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Genetic manipulation is often used to study gene function. However, unplanned genome changes (including single nucleotide polymorphisms [SNPs], aneuploidy, and loss of heterozygosity [LOH]) can affect the phenotypic traits of the engineered strains. Here, we compared the effect of classical deletion methods (replacing target alleles with selectable markers by homologous recombination) with CRISPR-Cas9 editing in the diploid human-pathogenic yeast Candida parapsilosis. We sequenced the genomes of 9 isolates that were modified using classic recombination methods and 12 that were edited using CRISPR-Cas9. As a control, the genomes of eight isolates that were transformed with a Cas9-expressing plasmid in the absence of a guide RNA were also sequenced. Following gene manipulation using classic homologous recombination, only one strain exhibited extensive LOH near the targeted gene (8.9 kb), whereas another contained multiple LOH events not associated with the intended modification. In contrast, large regions of LOH (up to 1,100 kb) were observed in most CRISPR-Cas9-edited strains. LOH most commonly occurred adjacent to the Cas9 cut site and extended to the telomere in four isolates. In two isolates, we observed LOH on chromosomes that were not targeted by CRISPR-Cas9. Among the CRISPR-edited isolates, two exhibited cysteine and methionine auxotrophy caused by LOH at a heterozygous site in

Blastobotrys aristata is a member of the Trichomonascaceae family in the order Saccharomycetales. Here, we present the genome sequence of B. aristata UCD613, which was isolated from soil in Dublin, Ireland. This genome is 13.3 Mb and was assembled into 4 chromosome-size scaffolds of >2.2 Mb in size plus a mitochondrial genome scaffold.

We analyzed the genomes of 170 C. parapsilosis isolates and identified multiple copy number variations (CNVs). We identified two genes, RTA3 (CPAR2_104610) and ARR3 (CPAR2_601050), each of which was the target of multiple independent amplification events. Phylogenetic analysis shows that most of these amplifications originated only once. For ARR3, which encodes a putative arsenate transporter, 8 distinct CNVs were identified, ranging in size from 2.3 kb to 10.5 kb with 3 to 23 copies. For RTA3, 16 distinct CNVs were identified, ranging in size from 0.3 kb to 4.5 kb with 2 to ~50 copies. One unusual amplification resulted in a DUP-TRP/INV-DUP structure similar to some human CNVs. RTA3 encodes a putative phosphatidylcholine (PC) floppase which is known to regulate the inward translocation of PC in Candida albicans. We found that an increased copy number of RTA3 correlated with resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine. Two genes, CPAR2_303950 and CPAR2_102700, coding for putative PC flippases homologous to S. cerevisiae DNF1 gained homozygous protein-disrupting mutations in the evolved strains. Overall, our results show that C. parapsilosis can gain resistance to miltefosine, a drug that has recently been granted orphan drug designation approval by the United States Food and Drug Administration for the treatment of invasive candidiasis, through both CNVs or loss-of-function alleles in one of the flippase genes.

Torulaspora quercuum is an ascomycete yeast first isolated in 2009. Here, we present the genome sequence of T. quercuum isolate UCD657, which was isolated from soil in Ireland. This genome is 10.4 Mb and was assembled into 8 chromosome-sized scaffolds of >1 Mb in size, plus a mitochondrial genome scaffold.

We analyzed the genomes of 170 C. parapsilosis isolates and identified multiple copy number variations (CNVs). We identified two genes

Genetic manipulation is often used to study gene function. However, unplanned genome changes (including Single Nucleotide Polymorphisms (SNPs), aneuploidy and Loss of Heterozygosity (LOH)) can affect the phenotypic traits of the engineered strains. Here, we show that CRISPR-Cas9 editing can induce LOH in the diploid human pathogenic yeast Candida parapsilosis. We sequenced the genomes of ten isolates that were edited with CRISPR-Cas9 and found that the designed changes were present in nine. However, we also observed LOH in all isolates, and aneuploidy in two isolates. LOH occurred most commonly downstream of the Cas9 cut site and extended to the telomere in three isolates. In two isolates we observed LOH on chromosomes that were not targeted by CRISPR-Cas9. Two different isolates exhibited cysteine and methionine auxotrophy caused by LOH at a heterozygous site in MET10, approximately 11 and 157 kb downstream from the Cas9 target site, respectively. C. parapsilosis isolates have relatively low levels of heterozygosity. However, our results show that mutation complementation to confirm observed phenotypes is important even when using CRISPR-Cas9, which is now the gold standard of genetic engineering.IMPORTANCECRISPR-Cas9 has greatly streamlined gene editing, and is now the gold standard and first choice for genetic engineering. However, we show that in diploid species extra care should be taken in confirming the cause of any phenotypic changes observed. We show that the Cas9-induced double strand break is often associated with loss of heterozygosity in the asexual diploid human fungal pathogen Candida parapsilosis. This can result in deleterious heterozygous variants (e.g. stop gain in one allele) becoming homozygous resulting in unplanned phenotypic changes. Our results stress the importance of mutation complementation even when using CRISPR-Cas9.

Antimicrobial drug resistance is a major problem in the prevention and treatment of infectious diseases. For patients on antimicrobial prophylaxis during cancer treatment and organ transplantation, breakthrough infections represent a significant and often unexplained cause of morbidity. Here, in high-risk patients on micafungin prophylaxis, we reveal that heteroresistance, the presence of a phenotypically unstable, low frequency subpopulation of resistant cells (∼1 in 10,000), causes breakthrough bloodstream infections by Candida parapsilosis strains that were misclassified as susceptible by standard antimicrobial susceptibility testing. Genome-matched micafungin heteroresistant strains were isolated from the intestinal reservoir prior to invasive disease. To identify heteroresistance, we harnessed whole genome sequence analysis of 118 clinical C. parapsilosis strains and extracted six genetic features into a predictive machine learning model that distinguished heteroresistant strains with high accuracy. These data directly implicate heteroresistance in unexplained prophylaxis failure and highlight a proof-of-principle approach to detect this phenomenon and inform clinical decisions.

Lager brewing first occurred in Bavaria in the 15th century, associated with restrictions of brewing to colder months. The lager yeast, Saccharomyces pastorianus, is cold tolerant. It is a hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus, and has been found only in industrial settings. Natural isolates of S. eubayanus were first discovered in Patagonia 11 years ago. They have since been isolated from China, Tibet, New Zealand, and North America, but not from Europe. Here, we describe the first European strains UCD646 and UCD650, isolated from a wooded area on a university campus in Dublin, Ireland. We generated complete chromosome level assemblies of both genomes using long- and short-read sequencing. The UCD isolates belong to the Holarctic clade. Genome analysis shows that isolates similar to the Irish strains contributed to the S. eubayanus component of S. pastorianus, but isolates from Tibet made a larger contribution.

Flippases and floppases are two classes of proteins that have opposing functions in the maintenance of lipid asymmetry of the plasma membrane. Flippases translocate lipids from the exoplasmic leaflet to the cytosolic leaflet, and floppases act in the opposite direction. Phosphatidylcholine (PC) is a major component of the eukaryotic plasma membrane and is asymmetrically distributed, being more abundant in the exoplasmic leaflet. Here we show that gene amplification of a putative PC floppase or double disruption of two PC flippases in the pathogenic yeast Candida parapsilosis results in resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism that has recently been granted orphan drug designation approval by the US FDA for treatment of invasive candidiasis. We analysed the genomes of 170 C. parapsilosis isolates and found that 107 of them have copy number variations (CNVs) at the RTA3 gene. RTA3 encodes a putative PC floppase whose deletion is known to increase the inward translocation of PC in Candida albicans. RTA3 copy number ranges from 2 to >40 across the C. parapsilosis isolates. Interestingly, 16 distinct CNVs with unique endpoints were identified, and phylogenetic analysis shows that almost all of them have originated only once. We found that increased copy number of RTA3 correlates with miltefosine resistance. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine over 26 days. Two genes, CPAR2_303950 and CPAR2_102700, gained homozygous protein-disrupting mutations in the evolved strains and code for putative PC flippases homologous to S. cerevisiae DNF1. Our results indicate that alteration of lipid asymmetry across the plasma membrane is a key mechanism of miltefosine resistance. We also find that C. parapsilosis is likely to gain resistance to miltefosine rapidly, because many isolates carry loss-of-function alleles in one of the flippase genes.Author summaryMiltefosine was developed as an anticancer drug but is commonly used to treat infections with the protozoan parasites Leishmania and Trypanosoma cruzi. More recently, it has been used to treat fungal infections, and in 2021 it was designated as an orphan drug by the US Food and Drug Administration for treatment of invasive candidiasis. Miltefosine is a derivative of phosphatidylcholine (PC), a major constituent of the cell membrane. PC and other phospholipids are asymmetrically distributed across the cell membrane. The mechanism of action of miltefosine is unknown. Here, we show that either increasing the activity of a putative floppase, which controls outward “flop” movement of phospholipids, or decreasing the activity of flippases, which control inward “flip” movement, results in increased resistance of the fungal pathogen Candida parapsilosis to miltefosine. This result suggests that miltefosine acts by controlling the localisation of PC or other phospholipids in the membrane. Importantly, we find that many C. parapsilosis isolates carry mutations in one flippase gene, which renders them partially resistant to miltefosine, and prone to easily acquiring increased resistance.

Candida metapsilosis is a member of the Candida parapsilosis species complex, a group of opportunistic human pathogens. Of all the members of this complex, C. metapsilosis is the least virulent, and accounts for a small proportion of invasive Candida infections. Previous studies established that all C. metapsilosis isolates are hybrids, originating from a single hybridization event between two lineages, parent A and parent B. Here, we use MinION and Illumina sequencing to characterize a C. metapsilosis isolate that originated from a separate hybridization. One of the parents of the new isolate is very closely related to parent A. However, the other parent (parent C) is not the same as parent B. Unlike C. metapsilosis AB isolates, the C. metapsilosis AC isolate has not undergone introgression at the mating type-like locus. In addition, the A and C haplotypes are not fully collinear. The C. metapsilosis AC isolate has undergone loss of heterozygosity with a preference for haplotype A, indicating that this isolate is in the early stages of genome stabilization.

Ogataea degrootiae is an ascomycete yeast that was first isolated in the Netherlands in 2017. It is a member of the Pichiaceae clade. Here, we present the genome sequence of O. degrootiae UCD465, which was isolated from soil in Ireland. This genome is 14.6 Mb and haploid.

Ogataea degrootiae is an ascomycete yeast that was first isolated in the Netherlands in 2017. It is a member of the Pichiaceae clade. Here, we present the genome sequence of O. degrootiae UCD465, which was isolated from soil in Ireland. This genome is 14.6 Mb and haploid.

Candida sanyaensis is a CUG-Ser1 clade yeast that is associated with soil. Assembly of short-read and long-read data shows that C. sanyaensis has a diploid and hybrid genome, with approximately 97% identity between the haplotypes. The haploid genome size is approximately 15.4 Mb.

Our study assesses the influence of differences in terrain and locomotor energetics on the land-use strategies and settlement patterns of Levantine Neanderthals and Modern Human – Early Upper Paleolithic groups through a digital application of site catchment analysis. Our findings indicate that Neanderthals habitually commanded smaller site exploitation territories (SETs), principally situated in the rugged Mediterranean Woodlands of the Levant, whereas early Upper Paleolithic groups generally enjoyed larger SETs and displayed a more generalized, wider settlement range encompassing both rugged woodland and more regular, level steppe landscapes. The broader implications of these findings may explain the biogeographic limits on the Neanderthal dispersal into Southwest Asia.

The vertical distribution and diel migration of Cryptomonas spp. were monitored continuously for 21 years in mesotrophic Cross Reservoir, northeast Kansas, USA. The movements of these motile algae were tracked on multiple dates during July–October of each year using in situ fluorometry and optical microscopy of Lugol’s iodine-preserved samples. Episodes of subepilimnetic diel vertical migration by Cryptomonas were detected and recorded on 221 different days between 1994 and 2014, with just 2 of these years (1998 and 2013) lacking any sampling events with deep peaks sufficiently large enough to track. Whenever a subepilimnetic layer of Cryptomonas was detectable, it was generally observed to ascend toward the bottom of the epilimnion beginning approximately at sunrise; to descend toward the lake bottom during the late afternoon and evening; and to remain as a deep-dwelling population until dawn of the following day. Moreover, there was high day-to-day consistency in the absolute water column depths at which the migrating algal cells would cease their ascending or descending movement. We believe this unique and remarkable dataset comprises the most detailed record of diel migratory behavior for any planktonic freshwater alga reported for a single freshwater lake.

The emergence of coupled natural and human landscapes marked a transformative interval in the human past that set our species on the road to the urbanized, industrial world in which we live. This emergence enabled technologies and social institutions responsible for human-natural couplings in domains beyond rural, agricultural settings. The Mediterranean Landscape Dynamics Project (MedLand) is studying the interacting social and biophysical processes associated with these novel socioecological systems and their long-term consequences using a new form of 'experimental socioecology' made possible by recent advances in computation. We briefly describe the MedLand modeling laboratory, a hybrid simulation environment that couples models of small-holder farming and herding, landscape evolution, and vegetation change managed through an interaction model. We then review three examples of experimental socioecology carried out in this laboratory. These examples offer new insights for scale-dependent thresholds in agropastoral productivity, long-term sustainability of alternative land-use strategies, and identifying signatures of human and climate-driven landscape dynamics. We conclude with an overview of new directions for this interdisciplinary research on Anthropocene human-earth systems, including: modeling more diverse decision-making strategies for land-use, developing more sophisticated models of vegetation dynamics and fire ecology, and generating digital proxy data for more robust model validation against the empirical record.

In previous research, the SE-NW time-trend in the age of the earliest Neolithic sites across Europe has been treated as a signal of a global-scale process that brought farming/herding economies to the continent. Residual variation from this global time-trend is generally treated as ‘noise’. A Complex Adaptive Systems perspective views this empirical record differently. The apparent time-trend is treated as an emergent consequence of the interactions of individuals and groups of different scale. Here, we examine the dynamics of agricultural dispersals, using the rich body evidence available from the Iberian Peninsula as a case study. We integrate two complementary approaches: (1) creating a high resolution Agent Based Modeling environment to simulate different processes that may have driven the spread of farming; (2) collecting and synthesizing empirical archeological data for the earliest Neolithic settlements that we use to evaluate our models results. Our results suggest that, (a) the source of radiocarbon data used to evaluate alternative hypotheses play an important role in the results; and (b) the model scenario that produces de best fit with archeological data implies a dispersal via northwestern and southern routes; a preference for leap-frog movement; an influence of ecological conditions (selecting most favorable agricultural land) and demographic factors (avoiding settled regions). This work represents a first attempt at high-resolution bottom-up modeling of this important dynamic in human prehistory. While we recognize that other social and environmental drivers could have also affected the dispersal of agropastoral systems, those considered here include many that have been widely considered important in prior research and so warrant inclusion.

The spread of agriculture from the Near East to Europe has long been a subject of intense archaeological study and debate in light of the social and economic changes that occurred and were set in motion as a result of this transition. Despite the attention paid to this important process, a consensus is far from being reached. Perhaps for these reasons, new methods and theoretical approaches have often been applied to the questions surrounding the spread of agriculture first. Recently, computational modeling has emerged as a promising technique for the study of the origins of agriculture. Our approach employs an agent-based computational model of agricultural spread for the Iberian Peninsula and utilizes a substantial radiocarbon database. This method allows for us to test multiple hypotheses about the manner in which agriculture spread, where it may have spread from and to focus on the critical evaluation of the available chronological record and its effects upon our results.

We present a repository for disseminating the computational models associated with publications in the social and life sciences. The number of research projects using computational models has been steadily increasing but the resulting publications often lack model code and documentation which hinders replication, verification of results and accumulation of knowledge. We have developed an open repository, the CoMSES Net Computational Model Library, to address this problem. Submissions to the library can be original models accompanying publications or replications of previous studies. Researchers can request that their models undergo a certification process that verifies that the model code successfully compiles and runs and that it follows documentation best practices. Models that pass the certification process are assigned persistent URLs and identifiers. We present the basic components of our repository, discuss our initial experiences with the library, and elaborate on future steps in the development of this cyberinfrastructure.

This study involves the high-resolution spatial analysis of a 9,500-year-old Early Neolithic site in an effort to reconstruct the social and economic organization of the settlement at household and community scales. We introduce an approach to distinguishing stratified occupational surfaces (floors) from intervening deposits (fills), to tracing the different formation processes associated with floors and fills, and to critically examining various factors (curation behaviors, palimpsests, and the Clarke Effect) that may have shaped house floor assemblages. The spatial analyses of behavioral residuals, features, and structures are then presented at intramural and intrasite scales, and the results are discussed as they relate to certain aspects of the social and economic organization of the community. These include family structure, control of resources, social differentiation, ritual participation, craft specialization, and gender-linked activities.

The archaeological record has been described as a key to the long-term consequences of human action that can help guide our decisions today. Yet the sparse and incomplete nature of this record often makes it impossible to inferentially reconstruct past societies in sufficient detail for them to serve as more than very general cautionary tales of coupled socio-ecological systems. However, when formal and computational modeling is used to experimentally simulate human socioecological dynamics, the empirical archaeological record can be used to validate and improve dynamic models of long term change. In this way, knowledge generated by archaeology can play a unique and valuable role in developing the tools to make more informed decisions that will shape our future. The Mediterranean Landscape Dynamics project offers an example of using the past to develop and test computational models of interactions between land-use and landscape evolution that ultimately may help guide decision-making.

Cross Reservoir, a small mesotrophic reservoir located at the University of Kansas Ecological Reserves (Kansas, USA), contained a dense metalimnetic community of algae and photosynthetic bacteria between early July and late October 1997-1999. Within this community, various Cryptomonas species, primarily C. erosa (Ehrenberg), C. erosa var. reflexa (Marsson), and C. rostratisformis (Skuja), diurnally migrated as indicated by in situ fluorescence monitoring and direct phytoplankton enumeration. The Cryptomonas spp. typically resided near the oxic-anoxic boundary of the water column; however, they actively migrated upward during the day and descended to lower anoxic locations at night, apparently responding to diurnal changes in their local habitat. Their nocturnal environment had moderate levels of sulfide, elevated secondary nutrients, and a community of anoxygenic phototrophic bacteria, whereas their daytime environment had higher light, lower nutrients, and no local photosynthetic bacteria. Monitoring indicated that the Cryptomonas spp. migration was generally linked to daily variations in absolute light intensity (e. g. sunny vs. cloudy days) and the level of other potentially growth-limiting resources, particularly nitrogen and phosphorus. However, further analyses showed that the primary factor that determined whether the Cryptomonas spp. migrated or not on a given day was the slope of the light gradient immediately above the Cryptomonas spp. peak.

The evolution of Mediterranean landscapes during the Holocene has been increasingly governed by the complex interactions of water and human land use. Different land-use practices change the amount of water flowing across the surface and infiltrating the soil, and change water’s ability to move surface sediments. Conversely, water amplifies the impacts of human land use and extends the ecological footprint of human activities far beyond the borders of towns and fields. Advances in computational modelling offer new tools to study the complex feedbacks between land use, land cover, topography and surface water. The Mediterranean Landscape Dynamics project (MedLand) is building a modelling laboratory where experiments can be carried out on the long-term impacts of agropastoral land use, and whose results can be tested against the archaeological record. These computational experiments are providing new insights into the socio-ecological consequences of human decisions at varying temporal and spatial scales.