A multilocus phylogeny of the Metarhizium anisopliae...
Transcript of A multilocus phylogeny of the Metarhizium anisopliae...
A multilocus phylogeny of the Metarhizium anisopliae lineage
Joseph F. BischoffAnimal and Plant Health Inspection Service, USDA,Beltsville, Maryland 20705-2350
Stephen A. Rehner1
Systematic Mycology and Microbiology Laboratory,USDA-ARS, Beltsville, Maryland 20705-2350
Richard A. HumberUSDA-ARS, RW Holley Center for Agriculture andHealth, Ithaca, New York 14853-2901
Abstract: Metarhizium anisopliae, the type species ofthe anamorph entomopathogenic genus Metarhi-zium, is currently composed of four varieties, includ-ing the type variety, and had been demonstrated to beclosely related to M. taii, M. pingshaense and M.guizhouense. In this study we evaluate phylogeneticrelationships within the M. anisopliae complex,identify monophyletic lineages and clarify the speciestaxonomy. To this end we have employed a multigenephylogenetic approach using near-complete sequenc-es from nuclear encoded EF-1a, RPB1, RPB2 and b-tubulin gene regions and evaluated the morphologyof these taxa, including ex-type isolates wheneverpossible. The phylogenetic and in some casesmorphological evidence supports the monophyly ofnine terminal taxa in the M. anisopliae complex thatwe recognize as species. We propose to recognize atspecies rank M. anisopliae, M. guizhouense, M.pingshaense, M. acridum stat. nov., M. lepidiotae stat.nov. and M. majus stat. nov. In addition we describethe new species M. globosum and M. robertsii,resurrect the name M. brunneum and show that M.taii is a later synonym of M. guizhouense.
Key words: biocontrol, Clavicipitaceae, Cordy-ceps, entomopathogen, green muscardine, Meta-cordyceps
INTRODUCTION
The genus Metarhizium Sorokin is composed ofanamorph entomopathogenic fungi that generallyare greenish when conidiating on the corpses of theirarthropod hosts or in axenic culture. They frequentlyare isolated from soils, parasitize a broad range ofinsect species representing numerous orders and arefound throughout the tropics and temperate regions.
Species from this genus are used as biological controlagents to manage and prevent infestations of variousspecies of superfamily Acridoidea, including locustsand grasshoppers (Lomer et al 1997, Milner 1997,Milner and Pereire 2000, Hunter et al 2001, Lomer etal 2001). In addition Metarhizium anisopliae(Metschn.) Sorokin, the type species of the genus,has been shown to be effective in the control ofmalaria-vectoring mosquitoes (Culicidae, Diptera).Two recent studies have estimated that applicationsof M. anisopliae could reduce the intensity of malariatransmission by 75% (Scholte et al 2005, Kanzok andJacobs-Lorena 2006).
Liang et al (1991) were the first to confirm theconnection of Metarhizium, long considered to beasexual, to the teleomorph genus Cordyceps (Fr.) Link(Clavicipitaceae, Hypocreales). They described C. taiiZ.Q. Liang & A.Y. Liu and linked it developmentallyto their newly described anamorph species, M. taiiZ.Q. Liang & A.Y. Liu. This genus-level anamorph-teleomorph connection was substantiated furtherwhen Liu et al (2001) described a Metarhiziumanamorph for C. brittlebankisoides Zuo Y. Liu, Z.Q.Liang, Whalley, Y.J. Yao & A.Y. Liu. This link has beensupported by subsequent molecular phylogeneticstudies (Liu et al 2002, Huang et al 2005a). Thesexual states of Metarhizium species have beentransferred from Cordyceps to Metacordyceps Sung etal (2007).
Before Tulloch’s (1976) revision of Metarhizium 13species and two varieties had been proposed (seewww.IndexFungorum.org). Tulloch rejected threespecies described by Sorokin (1883) as definedaccording to the morphology of the hyphal bodiesformed within the hemocoel of their respective insecthosts. Based on the illustrations accompanying thedescriptions of Metarhizium spp. by earlier authors,Tulloch determined that some species were notmembers of the genus and she concurred with thesynonymies proposed by others (Gams and Rozsypal1973, Latch 1965, Speare 1912, Veen 1968). In theend Tulloch (1976) rejected most of the publishednames and reduced the genus to two species (M.anisopliae and M. flavoviride W. Gams & Rozsypal)including the type and one other variety (M.anisopliae var. anisopliae and M. anisopliae var. majus[J.R. Johnst.] M.C. Tulloch).
In the most comprehensive morphological andmolecular phylogenetic treatment of Metarhizium todate Driver et al (2000) followed Tulloch’s (1976)lead in recognizing species complexes for M.
Accepted for publication 21 January 2009.1 Corresponding author. E-mail: [email protected]
Mycologia, 101(4), 2009, pp. 512–530. DOI: 10.3852/07-202# 2009 by The Mycological Society of America, Lawrence, KS 66044-8897
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anisopliae and M. flavoviride in addition to M. albumPetch, which was supported by Rombach et al (1987).However they were unable to assess the phylogeneticaffinities of several taxa associated with Metarhiziumthat had been described since Tulloch’s (1976)treatment. The taxa not studied by Driver et al(2000) included M. pingshaense Q.T. Chen & H.L.Guo, M. guizhousense Q.T. Chen & H.L. Guo and M.taii (anamorph of Metacordyceps taii). These taxa weredescribed from China and have not been available forstudy until recently.
Driver et al (2000) expanded the circumscriptionsof Metarhizium anisopliae and M. flavoviride. Applyingthe results of a phylogenetic analysis of the nuclearribosomal internal transcribed spacer (ITS), Driver etal (2000) recognized eight lineages as varieties andone undetermined species group (i.e. M. flavoviride‘‘Type E’’). They identified and described varietiesfor both species complexes including four varieties ofMetarhizium anisopliae, namely M. anisopliae var.acridum Driver & Milner, M. anisopliae var. anisopliae,M. anisopliae var. lepidiotae Driver & Milner (as M.anisopliae var. lepidiotum) and M. anisopliae var.majus. Driver et al (2000) restricted their descriptionsof new lineages to varieties due to the limitedresolution and support provided by the ITS sequenceanalysis. In their ITS phylogeny (Driver et al 2000FIG. 1) all the terminal lineages that defined thevarieties of M. flavoviride form an unresolvedpolytomy. Furthermore, the internal nodes withinthe clade containing M. anisopliae var. anisopliae andM. anisopliae var. majus were unresolved as well.Isolates associated with the invalidly described nomennudum ‘‘M. anisopliae var. frigidum’’ introduced byRath et al (1995) also were included in Driver et al(2000). The ITS phylogeny of Driver et al (2000)showed an affinity between ‘‘M. anisopliae var.frigidum’’ and M. flavoviride var. flavoviride. HoweverITS sequence data did not provide significant supportfor their reciprocal monophyly despite morphologicaldifferences that support their distinction, henceDriver et al (2000) considered them synonymous. Ina study that used a multigene phylogenetic approachBischoff et al (2006) unambiguously resolved thethree varieties of M. flavoviride and described M.frigidum J.F. Bisch. & S.A. Rehner (5 ‘‘M. anisopliaevar. frigidum’’) as a morphologically and phylogenet-ically well supported lineage within the M. flavoviridecomplex.
Huang et al (2005a, b) addressed the taxonomyand relationships of several taxa from China that werenot included by Driver et al (2000). In these papersMetarhizium guizhouense, M. pingshaense and M. taiiwere synonymized with M. anisopliae. However, as inDriver et al (2000), Huang et al (2005a, b) derived
their phylogenetic inferences exclusively from ITS,which performs poorly in resolving lineages withinMetarhizium, especially within the M. anisopliaecomplex. The results reported by Driver et al (2000)and Huang et al (2005a, b) suggest that ITSsequences by themselves are of limited use forresolving the phylogenetic history of Metarhizium.However the phylogenetic analysis used in Bischoff etal (2006) demonstrated the effectiveness of thismultigene approach and provides a more sensitiveand robust means to identify Metarhizium lineages.
In this study we address and clarify the taxonomyand phylogenetics of the Metarhizium anisopliaecomplex that we consider to include all of the fourcurrently recognized varieties of M. anisopliae as wellas M. guizhouense, M. pingshaense and M. taii. Toaccomplish this objective we have taken a multigenephylogenetic approach to determine whether thesepreviously recognized species and varieties are phylo-genetically distinct. Furthermore we examined themorphology of isolates from all inferred lineageswithin the complex, using ex-type isolates wheneverpossible. In the cases of several well supportedintrageneric lineages that are not morphologicallydistinct (Driver et al 2000, this study) we have adoptedthe genealogical concordance phylogenetic speciesrecognition criterion (GC-PSR, Taylor et al 2000) asimplemented by Dettman et al (2003) and O’Donnellet al (2000) to recognize species lineages within thiscomplex.
MATERIALS AND METHODS
Fungal isolates.—The 57 isolates used in this study(TABLE I) were obtained from ARSEF (Agricultural Re-search Service Collection of Entomopathogenic FungalCultures: Ithaca, New York) and CBS (Fungal BiodiversityCenter, Centraalbureau voor Schimmelcultures FungalBiodiversity Center, Utrecht, The Netherlands) and repre-sent a subset of the more than 200 isolates examined in thedevelopment of this study. These 57 isolates were selectedbecause they were ex-types and/or because they represent-ed the most inclusive phylogenetic sampling of the complexbased on analysis of the 59 end of EF-1a (tree not shown).Included among the isolates examined are ex-types forMetarhizium anisopliae var. anisopliae (ARSEF 7487, ex-neotype), M. anisopliae var. acridum (ARSEF 7486, ex-holotype; ARSEF 324, ex-paratype; ARSEF 3391, ex-para-type), M. anisopliae var. lepidiotae (ARSEF 7488, ex-paratype), M. flavoviride (ARSEF 2133, ex-holotype), M.frigidum (ARSEF 4124, ex-holotype), M. guizhouense (CBS258.90, ex-holotype), M. pingshaense (CBS 257.90, ex-holotype) and that of two newly described species describedherein (ARSEF 2596, ex-holotype; ARSEF 2575, ex-holo-type). There is no viable ex-type culture for M. brunneumPetch. However ARSEF 2107 is considered an authenticstrain because the taxon’s author, Petch (1935), identified it
BISCHOFF ET AL: METARHIZIUM ANISOPLIAE LINEAGE 513
and we designate it here as an ex-epitype. The ex-holotypeisolate of M. anisopliae var. majus (IMI 171404) is no longerviable (Aquino de Muro pers comm). However a broadgeographic sampling of M. anisopliae var. majus was used toassess the status of this taxon and an ex-epitype (BPI878297) derived from a living culture (ARSEF 1914) isdesignated for this taxon.
Morphological evaluations.—Isolates were grown underambient light/dark conditions in the laboratory underfluorescent illumination at approximately 23 C on one-quarter strength SDAY (SDAY/4) (SDAY: 10 2.5 g/LBactopeptone, 10 g/L dextrose, 2.5 g/L yeast extract,20 g/L agar). Observations and measurements were madeon 4–14 d old cultures with a Nikon Eclipse E600
FIGS. 1–20. Mature conidiogenous cells and conidia of Metarhizium species. Bar 5 10 mm. 1–2. Metarhizium majus (ARSEF1914). 1. Phialides with developing conidia. 2. Mature conidia. 3–4. M. guizhouense. 3. Phialides with developing conidia(ARSEF 7507). 4. Mature conidia (ARSEF 6238). 5–6. M. pingshaense. 5. Phialides with developing conidia (ARSEF 4342). 6.Mature conidia (ARSEF CBS 257.90). 7–8. M. anisopliae. 7. Phialides with developing conidia (ARSEF 7487). 8. Mature conidia(ARSEF 7487). 9–10. M. robertsii. 9. Phialides with developing conidia (ARSEF 727). 10. Mature conidia (ARSEF 727). 11–12.M. brunneum. 11. Phialides with developing conidia (ARSEF 4179). 12. Mature conidia (ARSEF 2107). 13–14. M. lepidiotae. 13.Phialides with developing conidia (ARSEF 4628). 14. Mature conidia (ARSEF 7412). 15–16. M. acridum. 15. Phialides (ARSEF6592); note that apices of conidiogenous cells thicken with successive conidial production. 16. Mature conidia (ARSEF 5736).17–18. M. globosum. 17. Phialides (ARSEF 2596). 18. Mature conidia (ARSEF 2596). 19. M. flavoviride. Mature conidia (ARSEF2025). 20. M. frigidum. Mature conidia (ARSEF 4124).
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compound microscope with differential interference con-trast illumination. Images were captured with ACT-1 version2.12 software (FIGS. 1–20, Nikon Corp.). Minimum andmaximum values are shown (TABLE I) and are based on aminimum of 20 measurements for each feature.
DNA extraction.—Isolates were grown in SDY/4 broth 5–7 don an orbital shaker set at 125 rpm and 25 C. The tissue wasremoved from the broth, rinsed twice with sterile water,blotted dry with filter paper, frozen at 280 C andlyophilized. Approximately 50 mg lyophilized myceliumwas ground into powder with the FastPrep tissue homoge-nizer (MP Biomedicals, Irvine, California) for 6 s at a speedsetting of 4.5. The pulverized tissue was lysed with 900 mLlysis buffer (2 M NaCl, 0.4% w/v deoxycholic acid, 1.0% w/vpolyoxyethylene ether) and incubated 10 min at 55 C.Cellular byproducts were extracted with 750 mL chloro-form : isoamyl alcohol (24 : 1) and centrifuged to separatethe aqueous and particulate phases. The 700 mL of clearedsolution containing DNA was removed, placed in a cleantube and mixed with an equal volume of 6 M guanidiniumisothiocyanate. DNA was bound to 40 mL 50 : 50 suspensionof acid-washed diatomaceous earth (Sigma, St Louis,Missouri) and flint glass powder (Minnesota Midwest Clay,Bloomington, Minnesota) prepared according Vogelsteinand Gillespie (1979). The silica-bound DNA was suspendedtwice in 75% ethanol, dried, eluted in sterile distilled waterby incubating 5 min at 55 C.
PCR amplification and nucleotide sequencing.—Genes usedin this study were translation elongation factor 1-alpha(EF-1a), RNA polymerase II largest subunit (RPB1), RNApolymerase second largest subunit (RPB2), b-tubulin (Bt)and the nuclear ribosomal intergenic spacer region (IGS).Only the 59 end of EF-1a was sequenced for all 57 isolates.Also, using the 59 end of EF-1a we determined thephylogenetic placement of two presumptive cryptic speciesof M. anisopliae reported by Bidochka et al (2001, 2005).Partial sequences of EF-1a, RPB1, RPB2 and Bt weresequenced for a subset of 33 isolates for the combinedmultigene analysis. In addition to the primers used inBischoff et al (2006) and the Bt primers T1 and T22 asdescribed by O’Donnell and Cigelnik (1997) these primerswere developed for sequencing purposes: RPB1: Mz1F1 (59-CGRACMYTRCCYCATTTCACAA), Mz1R1 (59-TTGAGCG-GAAGYTGCATCATCTCC), Mz1R2(59-TTCARRAARGC-CATSGCRCCWTC); Bt: Bt1F (59-GGTCCCTTCGGTC-AGCTCTTCC), Bt1R (59-CAGCCATCATGTTCTTAGGG-TC), Bt2R (59-GTAGTGACCCTTGGCCCAGT).
IGS was sequenced for exemplars validated in this studyfor these species: M. anisopliae (ARSEF 7450, ARSEF 7487),M. brunneum (ARSEF 2107), M. guizhouense (ARSEF 4303,ARSEF 4321, ARSEF 6238, ARSEF 7502, ARSEF 7507, CBS258.90), M. majus (ARSEF 1015, ARSEF 1914, ARSEF 1946,ARSEF 2808, ARSEF 4566, ARSEF 7505), M. pingshaense(ARSEF 3210, ARSEF 4342, ARSEF 7929, CBS 257.90) andM. robertsii sp. nov. (ARSEF 727, ARSEF 2575). IGS,including partial segments of the flanking large and smallribosomal subunits, was amplified in two overlappingsegments, designated here as IGSa and IGSb. IGSa wasamplified with primer pairs LSU4 (59-CCGTYCTTCGCCYC-
GATTTCC) and Ma-IGS1 (59-CGTCACTTGTATTGGCAC,Pantou et al 2003) and primer pairs 630 U (59-CTTTAGGG-TAGGCTGCTTGTT) and NS2 (59-GGCTGCTGGCACCA-GACTTGC, White et al 1990). Internal primers weredeveloped for sequencing IGSa (IGSaF12: 59-GTACCCGG-GACYCCRAGTAAG; IGSbR2: 59-GYTCCTGGTCGGGACT-TAYA) and IGSb (IGSbF4: 59-GCGYGYWGWATTRRATGG-TYT; IGSaR4: 59-ACCGGGCGCTCGTGKTCYATT) ampli-cons. All loci were amplified and sequenced according toBischoff et al (2006).
Sequence alignment and phylogenetic analyses.—Sequencher4.1 (Gene Codes Corp., Ann Arbor, Michigan) was used toassemble and edit sequence data. Alignments were madewith Clustal X (Thompson et al 1997) with the defaultsettings. Adjustment to the computer-assisted alignment wasneeded only in the case of the intron-containing 59 regionof EF-1a. These adjustments were made by eye and involvedthe elimination of ambiguous regions created by insertionsand deletions (indels).
Phylogenetic hypotheses were developed with maximumparsimony (MP), maximum likelihood (ML) and Bayesianinference (BI) methods. The MP analyses were performedwith PAUP* 4.0b10 (Swofford 2002) with heuristic searchesof 500 random-addition replicates with TBR branch-swap-ping and equal character weighting. Heuristic MP bootstrap(MP BS) analyses (Felsenstein 1985) with TBR branch-swapping included 1000 pseudoreplicates, and 10 randomaddition replicates were performed to provide bootstrapsupport values. The program GARLI 0.95 (Zwickl 2006) wasused to identify the most likely tree and to determinemaximum likelihood bootstrap support (ML BS). Thedefault settings in GARLI were used, and 1000 bootstraprepetitions were performed. All log-likelihood (2lnL)scores reported were calculated with PAUP.
MrBayes 3.1 (Huelsenbeck 2000, Ronquist and Huelsen-beck 2003) was used for Bayesian analyses to determineposterior probabilities (BI PP). The analysis was run fourtimes with each run including 4 MCMC chains (three cold,one heated), 1 000 000 generations, sampling every 100generations (including the first generation) for a total of40 004 trees. The first 20% of trees from each run werediscarded to allow the log-likelihood scores to achievestationarity (i.e. burn-in). The resulting trees were importedinto PAUP, and a 50% consensus tree was computed.
Clades that are supported with 95% BI PP or greater, 70%
ML BS or greater and 70% MP BS or greater values wereconsidered significantly supported by the data (Mason-Gamer and Kellogg 1996, Lutzoni et al 2004, Reeb et al2004). Mason-Gamer and Kellog (1996), Lutzoni et al(2004) and Reeb et al (2004) did not address a thresholdcriterion for determining significant support based on MLBS values. GARLI was not yet available when these twopapers were published. However because the ML BSsupport values from the GARLI analysis in this study wereclose in absolute value to those from the MP BS analysis weelected to use a 70% threshold to identify significantlysupported data for ML BS values. The phylograms (FIGS. 21,22) are the consensus trees with the mean branch lengthsfrom the Bayesian analyses.
BISCHOFF ET AL: METARHIZIUM ANISOPLIAE LINEAGE 515
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BISCHOFF ET AL: METARHIZIUM ANISOPLIAE LINEAGE 517
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ed518 MYCOLOGIA
EF-1a, RPB1, RPB2 and Bt first were analyzed in-dependently with MP and ML BS to identify areas ofconcordance and discordance among the phylogenies ofthe genes sampled (Dettman et al 2003) and to com-pare bootstrap support for clades resolved in the dif-ferent gene trees. In addition, to broaden the scope oftaxon sampling and to focus on the most recentlydiverged lineages, the rapidly evolving, intron-rich regionof the 59 end of EF-1a was analyzed independently (seeFIG. 22).
The results of Driver et al (2000), Spatafora et al (2007),Sung et al (2007) and our own unpublished work haveconfirmed that Metarhizium is a monophyletic group. Allphylogenetic analyses of the M. anisopliae complex in thisstudy used the closely related species M. flavoviride and M.frigidum as outgroup taxa.
TAXONOMY
Based on the results of the phylogenetic analyses andthe morphological data, we describe two new species,elevate three varieties of M. anisopliae to species level,resurrect the taxon M. brunneum and recognizeM. guizhouense as the anamorph of Metacordyceps taii.Despite our inability to delimit all these taxa basedon morphological characteristics, the molecular datasupport the recognition of these fungi at the specieslevel.
Metarhizium acridum (Driver & Milner) J.F. Bisch.,Rehner & Humber stat. nov. FIGS. 15–16
MycoBank MB512407; Metarhizium anisopliae var. acridum Driver & Milner,
Mycol. Res. 104:144 (2000).
Metarhizium anisopliae (Metsch.) Sorok., Plant para-sites of man and animals as causes of infectiousdiseases 2:268 (1883). FIGS. 7–8; Entomophthora anisopliae Metsch. Zapiski imperators-
kogo obshchestua sel’ska Khozyaistra yuzhnoi rossiip 45 (1879).
; Isaria anisopliae (Metsch.) Pettit, Cornell Univ. Agric.Exp. St. Bull. 97:356 (1895).
; Penicillium anisopliae (Metsch.) Vuill., Bull. Trimest.Soc. Mycol. Fr. 20:221 (1904).
5 Isaria destructor Metsch., Zool. Anz. 3:45 (1880).5 Oospora destructor (Metsch.) Delacroix, Bull. Trimest.
Soc. Mycol. Fr. 9:260 (1893).5 Isaria anisopliae var. americana Pettit, Cornell Univ.
Agric. Exp. St. Bull. 97:354 (1895)5 Penicillium cicadinum Hohn., Sber. Akad. Wiss. Wien
118:405 (1909).5 Metarhizium cicadinum (Hohn.) Petch, Trans. Br.
Mycol. Soc. 16:68 (1931).5 Sporotrichum paranense Marchionatto, Bol. Mens. Min.
Agric. Noac. Buenos Aires 34:241 (1933).Vo
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BISCHOFF ET AL: METARHIZIUM ANISOPLIAE LINEAGE 519
Metarhizium brunneum Petch, Trans. Br. Mycol. Soc.19:189 (1935). FIGS. 11–12In the absence of a readily accessible type for
Metarhizium brunneum, we designate a dried culturestored at the US National Fungus Collection (BPI878293) as an epitype. This epitype was preparedfrom a plate of the isolate ARSEF 2107, which also isdeposited elsewhere as CBS 316.51, IMI 014746,NRRL1944 and QM 191.
Metarhizium globosum J.F. Bisch., Rehner & Humbersp. nov. FIGS. 17–18Coloniae primum albae, transeuns griseolus-viridus.
Hyphae vegetativae 2.0–4.0 mm crasse. Conidiophorumerectus. Phialides clavatus, 5.0–12.0 mm longae et 3.0–4.0 mm crasse. Conidia globosus, 4.0–5.0 mm crasse.
Colony grown at 25 C on SDY/4 medium is firstwhite becoming pigmented within 5 d to a grayish-green (color plate 27C6, Kornerup and Wanscher1967). Vegetative hyphae are smooth and 2.0–4.0 mm.Conidiophores are erect, terminating in branches with2–3 phialides per branch, forming a palisade-like layer.Phialides (FIG. 15) are clavate, 5.0–12.0 mm long and3.0–4.0 mm at their broadest point with stronglytapering apices. Conidia are globose, 4.0–5.0 mm diam.
Typus: INDIA. On Pyrausta machaeralis (Lepidiop-tera, Pyralidae), 12 Sep 1988, collected by RC Rajak(ENT/12) (HOLOTYPE BPI 878294 [dried culture];ARSEF 2596 ex-type).
Etymology: M. globosum is named for its globoseconidia.
Metarhizium guizhouense Q.T. Chen & H.L. Guo, ActaMycologica Sinica 5:181 (1986). FIGS. 3–4; Metarhizium taii Z.Q. Liang & A. Y. Liu, Acta
Mycologica Sinica 10:260 (1991).
Teleomorph: Metacordyceps taii (Z.Q. Liang & A.Y.Liu) G.H. Sung, J.M. Sung, Hywel-Jones, Spatafora,Acta Mycologica Sinica 10:257 (1991).
Metarhizium lepidiotae (Driver & Milner) J.F. Bisch.,Rehner & Humber stat. nov. FIGS. 13–14
MycoBank MB512409; Metarhizium anisopliae var. lepidiotae Driver & Milner
(as Metarhizium anisopliae var. lepidiotum), Mycol. Res.104:145 (2000).
Metarhizium majus (J.R. Johnst.) J.F. Bisch., Rehner& Humber stat. nov. FIGS. 1–2
MycoBank MB512410; Metarhizium anisopliae f. major J.R. Johnst., Entomog-
enous Fungi of Porto Rico p 27 (1915).5 Metarhizium anisopliae f. oryctophagum Friederichs, Die
Grundfragen und Gesetzmassigkeiten der Land-undForstwirtschaflichen Zoologie p 199 (1930).
; Metarhizium anisopliae var. major (J.R. Johnst.) M.C.Tulloch, Trans. Brit. Mycol. Soc. 66:409 (1976).
In the absence of a useful type for Metarhiziummajus we designate a dried culture stored at the USNational Fungus Collection (BPI 878297) as anepitype. This epitype was prepared from a plate ofthe isolate ARSEF 1914.
Metarhizium pingshaense Q.T. Chen & H.L. Guo, ActaMycologica Sinica 5:181 (1986). FIGS. 5–6
Metarhizium robertsii J.F. Bisch., Rehner & Humbersp. nov. FIGS. 9–10
MycoBank MB512411Anamorphus in morphologia idem ac Metarhizium
anisopliae, sed distinguibilis characteribus sequentibusnucleotiditis fixationibus: translation elongation factorpositions 257 (gap), 258 (T), 287 (A), 447 (G) and 806 (C).
Metarhizium robertsii is morphologically indistin-guishable from M. anisopliae. It is diagnosed fromother members of the M. anisopliae complex by theseunique fixed nucleotide characters in the EF-1apositions: 257 (gap), 258 (T), 287 (A), 447 (G) and806 (C). Nucleotide positions can be evaluated bydownloading the alignment S2169 from www.TreeBase.org.
Typus: USA. SOUTH CAROLINA, on Curculiocaryae (Coleoptera, Curculionidae) (holotype BPI878819 is a specimen derived from ARSEF 2575 ex-type).
Etymology: M. robertsii is named in honor ofDonald W. Roberts.
Isolate ARSEF 2575 was selected as the ex-type ofM. robertsii because it is widely recognized as animportant isolate in insect biocontrol. Although thisstrain was not included in the phylogenetic treespresented the genes EF-1a, Bt, and RPB1 wereamplified and analyzed and found to be wellsupported within the Metarhizium robertsii clade.Furthermore ARSEF 2575 was found to exhibit the
TABLE II. Nodal support values for Metarhizium specieslineages from GARLI analyses of individual genes. Values inparentheses indicate support for conflicting topology (seeResults for details)
EF-1a RPB1 RPB2 B-tubulin
M. acridum 100 100 100 99M. anisopliae 100 98 100 100M. brunneum 100 100 — —M. globosum N/A N/A N/A N/AM. guizhouense 89 — (95) —M. lepidiotae — 100 98 —M. majus 96 — (99) —M. pingshaense 82 — — 100M. robertsii 100 — — 99
520 MYCOLOGIA
FIG. 21. Majority rule consensus phylogram from the Bayesian combined analysis of EF-1a, RPB1, RPB2, Bt (2lnL19284.18). Support values greater than 70% are shown for ML BS, BI PP and MP BS respectively (-- 5 support value , 70%).Ex-type isolates are denoted with a ‘‘T’’.
BISCHOFF ET AL: METARHIZIUM ANISOPLIAE LINEAGE 521
FIG. 22. Majority rule consensus phylogram from the Bayesian analysis of the 59 end of EF-1a for 57 isolates (2lnL2560.26). Support values greater than 70% are shown for ML BS, PP and MP BS respectively (-- 5 support value , 70%).Support values are included for the backbone and terminal taxa nodes only to prevent overcrowding the figure. Ex-typeisolates are denoted with a ‘‘T’’.
522 MYCOLOGIA
same informative EF-1a nucleotide bases used todistinguish this species from other members of the M.anisopliae complex. A single-spore isolate of ARSEF2575, accessioned as ARSEF 8020, has been designat-ed for full genome sequencing (D. Gibson perscomm).
Dong et al (2007) introduced the name Metarhi-zium anisopliae ‘‘var. dcjhyium’’. Their paper did notinclude a description or holotype designation andtherefore this taxon is an invalidly published nomennudum. Furthermore Dong et al (2007) provided nomorphological evidence nor did they evaluate M.anisopliae ‘‘var. dcjhyium’’ in the context of other M.anisopliae varieties. No isolates associated with thename M. anisopliae ‘‘var. dcjhyium’’ are available forstudy at this time, and we reject this taxon due to itsinvalid nomenclatural status and lack of any morpho-logical or molecular evidence that distinguishes itfrom other species of the genus.
RESULTS
Phylogenetic analyses.—Sequencing of the four nucle-ar genes from an exemplar set of 33 Metarhiziumisolates, after exclusion of 104 ambiguously alignedsequence positions, yielded a total of 7621 alignedcharacters that included a total of 741 parsimonyinformative characters: 1700 bp for EF-1a (163parsimony informative characters), 2783 bp forRPB1 (227 parsimony informative characters),1799 bp for RPB2 (201 parsimony informative char-acters), 1339 bp for Bt (150 parsimony informativecharacters). In a separate analysis of the 59 end of EF-1a for an expanded set of 57 isolates the alignedlength of this locus was 681 bp (139 parsimonyinformative characters). Alignments are availablethrough www.TreeBASE.org (accession numberS2169).
Preliminary GARLI ML analyses of the individualgenes were conducted to assess whether the inferredtree topologies from the different data partitions werephylogenetically congruent. These analyses yieldedsignificant support from at least two genes for sevenof the nine terminal clades that we interpret toconstitute phylogenetic species (single-gene trees notshown). Bootstrap support values for the nine speciesrecognized are provided (TABLE II). Topologicalconflict among the gene trees was confined to theclade containing M. majus and M. guizhouenseisolates and is discussed below.
A clade containing the ex-neotype isolate of M.anisopliae, ARSEF 7487, is designated here as thePARB clade and consists of four subclades that werecognize as the species M. anisopliae, M. pingshaense,M. robertsii and M. brunneum. M. anisopliae received
significant support from all four gene partitions andconflicted in none (TABLE II). The monophyly ofboth M. pingshaense and M. robertsii received signif-icant support from both EF-1a and Bt and did notconflict with either the RPB1 or RPB2 topologies. Themonophyly of M. brunneum, which is the most basallineage of the PARB clade, received significantsupport from EF-1a and RPB1 and conflicted in none.
The single instance of significant conflict amongthe individual gene analyses occurred in the place-ment and relationships of isolates of M. majus and M.guizhouense (5 M. taii), henceforth referred to as theMGT clade. EF-1a was the only gene phylogeny thatsignificantly supported the reciprocal monophyly ofM. majus and M. guizhouense (96% and 89% ML BSrespectively). RPB2 conflicted with the results of theEF-1a analysis in that M. majus isolates ARSEF 4566,ARSEF 7505 and ARSEF 2808 were placed in a cladealong with the ex-type of M. guizhouense (CBS258.90), ARSEF 7507, ARSEF 5714 and ARSEF 6238(95% ML BS), while the other isolates of M. majus(ARSEF 1015, 1946 and 1914) and M. guizhouense(ARSEF 4303, 4321 and 7502) were sister clades (99%
ML BS). The relationships among the M. majus andM. guizhouense isolates in the Bt analysis conflictedwith both the EF-1a and RPB2 analyses by groupingM. majus isolates ARSEF 4566, 7505 and 2808 in asingle distinct clade (94% ML BS) and all other M.majus and M. guizhouense isolates as a sister clade(81% ML BS). The ex-holotype of M. guizhouense,CBS 258.90, was placed in the same clade as theavailable M. taii isolates (ARSEF 5714 and ARSEF6238) in each of the single gene phylogenies, thussuggesting their synonymy as is discussed furtherbelow.
The monophyly of M. lepidiotae was supportedconcordantly by RPB1 and RPB2 and did not conflictwith either EF-1a or Bt. Metarhizium acridum wassignificantly supported by all four gene partitions.However the basal lineage of the M. acridum clade,ARSEF 2596, was phylogenetically distinct in allanalyses, differed morphologically from other M.acridum isolates and is described herein as the newspecies M. globosum. ARSEF 2596 is at present theonly isolate available for M. globosum, thus statisticalsupport for this lineage cannot be determined.Because topological conflict among individual genetrees was limited to sister lineages within a singlederived lineage, the MGT clade, we opted to combinethe datasets (FIG. 21).
In the combined dataset analyses the consensustree with mean branch lengths as determined byMrBayes (2lnL 19284.18, FIG. 21) and the best treeretrieved from the ML GARLI analysis (2lnL19284.18) were topologically identical. The combined
BISCHOFF ET AL: METARHIZIUM ANISOPLIAE LINEAGE 523
gene tree topology was consistent with the topologiesinferred from analyses of individual genes. The MPBS tree (not shown) did not significantly conflict witheither the BI or ML trees. The PARB clade (97% MLBS, 100% BI PP and 98% MP BS) comprises fourspecies lineages, including Metarhizium anisopliae(100% ML BS, 100% BI PP and 100% MP BS), M.pingshaense (99% ML BS, 100% BI PP and 99% MPBS), M. robertsii sp. nov. (74 ML BS, 100% BI PP, 62%
MP BS) and the most basal species lineage, M.brunneum (74% ML BS, 100% BI PP and 100% MPBS). The MGT clade (100% ML BS, 100% BI PP and100% MP BS) was highly supported as the sister of theM. anisopliae clade (100% ML BS, 100% BI PP and100% MP BS). However both species in this clade, M.majus and M. guizhouense, were paraphyletic witheach forming two subclades in the combined geneanalysis (FIG. 21). Metarhizium lepidiotae (99% ML BS,100% BI PP and 97% MP BS) is strongly supported asthe sister of the PARB and MGT clades (100 ML BS,100% BI PP, 100% MP BS). The most basal clade inthe M. anisopliae complex is that containing M.acridum and M. globosum (100% ML BS, 100% BI PPand 100% MP BS) with M. acridum strongly support-ed (100 ML BS, 100% BI PP, 100% MP BS) as distinctfrom M. globosum.
We further evaluated the relationships within theM. anisopliae complex by expanding the taxon set to57 isolates, focusing exclusively on the 59 end of EF-1a(FIG. 22). We selected this region for further analysisbecause, among the four loci analyzed in this study,the introns within 59 EF-1a provide the greatestconcentration of informative nucleotide variationand degree of phylogenetic resolution for terminalclades in Metarhizium (pers obs). In this analysis theconsensus tree with mean branch lengths inferredwith MrBayes (2ln L 2560.26, FIG. 22) and the besttree from the ML GARLI analysis (2ln L 2560.26)had identical topologies. The topology of the inferred59 EF-1a tree is consistent with the multigenephylogeny (FIG. 21), although nodal support values,particularly for internal nodes, are generally lower.This finding is not surprising given the fewer numberof variable characters provided by 59 EF-1a. However59 EF-1a alone provides strong support for all specieslineages in this study and thus is the most accurateand expedient locus for use in species identification.A unique and notable result of the 59 EF-1a phylogenyis that in the MGT clade (90% ML BS, 100% BI PP,89% MP BS) Metarhizium majus and M. guizhouenseare significantly supported as reciprocally monophy-letic (90% ML BS, 100% BI PP, 92% MP BS and 70%
ML BS, 97% BI PP, 78% MP BS respectively).Two M. anisopliae s.l. isolates from Bidochka et al
(2001, 2005), each representing one of the two
clades identified as ‘‘cold-active’’(43A-2i) and ‘‘heat-active’’(MAA1-2ii) were obtained from the authorand sequenced for the 59 region of EF-1a only. Basedon phylogenetic analysis, these isolates belong to M.robertsii and M. brunneum respectively (data notshown). Cultures and sequence accessions for theseisolates were deposited in the ARSEF collection andGenBank (43A-2i: ARSEF 8680, FJ229493; MAA1-2ii:ARSEF 8685, FJ229494 respectively).
In addition, in a characterization of rDNA andassociated spacer regions for M. anisopliae Pantou etal (2003) resolved three well supported clades, A–C,with IGS nucleotide sequences. IGS sequences weredetermined for 21 isolates (GenBank accessionsFJ228703–FJ228723) from the PARB and MGT cladesfrom the present study and compared to the data ofPantou et al (data not shown). These comparisonsrevealed that IGS Clade A includes M. anisopliae, M.pingshaense and M. robertsii. Within IGS Clade A M.anisopliae and M. pingshaense plus M. robertsii formedwell supported reciprocally monophyletic groups;however the relationship of M. pingshaense and M.robertsii is unresolved, which form a paraphyleticgrade. IGS Clade B corresponds to M. brunneum. IGSClade C of Pantou et al (2003) is most closely relatedto M. guizhouense Clade 1; however in the limitedsampling of IGS from isolates in this study the MGTclade is paraphyletic, in agreement with the resultsobtained for RPB1, RPB2 and Bt. Based on thepresent assessment, IGS by itself does not clearlyresolve relationships in either the PARB or MGTclades.
Morphological observations.—Colony pigmentation ofthe various isolates of Metarhizium anisopliae complexis initially white and usually becomes yellow duringthe early development of conidia (typically 4–7 d)and becomes greenish as the conidia mature (seeTABLE I). Yellow pigmentation is particularly conspic-uous in M. acridum, which tends to retain its color10–14 d. In addition we observed variation in colonycolor among the isolates within supported lineagesthat overlapped with color variation in other lineages,making it impossible to reliably assign diagnosticcolors to taxonomically defined groups. In generalthe mature colony color of isolates from the M.anisopliae complex is best described as olivaceous orsome slight variation of olive. Most isolates reachedmaturity 8–9 d. Likewise all examined members ofthe M. brunneum clade were olivaceous at maturityexcept for ARSEF 2107, which was pale yellow.Metarhizium globosum was greener than members ofthe M. acridum clade and did not approach the olivepigmentation of the other members of the complex.
Conidia are the only morphological feature that
524 MYCOLOGIA
reliably distinguishes several Metarhizium speciesconsidered in this study. The conidia of five of ninespecies treated here are closely overlapping in sizeand shape with those of M. anisopliae (5.0–7.0 3 2.0–3.5 mm, FIG. 8) and with each other, including M.brunneum (4.5–8.0 3 2.0–3.0 mm, FIG. 12), M. lepi-diotae (5.0–7.0 3 2.0–3.5 mm, FIG. 14), M. pingshaense(4.5–5.0 3 2.0–3.5 mm, FIG. 6) and M. robertsii (5.0–7.5 3 2.0–3.5 mm, FIG. 10). So it is not possible todistinguish these taxa based on conidial morphology.Isolates in the MGT clade have the largest conidia inthe M. anisopliae complex, and within the MGT cladeMetarhizium majus has the largest conidia (FIG. 2),which are cylindrical and 8.5–14.5 3 2.5–5.0 mm. Ingeneral the conidia of M. majus are rarely less than10 mm long and are usually 12–13 mm. The secondlargest conidia of the complex belong to the M.guizhouense (5 M. taii) clade. Conidia of this groupare 6.5–10.0 3 2.0–3.5 mm (FIG. 4), but rarely exceed9 mm long. Metarhizium acridum has the smallestconidia of the complex (FIG. 16). They are generallyovoid and are 4.0–5.5 3 2.0–3.0 mm. However theconidial measurements of M. acridum isolate ARSEF324 differed from its conspecifics. The conidia ofARSEF 324 were similar in size to M. lepidiotaeisolates, although often broader, 5.0–7.5 3 3.0–4.5 mm. In addition the globose conidia of M.globosum, which are 4.0–5.5 mm diam, are a distin-guishing characteristic of this species. They do notresemble the conidia from any other species in the M.anisopliae complex or the genus as a whole.
There was a great deal of variation in the size ofphialides within all recognized species. However theredid however appear to be a slight positive correlationbetween phialidic and conidial sizes, although thisassociation is too weak to be taxonomically diagnostic.
DISCUSSION
The species of Metarhizium, as in many hyphomyce-tous genera, can be difficult to distinguish morpho-logically (Crous et al 2005, Rehner and Buckley 2005,Tsui et al 2006). It has been argued recently thatmorphological species recognition (MSR) regularlyunder diagnoses evolutionarily meaningful species(Taylor et al 2000). Thus morphological similaritybetween sister and closely related species might be theresult of faster rates of reproductive isolation andgenetic divergence relative to the rate of morpholog-ical change (Taylor et al 2006). However, if rates ofmorphological evolution are heterogeneous amongrecently diverging species lineages, morphologicalcrypsis among non-sister lineages also could be due toretention of symplesiomorphic morphologies. On theother hand phenotypic similarities among non-sister
species might result from convergent morphologicalevolution (Crous et al 2005, Rehner and Buckley2005, Tsui et al 2006), perhaps due to occupation ofsimilar ecological niches. In the case of the M.anisopliae complex one or more of these phenomenamight occur because among the species consideredhere morphological crypsis occurs between both sisterand non-sister taxa.
Our approach to dealing with morphologicalcrypsis in M. anisopliae is to explore the use ofalternative methods of species recognition. Applica-tion of the biological species recognition criterion(BSR) to determine species boundaries as defined bythe ability to interbreed unfortunately is currently notan option with Metarhizium. While readily cultivatedin its mitotic state, neither sexual crosses nor sexualdevelopment have been successfully induced in vitro.Moreover BSR has been criticized because the abilityto mate represents a single, symplesiomorphic char-acteristic (Rosen 1979) that can be critically evaluatedonly in the context of a robust phylogenetic hypoth-esis. By contrast the genealogical concordance phylo-genetic species recognition criterion (GC-PSR, Tayloret al 2000) by itself has proven extremely effective atrecognizing diversity within the M. anisopliae com-plex in the absence of diagnostic morphologicalcharacters and the present inability to perform invitro mating tests. GC-PSR has been used regularly todelimit closely related and morphologically undiffer-entiated species in a wide range of fungi (Koufopa-nou et al 1997, Geiser et al 1998, O’Donnell et al1998, 2004, Adam et al 1999, Chaverri et al 2003,Dettman et al 2003, Weber et al 2003, Miller andHuhndorf 2004). Altogether our analyses providesupport for the recognition of nine distinct phyloge-netic species. As in several of the studies cited abovenot all gene partitions that we analyzed support thedelimitation of each Metarhizium species recognizedherein. However a minimum threshold of two-genesupport was achieved for the recognition of sevenphylogenetic species. Where a two-gene minimumsupport threshold was not obtained (e.g. delimitationof M. majus from M. guizhouense) fixed differences inconidia morphology and a single gene phylogenywere used as the basis for their recognition. We notethat congruence of only one sequenced gene andeither phenotype or geographic endemism is com-monly invoked as the basis to propose species status infungi (James et al 2001, Roets et al 2007, Zhao et al2008).
The core of the M. anisopliae complex is repre-sented by the PARB clade. The clade contains thetype species of the genus, M. anisopliae, and consistsof four well supported species lineages that we haverecognized here as M. pingshaense, M. anisopliae, M.
BISCHOFF ET AL: METARHIZIUM ANISOPLIAE LINEAGE 525
robertsii sp. nov. and M. brunneum (FIGS. 21–22). Allfour species are global in distribution, and the isolatesexamined in this study originated from Asia, Austra-lia, Europe and North and South America. Becausefew isolates from Africa were available at the time ofthis study our knowledge of Metarhizium from thiscontinent remains fragmentary. With the ability toseparate these cryptic species using objective phylo-genetic criteria it is now possible to mount systematicefforts to search for additional physiological andecological features that might further differentiatethese phylogenetic species. The genealogical datapresented here does not clarify the historical phylo-geography of any of these species, which is obscuredby their ubiquitous distributions. Determining thecenters of origin and dispersal histories of thesespecies will require further analysis requiring bothphylogenetic and population genetic approaches.
Metarhizium brunneum is the most basal lineage inthe PARB clade. As with other members of the cladewe found it impossible to differentiate isolates of M.brunneum from M. anisopliae, based on morpholog-ical characteristics, with the exception of the pre-sumptive color mutant ARSEF 2107. Petch (1935)designated a type collection from the Philippines,which he described as turning brown in maturecolonies. This color variant may occur regularly innature based on the fact that Petch had identifiedmultiple isolates as M. brunneum and from geograph-ically distant locations (see collections BPI 447601and BPI 447602). However because the type specimenof the species is not easily available for examinationand because no ex-type culture exists we havedesignated ARSEF 2107 (Oregon, USA), an isolatethat was authenticated by Petch, as the basis for anepitype. It is important to note that the majority of M.brunneum isolates examined here possess the typicalolivaceous M. anisopliae color instead of the buff ortan pigmentation as described for the type specimenor the ex-epitype culture, respectively.
The distinction between M. majus and M. guiz-houense is difficult to interpret because their relation-ship was not conclusively resolved with the moleculardata at hand. Based on morphological observationsand especially the results from the 59 end of EF-1a(FIG. 22), we conclude that there is sufficient evi-dence to segregate the associated isolates into the twospecies. From the standpoint of morphology theconidia of M. guizhouense were consistently smallerthan those of M. majus. No isolate of M. guizhouenseproduced conidia larger than 10 mm while all isolatesof M. majus produced conidia greater than 10 mmlong. Thus the morphological data support therecognition of M. majus and M. guizhouense. Howev-er the conflicting topologies between the RPB2 and
Bt gene trees (not shown) contradict the monophylyof these taxa. As mentioned above we recognize thatacceptance of M. majus and M. guizhouense at thespecies rank does not meet the molecular genealog-ical concordance criteria that we have implementedin recognizing the other taxa within the M. anisopliaecomplex. However the congruence of the morpho-logical grouping based on conidial size with the 59 EF-1a phylogeny provides a sufficient basis with which todistinguish and to accept these two species. It ispossible that the incongruity seen between the RPB2and EF-1a phylogenies might reflect incompletesorting of ancestral polymorphisms within this line-age. More detailed phylogenetic study of this cladeincorporating additional phylogenetic markers andincreased taxon sampling is needed to furtherevaluate the status of these two species.
We determined that M. taii is a synonym of theearlier published name M. guizhouense (MGT clade)and suggest that M. pingshaense (PARB clade) shouldbe resurrected. Huang et al (2005a, b) presented thefirst molecular phylogenetic studies to include M.guizhouense, M. pingshaense and M. taii in a contextthat included additional Metarhizium species. WithITS sequence data in both papers they reported ataxonomically broad polytomy for what we define asthe M. anisopliae complex and concluded that M.guizhouense, M. pingshaense and M. taii all weresynonyms of M. anisopliae. They also placed M. majuswithin a broadened concept of the M. anisopliae cladebut did not include it as part of the synonymy. Thesuperior resolution of the multilocus phylogenetichypothesis inferred in this study has enabled furtherinsight into the phylogenetic and taxonomic status ofthese species. For example in the present studyisolates previously identified as M. taii (ARSEF 5714and ARSEF 6238) were consistently placed in thesame clade as the ex-type of M. guizhouense. Toconfirm that our isolates were truly M. taii weevaluated their morphology in comparison to otheraccounts of the taxon and found that the conidialmeasurements of our isolates were in accordance withthe measurements described in the protolog for thespecies (Liang et al 1991, Huang et al 2005a).Furthermore the M. taii conidial measurements wereconsistent with those of the ex-type isolate of M.guizhouense. The holotypes of M. taii and M.guizhouense both were collected in the subtropicalGuizhou Province of China, and both were fromlepidopteran hosts. Together the morphological,molecular, host and geographic data lead us toconclude that M. taii is a synonym of M. guizhouenseand therefore the anamorph of Metacordyceps taiiwould be M. guizhouense, which has nomenclaturalpriority. Huang et al (2005b) synonymized M.
526 MYCOLOGIA
pingshaense with M. anisopliae because of their similarITS sequences and morphologies. However we resur-rect M. pingshaense (ex-type CBS 257.90) on the basisof the phylogenetic support for this species lineage.Also the conidial measurements of CBS 257.90, whilestill within much of the range of M. anisopliae, regu-larly surpassed the limits observed among its conspe-cifics.
Metarhizium lepidiotae is the most basal member ofthe M. anisopliae complex that is also morphologicallyindistinguishable from M. anisopliae. However themolecular data unequivocally support it as the sisterlineage of the PARB plus MGT clades. The similarconidial features of M. lepidiotae and M. anisopliaemight represent a symplesiomorphy that diverged in acommon ancestor of the MGT clade, whose speciesproduce larger conidia. On the other hand thesimilarity might have arisen through convergence. Ofnote, based on our limited sampling, M. lepidiotaeappears to have the most restricted geographic rangeof all members in the complex. Of the six M. lepidiotaeisolates included in this study two were from PapuaNew Guinea, two from Queensland, Australia, and twofrom the Australian island of Tasmania.
In our sampling of Metarhizium acridum a numberof isolates, including ARSEF 7486, are closely related.This species is used regularly as a biological controlagent in Africa against grasshoppers and locustsunder the trade name Green MuscleH and morerecently as Green GuardH. The ex-type of the species,ARSEF 7486 (originally collected from Niger), is theactive ingredient of Green MuscleH, while GreenGuardH is a formulated product based on anAustralian isolate (Milner 2002, CSIRO FI 48 5
ARSEF 324). Widespread release of Green MuscleH(i.e. ARSEF 7486) in many parts of Africa might leadone to think that isolates in this clade (FIG. 16) couldbe clones separated by multiple generations. Howeverfour of the nine isolates (ARSEF 3391, 6600, 324 and5736), for which 59 EF-1a sequences were deter-mined, are a different sequence haplotype thanARSEF 7486 and thus are unlikely to be its clonaldescendants. In addition isolates (ARSEF 6421, 6592,6597) that do share the same 59 EF-1a haplotype asARSEF 5748 either were collected before the releaseof Green MuscleH or were collected distant from thesites of localized applications in the Sahel andsouthern and western Africa (www.lubilosa.org).Although M. acridum is primarily known from Africaand Australia ARSEF 5748 was collected in Mexico in1992 before the widespread use of Green MuscleH(Lomer et al 2001). This suggests that M. acridum ismore geographically widespread and might occurnaturally in environments where locusts and grass-hoppers are endemic.
The newly described species Metarhizium globosumis distinguished from its sister taxon, M. acridum,based on the molecular, host affiliation and morpho-logical data. Both the multigene and 59 EF-1aphylogenetic analyses significantly support its distinc-tion (FIGS. 21–22). Metarhizium globosum is the onlytaxon within the genus to produce globose conidia.Furthermore M. acridum has been collected onlyfrom orthopterans and soil isolations while M.globosum was isolated from a lepidopteran host.Despite having only a single isolate of M. globosumthe molecular and morphological data support itsdistinction as a new species and hopefully willstimulate efforts on the Indian subcontinent to searchfor additional isolates of this species.
Bidochka et al (2001, 2005) used sequence-basedanalyses to propose the existence of cryptic specieswithin M. anisopliae s.l. from eastern Canada. Inaddition the usefulness of the nuclear ribosomalintergenic spacer (IGS) region as a phylogeneticmarker was evaluated by Pantou et al (2003) and usedto characterize the Metarhizium diversity associatedwith Neotropical leaf-cutter ant nests (Hughes et al2004). Cross-referencing the results of these investi-gations to each other and to the present study isdifficult due to the different genes and isolatessampled and to the lack of reference sequence datafor taxonomically validated isolates. We have ad-dressed this issue in two ways to determine theidentities of species in these studies. In the case ofthe Bidochka et al (2001, 2005) we sequenced isolatesobtained from the authors and determined that thetwo mutually exclusive groups they reported are M.robertsii and M. brunneum. Isolates from Pantou et al(2003) were not readily available, thus we determinedIGS sequences for the isolates of the six species in thePARB and MGT clades of this study and comparedthese to GenBank records submitted by Pantou et al(2003) and referenced by Hughes et al (2004).BLAST (Altschul et al 1990) analyses and analignment combining our sequences with those ofPantou et al (2003) revealed this correspondenceamong these studies: (i) Group A of Pantou et alcorresponds to our PARB clade, however the IGS ofM. pingshaense and M. robertsii are not reciprocallymonophyletic, and thus IGS is not informative fordifferentiating species within this clade; (ii) Group Bof Pantou et al is M. brunneum; and (iii) Group Ccorresponds to the MGT clade, however IGS does notsupport the monophyly of either M. guizhouense orM. majus, as with many of the loci sequenced in thisstudy. This comparison demonstrates the potentialand need for adoption of standardized molecularidentification methods to expedite communicationconcerning these organisms.
BISCHOFF ET AL: METARHIZIUM ANISOPLIAE LINEAGE 527
The multigene phylogenetic approach taken in thisstudy of the Metarhizium anisopliae complex has shedconsiderable light on this enigmatic group of fungi.The morphology of species in the complex provideslimited diagnostic information for the lineages re-solved in the molecular analyses. This is especially truewith colony pigmentation. Other than the extendedperiod of time in which M. acridum produces yellowpigmentation in culture, the vast majority of isolates ofall species showed various shades of olive at maturity.Rombach et al (1986) suggested that phialide mor-phology might be useful in discerning the taxa ofMetarhizium, but like Glare et al (1996) we foundphialide morphology to be plastic within lineages andnot particularly useful for taxonomic diagnoses. Weagree with Glare et al (1996) and Driver et al (2000)that conidial morphology, although useful in somecases (i.e. M. globosum) is also of limited use for speciesdelimitation and identification. Metarhizium majushad the largest conidia followed by M. guizhouense,whereas M. acridum generally had the smallest (exceptfor ARSEF 324). However distinguishing M. anisopliae,M. pingshaense, M. robertsii, M. brunneum and M.lepidiotae based on morphology alone was not possiblein our examinations of these taxa.
We have found that molecular tools and analyses arethe most reliable way to differentiate species within theMetarhizium anisopliae complex. Based on the resultsof this and a study of M. frigidum by Bischoff et al(2006), the 59 region of EF-1a is to date the mostinformative region to use for routine species identifi-cation within the genus. This region requires only twoprimers and is easily amplified. Although EF-1a aloneis not sufficient for fully resolving the genealogy ofMetarhizium, it is currently the most useful single locusfor diagnosing terminal groups as Geiser et al (2004)found for identifying Fusarium species. Future studieswill determine the use of this single locus for therecognition and identification of phylogenetic speciesin other fungal species complexes.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Karen Hansen andEllen Buckley for laboratory assistance throughout thisstudy, Amy Rossman for comments on an early version ofthe manuscript and Michael Bidochka for generouslyproviding isolates.
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