Frontiers in Fungal Biology

Introduction. Histoplasma capsulatum is an important fungal pathogen of humans, especially in immunocompromised patients, and the World Health Organisation has listed Histoplasma as a High Priority fungal pathogen. The taxonomy of the genus Histoplasma is still under development, but its only species H. capsulatum has at least of seven lineages, and a range of sublineages. Gap Statement. Allelic multilocus sequence typing (MLST) has been very successfully applied for typing and epidemiology of pathogenic bacteria, but its adoption for fungal pathogens is more limited. Genome sequence-based MLST approaches promise much a higher resolution for comparative phylogenetics, but there are only a small number of genome assemblies available for H. capsulatum. Aim. To develop and validate genome-based MLST typing for H. capsulatum. Methodology. A total of 406 genome assemblies were generated from publicly available Illumina short-read sequencing samples. Selected genome assemblies were subsequently used to generate MLST schemes with chewBBACA, based on whole genome sequences (wgMLST), NCBI-obtained annotations (annMLST) and annotations obtained with Funannotate (fanMLST), and used for lineage identification, and comparative analyses of population structure. Results. Genome assemblies were successfully generated for 406 H. capsulatum samples, with BUSCO completeness scores of over 98%. A subset of 60 genome assemblies was used to generate and validate MLST schemes based on the 60 genome assemblies, 5 genome annotations obtained from Genbank, and 13 genome annotations generated using Funannotate, resulting in schemes with > 8,000 markers. Each of the MLST schemes allowed reproducible separation of all 7 lineages and 6 H. capsulatum Suramericanum sublineages, without the need for high-performance clusters or powerful computing equipment. Comparison of the different schemes using tanglegrams and scatter plots showed good reproducibility (r2 values of 0.8-1.0). Comparison of total sizes of genome assemblies showed significant lineage-dependent size differences, with the Suramericanum, LAmB, India, Capsulatum and Mississippiense lineages averaging a total genome size of 30-33 million bp, and the Africa and Ohiense lineages averaging at 39 million bp. Conclusion. Genome sequence- and especially genome annotation-based MLST analyses are a promising approach that can be adapted as a rapid, high-throughput method for genomic clustering and epidemiological tracing of pathogenic fungi.

Phakopsora pachyrhizi, the causal agent of soybean rust, is among the most aggressive and economically impactful pathogens affecting soybean (Glycine max) production worldwide. As a dikaryotic rust fungus, P. pachyrhizi maintains its genetic information as two discrete haplotypes that are sequestered within separate nuclei for most of its life cycle as it reproduces asexually via the production of infectious urediniospores. Previous genomes of three P. pachyrhizi isolates collected in South America revealed a large genome with a high transposable element content, measuring up to 1.25 Gb and 93%, respectively. Although these efforts provided valuable foundational resources, the complexity of the genome resulted in highly fragmented assemblies. Here, we generated a highly contiguous, substantially improved, annotated genome resource from the P. pachyrhizi isolate LA04-1, collected during the initial outbreak of soybean rust in the continental United States in 2004. The assembly is haplotype-phased, with each haplotype assembled into 18 chromosomes totaling 572 Mb and 569 Mb in size. The isolate contains unique mating-type genes and does not share haplotype genomes with any available isolates with whole-genome sequencing datasets. This resource will be valuable to future investigations into the underlying mechanisms of virulence in this important plant pathogen.

The Fusarium oxysporum species complex (FOSC) comprises numerous plant pathogens infecting crops such as tomato, lettuce, melon, and banana. Despite its economic and ecological importance and decades of research, the species names linked to different phylogenetic lineages within this species complex remain largely controversial. Earlier taxonomic frameworks relied on a limited number of marker genes, variable taxon sampling and inconsistent species definitions, leading to a fragmented and unstable taxonomy. To address these challenges, we here analysed nearly 2,500 conserved marker genes from 660 genomes, including genome assemblies from 22 ex-type strains that were sequenced using long-read sequencing technology, representing nearly half of the 51 currently described species within FOSC. Maximum likelihood- and coalescence-based analyses of the full phylogenomic data matrix as well as subsets yielded a robust phylogeny of FOSC, which was furthermore corroborated by whole genome comparisons. Many previously described species are redundant, while two novel lineages remain undescribed. Direct comparisons to traditional marker gene-based approaches demonstrate their inadequacy for species delimitation in FOSC. We show that our phylogenomic framework can guide the detection of novel marker genes able to distinguish the major lineages within FOSC, which will be essential for taxon delineation and classification in future studies. Thus, our phylogenomic analyses provide a stable cornerstone for revising FOSC taxonomy and offers a critical resource for the broader research community.

Genetic rearrangement in the absence of selection is poorly understood. Long-read, high-depth sequencing has enabled analysis of the genetic landscape of spontaneous structural variants. We have applied PacBio Revio sequencing to identify the major classes of genetic alterations during long-term vegetative growth in Aspergillus flavus. These classes include a) precise deletions frequently associated with the flanking region of homology, b) clusters of short deletions that are heavily enriched within centromeric and subtelomeric regions that are typically 94% A+T, and c) deletions and expansions within a subset of homopolymer tracts. We find 70 homopolymer tracts longer than 44 bp, dispersed throughout the genome in noncoding regions and unassociated with transposable elements. A broader analysis of third-generation sequenced datasets from fungi found within the Phylum Ascomycota showed a high but variable prevalence of homopolymeric tracts across species. The adventitious identification of a putative flbA mutation, responsible for an altered conidial phenotype, by the Deep Variant AI system also demonstrates the power of this approach. These studies show that high-depth, long-read sequencing can probe SNPs, indels, homopolymeric tracts, and large-scale rearrangements in an unbiased fashion, and demonstrate that short deletions and homopolymer tract expansions arise spontaneously in A. flavus and other filamentous fungi.

Genomic structural variants (SVs) are important sources of genetic diversity and contribute to the adaptation and evolution of plant-pathogenic fungi. Colletotrichum graminicola, the causal agent of maize (Zea mays L.) anthracnose, causes significant yield losses and serves as an important model for genetic studies. To investigate genomic plasticity in this pathogen, long-read genome assemblies were generated for four C. graminicola field strains collected in France, Croatia, Brazil, and Argentina and compared with the chromosome-level reference genome from the USA. Comparative analyses revealed extensive structural variation across core chromosomes, including large-scale translocations, inversions, duplications, and insertions and deletions, whereas minichromosomes showed markedly reduced collinearity among strains. Secreted proteins and predicted effectors were located significantly closer to regions affected by SVs. Approximately 174 kb of segmental duplications lacking genes were shared between core chromosome 04 and minichromosomes 11 and 13 in the reference genome but were absent from the other field strains, revealing a strain-specific structural link between core and minichromosomes. Protein clustering identified 733 candidate strain-specific genes across the reference genome. Approximately 72% of the genes annotated on the minichromosomes were strain-specific, indicating that most of their annotated genes lacked orthologous gene models in the other strains. Of the 733 candidate strain-specific genes, 492 retained homologous genomic sequences in at least one other strain and many also showed evidence of gene disruption. This pattern is consistent with lineage-specific pseudogenization and suggests that apparent gene presence-absence variation may arise through several evolutionary processes, including gene gain, gene loss and loss of gene function. Insertions and deletions in potentially regulatory flanking regions were associated with plant-pathogen interaction genes. In addition, interpretable machine learning analyses revealed a co-occurrence of duplications and LTR retrotransposons in genomic regions associated with secreted genes. This study shows that structural variation, transposable elements, and lineage-specific pseudogenization are important features of genome evolution in C. graminicola and identifies associations between genomic variation and genes involved in plant-pathogen interactions.

Plant pathogen genomes adapt across coding and non-coding regions in response to their hosts, environments, and disease management practices. Venturia inaequalis, the causal agent of apple scab, has evolved alongside its Malus host throughout apple domestication. We generated 17 high-quality genome assemblies, including the first chromosome-scale assembly of V. inaequalis, to define the species' complete gene content and investigate the role of structural variation in genome evolution. Together with a previously published scaffold-level reference, these assemblies were used to construct a gene-based pangenome containing 215,301 predicted protein-coding genes clustered into 13,240 orthogroups, 88.11% of which were core (73.86%) or soft-core (14.25%), 11.88% shell (present in 2-16 isolates), and 0.1% private, indicating a highly conserved gene repertoire. Virulence-associated effectors were significantly enriched in the soft-core and shell components, and accessory apoplastic effectors displayed elevated nucleotide diversity and nonsynonymous-to-synonymous substitution ratios (Ka/Ks) relative to core effectors, consistent with diversification. We further identified a putative accessory chromosome of variable size (23-132.6 kb) in European and U.S. isolates encoding genes with virulence function. A graph-based pangenome identified 38,799 non-redundant structural variants (SVs), largely driven by Gypsy LTR retrotransposons. We used the graph-based pangenome to genotype 136 globally distributed V. inaequalis isolates and identified variants differentiating populations associated with domesticated apple (Malus domestica) from those associated with its Central Asian wild progenitor, M. sieversii. Together, these resources establish a foundation for future studies of diversity, host adaptation, and genome evolution in V. inaequalis.

Coinfections are common in a nature and often involve competition among coinfecting parasites to extract resources from the host. As a consequence of this competition, parasites can face trade-offs between investing in competition with other parasites and exploiting the host for their own growth and transmission. Such within-host interactions among parasites can either increase or decrease virulence depending on how they alter parasite density, resource use, and host damage. We tested the inhibitory effect of different species of the entomopathogenic fungus Metarhizium on one another in vitro and the impact of coinfection on virulence towards Tribolium castaneum. We found that Metarhizium pemphigi is able to inhibit all other isolates of Metarhizium tested, whereas Metarhizium pingshaense was relatively resistant to inhibition by other Metarhizium strains. Virulence towards the host, Tribolium castaneum, differed very little among isolates tested. We did, however, observe differences between virulence in single and coinfections in two cases. Where there were virulence differences, these were both consistent with in vitro inhibition patterns and interestingly resulted in reduced virulence in coinfection. This is inconsistent with broader patterns of virulence in coinfection, where virulence seems to be driven by the more virulent parasite. It is, however, consistent with data from other systems where there is evidence of interference competition. Our data thus support the idea that, in contrast to other forms of between parasite interaction, interference competition results in reduced virulence in coinfection.

Fungal domestication provides a powerful framework for understanding adaptation to human-made environments, yet genomic studies of cheese-associated fungi have largely focused on Penicillium molds. Here, we investigated cheese-associated Nectriaceae, a phylogenetically distant fungal lineage of ascomycetes, by comparing genomes of nine cheese isolates, belonging to five species, with 22 relatives from other environments. Comparative genomic analyses revealed strikingly contrasting evolutionary trajectories in the two cheese-associated genera. Longinectria showed extensive gene family expansions and duplications, particularly affecting nutrient transport, specialised metabolism and fungal recognition, whereas cheese-associated Bisifusarium exhibited genome reduction and contraction of many of the same functional systems. In Bisifusarium domesticum, these reductions extended to biosynthetic gene clusters, carbohydrate-active enzymes and peptidases, consistent with ecological specialisation. We further identified a 75-kb region with >98% nucleotide identity between two cheese-associated Bisifusarium species, strongly supporting recent horizontal transfer. This region contains several genes involved in ion transport and salt tolerance, including functions previously identified in horizontally transferred regions of cheese-associated Penicillium populations. Together, our results reveal multiple genomic routes to cheese association and highlight gene-family turnover, adaptive protein evolution and horizontal gene transfer as complementary mechanisms shaping fungal evolution in anthropogenic environments.

Clostridium botulinum Group I and Clostridium sporogenes are closely related genomic lineages for which historical toxin-based nomenclature can obscure species-level relationships. We developed BoNTax, a curated taxonomy-first framework that separates average nucleotide identity (ANI)-based species assignment from botulinum neurotoxin (BoNT) characterization while integrating assembly quality assessment, multilocus sequence typing (MLST), AMRFinderPlus-based detection of bont genes, other virulence-associated genes, and antimicrobial resistance genes into standardized outputs. Analysis of 966 genomes informed genome-size criteria and the selection of 46 representative references comprising 22 C. botulinum Group I and 24 C. sporogenes genomes. Pairwise ANI resolved these references into two distinct species clusters: within-species ANI values were >95%, whereas between-species values were <95%, supporting a 95% species-level assignment threshold. BoNTax was implemented in GalaxyTrakr as complementary FASTQ- and FASTA-input workflows that converge on a common downstream analysis. The workflow completed analysis of all 42 validation genomes. Among the 32 genomes with a reported taxonomic identity, 29 assignments were concordant and three prompted taxonomic re-evaluations. For the ten genomes reported as taxonomically unknown, BoNTax provided ANI-based assignments. Of the three discordances, BT-22100019 was assigned to C. sporogenes rather than the reported C. botulinum Group I, whereas R1125/03 and R1135/03 showed 99.96% ANI to C. caseinilyticum DSM 46279, supporting their identification as C. caseinilyticum. MLST assignments were concordant for genomes with established profiles except PNUSAB000061, for which ST183 was not assigned by the MLST database version used in this analysis. BoNT type and subtype detection was fully concordant across toxigenic genomes, including bivalent strains. Thirty-one genomes met the predefined assembly-size QC criterion, whereas 11 received QC warnings, including all eight datasets representing four known mixed cultures. BoNTax therefore provides a reproducible genomic framework for resolving closely related clostridial lineages, identifying taxonomic annotations requiring re-evaluation, and reporting clinically relevant toxin information without conflating toxin genotype with species identity.

Recombination suppression has evolved repeatedly across eukaryotes around sex-determining and mating-type loci. Recent theoretical models proposed that such suppression can evolve due to various effects of recessive deleterious mutations, predicting that it should only evolve in lineages experiencing extended diploid phases. Ascomycete fungi provide a powerful system to test this prediction, as pseudo-homothallic ascomycetes maintain prolonged diploid-like phases, while most other ascomycetes have a predominant haploid phase. Megabase-scale non-recombining regions have in fact been reported around the mating-type locus in the four pseudo-homothallic ascomycete lineages studied to date. Here, we studied a previously unexplored lineage of Sordariales, formerly referred to as Podospora australis and suggested to be pseudo-homothallic. Combining genome sequencing, phylogenomic analyses and life-cycle characterization, we identified and described two distinct species and reassigned them to the Cladorrhinum genus. We showed that the two species produce large proportions of self-fertile ascospore-derived mycelia, but the frequencies varied substantially among four strains, and with a selfing progeny, indicating large variation in the extent of the diploid-like phase. By analysing the genomes of five wild strains and of selfing progenies, we did not detect any footprints of large recombination suppression (no heterozygosity, rearrangements or repeated elements). This may be due to the smaller proportion of the life cycle spent in a diploid-like stage compared to other pseudo-homothallic species, or to other features of the life cycle, in agreement with theoretical models. In addition, our results decouple the evolution of pseudo-homothallism from that of recombination suppression, showing that there is no fundamental developmental constraint forcing their joint evolution.

Metabolic repertoires are continually remodeled by gene acquisition and loss, but how these contrasting processes jointly generate phenotypic diversity within bacterial species complexes remains poorly understood. Here, we elucidated the genetic and evolutionary basis of the classical biovar phenotypes of the Ralstonia solanacearum species complex (RSSC), which are defined by differential utilization of three hexitols (sorbitol, mannitol, and dulcitol) and three disaccharides (lactose, maltose, and cellobiose). Although this classification has been used for over half a century, the genetic determinants and evolutionary origins of these phenotypes have not been systematically resolved. Machine-learning analysis of genomic data from 250 RSSC strains with documented biovar phenotypes, combined with genome-wide association analysis and gene deletion, identified the smoEFGK gene cluster and flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (FADGDH) as the major determinants of hexitol and disaccharide utilization, respectively. Comparative phylogenomics revealed fundamentally different evolutionary histories of these loci. An ancestral smoEFGK system was lost early in RSSC evolution and subsequently reacquired by the ancestor of phylotype I through horizontal transfer from a Burkholderia-related lineage, whereas vertically inherited FADGDH underwent recurrent, independent losses across multiple RSSC lineages. Extending the analysis to 1,082 RSSC strains further showed that disaccharide-utilization capacity was significantly depleted among monocot-associated strains after accounting for phylogenetic relatedness and geographic origin. Together, these findings reveal how contrasting trajectories of gene gain and loss can reshape bacterial metabolic repertoires, generate lineage-associated phenotypic diversity, and potentially contribute to ecological differentiation.

Aedes aegypti, an important vector of several arboviral diseases, is highly adapted to human-associated environments and commonly exploits anthropogenic water-holding containers for breeding. Understanding the ecological factors governing oviposition-site selection is essential for developing sustainable vector control strategies. Previous studies have established that microbial communities in breeding waters influence mosquito oviposition-site selection through the production of volatile organic compounds. Importantly, rapid urbanization in India has increased the availability of artificial breeding habitats for Ae. aegypti, potentially facilitating the transmission and persistence of arboviral diseases. However, the composition of bacterial communities in these larval habitats and their potential role in shaping the oviposition behavior of Ae. aegypti remain poorly characterized in the Indian context. Here, we investigated the bacterial diversity and community composition of Aedes larval habitats during the 2024 Zika and chikungunya outbreaks in Pune, India. Water samples, collected from artificial urban containers and natural freshwater habitats, were subjected to 16S rRNA amplicon sequencing. Artificial breeding habitats exhibited significantly higher bacterial diversity and were enriched with Enterobacter spp. compared with natural habitats. Oviposition bioassays further demonstrated a strong preference of gravid Ae. aegypti females for Enterobacter-conditioned water. GCMS analysis of bacterial suspension identified indole as a prominent volatile metabolite associated with Enterobacter, suggesting its possible role in mediating oviposition site selection. Together, these findings provide insights into the microbial and chemical ecology of Ae. aegypti breeding habitats and establish a basis for investigating microbial volatile cues as potential targets for vector surveillance and environmentally sustainable attract-and-kill strategies.

Considering the increasing water and labor constraints in rice production, direct-seeded rice is becoming increasingly important. However, direct-seeded rice is vulnerable to water stress and inefficient nutrient use. Nitrogen use efficiency in rice is generally low, and improving nitrogen acquisition and assimilation is important for sustainable productivity. In this study, 48 recombinant inbred lines were evaluated under ammonium and nitrate nutrition in irrigated and rainfed environments across two cropping seasons. Agronomic, physiological, biochemical and molecular responses were assessed. Significant effects of genotype, nitrogen form, environments and their interactions were observed for several traits. Rainfed conditions reduced grain and biological yield compared with irrigated conditions. Ammonium produced a higher mean grain yield than nitrate by approximately 26.2 g m-2 under irrigated conditions and 154.2 g m-2 under rainfed conditions, although the responses varied among genotypes. Biological yield and SPAD values were generally higher with ammonium, whereas total chlorophyll showed season and environment-dependent responses. Glutamine synthetase activity was highest under ammonium treatment, while glutamate synthase activity was higher under nitrate treatment. Grain yield showed strong positive correlations with biological yield, but correlations between grain yield and GS or GOGAT activity were not significant. Expression of OsGln1.2, OsGln2 and OsGlt1 varied according to genotype, tissue and nitrogen treatment. Overall, the results demonstrate that responses to nitrogen forms are genotype- and environment-dependent, and that ammonium may provide a yield advantage under rainfed conditions. Integrating field performance with biochemical and molecular traits may help identify rice genotypes suited to water-limited systems.

Background: Surveillance tells laboratories which antimicrobial resistance genes are circulating now, but not which will change next. We built a model that forecasts, for each resistance gene and year, whether the following year brings a previously unseen allele (sequence variant), called emergence, or a change in the most common allele, called turnover. We tested it separately in Klebsiella pneumoniae and Acinetobacter baumannii. Results: We used genomes annotated with AMRFinderPlus: 2,385 gene-years across 331 genes for K.pneumoniae and 1,059 across 216 for A. baumannii. To remove the effect of sequencing effort, each gene-year was sampled to 10 records and the analysis repeated 25 times. Models were trained on earlier years and tested on later ones, and on genes held out of training. On later years every model ranked genes by risk better than simple baselines in both organisms, and results for held-out genes were close. In K.pneumoniae, turnover was predicted better than emergence (best area under the ROC curve, AUROC, 0.909 versus 0.819). In A. baumannii the order reversed (emergence 0.789, turnover 0.722). The two events also relied on different features. Emergence depended mainly on how many alleles a gene had already accumulated, in both organisms. Turnover in K. pneumoniae depended mainly on how dominant the leading allele already was, and a stability index added a small gain. In A. baumannii it was spread across many weaker features. There, 56% of changes in the leading allele involved only alleles already seen, against 23% in K. pneumoniae, which may make them harder to predict. Conclusions: Changes in resistance genes can be forecast a year ahead from routine genomic surveillance data, including for genes the model has not seen. How well it works, and what it depends on, differs between organisms, so models should be built and validated for each organism, not assumed to transfer. Such models could help laboratories decide which resistance genes to monitor most closely. Keywords: antimicrobial resistance; genomic surveillance; forecasting; machine learning; allele dynamics; Klebsiella pneumoniae; Acinetobacter baumannii

Genetic variation impacts biological response to chemical and environmental exposures, contributing to differences in resistance or susceptibility. Forward genetic screens of genetically diverse cell populations can be used to identify the precise genetic variants that drive response variation. Cell lines from laboratory mouse genetic reference populations like the Diversity Outbred (DO) population are well powered for genetic mapping. Diverse stem cell panels are especially beneficial for studying early developmental effects of chemical exposures like triphenyl phosphate (TPHP), an organophosphate flame retardant linked to adverse developmental effects, including altered cell cycle in stem cells. Self-renewal and pluripotency are defining properties of stem cells during development; therefore, it is important to understand how chemicals like TPHP may alter these critical characteristics. To better understand the influence of genetic variation on TPHP exposure response, we utilized a genetically diverse panel of DO induced pluripotent stem cells (DO iPSCs). Our analyses characterize interline variation in gene expression through differential gene expression analysis and expression quantitative trait locus (eQTL) mapping. We identified genomic loci that contribute to variation in gene expression following TPHP exposure, including a regulatory hotspot on chromosome 15. Gene set enrichment highlighted several pathways affected by TPHP including lipid metabolism, steroid hormone signaling, and cell cycle. Our study shows that genetic variation modulates the effects of TPHP on gene expression and cell cycle in stem cells. Additionally, our work demonstrates that genetically diverse cell panels like the DO iPSC resource offer a tractable and scalable approach for identifying the genetic determinants of chemical toxicity.

Diverse eukaryotes thrive under low oxygen conditions, in part through highly modified mitochondrion-related organelles (MROs) that use alternate metabolic pathways to support ATP production and cofactor recycling. Anaerobic lifestyles have evolved repeatedly across the eukaryotic tree of life, each providing an independent opportunity to understand how eukaryotes adapt to life in low oxygen conditions. Here, we use single-cell transcriptomics to reconstruct the MRO metabolism of PCE SSF, a benthic eukaryotrophic flagellate and the first cultivated representative of Novel Clade 12 (NC12; Rhizaria), an independently anaerobic rhizarian lineage. PCE SSF possesses an anaerobic hydrogen-producing mitochondrion capable of hydrogenosome-type substrate-level phosphorylation. It also retains a nearly complete but likely branched tricarboxylic acid pathway that lacks citrate synthase and malate dehydrogenase. The function of citrate synthase may instead be fulfilled by the typically cytosolic ATP citrate lyase, previously reported in this context only in the anaerobic cercozoan, Brevimastigomonas motovehiculus. Unlike B. motovehiculus, however, PCE SSF retains only Complex II and the NuoE/NuoF subunits of the electron transport chain and lacks a mitochondrial genome. Together, these features indicate an atypical and reduced mitochondrial metabolism, highlighting the diversity of evolutionary solutions to anaerobic energy metabolism in eukaryotes.

Genomic tools can support malaria control policy through surveillance of Plasmodium falciparum populations, tracking antimalarial drug resistance, pfhrp2/3 deletions that compromise rapid diagnostic tests, and selection at the circumsporozoite protein (PfCSP) vaccine target, as well as through molecular correction of therapeutic efficacy studies (TES). Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing (MAD4HatTeR), a comprehensive amplicon sequencing panel covering up to 276 targets, supports these applications but is tailored to research rather than routine programmatic use. We developed P. falciparum Public Health Amplicon Sequencing Tool (PfPHAST), a 56-target derivative of MAD4HatTeR spanning drug resistance loci, pfhrp2/3 deletion, PfCSP genotyping, non-falciparum species identification, and 20 high-heterozygosity microhaplotype loci for TES classification. We compared PfPHAST and MAD4HatTeR using laboratory strain controls, including two-strain dilution series and a five-strain mixture, across parasite densities of 100 to 10,000 parasites/L. At matched per-target depth, PfPHAST achieved a higher quality-control pass rate than MAD4HatTeR (94.4% versus 90.0%) and distributed reads more evenly across targets. The panels showed comparable recall and precision for drug resistance codons and microhaplotypes, reaching near-complete recall above 40% within-sample allele frequency (WSAF) at all densities, with reduced sensitivity for minor alleles below 10% WSAF at low parasite density in both panels. Observed and expected WSAF correlated strongly for both panels, and both resolved a five-strain polyclonal mixture, including a 5% minor strain. By concentrating sequencing capacity on targets of greatest programmatic relevance, PfPHAST offers a scalable, lower-cost alternative to comprehensive research panels without sacrificing performance on shared targets, complementing MAD4HatTeR for routine molecular malaria surveillance.

Carbohydrate-active enzymes (CAZymes) drive the turnover of polysaccharides in the oceans. However, the scale of diversity in polysaccharide utilization and genome organization strategies has yet to be characterized, particularly in marine ecosystems. In this paper, we introduce mpcgcdb.com as an interactive web catalog for marine CAZyme gene clusters, their associated metagenome-assembled genomes, and relevant enzyme families found in marine metagenomic samples. This database contains nearly 290,000 marine CAZyme gene clusters from more than 22,000 genomes, enabling user-initiated exploratory visualizations for genome organization networks and enzyme phylogeny. This tool will assist researchers in developing hypotheses about gene function, connecting species to metabolic niches, and identifying gaps where future work is needed to understand different pathways in marine glycan cycling.

Common tansy (Tanacetum vulgare), a significant agricultural invasive species, threatens forage productivity, livestock carrying capacity, and plant biodiversity across North American agroecosystems. Despite being a close relative to the ornamental Asteraceae Chrysanthemum, T. vulgare genomic studies are severely lacking, but remain critical to the development of novel weed management tools and the investigation of plant invasion evolution. In this study, we performed the assembly and annotation of the diploid genome (2n=2x=18) of an invasive North American T. vulgare genotype using long-read PacBio HiFi and Iso-Seq sequencing technology. The assembled haplotype genome comprised 9 pseudomolecules with a total size of ~4.8 Gb and a scaffold N50 of 546 Mb. In addition, both the plastid and mitochondrial genome sequences were assembled. BUSCO analysis revealed a high overall genome completeness with 69,343 predicted protein-coding genes (AED<0.6: 98%). A high-quality North American invasive reference T. vulgare genome, complementing a recent one assembled for a native European accession, is critical for the investigation of plant invasion biology, genome evolution, and the future development of novel genetic biocontrol weed management technologies.

Colistin (CST) is a critical last-line antibiotic for treating multidrug-resistant Pseudomonas aeruginosa infections, especially in chronic respiratory disease. Despite its clinical importance, the P. aeruginosa CST resistome remains incompletely characterised, with most studies focusing on the PAO1 prototypic strain. Here, we used experimental evolution to apply stepwise CST selective pressure to nine P. aeruginosa lineages representing different clinical presentations and diverse genetic backgrounds. Population-level whole-genome sequencing of broth cultures exposed to 2-fold increasing CST (0.0625 to 512 g/mL) was undertaken at multiple concentrations. We identified several known and novel mutational drivers of low- and high-level CST resistance, alongside associated compensatory mutations at allele frequencies (AFs) as low as 5%. Despite diverse clinical and genetic backgrounds, all lineages convergently evolved mutations within the two-component system pmrAB, with most accompanied by phoPQ variants. When canonical TCS remodelling plateaued, populations acquired novel secondary driver mutations, most notably in the lipid A-modifying gene, lpxO2. In addition to AMR driver mutations, strains evolved complex adaptive strategies to mitigate severe CST-induced oxidative and metabolic stress. Adaptations included the emergence of hypermutators and alterations to targeted DNA and protein repair networks. Furthermore, we observed convergent regulatory rewiring of the cyclic-di-GMP network and central metabolism pathways, likely acting as compensatory mechanisms to promote biofilm formation and offset the fitness costs of CST resistance. Using a mixture-aware population genomics approach, we captured intense clonal interference and transient tolerance mutations that failed to reach fixation, illustrating dynamic evolutionary complexities that would be missed with traditional end-point analysis methods.