The Oleaginous Yeast Metschnikowia pulcherrima Displays Killer Activity against Avian-Derived Pathogenic Bacteria.

Metschnikowia pulcherrima is a non-conventional yeast with potential to be used in biotechnological processes, especially those involving low-cost feedstock exploitation and biocontrol applications. The combination of traits that supports these industrial applications in M. pulcherrima also makes it an attractive option to study in the context of livestock health. In this study, we examined the specific interactions between M. pulcherrima and multiple avian pathogenic bacteria. We tested individual bacteria-yeast interactions and bacterial combinations in both solid and liquid media and in variable nutrient environments. Across multiple isolates of M. pulcherrima, we observed different levels of antimicrobial activity, varying from supporting the growth of competing bacteria through suppression and bacterial killing, and we found that these responses varied depending on the bacterial strains and media. We identified multiple molecular routes, including proteins produced by M. pulcherrima strains, that acted to control these microbial interactions. Furthermore, protein screening revealed that M. pulcherrima strains were induced to produce proteins specifically when exposed to bacterial strains, suggesting that fine-tuned mechanisms allow M. pulcherrima to function as a potential lynchpin in a microbial community.

Variation among Metschnikowia pulcherrima Isolates for Genetic Modification and Homologous Recombination.

Metschnikowia pulcherrima is a non-conventional yeast with the potential to be used in biotechnological processes, especially involving low-cost feedstock exploitation. However, there are a lack of tools for researching it at a molecular level and for producing genetically modified strains. We tested the amenability to genetic modification of ten different strains, establishing a transformation protocol based on LiAc/PEG that allows us to introduce heterologous DNA. Non-homologous integration was broadly successful and homologous recombination was successful in two strains. Chemical inhibition of non-homologous end joining recombination had a modest effect on the improvement of homologous recombination rates. Removal of selective markers via flippase recombinase was successful across integrated loci except for those targeted to the native URA3 locus, suggesting that the genome sequence or structure alters the efficacy of this system.

Semi-continuous pilot-scale microbial oil production with Metschnikowia pulcherrima on starch hydrolysate.

BACKGROUND: Heterotrophic microbial oils are potentially a more sustainable alternative to vegetable or fossil oils for food and fuel applications. However, as almost all work in the area is conducted on the laboratory scale, such studies carry limited industrial relevance and do not give a clear indication of what is required to produce an actual industrial process. Metschnikowia pulcherrima is a non-pathogenic industrially promising oleaginous yeast which exhibits numerous advantages for cost-effective lipid production, including a wide substrate uptake, antimicrobial activity and fermentation inhibitor tolerance. In this study, M. pulcherrima was fermented in stirred tank reactors of up to 350 L with 250-L working volume in both batch and semi-continuous operation to highlight the potential industrial relevance. Due to being food-grade, suitable for handling at scale and to demonstrate the oligosaccharide uptake capacity of M. pulcherrima, enzyme-hydrolysed starch in the form of glucose syrup was selected as fermentation feedstock. RESULTS: In batch fermentations on the 2-L scale, a lipid concentration of 14.6 g L-1 and productivity of 0.11 g L-1 h-1 were achieved, which was confirmed at 50 L (15.8 g L-1; 0.10 g L-1 h-1). The maximum lipid production rate was 0.33 g L-1 h-1 (daily average), but the substrate uptake rate decreased with oligosaccharide chain length. To produce 1 kg of dry yeast biomass containing up to 43% (w/w) lipids, 5.2 kg of the glucose syrup was required, with a lipid yield of up to 0.21 g g-1 consumed saccharides. In semi-continuous operation, for the first time, an oleaginous yeast was cultured for over 2 months with a relatively stable lipid production rate (around 0.08 g L-1 h-1) and fatty acid profile (degree of fatty acid saturation around 27.6% w/w), and without contamination. On the 250-L scale, comparable results were observed, culminating in the generation of nearly 10 kg lipids with a lipid productivity of 0.10 g L-1 h-1. CONCLUSIONS: The results establish the importance of M. pulcherrima for industrial biotechnology and its suitability to commercially produce a food-grade oil. Further improvements in the productivity are required to make M. pulcherrima lipid production industrial reality, particularly when longer-chain saccharides are involved.

Snf2 controls pulcherriminic acid biosynthesis and antifungal activity of the biocontrol yeast Metschnikowia pulcherrima.

Metschnikowia pulcherrima synthesises the pigment pulcherrimin, from cyclodileucine (cyclo(Leu-Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi. This yeast therefore has great potential for biocontrol applications against fungal diseases; particularly in the phyllosphere where this species is frequently found. To elucidate the molecular basis of the antifungal activity of M. pulcherrima, we compared a wild-type strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wild-type and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulator gene SNF2 in the mutant. Complementation of the mutant strain with the wild-type SNF2 gene restored pigmentation and recovered the strong antifungal activity. Mass spectrometry (UPLC HR HESI-MS) proved the presence of the pulcherrimin precursors cyclo(Leu-Leu) and pulcherriminic acid and identified new precursor and degradation products of pulcherriminic acid and/or pulcherrimin. All of these compounds were identified in the wild-type and complemented strain, but were undetectable in the pigmentless snf2 mutant strain. These results thus identify Snf2 as a regulator of antifungal activity and pulcherriminic acid biosynthesis in M. pulcherrima and provide a starting point for deciphering the molecular functions underlying the antagonistic activity of this yeast.

Elevated production of the aromatic fragrance molecule, 2-phenylethanol, using Metschnikowia pulcherrima through both de novo and ex novo conversion in batch and continuous modes.

BACKGROUND: 2-phenylethanol (2PE) is a fragrance molecule predominantly used in perfumes and the food industry. It can be made from petrochemicals inexpensively, however, this is unsuitable for most food applications. Currently, the main method of production for the bio-derived compound is to extract the trace amounts found in rose petals, which is extremely costly. Potentially fermentation could provide an inexpensive, naturally sourced, alternative. RESULTS: In this investigation, 2PE was produced from the yeast Metschnikowia pulcherrima, optimised in flasks before scaling to 2 L batch and continuous operation. 2PE can be produced in high titres under de novo process conditions with up to 1500 mg L-1 achieved in a 2 L stirred bioreactor. This is the highest reported de novo titre to date, and achieved through high sugar loadings coupled with low nitrogen conditions. The process successfully ran in continuous mode also, with a concentration of 650 mg L-1 of 2PE being maintained. The 2PE production was further increased by the ex novo conversion of phenylalanine and semi-continuous solid phase extraction from the supernatant. Under optimal conditions 14 000 mg L-1 of 2PE was produced. CONCLUSIONS: The work presented here offers a novel route to naturally sourced 2PE through a scalable fermentation with a robust yeast highly suited to industrial biotechnology. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Microevolutionary traits and comparative population genomics of the emerging pathogenic fungus Cryptococcus gattii.

Emerging fungal pathogens cause an expanding burden of disease across the animal kingdom, including a rise in morbidity and mortality in humans. Yet, we currently have only a limited repertoire of available therapeutic interventions. A greater understanding of the mechanisms of fungal virulence and of the emergence of hypervirulence within species is therefore needed for new treatments and mitigation efforts. For example, over the past decade, an unusual lineage of Cryptococcus gattii, which was first detected on Vancouver Island, has spread to the Canadian mainland and the Pacific Northwest infecting otherwise healthy individuals. The molecular changes that led to the development of this hypervirulent cryptococcal lineage remain unclear. To explore this, we traced the history of similar microevolutionary events that can lead to changes in host range and pathogenicity. Here, we detail fine-resolution mapping of genetic differences between two highly related Cryptococcus gattii VGIIc isolates that differ in their virulence traits (phagocytosis, vomocytosis, macrophage death, mitochondrial tubularization and intracellular proliferation). We identified a small number of single site variants within coding regions that potentially contribute to variations in virulence. We then extended our methods across multiple lineages of C. gattii to study how selection is acting on key virulence genes within different lineages.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'.

Species-specific PCR to describe local-scale distributions of four cryptic species in the Penicillium chrysogenum complex.

Penicillium chrysogenum is a ubiquitous airborne fungus detected in every sampled region of the Earth. Owing to its role in Alexander Fleming's serendipitous discovery of Penicillin in 1928, the fungus has generated widespread scientific interest; however its natural history is not well understood. Research has demonstrated speciation within P. chrysogenum, describing the existence of four cryptic species. To discriminate the four species, we developed protocols for species-specific diagnostic PCR directly from fungal conidia. 430 Penicillium isolates were collected to apply our rapid diagnostic tool and explore the distribution of these fungi across the London Underground rail transport system revealing significant differences between Underground lines. Phylogenetic analysis of multiple type isolates confirms that the 'Fleming species' should be named Penicillium rubens and that divergence of the four 'Chrysogenum complex' fungi occurred about 0.75 million yr ago. Finally, the formal naming of two new species, Penicillium floreyi and Penicillium chainii, is performed.

Transmission of Hypervirulence traits via sexual reproduction within and between lineages of the human fungal pathogen cryptococcus gattii.

Since 1999 a lineage of the pathogen Cryptococcus gattii has been infecting humans and other animals in Canada and the Pacific Northwest of the USA. It is now the largest outbreak of a life-threatening fungal infection in a healthy population in recorded history. The high virulence of outbreak strains is closely linked to the ability of the pathogen to undergo rapid mitochondrial tubularisation and proliferation following engulfment by host phagocytes. Most outbreaks spread by geographic expansion across suitable niches, but it is known that genetic re-assortment and hybridisation can also lead to rapid range and host expansion. In the context of C. gattii, however, the likelihood of virulence traits associated with the outbreak lineages spreading to other lineages via genetic exchange is currently unknown. Here we address this question by conducting outgroup crosses between distantly related C. gattii lineages (VGII and VGIII) and ingroup crosses between isolates from the same molecular type (VGII). Systematic phenotypic characterisation shows that virulence traits are transmitted to outgroups infrequently, but readily inherited during ingroup crosses. In addition, we observed higher levels of biparental (as opposed to uniparental) mitochondrial inheritance during VGII ingroup sexual mating in this species and provide evidence for mitochondrial recombination following mating. Taken together, our data suggest that hypervirulence can spread among the C. gattii lineages VGII and VGIII, potentially creating novel hypervirulent genotypes, and that current models of uniparental mitochondrial inheritance in the Cryptococcus genus may not be universal.

Chromosomal copy number variation, selection and uneven rates of recombination reveal cryptic genome diversity linked to pathogenicity.

Pathogenic fungi constitute a growing threat to both plant and animal species on a global scale. Despite a clonal mode of reproduction dominating the population genetic structure of many fungi, putatively asexual species are known to adapt rapidly when confronted by efforts to control their growth and transmission. However, the mechanisms by which adaptive diversity is generated across a clonal background are often poorly understood. We sequenced a global panel of the emergent amphibian pathogen, Batrachochytrium dendrobatidis (Bd), to high depth and characterized rapidly changing features of its genome that we believe hold the key to the worldwide success of this organism. Our analyses show three processes that contribute to the generation of de novo diversity. Firstly, we show that the majority of wild isolates manifest chromosomal copy number variation that changes over short timescales. Secondly, we show that cryptic recombination occurs within all lineages of Bd, leading to large regions of the genome being in linkage equilibrium, and is preferentially associated with classes of genes of known importance for virulence in other pathosystems. Finally, we show that these classes of genes are under directional selection, and that this has predominantly targeted the Global Panzootic Lineage (BdGPL). Our analyses show that Bd manifests an unusually dynamic genome that may have been shaped by its association with the amphibian host. The rates of variation that we document likely explain the high levels of phenotypic variability that have been reported for Bd, and suggests that the dynamic genome of this pathogen has contributed to its success across multiple biomes and host-species.

Using false discovery rates to benchmark SNP-callers in next-generation sequencing projects.

Sequence alignments form the basis for many comparative and population genomic studies. Alignment tools provide a range of accuracies dependent on the divergence between the sequences and the alignment methods. Despite widespread use, there is no standard method for assessing the accuracy of a dataset and alignment strategy after resequencing. We present a framework and tool for determining the overall accuracies of an input read dataset, alignment and SNP-calling method providing an isolate in that dataset has a corresponding, or closely related reference sequence available. In addition to this tool for comparing False Discovery Rates (FDR), we include a method for determining homozygous and heterozygous positions from an alignment using binomial probabilities for an expected error rate. We benchmark this method against other SNP callers using our FDR method with three fungal genomes, finding that it was able achieve a high level of accuracy. These tools are available at http://cfdr.sourceforge.net/.

Clonality despite sex: the evolution of host-associated sexual neighborhoods in the pathogenic fungus Penicillium marneffei.

Molecular genetic approaches typically detect recombination in microbes regardless of assumed asexuality. However, genetic data have shown the AIDS-associated pathogen Penicillium marneffei to have extensive spatial genetic structure at local and regional scales, and although there has been some genetic evidence that a sexual cycle is possible, this haploid fungus is thought to be genetically, as well as morphologically, asexual in nature because of its highly clonal population structure. Here we use comparative genomics, experimental mixed-genotype infections, and population genetic data to elucidate the role of recombination in natural populations of P. marneffei. Genome wide comparisons reveal that all the genes required for meiosis are present in P. marneffei, mating type genes are arranged in a similar manner to that found in other heterothallic fungi, and there is evidence of a putatively meiosis-specific mutational process. Experiments suggest that recombination between isolates of compatible mating types may occur during mammal infection. Population genetic data from 34 isolates from bamboo rats in India, Thailand and Vietnam, and 273 isolates from humans in China, India, Thailand, and Vietnam show that recombination is most likely to occur across spatially and genetically limited distances in natural populations resulting in highly clonal population structure yet sexually reproducing populations. Predicted distributions of three different spatial genetic clusters within P. marneffei overlap with three different bamboo rat host distributions suggesting that recombination within hosts may act to maintain population barriers within P. marneffei.

Emerging fungal threats to animal, plant and ecosystem health.

The past two decades have seen an increasing number of virulent infectious diseases in natural populations and managed landscapes. In both animals and plants, an unprecedented number of fungal and fungal-like diseases have recently caused some of the most severe die-offs and extinctions ever witnessed in wild species, and are jeopardizing food security. Human activity is intensifying fungal disease dispersal by modifying natural environments and thus creating new opportunities for evolution. We argue that nascent fungal infections will cause increasing attrition of biodiversity, with wider implications for human and ecosystem health, unless steps are taken to tighten biosecurity worldwide.

Sex, drugs and recombination: the wild life of Aspergillus.

Throughout the eukaryotes, sexual reproduction is an almost universal phenomenon. However, within the Kingdom Fungi, this relationship is not so clear-cut. Fungi exhibit a spectrum of reproductive modes and life-cycles; amongst the better known species, sexual reproduction is often facultative, can be rare, and in over half of the known Ascomycota (the moulds) is unknown (Taylor et al. 1999). However, over the last decade, it has become apparent that many of these asexual mitosporic taxa undergo cryptic recombination via unobserved mechanisms and that wholly asexual fungi are, in fact, a rarity (Taylor et al. 1999, 2001; Heitman 2010). This revolution in our understanding of fungal sexuality has come about in two ways: Firstly, sexual reproduction leaves an imprint on fungal genomes by maintaining genes required for mating and by generating patterns of mutation and recombination restricted to meiotic processes. Secondly, scientists have become better at catching fungi in flagrante delicto. The genus Aspergillus is one such fungus where a combination of population genetics, genomics and taxonomy has been able to intuit the existence of sex, then to catch the fungus in the act and formally describe their sexual stages. So, why are sexy moulds exciting? One species in particular, Aspergillus flavus, is notorious for its ability to produce a diverse array of secondary metabolites, of which the polyketide aflatoxins (AF) are carcinogenic and others (such as cyclopiazonic acid) are toxigenic. Because of the predilection of A. flavus to grow on crops, such as peanuts, corn and cotton, biocontrol is widely used to mitigate infection by pre-applying nonaflatoxigenic (AF-) strains to competitively exclude the wild-type AF+ strains. However, the eventual fate in nature of these biocontrol strains is not known. In this issue of Molecular Ecology, Olarte et al. (2012) make an important contribution by using laboratory crosses of A. flavus to show that not only is AF highly heritable, but AF- strains can become AF+ via crossing over during meiosis. This observation has raised the spectre of cross-breeding and non-mendelian inheritance of AF between native and biocontrol strains of the fungus, leading to an increase in the natural diversity of the fungus with perhaps unanticipated consequences.

The gut fungus Basidiobolus ranarum has a large genome and different copy numbers of putatively functionally redundant elongation factor genes.

Fungal genomes range in size from 2.3 Mb for the microsporidian Encephalitozoon intestinalis up to 8000 Mb for Entomophaga aulicae, with a mean genome size of 37 Mb. Basidiobolus, a common inhabitant of vertebrate guts, is distantly related to all other fungi, and is unique in possessing both EF-1α and EFL genes. Using DNA sequencing and a quantitative PCR approach, we estimated a haploid genome size for Basidiobolus at 350 Mb. However, based on allelic variation, the nuclear genome is at least diploid, leading us to believe that the final genome size is at least 700 Mb. We also found that EFL was in three times the copy number of its putatively functionally overlapping paralog EF-1α. This suggests that gene or genome duplication may be an important feature of B. ranarum evolution, and also suggests that B. ranarum may have mechanisms in place that favor the preservation of functionally overlapping genes.

Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage.

Batrachochytrium dendrobatidis (Bd) is a globally ubiquitous fungal infection that has emerged to become a primary driver of amphibian biodiversity loss. Despite widespread effort to understand the emergence of this panzootic, the origins of the infection, its patterns of global spread, and principle mode of evolution remain largely unknown. Using comparative population genomics, we discovered three deeply diverged lineages of Bd associated with amphibians. Two of these lineages were found in multiple continents and are associated with known introductions by the amphibian trade. We found that isolates belonging to one clade, the global panzootic lineage (BdGPL) have emerged across at least five continents during the 20th century and are associated with the onset of epizootics in North America, Central America, the Caribbean, Australia, and Europe. The two newly identified divergent lineages, Cape lineage (BdCAPE) and Swiss lineage (BdCH), were found to differ in morphological traits when compared against one another and BdGPL, and we show that BdGPL is hypervirulent. BdGPL uniquely bears the hallmarks of genomic recombination, manifested as extensive intergenomic phylogenetic conflict and patchily distributed heterozygosity. We postulate that contact between previously genetically isolated allopatric populations of Bd may have allowed recombination to occur, resulting in the generation, spread, and invasion of the hypervirulent BdGPL leading to contemporary disease-driven losses in amphibian biodiversity.

Genetic diversity, recombination, and divergence in animal associated Penicillium dipodomyis.

Penicillium dipodomyis is thought to be an exclusively asexual fungus associated with Kangaroo Rats, Dipodomys species, and is unique among Penicillium species in growing at 37°C but producing no known toxins. Lack of recombination within P. dipodomyis would result in limited adaptive flexibility but possibly enhance local adaptation and host selection via maintenance of favourable genotypes. Here, analysis of DNA sequence data from five protein-coding genes shows that recombination occurs within P. dipodomyis on a small spatial scale. Furthermore, detection of mating-type alleles supports outcrossing and a sexual cycle in P. dipodomyis. P. dipodomyis was a weaker competitor in in vitro assays with other Penicillium species found in association with Kanagaroo rats. Bayesian species level analysis suggests that the P. dipodomyis lineage diverged from closely related species also found in cheek pouches of Kangaroo Rats and their stored seeds about 11 million years ago, a similar divergence time as Dipodomys from its sister rodent taxa.

Population genetic structure of clinical and environmental isolates of Blastomyces dermatitidis, based on 27 polymorphic microsatellite markers.

Blastomyces dermatitidis, a thermally dimorphic fungus, is the etiologic agent of North American blastomycosis. Clinical presentation is varied, ranging from silent infections to fulminant respiratory disease and dissemination to skin and other sites. Exploration of the population genetic structure of B. dermatitidis would improve our knowledge regarding variation in virulence phenotypes, geographic distribution, and difference in host specificity. The objective of this study was to develop and test a panel of microsatellite markers to delineate the population genetic structure within a group of clinical and environmental isolates of B. dermatitidis. We developed 27 microsatellite markers and genotyped B. dermatitidis isolates from various hosts and environmental sources (n=112). Assembly of a neighbor-joining tree of allele-sharing distance revealed two genetically distinct groups, separated by a deep node. Bayesian admixture analysis showed that two populations were statistically supported. Principal coordinate analysis also reinforced support for two genetic groups, with the primary axis explaining 61.41% of the genetic variability. Group 1 isolates average 1.8 alleles/locus, whereas group 2 isolates are highly polymorphic, averaging 8.2 alleles/locus. In this data set, alleles at three loci are unshared between the two groups and appear diagnostic. The mating type of individual isolates was determined by PCR. Both mating type-specific genes, the HMG and α-box domains, were represented in each of the genetic groups, with slightly more isolates having the HMG allele. One interpretation of this study is that the species currently designated B. dermatitidis includes a cryptic subspecies or perhaps a separate species.

Low diversity Cryptococcus neoformans variety grubii multilocus sequence types from Thailand are consistent with an ancestral African origin.

The global burden of HIV-associated cryptococcal meningitis is estimated at nearly one million cases per year, causing up to a third of all AIDS-related deaths. Molecular epidemiology constitutes the main methodology for understanding the factors underpinning the emergence of this understudied, yet increasingly important, group of pathogenic fungi. Cryptococcus species are notable in the degree that virulence differs amongst lineages, and highly-virulent emerging lineages are changing patterns of human disease both temporally and spatially. Cryptococcus neoformans variety grubii (Cng, serotype A) constitutes the most ubiquitous cause of cryptococcal meningitis worldwide, however patterns of molecular diversity are understudied across some regions experiencing significant burdens of disease. We compared 183 clinical and environmental isolates of Cng from one such region, Thailand, Southeast Asia, against a global MLST database of 77 Cng isolates. Population genetic analyses showed that Thailand isolates from 11 provinces were highly homogenous, consisting of the same genetic background (globally known as VNI) and exhibiting only ten nearly identical sequence types (STs), with three (STs 44, 45 and 46) dominating our sample. This population contains significantly less diversity when compared against the global population of Cng, specifically Africa. Genetic diversity in Cng was significantly subdivided at the continental level with nearly half (47%) of the global STs unique to a genetically diverse and recombining population in Botswana. These patterns of diversity, when combined with evidence from haplotypic networks and coalescent analyses of global populations, are highly suggestive of an expansion of the Cng VNI clade out of Africa, leading to a limited number of genotypes founding the Asian populations. Divergence time testing estimates the time to the most common ancestor between the African and Asian populations to be 6,920 years ago (95% HPD 122.96 - 27,177.76). Further high-density sampling of global Cng STs is now necessary to resolve the temporal sequence underlying the global emergence of this human pathogen.

Common reservoirs for Penicillium marneffei infection in humans and rodents, China.

Human penicilliosis marneffei is an emerging infectious disease caused by the fungus Penicillium marneffei. High prevalence of infection among bamboo rats of the genera Rhizomys and Cannomys suggest that these rodents are a key facet of the P. marneffei life cycle. We trapped bamboo rats during June 2004-July 2005 across Guangxi Province, China, and demonstrated 100% prevalence of infection. Multilocus genotypes show that P. marneffei isolates from humans are similar to those infecting rats and are in some cases identical. Comparison of our dataset with genotypes recovered from sites across Southeast Asia shows that the overriding component of genetic structure in P. marneffei is spatial, with humans containing a greater diversity of genotypes than rodents. Humans and bamboo rats are sampling an as-yet undiscovered common reservoir of infection, or bamboo rats are a vector for human infections by acting as amplifiers of infectious dispersal stages.

Factors driving pathogenicity vs. prevalence of amphibian panzootic chytridiomycosis in Iberia.

Amphibian chytridiomycosis is a disease caused by the fungus Batrachochytrium dendrobatidis (Bd). Whether Bd is a new emerging pathogen (the novel pathogen hypothesis; NPH) or whether environmental changes are exacerbating the host-pathogen dynamic (the endemic pathogen hypothesis; EPH) is debated. To disentangle these hypotheses we map the distribution of Bd and chytridiomycosis across the Iberian Peninsula centred on the first European outbreak site. We find that the infection-free state is the norm across both sample sites and individuals. To analyse this dataset, we use Bayesian zero-inflated binomial models to test whether environmental variables can account for heterogeneity in both the presence and prevalence of Bd, and heterogeneity in the occurrence of the disease, chytridiomycosis. We also search for signatures of Bd-spread within Iberia using genotyping. We show (1) no evidence for any relationship between the presence of Bd and environmental variables, (2) a weak relationship between environmental variables and the conditional prevalence of infection, (3) stage-dependent heterogeneity in the infection risk, (4) a strong association between altitude and chytridiomycosis, (5) multiple Iberian genotypes and (6) recent introduction and spread of a single genotype of Bd in the Pyrenees. We conclude that the NPH is consistent with the emergence of Bd in Iberia. However, epizootic forcing of infection is tied to location and shaped by both biotic and abiotic variables. Therefore, the population-level consequences of disease introduction are explained by EPH-like processes. This study demonstrates the power of combining surveillance and molecular data to ascertain the drivers of new emerging infections diseases.