and genetic diversity can yield useful information for management and conservation of fish species but is complicated in Lake Malawi by the close genetic relatedness of species of the recent cichlid adaptive radiation. In this study, we analysed whole-genome sequencing data of “true utaka”, a group of cichlids previously common in fisheries, but that has faced strong decline due to overfishing. Our analysis of 223 individuals collected from fishermen’s catches along the western shoreline of Lake Malawi confirmed thatCopadichromis mloto(C. sp. “virginalis kajose”) is the true utaka most targeted by fisheries. Genetic principal component analysis, phylogenetic inference, and admixture analysis revealed complex patterns of population structure. The presence of at least three geographically widespread genetic clades that have remained separate despite gene flow and partial sympatry hints at the presence of currently undescribed, cryptic species diversity inC. mloto. This result leads us to suggest that, despite the lack of obvious habitat barriers, benthic and pelagic species of Malawi cichlids might harbour unidentified species diversity and calls for further genetic and taxonomic research to define appropriate conservation units.

Braichenko S., Borges R., Kosiol C.

AbstractThe role of balancing selection is a long-standing evolutionary puzzle. Balancing selection is a crucial evolutionary process that maintains genetic variation (polymorphism) over extended periods, however, detecting it poses a significant challenge. Building upon the polymorphism-aware phylogenetic models (PoMos) framework, we introduce PoMoBalance designed to disentangle the interplay of mutation, genetic drift, directional and balancing selection pressures influencing population diversity. Rooted in the Moran model, PoMos have demonstrated efficiency in species tree inference, capturing mutational effects, fixation biases, and GC-bias rates. Implemented in the open-source RevBayes Bayesian framework, PoMoBalance offers a versatile tool for multi-individual data analysis. This study extends PoMos’ capabilities to explore balancing selection and disentangle it from GC-biased gene conversion. The novel aspect of our approach in studying balancing selection lies in PoMos’ ability to account for ancestral polymorphisms and incorporate parameters that measure frequency-dependent selection. We implemented validation tests and assessed the model on the data simulated with SLiM and a custom Moran model simulator. Real sequence analysis ofDrosophilapopulations reveals insights into the evolutionary dynamics of regions subject to frequency-dependent balancing selection, particularly in the context of sex-limited colour dimorphism.