Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.

Toxoplasma and Plasmodium associate with host Arfs during infection.

The apicomplexans Toxoplasma gondii and Plasmodium are intracellular parasites that reside within a host-derived compartment termed the parasitophorous vacuole (PV). During infection, the parasites must acquire critical host resources and transport them across their PV for development. However, the mechanism by which host resources are trafficked to and across the PV remains uncertain. Here, we investigated host ADP ribosylation factors (Arfs), a class of proteins involved in vesicular trafficking that may be exploited by T. gondii and Plasmodium berghei for nutrient acquisition. Using overexpressed Arf proteins coupled with immunofluorescence microscopy, we found that all Arfs were internalized into the T. gondii PV, with most vacuoles containing at least one punctum of Arf protein by the end of the lytic cycle. We further characterized Arf1, the most abundant Arf inside the T. gondii PV, and observed that active recycling between its GDP/GTP-bound state influenced Arf1 internalization independent of host guanine nucleotide exchange factors (GEFs). In addition, Arf1 colocalized with vesicle coat complexes and exogenous sphingolipids, suggesting a role in nutrient acquisition. While Arf1 and Arf4 were not observed inside the PV during P. berghei infection, our gene depletion studies showed that liver stage development and survival depended on the expression of Arf4 and the host GEF, GBF1. Collectively, these observations indicate that apicomplexans use distinct mechanisms to subvert the host vesicular trafficking network and efficiently replicate. The findings also pave the way for future studies to identify parasite proteins critical to host vesicle recruitment and the components of vesicle cargo. IMPORTANCE: The parasites Toxoplasma gondii and Plasmodium live complex intracellular lifestyles where they must acquire essential host nutrients while avoiding recognition. Although previous work has sought to identify the specific nutrients scavenged by apicomplexans, the mechanisms by which host materials are transported to and across the parasite vacuole membrane are largely unknown. Here, we examined members of the host vesicular trafficking network to identify specific pathways subverted by T. gondii and Plasmodium berghei. Our results indicate that T. gondii selectively internalizes host Arfs, a class of proteins involved in intracellular trafficking. For P. berghei, host Arfs were restricted by the parasite's vacuole membrane, but proteins involved in vesicular trafficking were identified as essential for liver stage development. A greater exploration into how and why apicomplexans subvert host vesicular trafficking could help identify targets for host-directed therapeutics.

Elucidating the path to Plasmodium prolyl-tRNA synthetase inhibitors that overcome halofuginone resistance.

The development of next-generation antimalarials that are efficacious against the human liver and asexual blood stages is recognized as one of the world's most pressing public health challenges. In recent years, aminoacyl-tRNA synthetases, including prolyl-tRNA synthetase, have emerged as attractive targets for malaria chemotherapy. We describe the development of a single-step biochemical assay for Plasmodium and human prolyl-tRNA synthetases that overcomes critical limitations of existing technologies and enables quantitative inhibitor profiling with high sensitivity and flexibility. Supported by this assay platform and co-crystal structures of representative inhibitor-target complexes, we develop a set of high-affinity prolyl-tRNA synthetase inhibitors, including previously elusive aminoacyl-tRNA synthetase triple-site ligands that simultaneously engage all three substrate-binding pockets. Several compounds exhibit potent dual-stage activity against Plasmodium parasites and display good cellular host selectivity. Our data inform the inhibitor requirements to overcome existing resistance mechanisms and establish a path for rational development of prolyl-tRNA synthetase-targeted anti-malarial therapies.

Lessons Learned in a Large-Scale Mass Casualty Simulation.

BACKGROUND: Traditional Nursing Programs are required to provide student instruction regarding disaster care. The Brigham Young University College of Nursing, in conjunction with campus emergency medical services (EMS), holds two large-scale mass casualty simulations each year. Nursing students work alongside EMS to provide initial care to the victims. METHOD: After the mass casualty simulation, nursing students completed an anonymous survey evaluating their preparation and experience during the simulation. Students were also prompted to reflect on future implications of the exercise and give suggestions for simulation improvement. RESULTS: Nursing students felt the triage and communication skills they learned during this simulation will help them as future registered nurses. Interdisciplinary communication between nursing and EMS students presented a barrier to effective disaster response. CONCLUSION: Data gathered from this postsimulation survey will be used to improve future nursing student preparation and simulation participation. [J Nurs Educ. 2022;61(1):50-52.].

Substrate, temperature, and geographical patterns among nearly 2000 natural yeast isolates.

Yeasts have broad importance as industrially and clinically relevant microbes and as powerful models for fundamental research, but we are only beginning to understand the roles yeasts play in natural ecosystems. Yeast ecology is often more difficult to study compared to other, more abundant microbes, but growing collections of natural yeast isolates are beginning to shed light on fundamental ecological questions. Here, we used environmental sampling and isolation to assemble a dataset of 1962 isolates collected from throughout the contiguous United States of America (USA) and Alaska, which were then used to uncover geographic patterns, along with substrate and temperature associations among yeast taxa. We found some taxa, including the common yeasts Torulaspora delbrueckii and Saccharomyces paradoxus, to be repeatedly isolated from multiple sampled regions of the USA, and we classify these as broadly distributed cosmopolitan yeasts. A number of yeast taxon-substrate associations were identified, some of which were novel and some of which support previously reported associations. Further, we found a strong effect of isolation temperature on the phyla of yeasts recovered, as well as for many species. We speculate that substrate and isolation temperature associations reflect the ecological diversity of and niche partitioning by yeast taxa.

Characterization of the Tubovesicular Network in Plasmodium vivax Liver Stage Hypnozoites and Schizonts.

Plasmodium is a genus of apicomplexan parasites which replicate in the liver before causing malaria. Plasmodium vivax can also persist in the liver as dormant hypnozoites and cause clinical relapse upon activation, but the molecular mechanisms leading to activation have yet to be discovered. In this study, we use high-resolution microscopy to characterize temporal changes of the P. vivax liver stage tubovesicular network (TVN), a parasitophorous vacuole membrane (PVM)-derived network within the host cytosol. We observe extended membrane clusters, tubules, and TVN-derived vesicles present throughout P. vivax liver stage development. Additionally, we demonstrate an unexpected presence of the TVN in hypnozoites and observe some association of this network to host nuclei. We also reveal that the host water and solute channel aquaporin-3 (AQP3) associates with TVN-derived vesicles and extended membrane clusters. AQP3 has been previously shown to localize to the PVM of P. vivax hypnozoites and liver schizonts but has not yet been shown in association to the TVN. Our results highlight host-parasite interactions occur in both dormant and replicating liver stage P. vivax forms and implicate AQP3 function during this time. Together, these findings enhance our understanding of P. vivax liver stage biology through characterization of the TVN with an emphasis on the presence of this network in dormant hypnozoites.

A Systematic Analysis of Mosquito-Microbiome Biosynthetic Gene Clusters Reveals Antimalarial Siderophores that Reduce Mosquito Reproduction Capacity.

Advances in infectious disease control strategies through genetic manipulation of insect microbiomes have heightened interest in microbially produced small molecules within mosquitoes. Herein, 33 mosquito-associated bacterial genomes were mined and over 700 putative biosynthetic gene clusters (BGCs) were identified, 135 of which belong to known classes of BGCs. After an in-depth analysis of the 135 BGCs, iron-binding siderophores were chosen for further investigation due to their high abundance and well-characterized bioactivities. Through various metabolomic strategies, eight siderophore scaffolds were identified in six strains of mosquito-associated bacteria. Among these, serratiochelin A and pyochelin were found to reduce female Anopheles gambiae overall fecundity likely by lowering their blood-feeding rate. Serratiochelin A and pyochelin were further found to inhibit the Plasmodium parasite asexual blood and liver stages in vitro. Our work supplies a bioinformatic resource for future mosquito-microbiome studies and highlights an understudied source of bioactive small molecules.

Plasmodium vivax Liver and Blood Stages Recruit the Druggable Host Membrane Channel Aquaporin-3.

Plasmodium vivax infects hepatocytes to form schizonts that cause blood infection, or dormant hypnozoites that can persist for months in the liver before leading to relapsing blood infections. The molecular processes that drive P. vivax schizont and hypnozoite survival remain largely unknown, but they likely involve a rich network of host-pathogen interactions, including those occurring at the host-parasite interface, the parasitophorous vacuole membrane (PVM). Using a recently developed P. vivax liver-stage model system we demonstrate that host aquaporin-3 (AQP3) localizes to the PVM of schizonts and hypnozoites within 5 days after invasion. This recruitment is also observed in P. vivax-infected reticulocytes. Chemical treatment with the AQP3 inhibitor auphen reduces P. vivax liver hypnozoite and schizont burden, and inhibits P. vivax asexual blood-stage growth. These findings reveal a role for AQP3 in P. vivax liver and blood stages and suggest that the protein may be targeted for therapeutic treatment.

Postglacial migration shaped the genomic diversity and global distribution of the wild ancestor of lager-brewing hybrids.

The wild, cold-adapted parent of hybrid lager-brewing yeasts, Saccharomyces eubayanus, has a complex and understudied natural history. The exploration of this diversity can be used both to develop new brewing applications and to enlighten our understanding of the dynamics of yeast evolution in the wild. Here, we integrate whole genome sequence and phenotypic data of 200 S. eubayanus strains, the largest collection known to date. S. eubayanus has a multilayered population structure, consisting of two major populations that are further structured into six subpopulations. Four of these subpopulations are found exclusively in the Patagonian region of South America; one is found predominantly in Patagonia and sparsely in Oceania and North America; and one is specific to the Holarctic ecozone. Plant host associations differed between subpopulations and between S. eubayanus and its sister species, Saccharomyces uvarum. S. eubayanus is most abundant and genetically diverse in northern Patagonia, where some locations harbor more genetic diversity than is found outside of South America, suggesting that northern Patagonia east of the Andes was a glacial refugium for this species. All but one subpopulation shows isolation-by-distance, and gene flow between subpopulations is low. However, there are strong signals of ancient and recent outcrossing, including two admixed lineages, one that is sympatric with and one that is mostly isolated from its parental populations. Using our extensive biogeographical data, we build a robust model that predicts all known and a handful of additional regions of the globe that are climatically suitable for S. eubayanus, including Europe where host accessibility and competitive exclusion by other Saccharomyces species may explain its continued elusiveness. We conclude that this industrially relevant species has rich natural diversity with many factors contributing to its complex distribution and natural history.

Plasmodium chaperonin TRiC/CCT identified as a target of the antihistamine clemastine using parallel chemoproteomic strategy.

The antihistamine clemastine inhibits multiple stages of the Plasmodium parasite that causes malaria, but the molecular targets responsible for its parasite inhibition were unknown. Here, we applied parallel chemoproteomic platforms to discover the mechanism of action of clemastine and identify that clemastine binds to the Plasmodium falciparum TCP-1 ring complex or chaperonin containing TCP-1 (TRiC/CCT), an essential heterooligomeric complex required for de novo cytoskeletal protein folding. Clemastine destabilized all eight P. falciparum TRiC subunits based on thermal proteome profiling (TPP). Further analysis using stability of proteins from rates of oxidation (SPROX) revealed a clemastine-induced thermodynamic stabilization of the Plasmodium TRiC delta subunit, suggesting an interaction with this protein subunit. We demonstrate that clemastine reduces levels of the major TRiC substrate tubulin in P. falciparum parasites. In addition, clemastine treatment leads to disorientation of Plasmodium mitotic spindles during the asexual reproduction and results in aberrant tubulin morphology suggesting protein aggregation. This clemastine-induced disruption of TRiC function is not observed in human host cells, demonstrating a species selectivity required for targeting an intracellular human pathogen. Our findings encourage larger efforts to apply chemoproteomic methods to assist in target identification of antimalarial drugs and highlight the potential to selectively target Plasmodium TRiC-mediated protein folding for malaria intervention.

RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection.

The apicomplexan parasites Plasmodium spp. are the causative agents of malaria, a disease that poses a significant global health burden. Plasmodium spp. initiate infection of the human host by transforming and replicating within hepatocytes. This liver stage (LS) is poorly understood compared to other Plasmodium life stages, which has hindered our ability to target these parasites for disease prevention. We conducted an extensive transcriptome sequencing (RNA-Seq) analysis throughout the Plasmodium berghei LS, covering as early as 2 h postinfection (hpi) and extending to 48 hpi. Our data revealed that hundreds of genes are differentially expressed at 2 hpi and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using hierarchical clustering along with coexpression analysis, we identified clusters functionally enriched for important liver-stage processes such as interactions with the host cell and redox homeostasis. Furthermore, some of these clusters were highly correlated to the expression of ApiAP2 transcription factors, while showing enrichment of mostly uncharacterized DNA binding motifs. This finding indicates potential LS targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. Our work presents a window into the previously undescribed transcriptome of Plasmodium upon host hepatocyte infection to enable a comprehensive view of the parasite's LS. These findings also provide a blueprint for future studies that extend hypotheses concerning LS gene function in P. berghei to human-infective Plasmodium parasites.IMPORTANCE The LS of Plasmodium infection is an asymptomatic yet necessary stage for producing blood-infective parasites, the causative agents of malaria. Blocking the liver stage of the life cycle can prevent clinical malaria, but relatively less is known about the parasite's biology at this stage. Using the rodent model P. berghei, we investigated whole-transcriptome changes occurring as early as 2 hpi of hepatocytes. The transcriptional profiles of early time points (2, 4, 12, and 18 hpi) have not been accessible before due to the technical challenges associated with liver-stage infections. Our data now provide insights into these early parasite fluxes that may facilitate establishment of infection, transformation, and replication in the liver.

Pathogenic budding yeasts isolated outside of clinical settings.

Budding yeasts are distributed across a wide range of habitats, including as human commensals. However, under some conditions, these commensals can cause superficial, invasive, and even lethal infections. Despite their importance to human health, little is known about the ecology of these opportunistic pathogens, aside from their associations with mammals and clinical environments. During a survey of approximately 1000 non-clinical samples across the United States of America, we isolated 54 strains of budding yeast species considered opportunistic pathogens, including Candida albicans and Candida (Nakaseomyces) glabrata. We found that, as a group, pathogenic yeasts were positively associated with fruits and soil environments, whereas the species Pichia kudriavzevii (syn. Candida krusei syn. Issatchenkia orientalis) had a significant association with plants. Of the four species that cause 95% of candidiasis, we found a positive association with soil. These results suggest that pathogenic yeast ecology is more complex and diverse than is currently appreciated and raises the possibility that these additional environments could be a point of contact for human infections.

Close the ring to break the cycle: tandem quinolone-alkyne-cyclisation gives access to tricyclic pyrrolo[1,2-a]quinolin-5-ones with potent anti-protozoal activity.

Expanding the chemical space of quinolones led to a tandem quinolone-alkyne-cyclisation reaction allowing chemoselective control of the synthesis of tricyclic pyrrolo[1,2-a]quinolin-5-ones. Importantly, we discovered anti-protozoal activity against Plasmodium and Toxoplasma with specific potency of one of the compounds against the liver stage of the malaria parasite in the nanomolar range.

Synthesis and Analysis of Natural-Product-Like Macrocycles by Tandem Oxidation/Oxa-Conjugate Addition Reactions.

As traditional small-molecule drug discovery programs focus on a relatively narrow range of chemical space, most human proteins are viewed as unreachable targets. Consequently, there is a strong interest in expanding the chemical space in drug discovery beyond traditional small molecules. Here, a strategy for the preparation of a broad natural-product-like macrocyclic library by using the tandem allylic oxidation/oxa-conjugate addition and macrocyclization reactions is reported. Cheminformatic analyses demonstrate that this tetrahydropyran-containing macrocyclic library shows a significant overlap with natural products in the chemical space. This approach can be used for designing libraries that may probe more deeply into natural-product-like space.

In silico Screening and Evaluation of Plasmodium falciparum Protein Kinase†5 (PK5) Inhibitors.

An in silico screen of 350 000 commercially available compounds was conducted with an unbiased approach to identify potential malaria inhibitors that bind to the Plasmodium falciparum protein kinase 5 (PfPK5) ATP-binding site. PfPK5 is a cyclin-dependent kinase-like protein with high sequence similarity to human cyclin-dependent kinase 2 (HsCDK2), but its precise role in cell-cycle regulation remains unclear. After two-dimensional fingerprinting of the top scoring compounds, 182 candidates were prioritized for biochemical testing based on their structural diversity. Evaluation of these compounds demonstrated that 135 bound to PfPK5 to a similar degree or better than known PfPK5 inhibitors, confirming that the library was enriched with PfPK5-binding compounds. A previously reported triazolodiamine HsCDK2 inhibitor and the screening hit 4-methylumbelliferone were each selected for an analogue study. The results of this study highlight the difficult balance between optimization of PfPK5 affinity and binding selectivity for PfPK5 over its closest human homologue HsCDK2. Our approach enabled the discovery of several new PfPK5-binding compounds from a modest screening campaign and revealed the first scaffold to have improved PfPK5/HsCDK2 selectivity. These steps are critical for the development of PfPK5-targeting probes for functional studies and antimalarials with decreased risks of host toxicity.

Plasmodium parasite exploits host aquaporin-3 during liver stage malaria infection.

Within the liver a single Plasmodium parasite transforms into thousands of blood-infective forms to cause malaria. Here, we use RNA-sequencing to identify host genes that are upregulated upon Plasmodium berghei infection of hepatocytes with the hypothesis that host pathways are hijacked to benefit parasite development. We found that expression of aquaporin-3 (AQP3), a water and glycerol channel, is significantly induced in Plasmodium-infected hepatocytes compared to uninfected cells. This aquaglyceroporin localizes to the parasitophorous vacuole membrane, the compartmental interface between the host and pathogen, with a temporal pattern that correlates with the parasite's expansion in the liver. Depletion or elimination of host AQP3 expression significantly reduces P. berghei parasite burden during the liver stage and chemical disruption by a known AQP3 inhibitor, auphen, reduces P. falciparum asexual blood stage and P. berghei liver stage parasite load. Further use of this inhibitor as a chemical probe suggests that AQP3-mediated nutrient transport is an important function for parasite development. This study reveals a previously unknown potential route for host-dependent nutrient acquisition by Plasmodium which was discovered by mapping the transcriptional changes that occur in hepatocytes throughout P. berghei infection. The dataset reported may be leveraged to identify additional host factors that are essential for Plasmodium liver stage infection and highlights Plasmodium's dependence on host factors within hepatocytes.

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production.

BACKGROUND: Lignocellulosic biomass is a common resource across the globe, and its fermentation offers a promising option for generating renewable liquid transportation fuels. The deconstruction of lignocellulosic biomass releases sugars that can be fermented by microbes, but these processes also produce fermentation inhibitors, such as aromatic acids and aldehydes. Several research projects have investigated lignocellulosic biomass fermentation by the baker's yeast Saccharomyces cerevisiae. Most projects have taken synthetic biological approaches or have explored naturally occurring diversity in S. cerevisiae to enhance stress tolerance, xylose consumption, or ethanol production. Despite these efforts, improved strains with new properties are needed. In other industrial processes, such as wine and beer fermentation, interspecies hybrids have combined important traits from multiple species, suggesting that interspecies hybridization may also offer potential for biofuel research. RESULTS: To investigate the efficacy of this approach for traits relevant to lignocellulosic biofuel production, we generated synthetic hybrids by crossing engineered xylose-fermenting strains of S. cerevisiae with wild strains from various Saccharomyces species. These interspecies hybrids retained important parental traits, such as xylose consumption and stress tolerance, while displaying intermediate kinetic parameters and, in some cases, heterosis (hybrid vigor). Next, we exposed them to adaptive evolution in ammonia fiber expansion-pretreated corn stover hydrolysate and recovered strains with improved fermentative traits. Genome sequencing showed that the genomes of these evolved synthetic hybrids underwent rearrangements, duplications, and deletions. To determine whether the genus Saccharomyces contains additional untapped potential, we screened a genetically diverse collection of more than 500 wild, non-engineered Saccharomyces isolates and uncovered a wide range of capabilities for traits relevant to cellulosic biofuel production. Notably, Saccharomyces mikatae strains have high innate tolerance to hydrolysate toxins, while some Saccharomyces species have a robust native capacity to consume xylose. CONCLUSIONS: This research demonstrates that hybridization is a viable method to combine industrially relevant traits from diverse yeast species and that members of the genus Saccharomyces beyond S. cerevisiae may offer advantageous genes and traits of interest to the lignocellulosic biofuel industry.

Speciation driven by hybridization and chromosomal plasticity in a wild yeast.

Hybridization is recognized as a powerful mechanism of speciation and a driving force in generating biodiversity. However, only few multicellular species, limited to a handful of plants and animals, have been shown to fulfil all the criteria of homoploid hybrid speciation. This lack of evidence could lead to the interpretation that speciation by hybridization has a limited role in eukaryotes, particularly in single-celled organisms. Laboratory experiments have revealed that fungi such as budding yeasts can rapidly develop reproductive isolation and novel phenotypes through hybridization, showing that in principle homoploid speciation could occur in nature. Here, we report a case of homoploid hybrid speciation in natural populations of the budding yeast Saccharomyces paradoxus inhabiting the North American forests. We show that the rapid evolution of chromosome architecture and an ecological context that led to secondary contact between nascent species drove the formation of an incipient hybrid species with a potentially unique ecological niche.

Complex Ancestries of Lager-Brewing Hybrids Were Shaped by Standing Variation in the Wild Yeast Saccharomyces eubayanus.

Lager-style beers constitute the vast majority of the beer market, and yet, the genetic origin of the yeast strains that brew them has been shrouded in mystery and controversy. Unlike ale-style beers, which are generally brewed with Saccharomyces cerevisiae, lagers are brewed at colder temperatures with allopolyploid hybrids of Saccharomyces eubayanus x S. cerevisiae. Since the discovery of S. eubayanus in 2011, additional strains have been isolated from South America, North America, Australasia, and Asia, but only interspecies hybrids have been isolated in Europe. Here, using genome sequence data, we examine the relationships of these wild S. eubayanus strains to each other and to domesticated lager strains. Our results support the existence of a relatively low-diversity (π = 0.00197) lineage of S. eubayanus whose distribution stretches across the Holarctic ecozone and includes wild isolates from Tibet, new wild isolates from North America, and the S. eubayanus parents of lager yeasts. This Holarctic lineage is closely related to a population with higher diversity (π = 0.00275) that has been found primarily in South America but includes some widely distributed isolates. A second diverse South American population (π = 0.00354) and two early-diverging Asian subspecies are more distantly related. We further show that no single wild strain from the Holarctic lineage is the sole closest relative of lager yeasts. Instead, different parts of the genome portray different phylogenetic signals and ancestry, likely due to outcrossing and incomplete lineage sorting. Indeed, standing genetic variation within this wild Holarctic lineage of S. eubayanus is responsible for genetic variation still segregating among modern lager-brewing hybrids. We conclude that the relationships among wild strains of S. eubayanus and their domesticated hybrids reflect complex biogeographical and genetic processes.

Temperature and host preferences drive the diversification of Saccharomyces and other yeasts: a survey and the discovery of eight new yeast species.

Compared to its status as an experimental model system and importance to industry, the ecology and genetic diversity of the genus Saccharomyces has received less attention. To investigate systematically the biogeography, community members and habitat of these important yeasts, we isolated and identified nearly 600 yeast strains using sugar-rich enrichment protocols. Isolates were highly diverse and contained representatives of more than 80 species from over 30 genera, including eight novel species that we describe here: Kwoniella betulae f.a. (yHKS285(T) = NRRL Y-63732(T) = CBS 13896(T)), Kwoniella newhampshirensis f.a. (yHKS256(T) = NRRL Y-63731(T) = CBS 13917(T)), Cryptococcus wisconsinensis (yHKS301(T) = NRRL Y-63733(T) = CBS 13895(T)), Cryptococcus tahquamenonensis (yHAB242(T) = NRRL Y-63730(T) = CBS 13897(T)), Kodamaea meredithiae f.a. (yHAB239(T) = NRRL Y-63729(T) = CBS 13899(T)), Blastobotrys buckinghamii (yHAB196(T) = NRRL Y-63727(T) = CBS 13900(T)), Candida sungouii (yHBJ21(T) = NRRL Y-63726(T) = CBS 13907(T)) and Cyberlindnera culbertsonii f.a. (yHAB218(T) = NRRL Y-63728(T) = CBS 13898(T)), spp. nov. Saccharomyces paradoxus was one of the most frequently isolated species and was represented by three genetically distinct lineages in Wisconsin alone. We found a statistically significant association between Quercus (oak) samples and the isolation of S. paradoxus, as well as several novel associations. Variation in temperature preference was widespread across taxonomic ranks and evolutionary timescales. This survey highlights the genetic and taxonomic diversity of yeasts and suggests that host and temperature preferences are major ecological factors.