Spencermartinsiella nicolii sp. nov., a potential opportunistic pathogenic yeast species isolated from rotting wood in Brazil.

Nineteen isolates representing a candidate for a novel yeast species belonging to the genus Spencermartinsiella were recovered from rotting wood samples collected at different sites in Atlantic Rainforest and Amazonian Forest ecosystems in Brazil. Similarity search of the nucleotide sequence of the intergenic spacer (ITS)-5.8S and large subunit D1/D2 regions of the ribosomal gene cluster showed that this novel yeast is closely related to Spencermartinsiella cellulosicola. The isolates differ by four nucleotide substitutions in the D1/D2 domain and six substitutions and 31 indels in the ITS region from the holotype of S. cellulosicola. Phylogenomic analysis based on 1474 single-copy orthologues for a set of Spencermartinsiella species whose whole genome sequences are available confirmed that the novel species is phylogenetically close to S. cellulosicola. The low average nucleotide identity value of 83% observed between S. cellulosicola and the candidate species confirms that they are distinct. The novel species produced asci with hemispherical ascospores. The name Spencermartinsiella nicolii sp. nov. is proposed. The holotype is CBS 14238T. The MycoBank number is MB855027. Interestingly, the D1/D2 sequence of the S. nicolii was identical to that of an uncultured strain of Spencermartinsiella causing systemic infection in a male adult crocodile (Crocodylus niloticus). The characterization of some virulence factors and antifungal susceptibility of S. nicolii isolates suggest that this yeast may be an opportunistic pathogen for animals, including humans; the isolates grow at 37 °C.

Taxonomic and functional characterization of biofilms from a photovoltaic panel reveals high genetic and metabolic complexity of the communities.

AIMS: Biofilms are complex microbial cell aggregates that attach to different surfaces in nature, industrial environments, or hospital settings. In photovoltaic panels (PVs), biofilms are related to significant energy conversion losses. In this study, our aim was to characterize the communities of microorganisms and the genes involved in biofilm formation. METHODS AND RESULTS: In this study, biofilm samples collected from a PV system installed in southeastern Brazil were analyzed through shotgun metagenomics, and the microbial communities and genes involved in biofilm formation were investigated. A total of 2030 different genera were identified in the samples, many of which were classified as extremophiles or producers of exopolysaccharides. Bacteria prevailed in the samples (89%), mainly the genera Mucilaginibacter, Microbacterium, Pedobacter, Massilia, and Hymenobacter. The functional annotation revealed >12 000 genes related to biofilm formation and stress response. Genes involved in the iron transport and synthesis of c-di-GMP and c-AMP second messengers were abundant in the samples. The pathways related to these components play a crucial role in biofilm formation and could be promising targets for preventing biofilm formation in the PV. In addition, Raman spectroscopy analysis indicated the presence of hematite, goethite, and ferrite, consistent with the mineralogical composition of the regional soil and metal-resistant bacteria. CONCLUSIONS: Taken together, our findings reveal that PV biofilms are a promising source of microorganisms of industrial interest and genes of central importance in regulating biofilm formation and persistence.

Orthoporus fuscipes (PORAT, 1888) (Juliformia; Spirostreptidae): population structure and defensive secretion chemical analysis.

Diplopods are terrestrial arthropods important for the dynamics of terrestrial ecosystems. One of the reasons for that can be their low predation rate due to their defensive secretion. Thus, Orthoporus fuscipes, a species belonging to this group and endemic to northeastern Brazil, was investigated as to its population structure and chemical constituents of defensive secretion. The population structure showed that females are larger and have greater mass than do males, along with negative allometric growth between males and females. The defensive secretion hexane extract was submitted to fractionation using SiO2 open-column chromatography and the gas chromatographic coupled to mass spectrometric analysis was applied in the fraction possibilities to identify major fatty acid methyl esthers, along with minor alkanes, alkenes and fatty acids derivatives and the known quinoids 2-methoxy-3-methylhydroquinone, 2-methoxy-3-methyl-1,4-benzoquinone, 2,3-dimethoxy-1,4-benzoquinone and 2,3-dimethoxyhydroquinone. In addition, the cytochrome oxidase I sequence for the species was deposited for the first time.

Exploring the hidden hot world of long non-coding RNAs in thermophilic fungus using a robust computational pipeline.

Long noncoding RNAs (lncRNAs) are versatile RNA molecules recently identified as key regulators of gene expression in response to environmental stress. Our primary focus in this study was to develop a robust computational pipeline for identifying structurally identical lncRNAs across replicates from publicly available bulk RNA-seq datasets. In order to demonstrate the effectiveness of the pipeline, we utilized the transcriptome of the thermophilic fungus Thermothelomyces thermophilus and assessed the expression pattern of lncRNAs in conjunction with Heat Shock Proteins (HSP), a well-known protein family critical for the cell's response to high temperatures. Our findings demonstrate that the identification of structurally identical transcripts among replicates in this thermophilic fungus ensures the reliability and accuracy of RNA studies, contributing to the validity of biological interpretations. Furthermore, the majority of lncRNAs exhibited a distinct expression pattern compared to HSPs. Our study contributes to advancing the understanding of the biological mechanisms comprising lncRNAs in thermophilic fungi.

A new and threatened species of Bondarzewia from the Brazilian cloud forests.

A new and threatened polypore species, Bondarzewia loguerciae, is described from the cloud forests of southern Brazil. It is characterized by single-pileate basidiomata that grow on dead branches and along living stems of standing trunks and present a context with dark lines and resinous tubes. When growing in axenic culture, this species also develops chlamydospores. We provide an illustrated morphological description and molecular analysis. Our specimens from Brazil form a monophyletic group among other species of the Southern Hemisphere. The conservation status of B. loguerciae is assessed and published as "Critically Endangered" based on the International Union for Conservation of Nature  (IUCN) criteria. Additionally, a key to the species is provided.

Innovative infrastructure to access Brazilian fungal diversity using deep learning.

In the present investigation, we employ a novel and meticulously structured database assembled by experts, encompassing macrofungi field-collected in Brazil, featuring upwards of 13,894 photographs representing 505 distinct species. The purpose of utilizing this database is twofold: firstly, to furnish training and validation for convolutional neural networks (CNNs) with the capacity for autonomous identification of macrofungal species; secondly, to develop a sophisticated mobile application replete with an advanced user interface. This interface is specifically crafted to acquire images, and, utilizing the image recognition capabilities afforded by the trained CNN, proffer potential identifications for the macrofungal species depicted therein. Such technological advancements democratize access to the Brazilian Funga, thereby enhancing public engagement and knowledge dissemination, and also facilitating contributions from the populace to the expanding body of knowledge concerning the conservation of macrofungal species of Brazil.

Comparative genomics and stable isotope analysis reveal the saprotrophic-pathogenic lifestyle of a neotropical fungus.

In terrestrial forested ecosystems, fungi may interact with trees in at least three distinct ways: (i) associated with roots as symbionts; (ii) as pathogens in roots, trunks, leaves, flowers, and fruits; or (iii) decomposing dead tree tissues on soil or even on dead tissues in living trees. Distinguishing the latter two nutrition modes is rather difficult in Hymenochaetaceae (Basidiomycota) species. Herein, we have used an integrative approach of comparative genomics, stable isotopes, host tree association, and bioclimatic data to investigate the lifestyle ecology of the scarcely known neotropical genus Phellinotus, focusing on the unique species Phellinotus piptadeniae. This species is strongly associated with living Piptadenia gonoacantha (Fabaceae) trees in the Atlantic Forest domain on a relatively high precipitation gradient. Phylogenomics resolved P. piptadeniae in a clade that also includes both plant pathogens and typical wood saprotrophs. Furthermore, both genome-predicted Carbohydrate-Active Enzymes (CAZy) and stable isotopes (δ13C and δ15N) revealed a rather flexible lifestyle for the species. Altogether, our findings suggest that P. piptadeniae has been undergoing a pathotrophic specialization in a particular tree species while maintaining all the metabolic repertoire of a wood saprothroph. IMPORTANCE: This is the first genomic description for Phellinotus piptadeniae. This basidiomycete is found across a broad range of climates and ecosystems in South America, including regions threatened by extensive agriculture. This fungus is also relevant considering its pathotrophic-saprotrophic association with Piptadenia goanocantha, which we began to understand with these new results that locate this species among biotrophic and necrotrophic fungi.

Spathaspora marinasilvae sp. nov., a xylose-fermenting yeast isolated from galleries of passalid beetles and rotting wood in the Amazonian rainforest biome.

Four yeast isolates were obtained from rotting wood and galleries of passalid beetles collected in different sites of the Brazilian Amazonian Rainforest in Brazil. This yeast produces unconjugated allantoid asci each with a single elongated ascospore with curved ends. Sequence analysis of the internal transcribed spacer-5.8 S region and the D1/D2 domains of the large subunit ribosomal RNA (rRNA) gene showed that the isolates represent a novel species of the genus Spathaspora. The novel species is phylogenetically related to a subclade containing Spathaspora arborariae and Spathaspora suhii. Phylogenomic analysis based on 1884 single-copy orthologs for a set of Spathaspora species whose whole genome sequences are available confirmed that the novel species represented by strain UFMG-CM-Y285 is phylogenetically close to Sp. arborariae. The name Spathaspora marinasilvae sp. nov. is proposed to accommodate the novel species. The holotype of Sp. marinasilvae is CBS 13467 T (MycoBank 852799). The novel species was able to accumulate xylitol and produce ethanol from d-xylose, a trait of biotechnological interest common to several species of the genus Spathaspora.

Whole-genome sequencing analysis of multidrug-resistant Serratia marcescens isolates in an intensive care unit in Brazil.

INTRODUCTION: Serratia marcescens is an opportunistic pathogen found ubiquitously in the environment and associated with a wide range of nosocomial infections. This multidrug-resistant bacterium has been a cause of concern for hospitals and healthcare facilities due to its ability to spread rapidly and cause outbreaks. Next generation sequencing genotyping of bacterial isolates has proven to be a valuable tool for tracking the spread and transmission of nosocomial infections. This has allowed for the identification of outbreaks and transmission chains, as well as determining whether cases are due to endogenous or exogenous sources. Evidence of nosocomial transmission has been gathered through genotyping methods. The aim of this study was to investigate the genetic diversity of carbapenemase-producing S. marcescens in an outbreak at a public hospital in Cuiaba, MT, Brazil. METHODOLOGY: Ten isolates of S. marcenses were sequenced and antibiotic resistance profiles analyzed over 12 days. RESULTS: The isolates were clonal and multidrug resistant. Gentamycin and tigecycline had sensitivity in 90% and 80% isolates, respectively. Genomic analysis identified several genes that encode ß-lactamases, aminoglycoside-modifying enzymes, efflux pumps, and other virulence factors. CONCLUSIONS: Systematic surveillance is crucial in monitoring the evolution of S. marcescens genotypes, as it can lead to early detection and prevention of outbreaks.

Phylodynamics of avian influenza A(H5N1) viruses from outbreaks in Brazil.

Our study identified strains of the A/H5N1 virus in analyzed samples of subsistence poultry, wild birds, and mammals, belonging to clade 2.3.4.4b, genotype B3.2, with very high genetic similarity to strains from Chile, Uruguay, and Argentina. This suggests a migratory route for wild birds across the Pacific, explaining the phylogenetic relatedness. The Brazilian samples displayed similarity to strains that had already been previously detected in South America. Phylogeographic analysis suggests transmission of US viruses from Europe and Asia, co-circulating with other lineages in the American continent. As mutations can influence virulence and host specificity, genomic surveillance is essential to detect those changes, especially in critical regions, such as hot spots in the HA, NA, and PB2 sequences. Mutations in the PB2 gene (D701N and Q591K) associated with adaptation and transmission in mammals were detected suggesting a potential zoonotic risk. Nonetheless, resistance to neuraminidase inhibitors (NAIs) was not identified, however, continued surveillance is crucial to detect potential resistance. Our study also mapped the spread of the virus in the Southern hemisphere, identifying possible entry routes and highlighting the importance of surveillance to prevent outbreaks and protect both human and animal populations.

Pure lignin induces overexpression of cytochrome P450 (CYP) encoding genes and brings insights into the lignocellulose depolymerization by Trametes villosa.

Trametes villosa is a remarkable white-rot fungus (WRF) with the potential to be applied in lignocellulose conversion to obtain chemical compounds and biofuels. Lignocellulose breakdown by WRF is carried out through the secretion of oxidative and hydrolytic enzymes. Despite the existing knowledge about this process, the complete molecular mechanisms involved in the regulation of this metabolic system have not yet been elucidated. Therefore, in order to understand the genes and metabolic pathways regulated during lignocellulose degradation, the strain T. villosa CCMB561 was cultured in media with different carbon sources (lignin, sugarcane bagasse, and malt extract). Subsequently, biochemical assays and differential gene expression analysis by qPCR and high-throughput RNA sequencing were carried out. Our results revealed the ability of T. villosa CCMB561 to grow on lignin (AL medium) as the unique carbon source. An overexpression of Cytochrome P450 was detected in this medium, which may be associated with the lignin O-demethylation pathway. Clusters of up-regulated CAZymes-encoding genes were identified in lignin and sugarcane bagasse, revealing that T. villosa CCMB561 acts simultaneously in the depolymerization of lignin, cellulose, hemicellulose, and pectin. Furthermore, genes encoding nitroreductases and homogentisate-1,2-dioxygenase that act in the degradation of organic pollutants were up-regulated in the lignin medium. Altogether, these findings provide new insights into the mechanisms of lignocellulose degradation by T. villosa and confirm the ability of this fungal species to be applied in biorefineries and in the bioremediation of organic pollutants.

Comparative genetic and pathogenic approaches of Escherichia coli isolated simultaneously from pyometra and urine of bitches.

Escherichia coli (E. coli) are widely related to pyometra and cystitis in dogs, and these infections can occur simultaneously. The goal of this study was to determine genetic and pathogenic insights of 14 E. coli isolated simultaneously from pyometra content and bladder urine of seven bitches. To achieve this, in silico and in vitro comparative analyses were conducted. Whole-genome comparisons demonstrated that E. coli isolated from pyometra and urine of the same animal were predominantly genetic extraintestinal E. coli clones belonging to the same Sequence Type and phylogroup. The E. coli clones identified in this study included ST372, ST457, ST12, ST127, ST646, and ST961. Five isolates (35.7%) belonged to the ST12 complex. Except for two E. coli, all other isolates belonged to the B2 Clermont phylogroup. Interestingly, some genomes of E. coli from urine carried more virulence genes than those E. coli from pyometra. Both pyometra and urine E. coli isolates demonstrated a strong affinity for adhering to HeLa and T24 cells, with a low affinity for invading them. However, certain isolates from urine exhibited a greater tendency to adhere to T24 cells in qualitative and quantitative assays compared to isolates from pyometra. In conclusion, this study revealed the high genomic similarity between pyometra and urine E. coli isolates, as well as the virulent capacity of both to colonize endometrial and urothelial cells. The findings of this study underscore the importance of concurrently managing both infections clinically and could potentially contribute to future resources for the prevention of cystitis and pyometra.

Bacterial microbiome changes after fecal transplantation for recurrent Clostridioides difficile infection in the Brazilian center.

Clostridioides difficile infection (CDI) poses a significant global health threat owing to its substantial morbidity and associated healthcare costs. A key challenge in controlling CDI is the risk of multiple recurrences, which can affect up to 30% of patients. In such instances, fecal microbiota transplantation (FMT) is increasingly recognized as the optimal treatment. However, few related studies have been conducted in developing countries, and the microbiota composition of Brazilian patients and its dynamic modification post-FMT remain largely unexplored. This study aimed to evaluate the changes in the bacterial gut microbiome in Brazilian patients with recurrent CDI post-FMT. Ten patients underwent FMT, and the primary and overall CDI resolution rates were 80% and 90% after the first and second FMT, respectively. FMT was associated with an early increase in Shannon's diversity, evident as soon as 1 week post-FMT and persisting for at least 25 days post-treatment. Post-treatment, the abundance of Firmicutes increased and that of Proteobacteria decreased. Specifically, the abundance of the genera Ruminococcus, Faecalibacterium, Lachnospira, and Roseburia of the Firmicutes phylum was significantly higher 1 week post-transplantation, with Ruminococcus and Faecalibacterium remaining enriched 25 days post-transplantation. This study is the first of its kind in Brazil to evaluate the microbiota of a donor and patients undergoing FMT. Our findings suggest that FMT can induce remarkable changes in the gut microbiota, characterized by an early and sustained increase in diversity lasting at least 25 days. FMT also promotes enrichment of genera such as Ruminococcus spp., Faecalibacterium spp., and Roseburia spp., essential for therapeutic success.

Alternative amplicon-PCR protocol for maximizing bacterial and fungal sequencing in low-biomass samples.

Determining bacterial and fungal communities from low-biomass samples remains a challenge for high-throughput sequencing. Due to the low microbial load and host contamination, some sites, including the female upper reproductive tract and the lower respiratory tract, were even considered sterile until recent years. Despite efforts to improve sampling and DNA isolation protocols, some samples provide insufficient microbial DNA input for library preparation and sequencing. Herein, we propose an alternative amplicon-PCR protocol to be used in bacterial and fungal sequencing in low-biomass samples, targeting 16S-rDNA and the internal transcribed spacer region (ITS), respectively. Similar to a nested-PCR, we performed two sequential PCR reactions to maximise the target amplicon. We compared metagenomic results from the original Illumina protocol (Protocol 1 - P1) and the alternative one (Protocol 2 - P2), using a mock community and clinical samples with different microbial loads. Our findings showed no significant differences in data generated by P1 and P2, indicating that the second amplification round does not bias the microbiota diversity rates. Thus, the alternative protocol can be applied for low-biomass samples when the original protocol results in spurious output, preventing library preparation and sequencing.

Systematic review of reverse vaccinology and immunoinformatics data for non-viral sexually transmitted infections.

Sexually Transmitted Infections (STIs) are a public health burden rising in developed and developing nations. The World Health Organization estimates nearly 374 million new cases of curable STIs yearly. Global efforts to control their spread have been insufficient in fulfilling their objective. As there is no vaccine for many of these infections, these efforts are focused on education and condom distribution. The development of vaccines for STIs is vital for successfully halting their spread. The field of immunoinformatics is a powerful new tool for vaccine development, allowing for the identification of vaccine candidates within a bacterium's genome and allowing for the design of new genome-based vaccine peptides. The goal of this review was to evaluate the usage of immunoinformatics in research focused on non-viral STIs, identifying fields where research efforts are concentrated. Here we describe gaps in applying these techniques, as in the case of Treponema pallidum and Trichomonas vaginalis.

First report and genetic characterization of the highly pathogenic avian influenza A(H5N1) virus in Cabot's tern (Thalasseus acuflavidus), Brazil.

In 2021, the H5N1 virus lineage 2.3.4.4b spread to the Americas, causing high mortality in wild and domestic avian populations. South American countries along the Pacific migratory route have reported wild bird deaths due to A/H5Nx virus since October 2022. However, limited genomic data resulted in no cases reported in Brazil until May 2023. Brazil reported its first case of highly pathogenic avian influenza virus (HPAI A/H5N1) in May 2023. The virus was detected in Cabot's tern specimen in Marataízes, Espírito Santo. Cases were also found in backyard poultry and other wild birds, but no human or commercial poultry cases occurred. HPAI poses risks to the poultry industry, food security, and public health. Researchers used next-gen sequencing and phylogenetic analysis to study the Brazilian sample. It confirmed its affiliation with the 2.3.4.4b clade and proximity to sequences from Chile and Peru. This sheds light on the spread and evolution of HPAI A/H5N1 in the Americas, emphasizing continuous monitoring to mitigate risks for both avian and human populations. Understanding the virus's genetics and transmission allows implementing effective control measures to protect public health and the poultry industry.

Cactus Endophytic Fungi and Bioprospecting for their Enzymes and Bioactive Molecules: A Systematic Review.

Endophytic fungi are associated with plant health and represent a remarkable source of potential of enzymes and bioactive compounds, but the diversity of endophytes remains uncertain and poorly explored, especially in Cactaceae, one of the most species-rich families adapted to growing in arid and semi-arid regions. The aim of this study was to conduct a systematic review on the diversity and bioprospecting of endophytic fungi from Cactaceae. We analysed peer-reviewed articles from seven databases using PRISMA guidelines. The results showed that the Cactaceae family is a source of new taxa, but the diversity of endophytic fungi of Cactaceae is little explored, mainly the diversity among tissues and by metagenomics. Bioprospecting studies have shown that these microorganisms can be used in the production of enzymes and larvicidal and antifungal compounds. Our results are relevant as a starting point for researchers to develop studies that expand the knowledge of plant mycobiota in arid and semi-arid ecosystems, as well as comprising a remarkable source of fungal compounds with several biotechnological applications.

Yeasts from tropical forests: Biodiversity, ecological interactions, and as sources of bioinnovation.

Tropical rainforests and related biomes are found in Asia, Australia, Africa, Central and South America, Mexico, and many Pacific Islands. These biomes encompass less than 20% of Earth's terrestrial area, may contain about 50% of the planet's biodiversity, and are endangered regions vulnerable to deforestation. Tropical rainforests have a great diversity of substrates that can be colonized by yeasts. These unicellular fungi contribute to the recycling of organic matter, may serve as a food source for other organisms, or have ecological interactions that benefit or harm plants, animals, and other fungi. In this review, we summarize the most important studies of yeast biodiversity carried out in these biomes, as well as new data, and discuss the ecology of yeast genera frequently isolated from tropical forests and the potential of these microorganisms as a source of bioinnovation. We show that tropical forest biomes represent a tremendous source of new yeast species. Although many studies, most using culture-dependent methods, have already been carried out in Central America, South America, and Asia, the tropical forest biomes of Africa and Australasia remain an underexplored source of novel yeasts. We hope that this review will encourage new researchers to study yeasts in unexplored tropical forest habitats.

Systematic review and meta-analysis: the efficiency of bacteriophages previously patented against pathogenic bacteria on food.

Food-borne diseases are a global public health issue with 1 in 10 people falling ill after eating contaminated food every year. In response, the food industry has implemented several new pathogen control strategies, such as biotechnological tools using the direct application of bacteriophages for biological control. We have undertaken a systematic review and meta-analysis that evaluated the efficiency of patented phages as a biological control for food-borne pathogens and determined the physical-chemical characteristics of the antimicrobial effect. Included and excluded criteria was developed. Included criteria: Phage patent files with an application in biological control on food and scientific articles and book chapters that used phages patented for food biological control. Excluded criteria: Patent documents, scientific articles, and book chapters that included phage therapy in humans, animals, and biological control on plants but did not have an application on food were not considered in our study. The systematic analysis identified 77 documents, 46 scientific articles, and 31 documents of patents and 23 articles was included in the meta-analysis. Listeria monocytogenes and Salmonella sp. comprised most of the targets identified in the screening, so that we focused on these strains to do the meta-analysis. There are a total of 383 and 192 experiments for Listeria and Salmonella phages for quantitative data analysis.Indexing databases for the bibliographic search (Scopus, Web of Science (WoS) and PubMed (Medline) were addressed by an automated script written in Python 3 Python Core Team (2015) and deposited on GitHub ( https://github.com/glenjasper ).A random-effects meta-analysis revealed (i) significant antimicrobial effect of Listeria phages in apple, apple juice, pear, and pear juice, (ii) significant antimicrobial effect of Salmonella phages in eggs, apple, and ready-to-eat chicken, (iii) no heterogeneity was identified in either meta-analysis, (iv) publication bias was detected for Listeria phages but not for Salmonella phages. (v) ListShield and Felix01 phages showed the best result for Listeria and Salmonella biological control, respectively, (vi) concentration of phage and bacteria, time and food had significant effect in the biological control of Listeria, (vii) temperature and time had a significant effect on the antimicrobial activity of Salmonella phages. The systematic review and meta-analyses to determine the efficiency of bacteriophages previously patented against pathogenic bacteria on dairy products, meat, fruits and vegetables. Besides, the discovering of key factors for efficacy, so that future applications of phage biotechnology in foods can be optimally deployed.

Unveiling Fungal Community Structure along Different Levels of Anthropic Disturbance in a South American Subtropical Lagoon.

Studies of fungal communities through amplicon metagenomics in aquatic environments, particularly in freshwater ecosystems, are still relatively recent. Unfortunately, many of these water bodies are facing growing threats from human expansion, such as effluent discharge from various human activities. As a result, these effluents have the potential to significantly alter the characteristics of water bodies and, subsequently, impact the diversity of their resident microorganisms. In this context, our objective was to investigate whether the fungal community structure varies according to the presence of different anthropic disturbances. We expect (i) the diversity of fungi will be greater and (ii) more specific unique operational taxonomic units (OTUs) related to each ecotonal system will be found compared to other sites of a lagoon. The study was conducted in the Tramandaí Lagoon (subtropical southern Brazil) at four distinct sampling points (estuary, middle of the lagoon, crop field area, and near a residential area where the Tramandaí River flows into the lagoon). As expected, the estuary and residential zones, which are ecotones, exhibited greater fungal diversity and more specific OTUs compared to the middle of the lagoon and crop field area. Moreover, a substantial proportion of fungal taxa could not be identified at the genus level, with many only classified at the phylum level, indicating potential new lineages. These findings underscore our limited understanding of the subtropical freshwater mycobiota.