Mitochondrial genomes reveal recombination in the presumed asexual Fusarium oxysporum species complex

Abstract

Background

The Fusarium oxysporum species complex (FOSC) contains several phylogenetic lineages. Phylogenetic studies identified two to three major clades within the FOSC. The mitochondrial sequences are highly informative phylogenetic markers, but have been mostly neglected due to technical difficulties.

Results

A total of 61 complete mitogenomes of FOSC strains were de novo assembled and annotated. Length variations and intron patterns support the separation of three phylogenetic species. The variable region of the mitogenome that is typical for the genus Fusarium shows two new variants in the FOSC. The variant typical for Fusarium is found in members of all three clades, while variant 2 is found in clades 2 and 3 and variant 3 only in clade 2. The extended set of loci analyzed using a new implementation of the genealogical concordance species recognition method support the identification of three phylogenetic species within the FOSC. Comparative analysis of the mitogenomes in the FOSC revealed ongoing mitochondrial recombination within, but not between phylogenetic species.

Conclusions

The recombination indicates the presence of a parasexual cycle in F. oxysporum. The obstacles hindering the usage of the mitogenomes are resolved by using next generation sequencing and selective genome assemblers, such as GRAbB. Complete mitogenome sequences offer a stable basis and reference point for phylogenetic and population genetic studies.

Full text

Brankovics et al. BMC Genomics (2017) 18:735
DOI 10.1186/s12864-017-4116-5
RESEARCH ARTICLE Open Access
Mitochondrial genomes reveal
recombination in the presumed asexual
Fusarium oxysporum species complex
Balázs Brankovics1,2* , Peter van Dam3, Martijn Rep3,G.SybrendeHoog
1,2,TheoA.J.vanderLee
4,
Cees Waalwijk4and Anne D. van Diepeningen1,4
Abstract
Background: The Fusarium oxysporum species complex (FOSC) contains several phylogenetic lineages. Phylogenetic
studies identified two to three major clades within the FOSC. The mitochondrial sequences are highly informative
phylogenetic markers, but have been mostly neglected due to technical difficulties.
Results: A total of 61 complete mitogenomes of FOSC strains were de novo assembled and annotated. Length
variations and intron patterns support the separation of three phylogenetic species. The variable region of the
mitogenome that is typical for the genus Fusarium shows two new variants in the FOSC. The variant typical for
Fusarium is found in members of all three clades, while variant 2 is found in clades 2 and 3 and variant 3 only in
clade 2. The extended set of loci analyzed using a new implementation of the genealogical concordance species
recognition method support the identification of three phylogenetic species within the FOSC. Comparative analysis of
the mitogenomes in the FOSC revealed ongoing mitochondrial recombination within, but not between phylogenetic
species.
Conclusions: The recombination indicates the presence of a parasexual cycle in F. oxysporum. The obstacles
hindering the usage of the mitogenomes are resolved by using next generation sequencing and selective genome
assemblers, such as GRAbB. Complete mitogenome sequences offer a stable basis and reference point for
phylogenetic and population genetic studies.
Keywords: Comparative genomics, Mitochondrial genome, Mitochondrial recombination, Phylogenomics
Background
Members of the Fusarium oxysporum species complex
(FOSC) are important plant pathogens causing vascular
wilts, rots and damping-off on a broad range of agronom-
ically and horticulturally important crops [1, 2]. In addi-
tion, members of this species complex are also clinically
important, causing infections in both human and animal
hosts [3, 4]. Furthermore, despite the pathogenic poten-
tial of these fungi, not all strains are virulent. Putative
*Correspondence: b.brankovics@westerdijkinstitute.nl
1Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584CT Utrecht, The
Netherlands
2Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam,
Science Park 904, 1098 XH Amsterdam, The Netherlands
Full list of author information is available at the end of the article
non-pathogenic strains belonging to this group are used
as biocontrol agents against pathogens [5].
The taxonomy of Fusarium has historically been
based on the morphology of the asexual reproductive
structures, leading to a broad definition of F. o x ys -
porum. To capture intraspecific variability within the
morphospecies, formae speciales (ff. spp.) were intro-
duced based on the pathogenicity of the strains towards
particular plant hosts [6]. F. o x y sp o r um is closely related
to the F. f u j i k uro i species complex that contains several
heterothallic species. The genome of F. o xys p o rum resem-
bles the genome of heterothallic species, both mating
types can be found in populations, the mating type genes
are expressed and introns are correctly spliced from the
transcript [7]. However, since no sexual stage has been
found for this species nor could it be induced under
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Brankovics et al. BMC Genomics (2017) 18:735 Page 2 of 14
laboratory conditions, F. o x ysp o r um is considered to be
asexual [8, 9].
The use of molecular markers revealed that multiple
ff. spp. have evolved polyphyletically [1, 10, 11]. Phyloge-
netic studies by O’Donnell et al. [8, 10] identified three
major clades within the FOSC. However, later phyloge-
netic analysis conducted using genealogical concordance
phylogenetic species recognition (GCPSR) based on eight
loci supported the separation of only two phylogenetic
species within the complex: one species corresponding to
clade 1 and the other to the remaining clades [12].
The operational criteria for GCPSR for fungi were intro-
duced in the theoretical paper of Taylor et al. [13], and first
implemented as an operational framework using a two-
step methodology by Dettman et al. [14]. After genetic
isolation, two populations (or species) undergo the follow-
ing stages: shared polymorphism (apparent polyphyly),
loss of shared polymorphism (after fixation in one of
the species) and reciprocal monophyly (after fixation in
both species). According to GCPSR, the genetic isola-
tion between populations (phylogenetic species) can be
detected by a combination of genealogical concordance
and non-discordance. Genealogical concordance is meant
by the concordant reciprocal monophyly of multiple gene
genealogies. Genealogical non-discordance means that no
grouping supported by high support values for one of the
genes is contradicted by another gene with the same level
of support [13, 14].
Mitogenome sequences were used for resolving phylo-
genetic and evolutionary relationships between fungi at
all taxonomic levels [15–17]. The advances in sequencing
technologies and the reduction of costs involved, as well
as, the development of tools to selectively assemble target
genomic regions, like GRAbB, made it feasible to conduct
complete mitochondrial genome (mitogenome) sequence
analysis of a large number of strains [18].
The mitochondrial genomes of Fusarium spp. contain
a set of fourteen “standard” mitochondrial polypeptide-
encoding genes, two rRNA-encoding genes, rnl (mtLSU)
and rns (mtSSU), and more than twenty tRNA-encoding
genes [19]. The orientation and order of the genes are con-
served within the genus, with the exception of some of the
tRNA-encoding genes [17]. The mitochondrial genomes
of Fusarium spp. contain variable numbers of introns,
which causes significant size variation between the differ-
ent species [17, 19]. One of the introns is located in the
rnl gene and encodes a small ribosomal protein Rps3. This
intron is conserved in the Pezizomycotina [20].
In addition to the genes with functional predictions, a
large open reading frame (ORF) was found in all Fusarium
spp. except in F. oxy s p or u m (represented by strain F11)
[17, 19, 21]. This large ORF was found in a region that
is variable and contains several tRNA genes. For these
reasons the region was referred to as large variable (LV)
region and the large ORF with unknown function as
LV-uORF [19] (orf2229 in Fig. 1). The mitogenome of
the representative strain for F. ox y s p or u m was sequenced
using Sanger sequencing and primer walking [21]. This
sequence did not contained the LV-uORF, and this is
the only F. o x ysp o r um mitogenome that was used for
comparative studies so far [17, 19, 21]. Although several
F. ox y spor u m strains have been sequenced using next gen-
eration sequencing (NGS) methods, there are only two
strains for which the complete mitogenome was assem-
bled. Both of these mitogenome sequences, GenBank
accession no. KR952337 (unpublished) and LT571433 [18]
(Fig. 1), contain the large variable region with the large
variable ORF.
The goals of this study were (i) to prove that the
mitochondrial genomes of a large number of strains can
be analyzed, as we suggested in an earlier study [18],
(ii) to present a detailed analysis of these mitochondrial
genomes and (iii) to demonstrate how a detailed analysis
of these genomes can contribute to our understanding of
the biology of the given organism. To achieve these goals
we present the following in this study. First, the phyloge-
netic relationships between 61 strains of the F. o x y sp o r um
species complex is analyzed. For this, we used a revised
implementation of the GCPSR. Second, a detailed analy-
sis of the genetic diversity of the mitochondrial genomes
within the complex is given. Third, an interpretation of the
mitogenome diversity in the light of the phylogeny is pro-
vided. Lastly, the biological implications of our results are
discussed.
Methods
Fungal strains used and whole genome sequencing of the
strains
Sixty-one F. o x y spo r u m strains, two F. proliferatum strains
and one F. commune strain were analyzed in this study
(see Additional file 1). The F. commune strain (JCM 11502)
was previously identified as F. o xy s p oru m , but BLAST
and Fusarium-MLST (http://www.westerdijkinstitute.nl/
fusarium/) results showed that this strain belongs to
F. commune.
The F. o x y spo r u m f. sp. cumini strain F11, kindly pro-
vided by Dimitrios Tsirogiannis (Benaki Phytopathologi-
cal Institute, Kifissia, Greece), was used for re-sequencing.
For the two F. proliferatum strains (ITEM2287 and
ITEM2400), the two F. o x y spo r um f. sp. dianthi strains
(Fod001 and Fod008) and F. ox ys p o rum f. sp. cumini
strain F11 shotgun libraries were made using the Illumina
TruSeq nano DNA library prep kit, according to manu-
facturer’s protocols (Illumina). Each of the libraries was
loaded as (part of ) one lane of an Illumina paired-end
flowcell for cluster generation using a cBot. Sequencing
was done on an Illumina HiSeq2000 instrument using
101, 7, 101 flow cycles for forward, index and reverse
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Brankovics et al. BMC Genomics (2017) 18:735 Page 3 of 14
Fig. 1 Mitochondrial genome of Fusarium oxysporum f. sp. cubense race 4 (strain B2; LT571433). Green blocks: tRNA coding genes, blue arrows:
genes, yellow arrows: protein coding sequences, red arrows: rDNA coding sequence, purple arrows: intron encoded homing endonuclease genes,
gray segment: large variable (LV) region with orf2229 (LV-uORF)
reads respectively. De-multiplexing of resulting data was
done using Casava 1.8 software. The sequencing data has
been deposited into the European Nucleotide Archive
(ENA) with the following accession numbers: PRJEB18591
(ITEM2287 and ITEM2400), PRJEB18594 (F11) and
PRJEB18595 (Fod001 and Fod008) (see Additional file 1).
The remaining strains were sequenced by other research
groups or as part of previous studies [22–24]. The
sequencing reads for these strains were retrieved through
NCBI’s Sequence Read Archive (see Additional file 1).
Assembling sequences
The following nuclear protein coding genes were assem-
bled from NGS data for all 64 strains: γ-actin (act), ATP
citrate lyase (acl1), β-tubulin II (tub2), calmodulin (cal),
nitrate reductase (NIR), phosphate permease (PHO), 60S
ribosomal protein L10 (rpl10a), the largest and second
largest subunit of DNA-dependent RNA polymerase II
(RPB1 and RPB2, respectively), translation elongation fac-
tor 1α(tef1a), translation elongation factor 3 (tef3)and
topoisomerase I (top1). Besides the nuclear protein coding
genes, part of the mitochondrial SSU rRNA gene (mtSSU
or rns), the complete nuclear rDNA repeat region (18S
rRNA - ITS1 - 5.8S rRNA - ITS2 - 28S rRNA - IGS) and
the complete mitochondrial genome were also assembled
for all 64 strains.
The eight loci used by Laurence et al. [12] for genealog-
ical concordance phylogenetic species recognition were
only barcoding regions of the following genes: acl1,tub2,
cal,NIR,PHO,RPB1,RPB2 and tef1a.Asetofeightcom-
plete protein coding genes were used in this study, which
have been traditionally used as barcoding genes or have
recently been suggested as such [25]: act,tub2,cal,rpl10a,
RPB2,tef1a,tef3 and top1.
All of the regions listed above were assembled from
NGS reads using GRAbB (Genomic Region Assembly
by Baiting; [18]) by specifying the appropriate reference
sequence and employing SPAdes 3.6 [26, 27] as assem-
bler. The assembled sequences have been uploaded to
the European Nucleotide Archive under the following
accession numbers: LT841199-LT841268 and LT905535-
LT906358.
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Brankovics et al. BMC Genomics (2017) 18:735 Page 4 of 14
Sequence annotation
The initial mitogenome annotations were done using
MFannot (http://megasun.bch.umontreal.ca/cgi-bin/mfannot/
mfannotInterface.pl) and were manually curated: annota-
tion of tRNA genes was performed using tRNAscan-SE
[28], annotation of protein-coding genes and the rnl
gene was corrected by aligning intronless homologs to
the genome. Intron encoded proteins were identified
using NCBI’s ORF Finder (https://www.ncbi.nlm.nih.gov/
orffinder/) and annotated using InterPro [29] and CD-
Search [30]. The visualization of the annotated sequences
was produced using CLC Sequence Viewer 7.7.1 (https://
www.qiagenbioinformatics.com/products/clc-sequence-
viewer/), the graphical output was subsequently manually
adjusted.
Sequence analysis
All sequence alignments were done using MUSCLE
[31, 32]. The barcoding regions and genes were each
aligned per locus. The alignment of the mitochondrial
genome required a different approach. The mitochondrial
genome sequences were split into two regions: the large
variable region (between the trnT(tgt) and the nad2 genes)
and remaining part of the mitochondrial genome, which
we refer to as the conserved part of the mitochondrial
genome. Since the conserved part of the mitogenome and
thevariantsofthelargevariableregionweretoolong
forMUSCLEtohandleinasinglerun,weusedthefol-
lowing approach. First, the given sequences were divided
into non-overlapping homologous blocks (5000–7000 bp).
Second, each of these blocks were aligned using MUSCLE.
Finally, the individual aligned blocks were concatenated in
the original order.
Sequence variability of each region was calculated by
aligning the sequences, then the number of characters
with multiple character states was calculated and divided
by the total number of characters in the alignment. This
step was done using fasta_variability from the fasta_tools
package (https://github.com/b-brankovics/fasta_tools).
Phylogenetic analysis
The most appropriate substitution evolution model was
determined using jModelTest 2 [33] for each of the
single locus data sets. Phylogenetic reconstruction has
been conducted using MrBayes 3.2.5 [34]. The MCMC
algorithm was run for 4,000,000 generations with four
incrementally-heated chains, starting from random trees
and sampling one out every 400 generations. Burn-in was
set to a relative value, 0.25.
Majority-rule consensus (MRC) trees were calculated
based on the trees generated by the MrBayes run using
PAUP* 4.0a147 for Unix/Linux with “percent” set to 95
(corresponding to 0.95 posterior probability), using the
two F. proliferatum strains and the F. commune strain as
outgroups and rooting was set to “monophyl”.
Genealogical concordance phylogenetic species
recognition
Genealogical concordance phylogenetic species recogni-
tion (GCPSR) was implemented in two steps: (i) iden-
tifying independent evolutionary lineages (IELs) and
(ii) exhaustive subdivision of strains into phylogenetic
species. These were implemented using Perl scripts devel-
oped in house that are available at GitHub (https://github.
com/b-brankovics/GCPSR). The GCPSR method applied
in this study is a modified implementation of GCPSR
sensu Dettman et al. [14]. The revised implementation
of the GCPSR method is briefly described below (for
a detailed description as well as recommendations see
Additional file 2).
Identifying independent evolutionary lineages
Independent evolutionary lineages (IELs) have two crite-
ria: concordance and non-discordance. In our analysis, a
clade was considered concordant when it was supported
by at least two single locus MRC phylogenies (for explana-
tion on alternative settings, see Additional file 2). Clades
“A”and“B”arediscordantifA∩B=∅(they have common
elements) and neither one is a subset of the other. In our
implementation, we used these two criteria in a sequential
order: First, identifying concordant clades, then compar-
ing concordant clades and removing those that were dis-
cordant with each other. The concordant clades obtained
in this manner define an unambiguous tree topology. This
tree can be visualized and the number of loci supporting
each clade can be used as a support value for the given
clade. This tree can be used for exhaustive subdivision.
Exhaustive subdivision and phylogenetic species recognition
After identifying IELs, the IELs that are supported by a
large number of single locus genealogies (e.g. half of the
loci), are considered as putative phylogenetic species, the
rest of the IELs is removed (see Figure S2b in Additional
file 2). Each isolate must be classified within a putative
phylogenetic species. When an isolate is grouped within
a given clade (putative phylogenetic species), then all sub-
clades of the given clade are removed (see Figure S2c
in Additional file 2). This is referred to as exhaustive
subdivision, which ensures that all phylogenetic species
are monophyletic and there are no paraphyletic species.
The clades that are kept after this step are recognized as
phylogenetic species.
Results
Phylogenetic analysis and GCPSR
Sixty-one strains belonging to Fusarium oxysporum
species complex, one F. commune strain and two F.
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Brankovics et al. BMC Genomics (2017) 18:735 Page 5 of 14
proliferatum strains have been analyzed in this study.
Applying genealogical concordance phylogenetic species
recognition (GCPSR) on the eight single copy nuclear
(complete) protein coding genes (act,cal,RPB2,rpl10a,
tef1a,tef3,top1 and tub2) resulted in the recognition of
three phylogenetic species within the FOSC. All three
clades were present in at least half of the single locus
phylogenies with high support (BPP 0.95). Half of the
single locus phylogenies still supported the three species
when the rDNA repeat region and the conserved part
of the mitogenome were added besides the eight genes
(Fig. 2). The five loci that supported the recognition of all
three clades with high support were among the six most
variable loci included in the analysis (Table 2). The phy-
logeny of the conserved part of the mitogenome also sup-
ported the recognition of the three phylogenetic species
that correspond to clades 1, 2 and 3 sensu O’Donnell
et al. [10, 35].
In comparison, GCPSR based on the eight partial loci
(acl1,cal,NIR,PHO,RPB1,RPB2,tef1a and tub2)used
by Laurence et al. [12] had insufficient support to recog-
nize the three phylogenetic species within the FOSC. Only
tef1a showed high support for all three clades (data not
shown).
Mitogenomes
The mitogenomes of all 64 strains (61 FOSC and the
3 outgroup strains) were successfully assembled into
single contigs each showing circular topology (Fig. 3).
Some mitogenomes had identical sequences, in total there
were 42 unique haplotypes identified for mitochondrial
genome in this data set of 64 strains (Table 1).
Intron presence
All of the strains analyzed contained an intron in the rnl
gene (mtLSU) that is conserved in the Pezizomycotina
and contains a gene coding for a ribosomal protein, Rps3.
The two F. proliferatum mitogenomes contained no fur-
ther introns. The F. commune mitogenome contained an
intron in the nad1 gene, which was not found in the
other strains. The mitogenomes of the FOSC strains con-
tained intron positions in the following protein coding
genes: atp6,cob (this gene had two intron positions) and
nad5 (Table 1). The members of clade 2 could be differ-
entiated from all other strains based on their intron pat-
terns. Intron patterns showed almost no variation within
clades 2 and 3, the only exception being strain FOSC3-
a in clade 3. Clade 1 showed marked variation in intron
pattern.
Genetic diversity of the conserved part of the mitogenome
The mitogenomes of Fusarium spp. contain a region
that shows higher levels of variation than other parts of
the mitogenome [19]. This region is referred to as the
Fig. 2 Genealogical concordance phylogenetic species recognition
based on the 10 loci data set. The 10 loci used in the analysis were:
act,tub2,cal,tef1a,tef3,rpl10a,rpb2,top1, rDNA repeat and the
conserved part of the mitogenome. Only clades that were highly
supported (BPP 0.95) in at least two single locus phylogenies were
included in the analysis. The three clades within the FOSC were
recognized as phylogenetic species and shown in the tree. The
support values indicate how many single locus phylogenies
supported the given clade with BPP 0.95 out of the 10 loci
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Brankovics et al. BMC Genomics (2017) 18:735 Page 6 of 14
Fig. 3 Mitochondrial genome of Fusarium oxysporum f. sp. cumini strain F11. Green blocks: tRNA coding genes, blue arrows: genes, yellow arrows:
protein coding sequences, red arrows: rDNA coding sequence, purple arrows: intron encoded homing endonuclease genes, gray segment: large
variable (LV) region with orf2285 (LV-uORF)
largevariable(LV)regionanditislocatedbetweenrnl
(mitochondrial LSU rRNA gene) and nad2 (gray area in
Fig. 3). In this paragraph, we present the analysis of the
genetic diversity of the mitochondrial sequences located
outside the LV region, the analysis of the LV region can be
found in the next subsection.
Strains that belong to clades 2 and 3 had clearly differ-
ent lengths of the conserved region of the mitogenome.
This is partially due to the difference in intron patterns,
but even after these introns were excluded, the two popu-
lations, clades 2 and 3, had significantly different lengths,
33492.32 ±23.95 and 33688.68 ±14.42, respectively
(p-value <2.2 ∗10−16 according to two-sample t-test;
Table 1 and Additional file 3). The significant difference
between lengths of the conserved part of mitogenome
suggests that these two populations have been genetically
isolated. The variation observed in clade 1 was larger than
in the two other clades. There were only 5 strains within
clade 1, thus, further grouping of the strains based on
mitogenome length within this clade would not produce
statistically supported results.
Large variable region
Re-sequencing of F. o x ysp o r um f. sp. cumini strain F11
revealed that the LV-uORF (orf2285) gene, typical for
Fusarium spp., is present in the mitogenome of this strain
at the expected position, within the large variable (LV)
region located between rnl and nad2 (Fig. 3).
The LV region has three variants in the FOSC, which do
not appear to be homologous (Fig. 4). Although the three
variants contained tRNA genes with identical anticodon
sequences in a similar order (Fig. 4), there is a high level
of sequence variation between the three variants. BLAST
was unable to identify homology between the tRNA genes
of the different variants of the LV region. The variant
that is typical for Fusarium spp., variant 1, was 13424 or
13428 bp long in the F. proliferatum strains, 12422 bp in
the F. commune strain and 9833-12003 bp long in FOSC.
This variant was present in all three major clades of the
FOSC. Variant 2 of the LV region was 15816-16749 bp
long and it was present only in clades 2 and 3 within the
FOSC. Finally, variant 3 was 4570 or 5777 bp long and it
was found only in clade 2.
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Brankovics et al. BMC Genomics (2017) 18:735 Page 7 of 14
Table 1 Mitochondrial genome lengths and intron presence. The strains of clades 2 and 3 are ordered based on the corrected
mitochondrial genome length
Strain Species forma specialis Clade Introns LV mt length Correcteda
nad5 cob(1) cob(2) atp6 variant (bp) (bp)
Foc011 F. oxysporum f.sp. cucumerinum 1 yes yes - - 1 46476 33615
Foc013 F. oxysporum f.sp. cucumerinum 1 yes yes - - 1 46476 33615
Fov24500 F. oxysporum f.sp. vasinfectum 1 yes yes yes yes 1 48054 33727
B2 F. oxysporum f.sp. cubense 1 yes yes yes yes 1 49697 33419
II5 F. oxysporum f.sp. cubense 1 yes yes yes yes 1 49692 33414
Foc021 F. oxysporum f.sp. cucumerinum 2 yes - - - 2 50783 33449
Foc018 F. oxysporum f.sp. cucumerinum 2 yes - - - 2 50783 33449
Foc030 F. oxysporum f.sp. cucumerinum 2 yes - - - 2 50783 33449
NRRL25433 F. oxysporum f.sp. vasinfectum 2 yes - - - 1 44941 33458
N2 F. oxysporum f.sp. cubense 2 yes - - - 1 45639 33465
Foc035 F. oxysporum f.sp. cucumerinum 2 yes - - - 1 45706 33477
Fon002 F. oxysporum f.sp. niveum 2 yes - - - 1 45677 33477
Foc037 F. oxysporum f.sp. cucumerinum 2 yes - - - 1 45705 33479
Foc015 F. oxysporum f.sp. cucumerinum 2 yes - - - 1 45712 33486
Fom016 F. oxysporum f.sp. melonis 2 yes - - - 1 45538 33487
Fom013 F. oxysporum f.sp. melonis 2 yes - - - 1 45538 33487
Fom005 F. oxysporum f.sp. melonis 2 yes - - - 1 45538 33487
Fom012 F. oxysporum f.sp. melonis 2 yes - - - 1 45538 33487
NRRL54005 F. oxysporum f.sp. raphani 2 yes - - - 1 45536 33487
Fom004 F. oxysporum f.sp. melonis 2 yes - - - 1 45538 33487
Fom006 F. oxysporum f.sp. melonis 2yes - - - 1 45538 33487
NRRL37622 F. oxysporum f.sp. pisi 2 yes - - - 3 39941 33490
Fom009 F. oxysporum f.sp. melonis 2 yes - - - 3 38736 33492
Fom011 F. oxysporum f.sp. melonis 2 yes - - - 3 38736 33492
Fom010 F. oxysporum f.sp. melonis 2 yes - - - 3 38736 33492
Fod008 F. oxysporum f.sp. dianthi 2 yes - - - 1 45694 33498
Fod001 F. oxysporum f.sp. dianthi 2 yes - - - 1 44947 33498
Foc001 F. oxysporum f.sp. cucumerinum 2 yes - - - 1 45741 33509
Fon015 F. oxysporum f.sp. niveum 2 yes - - - 1 45708 33514
Fon019 F. oxysporum f.sp. niveum 2 yes - - - 2 50087 33514
Fon020 F. oxysporum f.sp. niveum 2 yes - - - 1 45752 33526
Fon005 F. oxysporum f.sp. niveum 2 yes - - - 1 45752 33526
Fon010 F. oxysporum f.sp. niveum 2 yes - - - 1 45752 33526
Fon021 F. oxysporum f.sp. niveum 2 yes - - - 1 45752 33526
Fon013 F. oxysporum f.sp. niveum 2 yes - - - 2 50795 33527
NRRL54008 F. oxysporum f.sp. conglutinans 2 yes - - - 2 50023 33534
Forc031 F. oxysporum f.sp. radicis-cucumerinum 3 yes yes - - 1 47541 33667
DF023 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52324 33667
Fo47 F. oxysporum 3 yes yes - - 1 47547 33667
Forc024 F. oxysporum f.sp. radicis-cucumerinum 3 yes yes - - 1 47541 33667
Forc016 F. oxysporum f.sp. radicis-cucumerinum 3 yes yes - - 1 47541 33667
NRRL54003 F. oxysporum f.sp. lycopersici 3 yes yes - - 1 47588 33674
UASWS AC1 F. oxysporum 3 yes yes - - 1 47598 33682
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Brankovics et al. BMC Genomics (2017) 18:735 Page 8 of 14
Table 1 Mitochondrial genome lengths and intron presence. The strains of clades 2 and 3 are ordered based on the corrected
mitochondrial genome length (Continued)
Strain Species forma specialis Clade Introns LV mt length Correcteda
nad5 cob(1) cob(2) atp6 variant (bp) (bp)
DF038 F. oxysporum f.sp. lycopersici 3 yes yes - - 1 47562 33688
DF062 F. oxysporum f.sp. lycopersici 3 yes yes - - 1 47562 33688
Fol016 F. oxysporum f.sp. lycopersici 3 yes yes - - 1 47562 33688
NRRL26406 F. oxysporum f.sp. melonis 3 yes yes - - 1 47444 33688
Fol004 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52352 33694
Fol014 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52352 33694
Fol4287 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52352 33694
Fol038 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52352 33694
DF041 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52352 33694
Fol018 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52352 33694
Fol002 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52352 33694
Fol026 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52352 33694
DF040 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52352 33694
Fol029 F. oxysporum f.sp. lycopersici 3 yes yes - - 2 52352 33694
MN14 F. oxysporum 3 yes yes - - 1 47573 33696
F11 F. oxysporum f.sp. cumini 3 yes yes - - 1 47409 33704
NRRL26381 F. oxysporum f.sp. radicis-lycopersici 3 yes yes - - 1 47263 33707
FOSC3-a F. oxysporum 3 yes yes yes - 2 53639 33727
JCM11502 F. commune - yes - - - 1 47526 33565
ITEM2287 F. proliferatum - - - - - 1 46555 33558
ITEM2400 F. proliferatum - - - - - 1 46549 33549
aThe length of the conserved part of the mitogenome after excluding the introns of protein coding genes
a)
b)
c)
Fig. 4 The three variants of the large variable region. aVariant 1 represented by F. oxysporum strain Fon015, bvariant 2 represented by F. oxysporum
strain FOSC3-a and cvariant 3 represented by F. oxysporum strain NRRL37622. Green blocks: tRNA coding genes, blue arrows: ORFs, yellow arrows:
ORFs that are not present in all representatives of the given variant
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Brankovics et al. BMC Genomics (2017) 18:735 Page 9 of 14
Variant 1 of the LV region
This variant contained thirteen tRNA genes and the LV-
uORF located between trnL(taa) and trnA( tgc) (Fig. 4).
LV-uORF has no known function. Domain predictions
returned low quality hits to a limited part of the pro-
tein sequence (see Additional file 4). Blast searches against
the NCBI and UniProt databases returned hits against
only Fusarium LV-uORF sequences. The GC-content of
LV-uORF was higher than the average of that of con-
served protein coding genes (both exonic and intronic
regions). This higher GC-content was similar to that of the
intergenic regions (see Additional file 4).
Variant 2 of the LV region
This variant contained fifteen tRNA genes, thirteen of
which are also found in variant 1 and two additional
tRNAs: trnG(acc) and trnL(aag). This region contains
eleven or twelve ORFs interleaved with the tRNA genes
(Fig. 4). Most of the variant 2 sequences contain twelve
ORFs, but strains Fon019 and NRRL54008 have lost the
ORF found between the second copy of trnM(cat) and
trnA(tgc),duetoadeletion.
Most of the ORFs have no functional prediction or
returned no hits using BLAST. Four ORFs appeared to
be homing endonuclease genes (HEGs) based on con-
served domain matches: two ORFs, the one between the
first and second trnM(cat) (orf536 in Fig. 4) and the
one between trnL(taa) and trnF(gaa) (orf323 in Fig. 4),
matched to LAGLIDADG endonucleases; the two ORFs
between trnL(tag) and trnQ( ttg) gene (orf274 and orf240
in Fig. 4) matched to the GIY-YIG endonucleases.
Variant 3 of the LV region
This variant contained a set of thirteen tRNA genes
that were also present in the other two variants. Strains
Fom009, Fom010 and Fom011 contained an additional
trnQ(ttg) gene. Besides the tRNA genes there were two
ORFs present in all four strains containing this variant,
while strain NRRL37622 contained an additional ORF
(Fig. 4).
The two ORFs present in all strains with variant 3 of the
LV region showed similarity to HEGs based on conserved
domain search results, one belonging to the LAGLIDADG
family and one to the GIY-YIG family. The putative LAGL-
IDADG HEG was located between trnT(tgt) and trnE(ttc)
gene. The second ORF, between trnL(tag) and trnQ(ttg)
gene, was a putative homolog of the GIY-YIG endonu-
clease gene upstream of trnQ(ttg) in variant 2, based on
BLASTp results.
F. ox y spor u m f.sp. pisi strain NRRL37622 contains a
third ORF, which returned partial hits to a hypotheti-
cal protein in Rhizophagus irregularis using BLASTp, and
CD-Search (Conserved Domain Search [30]) showed sim-
ilarity to a zinc-binding domain (zf-3CxxC). This ORF is
located at a homologous position to the second trnQ(ttg)
gene present in the other three strains possessing variant 3
of the large variable region (Fig. 4).
LV region phylogeny and conserved mitogenome phylogeny
In the phylogenetic tree based on variant 1 of the large
variable region, strain Fov24500 grouped with members
of clade 3, although this strain belonged to clade 1 based
on other markers (data not shown). Sequence compari-
son between the LV-uORF of strain Fov24500 and other
strains possessing the homologous region revealed that
there is a large deletion in the copy found in Fov24500
(see Additional file 4). Since this deletion was unique to
this strain, the phylogenetic reconstruction was also done
after removing the LV-uORF region from the alignment of
variant 1 sequences. The resulting tree and the phyloge-
netic tree of variant 2 are congruent with the phylogenetic
tree based on the conserved part of the mitochondrial
genome (presented as a tanglegram in Fig. 5 to high-
light the co-evolution of these sequences). The topological
incongruence is due to the differences in the resolution
power of the regions. The only topological incongru-
ence beyond the difference in resolution is the location
of strain Fon015 within clade 2 of the FOSC. Fon015 was
grouped with strains Fon019, Fom009, Fom010, Fom011
and NRRL54008 in the tree based on the conserved part of
the mitogenome, while it appeared separate from all other
clade 2 isolates in the tree based on variant 1 of the LV
region.
Highly similar conserved region irrespective of different
variants of the LV region
Sequence similarity of the conserved part of the mito-
chondrial genome was greater between members of the
same clade than between strains that shared the same vari-
ant of the LV region, but belonging to different clades.
The sequences of the conserved part of the mitogenome
of F. ox y spor u m f. sp. niveum strains Fon015 and Fon019
from clade 2 were completely identical, despite the fact
that they contain variants 1 and 2, respectively. A simi-
lar example was found in clade 3, where a high sequence
similarity was observed between the conserved part of
the mitogenome of the F. oxy s p or u m f. sp. melonis strain
NRRL 26406 and F. o x y spo r um f. sp. lycopersici strain
FOL4287 (99.85%), despite the fact that they contain vari-
ants 1 and 2, respectively.
Discussion
The theory of genealogical concordance phylogenetic
species recognition (GCPSR) for fungi was introduced by
Taylor et al. [13]. GCPSR consists of two steps: (i) iden-
tifying independent evolutionary lineages (IELs) based
on genetic isolation estimated from multiple single locus
phylogenies and (ii) exhaustive subdivision (classifying
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Brankovics et al. BMC Genomics (2017) 18:735 Page 10 of 14
a) b) c)
Fig. 5 Tanglegram of the trees based on athe large variable region variant 2, bthe conserved part of the mitogenome and cthe variant 1 of the LV
region, respectively. Clades with high Bayesian posterior probability (BPP) support are displayed with thicker branches. The support values are BPP
values. The three phylogenetic clades identified within the FOSC are highlighted in different shades of gray. The strains that contain variant 3 are
highlighted by blue boxes
eachisolateintocladessubstantiatedbyIELs).Previous
implementations of GCPSR [12, 14] presented no pro-
grammatic tools for performing the analysis, required
that balancing selection should not be influencing any
of the loci in the analysis, identified IELs based on
being at least concordant or non-discordant, and pre-
formed exhaustive subdivision after superimposing the
IELs on a maximum likelihood tree based on con-
catenated sequences. The last two characteristics make
the method difficult to automate, which means that it
is challenging to scale the method to a large num-
ber of loci. The scaling is further hindered by the
requirement that loci should not be under balancing
selection, which is difficult to detect before conducting
phylogenetic comparisons.
We have employed a new strategy for GCPSR that uses
sequential selection of clades: first identifying concor-
dant clades, and subsequently removing the discordant
clades from the selection. The programmatic implemen-
tationallowstheusertosettheminimumnumberof
phylogenies required to recognize a given clade as con-
cordant, which in combination with applying the two
criteria sequentially makes the detection indifferent to
some loci being under balancing selection. Our imple-
mentation of the GCPSR is scalable to any number of loci.
Since the criteria for clade selection is applied sequen-
tially and not in parallel, there can be no conflict left
between the clades that are kept, and these clades already
define a tree topology. The number of loci supporting
a given clade can be used to display the support in the
constructed tree. As a final selection, exhaustive subdivi-
sion can be used, by removing all subclades that would
make a clade paraphylic. Employing exhaustive subdi-
vision also ensures that no single strain can be recog-
nized as unique species, thereby adhering to the genetic
differentiation criterion.
Phylogenetic studies by O’Donnell et al. [8, 10] have
identified three major clades within the Fusarium oxys-
porum species complex. Further phylogenetic analysis
conducted using genealogical concordance phylogenetic
species recognition based on eight loci supported the sep-
aration of two phylogenetic species within the complex:
one species corresponding to clade 1 and the other to the
remaining three clades [12].
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Brankovics et al. BMC Genomics (2017) 18:735 Page 11 of 14
In the current study, the eight loci used by Laurence
et al. [12] were tested with our GCPSR approach. Only
one locus, tef1a, contained sufficient phylogenetic infor-
mation to support the grouping with sufficient Bayesian
posterior support (BPP 0.95) within the FOSC. In
contrast, when the eight complete protein coding genes,
the rDNA repeat region and the conserved part of the
mitochondrial genome were used for the GCPSR analy-
sis, three phylogenetic species were identified. All three
species were highly supported in half of the single locus
phylogenies. The three phylogenetic species correspond
to clades 1, 2 and 3 sensu O’Donnell within the FOSC, and
they were identified with low support (supported only by
tef1a) by the eight loci analysis. The eight loci set did not
contain sufficient phylogenetic information to separate
the three species. The ten loci used for GCPSR contained
more phylogenetic information, five out of the six most
informative loci could resolve the three species with high
support (BPP 0.95, Table 2), the exception being the
rDNA repeat region. Most of the variable sites in the
rDNA repeat region were located in the intergenic spacer
region (IGS), which is frequently the target of indel muta-
tions [36]. Indel mutations make it difficult to estimate
correctly which nucleotide characters are homologous,
and this can lead to incorrect phylogenetic tree estima-
tions. This could explain why the rDNA repeat sequence
did not support the clustering supported by the other
variable markers, despite the large number of parsimony
informative sites.
The recognition of two phylogenetic species corre-
sponding to clades 2 and 3 was further supported by
the fact that the conserved part of their mitogenomes
Table 2 The variability of the individual loci used for the GCPSR
based on the 10 loci data set. The loci are ordered based on the
number of variable sites
Locus Length Conserved PIaThe three clades
InterbIntracInterbIntracwere monophyletic
rpl10a 795 762 783 28 7 no
cal 979 920 963 52 8 no
act 1634 1572 1605 54 21 no
tub2 1671 1586 1634 78 27 no
tef1a 1776 1650 1711 99 43 yes
tef3 3588 3456 3532 111 43 yes
top1 3170 2981 3114 177 45 yes
rpb2 3907 3653 3812 213 61 yes
rDNA 7990 7437 7689 483 223 no
mt 40841 39121 40153 1501 464 yes
aParsimony informative sites
bOutgroup + FOSC (clades 1, 2, 3)
cFOSC (clades 1, 2, 3)
had significantly different lengths and their mitochon-
drial genomes had distinctly different intron patterns.
This length difference was statistically significant even
after excluding the introns of protein coding genes from
the analysis. This marked difference suggests that the two
populations have been genetically isolated. This genetic
isolation could not be explained by the geographic origin
of the strains.
The distribution of the variants of the large variable
(LV) region of the mitogenome across the clades seems
to contradict the recognition of the three phylogenetic
species. However, the trees based on the variant regions
and on the conserved part of the mitogenome are congru-
ent. This demonstrates that three phylogenetic species are
genetically isolated and differentiated from each other. It
is likely that variants 1 and 2 of the LV region appeared
in the common ancestor of clades 2 and 3, and they
were maintained by recombination during the separation
of the two lineages. This is supported by the fact that
the two variants are present in both clades 2 and 3, and
that the separation of clades 2 and 3 is supported by
the phylogeny of both the LV region as well as the con-
served part of the mitogenome. This hypothesis is also
supported by the complete sequence identity of the con-
served part of the mitogenome of F. o x ys p o rum f. sp.
niveum strains Fon015 and Fon019 from clade 2, despite
the fact that these strains contain variants 1 and 2, respec-
tively. A similar example in clade 3 is the high sequence
similarity between the conserved part of the mitogenome
of the F. ox y s por u m f. sp. melonis strain NRRL26406
and F. ox y spor u m f. sp. lycopersici strain FOL4287
(99.85%), while they contain variants 1 and 2 of the LV
region, respectively.
In this study strain F11, which was used for previ-
ous comparative mitogenome studies [17, 19], was re-
sequenced using next generation sequencing, and its
mitogenome was assembled from the sequencing reads.
The curated sequence contains the LV region, which is
typical for Fusarium spp. [17, 19]. In addition, two introns
were found that were absent from the original assem-
bly [37].
Al-Reedy et al. [19] reported that the LV-uORF may
represent a gene with unknown function and it is under
purifying selection. Because the LV-uORF region has a
higher GC content than the conserved protein encoding
genes or the intron encoded genes and because its codon
usage differs significantly from other mitochondrial genes,
they suggested that the LV-uORF was acquired via hori-
zontal gene transfer. However, examining the mitogenome
sequence presented by Al-Reedy et al. [19] reveals that the
GC contents of the LV-uORF and the intergenic regions
are similar. So the GC content does not necessarily suggest
horizontal gene transfer. Comparative genome analysis
conducted in this study revealed that within the FOSC
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Brankovics et al. BMC Genomics (2017) 18:735 Page 12 of 14
this region contains multiple indels as well as point muta-
tions, which in some strains lead to fragmentation of the
ORF into smaller ORFs (Additional file 4). The variability
of this region as well as its GC content is similar to inter-
genic regions of the mitochondrial genome. All these facts
combined suggest that the LV-uORF has lost its function
within the FOSC or it never had a function within Fusar-
ium. To understand whether the LV-uORF does have
a function in other species complexes within Fusar-
ium a study of the mitogenomes of these groups should
be undertaken.
The analysis of the 61 strain data set revealed two addi-
tional variants of the LV region, both of which had no
LV-uORF. Both contain at least thirteen tRNA genes con-
taining the same anticodons that are present in variant 1
which is present in F. proliferatum as well. The differ-
ent variants share only low sequence similarity and the
order of the tRNA genes is not completely syntenic. Vari-
ant 1 is present in all three clades of the FOSC and also in
other Fusarium spp. [17, 19], variant 2 is found in clades 2
and 3 within the FOSC, and variant 3 is confined to
clade 2.
The origin of the two new variants of the LV region
is unclear. They may have evolved from variant 1 by
the insertion of mobile elements similar to mitochondrial
introns, because the newly discovered variants contain
ORFs that have domains matching those present in hom-
ing endonuclease genes (HEGs). HEGs are commonly
associated with mitochondrial introns [38]. These may
have initiated double strand breaks inside this region,
which through homologous recombination repair could
have led to the rearrangement of the tRNA genes, while
still preserving some blocks of synteny.
F. ox y spor u m has been considered to be an asexual
species. In asexual species, genetic exchange between
strains is possible only between members of the same
vegetative compatibility group (VCG). We found recom-
bination of the mitochondrial genome, which indicates
the presence of either a sexual or a parasexual cycle.
The sexual cycle in haploid fungi involves the fusion of
two haploid nuclei leading to a diploid nucleus which
through meiosis results in haploid offspring. The para-
sexual cycle also begins by the fusion of two haploid
nuclei, but recombination occurs by mitotic crossing over
during multiplication of the diploid nucleus, and haploid
cells emerge through vegetative haploidization instead
of meiosis [39]. No sexual cycle could be induced in
F. ox y s por u m , but the fusion of the nuclei of strains
that were not members of the same VCG was shown
[23, 40]. Genome analysis of F. o x y sp o r um f. sp. lycop-
ersici strain FOL4287 revealed that pathogenicity factors
are located on accessory chromosomes and that trans-
fer of these chromosomes to a non-pathogenic recipient
conveyed pathogenicity to the recipient strain [23]. The
proposed underlying mechanism for this chromosome
transfer is that the nuclei of the two strains fuse, fol-
lowed by selective loss of chromosomes from one of the
fusion partners [40]. Genetic exchange may be restricted
to members of the same phylogenetic species since anas-
tomosis was observed between F. o x y sp o r um f.sp. lycop-
ersici Fol007 (clade 3) and F. ox ysp o r um Fo47 (clade 3),
between F. ox ysp o r um f.sp. lycopersici Fol007 (clade 3) and
F. ox y spor u m f.sp. melonis NRRL26406 (clade 3), but anas-
tomosis could not be induced between F. o x ys p o r um f.sp.
lycopersici Fol007 (clade 3) and F. ox ysp o r um f.sp. cubense
NRRL25603 (clade 1) [23, 35]. These results demonstrates
that recombination is possible between members of the
same clade, but there is genetic isolation between the
different clades.
Conclusions
In this study a programmatic implementation of
genealogical concordance phylogenetic species recog-
nition (GCPSR) strategy was introduced, which allows
themethodtoscaleeventoalargesetofloci.Itwas
shown to be robust in recognizing phylogenetic species.
Our new GCPSR approach revealed three phylogenetic
species within the Fusarium oxysporum species complex,
which correspond to clades 1, 2 and 3 sensu O’Donnell
et al. [10, 35]. We conclude that there has been genetic iso-
lation between the different phylogenetic species leading
to reciprocal monophyly of multiple loci. Thus, the phy-
logenetic species appear to define the limit of potential
genetic exchange between members of the FOSC.
Both the recombination identified in the mitochondrial
genome of the FOSC in our study and previous chro-
mosome transfer studies [23, 40] indicate that there is
a parasexual cycle in F. o x ysp o r um and recombination
is possible between members of the same phylogenetic
species.
Mitochondrial genomes are highly informative for
resolving phylogenetic relationships even between closely
related species and populations. Complete mitochondrial
genome sequences offer a stable basis and reference point
for phylogenetic and population genetic studies. Our
study demonstrates that a detailed comparative analysis of
the mitogenome may offer new insights into the biology
of the studied organism.
F. ox y spor u m strains contain the large variable region
containing the LV-uORF that is not a functional gene.
Furthermore, two new variants of the LV region were dis-
covered within the FOSC. The distribution pattern and
the sequence comparisons demonstrates that there has
been mitochondrial recombination during the separation
of clades 2 and 3. Clade 1 may contain more phyloge-
netic species. Extended sampling of this group may shed
more light on the composition of the Fusarium oxysporum
species complex.
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Brankovics et al. BMC Genomics (2017) 18:735 Page 13 of 14
Additional files
Additional file 1: Strains analyzed and accession numbers of the NGS
data used. (XLSX 9 kb)
Additional file 2: Scalable Genealogical Concordance Phylogenetic
Species Recognition: Detailed description of the new implementation of
the GCPSR method, including algorithm overview and recommended
workflow. (PDF 420 kb)
Additional file 3: Boxplot of the length of the conserved part of the
mitogenome of the three clades of the FOSC. (PDF 111 kb)
Additional file 4: Analysis of the LV-uORF region In depth comparative
analysis of the LV-uORF region. (PDF 434 kb)
Abbreviations
acl1: ATP citrate lyase; act:γ-actin; atp6: Mitochondrially encoded ATP synthase
6; atp8: Mitochondrially encoded ATP synthase 8; atp9: Mitochondrially
encoded ATP synthase 9; BLAST: Basic Local Alignment Search Tool; BPP:
Bayesian posterior probability; cal: Calmodulin; CD-Search: Conserved domain
search; cob: Mitochondrially encoded cytochrome b; cox1: Mitochondrially
encoded cytochrome c oxidase I; cox2: Mitochondrially encoded cytochrome
c oxidase II; cox3: Mitochondrially encoded cytochrome c oxidase III; DNA:
Deoxyribonucleic acid; ENA: European Nucleotide Archive; F: Fusarium;f.sp:
forma specialis; ff. spp: formae speciales;FOSC:Fusarium oxysporum species
complex; GCPSR: Genealogical concordance phylogenetic species
recognition; GRAbB: Genomic Region Assembly by Baiting; GTR: General time
reversible; HEG: Homing endonuclease; IEL: Independent evolutionary
lineages; IGS: Intergenic spacer; ITS: Internal transcribed spacer; LV: Large
variable; LV-uORF: Large variable open reading frame with unknown function;
MCMC: Markov chain Monte Carlo; MRC: Majority-rule consensus; mt:
Mitochondrial genome; nad1: Mitochondrially encoded NADH dehydrogenase
1; nad2: Mitochondrially encoded NADH dehydrogenase 2; nad3:
Mitochondrially encoded NADH dehydrogenase 3; nad4: Mitochondrially
encoded NADH dehydrogenase 4; nad4L: Mitochondrially encoded NADH
dehydrogenase 4L; nad5: Mitochondrially encoded NADH dehydrogenase 5;
nad6: Mitochondrially encoded NADH dehydrogenase 6; NCBI: National
Center for Biotechnology Information; NGS: Next generation sequencing; nir:
Nitrate reductase; ORF: Open reading frame; pho: Phosphate permease; rDNA:
Ribosomal DNA; rnl: Mitochondrially encoded 16S RNA; rns: Mitochondrially
encoded 12S RNA; rpb1: Largest subunit of DNA-dependent RNA polymerase
II; rpb2: Second largest subunit of DNA-dependent RNA polymerase II; rpl10a:
60S ribosomal protein L10; rps3: Ribosomal protein S3; sp: Species (singular
form); spp: Species (plural form); tef1a: Translation elongation factor 1α;tef3:
Translation elongation factor 3; top1: Topoisomerase I; tRNA: Transfer
ribonucleic acid; tub2:β-tubulin; VCG: Vegetative compatibility group
Acknowledgments
We would like to thank Dimitrios Tsirogiannis from the Benaki
Phytopathological Institute (Kifissia, Greece) for providing F. oxysporum f. sp.
cumini strain F11 for re-sequencing.
Funding
The investigations were supported by the Division for Earth and Life Sciences
(ALW) with financial aid from the Netherlands Organization for Scientific
Research (NWO, http://www.nwo.nl/) under grant number 833.13.006. This
work was further supported by the Horizon programme (project 93512007) of
the Netherlands Genomics Initiative (NGI) through a grant to MR. CW was
supported from the MycoKey project (Horizon2020, nr. 678781). The funding
organizations had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Availability of data and materials
The sequencing data has been deposited into the European Nucleotide
Archive (ENA) with the following accession numbers: PRJEB18591, PRJEB18594
and PRJEB18595. The assembled sequences have been uploaded to the
European Nucleotide Archive under the following accession numbers:
LT841199-LT841268 and LT905535-LT906358. Perl script, fasta_variability, is
available from the fasta_tools package (https://github.com/b-brankovics/
fasta_tools). GCPSR Perl scripts are available at GitHub (https://github.com/b-
brankovics/GCPSR).
Authors’ contributions
BB and AD designed the study. PD, MR, TAJL and CW sequenced large part of
the strains used for this study. BB wrote the scripts and carried out the analysis.
All authors (BB, PD, MR, GSH, TAJL, CW and AD) helped shaping the
manuscript. All authors approved the final version of the manuscript.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584CT Utrecht, The
Netherlands. 2Institute of Biodiversity and Ecosystem Dynamics, University of
Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
3Swammerdam Institute for Life Sciences, University of Amsterdam, Science
Park 904, 1098 XH Amsterdam, The Netherlands. 4Wageningen University and
Research Centre, Droevendaalsesteeg 4, 6708 PB Wageningen, The
Netherlands.
Received: 22 December 2016 Accepted: 5 September 2017
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