Phenotypic characterization of cryptic species in the fungal pathogen Histoplasma.

Histoplasmosis is an endemic mycosis that often presents as a respiratory infection in immunocompromised patients. Hundreds of thousands of new infections are reported annually around the world. The etiological agent of the disease, Histoplasma, is a dimorphic fungus commonly found in the soil where it grows as mycelia. Humans can become infected by Histoplasma through inhalation of its spores (conidia) or mycelial particles. The fungi transition into the yeast phase in the lungs at 37°C. Once in the lungs, yeast cells reside and proliferate inside alveolar macrophages. Genomic work has revealed that Histoplasma is composed of at least five cryptic phylogenetic species that differ genetically. Three of those lineages have received new names. Here, we evaluated multiple phenotypic characteristics (colony morphology, secreted proteolytic activity, yeast size, and growth rate) of strains from five of the phylogenetic species of Histoplasma to identify phenotypic traits that differentiate between these species: Histoplasma capsulatum sensu stricto, Histoplasma ohiense, Histoplasma mississippiense, Histoplasma suramericanum, and an African lineage. We report diagnostic traits for three species. The other two species can be identified by a combination of traits. Our results suggest that (i) there are significant phenotypic differences among the cryptic species of Histoplasma and (ii) those differences can be used to positively distinguish those species in a clinical setting and for further study of the evolution of this fungal pathogen.IMPORTANCEIdentifying species boundaries is a critical component of evolutionary biology. Genome sequencing and the use of molecular markers have advanced our understanding of the evolutionary history of fungal pathogens, including Histoplasma, and have allowed for the identification of new species. This is especially important in organisms where morphological characteristics have not been detected. In this study, we revised the taxonomic status of the four named species of the genus Histoplasma, H. capsulatum sensu stricto (ss), H. ohiense, H. mississippiense, and H. suramericanum, and propose the use of species-specific phenotypic traits to aid their identification when genome sequencing is not available. These results have implications not only for evolutionary study of Histoplasma but also for clinicians, as the Histoplasma species could determine the outcome of disease and treatment needed.

Genotypic diversity, virulence, and molecular genetic tools in Histoplasma.

SUMMARYHistoplasmosis is arguably the most common fungal respiratory infection worldwide, with hundreds of thousands of new infections occurring annually in the United States alone. The infection can progress in the lung or disseminate to visceral organs and can be difficult to treat with antifungal drugs. Histoplasma, the causative agent of the disease, is a pathogenic fungus that causes life-threatening lung infections and is globally distributed. The fungus has the ability to germinate from conidia into either hyphal (mold) or yeast form, depending on the environmental temperature. This transition also regulates virulence. Histoplasma and histoplasmosis have been classified as being of emergent importance, and in 2022, the World Health Organization included Histoplasma as 1 of the 19 most concerning human fungal pathogens. In this review, we synthesize the current understanding of the ecological niche, evolutionary history, and virulence strategies of Histoplasma. We also describe general patterns of the symptomatology and epidemiology of histoplasmosis. We underscore areas where research is sorely needed and highlight research avenues that have been productive.

Demographic history and the efficacy of selection in the globally invasive mosquito Aedes aegypti.

Aedes aegypti is the main vector species of yellow fever, dengue, zika and chikungunya. The species is originally from Africa but has experienced a spectacular expansion in its geographic range to a large swath of the world, the demographic effects of which have remained largely understudied. In this report, we examine whole-genome sequences from 6 countries in Africa, North America, and South America to investigate the demographic history of the spread of Ae. aegypti into the Americas its impact on genomic diversity. In the Americas, we observe patterns of strong population structure consistent with relatively low (but probably non-zero) levels of gene flow but occasional long-range dispersal and/or recolonization events. We also find evidence that the colonization of the Americas has resulted in introduction bottlenecks. However, while each sampling location shows evidence of a past population contraction and subsequent recovery, our results suggest that the bottlenecks in America have led to a reduction in genetic diversity of only ~35% relative to African populations, and the American samples have retained high levels of genetic diversity (expected heterozygosity of ~0.02 at synonymous sites) and have experienced only a minor reduction in the efficacy of selection. These results evoke the image of an invasive species that has expanded its range with remarkable genetic resilience in the face of strong eradication pressure.

Tracing histoplasmosis genomic epidemiology and species occurrence across the USA.

ABSTRACTHistoplasmosis is an endemic mycosis in North America frequently reported along the Ohio and Mississippi River Valleys, although autochthonous cases occur in non-endemic areas. In the United States, the disease is provoked by two genetically distinct clades of Histoplasma capsulatum sensu lato, Histoplasma mississippiense (Nam1) and H. ohiense (Nam2). To bridge the molecular epidemiological gap, we genotyped 93 Histoplasma isolates (62 novel genomes) including clinical, environmental, and veterinarian samples from a broader geographical range by whole-genome sequencing, followed by evolutionary and species niche modelling analyses. We show that histoplasmosis is caused by two major lineages, H. ohiense and H. mississippiense; with sporadic cases caused by H. suramericanum in California and Texas. While H. ohiense is prevalent in eastern states, H. mississipiense was found to be prevalent in the central and western portions of the United States, but also geographically overlapping in some areas suggesting that these species might co-occur. Species Niche Modelling revealed that H. ohiense thrives in places with warmer and drier conditions, while H. mississippiense is endemic to areas with cooler temperatures and more precipitation. In addition, we predicted multiple areas of secondary contact zones where the two species co-occur, potentially facilitating gene exchange and hybridization. This study provides the most comprehensive understanding of the genomic epidemiology of histoplasmosis in the USA and lays a blueprint for the study of invasive fungal diseases.

Phenotypic characterization of cryptic species in the fungal pathogen Histoplasma.

Histoplasmosis is an endemic mycosis that often presents as a respiratory infection in immunocompromised patients. Hundreds of thousands of new infections are reported annually around the world. The etiological agent of the disease, Histoplasma, is a dimorphic fungus commonly found in the soil where it grows as mycelia. Humans can become infected by Histoplasma through inhalation of its spores (conidia) or mycelial particles. The fungi transitions into the yeast phase in the lungs at 37°C. Once in the lungs, yeast cells reside and proliferate inside alveolar macrophages. We have previously described that Histoplasma is composed of at least five cryptic species that differ genetically, and assigned new names to the lineages. Here we evaluated multiple phenotypic characteristics of 12 strains from five phylogenetic species of Histoplasma to identify phenotypic traits that differentiate between these species: H. capsulatum sensu stricto, H. ohiense, H. mississippiense, H. suramericanum, and an African lineage. We report diagnostic traits for two species. The other three species can be identified by a combination of traits. Our results suggest that 1) there are significant phenotypic differences among the cryptic species of Histoplasma, and 2) that those differences can be used to positively distinguish those species in a clinical setting and for further study of the evolution of this fungal pathogen.

Aedes albopictus is present in the lowlands of southern Zambia.

Identifying the current geographic range of disease vectors is a critical first step towards determining effective mechanisms for controlling and potentially eradicating them. This is particularly true given that historical vector ranges may expand due to changing climates and human activity. The Aedes subgenus Stegomyia contains over 100 species, and among them, Ae. aegypti and Ae. albopictus mosquitoes represent the largest concern for public health, spreading dengue, chikungunya, and zika viruses. While Ae. aegypti has been observed in the country of Zambia for decades, Ae. albopictus has not. In 2015 we sampled four urban and three rural areas in Zambia for Aedes species. Using DNA barcoding, we confirmed the presence of immature and adult Ae. albopictus at two sites: Siavonga and Livingstone. These genotypes seem most closely related to specimens previously collected in Mozambique based on mtDNA barcoding. We resampled Siavonga and Livingstone sites in 2019, again observing immature and adult Ae. albopictus at both sites. Relative Ae. albopictus frequencies were similar between sites, with the exception of immature life stages, which were higher in Siavonga than in Livingstone in 2019. While Ae. albopictus frequencies did not vary through time in Livingstone, both immature and adult frequencies increased through time in Siavonga. This report serves to document the presence of Ae. albopictus in Zambia, which will contribute to understanding the potential public health implications of this disease vector in southern Africa.

The spread of Aedes albopictus (Diptera: Culicidae) in the islands of São Tomé and Príncipe.

The mosquito Aedes albopictus (Diptera: Culicidae) is a vector species of the causal agents of Dengue, yellow fever, and Zika among other diseases pathogens. The species originated in Southeast Asia and has spread widely and rapidly in the last century. The species has been reported in localities from the Gulf of Guinea since the early 2000s, but systematic sampling has been scant. We sampled Ae. albopictus twice, in 2013 and 2023 across the altitudinal gradient in São Tomé and found that the species was present in all sampled years at altitudes up to 680 m. We also found some evidence of increases in proportional representation compared to Ae. aegypti over time. We report the presence of the species in Príncipe for the first time, suggesting that the range of Ae. albopictus is larger than previously thought. Finally, we use bioclimatic niche modeling to infer the potential range of Ae. albopictus and infer that the species has the potential to spread across a large portion of São Tomé and Príncipe. Our results suggest that Ae. albopictus has established itself as a resident species of the islands of the Gulf of Guinea and should be incorporated into the list of potential vectors that need to be surveyed and controlled.

Ancestral neural circuits potentiate the origin of a female sexual behavior.

Courtship interactions are remarkably diverse in form and complexity among species. How neural circuits evolve to encode new behaviors that are functionally integrated into these dynamic social interactions is unknown. Here we report a recently originated female sexual behavior in the island endemic Drosophila species D. santomea, where females signal receptivity to male courtship songs by spreading their wings, which in turn promotes prolonged songs in courting males. Copulation success depends on this female signal and correlates with males' ability to adjust his singing in such a social feedback loop. Functional comparison of sexual circuitry across species suggests that a pair of descending neurons, which integrates male song stimuli and female internal state to control a conserved female abdominal behavior, drives wing spreading in D. santomea. This co-option occurred through the refinement of a pre-existing, plastic circuit that can be optogenetically activated in an outgroup species. Combined, our results show that the ancestral potential of a socially-tuned key circuit node to engage the wing motor program facilitates the expression of a new female behavior in appropriate sensory and motivational contexts. More broadly, our work provides insights into the evolution of social behaviors, particularly female behaviors, and the underlying neural mechanisms.

Aedes albopictus is present in the lowlands of southern Zambia.

Identifying the current geographic range of disease vectors is a critical first step towards determining effective mechanisms for controlling and potentially eradicating them. This is particularly true given that historical vector ranges may expand due to changing climates and human activity. The Aedes subgenus Stegomyia contains over 100 species, and among them, Ae. aegypti and Ae. albopictus mosquitoes represent the largest concern for public health, spreading dengue, chikungunya, and Zika viruses. While Ae. aegypti has been observed in the country of Zambia for decades, Ae. albopictus has not. In 2015 we sampled four urban and two rural areas in Zambia for Aedes species. Using DNA barcoding, we confirmed the presence of immature and adult Ae. albopictus at two rural sites: Siavonga and Livingstone. These genotypes seem most closely related to specimens previously collected in Mozambique based on CO1 sequence from mtDNA. We resampled Siavonga and Livingstone sites in 2019, again observing immature and adult Ae. albopictus at both sites. Relative Ae. albopictus frequencies were similar between sites, with the exception of immature life stages, which were higher in Siavonga than in Livingstone in 2019. While Ae. albopictus frequencies did not vary through time in Livingstone, both immature and adult frequencies increased through time in Siavonga. This report serves to document the presence of Ae. albopictus in Zambia, which will contribute to the process of determining the potential public health implications of this disease vector in Central Africa.

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Isotopic niches do not follow the expectations of niche conservatism in the bird genus Cinclodes.

Phenotypic traits are expected to be more similar among closely related species than among species that diverged long ago (all else being equal). This pattern, known as phylogenetic niche conservatism, also applies to traits that are important to determine the niche of species. To test this hypothesis on ecological niches, we analysed isotopic data from 254 museum study skins from 12 of the 16 species of the bird genus Cinclodes and measured stable isotope ratios for four different elements: carbon, nitrogen, hydrogen and oxygen. We find that all traits, measured individually, or as a composite measurement, lack any phylogenetic signal, which in turn suggests a high level of lability in ecological niches. We compared these metrics to the measurements of morphological traits in the same genus and found that isotopic niches are uniquely evolutionarily labile compared to other traits. Our results suggest that, in Cinclodes, the realized niche evolves much faster than expected by the constraints of phylogenetic history and poses the question of whether this is a general pattern across the tree of life.

The fitness of an introgressing haplotype changes over the course of divergence and depends on its size and genomic location.

The genomic era has made clear that introgression, or the movement of genetic material between species, is a common feature of evolution. Examples of both adaptive and deleterious introgression exist in a variety of systems. What is unclear is how the fitness of an introgressing haplotype changes as species diverge or as the size of the introgressing haplotype changes. In a simple model, we show that introgression may more easily occur into parts of the genome which have not diverged heavily from a common ancestor. The key insight is that alleles from a shared genetic background are likely to have positive epistatic interactions, increasing the fitness of a larger introgressing block. In regions of the genome where few existing substitutions are disrupted, this positive epistasis can be larger than incompatibilities with the recipient genome. Further, we show that early in the process of divergence, introgression of large haplotypes can be favored more than introgression of individual alleles. This model is consistent with observations of a positive relationship between recombination rate and introgression frequency across the genome; however, it generates several novel predictions. First, the model suggests that the relationship between recombination rate and introgression may not exist, or may be negative, in recently diverged species pairs. Furthermore, the model suggests that introgression that replaces existing derived variation will be more deleterious than introgression at sites carrying ancestral variants. These predictions are tested in an example of introgression in Drosophila melanogaster, with some support for both. Finally, the model provides a potential alternative explanation to asymmetry in the direction of introgression, with expectations of higher introgression from rapidly diverged populations into slowly evolving ones.

Strong Positive Selection in Aedes aegypti and the Rapid Evolution of Insecticide Resistance.

Aedes aegypti vectors the pathogens that cause dengue, yellow fever, Zika virus, and chikungunya and is a serious threat to public health in tropical regions. Decades of work has illuminated many aspects of Ae. aegypti's biology and global population structure and has identified insecticide resistance genes; however, the size and repetitive nature of the Ae. aegypti genome have limited our ability to detect positive selection in this mosquito. Combining new whole genome sequences from Colombia with publicly available data from Africa and the Americas, we identify multiple strong candidate selective sweeps in Ae. aegypti, many of which overlap genes linked to or implicated in insecticide resistance. We examine the voltage-gated sodium channel gene in three American cohorts and find evidence for successive selective sweeps in Colombia. The most recent sweep encompasses an intermediate-frequency haplotype containing four candidate insecticide resistance mutations that are in near-perfect linkage disequilibrium with one another in the Colombian sample. We hypothesize that this haplotype may continue to rapidly increase in frequency and perhaps spread geographically in the coming years. These results extend our knowledge of how insecticide resistance has evolved in this species and add to a growing body of evidence suggesting that Ae. aegypti has an extensive genomic capacity to rapidly adapt to insecticide-based vector control.

Patterns of Population Structure and Introgression Among Recently Differentiated Drosophila melanogaster Populations.

Despite a century of genetic analysis, the evolutionary processes that have generated the patterns of exceptional genetic and phenotypic variation in the model organism Drosophila melanogaster remains poorly understood. In particular, how genetic variation is partitioned within its putative ancestral range in Southern Africa remains unresolved. Here, we study patterns of population genetic structure, admixture, and the spatial structuring of candidate incompatibility alleles across a global sample, including 223 new accessions, predominantly from remote regions in Southern Africa. We identify nine major ancestries, six that primarily occur in Africa and one that has not been previously described. We find evidence for both contemporary and historical admixture between ancestries, with admixture rates varying both within and between continents. For example, while previous work has highlighted an admixture zone between broadly defined African and European ancestries in the Caribbean and southeastern USA, we identify West African ancestry as the most likely African contributor. Moreover, loci showing the strongest signal of introgression between West Africa and the Caribbean/southeastern USA include several genes relating to neurological development and male courtship behavior, in line with previous work showing shared mating behaviors between these regions. Finally, while we hypothesized that potential incompatibility loci may contribute to population genetic structure across the range of D. melanogaster; these loci are, on average, not highly differentiated between ancestries. This work contributes to our understanding of the evolutionary history of a key model system, and provides insight into the partitioning of diversity across its range.

A need for standardized reporting of introgression: Insights from studies across eukaryotes.

With the rise of affordable next-generation sequencing technology, introgression-or the exchange of genetic materials between taxa-has become widely perceived to be a ubiquitous phenomenon in nature. Although this claim is supported by several keystone studies, no thorough assessment of the frequency of introgression across eukaryotes in nature has been performed to date. In this manuscript, we aim to address this knowledge gap by examining patterns of introgression across eukaryotes. We collated a single statistic, Patterson's D, which can be used as a test for introgression across 123 studies to further assess how taxonomic group, divergence time, and sequencing technology influence reports of introgression. Overall, introgression has mostly been measured in plants and vertebrates, with less attention given to the rest of the Eukaryotes. We find that the most frequently used metrics to detect introgression are difficult to compare across studies and even more so across biological systems due to differences in study effort, reporting standards, and methodology. Nonetheless, our analyses reveal several intriguing patterns, including the observation that differences in sequencing technologies may bias values of Patterson's D and that introgression may differ throughout the course of the speciation process. Together, these results suggest the need for a unified approach to quantifying introgression in natural communities and highlight important areas of future research that can be better assessed once this unified approach is met.

Single nucleotide polymorphisms and chromosomal copy number variation may impact the Sporothrix brasiliensis antifungal susceptibility and sporotrichosis clinical outcomes.

Feline-transmitted sporotrichosis has garnered attention due to the recent high incidence and the lack of efficient control in the epicenter of the epidemic, Rio de Janeiro, Brazil. Sporothrix brasiliensis is the major pathogen involved in feline-to-human sporotrichosis in Brazil and displays more virulent genotypes than the closely related species S. schenckii. Over the last two decades, several reports of antifungal-resistant strains have emerged. Sequencing and comparison analysis of the outbreak strains allowed us to observe that the azole non-wild-type S. brasiliensis strain CFP 1054 had significant chromosomal variations compared to wild-type strains. One of these variants includes a region of 231 Kb containing 75 duplicated genes, which were overrepresented for lipid and isoprenoid metabolism. We also identified an additional strain (CFP 1055) that was resistant to itraconazole and amphotericin B, which had a single nucleotide polymorphism in the tac1 gene. The patients infected with these two strains showed protracted clinical course and sequelae. Even though our sample size is modest, these results suggest the possibility of identifying specific point mutations and large chromosomal duplications potentially associated with antifungal resistance and clinical outcomes of sporotrichosis.

Phylogenetic climatic niche conservatism in sandflies (Diptera: Phlebotominae) and their relatives.

Phylogenetic niche conservatism is a pattern in which closely related species are more similar than distant relatives in their niche-related traits. Species in the family Psychodidae show notable diversity in climatic niche, and present an opportunity to test for phylogenetic niche conservatism, which is as yet rarely studied in insects. Some species (in the subfamily Phlebotominae) transmit Leishmania parasites, responsible for the disease leishmaniasis, and their geographic range has been systematically characterized. Psychodid genus ranges can be solely tropical, confined to the temperate zones, or span both. We obtained observation site data, and associated climate data, for 234 psychodid species to understand which aspects of climate most closely predict distribution. Temperature and seasonality are strong determinants of species occurrence within the clade. Next, we built a phylogeny of Psychodidae, and found a positive relationship between pairwise genetic distance and climate niche differentiation, which indicates strong niche conservatism. This result is also supported by strong phylogenetic signals of metrics of climate differentiation. Finally, we used ancestral trait reconstruction to infer the tropicality (i.e., proportion of latitudinal range in the tropics minus the proportion of the latitudinal range in temperate areas) of ancestral species, and counted transitions to and from tropicality states. We find that tropical and temperate species produced almost entirely tropical and temperate descendant species, respectively. Taken together, our results imply that climate niches in psychodids are strongly predicted by phylogeny, and represent a formal test of a key prediction of phylogenetic niche conservatism in a clade with implications for human health.

The evolutionary trajectory of drosophilid walking.

Neural circuits must both execute the behavioral repertoire of individuals and account for behavioral variation across species. Understanding how this variation emerges over evolutionary time requires large-scale phylogenetic comparisons of behavioral repertoires. Here, we describe the evolution of walking in fruit flies by capturing high-resolution, unconstrained movement from 13 species and 15 strains of drosophilids. We find that walking can be captured in a universal behavior space, the structure of which is evolutionarily conserved. However, the occurrence of and transitions between specific movements have evolved rapidly, resulting in repeated convergent evolution in the temporal structure of locomotion. Moreover, a meta-analysis demonstrates that many behaviors evolve more rapidly than other traits. Thus, the architecture and physiology of locomotor circuits can execute precise individual movements in one species and simultaneously support rapid evolutionary changes in the temporal ordering of these modular elements across clades.

Diversity of cave Phlebotomines (Diptera: Psychodidae) from a Colombian cave.

Sandflies are vector species of Leishmania, among many other pathogens, with a global distribution and a variety of ecological niches. Previous samplings have found that karstic formations (i.e., caves, grottos, and folds formed by the erosion of limestone) serve as a natural habitat to sandfly species. The majority of samplings of cave sandfly diversity have occurred in Brazil and to date none have studied the species composition in a cave in the Northern Andes. We collected sandflies in the Cave "Los Guácharos", in the state of Antioquia, Colombia. The sampling was carried out during two consecutive nights in September 2019. CDC-type light traps were installed inside the cavern and in other surrounding karst systems (caves, rock-breaks, and folds). In total, we identified 17 species of sandfly from the cave and surrounding karst systems, including a new record for Colombia (Bichromomyia olmeca), and provide the first karstic reports for four other species (Lutzomyia gomezi, Lutzomyia hartmanni, Pintomyia ovallesi, and Psychodopygus panamensis). We then used the results of our survey and published literature to test two hypotheses. First, that sandfly diversity in Neotropical caves is richest nearer to the equator, and second that there is a phylogenetic signal of karstic habitat use in sandflies. Counter to our predictions, we found no evidence that diversity follows a latitudinal gradient. Further, we find no evidence of a phylogenetic signal of karstic habitat use, instead finding that the use of caves likely evolved multiple times across several genera. Our results highlight the importance of a wide sampling to understand the natural habitat of sandflies and other disease vectors.