Evaluation of two novel barcodes for species recognition of opportunistic pathogens in Fusarium

Accepted Manuscript Evaluation of two novel barcodes for species recognition of opportunistic pathogens in Fusarium Abdullah M.S. Al-Hatmi, A.H.G. Gerrits Van Den Ende, Benjamin Stielow, Anne D. Van Diepeningen, Keith A. Seifert, Wayne Mccormick, Rafik Assabgui, Tom Gräfenhan, G. Sybren De Hoog, C. André Levesque PII:

S1878-6146(15)00143-9

DOI:

10.1016/j.funbio.2015.08.006

Reference:

FUNBIO 614

To appear in:

Fungal Biology

Received Date: 14 June 2015 Revised Date:

28 July 2015

Accepted Date: 4 August 2015

Please cite this article as: Al-Hatmi, A.M.S., Gerrits Van Den Ende, A.H.G., Stielow, B., Van Diepeningen, A.D., Seifert, K.A., Mccormick, W., Assabgui, R., Gräfenhan, T., Sybren De Hoog, G., André Levesque, C., Evaluation of two novel barcodes for species recognition of opportunistic pathogens in Fusarium, Fungal Biology (2015), doi: 10.1016/j.funbio.2015.08.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Fungal Biol – Barcoding 26-07-2015

Evaluation of two novel barcodes for species recognition of opportunistic pathogens in Fusarium

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Abdullah M.S. AL-HATMI1,2,3**, A. H. G. GERRITS VAN DEN ENDE1, Benjamin STIELOW1, Anne D. VAN DIEPENINGEN1, Keith A. SEIFERT4, Wayne

McCORMICK4, Rafik ASSABGUI4, Tom GRÄFENHAN4,*, G. Sybren DE HOOG1,2,5,

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C. André LEVESQUE4

CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands

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Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam,

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The Netherlands

Directorate General of Health Services, Ibri Hospital, Ministry of Health, Oman

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Biodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa,

Canada 5

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Peking University Health Science Center, Research Center for Medical Mycology,

Beijing, China; Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou,

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China; Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military

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Medical University, Shanghai, China; Basic Pathology Department, Federal University of Paraná State, Curitiba, Paraná, Brazil; King Abdulaziz University, Jeddah, Saudi Arabia

*Present Address: Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, Canada **Corresponding author. CBS-KNAW Fungal Biodiversity Centre, 3485CT Utrecht, The Netherlands. E-mail address: [email protected]. 1

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Abstract The genus Fusarium includes more than 200 species of which 73 have been isolated from human infections. Fusarium species are opportunistic human pathogens with variable

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etiology. Species determination is best made with the combined phylogeny of proteincoding genes such as elongation factor (TEF1), RNA polymerase (RPB2) and the partial β-tubulin (BT2) gene. The internal transcribed spacers 1, 2 and 5.8S rRNA gene (ITS)

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have also been used, however, ITS cannot discriminate several closely related species and has nonorthologous copies in Fusarium. Currently, morphological approaches and tree-

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building methods are in use to define species and to discover hitherto undescribed species. Aftter a species is defined, DNA barcoding approaches can be used to identify species by the presence or absence of discrete nucleotide characters. We demonstrate the potential of two recently discovered DNA barcode loci, topoisomerase I (TOP1) and

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phosphoglycerate kinase (PGK), in combination with other routinely used markers such as TEF1, in an analysis of 144 Fusarium strains belonging to 52 species. Our barcoding study using TOP1 and PKG provided concordance of molecular data with TEF1. The

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currently accepted Fusarium species sampled were well supported in phylogenetic trees

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of both new markers.

Keywords: Fusarium, character-based DNA barcoding, topoisomerase I (TOP1), phosphoglycokinase (PGK), intra- and inter-specific variation.

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ACCEPTED MANUSCRIPT Introduction The genus Fusarium comprises more than 200 species, many occurring as pathogens on a wide range of important crop plants. They are also relevant to human health by causing opportunistic, superficial or disseminated infections, and by producing mycotoxins

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(Tortorano et al. 2014). The highly speciose genus is found in different climates zones, particularly in tropical and subtropical regions, and is associated with various habitats,

such as sheltered forests, harsh deserts or soils (Leslie & Summerell 2006). Al-Hatmi et

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al. (2014) noted that 71 taxonomic species, verified by multilocus sequence typing

(MLST), have been reported from human or animal infections. Recently two more species

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were added to this list: F. musae, reported from blood samples and biopsies of immunesuppressed patients in Belgium and France (Thrist et al. 2014), and F. langsethiae from keratitis in India (Ruban et al. 2015).

Morphological identification of Fusarium is largely based on macro- and

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micromorphological characters including colony color and appearance on specific culture media, and on size, shape, and development of sexual and asexual spores, as well as spore-forming structures (Moretti 2009). However, successful identification may be

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difficult because of similar, inconspicuous or degraded diagnostic characters in culture. Molecular methods have replaced traditional identification methods in most taxonomic

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research. DNA-based methods, particularly using geneological concordance analyses, frequently lead to the detection of novel sibling species (Scheel et al. 2013, Nucci et al. 2013). Standard diagnostic nuclear rDNA markers have limited resolution to distinguish currently recognized taxa in Fusarium (Geiser et al. 2004). The concept of DNA barcoding (Herbert et al. 2003) had a large impact on standardizing identification of eukaryotes. Short, easily amplified regions of DNA, based 3

ACCEPTED MANUSCRIPT on authoritatively identified vouchers, led to a more robust identification process (Riaz et al. 2011). Although tremendous progress has been made in terms of availability of molecular markers to study genetic diversity, there is a need to identify additional reliable and effective loci that can function at all taxonomic levels in Fusarium.

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A large number of molecular loci have been used in Fusarium phylogenetics, including nuclear and mitochondrial ribosomal rDNA (18S, 28S, ITS, IGS, mtSSU,

mtLSU) (O’Donnell et al. 1998a), although analysis of the ITS is confounded by the

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occurrence of nonorthologous copies (O’Donnell & Cigelnik 1997). Protein-coding genes are also in use, such as RNA polymerase (RPB2), β-tubulin (BT2), elongation factor

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(TEF1) (O’Donnell et al. 2012) and ATP citrate lyase (ACL1) (Gräfenhan et al. 2011). Geiser et al. (2004) concluded that TEF1 has high phylogenetic utility for the following reasons: (i) it is highly informative at the species level; (ii) non-orthologous copies of the gene have not been detected across the genus; and (iii) it is alignable across the genus

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Fusarium. Some single-copy protein-coding genes such as RPB1 and RPB2 are also promising for phylogeny and barcoding in Fusarium (O’Donnell et al. 2013). In this study, two newly characterized genes were used as molecular markers for

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Fusarium species genotyping, i.e. topoisomerase I (TOP1) and phosphoglycerate kinase (PGK). They were discovered during genome scans guided by protein families (Lewis et

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al. 2011), and tested extensively for their utility for a broad range of fungi, but mostly Ascomycetes, by Stielow et al. (2015). The DNA topoisomerases enzymes (Top1p & Top2p) were discovered in the 1970s and their function is to relax supercoiling that accumulates in DNA following polymerization. Polymerase causes supercoiling because it is fixed and cannot rotate. Topoisomerases relieve supercoiling by either cutting one strand (Top1p) or both strands (Top2p). Partial nucleotide sequences of TOP2 were used 4

ACCEPTED MANUSCRIPT for identifying both eukaryotes and prokaryotes (Li et al. 2007). Topoisomerase I has not yet been used for phylogenetic or barcoding studies of Fusarium, although some of the TOP1 and PGK data reported here was included and partially analysed in the large scale analysis of Stielow et al. (2015). Phosphoglycerate kinase (Pgkp) is a housekeeping

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enzyme involved in the Calvin cycle and glycolysis. It codes for the nuclear-encoded plastid enzyme (Huang et al. 2002) and, in common with TOP1, has not been used for phylogeny or identification of Fusarium species.

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In our study, marker assessment and comparison were carried out using a tree-

based DNA identification technique, as well as the distance-based technique for larger

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data sets, to assess the accuracy and robustness of DNA barcoding of Fusarium species using the sampled genes. Our hypothesis is that the genes topoisomerase I (TOP1) and phosphoglucokinase (PGK) may be reliable barcoding markers to overcome some of the identification problems for species in Fusarium inherent with the recognized barcode ITS

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(Schoch et al. 2012) and the informal but widely used secondary barcode TEF1 (Geiser et al. 2004). The present research addresses further testing of specific primer sets based on TOP1 and PGK presented by Stielow et al. (2015) to generate sequences that enhance

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discrimination of species of Fusarium.

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ACCEPTED MANUSCRIPT Materials and methods Strains Strains of different Fusarium species with clinical relevance and their environmental counterparts were chosen for analysis, including type and authentic strains. Particular

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attention was paid to the Fusarium fujikuroi species complex (FFSC), which includes molecular siblings separated by few bp differences in TEF1 (O’Donnell et al. 1998a). The set comprised 144 clinical (n=33) and environmental (n=111) isolates (Table S1), of

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which 80 were taken from the reference collection of the CBS-KNAW Fungal

Biodiversity Centre (CBS), Utrecht, The Netherlands, and 64 from the Department of

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Agriculture and Agri-Food (AAFC) culture collection, Ottawa, Canada (DAOMC). At CBS, stock cultures were maintained on slants of 2 % malt extract agar (MEA; Oxoid) and oatmeal agar (OA, home-made at CBS). Culture plates were incubated at 24 °C for one week. At AAFC, strains were grown on Difco Potato Dextrose agar (PDA) for 7−10

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days prior to DNA extraction.

Specific primer design strategy

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Topoisomerase I (TOP1) and phosphoglycerate kinase (PGK) were identified as potentially useful barcodes from a Pfam analysis of available fungal genomes by Lewis et

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al. (2011). As described in detail by Stielow et al. (2015), intensive universal primer testing and redesign for all fungi was undertaken for a 1 kb fragment of PGK and a 700 bp fragment of TOP1, with M13 primers added and tested for sequencing as an additional option to increase DNA sequencing success after PCR and facilitate high throughput when sequencing multiple markers.

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ACCEPTED MANUSCRIPT DNA extraction At CBS, DNA was extracted following the Quick CTAB protocol. 1–10 mm3 material was transferred to two mL screw-capped tubes filled with 490 µL CTAB-buffer 2× and 6– 10 acid-washed glass beads. 10 µL Proteinase K were added and mixed thoroughly on a

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MoBio vortex for 10 min. 500 µL Chloroform: isoamylalcohol (24:1) were added and shaken for 2 min after incubation for 60 min at 60 °C. Tubes were centrifuged for 10 min at 14,000 r.p.m. The supernatant was collected in a new Eppendorf tube. To ~400 µL

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DNA sample 2/3 vol (~270 µL) of ice-cold iso-propanol was added and centrifuged again at 14,000 r.p.m. for 10 min and the upper layer was dissolved in 1 mL ice-cold 70 %

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ethanol. Tubes were centrifuged again at 14,000 r.p.m. for 2 min, air-dried and resuspended in 50 µL TE-buffer. Quality of genomic DNA was verified by running 2–3 µL on a 0.8 % agarose gel. DNA was quantified with NanoDrop 2000 spectrophotometer (Thermo Fisher, Wilmington, U.S.A.). Samples were stored at −20 °C until use. The

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methods used at AAFC for DNA isolation was performed with the OmniPrep™ Genomic DNA Extraction Kit (G-Biosciences, St. Louis, Missouri, U.S.A.) following the procedure

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outlined in Stielow et al. (2015).

DNA amplification and sequencing

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Four gene regions were amplified directly from the genomic DNA: translation elongation factor-1α (TEF1), internal transcribed spacer regions of the nrDNA (ITS), topoisomerase I (TOP1) gene and the phosphoglycerate kinase (PGK) gene. Primers used for amplification and sequencing were PGK_480-F/R, PGK_533-F/R (only used for a few strains at AAFC) and TOP1_501-F/R from Stielow et al. (2015), EF1 and EF2 from O'Donnell et al. (1998), ITS1 and ITS4 from and White et al. (1990). 7

ACCEPTED MANUSCRIPT Methods for amplification and sequencing of TEF1, PGK and TOP1 at AAFC are detailed in Stielow et al. (2015). At CBS, PCR reaction mixture (12.5 µL final vol) contained 10× PCR buffer 1.25 µL, water 7.5 µL, dNTP mix (2.5 mM) 0.5 µL, 0.25 µL of each primer (10 pmol), Taq polymerase (5 U/µL) 0.05 µL, DMSO 0.7 µL, and template

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DNA (100 ng/µL) 1 µL. PCR reactions were performed in a Hybaid Touchdown PCR machine (Hybaid, Middlesex, U.K.). TEF1 and ITS were amplified and sequenced

following the methods of Al-Hatmi et al. (2015). For TOP1 and PGK, a touchdown PCR

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protocol consisted of two phases: phase 1 included an initial step of 95 °C for 5 min, followed by 10 cycles of denaturation at 95 °C for 45 sec, annealing at variable

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temperatures for 1 min, and extension at 72 °C for 1 min. In the first cycle, the annealing temperature was set to 68 °C and, at each of the 10 subsequent cycles, the annealing temperature was decreased by 1 °C (i.e., it varied from 68° to 58°C at 1°C decrements along the 10 cycles). Phase 2 consisted of 30 cycles of 95 °C for 45 sec, 58 °C for 45 sec,

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60 °C for 45 sec and 72 °C for 1 min and post elongation step of 5 min at 72 °C. After the last PCR cycle, the samples were cooled to 10 °C, and a 6-µL aliquot of the amplification products was electrophoresed on a 1 % agarose gel (1× TAE) and visualized under

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ultraviolet (UV) light. Amplicons were purified using exoSAP. Both strands of the PCR fragments were sequenced with M13 primers. Sequencing PCR was performed as

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follows: 1 min at 95 °C, followed by 30 cycles consisting of 10 sec at 95 °C, 5 sec at 50 °C and 2 min 60 °C. Reactions were purified with Sephadex G-50 fine (GE Healthcare Bio-Sciences, Uppsala, Sweden) and sequencing was done on an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, U.S.A.) with ABI PRISM BigDyeTM terminator cycle sequencing kit (Applied Biosystems). The ABI PrismH Big DyeTM Terminator v. 3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) was used 8

ACCEPTED MANUSCRIPT for sequencing PCR. Sequences were determined with an ABI PRISM™ 3,100 Genetic Analyzer.

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Barcoding resolution Barcoding gaps were evaluated by 1) analyses of pairwise distances, and 2) tree-based

Analyses of Pairwise distances

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analyses were carried out on the four alignments.

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analysis of barcodes of significant clades after tree reconstruction, for which parallel

Aligned sequences were analyzed in R (R Core Team 2014), APE to generate pairwise distances (Paradis et al. 2004), and using QQPLOT2 for generating graphics (Wickham

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2009). The raw pairwise distances were calculated for each pair of strains and categorized

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as being intra- or inter-specific.

Tree based assessment

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Sequences were edited and assembled SeqMan of the Lasergene package (DNAStar, Madison, WI, U.S.A.) and manually corrected. Sequences were aligned using the MAFFT server (www.ebi.ac.uk/Tools/msa/mafft/) under default parameters and whenever indicated, manually improved in BioEdit v. 7.0.5.2 (Hall 1999). The best-fit model of evolution was determined by ModelTest v. 2.3 (Nylander 2004). Neighbor-Joining (NJ) trees were generated based on the Kimura2-parameter model for each DNA marker to 9

ACCEPTED MANUSCRIPT provide visual displays of genetic variation within and between species. Additional trees were generated with Maximum Likelihood (ML) and Bayesian Inference (BI) with MrBayes v. 3.1.2 on the Cipres portal (http://www.phylo.org/ sub_sections/portal). Two parallel runs of four chains were run for 10,000,000 generations and trees were sampled

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every 1,000 generations. TRACER version 1.5 was used to verify that the mean likelihood value, effective sample size (ESS) and other parameters reached a plateau. For each run, 10 % of the trees were discarded as they were obtained during the burning phase.

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Maximum likelihood (ML) analysis was done with RAxML-VI-HPC v. 7.0.3 with nonparametric bootstrapping using 1000 replicates, a bootstrap percentage value as good

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support was regarded above 70 %, and the substitution model was Tamura-Nei and uniform rates among sites. To check the congruency of the data sets, a 70 % NeighbourJoining (NJ) reciprocal bootstrap method with maximum likelihood distance was performed on each individual locus (Mason-Gamer & Kellogg 1996). Trees were viewed

Results

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and edited with TreeView v. 1.6.6, FIGTREE v. 1.1.2 and MEGA6.2.

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Amplification and sequencing success rates

Strain designations and corresponding GenBank accession numbers of 144 Fusarium

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cultures (111 environmental and 33 clinical isolates; 80 sequenced at CBS and 64 at AAFC) representing the 52 species used in this study are listed in Table S1. The ITS, TEF1, TOP1 and PGK genes were selected as candidate markers to compare their suitability for the identification of the 52 Fusarium species. PCR amplification success rates evaluated at CBS for the four loci were 100 % for ITS, 96 % for TEF1, 95 % for TOP1 and 93 % for PGK. Properties of the sequences and 10

ACCEPTED MANUSCRIPT alignments of the tested loci are summarized in Table 1. No multiple bands were detected and sequencing success rates were 100 % of the isolates for ITS, 98 % for TEF1, 96 % for TOP1 and 95 % for PGK. ITS had the average shortest length of 469 bp followed by TEF1, which had an average total alignment length of 589 bp, TOP1 with 708 bp, and

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PGK at 828 bp. TEF1 had the highest number of variable sites (314/589 = 53.3 %), followed by TOP1 (359/708 =50.70 %), PGK (300/828 =36.2 %), and ITS (137/469

=29.2%). TOP1 had the most phylogenetically informative sites (327/700 = 46.1 %),

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followed by TEF1 (256/589 = 43.4.2 %), PGK (240.828 = 28.99 %) and ITS (105/469 22.5 %). The lengths and guanine-cytosine (G+C%) contents of the markers varied

50.7 % and 48.5 % with PGK.

Analyses of pairwise distances

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significantly and was highest at 57.8 % for PGK, followed by TEF1 at 52.5 %, ITS with

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The distributions of intra- and interspecific distances were compared for ITS, TEF1, TOP1 and PGK in order to better visualize the “barcode gap” (Fig. 1). Not surprisingly, ITS showed the greatest overlap between intra- and inter-specific distance. The barcode

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gap appeared to be similar for PGK and TEF1, however, the inter specific variation was

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much smaller in PGK. TOP1 and TEF1 had similar interspecific variation but TOP1 had less intraspecific variation. The interspecific variation was compared for each pair of markers (Fig. 2). The most important part of this graph is close to the origin on the axes. One can see that there are many instances where the ITS pairwise distances are zero but are above zero for the other markers. PGK and TEF1 appear to be similar close to the origin but TEF1 always showed a higher pairwise distance for more distant species.

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ACCEPTED MANUSCRIPT ITS yielded smallest barcoding gaps in Fusarium between sibling species at a minimum of 0.1 % for the species pairs F. succisae / F. anthophilum, F. andiyazi / F. anthophilum, F. circinatum / F. anthophilum, F. culmorum / F. crookwellense, F. proliferatum / F .mangiferae, and F. proliferatum / F. fujikuroi. The most divergent

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species in the ITS data set were F. petroliphilum and F. armeniacum (Fig.1S).

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Intra-specific variability

The most useful barcoding locus should have no overlap between inter- and intra-specific

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K2P distances. Twenty-three of the 52 Fusarium species had more than one strain in the data set and were selected for analysis (Fig. 3).

Phylogenetic analysis and congruence among loci

Bootstrap analyses were performed on each data set, with 70 % as cut-off. We used the

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program ModelTest v. 2.3.within MEGA6.2 to determine the nucleotide substitution model that best fit our data. K2+G+I was selected as the best model for the ITS and TEF1 data sets, TN93+G+I for TOP1, and TN93+G for PGK.

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The nodes, e.g. nodal distance from the root node to the branches on a

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phylogenetic tree obtained from the single gene data sets using ML and Bayesian analyses showed significant discordance with the ITS tree (not shown) and the other three markers, but revealed no conflicts between TEF1, TOP1, and PGK. Tree-based DNA identification was assessed using BI and ML without considering an outgroup. ML and BI analyses showed that the data sets for all terminal clades representing species were identical for TEF1, TOP1, and PGK.

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ACCEPTED MANUSCRIPT Based on the data generated here (Figs 4–6), we could delineate the Fusarium species using any one of the three protein coding genes, i.e. TEF1, TOP1, and PGK, individually. The currently accepted species were all well-supported in the trees of the three markers. Most species represented by several strains formed monophyletic groups

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with posterior probabilities higher than 1 and bootstrap values higher than 70 %.

Discussion

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The need for rapid identification of Fusarium species has always been apparent because of the frequency of diseases and mycotoxin production in plants. The genus is

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increasingly significant for human health, particularly because mortality rates from disseminated fusariosis are rising in severely immunocompromised patients. Molecular taxonomic studies have focused on resolving evolutionary relationships within species complexes or clades of agriculturally and medically important Fusarium

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(O'Donnell et al. 2012), where particularly RPB2 and RPB1 have been regarded significant (O'Donnell et al. 2013). The aim of the present study was to evaluate the efficacy of DNA barcoding using alternative markers in addition to ITS, i.e. the widely

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used translation elongation factor 1-α (TEF1, Geiser et al. 2004), and the newly identified potential barcodes topoisomerase I (TOP1) and phosphoglycokinase (PGK, Stielow et al.

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2015).

We obtained sequences from representatives of 52 species in 13 species

complexes of Fusarium, demonstrating the ability of the primers for all four markers to amplify a wide range of species within the genus. High-quality DNA was obtained from all strains and all amplicons were easily sequenced. ITS rDNA as the recommended barcode for fungi has many advantages, in particular the large number of reference 13

ACCEPTED MANUSCRIPT sequences available in GenBank (Schoch et al. 2012), but for most species complexes in Fusarium this gene is insufficiently variable to discriminate molecular siblings (Summerbell et al. 2005, Balajee et al. 2009). We analyzed potential barcode alternatives according to criteria listed by

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Letourneau et al. (2010) that the barcode should be short (500−800 bp), and easily amplifiable, with a low intraspecific variation and a higher interspecific than intraspecific variation. PCR amplification success rates were 98 % for TEF1, 96 % for TOP1 and 95 %

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for PGK. Generally, specific primer binding resulted in single bands and only a few

strains evidenced a complete lack of amplification. For both TOP1 and PGK, we used

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M13 primers for sequencing in our first trial and a 100 % success rate was obtained (Table 1). The identity of all strains with TEF1 sequences was confirmed using BLAST, Fusarium MLST database (http://www.cbs.knaw.nl/fusarium) (O’Donnell et al. 2010) and the Fusarium-ID database (Geiser et al. 2004). The identities of some unknown

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strains originally sequenced only for TOP1 or PGK sequences were inferred by correlation with similar sequences obtained for other strains identified using TEF1 sequences.

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Although amplification and sequencing success rates of ITS were high, close to 100% in our study, the gene could not discriminate between Fusarium species in this

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analysis. This could be because ITS sequences are identical in many Fusarium complexes and they do not tend to evolve at a rate correlated with speciation. In contrast, proteincoding genes usually have higher information content and tend to evolve at higher rate than the more commonly used ITS (Liu et al. 1999). In our data sets, TEF1 had the highest number of variable sites (53.3 %), followed by TOP1 (50.70 %), PGK (36.2 %), and ITS (29.2 %). In terms of phylogenetically informative sites among all four markers, 14

ACCEPTED MANUSCRIPT TOP1 had (46.1 %), followed by TEF1 (43.4.2 %), PGK (28.99 %) and ITS (22.5 %). Thus, the higher variability in intron-rich portions of protein coding genes generally allow these genes to do much better (Geiser et al. 2004), and could therefore represent better candidates for Fusarium DNA barcoding. These results demonstrate sufficient variability

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for species identification in Fusarium for all three protein-coding genes. According to concepts outlined by Jeffroy et al. (2006), this could reflect either degeneracy of the

genetic code, which means that even when a protein is completely conserved, the DNA

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sequence encoding that protein can vary, or invariant protein-coding regions combined with highly variable introns or intergenic spacer regions provide adequate barcode

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signals. Stielow et al. (2015) developed novel protein-based primers and recommended a far broader application of TEF1 for barcoding in fungi. TEF1 is already in use as barcode in Hypocreales, e.g. Fusarium, Tricholoma, Hypocrea and various genera in the Nectriaceae (Pino-Bodas et al. 2013).

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Lengths and guanine-cytosine contents (G+C%) of gene regions amplified varied among markers. The PGK data set had the highest G+C% content (57.8 % on average), followed by TEF1 (52.5 %) and TOP1 (48.5 %). Amplicons of TEF1, TOP1 and PGK

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ranged from 500−800 bp. Amplicon lengths and G+C% contents were generally speciesspecific, but similar G+C% contents do occur in different species. If G+C% contents are

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very different, however, the strains cannot be considered members of the same species (Baron 1996).

There are no absolute rules for the appropriate barcode gap distance between

species, but it is axiomatic that genes yielding wider inter-specific gaps are more reliable for delimiting species within a genus. There were no clear gaps between the minimum inter-specific variations for the four studied barcodes (Fig. 1). However, we have only 15

ACCEPTED MANUSCRIPT limited data for the intra-specific variation because many species were represented by single strain. In studies where intra-specific variability was included in more detail, it was noted that discrete barcode gaps are not always present. For example, in Ochroconis and related genera (Samerpitak et al. 2015), a remarkably high degree of divergence was

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noted in all markers, so that ITS and even LSU were useful for species distinction. This led to large inter-specific differences, but because intra-specific heterogeneity was also high, most markers did not demonstrate an ambiguous barcoding gap. In contrast,

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Scedosporium species outside the S. apiospermum complex (Chen et al. 2015) form

compact clusters, where ITS has insufficient resolution to separate the entities. Within the

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S. apiospermum complex no absolute barcoding gaps were present either, while other, unambiguously delimited species at larger phylogenetic distances did exist. In Fusarium, additional studies on intraspecific variability are necessary to confirm the performance of these loci, and whether or not unambiguous barcoding gaps are present among

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phylogenetic siblings. Considering our high success rates for amplification and sequencing, both TOP1 and PGK are promising supplementary barcodes for identifying Fusarium species.

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Our analyses of Fusarium show that TOP1 and PGK help to resolve large-scale genus level as well as detailed species level phylogenetic relationships. Sequences were

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alignable across the genus and had sufficient variability to resolve within-genus relationships. Phylogenetic results provided strong Bayesian and Maximum Likelihood support rates for a monophyletic ‘terminal Fusarium clade’ (TFC) (Figs 4–6). TFC was described previously in Fusarium by Gräfenhan et al. (2011), O'Donnell et al. (2013) and Lombard et al. (2015). TEF1, TOP1 and PGK genes produced congruent gene trees and we predict that TOP1 and PGK will have a higher potential to resolve phylogenetic 16

ACCEPTED MANUSCRIPT relationships among Fusarium species when analyzed in combination with other routinely used markers such as TEF1, RPB1 and RPB2. All loci resolved most of the terminal clades in their respective gene-trees. The loci can be combined to reach taxonomically sound conclusions about the relationships among species.

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We conclude that any of the three protein-coding loci tested in this study (TEF1, TOP1 and PGK) reliably identify all of the species studied. As noted in previous studies (e.g. O'Donnell et al. 2010), ITS is often insufficiently informative at the species level,

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but can be useful to identify species complexes. The ITS gene tree proved to be

incongruent with those from other loci. However, this does not preclude the use of this

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gene as a barcoding locus for species identification within the fungal kingdom and it is a good starting point to rapidly identify genera level within Ascomycota (Schoch et al. 2012). It is critical that ITS sequences continue to be generated for newly described Fusarium species, and that the species sharing identical ITS sequences be explicitly

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identified, so that information on Fusarium can be extracted from amplicon-based environmental sequencing at the maximum degree of resolution possible within the limitations of the marker. TEF1, TOP1 and PGK can more effectively be used as

species.

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barcoding markers for precise identification of cultures known to represent Fusarium

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Acknowledgments

This study was partially funded by the Ministry of Health, Oman. Formal Agreement no. 28/2014. Abdullah. M. S. AL-Hatmi received a PhD scholarship from the Ministry of Health, Oman. Genome mining at CBS and AAFC, and primer development and testing at AAFC, were supported by grants from the A.P. Sloan Foundation Programme on the Microbiology of the Built Environment. 17

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ACCEPTED MANUSCRIPT Table S1. GenBank sequences of the DNA regions: ITS, TEF1, TOP1 and PGK of the isolates of the Fusarium species included in the barcoding and phylogenetic analyses.

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TOP1 KR058993 KR058998 KR058997 KR058994 KR058995 KR058996 KT003328 KT003298 KT003299 KR059006 KR059007 KR059009 KR059008 KR058974 KR058975 KR058976 KR058977 KT003338 KT003339 KR058999 KR059002 KR059000 KR059001 KT003305 KT003304 KT003297 KT003354 KT003300 KT003335 KT003303 KR059016 KT003302 KT003344 KT003345 KT003346 KT003347 KT003349 KT003350

PGK KR058907 KR058908 KR058911 KR058909 KR058910 KR058912 KT070657 KT070682 KT070683 KR058892 KR058893 KR058895 KR058894 KR058903 KR058905 KR058904 KR058906 KT070677 KT070678 KR058896 KR058899 KR058897 KR058898 KT070662 KT070661 KT070681 KT070695 KT070636 KT070675 KT070659 KR058936 KT070658 KT070688 KT070689 KT070690 KT070691 KT070692 KT070693

SC

RI PT

TEF1 KR071754 KR071758 KR071756 KR071755 KR071757 KR071759 KR909360 KR909342 KR909343 KR071760 KR071761 KR071763 KR071762 KR071718 KR071720 KR071719 KR071721 KR909381 KR909382 KR071766 KR071767 KR071764 KR071765 KR909374 KR909373 KR909341 KR909337 KR909345 KR909378 KR909375 KR071776 KR909370 KR909331 KR909332 KR909333 KR909334 KR909335 KR909336

M AN U

ITS KR058682 KR058683 KR058684 KR058685 KR058686 KR058687 KR909445 KR909447 KR909449 KR071626 KR071628 KR071627 KR071629 KR071651 KR071652 KR071653 KR071657 KR909401 KR909402 KR071654 KR071655 KR071656 KR071658 KR909399 KR909398 KR909448 KR909423 KR909425 KR909391 KR909407 KR058665 KR909400 KR909416 KR909417 KR909418 KR909415 KR909420 KR909419

TE D

Species F. acutatum F. acutatum F. acutatum F. acutatum F. acutatum F. acutatum F. acutatum F. acuminatum F. acuminatum F. ananatum F. ananatum F. ananatum F. ananatum F. andiyazi F. andiyazi F. andiyazi F. andiyazi F. andiyazi F. andiyazi F. anthophilum F. anthophilum F. anthophilum F. anthophilum F. anthophilum F. anthophilum F. avenaceum F. armeniacum F. beomiforme F. brevicatenulatum F. bulbicola F. chlamydosporum F. circinatum F. culmorum F. culmorum F. culmorum F. culmorum F. crookwellense F. crookwellense

AC C

CBS Number CBS 402.97 CBS 131573 CBS 130548 CBS 401.97 CBS 739.97 CBS 113964 MRC 7544 DAOMC 194173 DAOMC 230348 CBS 118516 CBS 118517 CBS 118519 CBS 184.29 CBS 119857 CBS 119856 CBS 256.93 CBS 134430 MRC 6122 MRC 8046 CBS 222.76 CBS 245.59 CBS 119858 CBS 119859 MRC 2224 DAOM 235617 DAOMC 216323 DAOMC 235626 DAOMC 235779 BBA 69197 BBA 62628 CBS 635.76 MRC 7488 HLX 1503 IMI 014764 MRC 1823 MRC 7682 DAOMC 211718 DAOMC 235772

ACCEPTED MANUSCRIPT

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28

KT003356 KT003340 KR059015 KR059018 KT003296 KR058942 KR058947 KR058945 KR058943 KR058944 KR058946 KT003323 KT003324 KT003322 KT003348 KT003295 KT003309 KR059019 KT003294 KT003321 KT070567 KT003320 KT003331 KR058973 KR058980 KR058981 KR058982 KR058979 KR058983 KR058978 KT003343 KT003313 KR058984 KR058985 KR058987 KR058986 KT003332 KT003333 KR059010 KR059011 KT003314 KT003315 KT003316

KT070634 KT070673 KR058935 KR058939 KT070680 KR058862 KR058864 KR058865 KR058866 KR058867 KR058863 KT070648 KT070649 KT070650 KT070687 KT070679 KT070644 KR058938 KT070635 KT070652 KT070696 KT070651 KT070670 KR058887 KR058869 KR058870 KR058871 KR058873 KR058872 KR058868 KT070684 KT070637 KR058888 KR058891 KR058889 KR058890 KT070668 KT070669 KR058930 KR058931 KT070638 KT070639 KT070640

SC

RI PT

KR909326 KR909385 KR071777 KR071779 KR909340 KR071741 KR071745 KR071743 KR071744 KR071742 KR071746 KR909367 KR909368 KR909369 KR909330 KR909339 KR909346 KR071780 KR909344 KR909363 KT070566 KR909361 KR909362 KR071710 KR071717 KR071714 KR071716 KR071715 KR071713 KR071712 KR909327 KR909358 KR071722 KR071723 KR071725 KR071724 KR909386 KR909387 KR071771 KR071772 KR909355 KR909356 KR909357

M AN U

KR909389 KR909405 KR058664 KR058699 KR909450 KR058666 KR058669 KR058668 KR058670 KR058667 KR058671 KR909443 KR909444 KR909440 KR909421 KR909451 KR909426 KR058700 KR909446 KR909441 KR909438 KR909439 KR909432 KR071632 KR071646 KR071647 KR071648 KR071649 KR071650 KR071645 KR909424 KR909390 KR058693 KR058694 KR058695 KR058696 KR909434 KR909435 KR058660 KR058661 KR909394 KR909395 KR909396

TE D

F. decemcellulare F. denticulatum F. equiseti F. falciforme F. flocciferum F. fujikuroi F. fujikuroi F. fujikuroi F. fujikuroi F. fujikuroi F. fujikuroi F. fujikuroi F. fujikuroi F. globosum F. graminearum F. graminum F. hostae F. keratoplasticum F. lateritium F. mangiferae F. mangiferae F. mangiferae F. mangiferae F. musae F. napiforme F. napiforme F. napiforme F. napiforme F. napiforme F. napiforme F. nelsonii F. nisikadoi F. nygamai F. nygamai F. nygamai F. nygamai F. nygamai F. nygamai F. oxysporum F. oxysporum F. oxysporum F. oxysporum F. oxysporum

AC C

DAOMC 198607 BBA 67772 CBS 307.94 CBS 135796 DAOMC 235679 CBS 221.76 CBS 119855 CBS 121864 CBS 262.54 CBS 449.95 CBS 119825 DAOMC 235805 MRC 8532 MRC 6648 DAOMC 250220 DAOMC 235644 DAOMC 235655 CBS 135806 DAOMC 235741 MRC 2730 MRC 8101 MRC 7559 MRC 3105 CBS 115135 CBS 135139 CBS 135140 CBS 135141 CBS 673.94 CBS 674.94 CBS 748.97 MRC 8391 BBA 69015 CBS 749.97 CBS 120995 CBS 140.95 CBS 131377 MRC 8547 MRC 8546 CBS 132475 CBS 463.91 DAOMC 250221 DAOMC 250222 DAOMC 235623

ACCEPTED MANUSCRIPT

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29

KR059021 KT003308 KT003351 KT003352 KR059013 KR059012 KR058948 KR058953 KR058956 KR058949 KR058957 KR058954 KR058952 KR058950 KR058955 KR058951 KT003326 KT003327 KT003325 KT003336 KT003329 KT003330 KT003310 KT003312 KT003311 KR058958 KR058959 KR058960 KR058961 KR058962 KR058964 KR058963 KT003317 KT003318 KT003319 KR059017 KT003355 KR059020 KT003301 KT003334 KT003353 KR059003 KR059004

KR058941 KT070665 KT070685 KT070686 KR058933 KR058932 KR058913 KR058915 KR058919 KR058918 KR058922 KR058920 KR058914 KR058916 KR058921 KR058917 KT070654 KT070653 KT070655 KT070676 KT070656 KT070672 KT070641 KT070643 KT070642 KR058923 KR058924 KR058925 KR058926 KR058927 KR058929 KR058928 KT070645 KT070646 KT070647 KR058937 KT070633 KR058940 KT070660 KT070674 KT070694 KR058900 KR058901

SC

RI PT

KR071782 KR909359 KR909328 KR909329 KR071773 KR071774 KR071737 KR071735 KR071731 KR071734 KR071732 KR071733 KR071736 KR071738 KR071740 KR071739 KR909366 KR909364 KR909365 KR909379 KR909384 KR909377 KR909347 KR909349 KR909348 KR071753 KR071749 KR071748 KR071750 KR071751 KR071752 KR071747 KR909336 KR909351 KR909352 KR071781 KR909325 KR071778 KR909376 KR909380 KR909338 KR071768 KR071769

M AN U

KR058702 KR909430 KR909413 KR909414 KR071662 KR071663 KR058672 KR058673 KR058674 KR058675 KR058676 KR058677 KR058678 KR058679 KR058681 KR058680 KR909436 KR909437 KR909442 KR909412 KR909431 KR909397 KR909427 KR909429 KR909428 KR071639 KR071640 KR071659 KR071642 KR071641 KR071643 KR071644 KR909419 KR909411 KR909393 KR058698 KR909388 KR058699 KR909409 KR909406 KR909422 KR071623 KR071624

TE D

F. petroliphilum F. phyllophilum F. poae F. poae F. polyphialidicum F. polyphialidicum F. proliferatum F. proliferatum F. proliferatum F. proliferatum F. proliferatum F. proliferatum F. proliferatum F. proliferatum F. proliferatum F. proliferatum F. proliferatum F. proliferatum F. proliferatum F. pseudoanthophilum F. pseudonygamai F. ramigenum F. redolens F. redolens F. redolens F. sacchari F. sacchari F. sacchari F. sacchari F. sacchari F. sacchari F. sacchari F. sacchari F. sacchari F. sacchari F. solani (5) F. solani (5) F. solani (6) Fusarium sp. Fusarium sp. F. sporotrichioides F. subglutinans F. subglutinans

AC C

CBS 135955 MRC 7543 DAOMC 177423 DAOMC 213258 CBS 961.87 CBS 676.94 CBS 184.33 CBS 183.29 CBS 116324 CBS 182.32 CBS 131391 CBS 133030 CBS 480.77 CBS 125014 CBS 132761 CBS 130179 DAOMC 232117 DAOMC 232118 BBA 63629 MRC 7530 DAOMC 235762 BBA 68592 KAS 3766 KAS 4569 KAS 3767 CBS 134.73 CBS 135142 CBS 135143 CBS 135144 CBS 135145 CBS 131369 CBS 121683 KAS 3032 ATCC 201263 NRRL 20957 CBS 135785 DAOMC 250223 CBS 135789 MRC 8553 MRC 7537 DAOMC 181169 CBS 747.97 CBS 119831

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KR059005 KT003306 KT003307 KR058988 KR058991 KR058990 KR058989 KR058992 KT003337 KR059014 KR058965 KR058969 KR058970 KR058971 KR058966 KR058967 KR058968 KR058972 KT003341 KT003342

KR058902 KT070663 KT070664 KR058874 KR058875 KR058877 KR058878 KR058876 KT070671 KR058934 KR058879 KR058881 KR058882 KR058883 KR058880 KR058885 KR058886 KR058884 KT070666 KT070667

RI PT

KR071770 KR909371 KR909372 KR071726 KR071728 KR071730 KR071729 KR071727 KR909383 KR071775 KR071703 KR071704 KR071709 KR071705 KR071708 KR071706 KR071707 KR071711 KR909353 KR909354

SC

KR071625 KR909403 KR909404 KR058688 KR058689 KR058690 KR058691 KR058692 KR909433 KR058697 KR071630 KR071631 KR071633 KR071634 KR071635 KR071636 KR071637 KR071638 KR909408 KR909410

M AN U

F. subglutinans F. succisae F. succisae F. thapsinum F. thapsinum F. thapsinum F. thapsinum F. thapsinum F. thapsinum F. tricinctum F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides

TE D

CBS 136481 DAOMC 235796 BBA 63627 CBS 776.96 CBS 109077 CBS 733.97 CBS 113963 CBS 130176 MRC 8558 CBS 253.50 CBS 576.78 CBS 102699 CBS 108922 CBS 116665 CBS 123670 CBS 579.78 CBS 122159 CBS 131390 MRC 8560 MRC 8559

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Table 1. Amplification success, phylogenetic data and the substitution models used in the phylogenetic analyses, per locus. Gene data sets ITS

TEF1

TOP1

Best model used Number of aligned sites

K2+G+I 469

K2+G+I 589

TN93+G+I TN93+G 708 828

Amplification success (%) *

100%

98%

96%

95%

Conserved sites %

319 68.01

258 43.80

342 48.3

514 62.07

Variable sites %

137 29.2

314 53.3

359 50.70

300 36.2

105 22.3

256 43.4

327 46.1

240 28.9

32 6.82

49 8.31

31 4.37

60 7.24

50.7

52.5

48.5

57.8

G+C (average) %

TE D

Singleton sites %

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Parsimony informative sites %

PGK

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Markers

AC C

EP

* based only on the 80 strains sequenced at CBS since at AAFC the ITS and TEF1 had been done previously

31

ACCEPTED MANUSCRIPT Figure Legends: Fig.1. Distribution of intra- and inter-specific pairwise distances for all strains, for each marker. The average distance is represented by the dotted lines.

RI PT

Fig. 2. Comparison of interspecific pairwise distances between each marker pair. The points along the zero line for one axis represents the species pairs that cannot be differentiated by this primer. For each point, the coordinate with the higher number is the superior marker for species differentiation. The points that are along the line of x=y represent the species pairs where the distance is the same for both markers.

M AN U

SC

Fig. 3. Intra specific variation among all strains of each species pairs for the four markers. All strains had sequence data for the four markers. The line in the middle of the box is the median. When all markers of a species do not have a box, there was only a pair of strains for this species. When there is a mixture of lines and boxes for a species, the lines represent no variation in the intraspecific pairwise distance. The bottom and the top of a box represent the 25th and 75th percentiles and the whiskers are 1.5 times the interquartile range above and below the box limits. The dots are outliers, i.e. beyond ± 2.7 standard deviations.

EP

TE D

Fig. 4. Phylogenetic tree generated by Bayesian inference (BI) and maximum likelihood (ML) trees from 144 –TEF1 sequences, 589 characters, 10,000,000 generations, 4 mcmc runs. Numbers on the branches are Bayesian posterior probabilities (PP) and percentages of 1,000 bootstrap-replications of MEGA6-maximum likelihood (PP/ML). The tree was rooted with the F. solani complex

AC C

Fig. 5. Phylogenetic tree generated by Bayesian inference (BI) and maximum likelihood (ML) trees from 144 –TOP1 sequences, 708 characters, 10,000,000 generations, 4 mcmc runs. Numbers on the branches are Bayesian posterior probabilities (PP) and percentages of 1,000 bootstrap-replications of MEGA6-maximum likelihood (PP/ML). The tree was rooted with the F. solani complex

Fig. 6. Phylogenetic tree generated by Bayesian inference (BI) and maximum likelihood (ML) trees from 144 PGK sequences, 828 characters, 10,000,000 generations, 4 mcmc runs. Numbers on the branches are Bayesian posterior probabilities (PP) and percentages 32

ACCEPTED MANUSCRIPT of 1,000 bootstrap-replications of MEGA6-maximum likelihood (PP/ML). The tree was rooted with the F. solani complex

RI PT

Supplemental Table S1. GenBank sequences of the DNA regions: ITS, TEF1, TOP1 and PGK of the isolates of the Fusarium species included in the barcoding and phylogenetic analyses.

AC C

EP

TE D

M AN U

SC

Fig 1S: Inter-specific distances for the different markers for all pairs of species. Same approach as Fig. 3.

33

ACCEPTED MANUSCRIPT

1000

inter−specific

RI PT

500

0

SC

750

PGK

M AN U

500

250

0

TE D

800 600

TOP1

EP

400 200 0 500

AC C

count

ITS

intra−specific

400 300

TEF1 200 100 0 0.0

0.1

0.2

pairwise distance

0.3

ACCEPTED MANUSCRIPT

ITS

PGK

02

TOP1

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SC

00

TS

RI PT

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02

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TE D

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00

M AN U

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PGK

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AC C

pa rw se d stance

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01

TEF1

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TEF1

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03

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pa rw se d stance

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mangiferae acuminatum ●

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ACCEPTED MANUSCRIPT

oxysporum verticillioides crookwellense

RI PT

andiyazi

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subglutinans ●

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M AN U

sacchari

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nygamai poae

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TE D

Fusarium

proliferatum

SC

thapsinum

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fujikuroi

EP

napiforme

AC C

anthophilum

acutatum

TEF1

culmorum

TOP1

redolens

PGK

succisae

ITS

polyphialidicum ananatum 0.00

0.01

0.02

0.03

pairwise distance

0.04

AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

graminum−petroliphilum petroliphilum−sporotrichioides flocciferum−petroliphilum acuminatum−petroliphilum andiyazi−petroliphilum graminum−solani−2 brevicatenulatum−petroliphilum napiforme−petroliphilum graminum−solani−6 graminum−solani−5 acuminatum−solani−2 solani−6−sporotrichioides andiyazi−solani−5 armeniacum−petroliphilum petroliphilum−ramigenum pseudoanthophilum−solani−2 andiyazi−solani−2 solani−2−sporotrichioides petroliphilum−pseudoanthophilum petroliphilum−pseudonygamai anthophilum−petroliphilum petroliphilum−succisae pseudonygamai−solani−2 brevicatenulatum−solani−2 equiseti−petroliphilum petroliphilum−subglutinans pseudoanthophilum−solani−5 chlamydosporum−petroliphilum armeniacum−solani−6 solani−2−succisae napiforme−solani−5 brevicatenulatum−solani−5 petroliphilum−verticillioides pseudonygamai−solani−5 oxysporum−petroliphilum flocciferum−solani−2 bulbicola−solani−2 acuminatum−solani−5 falciforme−graminum avenaceum−petroliphilum ananatum−petroliphilum ramigenum−solani−2 oxysporum−solani−2 petroliphilum−tricinctum armeniacum−solani−2 circinatum−petroliphilum anthophilum−solani−2 solani−2−subglutinans cf−nygamai−petroliphilum nelsonii−petroliphilum cf−nygamai−solani−2 acuminatum−keratoplasticum solani−5−subglutinans andiyazi−falciforme napiforme−solani−2 circinatum−solani−2 bulbicola−petroliphilum acuminatum−solani−6 anthophilum−solani−5 solani−5−sporotrichioides ramigenum−solani−5 avenaceum−solani−2 graminum−keratoplasticum oxysporum−solani−5 petroliphilum−thapsinum flocciferum−solani−6 cf−nygamai−solani−5 denticulatum−solani−2 solani−5−succisae andiyazi−keratoplasticum circinatum−solani−5 avenaceum−solani−6 petroliphilum−sacchari flocciferum−solani−5 ananatum−solani−5 solani−6−tricinctum nelsonii−solani−6 globosum−solani−2 denticulatum−petroliphilum globosum−petroliphilum bulbicola−solani−5 graminearum−petroliphilum ananatum−solani−2 denticulatum−solani−5 petroliphilum−phyllophilum solani−5−verticillioides equiseti−solani−5 nelsonii−solani−2 equiseti−solani−6 falciforme−napiforme falciforme−pseudoanthophilum solani−5−tricinctum brevicatenulatum−keratoplasticum acuminatum−falciforme napiforme−solani−6 solani−2−verticillioides keratoplasticum−pseudoanthophilum flocciferum−keratoplasticum phyllophilum−solani−2 brevicatenulatum−falciforme solani−2−tricinctum petroliphilum−poae equiseti−solani−2 culmorum−petroliphilum falciforme−ramigenum andiyazi−solani−6 fujikuroi−petroliphilum nelsonii−solani−5 chlamydosporum−solani−6 crookwellense−petroliphilum falciforme−pseudonygamai solani−5−thapsinum keratoplasticum−tricinctum armeniacum−solani−5 keratoplasticum−ramigenum keratoplasticum−pseudonygamai keratoplasticum−napiforme sacchari−solani−5 cf−nygamai−falciforme falciforme−flocciferum globosum−solani−5 sacchari−solani−2 keratoplasticum−oxysporum ramigenum−solani−6 cf−nygamai−keratoplasticum phyllophilum−solani−5 petroliphilum−proliferatum avenaceum−solani−5 redolens−solani−2 decemcellulare−equiseti pseudoanthophilum−solani−6 avenaceum−keratoplasticum anthophilum−solani−6 anthophilum−falciforme acutatum−petroliphilum pseudonygamai−solani−6 falciforme−oxysporum beomiforme−solani−2 falciforme−subglutinans brevicatenulatum−solani−6 solani−2−thapsinum nygamai−petroliphilum keratoplasticum−sporotrichioides anthophilum−keratoplasticum solani−6−succisae acutatum−solani−5 keratoplasticum−subglutinans falciforme−succisae decemcellulare−subglutinans nisikadoi−petroliphilum solani−6−subglutinans falciforme−sporotrichioides hostae−solani−2 denticulatum−falciforme graminearum−solani−2 acutatum−solani−2 falciforme−verticillioides keratoplasticum−succisae cf−nygamai−solani−6 petroliphilum−redolens equiseti−falciforme circinatum−falciforme ananatum−falciforme chlamydosporum−solani−5 oxysporum−solani−6 keratoplasticum−verticillioides nisikadoi−solani−2 anthophilum−decemcellulare solani−6−thapsinum culmorum−solani−2 fujikuroi−solani−5 denticulatum−keratoplasticum solani−6−verticillioides crookwellense−solani−2 armeniacum−keratoplasticum proliferatum−solani−5 armeniacum−falciforme bulbicola−solani−6 circinatum−solani−6 equiseti−keratoplasticum avenaceum−falciforme bulbicola−falciforme keratoplasticum−phyllophilum chlamydosporum−decemcellulare crookwellense−solani−6 fujikuroi−solani−2 ananatum−keratoplasticum keratoplasticum−thapsinum decemcellulare−napiforme falciforme−globosum mangiferae−petroliphilum chlamydosporum−solani−2 redolens−solani−5 falciforme−tricinctum decemcellulare−ramigenum graminearum−solani−6 mangiferae−solani−2 culmorum−solani−6 falciforme−thapsinum ananatum−solani−6 globosum−solani−6 bulbicola−keratoplasticum hostae−petroliphilum acutatum−keratoplasticum proliferatum−solani−2 falciforme−nelsonii globosum−keratoplasticum nygamai−solani−5 circinatum−keratoplasticum acutatum−falciforme decemcellulare−sporotrichioides beomiforme−solani−5 decemcellulare−succisae nygamai−solani−2 keratoplasticum−sacchari falciforme−phyllophilum falciforme−sacchari culmorum−solani−5 ananatum−decemcellulare petroliphilum−polyphialidicum andiyazi−decemcellulare armeniacum−decemcellulare bulbicola−decemcellulare graminearum−solani−5 polyphialidicum−solani−5 decemcellulare−sacchari beomiforme−petroliphilum decemcellulare−verticillioides chlamydosporum−falciforme decemcellulare−pseudonygamai crookwellense−solani−5 fujikuroi−solani−6 culmorum−decemcellulare phyllophilum−solani−6 hostae−solani−5 poae−solani−2 nisikadoi−solani−5 decemcellulare−graminearum denticulatum−solani−6 sacchari−solani−6 circinatum−decemcellulare chlamydosporum−keratoplasticum poae−solani−6 keratoplasticum−nelsonii falciforme−fujikuroi falciforme−proliferatum polyphialidicum−solani−2 acutatum−solani−6 mangiferae−solani−5 lateritium−solani−6 proliferatum−solani−6 decemcellulare−graminum crookwellense−decemcellulare decemcellulare−pseudoanthophilum poae−solani−5 graminearum−keratoplasticum keratoplasticum−nygamai falciforme−graminearum falciforme−nygamai keratoplasticum−proliferatum nygamai−solani−6 keratoplasticum−redolens fujikuroi−keratoplasticum culmorum−keratoplasticum crookwellense−keratoplasticum culmorum−falciforme crookwellense−falciforme brevicatenulatum−decemcellulare beomiforme−keratoplasticum lateritium−petroliphilum decemcellulare−oxysporum keratoplasticum−nisikadoi falciforme−nisikadoi polyphialidicum−solani−6 keratoplasticum−poae redolens−solani−6 mangiferae−solani−6 falciforme−redolens beomiforme−solani−6 nisikadoi−solani−6 hostae−keratoplasticum keratoplasticum−mangiferae hostae−solani−6 falciforme−poae lateritium−solani−2 decemcellulare−denticulatum decemcellulare−thapsinum falciforme−mangiferae falciforme−hostae decemcellulare−polyphialidicum beomiforme−falciforme cf−nygamai−decemcellulare keratoplasticum−polyphialidicum falciforme−polyphialidicum acuminatum−decemcellulare decemcellulare−phyllophilum decemcellulare−fujikuroi decemcellulare−globosum avenaceum−decemcellulare decemcellulare−flocciferum acutatum−decemcellulare lateritium−solani−5 decemcellulare−nelsonii decemcellulare−tricinctum decemcellulare−nygamai keratoplasticum−lateritium falciforme−lateritium decemcellulare−proliferatum decemcellulare−nisikadoi decemcellulare−redolens decemcellulare−mangiferae decemcellulare−poae beomiforme−decemcellulare decemcellulare−hostae decemcellulare−lateritium armeniacum−flocciferum chlamydosporum−graminum equiseti−graminum equiseti−lateritium chlamydosporum−lateritium armeniacum−graminum flocciferum−poae graminum−sporotrichioides nelsonii−subglutinans graminum−poae poae−tricinctum armeniacum−lateritium nelsonii−sacchari lateritium−sporotrichioides acutatum−armeniacum flocciferum−subglutinans acuminatum−poae napiforme−nelsonii graminearum−graminum andiyazi−nelsonii avenaceum−poae flocciferum−sporotrichioides ananatum−nelsonii acuminatum−armeniacum nelsonii−verticillioides nelsonii−oxysporum graminum−polyphialidicum nelsonii−pseudoanthophilum acuminatum−subglutinans culmorum−graminum acuminatum−sacchari armeniacum−cf−nygamai chlamydosporum−flocciferum culmorum−lateritium avenaceum−napiforme acuminatum−chlamydosporum ananatum−flocciferum avenaceum−subglutinans avenaceum−equiseti acuminatum−equiseti avenaceum−sacchari armeniacum−tricinctum nelsonii−pseudonygamai acuminatum−napiforme graminearum−lateritium flocciferum−graminearum nelsonii−nisikadoi armeniacum−avenaceum armeniacum−phyllophilum chlamydosporum−phyllophilum brevicatenulatum−nelsonii acutatum−sporotrichioides subglutinans−tricinctum crookwellense−lateritium anthophilum−nelsonii acutatum−chlamydosporum anthophilum−flocciferum cf−nygamai−chlamydosporum acuminatum−sporotrichioides sacchari−tricinctum bulbicola−nelsonii avenaceum−chlamydosporum flocciferum−succisae circinatum−flocciferum armeniacum−hostae acuminatum−ananatum graminum−napiforme bulbicola−flocciferum lateritium−poae andiyazi−lateritium napiforme−tricinctum andiyazi−avenaceum nelsonii−succisae lateritium−napiforme ananatum−graminum cf−nygamai−sporotrichioides flocciferum−pseudonygamai bulbicola−tricinctum crookwellense−graminum acuminatum−bulbicola nelsonii−ramigenum graminum−subglutinans avenaceum−pseudoanthophilum armeniacum−redolens acuminatum−anthophilum avenaceum−succisae circinatum−nelsonii acutatum−culmorum flocciferum−pseudoanthophilum lateritium−verticillioides flocciferum−sacchari anthophilum−avenaceum acuminatum−pseudoanthophilum avenaceum−bulbicola anthophilum−graminum chlamydosporum−thapsinum acuminatum−succisae sporotrichioides−tricinctum pseudoanthophilum−tricinctum phyllophilum−sporotrichioides acuminatum−circinatum avenaceum−sporotrichioides lateritium−pseudonygamai circinatum−tricinctum avenaceum−ramigenum culmorum−globosum armeniacum−thapsinum crookwellense−flocciferum avenaceum−circinatum lateritium−subglutinans ananatum−avenaceum graminum−succisae acuminatum−andiyazi circinatum−graminum denticulatum−nelsonii culmorum−flocciferum andiyazi−graminum bulbicola−graminum pseudonygamai−tricinctum avenaceum−verticillioides armeniacum−globosum acuminatum−ramigenum acuminatum−graminearum flocciferum−verticillioides succisae−tricinctum cf−nygamai−nelsonii equiseti−phyllophilum equiseti−flocciferum andiyazi−tricinctum acuminatum−oxysporum equiseti−tricinctum avenaceum−oxysporum armeniacum−fujikuroi culmorum−fujikuroi ramigenum−tricinctum anthophilum−tricinctum graminum−verticillioides chlamydosporum−tricinctum armeniacum−beomiforme chlamydosporum−globosum ananatum−tricinctum acuminatum−pseudonygamai avenaceum−graminearum avenaceum−pseudonygamai nelsonii−polyphialidicum avenaceum−brevicatenulatum armeniacum−proliferatum acutatum−graminearum flocciferum−oxysporum globosum−graminearum acutatum−crookwellense lateritium−sacchari andiyazi−flocciferum culmorum−phyllophilum lateritium−pseudoanthophilum oxysporum−tricinctum lateritium−ramigenum brevicatenulatum−flocciferum graminum−pseudonygamai crookwellense−globosum graminearum−tricinctum acuminatum−verticillioides flocciferum−napiforme flocciferum−nisikadoi armeniacum−nygamai chlamydosporum−nygamai globosum−sporotrichioides chlamydosporum−fujikuroi acuminatum−brevicatenulatum cf−nygamai−equiseti graminum−sacchari circinatum−lateritium graminum−ramigenum brevicatenulatum−lateritium sporotrichioides−thapsinum chlamydosporum−proliferatum beomiforme−poae brevicatenulatum−tricinctum cf−nygamai−culmorum fujikuroi−graminearum armeniacum−mangiferae graminum−nelsonii culmorum−proliferatum acutatum−equiseti avenaceum−denticulatum ananatum−lateritium tricinctum−verticillioides acuminatum−denticulatum beomiforme−sporotrichioides acuminatum−culmorum graminearum−phyllophilum anthophilum−lateritium denticulatum−flocciferum equiseti−globosum equiseti−proliferatum denticulatum−tricinctum bulbicola−lateritium denticulatum−graminum fujikuroi−sporotrichioides graminearum−proliferatum crookwellense−fujikuroi culmorum−thapsinum globosum−nelsonii avenaceum−culmorum equiseti−fujikuroi hostae−sporotrichioides crookwellense−phyllophilum graminum−oxysporum proliferatum−sporotrichioides equiseti−thapsinum acuminatum−crookwellense flocciferum−ramigenum avenaceum−nisikadoi denticulatum−lateritium flocciferum−globosum graminum−pseudoanthophilum acuminatum−globosum acuminatum−nisikadoi lateritium−oxysporum brevicatenulatum−graminum cf−nygamai−crookwellense flocciferum−fujikuroi acuminatum−cf−nygamai redolens−sporotrichioides graminum−nisikadoi nisikadoi−tricinctum acutatum−nelsonii nelsonii−thapsinum avenaceum−crookwellense culmorum−tricinctum crookwellense−proliferatum nygamai−sporotrichioides avenaceum−phyllophilum crookwellense−thapsinum chlamydosporum−mangiferae acutatum−poae cf−nygamai−tricinctum acuminatum−fujikuroi crookwellense−tricinctum cf−nygamai−graminearum lateritium−nisikadoi decemcellulare−petroliphilum nelsonii−phyllophilum lateritium−succisae armeniacum−subglutinans culmorum−nygamai chlamydosporum−hostae globosum−tricinctum graminearum−thapsinum equiseti−mangiferae beomiforme−graminearum avenaceum−globosum poae−thapsinum hostae−poae mangiferae−sporotrichioides avenaceum−cf−nygamai acuminatum−acutatum nelsonii−proliferatum equiseti−nygamai acutatum−graminum beomiforme−culmorum acuminatum−nelsonii graminum−phyllophilum culmorum−mangiferae flocciferum−nelsonii flocciferum−proliferatum chlamydosporum−redolens acuminatum−phyllophilum armeniacum−oxysporum fujikuroi−nelsonii avenaceum−fujikuroi poae−redolens crookwellense−nygamai fujikuroi−tricinctum equiseti−hostae beomiforme−chlamydosporum acutatum−avenaceum globosum−poae culmorum−hostae globosum−lateritium flocciferum−phyllophilum graminearum−nygamai phyllophilum−tricinctum armeniacum−succisae graminearum−hostae avenaceum−polyphialidicum armeniacum−sacchari flocciferum−polyphialidicum avenaceum−thapsinum cf−nygamai−poae acuminatum−proliferatum acutatum−tricinctum beomiforme−nelsonii poae−proliferatum cf−nygamai−graminum ananatum−armeniacum lateritium−nelsonii fujikuroi−poae armeniacum−bulbicola acutatum−flocciferum cf−nygamai−flocciferum fujikuroi−graminum andiyazi−culmorum acuminatum−thapsinum decemcellulare−solani−6 armeniacum−circinatum beomiforme−equiseti crookwellense−hostae lateritium−thapsinum equiseti−redolens phyllophilum−poae lateritium−proliferatum lateritium−polyphialidicum chlamydosporum−sacchari graminearum−mangiferae nelsonii−nygamai culmorum−redolens mangiferae−nelsonii graminearum−redolens flocciferum−mangiferae armeniacum−brevicatenulatum beomiforme−crookwellense oxysporum−sporotrichioides anthophilum−armeniacum armeniacum−napiforme nygamai−poae fujikuroi−lateritium globosum−graminum avenaceum−nelsonii graminum−thapsinum proliferatum−tricinctum ACCEPTED MANUSCRIPT avenaceum−proliferatum acuminatum−mangiferae culmorum−napiforme crookwellense−mangiferae culmorum−subglutinans decemcellulare−solani−2 armeniacum−pseudoanthophilum armeniacum−pseudonygamai acutatum−lateritium culmorum−pseudoanthophilum crookwellense−redolens armeniacum−nisikadoi graminum−hostae culmorum−ramigenum acuminatum−polyphialidicum culmorum−pseudonygamai andiyazi−graminearum graminum−proliferatum cf−nygamai−lateritium sporotrichioides−subglutinans andiyazi−crookwellense mangiferae−poae acuminatum−nygamai flocciferum−lateritium hostae−nelsonii nelsonii−tricinctum andiyazi−armeniacum thapsinum−tricinctum nelsonii−redolens graminearum−napiforme beomiforme−tricinctum beomiforme−flocciferum beomiforme−graminum flocciferum−thapsinum andiyazi−sporotrichioides avenaceum−nygamai graminearum−subglutinans culmorum−sacchari avenaceum−mangiferae bulbicola−culmorum crookwellense−pseudoanthophilum crookwellense−ramigenum crookwellense−napiforme circinatum−sporotrichioides brevicatenulatum−culmorum nisikadoi−sporotrichioides mangiferae−tricinctum crookwellense−subglutinans graminearum−ramigenum pseudoanthophilum−sporotrichioides circinatum−culmorum crookwellense−pseudonygamai graminum−nygamai graminearum−pseudonygamai avenaceum−beomiforme ananatum−sporotrichioides flocciferum−nygamai sporotrichioides−succisae bulbicola−sporotrichioides graminum−lateritium brevicatenulatum−crookwellense ananatum−culmorum napiforme−sporotrichioides graminum−mangiferae armeniacum−ramigenum graminearum−pseudoanthophilum brevicatenulatum−sporotrichioides lateritium−nygamai culmorum−oxysporum avenaceum−lateritium pseudonygamai−sporotrichioides graminum−redolens chlamydosporum−subglutinans brevicatenulatum−graminearum culmorum−verticillioides bulbicola−graminearum anthophilum−sporotrichioides flocciferum−hostae armeniacum−verticillioides chlamydosporum−pseudonygamai crookwellense−sacchari circinatum−graminearum acuminatum−beomiforme chlamydosporum−napiforme polyphialidicum−tricinctum anthophilum−culmorum nygamai−tricinctum beomiforme−lateritium culmorum−succisae ananatum−graminearum culmorum−denticulatum crookwellense−oxysporum flocciferum−redolens bulbicola−chlamydosporum armeniacum−denticulatum brevicatenulatum−chlamydosporum crookwellense−verticillioides graminearum−sacchari bulbicola−crookwellense graminearum−verticillioides andiyazi−chlamydosporum graminearum−oxysporum sacchari−sporotrichioides chlamydosporum−pseudoanthophilum circinatum−crookwellense chlamydosporum−ramigenum ananatum−chlamydosporum ananatum−crookwellense acuminatum−lateritium decemcellulare−solani−5 culmorum−polyphialidicum graminearum−succisae anthophilum−graminearum sporotrichioides−verticillioides chlamydosporum−verticillioides lateritium−mangiferae culmorum−nisikadoi chlamydosporum−succisae crookwellense−denticulatum lateritium−tricinctum denticulatum−graminearum decemcellulare−keratoplasticum ramigenum−sporotrichioides denticulatum−sporotrichioides anthophilum−crookwellense crookwellense−nisikadoi equiseti−sacchari chlamydosporum−denticulatum lateritium−phyllophilum chlamydosporum−circinatum equiseti−subglutinans crookwellense−succisae anthophilum−chlamydosporum equiseti−polyphialidicum graminearum−polyphialidicum graminearum−nisikadoi chlamydosporum−polyphialidicum armeniacum−polyphialidicum hostae−tricinctum acuminatum−hostae avenaceum−hostae redolens−tricinctum equiseti−pseudonygamai chlamydosporum−nisikadoi acuminatum−redolens avenaceum−redolens ananatum−equiseti chlamydosporum−oxysporum bulbicola−equiseti brevicatenulatum−equiseti equiseti−nelsonii equiseti−succisae crookwellense−polyphialidicum equiseti−verticillioides anthophilum−equiseti decemcellulare−falciforme equiseti−pseudoanthophilum poae−subglutinans poae−polyphialidicum equiseti−nisikadoi circinatum−equiseti oxysporum−poae andiyazi−equiseti hostae−lateritium denticulatum−equiseti equiseti−napiforme equiseti−ramigenum nisikadoi−poae polyphialidicum−sporotrichioides andiyazi−poae chlamydosporum−nelsonii poae−pseudonygamai circinatum−poae bulbicola−poae lateritium−redolens polyphialidicum−thapsinum brevicatenulatum−poae poae−pseudoanthophilum ananatum−poae equiseti−oxysporum napiforme−poae poae−sacchari anthophilum−poae denticulatum−poae poae−succisae globosum−polyphialidicum polyphialidicum−proliferatum poae−verticillioides poae−ramigenum fujikuroi−polyphialidicum acutatum−polyphialidicum phyllophilum−polyphialidicum armeniacum−nelsonii graminearum−nelsonii cf−nygamai−polyphialidicum mangiferae−polyphialidicum nygamai−polyphialidicum culmorum−nelsonii flocciferum−graminum armeniacum−chlamydosporum beomiforme−polyphialidicum crookwellense−nelsonii polyphialidicum−ramigenum nelsonii−poae nelsonii−sporotrichioides armeniacum−equiseti napiforme−polyphialidicum hostae−polyphialidicum andiyazi−polyphialidicum avenaceum−graminum beomiforme−subglutinans nisikadoi−polyphialidicum graminum−tricinctum polyphialidicum−verticillioides polyphialidicum−succisae polyphialidicum−pseudonygamai anthophilum−polyphialidicum polyphialidicum−subglutinans polyphialidicum−pseudoanthophilum acuminatum−graminum beomiforme−ramigenum brevicatenulatum−polyphialidicum polyphialidicum−sacchari polyphialidicum−redolens bulbicola−polyphialidicum chlamydosporum−poae equiseti−graminearum ananatum−beomiforme circinatum−polyphialidicum ananatum−polyphialidicum chlamydosporum−sporotrichioides anthophilum−beomiforme chlamydosporum−graminearum beomiforme−succisae beomiforme−pseudoanthophilum beomiforme−pseudonygamai beomiforme−brevicatenulatum beomiforme−sacchari beomiforme−napiforme chlamydosporum−crookwellense beomiforme−bulbicola beomiforme−verticillioides equiseti−poae chlamydosporum−culmorum crookwellense−equiseti andiyazi−beomiforme beomiforme−circinatum culmorum−equiseti denticulatum−polyphialidicum equiseti−sporotrichioides beomiforme−nisikadoi oxysporum−polyphialidicum beomiforme−denticulatum beomiforme−phyllophilum beomiforme−oxysporum beomiforme−cf−nygamai beomiforme−globosum beomiforme−thapsinum beomiforme−proliferatum beomiforme−fujikuroi acutatum−beomiforme beomiforme−nygamai beomiforme−mangiferae ramigenum−redolens armeniacum−graminearum hostae−ramigenum graminearum−poae redolens−sacchari hostae−sacchari culmorum−poae hostae−subglutinans redolens−subglutinans napiforme−redolens andiyazi−redolens ananatum−redolens andiyazi−hostae anthophilum−redolens redolens−verticillioides redolens−succisae hostae−napiforme armeniacum−culmorum bulbicola−redolens anthophilum−hostae circinatum−redolens hostae−verticillioides crookwellense−poae ananatum−hostae hostae−succisae bulbicola−hostae pseudoanthophilum−redolens brevicatenulatum−redolens circinatum−hostae pseudonygamai−redolens hostae−pseudoanthophilum brevicatenulatum−hostae hostae−pseudonygamai armeniacum−crookwellense beomiforme−hostae beomiforme−redolens armeniacum−poae nisikadoi−thapsinum nisikadoi−phyllophilum graminearum−sporotrichioides denticulatum−hostae nisikadoi−proliferatum denticulatum−redolens globosum−nisikadoi fujikuroi−nisikadoi oxysporum−proliferatum oxysporum−thapsinum acutatum−nisikadoi globosum−oxysporum poae−sporotrichioides nisikadoi−redolens hostae−nisikadoi fujikuroi−subglutinans fujikuroi−oxysporum cf−nygamai−nisikadoi culmorum−sporotrichioides oxysporum−phyllophilum phyllophilum−subglutinans subglutinans−thapsinum globosum−napiforme proliferatum−subglutinans globosum−subglutinans proliferatum−ramigenum crookwellense−sporotrichioides globosum−ramigenum globosum−verticillioides napiforme−proliferatum cf−nygamai−oxysporum bulbicola−thapsinum sacchari−thapsinum bulbicola−fujikuroi ananatum−globosum andiyazi−proliferatum ananatum−thapsinum ananatum−fujikuroi hostae−phyllophilum acutatum−oxysporum globosum−hostae fujikuroi−napiforme nisikadoi−nygamai mangiferae−nisikadoi proliferatum−succisae globosum−succisae fujikuroi−ramigenum fujikuroi−succisae proliferatum−verticillioides succisae−thapsinum globosum−pseudonygamai redolens−thapsinum globosum−redolens brevicatenulatum−proliferatum circinatum−fujikuroi ananatum−proliferatum proliferatum−pseudonygamai ananatum−phyllophilum andiyazi−globosum proliferatum−redolens anthophilum−globosum mangiferae−subglutinans bulbicola−proliferatum phyllophilum−redolens circinatum−thapsinum anthophilum−thapsinum fujikuroi−verticillioides hostae−proliferatum anthophilum−proliferatum cf−nygamai−subglutinans anthophilum−fujikuroi circinatum−globosum mangiferae−oxysporum proliferatum−pseudoanthophilum fujikuroi−hostae hostae−thapsinum circinatum−proliferatum bulbicola−phyllophilum fujikuroi−pseudonygamai bulbicola−globosum phyllophilum−sacchari brevicatenulatum−globosum anthophilum−phyllophilum cf−nygamai−sacchari andiyazi−fujikuroi acutatum−sacchari fujikuroi−redolens oxysporum−redolens nygamai−oxysporum phyllophilum−succisae anthophilum−cf−nygamai ananatum−cf−nygamai cf−nygamai−hostae cf−nygamai−succisae bulbicola−cf−nygamai cf−nygamai−redolens avenaceum−flocciferum hostae−oxysporum brevicatenulatum−fujikuroi globosum−pseudoanthophilum circinatum−phyllophilum ananatum−mangiferae acutatum−redolens acutatum−hostae nygamai−sacchari acutatum−subglutinans mangiferae−ramigenum nisikadoi−ramigenum cf−nygamai−circinatum mangiferae−succisae acutatum−ramigenum andiyazi−phyllophilum fujikuroi−pseudoanthophilum bulbicola−mangiferae denticulatum−proliferatum anthophilum−mangiferae mangiferae−redolens mangiferae−napiforme hostae−mangiferae andiyazi−mangiferae circinatum−mangiferae phyllophilum−verticillioides napiforme−phyllophilum nygamai−redolens phyllophilum−ramigenum globosum−sacchari denticulatum−globosum fujikuroi−sacchari nygamai−subglutinans cf−nygamai−verticillioides mangiferae−verticillioides nisikadoi−sacchari andiyazi−cf−nygamai acutatum−succisae denticulatum−fujikuroi hostae−nygamai cf−nygamai−ramigenum nisikadoi−verticillioides mangiferae−sacchari acutatum−bulbicola nisikadoi−subglutinans brevicatenulatum−mangiferae acutatum−ananatum circinatum−nisikadoi acutatum−anthophilum mangiferae−pseudonygamai nisikadoi−succisae acutatum−brevicatenulatum proliferatum−sacchari acutatum−verticillioides brevicatenulatum−phyllophilum cf−nygamai−napiforme mangiferae−pseudoanthophilum nygamai−succisae phyllophilum−pseudonygamai napiforme−nisikadoi phyllophilum−pseudoanthophilum cf−nygamai−pseudoanthophilum brevicatenulatum−cf−nygamai brevicatenulatum−thapsinum ramigenum−thapsinum acutatum−pseudoanthophilum ananatum−nygamai acutatum−napiforme acutatum−circinatum acutatum−pseudonygamai brevicatenulatum−nisikadoi ananatum−nisikadoi flocciferum−tricinctum anthophilum−nygamai cf−nygamai−pseudonygamai bulbicola−nygamai andiyazi−nisikadoi oxysporum−ramigenum denticulatum−phyllophilum anthophilum−nisikadoi pseudoanthophilum−thapsinum nisikadoi−pseudoanthophilum nisikadoi−pseudonygamai acuminatum−flocciferum bulbicola−nisikadoi oxysporum−sacchari acutatum−andiyazi andiyazi−thapsinum pseudonygamai−thapsinum oxysporum−subglutinans keratoplasticum−petroliphilum oxysporum−succisae circinatum−nygamai thapsinum−verticillioides bulbicola−oxysporum globosum−thapsinum cf−nygamai−globosum napiforme−oxysporum proliferatum−thapsinum denticulatum−mangiferae brevicatenulatum−oxysporum anthophilum−oxysporum cf−nygamai−denticulatum circinatum−oxysporum sacchari−subglutinans oxysporum−pseudoanthophilum falciforme−petroliphilum oxysporum−verticillioides cf−nygamai−proliferatum nygamai−ramigenum ananatum−oxysporum petroliphilum−solani−2 petroliphilum−solani−5 acutatum−denticulatum denticulatum−nisikadoi andiyazi−oxysporum globosum−phyllophilum napiforme−thapsinum sacchari−succisae oxysporum−pseudonygamai fujikuroi−thapsinum ramigenum−sacchari brevicatenulatum−sacchari bulbicola−sacchari nygamai−pseudoanthophilum ramigenum−subglutinans circinatum−sacchari sacchari−verticillioides andiyazi−sacchari chlamydosporum−equiseti nygamai−pseudonygamai fujikuroi−phyllophilum cf−nygamai−fujikuroi phyllophilum−proliferatum nygamai−verticillioides brevicatenulatum−nygamai andiyazi−nygamai ananatum−sacchari anthophilum−sacchari denticulatum−thapsinum solani−2−solani−6 solani−5−solani−6 pseudoanthophilum−sacchari brevicatenulatum−subglutinans napiforme−sacchari acutatum−globosum napiforme−nygamai pseudoanthophilum−subglutinans brevicatenulatum−succisae ramigenum−succisae pseudonygamai−subglutinans andiyazi−subglutinans pseudoanthophilum−succisae keratoplasticum−solani−6 pseudonygamai−sacchari bulbicola−ramigenum acutatum−proliferatum anthophilum−ramigenum circinatum−ramigenum pseudonygamai−succisae acutatum−fujikuroi bulbicola−pseudonygamai bulbicola−pseudoanthophilum brevicatenulatum−bulbicola falciforme−solani−6 anthophilum−brevicatenulatum subglutinans−verticillioides circinatum−pseudoanthophilum anthophilum−pseudoanthophilum ananatum−ramigenum napiforme−subglutinans circinatum−pseudonygamai brevicatenulatum−circinatum anthophilum−pseudonygamai globosum−nygamai andiyazi−succisae ananatum−brevicatenulatum phyllophilum−thapsinum armeniacum−sporotrichioides ananatum−pseudonygamai acutatum−thapsinum cf−nygamai−mangiferae andiyazi−bulbicola ananatum−pseudoanthophilum napiforme−succisae nisikadoi−oxysporum succisae−verticillioides mangiferae−thapsinum circinatum−napiforme denticulatum−sacchari bulbicola−verticillioides fujikuroi−nygamai ananatum−andiyazi denticulatum−oxysporum andiyazi−circinatum andiyazi−anthophilum cf−nygamai−phyllophilum petroliphilum−solani−6 nygamai−proliferatum mangiferae−phyllophilum denticulatum−nygamai circinatum−verticillioides anthophilum−verticillioides bulbicola−napiforme anthophilum−napiforme acutatum−cf−nygamai ananatum−verticillioides acutatum−mangiferae ananatum−napiforme denticulatum−subglutinans denticulatum−succisae acutatum−phyllophilum cf−nygamai−thapsinum anthophilum−denticulatum bulbicola−denticulatum ananatum−denticulatum circinatum−denticulatum nygamai−phyllophilum acuminatum−avenaceum brevicatenulatum−verticillioides pseudoanthophilum−verticillioides mangiferae−nygamai brevicatenulatum−ramigenum acutatum−nygamai ramigenum−verticillioides brevicatenulatum−napiforme avenaceum−tricinctum globosum−mangiferae denticulatum−ramigenum napiforme−pseudoanthophilum pseudonygamai−verticillioides pseudoanthophilum−ramigenum pseudonygamai−ramigenum fujikuroi−mangiferae mangiferae−proliferatum cf−nygamai−nygamai brevicatenulatum−denticulatum napiforme−verticillioides nygamai−thapsinum keratoplasticum−solani−2 keratoplasticum−solani−5 denticulatum−pseudonygamai andiyazi−verticillioides andiyazi−denticulatum andiyazi−brevicatenulatum napiforme−pseudonygamai andiyazi−ramigenum denticulatum−pseudoanthophilum andiyazi−napiforme denticulatum−verticillioides denticulatum−napiforme acuminatum−tricinctum andiyazi−pseudonygamai brevicatenulatum−pseudonygamai falciforme−solani−2 falciforme−solani−5 andiyazi−pseudoanthophilum napiforme−ramigenum pseudoanthophilum−pseudonygamai falciforme−keratoplasticum circinatum−subglutinans fujikuroi−globosum fujikuroi−proliferatum culmorum−graminearum crookwellense−graminearum hostae−redolens globosum−proliferatum subglutinans−succisae ananatum−circinatum ananatum−succisae circinatum−succisae anthophilum−subglutinans brevicatenulatum−pseudoanthophilum ananatum−subglutinans bulbicola−subglutinans anthophilum−circinatum bulbicola−succisae ananatum−bulbicola ananatum−anthophilum crookwellense−culmorum bulbicola−circinatum anthophilum−bulbicola anthophilum−succisae solani−2−solani−5

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0.0

0.1

0.2

pairwise distance