Daniel Ricardo Matute

Genome sequencing is rapidly becoming a routine component of outbreak investigation and pathogen surveillance. In human fungal pathogens, however, the analytical framework has often remained centered on a single tree built from variants called against one reference genome. While that representation has been and will continue to be useful, epidemiological inference can also benefit from other approaches drawn from evolutionary genetics. Here, I review the transition from multilocus sequence typing to genome-scale epidemiology in human fungal pathogens, summarize representative successes in Coccidioides, Cryptococcus, Candida auris, Aspergillus fumigatus, Histoplasma, Sporothrix, Pneumocystis, and other fungi, and identify biological and technical sources of error that are still inconsistently addressed. More than a critique, this piece is a call to action. I argue that we need to start treating phylogenetic trees as one layer of evidence within a broader population-genetic framework and not as a stopping point. From a data collection point of view, we need a more robust epidemiological metadata collection scheme. From an analytical point of view, analyses of gene-tree discordance, demographic inference, ancestry, and structural variation can reveal features that a single tree derived from concatenated markers cannot. The amount of public sequence data is no longer the only limiting factor. The next pressing challenge is to translate the data generated by genomic and phylogenetic analyses into reproducible evolutionary inference while preserving and communicating the uncertainty that matters for public-health decisions.

The southern house mosquito Culex quinquefasciatus is a major disease vector for multiple arboviruses, including West Nile virus and St. Louis encephalitis virus. Insecticides are widely used to control Cx. quinquefasciatus populations, but resistant mosquitoes have emerged in numerous locations across the globe, including the United States. Much progress has been made in recent years in identifying insecticide resistance (IR) mutations in various pest species. In most species, these studies have focused primarily on SNPs as they are readily detectable. However, several genomic structural variants (SVs) have been experimentally shown to impact IR in Drosophila melanogaster, and structural variation has been observed in natural populations of vector species at a few genes that may be relevant to IR (e.g., cytochrome P450s). Because most population genomic data are derived from short sequencing reads, which fail to detect a large fraction of SVs, the contribution of structural variants to the evolution of IR and other adaptations in vector species is likely to be underestimated in the absence of long-read sequencing data. Here, we present the first genome-wide assessment of structural variants in Cx. quinquefasciatus from wild populations in Puerto Rico and Zambia using PacBio long-read sequencing. We constructed high-quality genome assemblies for each and found a total of 259,046 SVs which consisted predominantly of insertions and deletions. We found that 154,071 of these variants contained or overlapped genes. Of these SVs, 5,225 intersected IR genes and 56 of these were predicted to result in changes to downstream protein function. Several SVs predicted to severely impact protein-coding genes also coincided with signatures of selective sweeps, highlighting their possible role as targets of selection. Many of these variants would have gone undetected using short-read approaches, underscoring the importance of long-read sequencing for comprehensive population genetic analyses of adaptation to pesticides and other strong selective pressures, not only in insects but across the tree of life.

Paracoccidioidomycosis (PCM) is a systemic fungal infection caused by Paracoccidioides spp., predominantly affecting individuals in Latin America. While extensive epidemiological and molecular studies have been conducted in Brazil, Colombia, and Venezuela, where PCM is a recognized public health concern, Paraguay remains largely overlooked. The scarcity of epidemiological and molecular data hinders accurate disease surveillance, early diagnosis, and effective treatment strategies. This study provides the first comprehensive epidemiological and molecular characterization of PCM in Paraguay. We report the patient demographics, clinical presentations of PCM patients, and estimates of genetic diversity among isolates. A retrospective analysis of 66 confirmed PCM cases from the Mycology Section at the Center for Dermatological Specialties in San Lorenzo (2014–2024) was conducted. Diagnoses were confirmed through direct microscopy or fungal culture, and clinical isolates were genotyped using whole-genome sequencing. Findings indicate that PCM is more common among male patients, as 69% were agricultural workers representing high occupational risk. The chronic form was the most prevalent, commonly affecting the lungs and mucosa in males 40–60 years of age. Less frequent manifestations included the kidneys, central nervous system, gastrointestinal tract, and adrenal glands. Most PCM cases were treated with itraconazole; severe cases received amphotericin B. Molecular analysis demonstrated that Paraguayan isolates belong to both S1b and S1a clades; however they are distinct from Argentina and Mato Grosso do Sul, Brazil. These findings highlight the complexity of P. brasiliensis population structure in South America, emphasizing the need for enhanced diagnostic and treatment strategies in endemic regions.

The body size of adults and immature stages are fundamental animal traits that influence animal physiology, ecology, and range distribution. While the importance of egg size has been acknowledged as a proxy of parental investment in animals, little work has addressed the tempo and mode of evolution of egg size and shape. Here, we present a comparative study of this trait using a phylogeny based on genome-wide markers together with measurements of egg size and adult body size from 29 drosophilid species. Our analyses revisit the allometric relationship between egg size and body size and show that egg size scales negatively with respect to adult size, even after accounting for shared evolutionary history. In other words, larger species tend to produce proportionally smaller eggs. We also detect a moderate phylogenetic signal in both egg size and egg shape, indicating that closely related species resemble each other in these traits. Model comparisons show that the evolution of egg morphology in drosophilids is best described by gradual divergence through time driven by stochastic evolutionary change. This pattern contrasts with findings from other animal groups, including birds, cephalopods, and reptiles, where alternative evolutionary models better explain trait evolution. Together, these results suggest that the evolutionary dynamics shaping egg morphology in drosophilids differ from those operating in other major lineages and underscore the importance of comparative analyses of early developmental traits across taxa.

The Aedes aegypti mosquito is a vector for human arboviruses and zoonotic diseases and therefore poses a serious threat to public health. Understanding how Ae. aegypti adapts to environmental pressures—such as insecticides—is critical for developing effective mitigation strategies. However, most traditional methods for detecting recent positive selection search for signatures of classic “hard” selective sweeps, and to date no studies have examined soft sweeps in Ae. aegypti. This is a significant limitation as this is vital information for understanding the pace of adaptation—populations that can immediately respond to new selective pressures are expected to adapt more often via standing variation or recurrent adaptive mutations (both of which may produce soft sweeps) than via de novo mutations (which produces hard sweeps). To this end, we used a machine learning method capable of detecting hard and soft sweeps to investigate positive selection in Ae. aegypti population samples from Africa and the Americas. Our results reveal that soft sweeps are significantly more common than hard sweeps, which may imply that this species can respond quickly to environmental stressors. This is a particularly concerning finding for vector control methods that aim to eradicate Ae. aegypti using insecticides. We highlight genes under selection that include both well-characterized and putatively novel insecticide resistance genes. These findings underscore the importance of using methods capable of detecting and distinguishing hard and soft sweeps, implicate soft sweeps as a major selective mode in Ae. aegypti, and highlight genes that may aid in the control of Ae. aegypti populations.

Histoplasma spp. is a dimorphic fungal primary pathogen that infects people worldwide and frequently affects immunosuppressed patients. Previous studies have identified the AMY1 gene product, the α-amylase Amy1p, as essential for α-glucan production and virulence in Histoplasma capsulatum . We identified two new genes ( AMY2 and AMY3 ) in the Histoplasma genome that encode putative α-amylases and made mutants using CRISPR/Cas9 technology, followed by evaluation of their role in α-glucan biosynthesis and virulence. We also searched for AMY gene copies in 19 fungal genomes with the goals of identifying orthologs for AMY2 and AMY3 , and establishing how many AMY copies existed across different fungi. We found that the number and type of α-amylases vary depending on the fungal species; that all α-amylases related to Histoplasma Amy1p belong to the GH13_5 subfamily, and all orthologs related to Histoplasma ’s Amy2p and Amy3p belong to the GH13_1 subfamily. We performed phylogenetic analyses of the three paralogs and revealed that the Histoplasma AMY duplications are ancient. We further established Amy2 is an ortholog of Aspergillus niger AgtA, and Aspergillus nidulans AmyD, and that it is partially involved in Histoplasma α-glucan biosynthesis and virulence, while Amy3p is an ortholog of Aspergillus flavus Amy1, and it is dispensable for α-glucan biosynthesis and virulence.

Background: Temperature choice is a vector trait that influences microhabitat selection and can have important implications for vector species, as it may affect how often vectors encounter hosts. Aedes aegypti and Ae. albopictus are disease vectors whose geographic ranges continue to expand each year. One aspect that remains largely understudied is the altitudinal range of these species and the extent of differences in thermal behavior between lowland and highland populations. Methods: I collected Ae. aegypti and Ae. albopictus on the islands of Bioko and São Tomé. I compared the distribution of the two species along an altitudinal cline spanning 2,000 m of elevation. I then used live specimens to test temperature preference for both species in a laboratory thermocline. Results: I report the distribution of these two species on the island of Bioko and show that the abundance of immature stages of both species follows a negative exponential decay with altitude. I compare this distribution with that observed on the neighboring island of São Tomé, also in the Gulf of Guinea. Overall, the distribution patterns of the two species are similar, but models indicate a higher abundance at sea level in São Tomé than in Bioko. I used specimens from this survey to study temperature preference under controlled conditions. I found no significant differences between species or between sexes; however, I detected an altitudinal cline in temperature preference, with high-elevation populations preferring cooler temperatures on both islands. Conclusions: These results indicate the presence of phenotypic variation in a key trait—temperature choice—that may alter the likelihood of contact between these vectors and humans.

Sarocladium is a fungal genus containing diverse members with roles as pathogens of plants and animals, endophytes, and biocontrol agents. This study characterizes two Sarocladium spinificis strains isolated from patients diagnosed with coccidioidomycosis. Morphological, phylogenetic, and genomic analyses confirmed their classification within the Sarocladium genus, closely related to S. spinificis . Both strains exhibited mycelial growth at 24°C and 37°C, with distinct morphological features. Comparative genomic analysis identified unique orthologous clusters and functional gene families associated with diverse carbon utilization and potential pathogenicity. Notably, these strains’ genomes contain many secreted proteins, carbohydrate-activated enzymes, and peptidases, highlighting their metabolic versatility. Co-cultivation experiments revealed that S. spinificis strains CA16 and CA18 both inhibited the growth of Coccidioides posadasii , suggesting antagonistic interactions and potential antifungal properties. These findings underscore the ecological and clinical significance of S. spinificis and its impact on microbial communities, advancing our understanding of its diversity, pathogenic potential, and antifungal capabilities. IMPORTANCE: Accurate identification and characterization of emerging fungal pathogens are essential for improving diagnosis and treatment. Sarocladium spinificis , though not widely recognized as a human pathogen, exhibits traits suggesting a potential role in opportunistic infections. This study provides the first detailed morphological, phylogenetic, and genomic characterization of clinical S. spinificis isolates, highlighting its ability to survive at body temperature and its antifungal resistance. Additionally, we demonstrate that these strains inhibit Coccidioides posadasii , the agent of Valley fever, suggesting ecological competition or antifungal properties. These findings contribute to understanding fungal interactions in clinical and environmental settings, with implications for fungal pathogenesis and antifungal strategies. By uncovering new aspects of S. spinificis biology, this work expands knowledge of Sarocladium species in human health and lays the foundation for future research on its ecological role, pathogenic potential, and therapeutic applications.