Mycotoxins, 63 (2), 133-142 (2013) 133

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Molecular phylogeny and identification of Fusarium species based on nucleotide sequences

Maiko WATANABE＊

Department of Microiology, Nationa Institute of Health Sciences Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan

Key words：Fusarium species; genetic marker; molecular phylogenetic analysis; nucleotide sequence

(Received August 24, 2013)

Abstract

　Species of the genus Fusarium are well-researched in many fields, and a commonly problem by researchers inter-ested in Fusarium species is the probable taxonomic system and the identification method of this genus. The traditional taxonomic system for fungi has been proposed based on the mainly morphological species concept, including the genus Fusarium. Recently, many researchers have applied molecular markers to examine the taxonomy and identifi-cation of Fusarium species. This review shows some recent findings from our studies about molecular phylogeny and identification of Fusarium species based on analyses with nucleotide sequences. First, the genetic markers were evaluated for identifying Fusarium isolates by calculation of the homologies with pairwise comparison of all tested strains, and of the ratio of nucleotide substitution rate. It was suggested that aminoadipate reductase gene (lys2) is notionally the most appropriate genetic marker for identifying isolates among the six genes examined. Second, actual identification of food-borne isolates of the genus Fusarium based on the nucleotide sequence homology was performed, and the results were evaluated. In terms of accuracy and ease, b-tubulin gene, not lys2, is the most useful genetic maker among the six genes examined. Finally, the genetic markers were evaluated for the phylogenetic analysis of Fusarium species. It was suggested the lys 2 have a singular evolutionary history than other genes. To obtain a reliable phylogeny for Fusarium species, the lys2 sequences were excluded from the dataset, and the species tree was inferred. The reliable Fusarium species tree was reconstructed, and some interesting relationships were newly described.

Introduction

　　Species of the genus Fusarium are well-researched in many fields, such as ecology, plant pathology, medical-mycology and toxicology because they are important fungi with phytopathogenicity, human-infec-tivity and mycotoxin- productivity, which are associated with human and animal health hazards1, 2). One problem commonly encountered by researchers interested in Fusarium species is the probable taxonomic system and the identification method of this genus. 　　In general, species are recognized on the basis of the morphological species concept, the biological

Award Review

Corresponding Author＊ Department of Microiology, Nationa Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan. Tel: ＋81-xx-xxx-xxxx. Fax: ＋81-xx-xxx-xxxx. E-mail: mwatanabe@nifhs.go.jp

A full color PDF reprint of this article is available at the journal WEB site.

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species concept, the phylogenetic species concept or a combination of these3). The traditional taxonomic system for fungi has been proposed based on the mainly morphological species concept, including the genus Fusarium. The taxonomy of this genus has been debated for many years4-8). Recently, many researchers have applied molecular phylogenetic analysis to examine the taxonomy of Fusarium species, and have proposed new taxonomic systems based on the phylogenetic species concept. These molecular approaches achieved some positive results in the phylogenetic problems which could not be elucidated with morphological markers. Some previous studies, however, have shown phylogenetic trees with a low resolution in higher or lower taxonomy of Fusarium species, due to use of molecular markers which have a lack of suitable nucle-otide or amino acid substitution rates, and other factors. Therefore, it would appear that many phylogenetic relationships remain unclear, and there is no molecular marker for reconstruction of the reliable phylogenetic tree of all of Fusarium species. 　　Because current classification schemes of fungi are mainly based on the morphological species concept, described above, identification of the species primarily involves the use of morphological characteristic9). Recently, researchers have developed many strategies for rapid and easy molecular identification of fungal isolates, such as by the nucleotide sequences homology search with reference sequences in database10), recon-struction of phylogenetic tree with reference sequences11) and PCR-based assays with species-specific regions of genes12). There are also some problems in the field of molecular identification of Fusarium species. One problem is few species-specific diagnoses in sequences of the genetic markers commonly used for identifi-cation among the closely related species, such as species complexes13). Another problem is an inconsistency between morphological species concept and phylogenetic species concept14). This inconsistency sometimes hides the true phylogenetic position of a target-strain in the tree, making it impossible to be identified. This situation is thought to be one of the major difficulties in identification of Fusarium isolates with molecular methods. We must reconstruct a reliable species tree based on not only molecular but also morphological and other biological characteristics. 　　This review shows some recent findings from our studies about molecular phylogeny and identification of Fusarium species based on analyses with nucleotide sequences.

Evaluation of genetic markers for identifying isolates of the species of the genus Fusarium

　　Although many researchers studied about identification of Fusarium species with various genetic markers, a suitable marker for identification of all Fusarium species have yet to be revealed. Therefore, to overcome this issue, it is necessary to identify gene (s) that has a high evolutionary rate and have the species-specific diagnosis. To specify gene appropriate for identifying isolates of various Fusarium species, Watanabe et al.15) evaluated the 18S rRNA gene (rDNA), internal transcribed spacer region 1, 5. 8S rDNA, 28S rDNA, b-tubulin gene (β-tub), and aminoadipate reductase gene (lys2).　　The nucleotide sequences of these genes were determined and calculated homologies with pairwise comparison of all tested strains (Table 1). Inter-species nucleotide sequence homology of β-tub and lys2 ranged from 83.5 to 99.4% and 56.5 to 99.0%, respectively. The result indicated that sequence homologies of these genes against reference sequences in database have a high possibility to identify unknown Fusarium isolates when it is more than 99.0%, because these genes had no inter-species pairwise combinations that had 100% homologies, unlike with other markers. Subsequently, the ratio of the nucleotide substitution rates of

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each gene in genus Fusarium and related genera were inferred (Table 2). The higher this rate is, the easier it is to accumulate the species-specific diagnostic substitution in the nucleotide sequence of the genes. The result indicated that when the value of 18S rDNA was set to 1.0, the value of β-tub and lys2 were 10.1 and 26.3, meaning that the nucleotide substitution rate of lys2 was the highest among the six genes. 　　These results indicated that the lys2 is the most appropriate genetic marker with high resolution for identifying isolates of the genus Fusarium among the six genes examined. Therefore, when the lys2 is applied to identifying isolates of taxonomic groups which have been a subject of controversy for many years, this gene is expected to resolve difficulties for identification.

Identification of food-borne isolates of the genus Fusarium based on the nucleotide sequence homology

　　Fusarium species is known to be one of the most difficult species to be identified based on morpho-logical markers among fungal species. One of the main reasons for this difficulty is that genetic and morpho-logical characters vary among strains in a species and the ranges of character diversity are often overlapped among closely-related species. In such case, multiple species which are not easily distinguished each other are sometimes recognized as a “species complex”16). Many species complexes, such as F. equiseti/semitectum species complex, F. solani species complex, and Gibberella fujikuroi species complex, are often detected from numerous fruits and vegetables1). Therefore, it is important to develop an identification-method, which

Table 1.　 Comparison of nucleotide sequence homologies of nucleotide among six genes

GeneNucleotide sequence homology (%)

Intra-species Inter-species

18S rDNA 99.2-100.0 96.1-100.0ITS 1 84.0-100.0 65.0-100.05.8S rDNA 98.1-100.0 93.7-100.028S rDNA 98.9-100.0 91.1-100.0β-tuba 93.0-100.0 83.5- 99.4lys 2b 86.7-100.0 51.9- 99.0

aβ-tubulin genebAminoadipate reductase gene

Table 2.　 Comparison of ratios of nucleotide substitution rate of sequences among six genes

Gene Ratio of substitution rate

18S rDNA 1.0ITS 1 13.95.8S rDNA 1.528S rDNA 4.0β-tuba 10.1lys 2b 26.3

aβ-tubulin genebAminoadipate reductase gene

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Table 3.　 Identification of candidate species of Fusaium isolates based on mophological methods and nucleotide sequence homologies

IsolateIdentification based on morphological

methodsa

Identification based on nucleotide

sequence homology

Nucleotide sequence homology (%)

Reference strain 18SrDNA ITS1 5.8S

rDNA28S

rDNA β-tub lys2

Ap-1 F. oxysporumITS1, 28S rDNA,

β-tub, lys2→ F. oxysporum

F. oxysporumMAFF240304 100.00 100.00 100.00 99.78 98.64 96.72

F. oxysporumMAFF240321 100.00 100.00 100.00 99.78 98.64 96.72

F. proliferatum(F. phylophilum)

CBS216.76100.00 99.17 100.00 99.35 98.46 96.23

Ba-1 F. solani28S rDNA,β-tub, lys2→ F. solani

F. solaniMAFF238538 100.00 97.54 100.00 100.00 98.46 ―

F. solaniNBRC8505 100.00 94.22 100.00 99.14 97.95 ―

F. solaniMAFF239038 99.39 90.08 98.11 98.27 93.69 ―

F. decemcellulareMAFF238421 99.80 71.90 98.74 97.19 92.83

Ba-3 F. semitectum ITS1, β-tub, lys2→ semitectum

F. semitectumMAFF236521 100.00 100.00 100.00 100.00 99.83 90.37

F. equisetiMAFF236434 100.00 99.17 100.00 100.00 98.98 92.01

F. equisetiMAFF236723 100.00 99.17 98.73 100.00 98.46 90.98

Co-1 F. verticillioidesb

28S rDNA,→ F. verticillioidesb

F. verticillioidesb

(F. verticillioidesc)CBS576.78

100.00 100.00 100.00 100.00 100.00 100.00

b-tub, lys2→ F. verticillioidesc

F. proliferatumb

(F. phyllophilum)CBS216.76

100.00 100.00 100.00 99.57 98.64 97.32

F. verticillioidesb

(F. thapsinum)CBS100312

99.80 100.00 100.00 100.00 97.95 97.17

Co-2 F. subglutinansb β-tub, lys2→ F. subglutinansc

F. subglutinansb

(F. subglutinansc)ATCC38016

100.00 100.00 100.00 100.00 100.00 100.00

F. oxysporumMAFF240304 100.00 99.17 100.00 99.35 97.44 94.72

F. oxysporumMAFF240321 100.00 99.17 100.00 99.35 97.44 94.72

F. subglutinansb

(F. sacchari) 100.00 99.17 100.00 100.00 95.90 95.17

Eg-1 F. tricinctum28S rDNA,β-tub, lys2

→ F. tricinctum

F. tricinctumMAFF235551 100.00 100.00 100.00 100.00 100.00 ―

F. tricinctumATCC38183 100.00 100.00 100.00 100.00 100.00 ―

F. avenaceumMAFF239206 100.00 100.00 100.00 100.00 97.95 ―

aNelson, et al. Fusarium species: An illustrated manual for identification. 1983.bsensu lato.csensu strict.The grey box indicates that the nucleotide sequence homology leaded to misidentification.The open box indicates that the nucleotide sequence homology leaded to accurate identification.Species in parenthesis is re-identified by nucleotide sequence homology in the new taxonomic systems.

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can distinguish a species forming species complex.　　The genetic markers commonly used can not sometimes identify the closely related species, such as species complexes, due to few species-specific diagnoses in sequences10,15). Watanabe et al.15) reported that the lys2 is the most appropriate genetic marker for identifying Fusarium isolates with the fastest nucleotide substitution rate among the six genes examined in their study. However, there is a possibility that their study did not cover all species which are frequently isolated, because of a limit to the number of the tested strains. Therefore, as mentioned by authors, it is necessary to be examined whether the genetic makers including lys2 can identify the actual isolates with accuracy.　　Watanabe et al.17) evaluated the usability of some genetic markers which were previously reported the capability to identify Fusarium isolates based on barcording with nucleotide sequences, and to clear up the questionable points of application for actual isolates. A local database were constructed with 46 references of nucleotide sequences of Fusarium strains already-identified, and nucleotide sequences of six genetic regions of 19 food-borne Fusarium isolates were determined. And then, the nucleotide sequence homologies of each genetic region were calculated pairwisely between an isolate and a reference strain. Their extracted result was indicated in Table 3 . The 18 S rDNA, 5.8 S rDNA, 28 S rDNA and ITS 1 sequences leaded to the accurate identification of only three to 13 isolates, respectively, because of perfect matches to sequences of more than two species of reference strains, or mis-identification. The lys2 sequences leaded to the accurate identification of several isolates not identified by these four regions. However, other five isolates could not be identified because of non-amplification of lys2 by PCR. The β-tub sequences leaded to the accurate identification of all tested-isolates. Thus, the β-tub is more useful genetic marker for identifying Fusarium isolates in a wide range than other five loci including lys2.

Reconstruction of the higher and lower taxonomic tree of the genus Fusarium

　　Although some researchers have applied molecular phylogenetic analysis to examine the taxonomy of Fusarium species, many trees showed a low resolution, and phylogenetic relationships remain unclear, especially in the deep lineages. This is due to partial taxon-sampling of Fusarium species for phylogenetic analyses, and to a lack of suitable nucleotide and amino acid substitution rates and other factors. Furthermore, whole genome comparison among Fusarium species revealed the possibility that each gene in Fusarium genomes has a unique evolutionary history18), and such gene may bring difficulty to the reconstruction of phylogenetic tree of Fusarium. There is a need for comprehensive taxon-sampling and use of appropriate genetic markers to reveal reliable phylogenetic relationships. Furthermore, to select the appropriate genes, it is necessary to consider substitution rates and gene-evolution of each gene.　　Watanabe et al.19) performed phylogenetic analyses based on the nucleotide sequences of the rDNA cluster region, β-tub, EF-1α, and lys2. Although all fore gene trees supported the classification of Fusarium species into 7 major clades, I to VII. However, the incongruence of the tree topologies between lys2 and the other genes was detected (Figure 1). Additional analyses confirmed that this incongruence among gene trees was not due to an analytical artifact, such as long-branch attraction, composition bias of nucleotide and amino acid, and convergent evolution. Instead, many branches which displayed evidence of positive selection, indicated by bold branches in Figure 1 d, were detected in the lys2 tree. The adaptive evolution of the lys2 gene within the genus Fusarium may be occurred. It means that the differences in the tree topology may

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reflect differences in the evolutionary histories among the genes. Therefore, to obtain a reliable phylogeny for Fusarium species, the lys2 sequences were excluded from the dataset. 　　Subsequently, the evolutionary distances for each gene were compared. Each gene divided into cording/non-cording regions, followed by dividing the substitutions into synonymous/non-synonymous. Then, pairwise comparisons of substitution distances were exhaustively performed. With regard to the non-synon-ymous substitutions of β-tub and EF-1α, the substitution rates of these regions are slow, therefore, the numbers of non-synonymous substitutions in these genes were too few to reconstruct the phylogenetic relationships especially among clades I to VII (Figure 1 ) and among Fusarium and its related genera (the higher taxa). As for the synonymous substitutions of β-tub and the introns within EF-1α, the rates are fast, and substitutions are completely saturated among the higher taxa. However, substitutions like these are useful for examining among the lower taxa. The rDNA cluster region and the synonymous substitutions of EF-1α can provide information for phylogenetic reconstruction for both the higher and lower taxa, because the substitution rate of these regions are moderate for the lineage in the genus Fusarium. Therefore, it was shown that information obtained from multiple substitutions in multiple genes are required to reconstruct a compre-

Figure 1.　 Maximum likelihood trees for the genus Fusarium and related genera inferred from each gene. The GTR+I+G model was used as the model for nucleotide substitution. Branch lengths are proportional to the estimated number of nucle-otide substitutions. For protein-coding genes (β-tub, EF-1α, and lys2), each codon position was analysed separately. Panel A: rDNA cluster region; panel B: β-tub; panel C: EF-1α; and panel D: lys2. The BP values over 75 % are displayed on the nodes (BP; 1000 replicates). In panel D, the branches with bold lines indicate the lineages in which positive selection has occurred with the p-value under the null hypothesis that the ω ratio of the positively selected sites is equal to 1.0. (p<0.001).

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hensive description of the phylogenetic relationships among Fusarium species and closely related species including both the higher and lower taxa. 　　A maximum likelihood (ML) tree was reconstructed based on the combined data of the rDNA cluster region, β-tub, and EF-1α. The ML tree indicated some interesting relationships in the higher and lower taxa of Fusarium species and related genera (Figure 2 ). The monophyly of the sections of the genus Fusarium, which have previously been defined based upon morphological characteristics, were tested. In the ML tree, only Discolor was recovered as a monophyletic clade. Many sections, namely Arachnites, Eupionnotes, Gibbosum, and Sporotrichiella, were not verified as monophyly. They formed the paraphyletic and polyphy-letic groups, respectively. The taxonomic groups, which were traditionally classified based on the morpho-logical species concept, are not always recovered in molecular phylogenetic analyses of the genus Fusarium. The result means that some morphological characteristics recognized as section-specific ones, are actually not synapomorphies shared among only species in a section.

Figure 2.　 Maximum likelihood tree of the genus Fusarium and related genera inferred from the combined sequences of the rDNA cluster region and the β-tub, and EF-1α genes. Taking into account the different tempos and modes of nucle-otide substitutions, all parameters of the substitution model were separately estimated for each gene using the GTR+I+G model. The branch lengths are proportional to the estimated number of nucleotide substitutions. For the protein-coding genes β-tub and EF-1α, each codon position was analysed separately. The bootstrap probability (BP; 1000 replicates) values over 75% are displayed on the nodes. Sections supported by morphological taxonomic systems are described, and include Arachnites, Arthrosporiella, Discolor; Elegans, Eupionnotes, Gibbosum, Lateritium, Liseola, Martiella and Ventricosum, Roseum, Spicarioides, and Sporotrichiella.

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　　The ML tree demonstrated convoluted and nested structures of species as some “species complexes” (Figure 2 ). The Gibberella fujikuroi species complex was revealed in the ML tree and contained F. subglu-tinans, F. proliferatum, and F. verticillioides, consistent with the previous molecular studies20, 21). These three species have been recognized by the traditional morphological species concept, and some researchers indicated that the monophyly of each species was not recovered in molecular phylogenetic trees. Therefore, these species have been re-classified and divided into new multiple species based on the novel taxonomic system by mainly molecular method, such as F. fujikuroi and F. circinatum20-22). The ML tree in Figure 2 could not resolve the phylogenetic relationships among re-identified species in this species complex. This difficulty of resolving phylogenetic relationships in this complex is probably caused by rapid divergence events occurred in this species complex, implied by the shortened internal branches in this species complex in that ML tree. Furthermore, an additional complex containing the F. avenaceum, F. tricinctum and F. lateritium were newly detected by Watanabe et al.19)

　　Recently, more exhaustive Fusarium tree was reported23), and it provided us more new facts about phylo-genetic relationships among Fusarium speices. However, many controversial points remain nclear, such as phylogenetic positions of basal lineages; F. solani, F. decemcellulare, F. dimerum, and phylogenetic relation-ships with the genus Fusarium and closely-related genera. There is a need to have more studies about methods and procedures for phylogenetic analyses, and to reconstruct a more reliable species tree for further understanding of the Fusarium phylogeny. 　　Although the morphological species concept does not reflect the phylogenetic tree of the genus Fusarium, this does not imply that morphological characteristics are not useful for identification and taxonomy. Because, identifying unknown species because morphological characteristics can be widely applied to any species, not only those of the genus Fusarium but also other fungi3, 24). Fusarium isolates can be initially classified on the basis of morphological similarity, with the awareness that sections are in fact a means of artificial grouping. We need to construct more reliable taxonomic system in combination with the morphological, phylogenetic, toxicological, biological, and other recognition methods.　　In the future study, we plan to analyze the states of mycotoxin production of each Fusarium species based on phylogenetic tree topology for understanding how the potential productivity of a mycotoxin is acquired in the Fusarium species evolution. We think it would give us some insights into mechanisms of acquisition and biological implication for Fusarium species of mycotoxin-producivity, and prediction of potential toxin-production of productivity-unknown speices. Fusarium.

Acknowledgements

　　I am deeply grateful to Dr. Ichinoe of Tokyo Kasei University and Dr. Takatori of Center for Fungal Consultasion Japan for much guidance and giving great knowledges about mycotoxin-producing fungi since a time early in my career as a researcher about mycology. I credit my accomplishment of my researches to Dr. Sugita-Konishi, Dr. Hara-Kudo, Dr. Kamata, Dr. Takahashi and co-workers of National Institute of Health Sciences, who have been offered their support and encouragement. Finally, I wish to express my special thanks to all of my collaboraters for their help and cooperation.

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