Lindetal 2017

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Drivers of genetic diversity in secondary metabolic gene clusters within a fungal

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RESEARCH ARTICLE
Drivers of genetic diversity in secondary

metabolic gene clusters within a fungal
species
Abigail L. Lind1¤a, Jennifer H. Wisecaver2, Catarina Lameiras3, Philipp Wiemann4¤b,
Jonathan M. Palmer5, Nancy P. Keller4, Fernando Rodrigues6,7, Gustavo H. Goldman8,
Antonis Rokas1,2*
1 Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, Tennessee,
United States of America, 2 Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee,
a1111111111 United States of America, 3 Department of Microbiology, Portuguese Oncology Institute of Porto, Porto,
a1111111111 Portugal, 4 Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison,
Wisconsin, United States of America, 5 Center for Forest Mycology Research, Northern Research Station,
a1111111111
US Forest Service, Madison, Wisconsin, United States of America, 6 Life and Health Sciences Research
a1111111111 Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal, 7 ICVS/3B0 s - PT Government
a1111111111 Associate Laboratory, Braga/Guimarães, Portugal, 8 Faculdade de Ciências Farmacêuticas de Ribeirão
Preto, Universidade de São Paulo, São Paulo, Brazil
¤a Current address: Gladstone Institutes, San Francisco, California, United States of America
¤b Current address: Hexagon Bio, Menlo Park, California, United States of America
* [email protected]
OPEN ACCESS
Citation: Lind AL, Wisecaver JH, Lameiras C,

Wiemann P, Palmer JM, Keller NP, et al. (2017)
Drivers of genetic diversity in secondary metabolic Abstract
gene clusters within a fungal species. PLoS Biol
15(11): e2003583. https://doi.org/10.1371/journal. Filamentous fungi produce a diverse array of secondary metabolites (SMs) critical for
pbio.2003583 defense, virulence, and communication. The metabolic pathways that produce SMs are
Academic Editor: Sophien Kamoun, The Sainsbury found in contiguous gene clusters in fungal genomes, an atypical arrangement for metabolic
Laboratory, UNITED KINGDOM pathways in other eukaryotes. Comparative studies of filamentous fungal species have
Received: July 12, 2017 shown that SM gene clusters are often either highly divergent or uniquely present in one or a
handful of species, hampering efforts to determine the genetic basis and evolutionary driv-
Accepted: November 2, 2017
ers of SM gene cluster divergence. Here, we examined SM variation in 66 cosmopolitan
Published: November 17, 2017
strains of a single species, the opportunistic human pathogen Aspergillus fumigatus. Investi-
Copyright: This is an open access article, free of all gation of genome-wide within-species variation revealed 5 general types of variation in SM
copyright, and may be freely reproduced,
gene clusters: nonfunctional gene polymorphisms; gene gain and loss polymorphisms;
distributed, transmitted, modified, built upon, or
otherwise used by anyone for any lawful purpose. whole cluster gain and loss polymorphisms; allelic polymorphisms, in which different alleles
The work is made available under the Creative corresponded to distinct, nonhomologous clusters; and location polymorphisms, in which a
Commons CC0 public domain dedication. cluster was found to differ in its genomic location across strains. These polymorphisms
Data Availability Statement: Short-read affect the function of representative A. fumigatus SM gene clusters, such as those involved
sequences for the 8 strains sequenced in this study in the production of gliotoxin, fumigaclavine, and helvolic acid as well as the function of clus-
are available in the NCBI Sequence Read Archive
ters with undefined products. In addition to enabling the identification of polymorphisms, the
(SRA) under accession SRP109032 (https://trace.
ncbi.nlm.nih.gov/Traces/sra/?study=SRP109032). detection of which requires extensive genome-wide synteny conservation (e.g., mobile
All sequence alignments and phylogenies gene clusters and nonhomologous cluster alleles), our approach also implicated multiple
generated in this study are available on the underlying genetic drivers, including point mutations, recombination, and genomic deletion
Figshare repository (https://figshare.com/projects/
and insertion events as well as horizontal gene transfer from distant fungi. Finally, most of
Data_for_Drivers_of_genetic_diversity_in_
secondary_metabolic_gene_clusters_within_a_ the variants that we uncover within A. fumigatus have been previously hypothesized to
fungal_species_/26089).
PLOS Biology | https://doi.org/10.1371/journal.pbio.2003583 November 17, 2017 1 / 26

Population genomics of fungal secondary metabolic pathways
Funding: National Science Foundation (grant contribute to SM gene cluster diversity across entire fungal classes and phyla. We suggest
number DEB-1442113). Received by AR. U.S. that the drivers of genetic diversity operating within a fungal species shown here are suffi-
National Library of Medicine training grant (grant
number 2T15LM007450). Received by ALL.
cient to explain SM cluster macroevolutionary patterns.
Conselho Nacional de Desenvolvimento Cientı́fico e
573 Tecnológico. Northern Portugal Regional
Operational Programme (grant number NORTE-01-
0145-FEDER-000013). Received by FR. Fundação
de Amparo à Pesquisa do 572 Estado de São Author summary
Paulo. Received by GHG. National Institutes of
All organisms produce metabolites, which are small molecules important for growth,
Health (grant number R01 AI065728-01). Received
by NPK. National Science Foundation (grant
reproduction, and other essential functions. Some organisms, including fungi, plants, and
number IOS-1401682). Received by JHW. The bacteria, make specialized forms of metabolites known as “secondary” metabolites that
funders had no role in study design, data collection are ecologically important and improve their producers’ chances of survival and repro-
and analysis, decision to publish, or preparation of duction. In fungi, the genes in pathways that synthesize secondary metabolites are typi-
the manuscript. cally located next to each other in the genome and organized in contiguous gene clusters.
Competing interests: The authors have declared These gene clusters, along with the metabolites they produce, are highly distinct, even
that no competing interests exist. between otherwise similar fungi, and it is often difficult to reconstruct how these differ-
Abbreviations: ABC, ATP-binding cassette; HGT, ences evolved. To understand how secondary metabolic pathways evolve in fungi, we
horizontal gene transfer; HR, highly reducing; NR, compared secondary metabolic gene clusters in 66 strains of one species of filamentous
nonreducing; NRPS, nonribosomal peptide fungus, the human pathogen Aspergillus fumigatus. We show that these gene clusters vary
synthase; ORF, open reading frame; PKS, extensively within this species, and describe the genetic processes that cause these differ-
polyketide synthase; SM, secondary metabolite;
ences. We identify 5 types of variants: single nucleotide changes, gene and gene cluster
SNP, single nucleotide polymorphism; SNV, single
nucleotide variant; TE, transposable element. gain and loss, different gene clusters at the same genomic position, and mobile gene clus-
ters that “jump” around the genome. These results provide a road map to the types and
frequencies of genomic changes underlying the extensive diversity of fungal secondary
metabolites.
Introduction
Filamentous fungi produce a diverse array of small molecules that function as toxins, antibiot-
ics, and pigments [1]. Although by definition, these so-called specialized or secondary metabo-
lites (SMs) are not strictly necessary for growth and development, they are critical to the
lifestyle of filamentous fungi [2]. For example, antibiotic SMs give their fungal producers a
competitive edge in environments crowded with other microbes [3]. SMs can additionally
mediate communication between and within species as well as contribute to virulence on ani-
mal and plant hosts in pathogenic fungi [4,5].
A genomic hallmark of SMs in filamentous fungi is that the biosynthetic pathways that pro-
duce them are typically organized into contiguous gene clusters in the genome [6]. These gene
clusters contain the chemical backbone synthesis genes whose enzymatic products produce a
core metabolite, such as nonribosomal peptide synthases (NRPSs) and polyketide synthases
(PKSs), tailoring enzymes that chemically modify the metabolite, transporters involved in
product export, and, often, transcription factors that control the expression of the clustered
genes [6]. These gene clusters also occasionally contain resistance genes that confer self-protec-
tion against reactive or toxic metabolites [6]. Filamentous fungal genomes, particularly those
in the phylum Ascomycota [6], typically contain dozens of SM gene clusters. However, most
individual SM gene clusters appear to be either species specific or narrowly taxonomically dis-
tributed in only a handful of species [6,7]. SM gene clusters that are more broadly distributed
show discontinuous taxonomic distributions and are often highly divergent between species.
Consequently, the identity and total number of SM gene clusters can vary widely even between

very closely related species whose genomes exhibit very high sequence and synteny conserva-
tion [8,9].
In the last decade, several comparative studies have described macroevolutionary patterns
of SM gene cluster diversity. For example, studies centered on genomic comparisons of closely
related species have identified several different types of interspecies divergence, from single
nucleotide substitutions (e.g., differences in fumonisins produced by Fusarium species are
caused by variants in 1 gene [10]) to gene gain/loss events (e.g., the trichothecene gene clusters
in Fusarium species and the aflatoxin family SM gene clusters in Aspergillus species) [11–16]
and genomic rearrangements (e.g., the trichothecene gene clusters in Fusarium) [11]. Addi-
tionally, genetic and genomic comparisons across fungal orders and classes have identified sev-
eral instances of gene gain or loss [17–19] and horizontal gene transfer (HGT) [13,20–23]
acting on individual genes or on entire gene clusters, providing explanations for the diversity
and discontinuity of the taxonomic distribution of certain SM gene clusters across fungal
species.
Although interspecies comparative studies have substantially contributed to our under-
standing of SM diversity, the high levels of evolutionary divergence of SM clusters make infer-
ence of the genetic drivers of SM gene cluster evolution challenging; put simply, it has been
difficult to “catch” the mechanisms that generate SM gene cluster variation “in the act.” Several
previous studies have examined intraspecies or population-level differences in individual SM
gene clusters, typically focusing on the presence and frequency of nonfunctional alleles of clus-
ters involved in the production of mycotoxins. Examples of clusters exhibiting such polymor-
phisms include the gibberellin gene cluster in F. oxysporum [24], the fumonisin gene cluster in
F. fujikuroi [25], the aflatoxin and cyclopiazonic acid gene clusters in A. flavus [26], and the
bikaverin gene cluster in Botrytis cinerea [27]. While these studies have greatly advanced our
understanding of SM gene cluster genetic variation and highlighted the importance of within-
species analyses, studies examining the entirety of SM gene cluster polymorphisms within fun-
gal species are so far lacking. We currently do not know the types and frequency of SM gene
cluster polymorphisms within fungal species, whether these polymorphisms affect all types of
SM gene clusters, or the genetic drivers of SM gene cluster evolution.
To address these questions, we investigated the genetic diversity of all 36 known and pre-
dicted SM gene clusters in whole genome sequence data from 66 strains, 8 of which were
sequenced in this study, of the opportunistic human pathogen A. fumigatus, a species with cos-
mopolitan distribution and panmictic population structure [28]. We found that 13 SM gene
clusters were generally conserved and harbored low amounts of variation. In contrast, the
remaining 23 SM gene clusters were highly variable and contained 1 or more of 5 different
types of genetic variation: single nucleotide polymorphisms (SNPs), including nonsense and
frameshift variants, individual gene gain and loss polymorphisms, entire cluster gain and loss
polymorphisms, polymorphisms associated with changes in cluster genomic location, and
clusters with nonhomologous alleles resembling the idiomorphs of fungal mating loci. Many
clusters contained interesting combinations of these types of polymorphisms, such as pseudo-
genization in some strains and entire cluster loss in others. The types of variants we find are
likely generated by a combination of DNA replication and repair errors, recombination, geno-
mic insertions and deletions, and horizontal transfer. We additionally find an enrichment for
transposable elements (TEs) around horizontally transferred clusters, clusters that change in
genomic locations, and idiomorphic clusters. Taken together, our results provide a guide to
both the types of polymorphisms and the genetic drivers of SM gene cluster diversification in
filamentous fungi. As most of the genetic variants that we observe have been previously associ-
ated with SM gene cluster diversity across much larger evolutionary distances and timescales,

we argue that processes influencing SM gene cluster diversity within species are sufficient to
explain SM cluster macroevolutionary patterns.
Results
We analyzed the genomes of 66 globally distributed strains of A. fumigatus for polymorphisms
in SM gene clusters. We performed whole genome sequencing on 8 strains and collected the
remaining 58 strains from publicly available databases, including NCBI Genome and NCBI
Sequence Read Archive (Fig 1, S1 Table) [28–32]. All publicly available strains of A. fumigatus
with sequencing data passing quality thresholds (see Materials and methods) or with assem-
bled genomes were included in our analysis. The resulting dataset contains strains sampled
from 12 sites worldwide and from clinical and environmental sources (S1 Table).
We analyzed all strains for polymorphisms in 33 curated SM gene clusters present
in the reference Af293 genome and additionally searched for novel SM gene clusters
Fig 1. Genetic diversity of secondary metabolic gene clusters within a fungal species. The phylogeny was constructed
using 15,274 biallelic SNPs with no missing data. The tree is midpoint rooted and all branches with bootstrap support less than
80% are collapsed. This phylogeny does not include strains Af10, Af210, Z5, or RP-2014, as short-read data were not available.
Superfixes following strain names indicate publications associated with DNA sequencing. * indicates strains sequenced in this study,
† indicates strains sequenced at JCVI with no associated publication, and ‡ indicates strains sequenced by Rikenwith no associated
publication. Heat maps show presence, absence, and polymorphisms in SM gene clusters. Black indicates the cluster is present in a
strain with no polymorphisms, aside from missense variants, light gray indicates 1 or more genes in the cluster are pseudogenized,
and dark gray indicates the cluster is partially or entirely absent (see Fig 2). Colors for cluster 4 indicate which pseudogenizing
variants are present (see Fig 3) and colors for cluster 10 indicate which allele of the cluster is present (see Fig 4). Chromosomal
location of clusters 1 and 33 are indicated. If more than one type of polymorphism is present within a cluster in a strain, only 1 is
depicted. Types of polymorphisms found in each cluster are summarized below the cluster heat map. DHN, dihydroxynaphthalene;
JCVI, J. Craig Venter Institute; NRPS, nonribosomal peptide synthase; PKS, polyketide synthase; SM, secondary metabolite; SNP,
single nucleotide polymorphism.
https://doi.org/10.1371/journal.pbio.2003583.g001

Table 1. Types and rates of SM gene cluster variants in Aspergillus fumigatus strains.
Description Phenotype Drivers Frequency at cluster Frequency at Previous reports
level strain level
Single-nucleotide Potential for protein function DNA replication errors; 100% (33/33 clusters; Every strain Bikaverin in Botrytis [17,27],
polymorphisms and change (missense); relaxation of purifying missense); 70% (23/ affected aflatoxin in A. oryzae and A.
indels abrogation of protein selection 33 clusters; nonsense flavus [26], fumonisins in
function (nonsense and and frameshift) Fusarium [10], many others
frameshift)
Gene content Loss of gene cluster Deletion and insertion 6 clusters 27/66 strains Trichothecene in Fusarium,
polymorphisms function; structural changes events; recombination; aflatoxin and sterigmatocyst in
in the metabolite; change in transposable elements Aspergillus [11–15], HC toxin in
cluster expression or Cochliobolus carbonarum [33]
metabolite transport
Whole gene cluster Loss or gain of novel Deletion and insertion 6 clusters 13/66 strains Gibberellin and fumonisin in
polymorphisms metabolites events; horizontal gene Fusarium [24,25]
transfer; transposable
elements
Cluster idiomorphs Changes in metabolites Transposable elements; 1 gene cluster 8 unique Putative SM gene clusters in
produced or structure of recombination; other identified dermatophytes; putative SM
metabolites mechanisms? alleles gene cluster in A. flavus and A.
oryzae [34,35]
Mobile gene Potential for change in gene Transposable elements; 2 gene clusters 8/66 strains None
clusters regulation horizontal gene transfer;
other mechanisms?
Abbreviation: SM; secondary metabolite.
https://doi.org/10.1371/journal.pbio.2003583.t001
(see Materials and methods). These examinations revealed 5 distinct types of polymor-
phisms in SM gene clusters (Fig 1, Table 1):
1. SNPs and short indel polymorphisms. Thirty-three of 33 SM gene clusters (present in the
reference Af293 strain) contained multiple genes with missense SNPs and short indel vari-
ants in 1 or more strains. Twenty-three of 33 SM gene clusters contained 1 or more genes
with frameshift or nonsense variants.
2. Gene content polymorphisms involving loss or gain of 1 or more genes. Six of 33 SM gene
clusters contained a gene content polymorphism.
3. Whole SM gene cluster gain and loss polymorphisms. Three of 33 SM gene clusters were
entirely absent in 1 or more strains and an additional 3 previously unknown SM gene clus-
ters were discovered.
4. Idiomorphic polymorphisms. One locus contained multiple nonhomologous SM gene clus-
ter alleles in different strains.
5. Genomic location polymorphisms. Two of 33 SM gene clusters were found on different
chromosomes between strains.
Both genomic location polymorphisms and idiomorphic polymorphisms are novel types of
variants that have not been previously described for secondary metabolic gene clusters, likely
because they can only be identified when genome-wide synteny and sequence conservation
are high. The remaining types of variants, including single-nucleotide changes and gene gain
and loss events, have been implicated at the species level as major drivers of secondary meta-
bolic gene cluster evolution (Table 1), suggesting that the diversity-generating processes
observed within a species are sufficient to explain SM gene cluster evolution across species.

SNPs and indel polymorphisms

It is well established that SNPs and short indel polymorphisms, which are caused by errors in
DNA replication and repair, are a major source of genomic variation [36]. Nonsynonymous
SNPs and indels with missense, frameshift, and nonsense effects were widespread across the 33
SM reference gene clusters (Fig 1, S2 Table). Every strain contained numerous missense muta-
tions and at least 1 nonsense or frameshift mutation in its SM gene clusters. Although missense
mutations are likely to influence SM production, the functional effects of nonsense and frame-
shift mutations are comparatively easier to infer from genomic sequence data because they
often result in truncated proteins and loss of protein function.
SNPs and short indel polymorphisms can affect secondary metabolite production, as in the
case of the lack of trypacidin production in the A1163 strain because of a previously identified
frameshift mutation in the PKS of the trypacidin gene cluster [37]. Interestingly, we identified
a premature stop codon (Gln273 ) in a transcription factor required for trypacidin production,
tpcD (Afu4g14550), in a strain sequenced in this study (MO79587EXP) (S2 Table). These data
suggest that function of this SM gene cluster has been lost at least twice, independently, in A.
fumigatus.
Individual nonsense or frameshift variants varied in frequency. For example, the NRPS pes3
gene (Afu5g12730) in SM gene cluster 21 harbors 16 nonsense or frameshift polymorphisms
in 55 strains, 7 of which are common (present in 10 strains) and another 7 of which are rare
(5 strains). Strains with lab-mutated null alleles of the pes3 gene are more virulent than
strains with functional copies [38], which may explain the widespread occurrence of null pes3
alleles within A. fumigatus.
Gene content polymorphisms

We additionally identified several SM gene clusters that gained or lost genes in some strains.
These gene content polymorphisms were most likely generated through genomic deletion or
insertion events and were sometimes found at high frequencies among strains (Fig 1, Table 1).
In 3 cases, these polymorphisms impacted backbone synthesis genes, rendering the SM gene
cluster nonfunctional.
One example involves SM gene cluster 14, whose standard composition includes a pyover-
dine synthase gene, an NRPS-like gene, an NRPS backbone gene, and several additional modi-
fication genes (Fig 2A). Four of the 66 strains examined lack an 11-kb region on the 30 end of
the cluster, which normally contains an NRPS gene and 2 additional cluster genes, and the
first non-SM genes on the 30 end flanking the cluster. All A. fumigatus strains contain a copia
family TE [39,40] at the 30 end of the cluster, suggesting that TEs may have been involved in
the generation of this polymorphism. While this polymorphism could have arisen through a
deletion event, a homologous cluster lacking the 11-kb region is also present in the reference
genomes of A. lentulus and A. fischeri, close relatives of A. fumigatus (Fig 2A). The most parsi-
monious explanation is that the genome of the A. fumigatus ancestor contained an SM gene
cluster that lacked the 11-kb region and that this genomic region was subsequently gained and
increased in frequency within A. fumigatus.
The remaining 2 gene content polymorphisms affecting SM backbone genes were restricted
to 1 strain each and appear to have arisen through genomic deletion events. Specifically, strain
IF1SWF4 lacks an 8-kb region near the helvolic acid SM gene cluster, resulting in the loss of
the backbone oxidosqualene cyclase gene as well an upstream region containing 2 non-SM
genes (S1 Fig). Strain LMB35Aa lacks a 54-kb region on the end of chromosome 2, which
includes 5 genes from the telomere-proximal fumigaclavine C cluster (S1 Fig).

Fig 2. Gene gains and deletions in SM gene clusters. (A) Differences in gene content in SM gene cluster 14 in Aspergillus fumigatus strains and
closely related species. Four A. fumigatus strains lack an 11-kb region in this cluster, including an NRPS backbone gene, highlighted in yellow. Regions
upstream and downstream of this cluster are syntenic. LMB35Aa also contains a large inversion that moves a transcription factor, oxidoreductase, and
hypothetical protein 275 kb away from the cluster. A. fischeri and A. lentulus, close relatives of A. fumigatus, contain a cluster lacking the 11-kb region.
(B) SM gene cluster found in most A. fumigatus strains but absent from the Af293 reference and from the F7763 strain. EOS, end of scaffold; MFS,
major facilitator superfamily; NRPS, nonribosomal peptide synthase; SM, secondary metabolite.
Three other cases of gene content polymorphisms involved gene loss or truncation events
of non-backbone structural genes. The second half of the open reading frame (ORF) of the
gliM O-methyltransferase gene in the gliotoxin gene cluster has been lost in 2 of 66 strains (S1
Fig) and the first half of the permease fmqE in the fumiquinazoline gene cluster has been lost

in 4 of 66 strains (S1 Fig). Finally, an ATP-binding cassette (ABC) transporter gene in SM clus-
ter 21 has been almost entirely lost in 21 of 66 strains (S1 Fig). This deletion event is found in
strains that are related in the SNP-based strain phylogeny but does not perfectly mirror the
phylogeny (Fig 1).
Whole gene cluster loss polymorphisms

Several SM gene clusters were gained or lost entirely across strains. We observed several
instances in which a cluster present in the genome of either the reference Af293 or A1163 (also
known as CEA10) strain was absent or pseudogenized in other strains, which we present in
this section.
One of the novel SM gene clusters, cluster 34, was present in all but 2 of the strains (Af293
and F7763). Cluster 34 contains a PKS backbone gene, 1 PKS-like gene with a single PKS-asso-
ciated domain, 9 genes with putative biosynthetic functions involved in secondary metabolism,
and 6 hypothetical proteins (Fig 2B). The 2 strains that lack cluster 34 contain a likely nonfunc-
tional cluster fragment that includes the PKS-like gene, 2 biosynthetic genes, and 3 hypotheti-
cal proteins. Interestingly, the 30 region flanking cluster 34 is syntenic across all 66 strains but
the 50 region is not, suggesting that a recombination or deletion event may have resulted in its
loss in the Af293 and F7763 strains. These 2 strains form a clade in the strain phylogeny (Fig
1), so it is likely that this deletion or recombination event occurred once.
One notable example of an SM gene cluster present in the Af293 reference genome but
absent or pseudogenized in others was SM cluster 4. This cluster contains 5 genes on the tip of
the Af293 chromosome 1 and contains orthologs to 5 of the 6 genes in the fusarielin-producing
gene cluster in F. graminearum [41]. Cluster 4 is also present in several other Aspergillus spe-
cies, including A. clavatus and A. niger [41], as well as in whole or in part in other non-Asper-
gillus fungi in the class Eurotiomycetes and in fungi in the class Sordariomycetes (S3 Fig)
[30,42–50]. Phylogenetic analysis of the genes in cluster 4 does not provide a clear view of the
origin of this cluster, which is consistent either with extensive gene loss in both Sordariomy-
cetes and Eurotiomycetes or, alternatively, with HGT between fungi belonging to the 2 classes
(S2 and S3 Figs).
Cluster 4 is entirely absent in 4 of 66 strains, and its genes are undergoing pseudogenization
in an additional 43 strains via multiple independent mutational events (Fig 3). The 4 strains
lacking the cluster form a single clade on the strain phylogeny, suggesting that the cluster was
lost in a single deletion event (Fig 1). Further, 19 strains shared a single frameshift variant in
the PKS gene (4380_4381insAATGGGCT; frameshift at Glu1461 in Afu1g17740) and an addi-
tional 13 strains shared a single frameshift variant (242delG; frameshift at Gly81) in an aldose
1-epimerase gene (Afu1g17723) (Fig 3A, S2 Table). Eleven other strains each contained 1 to
several frameshift or nonsense polymorphisms involving 9 unique mutational sites. Five of
these strains contained multiple distinct frameshifts and premature stop codons in more than
1 gene in the cluster, indicating that the entire pathway is pseudogenized in these strains.
A phylogeny of the entire cluster 4 locus across all 62 strains with short-read data shows
that 2 pseudogenizing variants shared across multiple strains, one in the aldose 1-epimerase
gene and one in the PKS, are found in loci that form well-supported clades (Fig 3B), suggesting
that these variants arose once. Similarly, a set of variants shared across 3 strains and 1 variant
shared in 2 strains are found in loci that form well-supported clades in the locus phylogeny.
Two strains sharing a pseudogenizing variant in the PKS do not group together in the locus
phylogeny, a discordance likely stemming from within-locus recombination events. Finally,
functional alleles of cluster 4 are distributed throughout the locus phylogeny, suggesting that
the functional allele is ancestral and the pseudogenized variants are derived.

Fig 3. Pseudogenization in the fusarielin-like SM gene cluster. (A) Positions of frameshift variants and nonsense variants in the fusarielin-like SM
gene cluster 4. (B) Locus phylogeny of the fusarielin-like SM gene cluster based on a nucleotide alignment of the entire gene cluster, including
intergenic and noncoding regions. The phylogeny is midpoint rooted and branches with bootstrap support <80% are collapsed. Two branches were
shortened for visualization purposes. Strains with pseudogenizing variants are indicated with colored boxes. Colors correspond to variants shown in
(A). PKS, polyketide synthase; SM, secondary metabolite; SNV, single nucleotide variant.

Perhaps surprisingly, loss-of-function polymorphisms (from nonsense and frameshift

mutations to wholesale cluster loss) are common and sometimes frequent within A. fumigatus.
The majority of these polymorphisms are presumably neutral and reflect the fact that any
mutation is more likely to result in loss of a function than in gain. Consistent with this hypoth-
esis is our observation that these loss events were often found at low frequencies. However, the
possibility also exists that some of the high-frequency, recurrent loss-of-function polymor-
phisms may be adaptive. Given that many secondary metabolites are primarily secreted in the
extracellular environment and can benefit nearby conspecifics that are not themselves produc-
ing the metabolite [51], individual strains may be circumventing the energetically costly pro-
cess of producing the metabolite themselves in a situation analogous to the Black Queen
Hypothesis [52].
Whole gene cluster gain polymorphisms

By searching for novel SM gene clusters in the genomes of the other 65 A. fumigatus strains,
we found 3 SM gene clusters that were absent from the genome of the Af293 reference strain.
As SM gene clusters are often present in repeat-rich and subtelomeric regions that are chal-
lenging to assemble [53,54], the strains analyzed here might harbor additional novel SM gene
clusters that were not captured here.
One of these SM gene clusters, cluster 34, was mentioned earlier as an example of whole
gene cluster loss polymorphism (Fig 2B) and is present in most strains but has been lost in 2
strains. The other 2 SM gene clusters absent from the Af293 genome are present at lower fre-
quencies and likely reflect gene cluster gain events; cluster 35 is present in 2 of 66 strains and
cluster 36 in 4 of 66 strains. Cluster 35 is located in a region syntenic with an Af293 chromo-
some 4 region and is flanked on both sides by TEs (S4 Fig). Eight of the 14 genes in this SM
gene cluster are homologous to genes in an SM gene cluster in the genome of the insect patho-
genic fungus Metarhizium anisopliae (S4 Fig) [55]. Phylogenetic analysis of these 8 genes is
consistent with a horizontal transfer event (S5 Fig). The 2 strains that contain this novel cluster
are not sister to each other on the strain phylogeny (Fig 1).
Cluster 36 is an NRPS-containing cluster located on shorter genomic scaffolds that lack
homology to either the Af293 or A1163 genomes, making it impossible to determine on which
chromosome this cluster is located (S4 Fig). Two of the strains containing this novel cluster
are sister to each other on the strain phylogeny, while the third is distantly related to these 2
(Fig 1). The evolutionary histories of the genes in the cluster are consistent with vertical inheri-
tance, and these genes are present in multiple Aspergillus species.
Idiomorph polymorphisms
One of the most peculiar types of polymorphisms that we identified is a locus containing dif-
ferent unrelated alleles of SM gene clusters, reminiscent of the idiomorph alleles at the fungal
mating loci [56]. This locus, which resides on chromosome 3 and corresponds to cluster 10 in
the Af293 genome (Fig 4), was previously described as being strain specific in a comparison
between Af293 and A1163 strains [30] and is thought to reside in a recombination hot spot
[28]. Our analysis showed that there are at least 6 different alleles of this cluster in A. fumigatus
containing 4 different types of key enzymes involved in natural product biosynthesis: a
PKS-NRPS hybrid, a highly reducing (HR) PKS, a nonreducing (NR) PKS, and an NRPS-like
enzyme (Fig 4). Two additional alleles were present in only 1 strain each (S6 Fig).
In the Af293 reference genome, the cluster present at the idiomorph locus contains 1
NR-PKS along with an NRPS-like gene (allele B). In the A1163 reference genome and 17 other
strains, there is a PKS-NRPS and an HR-NRPS at this locus (allele E). These alleles show an

Fig 4. Six alleles of an idiomorphic SM gene cluster. (A) Alleles of SM gene cluster 10 on chromosome 3. Red boxes denote
transposable elements. Green arrows denote backbone genes (PKS or NRPS). (B) Locus phylogeny of conserved downstream of the
idiomorph cluster (highlighted in gray in [A]). Phylogeny was constructed using a 48-mb nucleotide alignment with the GTRGAMMA model
and midpoint rooted. Branches with bootstrap support <80% were collapsed. HR, highly reducing; NR, nonreducing; NRPS, nonribosomal
peptide synthase; PKS, polyketide synthase; SM, secondary metabolite.

almost complete lack of sequence similarity except for a conserved hypothetical protein and a
fragment of the HR-PKS in the Af293 allele; in contrast, the upstream and downstream flank-
ing regions of the 2 alleles, which do not contain any backbone genes, are syntenic. Remark-
ably, another allele, present in 12 strains, contains all of the genes from both the Af293 and
A1163 clusters (allele D). The remaining 3 alleles contain various combinations of these genes.
One allele found in 22 strains contains some A1163-specific genes, including the HR-PKS, and
no Af293-specific genes (allele F), while another allele found in 3 strains contains some
Af293-specific genes, including the NRPS-like gene, but no A1163 genes (allele A). The final
allele, present in 8 strains, contains the entire Af293 allele as well as part of the A1163 allele
containing the HR-PKS (allele C). Every allele is littered with multiple long terminal repeat
sequence fragments from gypsy and copia TE families as well as with sequence fragments from
DNA transposons from the mariner family [39]. In some cases, these TEs correspond with
break points in synteny between alleles, suggesting that the diverse alleles of this SM gene clus-
ter may have arisen via TE-driven recombination. Furthermore, both of the alleles that are
restricted to a single strain have an insertion event of several genes near a TE, while the rest of
the locus is highly similar to one of the more common alleles (S6 Fig).
Untargeted XCMS analysis [57] of an allele D strain (08-19-02-30) and 2 allele F strains
(08-12-12-13 and 08-19-02-10) and comparison of their metabolite profiles revealed the pres-
ence of 2 unique masses in 08-19-02-30 (S4 Table; S7 Fig), raising the possibility that variation
at the idiomorph locus is functional. Further analysis is underway to investigate whether any
of these mass to charge ratios can be directly linked to the allele D sequence.
To gain insight into the evolutionary history of this locus, we constructed a phylogeny
based on its conserved downstream flanking region (Fig 4B). The resulting phylogeny shows
some grouping of strains that share alleles, but there are no clades that contain all instances of
a particular allele. This is likely to be the consequence of within-locus recombination between
strains of A. fumigatus, which has been previously described at this locus [28] and which is
potentially driven by the high number of repetitive sequences at this locus.
While it is tempting to speculate that allele D, the longest allele containing all observed
genes, represents the ancestral state, this does not explain the presence of a shared hypothetical
protein and PKS gene fragment between allele C and allele B. Furthermore, 2 close relatives of
A. fumigatus, A. lentulus and A. fischeri, contain a similar region with conserved upstream and
downstream flanking genes that is highly dissimilar to any of the alleles observed in A. fumiga-
tus (S8 Fig). In both species, this locus contains numerous TEs as well as genes homologous to
portions of allele E in A. fumigatus (S8 Fig). A. fischeri additionally contains 2 hypothetical pro-
teins from the PKS-NRPS region of A. fumigatus and an additional hybrid PKS-NRPS-con-
taining gene cluster not found in either A. lentulus or any A. fumigatus strain (S8 Fig). Other
genes at this locus in both A. lentulus and A. fischeri have functions likely not related to SM.
Interestingly, A. lentulus contains a gene with a heterokaryon incompatibility protein domain,
which may be involved in determining vegetative incompatibility [58]. Only 1 representative
genome from each species has been sequenced, but based on the high concentration of TEs
and lack of sequence similarity with any A. fumigatus alleles, it is likely that this locus is highly
variable within both A. lentulus and A. fischeri.
It is possible that polymorphism at this locus originated via SM gene cluster fusion or fis-
sion events driven by TEs, which are present in large numbers. Interestingly, 2 other previ-
ously described instances of SM gene cluster variation bear some resemblance to the A.
fumigatus idiomorphic SM gene cluster 10 locus. The first is the presence of 2 nonhomologous
A. flavus alleles, for which some strains contain a 9-gene sesquiterpene-like SM gene cluster
and others contain a nonhomologous 6-gene SM gene cluster at the same genomic location
[35]. The second is the presence of 2 nonhomologous SM gene clusters at the same well-

conserved locus in a comparison of 6 species of dermatophyte fungi [34]. Based on these

results, we hypothesize that idiomorphic clusters may be common in fungal populations and
contribute to the broad diversity of SM gene clusters across filamentous fungi.
Genomic location polymorphisms

The final type of polymorphism that we observed is associated with SM gene clusters that are
found in different genomic locations in different strains, suggesting that these SM gene clusters
are behaving like mobile genetic elements. This type of polymorphism was observed in SM
gene clusters 1 and 33, both of which produce as-yet-identified products and are present at
low frequencies in A. fumigatus strains.
SM gene cluster 1, which is present in 6 strains at 3 different genomic locations (Fig 5A),
consists of a PKS and 4 other structural genes that are always flanked by a 15-kb region
(upstream) and a 43-kb region (downstream) containing TEs. In the reference Af293 strain
and in strain F7763, cluster 1 and its flanking regions are located on chromosome 1, while in
strains 08-31-08-91, F13619, and Z5 they are located between Afu4g07320 and Afu4g07340 on
chromosome 4. In contrast, in strain JCM10253, the cluster and flanking regions are located
on chromosome 8 immediately adjacent to the 30 end of the intertwined fumagillin and pseur-
otin SM gene supercluster [59]. The strains containing the allele on chromosome 1 are sister to
each other on the strain phylogeny, while the other strains are scattered across the tree and do
not reflect the phylogeny (Fig 1).
In 5 of 6 strains, cluster 1 appears to be functional and does not contain nonsense SNPs or
indels. However, the cluster found on chromosome 1 in strain F7763 contains 2 stop codons
in the oxidoreductase gene (Gln121 and Gln220 ) and 2 premature stop codons in the PKS
(Gln1156 and Gln1542 ), suggesting this strain contains a null allele.
This “jumping” gene cluster is not present in any other sequenced genome in the genus
Aspergillus, and phylogenetic analysis of its constituent genes is consistent with HGT between
fungi (S9 Fig). Specifically, this gene cluster is also present in Phaeosphaeria nodorum [60], a
plant pathogen from the class Dothideomycetes, Pseudogymnoascus pannorum [61], a fungus
isolated from permafrost from the Leotiomycetes, and Escovopsis weberi [62], a fungal parasite
of fungus-growing ants from the Sordariomycetes (Fig 5B). One additional species, the endo-
phyte Hypoxylon sp. CI4A from the class Sordariomycetes [63], contains 4 of the 5 cluster
genes but is missing Afu1g00970, an MFS drug transporter. However, this species contains a
gene unrelated to Afu1g00970 that is annotated as an MFS drug transporter immediately adja-
cent to this cluster (Fig 5B). None of these fungi contain the upstream or downstream TE-rich
flanking regions present in A. fumigatus, and each fungus contains additional unique genes
with putative biosynthetic functions adjacent to the transferred cluster. The most likely expla-
nation for this change in flanking regions is that this SM gene cluster was transferred into A.
fumigatus once and has subsequently “jumped” in different genomic locations in different
strains.
The second SM gene cluster that shows variation in its genomic location across strains,
cluster 33, contains a terpene synthase. This cluster is present in only 5 strains at 3 distinct
locations (Fig 5C). Similar to cluster 1, cluster 33 is also flanked by TEs, and in 1 strain the
cluster is located in a new region 58 Kb from SM gene cluster 34. Two strains that contain the
cluster in the same genomic location are sister to each other on the strain phylogeny, while the
placement of the other 3 strains containing the cluster does not reflect the phylogeny (Fig 1).
In contrast to cluster 1, cluster 33 does not appear to have been horizontally transferred
between fungi and its genes are present in other sequenced Aspergillus species [64], suggesting
that the mobility of clusters 1 and 33 may be driven by different mechanisms.

Fig 5. Multiple genomic locations of 2 SM gene clusters. (A) SM gene cluster 1 (Afu1g00970-01010) and flanking region are
found in different genomic locations. The flanking regions contain transposon-derived open reading frames, including 2 putative
reverse transcriptases. In one strain, SM gene cluster 1 is found adjacent to SM gene cluster 30. (B) Synteny of A. fumigatus SM
gene cluster 1 with clusters in Phaeosphaeria nodorum, Pseudogymnoascus pannorum, Escovopsis weberi, and Hypoxylon sp.
CI4A. All species contain nonsyntenic genes predicted by antiSMASH to be part of a biosynthetic gene cluster. (C) SM gene cluster
33 (Afu5g00100-00135) is found in different genomic locations in different strains. In one strain, the cluster is adjacent to SM gene
cluster 34. Multiple transposable elements flank the cluster in each strain. EOS, end of scaffold; FAD, flavin adenine dinucleotide;
MFS, major facilitator superfamily; ORF, open reading frame; PKS, polyketide synthase; SM, secondary metabolite.

Interestingly, both cases of mobile gene clusters are located near or immediately adjacent to
other SM gene clusters in some strains. Cluster 33 is located 58 kb away from cluster 34 in one
strain, and cluster 1 is located immediately adjacent to the intertwined fumagillin and pseuro-
tin supercluster [59] in another. This supercluster is regulated by the transcriptional factor
fapR (Afu8g00420) and is located in a chromosomal region controlled by the master SM regu-
lators laeA (Afu1g14660) and veA (Afu1g12490) [59,65], raising the hypothesis that mobile
gene clusters might be co-opting the regulatory machinery acting on adjacent SM gene clus-
ters. Previous work has hypothesized that the fumagillin and pseurotin supercluster formed
through genomic rearrangement events, placing the once-independent gene clusters in close
proximity to each other [59]. Our observation that the mobile cluster 1 is located in this same
region not only supports this hypothesis but also implicates TEs as one of the mechanisms by
which superclusters are formed. These superclusters may also represent an intermediate stage
in the formation of new SM gene clusters. Supercluster formation, potentially mediated by
mobile gene clusters and followed by gene loss, could explain macroevolutionary patterns of
SM gene clusters in which clustered genes in one species are found to be dispersed over multi-
ple gene clusters in other species [9,11].
Discussion
Our examination of the genomes of 66 strains of A. fumigatus revealed 5 general types of poly-
morphisms that describe variation in SM gene clusters. These polymorphisms include varia-
tion in SNPs and short indels, gene and gene cluster gains and losses, nonhomologous
(idiomorph) gene clusters at the same genomic position, and mobile clusters that differ in
their genomic location across strains (Fig 6). Previous work has demonstrated that SM gene
clusters, like the metabolites that they produce, are highly divergent between fungal species
[8,9,19,64]. Our examination of genome-wide variation shows that these SM gene clusters are
also diverse across strains of a single fungal species. These results also demonstrate that the
diversity of SM gene clusters within A. fumigatus cannot be captured by sequencing a single
representative strain, which is the current standard practice for determining the SM gene clus-
ter content of a fungal species.
The quantification of diversity in SM gene clusters within a species is dependent on both
numbers and types of strains analyzed. The types of polymorphisms detected as well as their
observed frequency, especially for rare polymorphisms, will increase with the number of
genomes examined. In addition, both the frequencies of the different types of polymorphisms
and the polymorphisms themselves may also change with sampling design or in a manner cor-
responding to the population structure or ecology of the species under study. A. fumigatus is a
cosmopolitan species with panmictic population structure [28], characteristics that do not
always apply to other filamentous fungi. Fungi exhibiting strong population structure or fungi
adapted to different ecological niches might contain different patterns of genetic diversity.
Nevertheless, the variants and genetic drivers we observe at the within-species level are
also implicated as driving SM gene cluster variation at the between-species level, suggesting
that the observed microevolutionary processes are sufficient to explain macroevolutionary
patterns of SM gene cluster evolution. For example, the narrow and discontinuous distribu-
tion of SM gene clusters across the fungal phylogeny has been attributed to HGT as well as
to gene cluster loss [13,15,20,22,30,66–68]. Here, we find evidence that both processes also
influence the distribution of SM gene clusters within a species (Figs 2 and 5, S2–S5 Figs).
Interestingly, the fraction of SM gene clusters within A. fumigatus that harbor loss of func-
tion polymorphisms is substantial, consistent with the macroevolutionary view that SM
gene cluster loss is rampant [18,19,68]. However, our within-species observations are also

Fig 6. Types and frequencies of all SM gene cluster variants within A. fumigatus. SM, secondary metabolite.
consistent with the macroevolutionary importance of HGT to SM gene cluster evolution.

Once thought to be nonexistent in eukaryotes, HGT is now considered to be responsible for
the presence of several different SM gene clusters in diverse filamentous fungi [13,68,69].
The instances of HGT of SM gene clusters within A. fumigatus suggests that acquisition of
foreign genetic material containing SM gene clusters is likely a common and ongoing occur-
rence in fungal populations.
One recurring theme across different types of SM gene cluster polymorphisms in A. fumiga-
tus was the perpetual presence of TEs adjacent to or within clusters. One particularly striking
case is the “idiomorphic” cluster 10, in which TEs seem to correspond with break points in
synteny both within A. fumigatus and also between A. fumigatus and its close relatives (Fig 4,
S8 Fig). TEs were also present flanking mobile and horizontally transferred SM gene clusters
and were located adjacent to gene gain sites. There are several potential explanations for the
observed TE enrichment. First, TE presence may promote repeat-driven recombination and
gene rearrangement, or the TEs themselves may be the agents of horizontally transferred clus-
ters (either on their own or through a viral vector). Alternatively, it may simply be the case that
SM gene clusters preferentially reside in TE-rich genomic regions.
In summary, examination of SM gene cluster variation within a single fungal species
revealed 5 distinct types of polymorphism that are widespread across different types of SM

gene clusters and are caused by many underlying genetic drivers, including errors in DNA
transcription and repair, nonhomologous recombination, gene duplication and loss, and
HGT. The net effect of the observed variation raises the hypothesis that the chemical products
of filamentous fungal species are in a state of evolutionary flux, each population constantly
altering its SM gene cluster repertoire and consequently modifying its chemodiversity.
Materials and methods

Strains analyzed
Eight strains of A. fumigatus were isolated from 4 patients with recurrent cases of aspergillosis
in the Portuguese Oncology Institute in Porto, Portugal. Each strain was determined to be A.
fumigatus using macroscopic features of the culture and microscopic morphology observed in
the slide preparation from the colonies with lactophenol solution [70]. Based on the morpho-
logical characterization, all clinical strains were classified as A. fumigatus complex-Fumigati.
After whole genome sequencing, retrieval and examination of the beta tubulin and calmodulin
sequences of each strain confirmed that all strains belonged to A. fumigatus (see Phylogenetic
analysis and S9 Fig). The genomes of all 8 strains were sequenced using 150-bp Illumina
paired-end sequence reads at the Genomic Services Lab of Hudson Alpha (Huntsville, Ala-
bama, USA). Genomic libraries were constructed with the Illumina TruSeq library kit and
sequenced on an Illumina HiSeq 2500 sequencer. Samples of all 8 strains were sequenced at
greater than 180X coverage or depth (S1 Table). Short-read sequences for these 8 strains are
available in the NCBI Sequence Read Archive (SRA) under accession SRP109032 (https://
trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP109032).
In addition to the 8 strains sequenced in this study, we retrieved 58 A. fumigatus strains
with publicly available whole genome sequencing data, resulting in a dataset of 66 strains (S1
Table). The strains used included both environmental and clinical strains and were isolated
from multiple continents. Genome assemblies for 10 of these strains, including the Af293 and
A1163 reference strains, were available for download from GenBank [28–32,71]. For 6 of these
strains, short-read sequences were also available from the NCBI SRA, which were used for var-
iant discovery only (see Single nucleotide variant [SNV] and indel discovery) and not for
genome assembly. Short-read sequences were not available for the remaining 4 strains. Short-
read sequences were downloaded for an additional 48 strains from the NCBI SRA if they were
sequenced with paired-end reads and at greater than 30X coverage.
Single nucleotide variant (SNV) and indel discovery

All strains with available short-read data (62 of 66 strains) were aligned to both the Af293 and
A1163 reference genomes using BWA mem version 0.7.12-r1044 [72]. Coverage of genes
present in the reference genome was calculated using bedtools v2.25.0 [73]. SNV and indel dis-
covery and genotyping were performed relative to the Af293 reference genome and were con-
ducted across all samples simultaneously using the Genome Analysis Toolkit version 3.5-0-
g36282e4 with recommended hard filtering parameters [74–76] and annotated using snpEff
version 4.2 [77].
De novo genome assembly and gene annotation

All 56 strains without publicly available genome assemblies were de novo assembled using
the iWGS pipeline [78]. Specifically, all strains were assembled using SPAdes v3.6.2 and
MaSuRCA v3.1.3 and resulting assemblies were evaluated using QUAST v3.2 [79–81]. The
average N50 of assemblies constructed with this strategy was 463 kb (S1 Table). Genes were

annotated in these assemblies as well as in 5 GenBank assemblies with no predicted genes

using augustus v3.2.2 trained on A. fumigatus gene models [82]. Repetitive elements were
annotated in all assemblies using RepeatMasker version open-4.0.6 [83].
Secondary metabolic gene cluster annotation and discovery

Secondary metabolic gene clusters in the Af293 reference genome were taken from 2 recent
reviews, both of which considered computational and experimental data to delineate cluster
boundaries [84,85] (S3 Table). The genomes of the other 65 strains were scanned for novel SM
gene clusters using antiSMASH v3.0.5.1 [86]. To prevent potential assembly errors from con-
founding the analysis, any inference about changes in genomic locations of genes or gene clus-
ters was additionally verified by manually inspecting alignments and ensuring that paired end
reads supported an alternative genomic location (see Single nucleotide variant [SNV] and
indel discovery). Cases in which paired end reads did not support the change in genomic loca-
tion (i.e., all 30 read mapping to chromosome 1 and all 50 pairs mapping to chromosome 8) or
mapping was ambiguous or low quality were discarded.
Phylogenetic analysis
To confirm all strains in this analysis belonged to the species A. fumigatus, the genomic
sequences of the beta tubulin and calmodulin genes were extracted from the assembled
genomes of all strains. Gene phylogenies were constructed using A. fischerianus as an out-
group using RAxML v8.0.25 with the GTRGAMMA substitution model [87]. The tree was
midpoint rooted and all branches with bootstrap support less than 80% were collapsed (S10
Fig).
To construct an SNP-based strain phylogeny, biallelic SNPs with no missing data were
pruned using SNPRelate v1.8.0 with a linkage disequilibrium threshold of 0.8 [88]. A total of
15,274 SNVs were used to create a phylogeny using RAxML v8.0.25 with the ASC_BIN-
GAMMA substitution model [87]. The tree was midpoint rooted and all branches with boot-
strap support less than 80% were collapsed. The phylogeny was visualized using ITOL version
3.0 [89].
To understand the evolutionary histories of specific SM gene clusters showing unusual tax-
onomic distributions, we reconstructed the phylogenetic trees of their SM genes. Specifically,
SM cluster protein sequences were queried against a local copy of the NCBI nonredundant
protein database (downloaded May 30, 2017) using phmmer, a member of the HMMER3 soft-
ware suite [90], using acceleration parameters—F1 1e-5—F2 1e-7—F3 1e-10. A custom perl
script sorted the phmmer results based on the normalized bitscore (nbs), in which nbs was cal-
culated as the bitscore of the single best-scoring domain in the hit sequence divided by the best
bitscore possible for the query sequence (i.e., the bitscore of the query aligned to itself). No
more than 5 hits were retained for each unique NCBI Taxonomy ID. Full-length proteins cor-
responding to the top 100 hits (E-value < 1 × 10 − 10) to each query sequence were extracted
from the local database using esl-sfetch [90]. Sequences were aligned with MAFFT v7.310
using the E-INS-i strategy and the BLOSUM30 amino acid scoring matrix [91] and trimmed
with trimAL v1.4.rev15 using its gappyout strategy [92]. The topologies were inferred using
maximum likelihood, as implemented in RAxML v8.2.9 [87], using empirically determined
substitution models and rapid bootstrapping (1,000 replications). The phylogenies were mid-
point rooted and branches with less than 80% bootstrap support were collapsed using the ape
and phangorn R packages [93,94]. Phylogenies were visualized using ITOL version 3.0 [89].
To understand the evolutionary histories of SM gene clusters 4 and 10, full-length nucleo-
tide sequences of all 62 strains with short-read sequence data were extracted for the entire

cluster region (SM gene cluster 4) or the downstream flanking region (SM gene cluster 10)
using the previously described SNV analysis procedure followed by Genome Analysis Toolkit’s
“ExtractAlternativeReferenceFasta” tool [75]. The resulting nucleotide sequences were aligned
using MAFFT v7.310 [91]. Phylogenies were constructed using maximum likelihood as imple-
mented in RAxML v 8.0.25, using the GTRGAMMA substitution model and rapid bootstrap-
ping (1,000 replications) [87]. Phylogenies were midpoint rooted and branches with less than
80% bootstrap support were collapsed. Phylogenies were visualized using ITOL version 3.0
[89].
All sequence alignments and phylogenies generated in this study are available on the Fig-
share repository (https://figshare.com/projects/Data_for_Drivers_of_genetic_diversity_in_
secondary_metabolic_gene_clusters_within_a_fungal_species_/26089).
Differential metabolite analysis

For natural product analysis, 5 × 106 spores/mL for the indicated strains were grown in 50 mL
liquid GMM [95] for 5 days at 25˚C and 250 rpm in duplicates. Supernatants were extracted
with equal volumes of ethyl acetate, dried down and resuspended in 20% acetonitrile (ACN).
Each sample was analyzed by ultra high-performance liquid chromatography (UHPLC)
coupled with mass spectrometry (MS). The samples were separated on a ZORBAX Eclipse
XDB-C18 column (Agilent, 2.1 × 150 mm with a 1.8 μM particle size) using a binary gradient
of 0.5% (v/v) formic acid (FA) as solvent A and 0.5% (v/v) FA in ACN as solvent B that was
delivered by a VanquishTM UHPLC system (Thermo Scientific) with a flow rate of 0.2 mL/
min. The binary gradient started with 20% B that was increased with a linear gradient to 100%
B in 15 min followed by an isocratic step at 100% B for 5 min. Before every run, the system was
equilibrated for 5 min at 20% B. The UHPLC system was coupled to a Q Exactive hybrid quad-
rupole OritrapTM MS (Thermo Scientific). For electrospray ionization, the ion voltage was set
at ±3.5 kV in positive and negative mode. Nitrogen was used as sheath gas at a flow rate of 45
and as sweep gas at a flow rate of 2. Data analysis was performed using XCMS [57] and Maven
[96] software.
Supporting information
S1 Fig. Alignments showing deletion of genes in SM gene clusters. (A) Deletion of helvolic
acid genes in IF1SWF4. (B) Deletion of fumigaclavine genes in LMB35Aa. (C) Partial deletion
of gliM in the gliotoxin gene cluster in 2 strains. (D) Partial deletion of fmqE in the fumiquina-
zoline gene cluster in 3 strains. (E,F) Coverage of 21 strains with partial deletion of ABC trans-
porter gene in SM gene cluster 21. ABC, ATP-binding cassette; SM, secondary metabolite.
(PDF)
S2 Fig. Gene phylogenies of the fusarielin-like SM gene cluster 4. These phylogenies are con-
sistent with horizontal transfer between Eurotiomycete and Sordariomycete fungi or with
extensive gene loss. SM, secondary metabolite.
(PDF)
S3 Fig. Fusarielin-like clusters in Eurotiomycetes and Sordariomycetes. All species with
genes grouping together with each Aspergillus fumigatus gene from the fusarielin-like cluster
(see S2 Fig). Beauveria bassiana and A. udagawae were excluded, as they only contained the
transcription factor from the cluster. The gene cluster in Fusarium graminearum has been
functionally characterized as producing fusarielin.
(PDF)

S4 Fig. Novel SM gene clusters in Aspergillus fumigatus strains. (A) Synteny between a
novel PKS-containing cluster in 2 strains with an SM gene cluster in Metarhizium anisopliae.
This novel PKS cluster is located between transposable elements in a region syntenic with
the reference Af293 chromosome 4. (B) Novel SM gene cluster in MO54056EXP and 3 addi-
tional strains. This cluster is only located on 1 scaffold in MO540556EXP and is fragmented
across the other strains (ends of scaffolds are marked). (C) Coverage data from short-read
alignments for MO54056EXP, 12–7504462, and 08-19-02-30 relative to the MO54056EXP
scaffold containing the novel SM gene cluster. PKS, polyketide synthase; SM, secondary
metabolite.
(PDF)
S5 Fig. Gene phylogenies of SM gene cluster 24. The phylogenies of several genes in this clus-
ter are consistent with horizontal transfer between Aspergillus fumigatus and Metarhizium
fungi. SM, secondary metabolite.
(PDF)
S6 Fig. Two alleles of the idiomorphic SM gene cluster 10 present in 1 strain each. (A)
This allele contains an insertion of genes from chromosome 6 immediately upstream of allele
C (see main text Fig 4). None of these genes is likely an SM gene cluster backbone gene. An
additional transposable element is found flanking this insertion. (B) This allele contains an
insertion of genes present in the A1163 reference but not in the Af293 reference in the middle
of allele A (see main text Fig 4). None of these genes is likely an SM gene cluster backbone
gene. One additional transposable element is contained in this insertion. SM, secondary
metabolite.
(PDF)
S7 Fig. Metabolomics analysis of strains with different alleles of the idiomorphic cluster
indicates the presence of different metabolites. Extracted ion chromatograms for the 2 mass
to charge ratios identified in negative mode from XCMS analysis comparing extracts from
strains with alleles D and F.
(PDF)
S8 Fig. Idiomorph locus in other species. Structure of the idiomorph locus in (A) Aspergillus
lentulus and (B) A. fischeri and homology with A. fumigatus allele E (main text Fig 4). Green
arrows denote backbone biosynthetic genes and red boxes denote transposable elements as
detected by RepeatMasker. A. fischeri contains a novel SM gene cluster not found in A. fumiga-
tus strains. Other genes at this locus have various functions that may not be related to second-
ary metabolism. A. lentulus contains 1 gene with a heterokaryon incompatibility domain,
which may play a role in vegetative incompatibility. SM, secondary metabolite.
(PDF)
S9 Fig. Gene phylogenies of the mobile SM gene cluster 1. These phylogenies are consistent
with horizontal transfer between Eurotiomycete, Dothidiomycete, Leotiomycete, and Sordar-
iomycete fungi. SM, secondary metabolite.
(PDF)
S10 Fig. Marker gene phylogenies of all strains and Aspergillus fischeri. (A) Phylogeny of
beta tubulin gene. (B) Phylogeny of calmodulin gene.
(PDF)
S1 Table. Aspergillus fumigatus strain information.
(XLSX)

S2 Table. Nonsynonymous variants in Aspergillus fumigatus strains.

(XLSX)
S3 Table. Secondary metabolic gene clusters in Aspergillus fumigatus Af293.
(XLSX)
S4 Table. Metabolite analysis.
(XLSX)
Acknowledgments
This work was conducted in part using the resources of the Advanced Computing Center for
Research and Education at Vanderbilt University (http://www.accre.vanderbilt.edu/).
Author Contributions
Conceptualization: Abigail L. Lind, Gustavo H. Goldman, Antonis Rokas.
Data curation: Abigail L. Lind.
Formal analysis: Abigail L. Lind.
Funding acquisition: Abigail L. Lind, Jennifer H. Wisecaver, Nancy P. Keller, Gustavo H.
Goldman, Antonis Rokas.
Investigation: Abigail L. Lind.
Methodology: Abigail L. Lind, Jennifer H. Wisecaver, Catarina Lameiras, Philipp Wiemann,
Jonathan M. Palmer, Nancy P. Keller.
Resources: Jennifer H. Wisecaver, Catarina Lameiras, Nancy P. Keller, Fernando Rodrigues,
Gustavo H. Goldman.
Supervision: Antonis Rokas.
Visualization: Abigail L. Lind, Jennifer H. Wisecaver, Philipp Wiemann.
Writing – original draft: Abigail L. Lind, Antonis Rokas.
Writing – review & editing: Abigail L. Lind, Jennifer H. Wisecaver, Philipp Wiemann, Jona-
than M. Palmer, Nancy P. Keller, Gustavo H. Goldman, Antonis Rokas.
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Drivers of genetic diversity in secondary metabolic gene clusters within a fungal

Preprint · October 2017

Abigail Lind Catarina Lameiras

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Philipp Wiemann Jonathan Mark Palmer

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infection biology View project

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Drivers of genetic diversity in secondary

Citation: Lind AL, Wisecaver JH, Lameiras C,

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Abbreviation: SM; secondary metabolite.

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SNPs and indel polymorphisms

Gene content polymorphisms

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Whole gene cluster loss polymorphisms

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Perhaps surprisingly, loss-of-function polymorphisms (from nonsense and frameshift

Whole gene cluster gain polymorphisms

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conserved locus in a comparison of 6 species of dermatophyte fungi [34]. Based on these

Genomic location polymorphisms

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consistent with the macroevolutionary importance of HGT to SM gene cluster evolution.

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Materials and methods

Single nucleotide variant (SNV) and indel discovery

De novo genome assembly and gene annotation

PLOS Biology | https://doi.org/10.1371/journal.pbio.2003583 November 17, 2017 17 / 26

annotated in these assemblies as well as in 5 GenBank assemblies with no predicted genes

Secondary metabolic gene cluster annotation and discovery

PLOS Biology | https://doi.org/10.1371/journal.pbio.2003583 November 17, 2017 18 / 26

Differential metabolite analysis

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S2 Table. Nonsynonymous variants in Aspergillus fumigatus strains.

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