Genomic characterization of Parengyodontium americanum sp. nov.

Abstract

Modern genome analysis and phylogenomic methods have increased the number of fungal species, as well as enhanced appreciation of the degree of diversity within the fungal kingdom. In this context, we describe a new Parengyodontium species, P. americanum, which is phylogenetically related to the opportunistic human fungal pathogen P. album. Five unusual fungal isolates were recovered from five unique and confirmed coccidioidomycosis patients, and these isolates were subsequently submitted to detailed molecular and morphological identification procedures to determine identity. Molecular and morphological diagnostic analyses showed that the isolates belong to the Cordycipitaceae. Subsequently, three representative genomes were sequenced and annotated, and a new species, P. americanum, was identified. Using various genomic analyses, gene family expansions related to novel compounds and potential for ability to grow in diverse habitats are predicted. A general description of the genomic composition of this newly described species and comparison of genome content with Beauveria bassiana, Isaria fumosorosea and Cordyceps militaris shows a shared core genome of 6371 genes, and 148 genes that appear to be specific for P. americanum. This work provides the framework for future investigations of this interesting fungal species.

1. Introduction

The taxonomic history of the genus Parengyodontium is complex. One common type-species is Parengyodontium album, which was originally assigned to the Beauveria genus (Vuillemin 1912) and later to the Tritirachium genus (Limber 1940). Subsequently, it was assigned to the genus Engyodontium, which was thought to harbor three species: E. album, E. parvisporum and E. rectidentatum (Hoog 1978; Gams, Hoog, and Samson 1984). However, a close evolutionary relationship was discovered with the genus Lecanicillium, and therefore this species was again reclassified as L. tenuipes (Gams, Hoog, and Samson 1984). Finally, the genus was reclassified as Parengyodontium (Tsang et al. 2016). Parengyodontium spp. may be confounded with many other fungi due to shared macroscopic morphological characteristics such as a white, floccose to cottony macroscopic colony when grown on Sabouraud agar at 25°C, a trait that is widely polyphyletic across the Cordycipitaceae. Shared microscopic features with Lecanicillium spp. include narrow vegetative and fertile hyphae that are apically dichotomously branched (Tsang et al. 2016). Conidiogenous cells are displayed in whorls and born singly or in pairs on a lateral stalk/stem, similar to the genus Beauveria. Conidia have an elongated oval to cylindrical shape lacking pigmentation (Hoog 1972, 1978).

In the following report, we characterize a novel fungal species that we name Parengyodontium americanum sp. nov., which is phylogenetically related to the afore mentioned opportunistic human fungal pathogen Parengyodontium album. Five unusual fungal isolates were recovered from clinical samples of confirmed coccidioidomycosis cases. These isolates were obtained from patients residing in the U.S. states of Arizona and California. It was hypothesized these isolates were either contaminants, or the result of a co-infection with Coccidioides immitis or C. posadasii. The novel fungi appeared to be closely related to Parengyodontium album. The macro and microscopic features of this new species were characterized. A preliminary phylogenetic-based classification using available Multi Locus Sequencing Typing (MLST) data for Cordycipitaceae (Kepler et al. 2017) was completed. Additionally, 490 ribosomal DNA sequences derived from clinical and metagenomics projects were retrieved from existing data deposited at NCBI, and compared with our novel sequences. Next, the genomes from three isolates were sequenced using the Illumina MiSeq platform, and then assembled and annotated. Finally, a general description of the genomic composition of this newly described species designated Parengyodontium americanum sp. nov. is presented. Preliminary insights into the biology of this species compared with other whole genome sequenced Cordycipitaceae fungi reveal the potential for occupation of broad ecological niches including human hosts, and production of novel compounds.

2. Materials and Methods

2.1. Isolates and cultivation.

P. americanum isolates (Table 1) were recovered from Coccidioides spp. isolation slants from the Fungus Testing Laboratory at University of Texas Health San Antonio, TX. These isolates were all originally obtained from ACCUPROBE confirmed cases of coccidioidomycosis for phylogenomic analysis of Coccidioides. Monosporic isolation was performed in a BSL-3 facility at Northern Arizona University as presumed risk group 3 fungi. Mycelia were grown on 2X GYE (1% glucose, 2% yeast extract) agar, 25°C for 10 days. Conidia were harvested using a cell scraper and 1X PBS, and filtered in a sterile Miracloth filter assembled in a funnel. Harvested conidia were washed once in 1X PBS, counted with a hemocytometer and plated on 2X GYE for monosporic isolation. To assess macroscopic morphology, each fungus was inoculated on 2% Malt Extract Agar (MEA), Potato Dextrose Agar (PDA), Oatmeal Agar (OA), and 2%Glucose-1%Yeast Extract (2XGYE) plates and incubated for seven days at 25°C. A 2X GYE plate was also incubated at 37°C for seven days, and morphology evaluated for all media. For the microscopic characterization, slide culture method was applied; mycelia from macrocolonies were inoculated into 2X GYE, MEA, PDA and OA agar blocks. The blocks were then covered with glass coverslips and incubated for two weeks in a moist chamber. Slides were mounted in clear lactic acid and examined under a Nikon Eclipse 80i microscope with Differential Interference Contrast (DIC) optics. Micrographs were taken with a calibrated Nikon digital sight DS-5M camera and processed in NIS-elements D3.0 software. A type strain of specimen AZ2 has been deposited the Westerdijk Fungal BioDiversity Institute in the CBS-KNAW collection (CBS 144514)

Table 1 –

General Characteristics of Parengyodontium americanum genomes and close related Clavicipitaceae.

Species	Genome Size (Mb)	GC (%)	Predicted proteins	Total Pfam families	Total Pfam counts	Repetitive DNA content	Gene density (gene per Mb)
P. americanum - AZ2	33.0	52.9	10,354	3699	12191	1.63	314
P. americanum - CA11	33.0	52.9	10,347	3695	12188	1.63	314
P. americanum - CA13	33.0	52.9	10,346	3694	12186	1.59	314
B. bassiana	33.7	51.5	10,366	3689	11290	2.03	308
I. fumosorosea	33.5	53.6	10,060	3625	10901		300
C. militaris	32.2	51.4	9,684	3581	10411	3.04	301

2.2. DNA purification and molecular identification

DNA was obtained from approximately 100 mg of fresh harvested mycelial tissue collected after seven days post conidia inoculation on 2X GYE plates. DNA was extracted using the UltraClean® Microbial DNA Isolation Kit (MoBio, Qiagen). Lysis of the microorganism was performed using MicroBead tubes provided in the kit and horizontally agitated vigorously using a vortex adapter tube holder (MoBio, Qiagen) for 15 minutes. The protocol was performed according to the manufacturer’s instructions. Purified DNA was inspected via electrophoresis with a 0.8% agarose gel stained with SYBR Safe (Thermo Fisher Scientific) and quantity estimated. Quality was assessed using spectrophotometry on a NanoDrop® ND-1000 system (Thermo Fisher Scientific). Because these isolates were initially diagnosed as Coccidioides spp., we applied a real-time-based test that targets a specific repetitive region found only in Coccidioides spp. genomes following previously published protocols (Bowers et al. 2018). Five samples tested did not amplify Coccidioides spp.

These five fungal isolates were then classified using the universal rDNA for fungal barcoding. About 20ng of DNA was used as input for the PCR amplifications of the ITS1/2 + 5.8S using the universal ITS 1 and 4 primers (White et al. 1990) as follows: 25ul of MyFi™ Mix (Bioline), 0.2 μM of each primer, DNA template and ultrapure water to a final volume of 50μl. The thermocycling steps were: initial denaturation for 2 min at 95°C, followed by 35 cycles of 15 s at 95°C, 15 s at 55°C and 30 s at 72°C. A final elongation step of 5 m at 72 °C was applied. PCR fragments were purified using QIAquick PCR Purification kit (Qiagen) and sequenced via Sanger capillary sequencing method using the BigDye v3.1Terminator Cycle Sequencing Kits on an ABI 3130xl Genetic Analyzer instrument (Thermo Fisher Scientific). Electropherograms were inspected using the Phred algorithm in a tool implemented in the webpage http://www.biomol.unb.br/phph/. Sequences were compared to the NCBI nucleotide databank using Blastn algorithm (Altschul et al. 1990) and the top 490 sequences hits were retrieved for phylogenetic analysis. Sequences were aligned using the MAFFT online server http://mafft.cbrc.jp/alignment/server/ using G-INS-i (allowing gaps) as the alignment strategy and manually inspected. Both left and right flanks of the alignments were trimmed and sequences with low coverage in the alignment were removed for the final data set, that resulted in an aligned DNA matrix of 502 bp covering 493 taxa (See Table S1). Additionally, phylogenetic relationships were assessed among Cordycipitaceae species using the Multi Locus Sequencing Typing (MLST) scheme: small and large subunits of ribosomal DNA genes (SSU and LSU), translation elongation factor 1 alpha (TEF) and the two largest subunits of RNA polymerase II (RPB1) and (RPB2) (Kepler et al. 2017). Maximum likelihood trees were produced using an effective stochastic algorithm implemented in IQ-TREE (Nguyen et al. 2015) using -m TEST option (model selection) and 1,000 nonparametric bootstrap pseudoreplicates were performed for branch confidence (Minh, Nguyen, and von Haeseler 2013). Trees were visualized and compared using the FigTree v1.4.2 http://tree.bio.ed.ac.uk/software/figtree/.

Whole genome sequencing, assembly and annotation

The genomes of three P. americanum isolates were sequenced using the Illumina MiSeq sequencing platform. One μg of double-stranded DNA was used as input for paired-end sequencing. DNA was sheared by sonication and size selected at 500bp. Libraries were prepared using a Kapa Biosystems kit (catalog number kk8201; Kapa Biosystems, Woburn, MA) protocol with an 8-bp index multiplexing (Kozarewa and Turner 2011). Sequencing libraries were quantified using quantitative PCR (qPCR) in a 7900HT system (Life Technologies Corporation, Carlsbad, CA), and a Kapa library quantification kit (Kapa, Woburn, MA). Paired-end libraries were sequenced to a read length of 300bp. Genomes were assembled using UGAP suite (https://github.com/jasonsahl/UGAP) which utilizes the universal A-Bruijn assembler SPAdes (Bankevich et al. 2012). Gene models were predicted using two different approaches: AUGUSTUS (Stanke et al. 2004) and GeneMark-ES fungal version (Ter-Hovhannisyan et al. 2008). Gene predictions were used as input to identify consensual Open Read Frames (ORF’s) using EVidenceModeler (Haas et al. 2008). Functional prediction of consensus ORF’s were performed using PFAM (version 30.0) and Interpro (version 5.20–59.0). Predicted genes were categorized according to biological process, cellular component and molecular function using GeneOntology (GO) using Blast2GO tool (Conesa et al. 2005).

2.3. Comparative Genomics and Phylogenomics

The genomes of P. americanum were compared with closely related Cordycipitaceae genomes of Beauveria bassiana, Isaria fumosorosea and Cordyceps militaris (Table S2). OrthoVenn was used to identify the orthologous clusters across the evaluated species (Wang et al. 2015) via the OrthoMCL approach (Li, Stoeckert, and Roos 2003). Unique and shared clusters were subjected to blastp against the nonredundant protein UniProt database for function prediction and annotation. Any sequence hit with an e-value <1e–5 was queried for terms associated with biological process, molecular function, and cellular component categories via GOSlim (Gene Ontology 2015). Gene ontologies expanded in P. americanum were visualized using semantic similarity-based scatterplots implemented in the REVIGO platform (Supek et al. 2011).

Peptidases were classified into families using the MEROPS database (Rawlings et al. 2014) and Carbohydrate-degrading Enzymes (CAZy) via dbCAN (Yin et al. 2012) by sequence similarity methods. Transposons and other repetitive sequences were identified and annotated using both de novo and homology-based approaches. Transposable elements (TEs) with terminal inverted repeats were identified de novo with an inverted repeat finding tool irf (Warburton et al. 2004) whereas repetitive sequences including transposons were identified using RepeatModeler (Smit and Hubley 2008–2015). All TE candidate sequences were clustered with cd-hit, and subsequently scanned for protein domains related to transposons using PFAM (Finn et al. 2016) and CDD protein domains (Marchler-Bauer et al. 2011). Only those TE-candidates with similarity to transposons were retained and merged with RepBase (Jurka et al. 2005) as a custom library for RepeatMasker (Smit and Hubley 2008–2015). RepeatMasker output was parsed with in-house scripts filtering out hits with scores below 200. This threshold was set by plotting scores for manually checked elements. Two datasets were created i) all TEs with RepeatMasker scores better than 200 and ii) TEs additionally retaining similarity to typical TE-coding regions. Secondary metabolite clusters were predicted using antiSMASH for fungi (Weber et al. 2015). A phylogenetic birth-and-death model implemented in the Count software (Csuros 2010) was applied to determine the evolution of homologous family size of MEROPS, CAZy and PFAM classes based on ancestral reconstruction.

A genome-level phylogenetic tree was inferred based on 50 Hypocreales genomes previously published covering the diversity of the order. OrthoMCL (Li, Stoeckert, and Roos 2003) was used to infer orthologous groups across all Hypocreales genomes and paralogs were removed using INPARANOID (Sonnhammer and Ostlund 2015). Individual alignments of 319 single clusters were trimmed and concatenated into a single matrix. Phylogenomic trees were generated by IQ-TREE software (Nguyen et al. 2015) using -m TEST option (model selection) and 1,000 nonparametric bootstrap pseudoreplicates were performed for branch fidelity (Minh, Nguyen, and von Haeseler 2013). Phylogenomic trees were visualized using FigTree v1.4.2.

3. Results and Discussion

3.1. Morphological characterization

The five atypical clinical isolates obtained from five different diagnosed coccidioidomycosis cases all grew white and floccose mycelial irregular colony morphology at 25°C on 2X GYE media (Figure 1). The surface of the colony differed from typical Coccidioides spp. with a rough, irregular, undulated and raised colony-type morphology, although colonies of Coccidioides spp. can be very diverse (Friedman et al. 1953; Baker, Mrak, and Smith 1943). The reverse side of the macrocolonies were pale yellow and wrinkled, presenting cavitations (Figure S1). Moreover, those isolates were unable to grow at 37°C (Figure S1). Commonly, microorganisms are grown at lower temperatures in clinical laboratories, and it has been observed that Coccidioides immitis does not grow well as a mycelium at 37°C (Mead et al. in prep). Additionally, a wide variety of colony morphologies are observed among various isolates of Coccidioides spp., so microscopic confirmation was warranted (Friedman et al. 1953). The microscopic characteristics of these five isolates included narrow vegetative fertile hyphae that raise phialides and/or aphanophialides, usually verticillate, which are displayed either in pairs or solitary on a lateral stalk/stem (Figure 1). Conidia vary from elongated cylindrical to oval. These morphological characteristics are commonly observed in Lecanicillium spp., as opposed to the zigzag structure of phialides on terminal hyphae from P. album, or barrel shaped arthroconidia produced by Coccidioides (Tsang et al. 2016; Sun, Sekhon, and Huppert 1979).

Figure 1.

Macro- and microscopic characteristics of monosporic cultures from atypical clinical isolates recovered from different diagnosed coccidioidomycosis cases. Macro-morphological characteristics of the isolates include an irregular, undulated and raised colony-type morphology after 2 weeks of incubation at 24°C on A) Malt Extract Agar, B) Potato Dextrose Agar and C) Oatmeal Agar. The microscopic characteristics of these isolates include narrow vegetative fertile hyphae that raise phialides and/or aphanophialides, usually verticillate, which are displayed either in pairs or solitary on a lateral stalk/stem (D-M). Conidia vary from elongated cylindrical to oval and the pictures are all scaled to 10 μm.

3.2. Molecular identification of the fungal specimens

The DNA extracted from the five monosporic fungal cultures did not contain any Coccidioides DNA, as no amplification was detected in the Coccidioides-specific qPCR assay (data not shown (Bowers et al. 2018)). Internal Transcribed Spacer (ITS) regions of AZ2, CA11, CA13, CA19 and CA21 isolates were Sanger sequenced using flanking ITS1 and ITS4 primers (White et al. 1990), which revealed a single operational taxonomical unit (OTU) and no single nucleotide polymorphisms (SNPs) in the ITS sequence region among those five isolates (KY683770-KY683774 – Table S1). BlastN analysis revealed that all five isolates were 100% identical to fungal sequences obtained from fungi associated with corals in Xuwen, China (JQ717351), bat guano in Heshang cave, central China (KP216954/ KP216955), deep-sea sediments of the Central Indian Basin (EU729707) (Singh et al. 2010), entomopathogens of Lecanicillium spp. infecting the Asian citrus psyllid in China (KM013699), and an E. album isolated from dipteran insects trapped by adhesive sheets in Shiitake sawdust-block cultivation facility (LC035053). We therefore analyzed the phylogenetic relationships of the top ITS blast hits, and then performed a refined systematic analysis (Table S1). Maximum likelihood trees revealed seven supported species-level clades found within the Parengyodontium album/Lecanicillium sp. complex (Figure S2). The Parengyodontium album subclades I-III and Parengyodontium clade I are similar to those previously reported ((Tsang et al. 2016) Figure S2). Our five isolates grouped within the Parengyodontium clade II along with other 46 additional taxa. Along with Parengyodontium clades II and III, this genetic cluster appears to be distinct from Parengyodontium album subclades I-III and Lecanicillium kalimantanense.

Additionally, an MSLT-based scheme analysis was employed to verify the phylogenetic distribution of Parengyo./Lecanicillium among other genera within the Cordycipitaceae family. Akanthomyces (syn: Lecanicillium), Blackwelliella, Engyodontium, Hevansia, Gibbellula, Ascopolyporus, Parengyodontium, and Simplicillium are all represented by their type species (Figure S3). Our isolates grouped in a sister clade of Parengyodontium album. We therefore classified these isolates as Parengyodontium americanum sp. nov. as they share genetic traits with P. album, and share morphological similarities with the now extinct Lecanicillium sp. Based on metadata from cultures and metagenomic sequences, P. americanum may be distributed across 4 continents (Asia, South and North America and Europe), and potentially occupies different ecological niches (Figure S2). In addition to our human associated isolates from North America, members of the P. americanum clade are found in very diverse habitats, such as subaerial biofilms in Spain (Vazquez-Nion et al. 2016), caves in China (Man et al. 2015), deep ocean sediments in India (Singh et al. 2010), in a marine sponge Tethya aurantium in China (Wiese et al. 2011), insect associations (China and Japan), plant associations (USA, Brazil, Finland and Thailand), and as extremophiles isolated from 3M potassium chloride-containing media in Germany (Table S1).

3.3. Genome characteristics of Parengyodontium americanum.

The genomes of three isolates of P. americanum were assembled into ~33Mb distributed among 329–429 contigs (Table S3), which is similar in size to other Cordycipitaceae species. The whole genome sequence data of the isolates AZ2, CA11 and CA13 were deposited with GenBank under deposit numbers as follows: PXYO00000000, PXYN00000000 and PXYM00000000. N95 values for each assembly were calculated and were on average 350Kb; the largest contig spanned 880kb (Table S3). The G + C content averaged 52.88%, which is compatible with other Ascomycota genomes (Li and Du 2014). The CA11 and CA13 assemblies were mapped into AZ2 reference and the genomes shared about 97.13% of genetic content, revealing that these isolates are not clones. Thus, although possible, it is unlikely these isolates are the result of laboratory contamination during isolation procedures (Table S4). A range of 10,346 to 10,354 protein-coding genes were predicted in genomes of P. americanum, which is similar to other closely related species (Table 1). The three analyzed assemblies contain less than 1.7% of repetitive sequences. Moreover, only ~2% of those repetitive sequences retain similarity to protein domains typical of transposons, the remaining are either simple repeats or remnants of ancient transposons.

The mobile elements in P. americanum belong to the common LINE (I and Tad1) and LTR retrotransposon (Ty1/Copia) families found in many eukaryotic genomes. Most fungal genomes have more Ty3/Gypsy elements compared to Ty1/Copia (Muszewska, Hoffman-Sommer, and Grynberg 2011), but the analyzed genomes contain double the number of Ty1/Copia elements compared to Ty3/Gypsy elements (Table S5). All three genomes contain DIRS elements, Helitrons and a variety of DNA and LINE family remnants. DNA transposons with DDE transposases from super-families Mutator-like and hAT retain coding regions, but all others are present as sequence fragments (Table S5).

The low relative abundance of repetitive DNA content found in P. americanum also was reported for Metarhizium acridum, B. bassiana and M. anisopliae (Zheng et al. 2011; Gao et al. 2011; Xiao et al. 2012). However, the genomes of C. militaris contain slightly higher repetitive content (3.04%) with a decrease of total Pfam counts, Pfam families, and gene density per 10⁷ bases (Table 1). The repetitive content is low considering the assembly quality measured by N50 (Table S3). The P. americanum genomes have a relatively high G + C content of 53%, which can result from low repeat content and lead to lower repeat proliferation as repeats are usually AT rich and propagate in AT rich regions. Hence, P. americanum might have an efficient genome defense mediated by Repeat-induced point mutation (RIP). It is thought that RIP evolved to avoid transposon replication and proliferation in fungal genomes and occurs during meiosis, suggesting that P. americanum is a sexually recombining species (Fierro and Martin 1999; Li and Du 2014). We therefore investigated the mating type loci in the genome.

The MAT locus of P. americanum was characterized based on MAT1–1 and MAT1–2 alleles of closely-related Cordycipitaceae. BLAST analysis revealed that all three P. americanum genomes assembled harbor a single copy of MAT1-1-1 gene containing the MAT alpha1 HMG-box domain (pfam04769/ IPR006856). This suggests a heterothallic mating system, although the MAT1–2 locus was not identified among the genomes we sequenced. The canonical structure of MAT1–1 locus of Sordariomycetes contains MAT1-1-1, MAT1-1-2 and MAT1-1-3 genes that are usually flanked by the SLA2 and APN2 genes (Figure 2). Genomes of C. militaris and B. bassiana have lost the MAT1-1-3 gene (Xiao et al. 2012), and the same was observed for the P. americanum genome. Interestingly, the entomopathogenic fungus Metarhizium robertsii has lost the MAT1-1-2 gene suggesting different evolutionary trajectories of the MAT1–1 locus among Cordycipitaceae fungi (Pattemore et al. 2014).

Figure 2.

Mating Type representation of P. americanum MAT1–1 locus compared to other Clavicipitaceae species. All three P. americanum isolates harbor the MAT 1-1-1 and MAT1-1-2 genes suggesting a heterothallic mating system, and gene organization is conserved.

3.4. Phylogenomics of Hypocreales

The Sordariomycetes are one of the most diverse clades of fungi in the Ascomycota, containing more than 600 genera and 3000 described species (Kirk et al. 2001). To evaluate the taxonomic relationships of Parengyodontium americanum to other Hypocreales, we performed a phylogenetic analysis using a group of 319 orthologous proteins across 54 taxa (Table S2). Insertae sedis Sordariomycetes taxa were included in this genome-scale tree for a more complete understanding of family/order-level clades for these diverse fungi. Results indicate Ustilaginoidea virens represents a basal lineage of the Hypocreales order along with Aschersonia aleyrodis (former Oomycetoideae), which is not associated with Clavicipitaceae or related sub-families (Liu, Chaverri, and Hodge 2006) (Figure 5). The subfamily Clavicipitoideae forms a strongly supported monophyletic clade, and may be a distinct family, as well as Metacordyceps clade (C. taii) (Sung et al. 2007).

Figure 5.

Enriched GOSlim terms for biological process and molecular function in P. americanum genomes. The Gene Ontology terms were summarized by purging redundant GO terms that were deduced by orthologous searches coupled with semantic similarity-based scatterplots using the REVIGO platform (Supek et al., 2011).

We observe that the isolates CA11, CA13 and AZ2 form a monophyletic clade within the Cordycipitaceae, clustering apart from Beauveria bassiana, Isaria fumosorosea and Cordyceps militaris (Figure 3). Acremonium chrysogenum is considered an Insertae sedis taxa among Sordariomycetes, and we show that this species appears in a branch between Nectriaceae and Stachybotryaceae families. Lastly, we show that Madurella mycetomatis (Insertae sedis) is also positioned within Soradariales, and related to Lasiosphaeriaceae and Chaetomiaceae families ((van de Sande 2012) Figure 3).

Figure 3.

Phylogenomic Maximum Likelihood tree representing the evolutionary placement among Hypocrealean fungi based on 319 single orthologous clusters. The P. americanum isolates CA11, CA13 and AZ2 are placed in a monophyletic clade within the Cordycipitaceae family, apart from Beauveria bassiana, Isaria fumosorosea and Cordyceps militaris. Scales represents branch length equivalent to nucleotide substitutions per site.

3.5. Comparative genomics

We compared P. americanum genome content with three related Clavicipitaceae: B. bassiana, I. fumosorosea and C. militaris. According to orthologous predictions, the core genome of these species contains 6371 gene clusters (Figure S4). At least three species share 9688 gene clusters, at least two species contain 9410 orthologous groups, and 6055 single-copy gene clusters were assigned to a single lineage. We identified 1913 singletons that are represented by 148 clusters in P. americanum. Those unique clusters were classified via AMIGO and hypergeometric tests, showing enrichment (p-value < 0.05) of the following GO terms: inorganic phosphate transmembrane transporter activity, symporter activity, xenobiotic-transporting ATPase activity, amino acid transmembrane transporter activity (Molecular Function) and Phosphate ion transport (Biological Process) (Table 2). We hypothesize that P. americanum may be adept at uptake of exogenous macro- and microcomponents via a repertoire of transmembrane transporters/symporters of inorganic phosphate, xenobiotic compounds and amino acids, and these fungi may play a role in nutrient cycling and soil decontamination. For example, L. saksenae (Figure S2) metabolizes different pesticides via biotransformation of xenobiotic compounds and may play a role in biopurification (Pinto et al. 2012).

Table 2:

Gene ontology classes enriched in Parengyodontium americanum

	Name	Namespace	p-value
GO:0005315	inorganic phosphate transmembrane transporter activity	molecular_function	8.138802683590708E-4
GO:0015293	symporter activity	molecular_function	0.007996024681548738
GO:0008559	xenobiotic-transporting ATPase activity	molecular_function	0.013527867129640405
GO:0015171	amino acid transmembrane transporter activity	molecular_function	0.013765953069964544
GO:0006817	phosphate ion transport	biological_process	0.01819381333252431

Because Lecanicillium/Parengyodontium spp. are widespread and ubiquitous in many different biomes, we cannot establish a specific ecological function based solely on genomic comparisons. It is often observed that nitrogen and phosphate transporters are overrepresented in mycorrhizal fungi. However, it is unlikely that P. americanum is a mycorrhizal fungus. Based on comparisons to close relatives, we suggest that P. americanum is an entomopathogenic or opportunistic pathogenic fungus of animals, and increased phosphate or nitrogen transport may contribute to virulence by sequestering macromolecules from hosts.

We quantified gene gain and loss among a select group within the Clavicipitaceae using Pfam, CAZy and MEROPS protein classification methods using gene birth and death models. Protein family history revealed that P. americanum gained more Pfam, CAZy, MEROPS families than B. bassiana, I. fumosorosea, and C. militaris, which appear to have significant gene family losses (Figure 4). We identified expansion of eight MEROPS families, nine CAZy and 80 Pfam gene families in P. americanum (Table S6). The MEROPS analysis of P. americanum expanded families includes the inhibitor (I) peptidase inhibitor LMPI (Orthoptera) inhibitor unit 1 (I19), Aspergillus elastase inhibitor (I78), cvSI-2 serine peptidase inhibitor (I84), metalloproteases (M) carboxypeptidase E (M14B), IMPa endopeptidase (M88), mixed type peptidase (P) EGF-like module containing a mucin-like hormone receptor-like homolog of Homo sapiens (P2A), serine proteases (S) dipeptidase E (S51) and LD-carboxypeptidase (S66). These proteases are not characterized in fungi and MEROPS has no fungal homologs listed. The expanded CAZy families include the glycosyl hydrolases (GH) as follows: lysozyme (GH24), GH30, α-L-arabinofuranosidase (GH51), α-glucuronidase (GH67), α-L-rhanmosidase (GH78), exo-α-L-1,5-arabinase (GH93), xylan α−1,2-glucuronidase (GH115), α−1,3-L-neoagarooligosaccharide hydrolase (G117). A single lyase – alginate lyase (PL5) CAZy family was found in P. americanum as well.

Figure 4.

Gene gain and loss analysis of A) Pfam, B) CAZy and C) MEROPS homolog gene family sizes among P. americanum and other Clavicipitaceae species. The species tree was inferred based on phylogenomic analyses, and used to define the identification of gene expansions and contractions of the above mentioned functional categories. Bar plots related to each tree node represents the number of ancestral orthologues in each tree taxon position.

The expanded Pfam gene families were transformed into 61 GO categories (Molecular Function – 30, Cellular Component - 10 and Biological Process - 21). These categories harbor 127 “GO_Slim” ancestor terms, and after filtering the redundant GO terms, 32 Molecular Function and Biological Process (Table S7) categories were visualized via a semantic similarity-based scatterplots approach (REVIGO – Figure 5). We observed an overlap of GO enrichment classes between orthology-based (OrthoVenn) methods and the gene birth and death models implemented in Count software. For example, within the GO category “Biological Process” we identified expanded Pfam domains associated with antibiotic response, siderophore biosynthesis, nitrate metabolism, ion transport, cell wall macromolecule catabolism and aflatoxin (secondary metabolites) biosynthesis (Figure 5). Within the GO “Molecular Function” category, we identified an enrichment of calcium/zinc binding, electron carrier and siderophore transmembrane transporter that were similar to GO classes identified in the OrthoVenn analysis (Figure S4, Table 2).

Moreover, we found several gene family expansions within P. americanum involved in carbohydrate metabolism as well hydrolase and peptidase functions that support the CAZy and MEROPS results. For example, we found a significant expansion of genes containing α-glucoronidase, lysozyme and α-L-arabinofuranosidase corresponding to GH67, GH54 and GH51 respectively (Figure 4, Table S6). Comparative genomic analyses suggest that L-arabinose may be an important nutrition source because this compound is abundant in plant polysaccharides, hemicellulose, and pectin (Seiboth and Metz 2011). Lastly, we identified GO semantic terms associated with TOR signaling, organic acid phosphorylation, oxidative stress and 3-phytase activity. Phytic acid is an important form of phosphorus storage in plants, fungi and bacteria. Inorganic phosphate is usually absorbed after the break down of phytic acid (Pandey et al. 2001). These distinct genes are unique to this fungus when compared to other Clavicipitaceae species, and warrant further investigation of their role in general metabolism and ecology of P. americanum.

3.6. Secondary metabolites and biotechnology prospects

Interestingly, P. americanum is related to entomopathogenic species nested within Clavicipitaceae family and may be relevant to investigate as a biopesticide, and may produce important protein/carbohydrate-degrading enzymes. Sequences from this species, or at least a very closely related species based on ITS similarity, has been detected in oceanic sediments, and may therefore also play an important role in marine ecosystems. A close relative of P. americanum, P. album, has been used for biotechnological purposes (Suresh and Muthusamy 1999; Molitoris and Schaumann 1986). Because P. americanum may be economically and ecologically important, these genomic studies will help to guide future functional investigations.

Using the fungal version of antiSMASHv3, we characterized the biosynthetic gene cluster composition of P. americanum and other Clavicipitaceae, which are potentially responsible for secondary metabolite production. P. americanum harbors 29 gene clusters associated with secondary metabolite production (Table S8). We observed a similar number of clusters in C. militaris and an increased number in B. bassiana and I. fumosorosea. P americanum has fewer modular nonribosomal peptide synthetase (NRPS) clusters, and a greater number of phosphonate and siderophore cluster genes compared to the other insect-associated fungi (Table S8).

Several conserved biosynthetic clusters previously identified in other fungi were annotated. For example, P. americanum may produce analog compounds of equisetin (Kakule et al. 2013), a natural product produced by Fusarium heterosporum (Figure S5). We identified a biosynthetic gene cluster sharing 35% gene similarity to a cluster found in Pestalotiopsis fici, which is responsible for pestheic acid production (Xu et al. 2014). This gene cluster also is similar to biosynthetic clusters involved in the synthesis of emericellin (Xu et al. 2014) and trypacidin (Throckmorton et al. 2016) in Aspergillus nidulans and A. fumigatus, respectively. Lastly, the genes responsible for alternariol production in A. fumigatus were identified in the genome of P. americanum (Ahuja et al. 2012). Those compounds generally have antibiotic, cytotoxic and phytotoxic activities. This analysis provides strong evidence that P. americanum is a good candidate for natural product discovery. A series of natural compounds, such as cytotoxic polyketides from P. album isolated from a deep sea in south China have been characterized (Yao et al. 2014). Some of these compounds have antibacterial activity against Escherichia coli and Bacillus subtilis or antilarval activity against barnacle Balanus amphitrite. Recently, new polyketide compounds were characterized from the P. album strain LF069 showing inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA), and these compounds are 10-fold more efficient than chloramphenicol (Wu et al. 2016). Beyond using filamentous fungi for natural product production and biotechnological purposes, little is known about the potential production of such biomolecules within Clavicipitaceae (Wang et al. 2014). We hope the data from this study will open new avenues for exploration in the Parengyodontium/Lecanicillium clade.

4. Conclusions

A new fungal species, herein named Parengyodontium americanum sp. nov., was described based on in-depth and complementary morphological and genomic studies. Morphologically, the genus Lecanicillium is polyphyletic and should not be used according to Kepler et al., 2017. Parengyodontium americanum sp. nov is phylogenetically related to P. album and has a genome similar to those of other species in the Cordycipitaceae. Comparative genomic analyses predict phenotypes such as natural product production and diverse habitat range. This fungus may be an opportunistic or co-infecting fungal pathogen, as isolates were recovered from clinical coccidioidomycosis cases. As P. americanum cannot grow at 37°C, it is possible that this was a contamination during the isolation procedure, such as spores of the organisms present in the lung airways, or in tubing or receptacles used during BALF collection. Alternatively, the organism may need the host specific milieu to grow at higher temperatures. The genomic architecture of this fungus was described, which gives insight into the metabolic profile of the fungus even though these specific molecules remain untested in vitro. Finally, we provide a genomic and taxonomic framework to help to avoid future fungal misclassification and expand knowledge of an understudied fungal species.

5. Species description:

Parengyodontium americanum Teixeira, Wiederhold & Barker, sp. nov. – Figures 1, S2 and S3, MycoBank MB: 828553

Etymology: referring to the continent that this fungus was isolated.

Holotype: United States of America (Texas), specimen of culture AZ2 (CBS 144514) (preserved in liquid nitrogen) acquired from patient with pulmonary infection, initially diagnosed with coccidioidomycosis. Colonies on 2X-GYE 24°C after 2 weeks are white and flat, reaching 34 mm diameter, with a compact, cottony and margin irregular. This fungus is unable to grow at 37°C. No aerial mycelium and or hyphal tufts were observed. The reverse is pale yellow and sulcated. Transparent and water-like droplets are present on the surface of the colonies. Hyphal structures are hyaline, septated and irregularly branched. Narrow vegetative fertile hypha raises right-angled phialides and/or aphanophialides, usually verticillate, which are displayed either in pairs or solitary on a lateral stalk/stem. These morphological characteristics are commonly observed in Lecanicillium spp., as opposed to the zigzag structure of phialides on terminal hyphae from P. album. Conidia vary from elongated cylindrical to globose ranging from 1.71μm to 3.43μm (± 2.35) in diameter and lack pigmentation under the conditions tested.

Supplementary Material

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Figure S1. Thermal tolerance of the atypical clinical isolates evaluated at both 24 °C and 37 °C after seven days of growth. Macromorphology of these isolates include an irregular, undulated and raised colony-type morphology. The reverse side of the macrocolonies are pale yellow and wrinkled, presenting cavitations. These isolates were unable to grow at 37 °C.

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Figure S2. Molecular diversity of the Parengyodontium/Lecanicillium sp. complex based on the Maximum Likelihood phylogenetic analysis of the partial 5.8S, ITS 1 and ITS 2 sequences. The Parengyodontium sp. II clade is supported by bootstrap analysis, and represents the newly discovered species Parengyodontium americanum. The figure inset shows the geographic distributions of P. americanum and the origin of each taxon analyzed is displayed in the legend inset.

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Figure S3. Molecular systematics of Parengyodontium/Lecanicillium sp. complex deduced by the MLST phylogenetic scheme proposed by (Kepler et al. 2017) as follows: small and large subunits of ribosomal DNA genes (SSU and LSU), translation elongation factor 1 alpha (TEF) and the two largets subunits of RNA polymerase II (RPB1) and (RPB2). The Parengyodontium clade harbors the both P. album and the newly described species Parengyodontium americanum.

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Figure S4. Orthologous gene clustering representation by Venn diagrams among Clavicipitaceae species. Numbers within intersections represents orthologous shared between individual genomes.

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Figure S5. Biosynthetic clusters previously identified in other fungi identified with the AntiSMASH platform and conserved within P. americanum genomes as follows: A) equisetin; B) pestheic acid, emericellin, and trypacidin; and C) alternariol.

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Table S1. Accession numbers of analyzed isoaltes for P. americanum and related taxa collected at Genbank

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Table S2. Genomes evaluated in the current study and accession numbers for each individual project

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Table S3. Genome assembly statistics for the P. americanum isolates

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Table S4. Polymorphism analysis within P. americanum isolates

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Table S5. Transposable element profile of P. americanum genomes

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Table S6. Gene family expansion in P. americanum according to CAZy, Merops and Pfam protein classifications

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Table S7. Enriched GO categories in P. americanum

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Table S8. Secondary metabolite clusters identified in P. americanum and closely related Clavicipitaceae fungi. Blue cells represent enriched classes of secondary metabolites while red cells represents reduced ones

Highlights.

• A new Hypocreales fungal species Parengyodontium americanum is proposed

• P. americanum is phylogenetically related the opportunistic fungal pathogen P. album

• The genome of P. americanum was sequenced, assembled and annotated

• Comparative genomic studies of Hypocreales reveals unique metabolic profile

• Genomic and taxonomic tools should be used in concert with traditional morphological characteristics for fungal species discovery