Processing and Visualization of Protec-Seq Data for the Generation of Calibrated, Genome-Wide Double Double-Strand Break (dDSB) Maps.

This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.

Strain heterogeneity in a non-pathogenic Aspergillus fungus highlights factors associated with virulence.

Fungal pathogens exhibit extensive strain heterogeneity, including variation in virulence. Whether closely related non-pathogenic species also exhibit strain heterogeneity remains unknown. Here, we comprehensively characterized the pathogenic potentials (i.e., the ability to cause morbidity and mortality) of 16 diverse strains of Aspergillus fischeri, a non-pathogenic close relative of the major pathogen Aspergillus fumigatus. In vitro immune response assays and in vivo virulence assays using a mouse model of pulmonary aspergillosis showed that A. fischeri strains varied widely in their pathogenic potential. Furthermore, pangenome analyses suggest that A. fischeri genomic and phenotypic diversity is even greater. Genomic, transcriptomic, and metabolic profiling identified several pathways and secondary metabolites associated with variation in virulence. Notably, strain virulence was associated with the simultaneous presence of the secondary metabolites hexadehydroastechrome and gliotoxin. We submit that examining the pathogenic potentials of non-pathogenic close relatives is key for understanding the origins of fungal pathogenicity.

A putative scenario of how de novo protein-coding genes originate in the Saccharomyces cerevisiae lineage.

BACKGROUND: Novel protein-coding genes were considered to be born by re-organization of pre-existing genes, such as gene duplication and gene fusion. However, recent progress of genome research revealed that more protein-coding genes than expected were born de novo, that is, gene origination by accumulating mutations in non-genic DNA sequences. Nonetheless, the in-depth process (scenario) for de novo origination is not well understood. RESULTS: We have conceived bioinformatic analysis for sketching a scenario for de novo origination of protein-coding genes. For each de novo protein-coding gene, we firstly identified an edge of a given phylogenetic tree where the gene was born based on parsimony. Then, from a multiple sequence alignment of the de novo gene and its orthologous regions, we constructed ancestral DNA sequences of the gene corresponding to both end nodes of the edge. We finally revealed statistical features observed in evolution between the two ancestral sequences. In the analysis of the Saccharomyces cerevisiae lineage, we have successfully sketched a putative scenario for de novo origination of protein-coding genes. (1) In the beginning was GC-rich genome regions. (2) Neutral mutations were accumulated in the regions. (3) ORFs were extended/combined, and then (4) translation signature (Kozak consensus sequence) was recruited. Interestingly, as the scenario progresses from (2) to (4), the specificity of mutations increases. CONCLUSION: To the best of our knowledge, this is the first report outlining a scenario of de novo origination of protein-coding genes. Our bioinformatic analysis can capture events that occur during a short evolutionary time by directly observing the evolution of the ancestral sequences from non-genic to genic. This property is suitable for the analysis of fast evolving de novo genes.

​Fusarium Protein Toolkit: a web-based resource for structural and variant analysis of Fusarium species.

BACKGROUND: ​​The genus Fusarium poses significant threats to food security and safety worldwide because numerous species of the fungus cause destructive diseases and/or mycotoxin contamination in crops. The adverse effects of climate change are exacerbating some existing threats and causing new problems. These challenges highlight the need for innovative solutions, including the development of advanced tools to identify targets for control strategies. DESCRIPTION: In response to these challenges, we developed the Fusarium Protein Toolkit (FPT), a web-based tool that allows users to interrogate the structural and variant landscape within the Fusarium pan-genome. The tool displays both AlphaFold and ESMFold-generated protein structure models from six Fusarium species. The structures are accessible through a user-friendly web portal and facilitate comparative analysis, functional annotation inference, and identification of related protein structures. Using a protein language model, FPT predicts the impact of over 270 million coding variants in two of the most agriculturally important species, Fusarium graminearum and F. verticillioides. To facilitate the assessment of naturally occurring genetic variation, FPT provides variant effect scores for proteins in a Fusarium pan-genome based on 22 diverse species. The scores indicate potential functional consequences of amino acid substitutions and are displayed as intuitive heatmaps using the PanEffect framework. CONCLUSION: FPT fills a knowledge gap by providing previously unavailable tools to assess structural and missense variation in proteins produced by Fusarium. FPT has the potential to deepen our understanding of pathogenic mechanisms in Fusarium, and aid the identification of genetic targets for control strategies that reduce crop diseases and mycotoxin contamination. Such targets are vital to solving the agricultural problems incited by Fusarium, particularly evolving threats resulting from climate change. Thus, FPT has the potential to contribute to improving food security and safety worldwide.

Complete genome sequence analysis of Pestalotiopsis microspora, a fungal pathogen causing kiwifruit postharvest rots.

BACKGROUND: The postharvest rot of kiwifruit is one of the most devastating diseases affecting kiwifruit quality worldwide. However, the genomic basis and pathogenicity mechanisms of kiwifruit rot pathogens are lacking. Here we report the first whole genome sequence of Pestalotiopsis microspora, one of the main pathogens causing postharvest kiwifruit rot in China. The genome of strain KFRD-2 was sequenced, de novo assembled, and analyzed. RESULTS: The genome of KFRD-2 was estimated to be approximately 50.31 Mb in size, with an overall GC content of 50.25%. Among 14,711 predicted genes, 14,423 (98.04%) exhibited significant matches to genes in the NCBI nr database. A phylogenetic analysis of 26 known pathogenic fungi, including P. microspora KFRD-2, based on conserved orthologous genes, revealed that KFRD-2's closest evolutionary relationships were to Neopestalotiopsis spp. Among KFRD-2's coding genes, 870 putative CAZy genes spanned six classes of CAZys, which play roles in degrading plant cell walls. Out of the 25 other plant pathogenic fungi, P. microspora possessed a greater number of CAZy genes than 22 and was especially enriched in GH and AA genes. A total of 845 transcription factors and 86 secondary metabolism gene clusters were predicted, representing various types. Furthermore, 28 effectors and 109 virulence-enhanced factors were identified using the PHI (pathogen host-interacting) database. CONCLUSION: This complete genome sequence analysis of the kiwifruit postharvest rot pathogen P. microspora enriches our understanding its disease pathogenesis and virulence. This study establishes a theoretical foundation for future investigations into the pathogenic mechanisms of P. microspora and the development of enhanced strategies for the efficient management of kiwifruit postharvest rots.

Comparative genomic analysis of thermophilic fungi reveals convergent evolutionary adaptations and gene losses.

Thermophily is a trait scattered across the fungal tree of life, with its highest prevalence within three fungal families (Chaetomiaceae, Thermoascaceae, and Trichocomaceae), as well as some members of the phylum Mucoromycota. We examined 37 thermophilic and thermotolerant species and 42 mesophilic species for this study and identified thermophily as the ancestral state of all three prominent families of thermophilic fungi. Thermophilic fungal genomes were found to encode various thermostable enzymes, including carbohydrate-active enzymes such as endoxylanases, which are useful for many industrial applications. At the same time, the overall gene counts, especially in gene families responsible for microbial defense such as secondary metabolism, are reduced in thermophiles compared to mesophiles. We also found a reduction in the core genome size of thermophiles in both the Chaetomiaceae family and the Eurotiomycetes class. The Gene Ontology terms lost in thermophilic fungi include primary metabolism, transporters, UV response, and O-methyltransferases. Comparative genomics analysis also revealed higher GC content in the third base of codons (GC3) and a lower effective number of codons in fungal thermophiles than in both thermotolerant and mesophilic fungi. Furthermore, using the Support Vector Machine classifier, we identified several Pfam domains capable of discriminating between genomes of thermophiles and mesophiles with 94% accuracy. Using AlphaFold2 to predict protein structures of endoxylanases (GH10), we built a similarity network based on the structures. We found that the number of disulfide bonds appears important for protein structure, and the network clusters based on protein structures correlate with the optimal activity temperature. Thus, comparative genomics offers new insights into the biology, adaptation, and evolutionary history of thermophilic fungi while providing a parts list for bioengineering applications.

Evolution of unexpected diversity in a putative mating type locus and its correlation with genome variability reveals likely asexuality in the model mycorrhizal fungus Rhizophagus irregularis.

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) form mutualistic partnerships with approximately 80% of plant species. AMF, and their diversity, play a fundamental role in plant growth, driving plant diversity, and global carbon cycles. Knowing whether AMF are sexual or asexual has fundamental consequences for how they can be used in agricultural applications. Evidence for and against sexuality in the model AMF, Rhizophagus irregularis, has been proposed. The discovery of a putative mating-type locus (MAT locus) in R. irregularis, and the previously suggested recombination among nuclei of a dikaryon R. irregularis isolate, potentially suggested sexuality. Unless undergoing frequent sexual reproduction, evolution of MAT-locus diversity is expected to be very low. Additionally, in sexual species, MAT-locus evolution is decoupled from the evolution of arbitrary genome-wide loci. RESULTS: We studied MAT-locus diversity of R. irregularis. This was then compared to diversification in a phosphate transporter gene (PTG), that is not involved in sex, and to genome-wide divergence, defined by 47,378 single nucleotide polymorphisms. Strikingly, we found unexpectedly high MAT-locus diversity indicating that either it is not involved in sex, or that AMF are highly active in sex. However, a strongly congruent evolutionary history of the MAT-locus, PTG and genome-wide arbitrary loci allows us to reject both the hypothesis that the MAT-locus is involved in mating and that the R. irregularis lineage is sexual. CONCLUSION: Our finding shapes the approach to developing more effective AMF strains and is highly informative as it suggests that introduced strains applied in agriculture will not exchange DNA with native populations.

Histone variant H2A.Z is needed for efficient transcription-coupled NER and genome integrity in UV challenged yeast cells.

The genome of living cells is constantly challenged by DNA lesions that interfere with cellular processes such as transcription and replication. A manifold of mechanisms act in concert to ensure adequate DNA repair, gene expression, and genome stability. Bulky DNA lesions, such as those induced by UV light or the DNA-damaging agent 4-nitroquinoline oxide, act as transcriptional and replicational roadblocks and thus represent a major threat to cell metabolism. When located on the transcribed strand of active genes, these lesions are handled by transcription-coupled nucleotide excision repair (TC-NER), a yet incompletely understood NER sub-pathway. Here, using a genetic screen in the yeast Saccharomyces cerevisiae, we identified histone variant H2A.Z as an important component to safeguard transcription and DNA integrity following UV irradiation. In the absence of H2A.Z, repair by TC-NER is severely impaired and RNA polymerase II clearance reduced, leading to an increase in double-strand breaks. Thus, H2A.Z is needed for proficient TC-NER and plays a major role in the maintenance of genome stability upon UV irradiation.

Discovering the hidden function in fungal genomes.

New molecular technologies have helped unveil previously unexplored facets of the genome beyond the canonical proteome, including microproteins and short ORFs, products of alternative splicing, regulatory non-coding RNAs, as well as transposable elements, cis-regulatory DNA, and other highly repetitive regions of DNA. In this Review, we highlight what is known about this 'hidden genome' within the fungal kingdom. Using well-established model systems as a contextual framework, we describe key elements of this hidden genome in diverse fungal species, and explore how these factors perform critical functions in regulating fungal metabolism, stress tolerance, and pathogenesis. Finally, we discuss new technologies that may be adapted to further characterize the hidden genome in fungi.

Highly Repetitive Genome of Coniella granati (syn. Pilidiella granati), the Causal Agent of Pomegranate Fruit Rot, Encodes a Minimalistic Proteome with a Streamlined Arsenal of Effector Proteins.

This study describes the first genome sequence and analysis of Coniella granati, a fungal pathogen with a broad host range, which is responsible for postharvest crown rot, shoot blight, and canker diseases in pomegranates. C. granati is a geographically widespread pathogen which has been reported across Europe, Asia, the Americas, and Africa. Our analysis revealed a 46.8 Mb genome with features characteristic of hemibiotrophic fungi. Approximately one third of its genome was compartmentalised within 'AT-rich' regions exhibiting a low GC content (30 to 45%). These regions primarily comprised transposable elements that are repeated at a high frequency and interspersed throughout the genome. Transcriptome-supported gene annotation of the C. granati genome revealed a streamlined proteome, mirroring similar observations in other pathogens with a latent phase. The genome encoded a relatively compact set of 9568 protein-coding genes with a remarkable 95% having assigned functional annotations. Despite this streamlined nature, a set of 40 cysteine-rich candidate secreted effector-like proteins (CSEPs) was predicted as well as a gene cluster involved in the synthesis of a pomegranate-associated toxin. These potential virulence factors were predominantly located near repeat-rich and AT-rich regions, suggesting that the pathogen evades host defences through Repeat-Induced Point mutation (RIP)-mediated pseudogenisation. Furthermore, 23 of these CSEPs exhibited homology to known effector and pathogenicity genes found in other hemibiotrophic pathogens. The study establishes a foundational resource for the study of the genetic makeup of C. granati, paving the way for future research on its pathogenicity mechanisms and the development of targeted control strategies to safeguard pomegranate production.

Structure and distribution of sensor histidine kinases in the fungal kingdom.

Two-component systems (TCSs) are diverse cell signaling pathways that play a significant role in coping with a wide range of environmental cues in both prokaryotic and eukaryotic organisms. These transduction circuitries are primarily governed by histidine kinases (HKs), which act as sensing proteins of a broad variety of stressors. To date, nineteen HK groups have been previously described in the fungal kingdom. However, the structure and distribution of these prominent sensing proteins were hitherto investigated in a limited number of fungal species. In this study, we took advantage of recent genomic resources in fungi to refine the fungal HK classification by deciphering the structural diversity and phylogenetic distribution of HKs across a large number of fungal clades. To this end, we browsed the genome of 91 species representative of different fungal clades, which yielded 726 predicted HK sequences. A domain organization analysis, coupled with a robust phylogenomic approach, led to an improved categorization of fungal HKs. While most of the compiled sequences were categorized into previously described fungal HK groups, some new groups were also defined. Overall, this study provides an improved overview of the structure, distribution, and evolution of HKs in the fungal kingdom.

CRISPR/Cas9-based genome engineering in the filamentous fungus Rhizopus oryzae and its application to L-lactic acid production.

The filamentous fungus Rhizopus oryzae is one of the main industrial strains for the production of a series of important chemicals such as ethanol, lactic acid, and fumaric acid. However, the lack of efficient gene editing tools suitable for R. oryzae makes it difficult to apply technical methods such as metabolic engineering regulation and synthetic biology modification. A CRISPR-Cas9 system suitable for efficient genome editing in R. oryzae was developed. Firstly, four endogenous U6 promoters of R. oryzae were identified and screened with the highest transcriptional activity for application to sgRNA transcription. It was then determined that the U6 promoter mediated CRISPR/Cas9 system has the ability to efficiently edit the genome of R. oryzae through NHEJ and HDR-mediated events. Furthermore, the newly constructed CRISPR-Cas9 dual sgRNAs system can simultaneously disrupt or insert different fragments of the R. oryzae genome. Finally, this CRISPR-Cas9 system was applied to the genome editing of R. oryzae by knocking out pyruvate carboxylase gene (PYC) and pyruvate decarboxylase gene (pdcA) and knocking in phosphofructokinase (pfkB) from Escherichia coli and L-lactate dehydrogenase (L-LDH) from Heyndrickxia coagulans, which resulted in a substantial increase in L-LA production. In summary, this study showed that the CRISPR/Cas9-based genome editing tool is efficient for manipulating genes in R. oryzae.

Complete Genome Sequence of Candida mucifera from an Otitis Media Patient.

We describe for the first time, a high-quality genome for a rare human yeast pathogen Candida mucifera, from a patient with chronic suppurative otitis media. This pathogen exhibited reduced azole susceptibility, similar to its close relatives within the Trichomonascus ciferrii species complex.

Comparative analysis of the genomes and aflatoxin production patterns of three species within the Aspergillus section Flavi reveals an undescribed chemotype and habitat-specific genetic traits.

Aflatoxins are the most dangerous mycotoxins for food safety. They are mainly produced by Aspergillus flavus, A. parasiticus, and A. minisclerotigenes. The latter, an understudied species, was the main culprit for outbreaks of fatal aflatoxicosis in Kenya in the past. To determine specific genetic characteristics of these Aspergillus species, their genomes are comparatively analyzed. Differences reflecting the typical habitat are reported, such as an increased number of carbohydrate-active enzymes, including enzymes for lignin degradation, in the genomes of A. minisclerotigenes and A. parasiticus. Further, variations within the aflatoxin gene clusters are described, which are related to different chemotypes of aflatoxin biosynthesis. These include a substitution within the aflL gene of the A. parasiticus isolate, which leads to the translation of a stop codon, thereby switching off the production of the group 1 aflatoxins B1 and G1. In addition, we demonstrate that the inability of the A. minisclerotigenes isolates to produce group G aflatoxins is associated with a 2.2 kb deletion within the aflF and aflU genes. These findings reveal a relatively high genetic homology among the three Aspergillus species investigated. However, they also demonstrate consequential genetic differences that have an important impact on risk-assessment and food safety.

Natural Transposable Element Insertions Contribute to Host Fitness in Model Yeasts.

Transposable elements (TEs) are ubiquitous in the eukaryote genomes, but their evolutionary and functional significance remains largely obscure and contentious. Here, we explore the evolution and functional impact of TEs in two model unicellular eukaryotes, the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, which diverged around 330 to 420 million years ago. We analyze the distribution of LTR retrotransposons (LTR-RTs, the only TE order identified in both species) and their solo-LTR derivatives in 35 strains of S. pombe and 128 strains of S. cerevisiae. We find that natural LTR-RT and solo-LTR insertions exhibit high presence-absence polymorphism among individuals in both species. Population genetics analyses show that solo-LTR insertions experienced functional constraints similar to synonymous sites of host genes in both species, indicating a majority of solo-LTR insertions might have evolved in a neutral manner. When knocking out nine representative solo-LTR insertions separately in the S. pombe strain 972h- and 12 representative solo-LTR insertions separately in the S. cerevisiae strain S288C, we find that one solo-LTR insertion in S. pombe has a significant effect on the fitness and transcriptome of its host. Together, our findings indicate that a fraction of natural TE insertions likely shape their host transcriptomes and thereby contribute to their host fitness, with implications for understanding the functional significance of TEs in eukaryotes.

Evolutionary origin and population diversity of a cryptic hybrid pathogen.

Cryptic fungal pathogens pose disease management challenges due to their morphological resemblance to known pathogens. Here, we investigated the genomes and phenotypes of 53 globally distributed isolates of Aspergillus section Nidulantes fungi and found 30 clinical isolates-including four isolated from COVID-19 patients-were A. latus, a cryptic pathogen that originated via allodiploid hybridization. Notably, all A. latus isolates were misidentified. A. latus hybrids likely originated via a single hybridization event during the Miocene and harbor substantial genetic diversity. Transcriptome profiling of a clinical isolate revealed that both parental subgenomes are actively expressed and respond to environmental stimuli. Characterizing infection-relevant traits-such as drug resistance and growth under oxidative stress-revealed distinct phenotypic profiles among A. latus hybrids compared to parental and closely related species. Moreover, we identified four features that could aid A. latus taxonomic identification. Together, these findings deepen our understanding of the origin of cryptic pathogens.

Ustilaginoidea virens, an emerging pathogen of rice: the dynamic interplay between the pathogen virulence strategies and host defense.

MAIN CONCLUSION: The Ustilaginoidea virens -rice pathosystem has been used as a model for flower-infecting fungal pathogens. The molecular biology of the interactions between U. virens and rice, with an emphasis on the attempt to get a deeper comprehension of the false smut fungus's genomes, proteome, host range, and pathogen biology, has been investigated. Meta-QTL analysis was performed to identify potential QTL hotspots for use in marker-assisted breeding. The Rice False Smut (RFS) caused by the fungus Ustilaginoidea virens currently threatens rice cultivators across the globe. RFS infects rice panicles, causing a significant reduction in grain yield. U. virens can also parasitize other hosts though they play only a minor role in its life cycle. Furthermore, because it produces mycotoxins in edible rice grains, it puts both humans and animals at risk of health problems. Although fungicides are used to control the disease, some fungicides have enabled the pathogen to develop resistance, making its management challenging. Several QTLs have been reported but stable gene(s) that confer RFS resistance have not been discovered yet. This review offers a comprehensive overview of the pathogen, its virulence mechanisms, the genome and proteome of U. virens, and its molecular interactions with rice. In addition, information has been compiled on reported resistance QTLs, facilitating the development of a consensus genetic map using meta-QTL analysis for identifying potential QTL hotspots. Finally, this review highlights current developments and trends in U. virens-rice pathosystem research while identifying opportunities for future investigations.

Protocol for rapid and cost-effective extraction of genomic DNA from a wide range of wild yeast species for use in PCR-based applications.

Isolation of amplifiable genomic DNA is a prerequisite for the implementation of PCR-based techniques. Here we present a protocol for isolating the genomic DNA from a variety of wild yeast species. This can be completed in approximately 1 h and does not require sophisticated laboratory equipment. We describe steps for growing yeast cells, genomic data extraction, and downstream assay for amplification of specific sequences from the genomic DNA. We then detail procedures for gel electrophoresis and analysis of the results. For complete details on the use and execution of this protocol, please refer to Kristjuhan et al.1.

Two near-chromosomal-level genomes of globally-distributed Macroascomycete based on single-molecule fluorescence and Hi-C methods.

Discinaceae holds significant importance within the Pezizales, representing a prominent group of macroascomycetes distributed globally. However, there is a dearth of genomic studies focusing on this family, resulting in gaps in our understanding of its evolution, development, and ecology. Here we utilized state-of-the-art genome assembly methodologies, incorporating third-generation single-molecule fluorescence and Hi-C-assisted methods, to elucidate the genomic landscapes of Gyromitra esculenta and Paragyromitra xinjiangensis. The genome sizes of two species were determined to be 47.10 Mb and 48.20 Mb, with 23 and 22 scaffolds, respectively. 10,438 and 11,469 coding proteins were identified, with functional annotations encompassing over 96.47% and 94.40%, respectively. Assessment of completeness using BUSCO revealed that 98.71% and 98.89% of the conserved proteins were identified. The application of comparative genomic technology has helped in identifying traits associated with of heterothallic life cycle traits and elucidating unique patterns of chromosomal evolution. Additionally, we identified potential saprotrophic nutritional modes and systematic phylogenetic relationships between the two species. Therefore, this study provides crucial genomic insights into the evolution, nutritional type, and ecological roles of species within the Pezizales.

A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa.

As a widely used eukaryotic model organism, Neurospora crassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N. crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamine amidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N. crassa compared to traditional methods.