Microbiology Resource Announcements

Thrips are known for being pests of many agricultural crops, as well as vectors of plant viruses. Yet despite their economic importance, genomic resources for this group remain limited, and no chromosome-level genome has been available for the genus Scirtothrips. One such species is the chilli thrips, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae). This invasive and highly polyphagous pest is established across much of the world and transmits several tospoviruses. Genomic resources would provide a new means to understand the traits that make thrips difficult to manage. Here, we present a chromosome-level genome assembly for S. dorsalis. The assembly is 266.5 Mb, with 96.9% of the sequence anchored in 16 chromosome-scale scaffolds. It contains 20,557 genes, and repetitive elements account for 28.8% of the sequence, which is comparable to those of other sequenced Thripidae. A species tree based on 3,019 single copy orthologs placed S. dorsalis as sister to Scirtothrips hansoni, with Scirtothrips most closely related to Frankliniella, Megalurothrips, and Odontothrips. The mitochondrial genome is a single circular molecule of 15,207 bp in which the sequence of the published mini circle is integrated, in contrast to the bipartite mitogenome previously reported for the same cryptic species.

Pectobacterium comprises Gram-negative, pectinolytic plant pathogens that cause soft rot and blackleg diseases on numerous economically important crops. During sampling in 2018 in taro (Colocasia esculenta) fields in Hawaii, two bacterial strains PL152T and PL155 were isolated from taro. Peliminary molecular characterization placed both strains in the genus Pectobacterium. However, their taxanomic postions could not be resoved at the species level. A polyphasic taxonomic approach incorporating phenotypic characterization, 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), whole genome based phylogeny, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) was therefore employed to determine their taxonomic status. Phylogenetic and phylogenomic analyses consistently placed PL152 and PL155 together in a distinct and well supported lineage within the genus Pectobacterium, separate from currently recognized species. The two strains exhibited high genomic relatedness to each other, whereas the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, were lower than the accepted species delineation thresholds of 95% and 70%, respectively, supported the delineation of a novel bacterial species. The genomic characteristic of PL152 included a genome size of 5.02 Mb and a DNA G+C content of 52%. Collectively, based on a polyphasic approach including phylogenetic, phlyogenomic, biochemical, and phenotypic evidences supports the classification of strains PL152 and PL155 as representatives of a novel species within the genus Pectobacterium, for which the name Pectobacterium pacificum sp. nov. is proposed. Strain PL152 (ICMP 2436274, LMG 32536) is designated as the type strain.

Abstract The Bacillus cereus group is a complex of closely related species, which vary in terms of their geographic spread, industrial utility, and pathogenic potential. In addition to encompassing well-known species represented by thousands of genomes (e.g., bioterrorism agent B. anthracis, foodborne pathogen B. cereus, biopesticide B. thuringiensis), the B. cereus group houses "rare" species discovered/first sequenced over a decade ago. Until recently, four rare species (B. manliponensis, B. bingmayongensis, B. gaemokensis, "B. clarus") were each represented by a single unique genome; as such, virtually nothing is known about these species beyond their type strains. We hypothesized that additional representatives of rare B. cereus group species may already be present in public databases as yet-unassembled metagenomes. To this end, we used species-specific markers to identify 7 metagenomic samples in NCBI's Sequence Read Archive, which harbored B. manliponensis, B. bingmayongensis, or B. gaemokensis. Metagenomic assembly and binning yielded an isolate-quality B. manliponensis metagenome-assembled genome (MAG) from avocado orchard soil in Israel, plus a partial B. bingmayongensis MAG from commercial spinach spiked with Shiga toxin-producing Escherichia coli. For rare species represented by >1 unique genome, we compared our novel MAGs to all available genomes and identified strain- and lineage-specific genomic elements (e.g., sphingomyelinase C and hemolysin BL homologs, variably present in B. manliponensis and B. bingmayongensis, respectively). Altogether, our study indicates that some rare B. cereus group species may be more common and geographically widespread than previously thought, and highlights the utility of culture-independent methods for recovering rare/under-isolated taxa. Impact Statement Individual B. cereus group strains vary in terms of their pathogenic potential and industrial utility. While some strains cause illness in humans and/or animals (e.g., anthrax, emetic intoxication, diarrheal toxicoinfection, non-gastrointestinal illnesses), other strains have important biotechnological applications (e.g., biocontrol, plant growth promotion, production of antimicrobials and other natural products). Because many virulence- and biotechnological-related phenotypes are strain-dependent (rather than species-dependent), phenotypes associated with an individual strain (e.g., a species type strain) cannot be extrapolated to an entire B. cereus group species. Within this context, rare B. cereus group species represent a major gap in our understanding of B. cereus group evolution, ecology, and pathogenicity, as until 2021, each rare species was represented solely by its type strain genome. Here, we aggregate all publicly available genomes/MAGs of rare B. cereus group species and supplement them with their first novel representatives since 2024. Our efforts bring the total number of unique B. manliponensis and B. bingmayongensis genomes/MAGs to >2, allowing us to conduct the first-ever phylogeographic survey and strain-level assessment of functional potential for each species. Overall, our study represents a critical first step toward understanding rare B. cereus group species beyond their type strains.

Vibrio coralliilyticus (V. coralliilyticus) is an emerging coral pathogen associated with tissue-loss diseases in reef-building corals and diseases in other marine organisms, such as oyster larvae. Despite the great ecological and economic significance of this pathogenic bacterium, characterization of bacteriophages infecting V. coralliilyticus remains limited, particularly of temperate phages. In this study, we describe the isolation and characterization of four novel phages - Vibrio phages Satoshi, Kolob, Ushijima, and Kane - obtained from coastal waters near L[a]ie, Hawaii, using the indigenous Hawaiian V. coralliilyticus strain OCN008 as the host. All four phages produced small, turbid plaques and encoded integrases, consistent with temperate lifestyles. The genome organization and taxonomic relationships of the viruses were examined using whole-genome sequencing and comparative genomics, while representative virion morphologies were examined by transmission electron microscopy. To the best of the authors' knowledge, these are the first V. coralliilyticus phages reported from Oahu, Hawaii. Comparative genomic analyses support the placement of all four phages within the class Caudoviricetes, but outside of any previously described orders, families, or genera within the class. Satoshi, Kolob, and Ushijima, along with previously described V. coralliilyticus phage CKB-S2, cluster within one genus-level lineage, while Kane, which has no close relatives in available databases, represents a distinct, previously undescribed lineage. All four phages were also found to contain homologs to experimentally established virulence factors, which may contribute to the pathogenicity of the host bacterium during lysogeny. Together, these isolates expand the diversity of phages infecting V. coralliilyticus and support the proposal of two new genera of Caudoviricetes: the genus Mokushimavirus, containing Satoshi, Kolob, Ushijima, and CKB-S2, and the genus Laievirus, containing Kane.

The Olympia oyster (Ostrea lurida) was once a major food source and a key component of coastal marine ecosystems and culture on the US West Coast. Overfishing and habitat contamination in the early to mid-20th century severely depleted populations of Olympia oysters, and the species is currently considered a species of concern. Although multiple restoration efforts are underway, progress understanding the species biology and recovery potential has been limited by the historical lack of genomic resources. Here, we report a chromosome-level assembly of the Olympia oyster. The assembly was built using Oxford Nanopore reads and Hi-C to obtain a highly contiguous 1.03 Gb-assembly consisting of 10 chromosomes. RNA from gills, mantle, adductor muscle, and a mix of brooding eggs and larvae were also sequenced as 150 nt long paired-end reads which were used to guide the genome annotation. Over 52 K genes were predicted in the genome, of which 34K were protein-coding. A BUSCO analysis on the assembly showed 98.9% of the mollusca_odb12 gene set were complete (with 2.1% duplicated, and 0.8% missing). The short RNA reads were also used to identify tissue-level gene expression signatures in adductor muscles, egg and brooding larvae, and mantle. Development-related genes were highly expressed in eggs and brooding larvae whereas many xenobiotic metabolism genes were present in mantle tissues, highlighting mantles role in the interaction of the oyster with external environments. This new genomic resource will greatly advance our understanding of this keystone mollusk and provide a critical tool for guiding restoration efforts and the development of sustainable commercial production.

Lateral gene transfer (LGT) from organellar to eukaryotic genomes is ubiquitous, resulting in the presence of widespread nuclear mitochondrial DNA segments (NUMTs) and/or plastid gene transfers. While LGT from bacterial associates of eukaryotes is generally less common, many arthropod genomes are littered with LGT originating from Wolbachia (order Rickettsiales) - a vertically transmitted, obligate intracellular symbiont of invertebrates. This contrasts with Rickettsia, a related genus including many important human pathogens, which has not been shown to promulgate LGT within its arthropod vectors. Here, we present the 8.6 Gb genome of a cell line derived from A. variegatum (the tropical bont tick), which is the primary vector of Rickettsia africae (agent of African tick-bite fever). In addition to numerous NUMTs and diverse families of transposable elements, an almost-complete chromosome of R. africae origin was identified in the cell line genome, localised to the putative sex chromosome. Sequencing of field-collected A. variegatum confirmed the presence of this large LGT in wild ticks, although the absence of transferred genes from the R. africae plasmid provided a means to differentiate LGT from genuine rickettsial infections. These findings highlight the imperative to consider the possibility of LGT when screening vectors for pathogens by PCR.

Microsporidia are a group of parasites that exhibit vast morphological, ecological, and genetic diversity. To catalog this diversity, we previously generated a database of attributes for microsporidia species. Here we expand this database to include species described since 2021, previously missed descriptions, and provisional species defined by clustering GenBank 18S rRNA sequences, resulting in 1,795 species. Using these data, we describe when and where microsporidia have been reported, their hosts and host habitats, the tissues they infect, and their spore morphology. A phylogeny of 18S sequence clusters shows that closely related microsporidia tend to infect closely related hosts, such that sequence identity is predictive of host taxonomy. Clustering also reveals previously unappreciated genetic diversity within some named species, in particular the human pathogen Vittaforma corneae, suggesting that these may comprise multiple species. We have also generated a web portal through which new species information can be submitted, which will facilitate regular updates. This database provides a resource for systematic analysis of microsporidia properties and a reference for taxonomic descriptions.

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.

Precise identification depends on highly resolved classification. Therefore, whole genome-based strain typing schemes have been implemented for identification of the most important microbial human pathogens. However, there are no dedicated typing schemes for the majority of prokaryotic human, animal, and plant pathogens, or for non-pathogenic microbes, often limiting their identification to taxonomic classification tools that have the species as the smallest unit. To address this deficiency, we created the genomeRxiv web server which contains over 350,000 genomes and leverages the average nucleotide identity (ANI)-based Life Identification Number (LIN) approach, allowing users to identify prokaryotic genomes in real time from approximately genus-rank to near strain-level resolution. LINs are overlaid with circumscriptions of species and infraspecies classes, integrating taxonomy with strain typing. Metadata and distance trees are visualized to complement inferred phylogenetic relationships with epidemiological and ecological information. A command line tool is provided for users to upload k-mer sketches of unpublished genomes and associated metadata, facilitating epidemiological investigations without the need to share complete DNA sequences. In conclusion, genomeRxiv constitutes a much-needed resource for precise, rapid identification of the many prokaryotes not included in any pathogen-specific typing scheme.

Background: Following the global resurgence of cholera in 2022, 51 cholera cases were reported by European countries. We previously characterised 49 Vibrio cholerae O1 isolates from these cases using whole genome sequencing. In 2023 and 2024, 59 additional cholera cases were reported by 11 European countries. Aim: We aimed to confirm that V. cholerae O1 isolates associated with cholera cases reported in Europe in 2023-2024 belonged to the epidemic seventh pandemic El Tor (7PET) lineage rather than to non-epidemic V. cholerae O1 lineages, and to characterise their virulence, antimicrobial resistance (AMR) determinants and phylogenetic relationships. Methods: Fifty V. cholerae O1 isolates were available for whole genome sequencing, of which 49 yielded genomes of sufficient quality for analysis. Genomes were analysed together with more than 1,500 publicly available 7PET genomes to place the European isolates into a global phylogenetic context. Results: All 49 genomes belonged to the 7PET lineage and Wave 3. Four sub-lineages were identified: BD1.2 (2/49), AFR12 (6/49), AFR13 (8/49) and Pre-AFR15 (33/49). The predominance of Pre-AFR15 confirms its continued contribution to the ongoing global cholera resurgence. In contrast to our 2022 survey, eight highly drug-resistant AFR13 isolates were detected, documenting the international spread of this highly drug-resistant clone resistant to multiple first-line antimicrobial agents. Conclusion: Whole genome sequencing should be routinely used by reference laboratories to distinguish epidemic 7PET from non-epidemic V. cholerae O1 lineages and to monitor the emergence and international spread of AMR clones. Continued collaborative genomic surveillance of travel-associated cholera cases can provide early insights into the emergence, international spread and antimicrobial resistance of 7PET sub-lineages.

A diverse array of photosynthetic phytoplankton drives primary production in equatorial surface waters. Among these, the cyanobacterium Prochlorococcus is an important contributor to net primary production in these typically iron-limited, high-nutrient and low-chlorophyll (HNLC) regions. Here, we explore the diversity of these organisms, in part, through targeted enrichment of Prochlorococcus cells using field-based high-speed cell sorting techniques. We demonstrate that the genomes of Prochlorococcus belonging to the low-light adapted LLI clade, and isolated from the surface of the Equatorial Pacific Ocean, are depleted in functions related to the assimilation of urea, nitrite, and amino acids. These are the first examples of LLI Prochlorococcus that have lost the ability to use nitrite, a trait considered to be a core feature of this clade. All new equatorial cultures of LLI Prochlorococcus appear to use a distinct isoform of protoporphyrinogen IX oxidase (HemG), for the biosynthesis of a chlorophyll precursor, that does not require the use of iron-containing heme. In contrast, the heme-dependent HemJ isoform is typically used by Prochlorococcus found outside equatorial HNLC waters. Together, these findings suggest that low-light adapted Prochlorococcus in the equatorial ocean possess accessory gene content that reflects adaptation to the generally iron-limited but nitrogen-replete conditions of surface waters.

The Gardnerella genus includes a complex sub-taxonomy of bacterial species and subgroups which have been implicated in bacterial vaginosis. We analyzed G. leopoldii, G. swidsinski, G. piotii, and G. vaginalis genomes available in public databases and predicted proviral sequences using Cenote-Taker3. We clustered 266 prophage sequences into 140 viral genera level groupings that encode four distinct gene clusters and 19 different lysin proteins. Analysis of the bacterial and prophage sequence phylogenies reveals that while there were no specific prophage sequence lineages that infected each bacterial host, there is clustering of the prophage sequences by bacterial species within the Gardnerella genus.

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.

Four rhizobial strains isolated from root nodules of tropical leguminous plants in Costa Rica were characterized. Strain LEGMi-135b was isolated from Lonchocarpus felipei root nodules, LEGMi-12c from Cojoba arborea, LEGMi-198b from Calliandra haematocephala and Rhizobium altiplani LEGMi-166a from Mimosa pigra. Phylogenetic analyses based on the 16S rRNA gene and four concatenated housekeeping genes (recA, atpD, GATase and gyrB) placed these strains within the genera Martinezella (LEGMi-12c, LEGMi-135b, and LEGMi-198b) and Rhizobium (LEGMi-166a). Genome-based analyses showed that strain LEGMi-135b exhibited an average nucleotide identity (ANI) value of 94% and a digital DNA-DNA hybridization (dDDH) value of 53.2% compared with its closest related species, Martinezella hainanensis CCBAU 57015T, supporting its classification as a distinct species. LEGMI-12c exhibited ANI and dDDH values of 98% and 87%, respectively, compared with Martinezella hainanensis CCBAU 57015T. LEGMi-198b showed the highest similarity to Rhizobium cerradonense CNPSo 3464, with ANI and dDDH values of 98% and 81.4%, respectively; however, this taxon has not been validly published. LEGMi-166a exhibited ANI and dDDH values of 100% and 99.3%, respectively, compared with Rhizobium altiplani BR 10423, confirming its assignment to this species. Additionally, differentiation of strains LEGMi-135b and LEGMi-198b from their closest phylogenetic relatives was supported by phenotypic, physiological, and biochemical analyses. Based on the combined genomic, phenotypic, and biochemical evidence, we propose the establishment of the novel rhizobial species Martinezella legmii sp. nov., with strain LEGMi-135b designated as the type strain (LEGMi-135b), and Martinezella ivanii sp. nov., with strain LEGMi-198b designated as the type strain (LEGMi-198b). This study expands our understanding of rhizobial diversity associated with nitrogen-fixing symbioses, particularly among tropical leguminous plants from Costa Rica

Microalgae are promising biological platforms for sustainable energy production because they can convert photosynthetic activity into electricity in biophotovoltaic (BPV) systems. Identifying strains that grow well at elevated temperatures and under reduced irradiance is an important step toward BPV applications in seasonally variable environments. Microalgae were isolated from Seondong Lake (Seoul, Republic of Korea) during three sampling periods and screened for growth at different temperatures. Among 45 isolates, KHUC-11 showed the highest weighted growth index based on growth rates at 25, 30, and 35 under dark conditions and was selected for further characterization. Phylogenetic reconstruction of the 45S rRNA gene cluster, internal transcribed spacer 2 (ITS2) secondary-structure comparison, and morphological characterization supported the description of KHUC-11 as Desmodesmus ecotolerans sp. nov. D. ecotolerans produced more biomass than Chlamydomonas reinhardtii CC-125 under both moderate- and low-light conditions. Under low light, its final dry biomass concentrations were 1.81 and 1.58 times those of CC-125 under mixotrophic and photoautotrophic conditions, respectively. Despite its smaller cell size, D. ecotolerans showed higher photosynthetic performance than CC-125, and screen-printed electrode (SPE)-based electrochemical assays demonstrated enhanced anodic current generation and CV-derived estimated power output. Taken together, these findings identify D. ecotolerans as a novel climate-resilient Desmodesmus species with bioelectrochemical properties that support further evaluation for BPV applications.

Stratified benthic deposits offer crucial modern analogs for Precambrian biogeochemical dynamics, yet whether the vertical organization of deep sedimentary columns reflects depositional history, present-day geochemical conditions, or both remains poorly understood, particularly under extreme oligotrophy. Here, we present a depth-resolved exploratory survey of a continuous 34-cm sediment core partitioned into 11 discrete horizons (L1 to L11) from Poza Roja 2, an alkaline, sub-hypersaline pond in the Cuatro Cienegas Basin, Mexico. Integrating physical stratigraphy, organic carbon and nutrient stoichiometry, and 16S rRNA gene metabarcoding, we evaluated down-core microbial community structure alongside acute phosphorus scarcity. Molar C:Po and N:Po ratios exceeded canonical Redfield proportions (106:16:1) in every layer, by up to 48.5-fold and 87.8-fold, respectively, both peaking in layer L4. Unconstrained ordination (PCoA) and hierarchical clustering grouped the layers into three compositional zones: upper (L2-L4), intermediate (L5-L6), and basal (L8-L11), with L1 and L7 occupying transitional positions. Archaea accounted for 31.0% of prokaryotic reads, ranging from 8.3% (L5) to 43.7% (L9), and were dominated by SM1-50 (Thermoplasmatota) and Halobacteriota. The phototrophic heliobacterium Heliorestis peaked in the intermediate zone (47.6% of bacterial reads in L6). Constrained Redundancy Analysis models lacked statistical significance (P >= 0.12), highlighting the exploratory nature of a single core (n = 11). This pilot characterization provides a baseline for understanding deep biogenic archives under severe nutrient limitation.

The molecular epidemiology of Acinetobacter, which is almost exclusively culture-dependent, has centered mainly on clinical isolates of A. baumannii. Yet, this overlooks sources not amenable to microbial culture and many non-A. baumannii species. Here, by analysing metagenomic projects from public transport systems worldwide, we show that metagenome-assembled genomes (MAGs) are highly relevant for assessing the unrecognized diversity of Acinetobacter. We recovered 839 Acinetobacter MAGs, with 269 being high-quality MAGs. The high-quality MAGs were across more than 20 well-defined Acinetobacter species; however, five species (A. nosocomilalis, A. ursingii, A. radioresistens, A. schindleri, and A. variabilis) had the most MAGs. Pangenome and phylogenetic diversity analyses of 685 genomes plus the 269 high-quality MAGs from those 5 species showed that MAGs are an important source of both lineage and gene diversity. Also, FST analyses and genome phylogenies show that for some species there is no genetic differentiation between MAGs and clinical isolates, implying transmission between hospitals and public transport settings. In addition to finding the antibiotic resistance genes (ARGs) in MAGs, we implemented an ad hoc strategy (leveraging the use of genomes) to recover considerably more ARGs. PCoA and PERMANOVA analyses show that, whereas in A. nosocomialis and A. ursingii, the ARG profiles of MAGs and clinical genomes are different, in A. radioresistens and A. schindleri, the ARG profiles are not different. Finally, some clinically important ARGs, such as sul1 and sul2, aadA, and blaOXA23, were found in all or some of the 5 species. Our study shows how combining MAGs and regular genomes is a powerful strategy for better appraising the genomic diversity (including the resistome) of Acinetobacter. On a broader level, our study showcases the importance of MAGs for genomic surveillance of man-made environments with clear public health implications.

Mitochondrial genomes of angiosperms are unusual due to their large size, low gene content, and high structural variation. In the palm family (Arecaceae), mitochondrial architecture is highly dynamic, characterized by expansive sizes, fragmentation, and extensive recombination activity. This biological complexity presents a significant bioinformatic challenge, especially since only three complete palm mitochondrial genomes are publicly available to date. Here, we present the complete mitochondrial genome sequence for the Amazonian palm Euterpe precatoria, assembled de novo using PacBio HiFi long-read sequencing data. The assembly required extensive manual curation and graph tracing to resolve a complex recombination topology. The final resolved sequence spans 1,268,308 base pairs and is supported by a mean read coverage depth estimated at 746X. Annotation revealed a high degree of structural complexity, including 177 total gene features corresponding to 76 unique genes. This gene set includes 41 unique protein-coding genes (56 features), 32 unique tRNA genes (118 features), and three unique rRNA genes, reflecting the characteristic gene duplication and fragmentation documented in Arecaceae. The complete mitochondrial genome sequence is publicly available and provides a high-quality resource for understanding the mechanisms of plant mitochondrial genome expansion and structural evolution.

Bats are major natural reservoirs for coronaviruses, yet complete viral genomes from South America remain scarce, limiting evolutionary and taxonomic understanding. Here, we conducted metatranscriptomic sequencing of coronavirus-positive bat samples collected across two ecologically distinct Brazilian biomes: the Atlantic Forest and the semi-arid Caatinga. We recovered seven complete or near-complete genomes belonging to Alphacoronavirus and Betacoronavirus. Phylogenetic and comparative similarity analyses of conserved replicase domains (3CLpro, NiRAN, RdRp, ZBD, HEL1), following International Committee on Taxonomy of Viruses (ICTV) demarcation criteria, revealed significant viral diversity. Within Alphacoronavirus, two genomes from Atlantic Forest phyllostomid bats (Artibeus lituratus and Carollia perspicillata) formed a deeply divergent sister lineage to Amalacovirus, exhibiting a mean amino acid similarity of 76.7% with the reference genome. Within Betacoronavirus, one genome from a Caatinga phyllostomid bat (Artibeus planirostris) clustered within the recently described Ambecovirus clade, displaying 75.9% mean amino acid similarity with mormoopid-associated reference sequences. Based on these divergence levels and non-recombinant genomic architectures, we propose two novel candidate subgenera, Phyllacovirus and Phyllobecovirus, alongside potential novel viral species. Furthermore, our findings demonstrate strong host-associated structuring and biogeographical partitioning of viral lineages across Neotropical biomes. Overall, this study expands the genomic landscape of South American bat coronaviruses and underscores the importance of continuous genomic surveillance at human-wildlife interfaces.

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.