cryptic speciation in fusarium subglutinans

1032

Mycologia, 94(6), 2002, pp. 1032–1043.q 2002 by The Mycological Society of America, Lawrence, KS 66044-8897

Cryptic speciation in Fusarium subglutinans

Emma T. Steenkamp1

Brenda D. WingfieldDepartment of Genetics, Forestry and AgriculturalBiotechnology Institute, University of Pretoria, Pretoria0002, South Africa

Anne E. DesjardinsMycotoxin Research Unit, National Center forAgricultural Utilization of Research, USDA,Agricultural Research Services, 1815 N UniversityStreet, Preoria, Illinois 61604

Walter F.O. MarasasPROMEC, Medical Research Council, P.O. Box19070, Tygerberg 7505, South Africa

Michael J. WingfieldForestry and Agricultural Biotechnology Institute,University of Pretoria, Pretoria 0002, South Africa

Abstract: Fusarium isolates that form part of theGibberella fujikuroi species complex have been classi-fied using either a morphological, biological, or phy-logenetic species concept. Problems with the taxon-omy of Fusarium species in this complex are mostlyexperienced when the morphological and biologicalspecies concepts are applied. The most consistentidentifications are obtained with the phylogeneticspecies concept. Results from recent studies have pre-sented an example of discordance between the bio-logical and phylogenetic species concepts, where agroup of F. subglutinans sensu stricto isolates, i.e., iso-lates belonging to mating population E of the G. fu-jikuroi complex, could be sub-divided into more thanone phylogenetic lineage. The aim of this study wasto determine whether this sub-division representedspecies divergence or intraspecific diversity in F.subglutinans. For this purpose, we included 29 F.subglutinans isolates belonging to the E-mating pop-ulation that were collected from either maize or te-osinte, from a wide geographic range. DNA sequencedata for six nuclear regions in each of these isolateswere obtained and used in phylogenetic concordanceanalyses. These analyses revealed the presence of twomajor groups representing cryptic species in F. sub-

Accepted for publication April 28, 2002.1 Corresponding author, Email: [email protected]; current address:Department of Biology, University of York, PO Box 373, York YO105YW, UK.

glutinans. These cryptic species were further sub-di-vided into a number of smaller groups that appearto be reproductively isolated in nature. This suggestsnot only that the existing F. subglutinans populationsare in the process of divergence, but also that eachof the resulting lineages are undergoing separationinto distinct taxa. These divergences did not appearto be linked to geographic origin, host, or phenotyp-ic characters such as morphology.

Key Words: Fusarium subglutinans, maize, repro-ductive isolation, teosinte

INTRODUCTION

Gibberella fujikuroi (Sawada) Wollenw. is a speciescomplex that encompasses many Fusarium species(Nirenberg and O’Donnell 1998, O’Donnell and Cig-elnik 1997, O’Donnell et al 1998a, 2000a, Steenkampet al 1999, 2000a, 2001). In the global environment,species in this complex are important, because oftheir association with diseases of agronomically im-portant plants (Correll et al 1991, Leslie 1995, Leslieet al 1990, Sun and Snyder 1981, Varma et al 1974,Ventura et al 1993). These fungi also affect humanand animal health, since many species produce toxicsecondary metabolites such as moniliformin, beau-vericin, fumonisin, fusaproliferin, and fusaric acid(Leslie et al 1992, Logrieco et al 1993, Marasas et al1983, Sewram et al 1999a, 1999b, Shephard et al1999, Vesonder et al 1995).

The taxonomy of Fusarium species in the G. fujiku-roi complex has been subject to much controversy(Leslie 1995). This is mainly due to a lack of consen-sus among researchers on how to define a morpho-logical species for the fungi in this complex (Gerlachand Nirenberg 1982, Nirenberg and O’Donnell 1998,Snyder and Hansen 1945). In an attempt to resolvethis problem, a biological species concept (Dobzhan-sky 1937, Mayr 1940) was introduced, whereby eightbiological species have been identified (Britz et al1999, Hsieh et al 1977, Klittich and Leslie 1992, Kuhl-man 1982, Leslie 1991, 1995, 1996). These biologicalspecies were designated as mating populations A toH, where mating population E, for example, repre-sents F. subglutinans (Wollenw. & Reinking) Nelson,Toussoun & Marasas sensu stricto (Britz et al 1999,Hsieh et al 1977, Klittich and Leslie 1992, Kuhlman

1033STEENKAMP ET AL: CRYPTIC SPECIATION IN FUSARIUM

1982, Leslie 1991, 1995, 1996). The eight biologicalspecies, however, exclude the more than 80% of spe-cies in this complex with no apparent sexual repro-ductive cycle. Currently, the only method for classi-fying all the fungal strains in the G. fujikuroi speciescomplex is through the application of the phyloge-netic species concept (Cracraft 1983, Taylor et al2000). With this method, fungi in the G. fujikuroicomplex are classified into more than 40 differentphylogenetic species (O’Donnell et al 1998a, Steen-kamp et al 1999, 2000a).

In a recent study, ten additional phylogeneticallydistinct species in the G. fujikuroi complex were re-ported (O’Donnell et al 2000a). Among these was aF. subglutinans strain associated with maize in SouthAfrica, that had previously been classified in matingpopulation E of the G. fujikuroi species complex us-ing the biological species concept (Steenkamp et al1999). Application of the phylogenetic species con-cept has, however, indicated that this biological spe-cies is subdivided into more than one phylogeneticlineage (O’Donnell et al 2000a, Steenkamp et al1999, 2001). These lineages may either reflect speciesdivergence within F. subglutinans or are simply theresult of intraspecific diversity. However, all of thestudies dealing with the molecular classification ofstrains representing F. subglutinans were based onsmall sets of different isolates (O’Donnell et al 2000a,Steenkamp et al 1999, 2001) and no definite conclu-sions could be drawn on the taxonomic status ofthese fungi. Clarification of the relationships amongdifferent F. subglutinans isolates would undoubtedlyshed light on how the phylogenetic species conceptmight influence our interpretation of the biologicalspecies concept in the G. fujikuroi species complex.The aim of this study was, therefore, to address thephylogenetic status of members of F. subglutinans by(i) including strains isolated from maize and its wildteosinte relatives from a wide geographic range; (ii)obtaining DNA sequence data from six nuclear re-gions for these strains; and (iii) using phylogeneticconcordance analysis (Avise and Ball 1990, Taylor etal 2000) to determine whether these isolates are in-terbreeding in nature and, if not, to define sub-groups within F. subglutinans.

MATERIAL AND METHODS

Fungal isolates. Twenty-nine F. subglutinans sensu strictoisolates belonging to the E-mating population of the G. fu-jikuroi species complex were included in this study (TABLE

I). The six South African, ten United States and six Mexicanstrains were isolated from maize. The remaining five Mex-ican strains and the two Guatemalan strains were isolatedfrom teosinte. For outgroup purposes we also included two

isolates of F. circinatum Nirenberg et O’Donnell that wereisolated from pines.

DNA isolation, PCR amplification and sequencing. DNAwas isolated using a CTAB (N-cetyl-N,N,N-trimethyl-ammo-nium bromide) extraction method (Steenkamp et al 1999).A portion of three nuclear genes, histone H3 (Glass andDonaldson 1995), calmodulin (Carbone and Kohn 1999)and b-tubulin (Glass and Donaldson 1995), were amplifiedfrom all 31 isolates. We also used an additional set of prim-ers that amplify three unlinked nuclear regions of unknownfunction (H. Britz unpubl). The first primer set is HB9-a(59-tcaatacccctcgcctagaa-39) and HB9-b (59-gaccacagcctcga-gaacat-39), the second is HB14-a (59-ttccaccatgagaggaaaccc-39) and HB14-b (59-ccattgccaatcttgatcct-39), and the thirdHB26-a (59-gacttgagtatctgcactgc-39) and HB26-b (59-gaatgt-actactcgacgtcg-39).

For amplification of all these loci, the PCR mixture con-tained 1 mM deoxynucleotide triphosphates (0.25 mMeach), 2.5 mM MgCl2, 0.2 mM of each primer, 0.25 ng/mLDNA, 0.05 U/mL of Super-Therm DNA polymerase [South-ern Cross biotechnology (Pty.) Ltd., Cape Town, South Af-rica] and 1 3 Super-Therm reaction buffer. PCR-cyclingconditions were as follows: denaturation at 92 C for 20 s,annealing for 20 s at 55 C (calmodulin, tubulin, and his-tone) or 47 C (HB9, HB14, and HB26), and elongation for20 s at 72 C. This was repeated 30 times and was precededby an initial denaturation at 92 C for 1 min and followedby a final elongation step at 72 C for 5 min.

After PCR, the products were purified with a QIAquickPCR Purification Kit (Qiagen GmbH, Hilden, Germany)and sequenced in both directions with the respective prim-ers. Reactions were performed on an ABI PRISMy 377 au-tomated DNA sequencer, with an ABI PRISMy Dye Ter-minator Cycle Sequencing Ready Reaction Kit (Perkin El-mer, Warrington, United Kingdom). Sequences were ana-lyzed with Sequence Navigator version 1.0.1.y (PerkinElmer Applied BioSystems, Inc., Foster City, California).

Phylogenetic analyses. The data sets obtained for eachprimer set were aligned manually by inserting gaps(TreeBase accession numbers S789 and M1249). Phyloge-netic analyses were performed with PAUP (PhylogeneticAnalysis Using Parsimony) version 4.0b1 (Swofford 1998),with gaps treated as fifth characters in heuristic parsimonysearches, and with tree-bisection-reconnection (TBR)branch swapping and MULTREES (saving of all optimaltrees) effective. For bootstrap analyses 1000 replicationswere performed.

RESULTS

PCR amplification and sequencing. With the primersused, we were able to amplify and sequence 480 basepairs (bp), 456 bp, and 332 bp of the calmodulin, b-tubulin, and histone H3 genes, respectively. For thethree regions of unknown function, 250 bp, 235 bp,and 236 bp were sequenced for HB9, HB14, andHB26, respectively. Of the 1989 nucleotides (nc) se-quenced, 17 nc (0.9%) were polymorphic in the dif-

1034 MYCOLOGIAT

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1036 MYCOLOGIA

TABLE II. Summary of polymorphic nucleotides (nc) in the six nuclear regions sequenced among the Fusarium subglutinansisolates from maize and teosinte

Isolates

Polymorphisms (nc)a

CAL

33 377

TUB

144 204 285

HIS

27 99 275 286 351 426

HB14

99 230 231 233

HB26

69

HB9

101 204Geno-type

MRC7828, MRC7833,MRC837, MRC1077,MRC1084 T C A T T C C T G C T T 2 2 2 A C C 1-1

MRC7786MRC7803

TT

CC

AA

TT

TC

CC

TT

CC

GG

CC

CC

TT

22

22

22

AA

CC

CC

1-21-3

MRC7862, MRC7861,MRC7838, MRC7849,MRC756 T C G T T C C T G C T T 2 2 2 A C C 1-4

MRC7787, MRC7790,MRC7794, MRC7799,MRC115, KSU1257,KSU1417, KSU3815 C C A C T C C T A T T C T A A G T G 2-1

KSU434, KSU731 C T A C T T C T A T T C T A A G T G 2-2MRC7817, MRC714,

MRC6512, MRC6483,KSU507, KSU993,KSU2921 C T A C T C C T A T T C T A A G T G 2-3

a Numbering of polymorphic nucleotides corresponds with their nucleotide positions in the respective DNA sequences.Horizontal lines (2) indicate nucleotide deletions.

TABLE III. Number of polymorphic and parsimony infor-mative characters, as well as the actual length of trees, gen-erated from the individual and combined sequence datasets

LocusInformativecharacters

Polymorphiccharacters

Treelength

CalmodulinHistone H3b-tubulinHB9HB14HB26Combined

262241

17

263241

18

262241

17

ferent F. subglutinans strains and none of the siteshad more than two different nucleotide characterstates. Among these strains, between one and sixpolymorphic nucleotides in each of the six regionswere identified (TABLE II). Upon combination of thepolymorphisms for each individual, we recognizedseven different genotypes within the set of 29 F. sub-glutinans isolates (TABLE II). The most frequentlysampled genotype was 2-1, which was represented byeight strains associated with maize in South Africaand the United States and teosinte in Mexico andGuatemala. Seven strains displayed genotype 2-3, andwere associated with maize in the United States and

South Africa, as well as teosinte in Mexico. Five iso-lates displayed genotype 1-1 and were collected frommaize in South Africa and Mexico, as well as Mexicanteosinte. Genotype 1-4 was also represented by fivestrains, all of which were isolated from maize inSouth Africa and Mexico. Genotype 2-2 was repre-sented by two strains associated with maize in theUnited States and both genotypes 1-2 and 1-3 wererepresented by single strains that were isolated fromteosinte in Mexico.

Phylogenetic analyses. The number of parsimony in-formative characters in the six data sets ranged fromone for HB26 to six for histone H3 (TABLE III). Inall the data sets, only the b-tubulin sequence har-bored a parsimony uninformative character (TABLE

III, FIG. 1). This variable character was present onlyin isolate MRC7803. Phylogenetic analyses generatedunique gene genealogies for each of the individualdata sets (FIG. 1). In each case, a single tree was gen-erated and the consistency (CI) and retention (RI)indices for each were 1.00 and 1.00, respectively, in-dicating no homoplastic characters in any of the sixindividual data sets. As a result, all of the single-genegenealogies were of minimal length, i.e., equal to thenumber of parsimony informative sites. A single mostparsimonious tree was also obtained from the com-bined data sets (FIG. 2). The length of this tree was


FIG. 1. Single-gene genealogies generated from sequence data sets for six different loci studied in 29 F. subglutinansstrains associated with maize and teosinte. In each case only one most parsimonious tree was obtained. The branch associatedwith the single parsimony uninformative character in the b-tubulin data set is indicated with an asterisk (*). The consistency(CI) and retention (RI) indices for each were 1.00 and 1.00, respectively. The different geographic origins are indicated incolor (red 5 South Africa; black 5 United States; blue 5 Mexico and Guatemala). A: b-tubulin gene genealogy consistingof 2 parsimony informative characters and 2 steps. B: Histone H3 genealogy consisting of 6 parsimony informative charactersand 6 steps. C: The single-gene genealogy for each of the HB9, HB14, and HB26 nuclear regions. Because the clusteringfor each of these regions was identical, they are represented by a single tree with lengths 2, 4, and 1, respectively. D:Calmodulin gene genealogy consisting of 2 steps.

equal to the number of parsimony informative char-acters (TABLE III), since homoplastic characters werealso absent in the combined data set (CI 5 1.00, RI5 1.00). The length of this tree was equal to thesummed lengths of the individual gene trees (TABLE

III), which is a distinctive feature of absolute congru-

ence among individual gene genealogies (Dykhuizenand Green 1991, Maynard Smith and Smith 1998,Taylor et al 1999b).

Clustering within the different genealogies wasvery similar. For the data sets HB9, HB14, and HB26the isolates were separated into two groups that al-

1038 MYCOLOGIA

FIG. 2. A: Genealogy generated from the combined data sets (color coding same as in FIG. 1). The branch associatedwith the single parsimony uninformative character in the b-tubulin data set is indicated with an asterisk (*). One single mostparsimonious tree with a length of 17 steps was obtained (CI 5 1.00; RI 5 1.00). B: The individuals included in each of theseven clusters correspond with the individuals displaying each of seven genotypes. Bootstrap values based on 1000 replicationsare indicated in parentheses.

ways included the same isolates (FIG. 1). The b-tu-bulin and calmodulin genealogies consisted of fourand three clusters of isolates, respectively. Althoughthere was some overlap, the clustering patterns forthe b-tubulin and calmodulin genes were unique.The genealogy generated from the histone H3 datagenerated four groups of isolates, some of whichshowed some resemblance to those generated for theother data sets.

Phylogenetic analysis of the combined data setfrom all the isolates included in this study revealedthe presence of two distinct groups among the iso-lates associated with maize and teosinte (FIGS. 2 and3). They were designated as Group 1 and 2 (FIG. 3).The genotypes 1-1, 1-2, 1-3 and 1-4 were present inGroup 1 and the genotypes 2-1, 2-2 and 2-3 were pres-

ent in Group 2 (FIGS. 2 and 3). Although this clus-tering pattern was not immediately detectable fromthe individual gene trees, the combined gene gene-alogy was not discordant with any of them.

DISCUSSION

In this study, we set out to use phylogenetic tools toanswer what appeared to be either a population orspecies level question. Although this approach is notwidely used in fungal taxonomy, it is not without pre-cedence and a number of researchers have reportedon the value of using phylogenetic concordance anal-ysis (Carbone et al 1999, Geiser et al 1998, Koufo-panou et al 1997, O’Donnell et al 1998b, 2000a, b,Taylor et al 1999a, b, 2000). Based on previous work


FIG. 3. A single most parsimonious phylogram generated from the combined sequence data sets. Parsimony informativeand parsimony uninformative characters (indicated with an asterisk) were included in the analysis. In parentheses are indi-cated the different hosts followed by their specific geographic origins within South Africa, the United States, or Mexico andGuatemala. The tree is rooted to F. circinatum. Branch lengths are indicated above the branches and bootstrap values basedon 1000 replications are indicated in bold. (N/a 5 not available)

(Steenkamp et al 1999, 2001), our null hypothesiswas that the set of isolates associated with maize andteosinte, from a wide geographic range, would formpart of the existing E-mating population of the G.fujikuroi species complex (i.e., F. subglutinans sensustricto), and that the observed sequence variationmerely reflect intraspecific diversity. However, consid-ering the data generated in this study, we had to re-ject this hypothesis. The results clearly showed thatF. subglutinans is separated into reproductively isolat-ed populations that probably constitute separate sib-ling species.

The basic rationale behind the use of phylogeneticconcordance analyses involves the detection of con-gruence or the lack thereof, among different genetrees (Avise and Ball 1990, Dykhuizen and Green1991, Taylor et al 1999a, 1999b, 2000). Incongruenceamong gene trees from different loci indicates inter-breeding among individuals, since sexual recombi-nation ‘reshuffles’ their genomes. In an interbreed-

ing population this ‘reshuffling’ results in uniqueevolutionary histories for the genes in a specific in-dividual, while the genes from different individualsshare numerous characteristics or polymorphisms. Inreproductively isolated populations genes do not nor-mally flow among individuals, resulting in a lack ofshared polymorphisms and consequently perfectlycongruent gene trees. Our analyses showed that theevolutionary histories of the six nuclear regions wereperfectly congruent in each of the F. subglutinans in-dividuals studied. This is because every gene tree, aswell as the one generated from the combined data,was of minimum length (FIGS. 1 and 2). The F. sub-glutinans isolates from the United States, South Af-rica, Mexico, and Guatemala thus appear to repre-sent reproductively isolated populations.

The tree generated from the combined data sep-arated the 29 F. subglutinans isolates into sevengroups (FIG. 2). These groups corresponded to theseven multilocus genotypes identified from the se-

1040 MYCOLOGIA

quence comparisons (TABLE II). The evolutionaryhistories for each of the six nuclear regions in all theisolates from genotype 1-1, for example, are identical,indicating a lack of genome ‘reshuffling’ throughsexual reproduction. All the isolates in genotype 1-1can, therefore, be considered as clones of one anoth-er, since there is no evidence of interbreeding. Thesame is also true for genotypes 1-4, 2-1, 2-2 and 2-3.Although genotypes 1-2 and 1-3 are represented bysingle isolates, analysis of additional isolates will mostlikely reveal a similar trend. In theory, the overall lackof shared polymorphisms in the six nuclear regionsamong the seven groups or genotypes, separate the29 isolates into seven clones, a notion that is probablynot entirely correct (see below).

In light of similar studies on other fungi (reviewedby Taylor et al 1999a, 1999b, 2000) our results wererather unexpected. In most of these studies, phylo-genetic concordance analysis revealed the presenceof so-called cryptic species where individuals fromone species were shown to be reproductively isolatedfrom those in the other species. However, these testsalso detected that individuals within some of thesecryptic species were interbreeding in nature, becauseof the presence of numerous shared polymorphisms.As a result, generation of consensus trees from com-bined sequence data resulted in trees that were lon-ger than the expected minimum (observed treelength . number of polymorphic characters). Incontrast, the observed tree length in our study wasequal to the number of polymorphic characters (TA-BLE III), providing no evidence for interbreedingamong any of the seven F. subglutinans genotypes. Byemploying phylogenetic concordance analysis, ourstudy is one of few, and perhaps the first to suggestthat a group of fungi capable of interbreeding in thelaboratory appears to be propagating exclusivelyasexually in nature.

The idea that isolates of F. subglutinans exist inclonal populations is not congruent with previous re-ports. Based on analyses of phenotypic characters,isolates of a specific genotype do not always displaysimilar degrees of resistance to antimicrobial agentsand demonstrate similar abilities to produce myco-toxins (Desjardins et al 2000, Marasas et al 1983,1984, Sewram et al 1999a, 1999b, Shephard et al1999, Yan et al 1993). For example, the two isolatesin genotype 2-2 are significantly different in their re-sistance to hygromycin B (Yan et al 1993) and thethree South African isolates of genotype 1-1 producemarkedly different levels of moniliformin and fusa-proliferin (Sewram et al 1999a, 1999b, Shephard etal 1999). Another trait suggesting that isolates withsimilar genotypes are not ‘true clones’ is the distri-bution of mating type. If all the isolates with a specific

genotype are clones of one another, their matingtypes determined using either laboratory crosses orPCR-based methods (Steenkamp et al 2000b) shouldbe the same. Among the F. subglutinans isolates stud-ied, this was not the case, because each of the fivemulti-isolate genotypes was represented by bothMAT-1 and MAT-2 isolates (Desjardins et al 2000,Steenkamp et al 2000a, Yan et al 1993). This variationwithin groups of isolates with the same genotype, wasundoubtedly generated by earlier sexual recombina-tion events. Even though we did not include suffi-cient isolates and polymorphic loci to address ques-tions on the reproductive mode of F. sublutinans, ourresults suggest that sexual reproduction is not a com-mon phenomenon. Among the 17 polymorphic sitesanalyzed, all were fixed within isolates associated witha specific genotype. It is possible that the inclusionof additional data, especially those associated withmycotoxin production, antibiotic resistance and mat-ing, will result in the identification of non-fixed orshared polymorphism. However, the ongoing ab-sence of sex will also bring about fixation in thesecharacters and eventually the absence of shared poly-morphisms. Our results, therefore, suggest F. subglu-tinans populations are diverging into various repro-ductively isolated lineages that will most probablyeach eventually constitute separate species.

Classifying the isolates used in this study into theirsmallest diagnosable units or redefining species limitsin the existing F. subglutinans sensu stricto is problem-atic. Given that sexual reproduction between theseisolates appears to be absent in nature, the biologicalspecies concept cannot be applied. The phylogeneticspecies concept assists to some extent, but as shownabove, it identifies ‘clones’. Determining whetherthese ‘clones’ represent separate species is an arbi-trary exercise, since it is impractical to designate eachclonal population as a distinct species. Likewise, toclassify each as the same species would be incorrectand would not reflect the natural situation. Separat-ing F. subglutinans into smaller units/taxa is furthercomplicated by the fact that some isolates differ byno more than a single nucleotide at the six nuclearregions analyzed and display no known or diagnos-able phenotypic differences. Among the isolates stud-ied, we have, however, observed two major phyloge-netic groups (groups 1 and 2) (FIG. 3), which mayrepresent a species partition where groups 1 and 2represent cryptic species. Genotypes 1-1, 1-2, 1-3 and1-4 would belong to group 1 and 2-1, 2-2 and 2-3would belong to group 2. Whether this partition willprove to be diagnosable using phenotypic charactersremains to be tested.

There are no apparent links between the genotypeto which the F. subglutinans isolates belong and geo-


graphic origins or host (FIG. 3). South African iso-lates were present in all genotypes except 1-2, 1-3 and2-2, while Mexican/Guatemala isolates were presentin all but genotype 2-2. The United States isolatesappeared to be restricted to genotypes 2-1, 2-2 and2-3. Isolates from maize were present in all genotypesexcept 1-2 and 1-3, while those from teosinte wereonly absent from genotype 2-2. From the limited dataavailable, the separation of F. subglutinans into theseven genotypes/groups also does not appear to belinked to mycotoxin production (Desjardins et al2000, Marasas et al 1983, 1984, Sewram et al 1999a,1999b, Shephard et al 1999) or morphology (Desjar-dins et al 2000, Steenkamp et al 1999, 2001). Therewere also no apparent links between the Groups 1and 2 separation and geographic origin, host or my-cotoxin production (FIG. 3) (Desjardins et al 2000,Marasas et al 1983, 1984, Sewram et al 1999a, 1999b,Shephard et al 1999, Steenkamp et al 1999, 2001).Noticeably, maize in the United States is associatedonly with group 2, but this is probably a samplingeffect. The 17 polymorphic sites, therefore, appearto be the only diagnosable features separating theseven genotypes and Groups 1 and 2. As with otherfungi (Geiser et al 2000, Peng et al 1999, Taylor et al2000), our results may lead to the future identifica-tion of morphological, physiological, pathogenic, ortoxigenic characters supporting either the presenceof Groups 1 and 2 or their sub-lineages.

Based on the results presented here, we have at-tempted to interpret the findings of previous matingstudies (Desjardins et al 2000, Steenkamp et al 1999,2001). Again, there was no relationship between thegenotype represented and ability to sexually interactin the laboratory. The genotype 1-1 isolate MRC1084from South African maize, for example, was capableof producing fertile offspring when mated with ge-notype 1-3 and 2-1 isolates. Many other isolates werealso capable of successful sexual interaction with iso-lates displaying genotypes other than their own. Thepresence of the two proposed cryptic species helpedto clarify some of the problems that were encoun-tered in previous mating studies (Desjardins et al2000, Steenkamp et al 1999, 2001). For example,some of the F. subglutinans strains that were collectedfrom maize and teosinte in Mexico and CentralAmerica were sexually incompatible with those fromthe United States (Desjardins et al 2000). As revealedhere all the available F. subglutinans isolates from theUnited States belong to Group 2, whereas those fromMexico and Central America belong to both Groups1 and 2, which may partially explain their incompat-ibility. Our results can, however, not explain why theUnited States isolates were sexually incompatibleeven with the Mexican strains from Group 2, or why

most of the isolates collected from Mexico and Cen-tral America are capable of fertile sexual interaction,even though they represent separate cryptic species.Inclusion of all 29 isolates in a mating study mighteventually shed light on the connection between theability to reproduce sexually and genotype/crypticspecies, but conditions in the laboratory do not nec-essarily mimic those in nature. Many fungi are ableto sexually interact across the species barrier (e.g.,Vilgalys and Sun 1994), which was also demonstratedfor strains of F. subglutinans and F. circinatum (Des-jardins et al 2000, Steenkamp et al 2001).

The inconsistency between the biological and phy-logenetic species concepts illustrated in this study in-troduces serious complications for the classificationof fungi, especially those in the G. fujikuroi speciescomplex, since classification relies heavily on bothconcepts. Problems with using the morphologicalspecies concept have been reported by many workers(O’Donnell et al 1998a, 2000a, Steenkamp et al 1999,2001), but the current study is the first to report dis-parities using the biological species concept for clas-sifying fungi in the G. fujikuroi species complex. Un-doubtedly, species recognition using phylogeneticconcordance analysis will yield the most informativeresults, but there are two major problems associatedwith the use of this approach. Firstly, even though wehave considerable knowledge on the subject of phy-logenetics, new ideas are constantly emerging and weare still in the learning-phase with respect to its meth-odologies and the interpretation of results. Secondly,we are still in the process of developing a definitionfor a fungal species and how to recognize this unit.Although we have attempted to address the taxonom-ic status and reproductive mode of isolates residingin F. subglutinans sensu stricto, many questions remainunanswered. These include an understanding of howan asexual species should be defined, how many nu-cleotide differences constitute a species divergence inFusarium and others. Our study and similar cases willthus represent an interesting topic for discussion asthe fields of fungal reproduction and phylogenetictaxonomy advance.

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cryptic speciation in fusarium subglutinans

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