Phylogeny and systematics of the anamorphic,entomopathogenic genus Beauveria

Stephen A. Rehner1

Systematic Mycology and Microbiology Laboratory,USDA-ARS, Beltsville, Maryland 20705

Andrew M. MinnisSystematic Mycology and Microbiology Laboratory,USDA-ARS, Beltsville, Maryland 20705

Gi-Ho SungMushroom Research Division, National Institute ofHorticultural and Herbal Science, Rural DevelopmentAdministration, Suwon, 404-707, Republic of Korea

J. Jennifer Luangsa-ardPhylogenetics Laboratory, National Center for GeneticEngineering and Biotechnology, Science Park, PathumThani, Thailand

Luis DevottoInstituto de Investigaciones Agropecuarias (INIA),Centro Regional de Investigacion Quilamapu, Chillan,Chile

Richard A. HumberBiological Integrated Pest Management, Research,Robert Holley Center for Agriculture and Health, USDA-ARS, Tower Road, Ithaca, New York 14853

Abstract: Beauveria is a cosmopolitan anamorphicgenus of arthropod pathogens that includes theagronomically important species, B. bassiana and B.brongniartii, which are used as mycoinsecticides forthe biological control of pest insects. Recent phylo-genetic evidence demonstrates that Beauveria ismonophyletic within the Cordycipitaceae (Hypo-creales), and both B. bassiana and B. brongniartiihave been linked developmentally and phylogeneti-cally to Cordyceps species. Despite recent interest inthe genetic diversity and molecular ecology ofBeauveria, particularly as it relates to their role aspathogens of insects in natural and agriculturalenvironments, the genus has not received criticaltaxonomic review for several decades. A multilocusphylogeny of Beauveria based on partial sequences ofRPB1, RPB2, TEF and the nuclear intergenic region,Bloc, is presented and used to assess diversity withinthe genus and to evaluate species concepts and theirtaxonomic status. B. bassiana and B. brongniartii,both which represent species complexes and whichheretofore have lacked type specimens, are rede-

scribed and types are proposed. In addition six newspecies are described including B. varroae and B.kipukae, which form a biphyletic, morphologicallycryptic sister lineage to B. bassiana, B. pseudobassiana,which also is morphologically similar to but phyloge-netically distant from B. bassiana, B. asiatica and B.australis, which are sister lineages to B. brongniartii,and B. sungii, an Asian species that is linked to anundetermined species of Cordyceps. The combinationB. amorpha is validly published and an epitype isdesignated.

Key words: Ascomycota, Beauveria bassiana com-plex, B. brongniartii complex, cryptic species, insectfungi, phylogeny

INTRODUCTION

Beauveria is a cosmopolitan anamorphic genus ofsoilborne facultative necrotrophic arthropod-patho-genic fungi (Roberts and Hajek 1992, Goettel et al.2005) that occur also as saprotrophs and plantendophytes (Vega et al. 2008). Worldwide numerousregistered mycoinsecticide formulations based on B.bassiana and B. brongniartii are used for control ofinsect pests (Faria and Wraight 2007). In additionBeauveria produces a diverse array of biologicallyactive secondary metabolites that include non-peptidepigments and polyketides, nonribosomally synthe-sized peptide antibiotics and other secreted metabo-lites implicated in insect pathogenesis and virulencethat have potential industrial, pharmaceutical andagricultural uses (Vey et al. 2001).

Genus Beauveria is characterized morphologicallyby globose to flask-shaped conidiogenous cells fromwhich one-celled, terminal holoblastic conidia areproduced in sympodial succession on an indetermi-nate, denticulate rachis. Beauveria species are distin-guished principally according to characteristics oftheir conidia, which are typically smooth-walled,hyaline, 1.5–5.5 mm and globose to cylindrical orvermiform.

In the most recent taxonomic treatment of thegenus, de Hoog (1972) recognized three speciesincluding B. bassiana, B brongniartii and B. alba(Limber) Saccas (now Engyodontium album (Limber)de Hoog (de Hoog 1978). Four additional Beauveriaspecies have been recognized since including B.vermiconia de Hoog & Rao (1975), B. amorphaSamson & Evans (1982), B. caledonica Bisset &

Submitted 30 Sep 2010; accepted for publication 14 Feb 2011.1 Corresponding author. E-mail: stephen.rehner@ars.usda.gov

Mycologia, 103(5), 2011, pp. 1055–1073. DOI: 10.3852/10-302# 2011 by The Mycological Society of America, Lawrence, KS 66044-8897

1055

Widden (1986) and B. malawiensis Rehner et al.(2006b). Although previously classified with otheranamorphic taxa in the now defunct Deuteromycota,cultural studies have linked B. bassiana and B.brongniartii developmentally to Cordyceps Fr. teleo-morphs (Shimazu et al. 1988, Li et al. 2001, Huang etal. 2002) and molecular phylogenetic analyses confirmthe placement of Beauveria within Cordycipitaceae(Hypocreales) (Sung et al. 2007). A combined ITS andTEF phylogeny of the genus (Rehner and Buckley2005) corroborated its monophyly and delineatedseven well supported terminal lineages correspondingto B. amorpha, B. bassiana, B. brongniartii, B.caledonica and B. vermiconia and two novel lineages,clade C, which is phylogenetically distinct from butmorphologically similar to B. bassiana, and clade E, ataxon associated with a Cordyceps teleomorph. Inaddition this phylogenetic analysis also revealed thatB. bassiana and B. brongniartii each embody crypticphylogenetic species complexes. In particular B.bassiana includes an as yet undetermined number ofcryptic lineages, many with intercontinental distribu-tions (Rehner et al. 2006a, Ghikas et al. 2010, Rehnerunpubl), and which occur as multispecies assemblagesin both natural and agricultural habitats (Rehner et al.2006a, Meyling et al. 2009).

As background to the present study, 1192 Beauveriastrains mostly from the ARSEF culture collection(TABLE I) and representing all continents exceptAntarctica were analyzed for species diversity withBloc, a nuclear intergenic region developed as aphylogenetic marker for Beauveria (Rehner et al.2006a). Here a 68-strain ingroup dataset encompass-ing the range of phylogenetic diversity detected in theBloc screen was subjected to multilocus phylogeneticanalysis with an expanded nuclear genome samplingthat included the Bloc nuclear intergenic region andthree nuclear protein-encoding genes, translationelongation factor-1a (TEF), RNA polymerase IIlargest subunit (RPB1) and RNA polymerase IIsecond largest subunit (RPB2). The principal objec-tives of this study were to (i) determine thephylogenetic structure of Beauveria, (ii) evaluateexisting species concepts, (iii) revise the circumscrip-tion of B. bassiana and B. brongniartii and (iv)describe new species corresponding to novel lineagesresolved in the phylogenetic analyses herein.

MATERIALS AND METHODS

Taxon sampling.—Exemplar strains of Beauveria used inthe phylogenetic analysis included available ex-type culturesof currently recognized species, selected strains represent-ing novel phylogenetic terminals discovered in a prelimi-nary diversity screen with Bloc (data not shown) and

outgroup taxa (TABLE I). Additional strains included formorphological examinations are listed under speciestreated in TAXONOMY. Strains were stored as small blocksof agar cultures and conidia in 10% glycerol at 280 C.

Colony growth and morphology.—Colony descriptions andmeasurements were determined from cultures grown on fullstrength Sabouraud’s dextrose agar and potato dextrose agar(DifcoTM) at 23 C in darkness at 10 d from inoculation. Termsand notations used to describe colony coloration are those ofKornerup and Wanscher (1961). Microscopic measurementsof conidiogenous cells and conidia were taken from agarcultures at 10–15 d and images acquired with a Nikon DSFi1digital camera on a Nikon Eclipse E600 microscope with NISElements Basic Research 2.30, SP4 (Nikon) software. Lengthto width ratios are given as Q. Mean values for length, widthand Q are indicated respectively by Lm, Wm and Qm.Terminology for conidial shape follows Vellinga (1988).The images of conidia and conidiogenous cells werephotographed in multiple focal planes with a JenoptikCFscan digital camera (Jena, Germany) on an Olympus BS-51 microscope, and converted to photomontage to enhancedepth of focus with Helicon Focus Pro 4.2.1.5 Pro software(Helicon Soft; www.heliconfocus.com).

DNA extraction, PCR amplification and sequencing.—Geno-mic DNA was prepared either from ground lyophilizedmycelium and purified according to Rehner and Buckley(2005) or extracted with PrepMan Ultra Sample Prepara-tion ReagentH (Applied Biosystems, Foster City, California)according to the manufacturer’s instructions.

We used partial sequences of four nuclear loci in themolecular phylogenetic analysis including RNA polymeraseII largest subunit (RPB1), RNA polymerase II second largestsubunit (RPB2), translation elongation factor-1a (TEF) andthe nuclear intergenic region Bloc. PCR amplification andsequencing primers for these loci are provided (TABLE II).The loci RPB1, RPB2 and TEF were selected for analysisbecause of their wide usage in phylogenetic reconstructionsof fungi (AFTOL, http://aftol.org/data.php) includingtheir demonstrated utility in phylogenetic studies ofBeauveria and related clavicipitaceous fungi (Rehner andBuckley 2005, Sung et al. 2007). Bloc is a nuclear intergenicregion initially developed for cryptic speciation investigationswithin B. bassiana (Rehner et al. 2006a). Sequences for thenuclear ribosomal internal transcribed spacer region (ITS)also were determined for available Beauveria ex-type cultureswith ITS5 and ITS4 primers (White et al. 1990) to serve asauthenticated reference sequences for these species. Howev-er the ITS was not included in the present phylogenetic datamatrix due to its limited information within the ingroup andbecause of difficulties in resolving alignment ambiguities,particularly between outgroup and ingroup taxa.

PCR amplification, sequencing template preparation andsequencing methods are described in Rehner and Buckley(2005), Rehner et al. (2006a) and Rojas et al. (2010).

Data editing and phylogenetic analysis.—Raw sequencechromatograms were assembled and edited with Se-quencher 4.8 (Gene Codes Corp., Ann Arbor, Michigan).Multiple sequence alignments were created with MAFFT

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REHNER ET AL.: BEAUVERIA PHYLOGENY, TAXONOMY 1057

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1058 MYCOLOGIA

(Katoh et al. 2005, Katoh and Toh 2008) using the FFT-NS-2alignment option. Length variable regions in RPB1 andRPB2 introns and Bloc intergenic regions were excludedfrom phylogenetic analyses; thus no attempt was made tooptimize alignments for these regions.

Maximum parsimony (MP) analyses were performed onindividual and concatenated datasets. MP and nonparamet-ric MP bootstrapping (MP BS) analyses were conducted withPAUP* 4b10 (Swofford 2003; Sinauer Associates, Sunder-land, Massachusetts) with all nucleotide characters unor-dered, equally weighted and gaps excluded. MP searches forthe shortest trees for all datasets implemented tree-bisectionand reconnection branch-swapping (TBR) and 1000 randomtaxon-addition sequence replicates. Nonparametric MP BSanalyses (Felsenstein 1985) were performed to assess cladesupport with 1000 BS replicates. We used a conditionalcombination criterion (Mason-Gamer and Kellogg 1996) todetermine whether different data partitions could beconcatenated and analyzed jointly. Data partitions werecombined if monophyly statements were congruent or whereeither one or both conflicting monophyletic groups receivedless than 70% bootstrap support.

Selection of models of nucleotide substitution for themaximum likelihood (ML) and Bayesian (BI) analyses,implementing the Akaike information criterion (AIC), wasdetermined with Modeltest 3.7 (Posada and Crandall 1998).The four-gene dataset was divided into 10 partitions, andthese and their substitution models are a single partition forBloc (HKY + G), TEF first and second codon positions (F81+ I + G), TEF 3rd codon positions (TrN + I + G), RPB1 firstcodon positions (TrN + I + G), RPB1 second codonpositions (TVM + I), RPB1 third codon positions (TVM +G), RPB1 intron (K80 + G), RPB2 first and second codonpositions (TIM + I), RPB2 third codon positions (TVM + G)and RPB2 intron (TIM + G). ML (Felsenstein and Churchill1996) searches and 800 ML bootstrap (ML BS) replicateswere conducted with GARLI 0951 (Zwickl 2006).

Partitioned Bayesian analyses were carried out with MrBayes3.1.2 (Ronquist and Huelsenbeck 2003) with nucmodel 5

4by4 and samplefreq 5 500 for 25 3 106 generations. Allparameters were unlinked across partitions except branchlengths and topology. Branch-length rate multipliers wereunlinked (prset ratepr 5 variable). All analyses were carriedout with parallel processing (one chain per CPU) with eightchains per run and two runs per analysis (nruns 5 2) andbrlenspr 5 unconstrained:Exp (100). The props commandwas used to increase the proposal rate from 1000 to 10 000 andto decrease the Dirichlet alpha parameter from 500 to 250 forthe rate multipliers (proposal mechanism 26 in MrBayes).Burn-in and convergence were assessed with Tracer 1.5(Rambaut A, Drummond AJ [2007].Tracer 1.4, available athttp://beast.bio.ed.ac.uk/Tracer) by examining PSRF valuesin the MrBayes.stat output files and by using Bayes Factorcomparisons of marginal likelihoods of pairs of runs in Tracer.

RESULTS

Data matrix and phylogenetic analysis.—The com-bined Beauveria data matrix included four loci, 72T

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REHNER ET AL.: BEAUVERIA PHYLOGENY, TAXONOMY 1059

isolates and 7628 characters (Bloc: 1574, TEF: 992,RPB1: 2928, RPB2: 2166,) and is available fromTreeBASE as submission 11129. After removal of 56ambiguously aligned and gapped characters the datamatrix contained 7437 characters (Bloc: 1425; TEF:986; RPB1: 2916; RPB2: 2166), including 1603parsimony informative characters (Bloc: 454; TEF:105; RPB1: 580; RPB2: 464). Character and MP treestatistics for the individual genes and the concatenat-ed dataset are provided (TABLE III). The log-likeli-hood score of the best tree from the GARLI MLanalysis was 228973.86. All Bayesian analyses con-verged on a stationary phase that yielded topologicallyidentical 50% majority rule consensus trees (data notshown). The BS values from MP BS and ML BSanalyses and the PPs from one of the Bayesiananalyses are provided (FIG. 1).

Phylogenetic diversity and relationships within Beau-veria.—ML, MP and Bayesian phylogenetic analyses ofBeauveria yielded compatible trees that each resolved 12well supported terminal lineages or branches (FIG. 1).We summarized the MP BS support values for speciesclades (TABLE IV). Six terminal clades correspond topreviously described species that include B. amorpha, B.bassiana, B. brongniartii, B. caledonica, B. malawiensisand B. vermiconia. B. bassiana represents a highlyspeciose complex of morphologically cryptic phyloge-netic species (Rehner unpubl). Details of the compo-sition and structure of the B. bassiana complex arebeyond the scope of the taxon and gene samplingpresented here and will be described elsewhere. Sixadditional clades or lineages represent novel speciesthat we describe herein and include B. asiatica, B.australis, B. kipukae, B. pseudobassiana, B. sungii and B.

TABLE II. PCR amplification (3) and sequencing primers

Locus Primer PCR Sequence (59–39)a Reference

Bloc B5.1F 3 CGACCCGGCCAACTACTTTGA Rehner et al. 2006aB3.1R 3 GTCTTCCAGTACCACTACGCC Rehner et al. 2006aB822Ldg AGATYCGYAACGTCAACTTT Rehner et al. 2006aB22Udg GTCGBAGCCAGAGCAACT this studyB5.4F CATTCGMGGCYTGTTCTTTGG this studyBRn2 CTCCACGCATTCCGCACCAG this studyBfint GTTCCTTGCCCTCGGTAATGAA this studyBFn2 TCTCGATGCCGTTACCTACA this studyBrint AGCATATCGGGCATGACTGA this studyBFn6 TGGTGCGGAATGCGTGGAGC this studyB3.3R TTCCAGTACCACTACGCCGGC Rehner et al. 2006a

rbp1 RPB1A 3 GARTGYCCDGGDCAYTTYGG Matheny et al. 2002RPB1A_VH6R 3 ATGACCCATCATRGAYTCCTTRTG Stiller and Hall 1997RPB1B_VH6Fa 3 CAYAAGGARTCYATGATGGGTCAT Hofstetter et al. 2007RPB1B_G2R 3 GTCATYTGDGTDGCDGGYTCDCC Hoffstetter et al. 2007Bv-RPB1b_R2 TTGAGBARRCCRTACATRAGC this studyBv-RPB1B_R4 GGGCTRACRAARGCYCKRTCCC this studyBv-RPB1B_F1 CCHTACAAYGCYGATTTYGACG this studyBv-RPB1B_F2 GCTYATGTAYGGYYTVCTCAA this studyBv-RPB1B_F4 GCTYTDCCKGGYATGAAYGTYCG this studyBv-RPB1B_R7 CMGCWGTATCAATGAGACCYTC this study

rpb2 fRPB2_5F 3 GACGAYAGAGAYCAYTTYGG Liu et al. 1999RPB2A_7cR 3 CCCATRGCTTGYTTRCCCAT Liu et al. 1999fRPB2-7cF 3 ATGGGYAARCAAGCYATGGG Liu et al. 1999RPB2-3053bR 3 TGRATYTTRTCRTCSACCAT Reeb et al. 2004Bv-RPB2A_R1 CCCCTGTTGATCATRAAGTCA this studyBv-RPB2A_F3 CCMGCCGARCCRCTYATTGA this studyBv-RPB2A_F4 CGCCTGAAGACDAARACMAACC this studyBv-RPB2B_R4 CRGCGTTRACAGRCACRATGA this studyBv-RPB2B_F1 AAGCGTCTTGATTTRGCRGGYCC this studyBv-RPB2B_R2 GCGTGAATYTTRTCRTCCAC this study

tef 983F 3 GCYCCYGGHCAYCGTGAYTTYAT Rehner and Buckley 20052218R 3 ATGACACCRACRGCRACRGTYTG Rehner and Buckley 20051567RintB ACHGTRCCRATACCACCRAT Rehner and Buckley 20051577F CARGAYGTBTACAAGATYGGTGG Rehner and Buckley 2005

a IUPCA degenerate nucleotides: D, AGT; H, ACT; N, ACGT; S, CG; R, AG; Y, CT.

1060 MYCOLOGIA

varroae. B. varroae and B. kipukae form a biphyleticclade that is the sister to the B. bassiana clade. B.asiatica and B. australis are sisters to B. brongniartii, andthese three species comprise a lineage that is sister tothe B. bassiana/varroae/kipukae clade. In all analyses B.amorpha was the sister to the B. bassiana/brongniartiigroup clade, although this relationship received signif-icant bootstrap support only from RPB1 and RPB2. B.malawiensis was placed as the basal-most taxon withsignificant support from Bloc, RPB1 and RPB2. Sistergroup relationships and branching order among theremaining basal taxa, including B. pseudobassiana, B.sungii and the sister pair B. vermiconia/B. caledonica,were not unanimously supported by the three analyticalmethods used and thus remain equivocal.

Colony characteristics and micromorphology of Beau-veria.—Beauveria presents few morphological char-acteristics that are taxonomically diagnostic at speciesrank. Colony growth characteristics and appearanceare similar among the majority of Beauveria specieswith colonies appearing white to light yellow andhaving a lanose or velutinous texture that frequentlybecomes farinaceous as conidia accumulate on thesurface of aging cultures. Exceptions are B. mala-wiensis with colonies that are light to intensely pinkand B. sungii, which becomes strongly yellow.Conidiogenous cells, although diagnostic for thegenus, are largely uninformative for distinguishingspecies. The only species uniquely characterized onthe basis of conidial features is B. vermiconia, whichproduces short comma- or sickle-shaped conidia.Otherwise conidial shapes in other Beauveria speciesare more or less either globose/subglobose/broadlyellipsoid or ellipsoid/oblong/cylindrical. Species withineach conidial shape category overlap in size variationand are difficult to differentiate. B. bassiana groupspecies are characterized by globose/subglobose/broad-ly ellipsoid conidia, as are the related B. varroae and B.kipukae. In addition B. australis and B. pseudobassiana,

which are not immediately related to B. bassiana or toone another, also produce similar-sized globose, sub-globose to broadly ellipsoid conidia that cannot bereadily distinguished from those of B. bassiana or eachother. The remaining species, B. amorpha, B. asiatica, B.brongniartii, B. caledonica, B. malawiensis and B. sungii,are characterized by ellipsoid/oblong/cylindrical conid-ia but are hard to separate diagnostically due tooverlapping size and shape variation.

TAXONOMY

B. bassiana and B. brongniartii are redescribed, andtypifications are proposed for these species. Genealog-ical criteria were used to delimit six novel clades orphylogenetic terminals that are described here as newspecies including B. asiatica, B. australis, B. kipukae, B.pseudobassiana, B. sungii and B. varroae. B. amorpha isvalidly published and an epitype specimen is proposed.

Beauveria Vuill., Bull Soc Bot France 59:40. 1912.Typus genericus. Beauveria bassiana (Bals.-Criv.)

Vuill.Colonies on full-strength Sabouraud’s dextrose and

potato dextrose agar at 10 d at 23 C 15–42 mm diam,cottony, powdery, velutinous, or woolly, white oryellow or rarely pinkish, margin white, reverseuncolored or yellow and occasionally with pinkish tored soluble pigment that discolors the medium, attimes producing synnemata or irregular synnema-likestructures. Aerial hyphae branching, hyaline, septate,walls thin and smooth. Conidiogenous cells scatteredor gregarious in dense clusters or whirls, sessile onaerial hyphae or formed one or more at a time onsubtending cells borne on aerial hyphae, hyaline, basetypically subspherical or ampulliform, with walls thinand smooth, apex proliferating sympodially as ageniculate rachis with prominent denticles resultingfrom conidial secession. Cells subtending the conid-

TABLE III. Character, MP tree statistics for Bloc, TEF, RPB1, RPB2 and combined gene dataset

Locus No. charactersNumber PICa

(PIC/bp)b

MPTc

Number Length CId RCe

Bloc 1574 454 (28.8) 192 1326 0.5226 0.4454TEF 986 105 (10.6) 20 229 0.5240 0.4458RPB1 2928 580 (19.8) 14 1318 0.5622 0.4485RPB2 2166 464 (21.4) 8 899 0.6585 0.5821Combined 7437 1603 (21.6) 12 3844 0.5583 0.4797

a PIC, parsimony-informative base pairs.b (PIC/bp), parsimony-informative characters/total base pairs.c MPTs, most parsimonious trees.d CI, consistency index.e RI, retention index.

REHNER ET AL.: BEAUVERIA PHYLOGENY, TAXONOMY 1061

FIG. 1. Phylogeny of Beauveria illustrating species relationships inferred from joint ML analysis of Bloc, TEF, RPB1 andRPB2. The series of three values above internal branches correspond respectively to MP BS proportions ($ 70), ML BSproportions ($ 70) and PPs ($ PP 95) from Bayesian analyses, respectively. Species clades are indicated by vertical bars.Strains associated with type material are indicated in boldface.

1062 MYCOLOGIA

iogenous cells subspherical, fusiform, cylindrical orshield-shaped, forming short chains that sometimesbranch, hyaline, walls thin and smooth. Conidiaholoblastic, acrogenous, produced singly, globose,subglobose, ellipsoid, oblong, cylindrical, reniform orsickle-shaped, hyaline, aseptate, walls thin andsmooth, at times with inconspicuous hilum at base.Chlamydospores absent. Some species known to beassociated with Cordyceps teleomorphs.

Beauveria bassiana (Bals.-Criv.) Vuill., Bull Soc BotFrance 59 40. 1912. FIGS. 2–4; Botrytis bassiana Bals.-Criv., Linnaea 10:611. 1835

(basionym).

See de Hoog (1972) for a nomenclatural discussionand additional synonyms.

Neotypus of Botrytis bassiana (hic designatus). IT-ALY. Emilia-Romagna: Villa Cade, isolated from pupaof Hyphantria cunea (Lepidoptera: Arctiidae), Mar1984, leg. KV Deseo, neotype BPI 880999 is a driedagar culture of ARSEF 1564.

Colony characteristics similar on full strengthSabouraud’s dextrose and potato dextrose agar(DifcoTM), 15–32 mm diam at 10 d at 23 C,appearance densely to loosely lanose, either closelyappressed to agar surface or cushion-like and up to5 mm thick, at first white and often becomingyellowish white (4A2). Conidia aggregated as ,

0.1 mm spherical clusters among aerial hyphae andwhite in mass. Colony reverse uncolored to yellowishwhite (4A2) or tinted by a diffuse to intense dull red(84C, 84D) soluble pigment that discolors themedium. Odor indistinct. Vegetative hyphae septate,branched, hyaline, smooth-walled, 1–2 mm wide.Conidiogenous cells solitary but usually in denseclusters of five or more, base subspherical to ampulli-form and 3–6 mm wide, apex with an indeterminate1 mm wide geniculate, denticulate rachis, producedlaterally on aerial hyphae or from subtending cells

mostly 2.5–6 3 3–6 mm, (FIGS. 2–4). Conidia 2–3 3 2–3 mm, Q 5 0.9–1.5 (Lm 5 2.7 mm, Wm 5 2.2 mm, Qm 5

1.2), globose, subglobose or broadly ellipsoid, rarelyellipsoid, sometimes with inconspicuous hilum atbase, hyaline, aseptate, walls thin and smooth.

Habitat and distribution. B. bassiana is cosmopoli-tan and is isolated from soil, insects, phylloplanes andoccasionally from living plant tissues.

Additional isolates examined. (TABLE I.) BRAZIL: MatoGrosso do Sul, Mato Grosso, isolated from Zulia entreriana(Homoptera: Cercopidae), 26 Jan 1980, leg. RA Daoust(ARSEF 477); Londrina, Parana, isolated from Diabroticaspeciosa (Coleoptera: Chrysomelidae), 6 Jan 1983, leg. RADaoust (ARSEF 958). REPUBLIC OF KOREA: DaeryongMountain, Kangwon-do, isolated from mantid (Dictyoptera:Mantidae), 16 Sep 1996, leg. JM Sung (culture ARSEF 72385 EFCC 1037). MOROCCO: Jramma, isolated from Sitonadiscoideus (Coleoptera: Curculionidae), Jan 1984, leg. TJPoprawski (culture ARSEF 1164); Achouria, isolated fromSitona discoideus (Coleoptera: Curculionidae), Feb 1984,leg. TJ Poprawski (culture ARSEF 1174 5 A6). HUNGARY:Godollo, isolated from unidentified larva (Lepidoptera:Noctuidae), 13 Jun 1984, leg. TJ Poprawski (culture ARSEF1628). CANADA: Magrath, Alberta, soil, 12 Jul 1991, leg. MSGoettel (culture ARSEF 3462). USA: Randolph, Vermont,isolated from Paraclemensia acerifoliella (Lepidoptera: In-curvariidae), 16 May 1997, leg. M Brownbridge (cultureARSEF 5492).

Notes. The concept of B. bassiana is based on aphylogenetic species that is prevalent in Europe(Meyling et al. 2009) and Asia and which also occursin Africa and the western hemisphere (Rehnerunpubl). A dried herbarium specimen of ARSEF1564 is designated to neotypify this species becausethis isolate originated from a location in northern Italyand a lepidopteran host phylogenetically related tosilkworms on which B. bassiana originally was observed(Bassi 1835; Balsamo-Crivelli 1835a, b). In addition theex-type of Cordyceps bassiana Z.Z. Li, C.R. Li, B. Huang& M.Z. Fan, ARSEF 7047, is placed in the same clade asARSEF 1564, definitively linking this phylogenetic

TABLE IV. MP bootstrap support (. 50%) for species by individual gene partitions and combined (All) dataset for 72-isolateBeauveria analysis. Bootstrap support for B. kipukae and B. vermiconia were not evaluated because only single isolates wereavailable for these species

Species Bloc TEF RPB1 RPB2 All

B. amorpha 98 71 99 99 99B. asiatica 100 91 100 100 100B. australis 100 97 100 100 100B. bassiana 100 75 100 100 100B. brongniartii 79 NA 100 99 99B. caledonica 100 97 100 100 100B. malawiensis 100 100 100 100 100B. pseudobassiana 99 100 99 100 100B. sungii 100 100 100 100 100B. varroae 100 99 100 100 100

REHNER ET AL.: BEAUVERIA PHYLOGENY, TAXONOMY 1063

FIGS. 2–10. Conidiogenous cells and conidia of Beauveria species. 2–4. B. bassiana (ARSEF 1564). 5, 6. B. amorpha (ARSEF2641). 7. B. asiatica (ARSEF 4850). 8–10. B. australis (ARSEF 4598). All images at the same magnification from materialmounted in 50% acetic acid plus cotton blue.

1064 MYCOLOGIA

species to a teleomorph. B. bassiana is hardlydistinguishable morphologically from B. australis, B.kipukae, B. pseudobassiana and B. varroae, which alsoproduce globose/sublglobose/broadly ellipsoid conid-ia, but its conidia may be larger than some of thesespecies.

Additional species of Beauveri:

Beauveria amorpha (Hohn.) Minnis, S.A. Rehner &Humber, comb. nov. FIGS. 5, 6

MycoBank MB519121; Isaria amorpha Hohn., Sitzungsber Kaiserl Akad Wiss,

Math-Naturwiss Kl, Abt 1 118:415. 1909 (basionym).; Beauveria amorpha (Hohn.) Samson & H.C. Evans, J

Invertebr Pathol 39:95. 1982. Nom. inval. via ICBNArt.33.

Holotypus of Isaria amorpha. INDONESIA: Java,Sukabumi, on a large cicada, leg. Major Ouwens, inFH.

Epitypus of Isaria amorpha (hic designatus). BRA-ZIL: Alambari, Sao Paulo, on Solenopsis sp. (Hyme-noptera: Formicidae), received at ARSEF 23 Nov 1988,leg. SB Alves (621), epitype BPI 880996 is a dried agarculture of ARSEF 2641.

Colony growth and appearance on full-strengthSabouraud’s dextrose and potato dextrose agars(DifcoTM) similar, 16–24 mm diam at 10 d at 23 C,velutinous and closely appressed to agar surface andup to 4 mm thick, white, changing to yellowish white(4A2) in older portions of the colony. Conidiaaggregated as , 0.1 mm spherical clusters and whitein mass. Colony reverse uncolored or yellowish white(4A2) to grayish orange (5B5, 5B6). Odor indistinct.Vegetative hyphae septate, branched, hyaline,smooth-walled, 1–2 mm wide. Conidiogenous cellssolitary but usually in dense lateral clusters, basesubspherical to ampulliform and 3–6 mm wide, apexwith an indeterminate 1 mm wide geniculate, dentic-ulate rachis, produced laterally on aerial hyphae orfrom subtending cells mostly 2.5–6 3 3–6 mm(FIGS. 5–6). Conidia 2.5–4 3 1.5–2.5 mm, Q 5 1.3–2.2 (Lm 5 3.3 mm, Wm 5 2.0 mm, Qm 5 1.6), ellipsoid,oblong or cylindrical, at times slightly curved toreniform, occasionally with inconspicuous hilum atbase, hyaline, aseptate, walls smooth and thin.

Habitat and distribution. Isolated from Solenopsis sp.in Sao Paulo Brazil. Also known from North Americaand Australia and to infect Coleoptera.

Additional isolates examined. (TABLE I)

Notes. This species was described originally frommaterial on a cicada found in Java (Hohnel 1909).Samson and Evans (1982) examined the holotype atthe Farlow Herbarium (FH) and studied what theybelieved to be conspecific isolates from South Amer-ica. Unfortunately they did not validly publish the new

combination in Beauveria. Thus we have done so hereand epitypify the species with an isolate that is similarin appearance to those of Samson and Evans (1982)and the synonym, Isaria orthopterorum Petch asdescribed in de Hoog (1972), to stabilize theapplication of this name. We examined the Samsonand Evans (1982) isolates from CBS, but they were notBeauveria; thus these isolates might be contaminants.This species is characterized by its versiform, ellipsoidto cylindrical conidia, which may be slightly curved toreniform.

Beauveria asiatica S.A. Rehner & Humber, sp.nov. FIG. 7

Mycobank MB519122Beauveriae brongniartii similis, sed conidiis majoribus,

2.5–4 3 2–3 mm, et Q minoribus, 1.2–2.0 (late ellipticis,ellipticis vel oblongis; non cylindricis) differt.

Holotypus. REPUBLIC OF KOREA: Kangwon-do,Chiag Mountain, isolated from Coleoptera: Ceramby-cidae, 14 Jul 1994, leg. JM Sung, holotype BPI 880997is a dried agar culture of ARSEF 4850.

Etymology. The epithet refers to the Asian origin ofthe type material.

Colony growth and appearance on full-strengthSabouraud’s dextrose and potato dextrose agars(DifcoTM) similar, 26–30 mm diam at 10 d at 23 C,mycelium dense, velutinous and closely appressed toagar surface or cottony and up to 5 mm thick, white,changing to yellowish white (4A2) in older portionsof the colony. Conidia aggregated as , 0.1 mmspherical clusters and white in mass. Colony reverseuncolored or yellowish white (4A2). Odor indistinct.Vegetative hyphae septate, branched, hyaline,smooth-walled, 1–2 mm wide. Conidiogenous cellssolitary but usually in dense lateral clusters, basesubspherical to ampulliform and 3–6 mm wide, apexwith an indeterminate 1 mm wide geniculate, dentic-ulate rachis, produced laterally on aerial hyphae orfrom subtending cells mostly 2.5–6 3 3–6 mm(FIG. 7). Conidia 2.5–4 3 2–3 mm, Q 5 1.2–2 (Lm

5 3.2 mm, Wm 5 2.3 mm, Qm 5 1.4), broadly ellipsoid,ellipsoid or oblong, occasionally with inconspicuoushilum at base, hyaline, aseptate, walls smooth andthin.

Habitat and distribution. On longicorn beetle (Co-leoptera: Cerambycidae) in forest habitat, ChiagMountain, Kangwon-do, Republic of Korea. Alsoknown from Holotrichia parallela (Coleoptera: Scar-abaeidae), Cangzhou, China.

Additional isolates examined. (TABLE I)

Notes. A member of the B. brongniartii complex, B.asiatica is characterized by broadly ellipsoid, ellipsoidor oblong conidia that are slightly larger than those ofB. brongniartii.

REHNER ET AL.: BEAUVERIA PHYLOGENY, TAXONOMY 1065

Beauveria australis S.A. Rehner & Humber, sp.nov. FIGS. 8–10

Mycobank MB519123A speciebus in Acrididae et similis Beauveriae bassianae,

sed conidiis minoribus, 2–2.5 3 1.5–2.5 mm differt.

Holotypus. AUSTRALIA: Benham, Avoca, Tasmania,soil, 18 Feb 1988, leg. AC Rath, holotype BPI 880998 isa dried agar culture of ARSEF 4598 5 F308.

Etymology. The epithet australis, meaning southern,refers to the geographic origin of the type in thesouthern hemisphere.

Colony growth and appearance on full-strengthSabouraud’s dextrose and potato dextrose agarssimilar, 26–30 mm diam at 10 d at 23 C, surfacemycelium velutinous and closely appressed to agarsurface, becoming yellowish white (4A2) in interiorportions of the colony with margin white. Conidiaaggregated in , 0.1 mm spherical clusters and whitein mass. Colony reverse uncolored to light yellow(4A5) or light orange (5A5). Odor indistinct.Vegetative hyphae septate, branched, hyaline,smooth-walled, 1–2 mm wide. Conidiogenous cellssolitary but usually in dense lateral clusters, basesubspherical to ampulliform and 3–6 mm wide, apexwith an indeterminate 1 mm wide geniculate, dentic-ulate rachis, produced laterally on aerial hyphae orfrom subtending cells mostly 2.5–6 3 3–6 mm (FIGS. 8,10). Conidia 2–2.5 3 1.5–2.5 mm, Q 5 1–1.6 (Lm 5

2.3 mm, Wm 5 1.9 mm, Qm 5 1.2), primarilysubglobose, broadly ellipsoid or ellipsoid, less com-monly globose, occasionally with inconspicuous hi-lum at base, hyaline, aseptate, walls smooth and thin.

Habitat and distribution. Isolated from soil andgrasshoppers (Orthoptera: Acrididae). Known onlyfrom Australia.

Additional isolates examined. (TABLE I)

Notes. The sister lineage to B. brongniartii, B.australis is differentiated from the former by itsconidia with smaller Q values that overlap in size andshape with other Beauveria species with subgloboseconidia, including B. bassiana, B. kipukae, B. pseudo-bassiana and B. varroae.

Beauveria brongniartii (Sacc.) Petch, Trans Brit MycolSoc 10:249. 1926. FIGS. 11–13; Botrytis brongniartii Sacc., Sylloge Fungorum 10:540.

1982 (basionym).

De Hoog (1972) listed additional synonyms.Typus of Botrytis brongniartii. France: On larva of

Melolontha melolontha [Coleoptera: Scarabaeidae],ARSEF 617 received 27 May 1981, leg. P. Ferron, BPI881000 (dried culture of ARSEF 617 5 ATCC 26156),proposed as a conserved type (Minnis et al. 2011).

Colony characteristics on full-strength Sabouraud’sdextrose and potato dextrose agars (DifcoTM) similar,

20–30 mm diam at 10 d at 23 C, appearance denselyto loosely lanose, either closely appressed to agarsurface or cushion-like and up to 7 mm thick, colorwhite and often becoming yellowish white (4A2, 4A3)to pale yellow (4A4). Conidia aggregated as , 0.1 mmspherical clusters and white in mass. Colony reverseoff-white to pale yellow (4A4) and in some isolatesdiscolored by a diffuse to strongly dull red (84C, 84D)soluble pigment. Odor indistinct. Vegetative hyphaeseptate, branched, hyaline, smooth-walled, 1–2 mmwide. Conidiogenous cells solitary but usually indense lateral clusters, base subspherical to ampulli-form and 3–6 wide, apex with an indeterminate 1 mmwide geniculate, denticulate rachis, produced laterallyon aerial hyphae or from subtending cells mostly 2.5–6 3 3–6 mm (FIGS. 11–13). Conidia 2.5–3.5 3 1–2 mm,Q 5 1.6–2.4 (Lm 5 3 mm, Wm 5 1.6 mm, Qm 5 1.9),primarily oblong or cylindrical, occasionally withinconspicuous hilum at base, hyaline, aseptate, wallsthin and smooth.

Habitat and distribution. Occurring in soils innatural and agricultural habitats, pathogen principallyof Coleoptera but also infects other insect taxa. Knowngeographic range includes Asia, Europe and NorthAmerica.

Additional isolates examined. (TABLE I) MOROCCO:Achouria, isolated from adult Sitona discoideus (Coleoptera:Curculionidae), Feb 1984, leg. TJ Poprawski (culture ARSEF1174). FRANCE: isolated from Melolontha melolontha(Coleoptera: Scarabaeidae), 17 Oct 1983, leg. RA Hall andJ Fargues (culture ARSEF 979). HUNGARY: Godollo,isolated from larva, (Lepidoptera: Noctuidae), 13 Jun1984, leg. TJ Poprawski (culture ARSEF 1628). CANADA:Magrath, Alberta, soil, 12 Jul 1991, leg. MS Goettel (cultureARSEF 3462).

Notes. The original concept of B. brongniartii wasbased on material isolated from locusts in North Africa(Saccardo 1892). However in Europe, where B.brongniartii frequently occurs, the prevailing conceptof this species corresponds to the principal Beauveriapathogen of the European cockchafer, Melolonthamelolontha. Although the host sources for isolatesexamined here indicate a wider host range for thisspecies, there is no evidence that B. brongniartii occursspecifically on Orthoptera. In addition conidia in theoriginal material were described as being oval orellipsoid with a smaller Q value (1.6) than theprevailing concept. Only a single clade is known toexist in Europe. Because there is a conflict between theprevailing, economically important concept of thisspecies and the application of this species dictated bythe protolog this name has been proposed forconservation with a conserved type to preserve currentusage (Minnis et al. 2011). The known species rangeincludes the Holarctic and southeastern Asia. Its colonygrowth characteristics are similar to other Beauveria

1066 MYCOLOGIA

FIGS. 11–21. Conidiogenous cells and conidia of Beauveria species. 11–13. B. brongniartii (ARSEF 617). 14, 15. B.caledonica (ARSEF 2567 5 DAOM 191855). 16–18. B. kipukae (ARSEF 7032). 19–21. B. malawiensis (ARSEF 7760 5 IMI228343). All images at the same magnification from material mounted in 50% acetic acid plus cotton blue.

REHNER ET AL.: BEAUVERIA PHYLOGENY, TAXONOMY 1067

species including other members of both the B.bassiana and B. brongniartii morphological complexes.Most isolates have oblong or cylindrical conidia and canbe distinguished by having among (see B. caledonicaand B. malawiensis) the largest Q value of species withellipsoid/oblong/cylindrical conidia.

Beauveria caledonica Bissett & Widden, Can J Bot66:361. 1988. FIGS. 14, 15Comments. See Bissett and Widden (1988) for adescription and Rehner and Buckley (2005) foradditional discussion of this species.

Beauveria kipukae S.A. Rehner & Humber, sp.nov. FIGS. 16–18

Mycobank MB519124Similis Beauveriae bassianae, sed ab eo propter sequentia

Bloc dicta (GenBank HQ880734) differt.

Holotypus. USA. HAWAII: Bird Park (Kipuka Puaulu,Mauna Loa, Hawaii, isolated from unidentified Ho-moptera (Delphacidae), 26 Jan 2002, leg. RG Hollings-worth, holotype BPI 881001 is a dried culture ofARSEF 7032.

Etymology. Hawaiian, from kipuka, which refers tonative montane Hawaiian forests isolated by recentlava flows.

Colony growth and appearance on full-strengthSabouraud’s dextrose and potato dextrose agars(DifcoTM) similar, 21–26 mm diam at 10 d at 23 C,velutinous, closely appressed to agar surface, marginwhite and colony interior white or changing toyellowish white (4A2). Conidia aggregated in ,

0.1 mm spherical clusters and white in mass. Colonyreverse uncolored to yellowish white (4A2). Colonyodor indistinct. Vegetative hyphae septate, branched,hyaline, smooth-walled, 1–2 mm wide. Conidiogenouscells solitary but usually in dense clusters of five ormore, base subspherical to ampulliform and 3–6 mmwide, apex with an indeterminate 1 mm widegeniculate, denticulate rachis, produced laterally onaerial hyphae or from subterminal cells mostly 2.5–63 3–6 mm, (FIGS. 16–18). Conidia 2–3.5 3 1.5–3 mm,Q 5 0.9–1.4 (Lm 5 2.5 mm, Wm 5 2.2 mm, Qm 5 1.2),globose, subglobose or broadly ellipsoid, rarelyellipsoid, sometimes with inconspicuous hilum atbase, hyaline, aseptate, walls smooth and thin.

Habitat and distribution. Known from a singleisolate from an unidentified homopteran in remnantnative montane forest surrounded by recent lava flowsin Bird Park, Mauna Loa, Hawaii.

Notes. B. kipukae has conidia that are smaller, basedon means, than B. bassiana but cannot be distin-guished from B. australis, B. pseudobassiana and B.varroae in the absence of phylogenetically informativenucleotide characters. Known only from a single

specimen; it is not known whether B. kipukae is aHawaiian endemic.

Beauveria malawiensis S.A. Rehner & Aquino deMuro, Mycotaxon 98:140. 2007 [2006]. FIGS. 19–21Notes. See Rehner et al. (2006b) for a description

and discussion of this taxon.

Beauveria pseudobassiana S.A. Rehner & Humber, sp.nov. FIGS. 22–24

Mycobank MB519125Similis Beauveriae bassianae, sed conidiis minoribus, 2–3

3 1.5–2.5 mm, et ab eo propter sequentias Bloc dictas(GenBank HQ880723) differt.

Holotypus. USA Virginia: Page County, KoontzLokey, isolated from larva of Lymantria dispar (Lep-idoptera: Lymantriidae), 10 Jun 1991, leg. AE Hajek,holotype BPI 881002 is a dried culture of ARSEF 3405.

Etymology. Pseudobassiana refers to the close phe-notypic similarity of this species to B. bassiana.

Colony growth and appearance on full-strengthSabouraud’s dextrose and potato dextrose agars(DifcoTM) similar, 21–34 mm diam at 10 d at 23 C.Surface mycelium subvelutinous, velutinous to cot-tony, closely appressed to agar surface or greaterthan 5 mm thick; margin white with colony interiorwhite or changing to yellowish white (4A2) to paleyellow (4A3). Conidia aggregated as , 0.1 mmspherical clusters, white in mass, conidia occasion-ally forming a farinaceous surface layer in oldercultures. Colony reverse either uncolored or yellow-ish white (4A2, 4A3). Odor indistinct. Vegetativehyphae septate, branched, hyaline, smooth-walled,1–2 mm wide. Conidiogenous cells solitary butusually in dense lateral clusters, base subsphericalto ampulliform and 3–6 wide, apex with anindeterminate 1 mm wide geniculate, denticulaterachis, produced laterally on aerial hyphae or fromsubtending cells mostly 2.5–6 3 3–6 mm (FIGS. 22–24). Conidia 2–3 3 1.5–2.5 mm, Q 5 1.1–1.4 (Lm 5

2.3 mm, Wm 5 2 mm, Qm 5 1.2), primarily subgloboseor broadly ellipsoid, rarely ellipsoid, occasionallywith inconspicuous hilum at base, hyaline, aseptate,walls thin and smooth.

Habitat and distribution. B. pseudobassiana is aglobally distributed soilborne, broad host-range gen-eralist entomopathogen that occurs in a wide range ofhabitats. Its conidia are generally smaller than B.bassiana but it is indistinguishable from B. varroae, B.kipukae and B. australis, which also have globose/subglobose/broadly ellipsoid conidia.

Additional isolates examined. (TABLE I)

Notes. B. pseudobassiana is similar phenotypically toB. bassiana in all respects except for its slightly smaller

1068 MYCOLOGIA

FIGS. 22–32. Conidiogenous cells and conidia of Beauveria species. 22–24. B. pseudobassiana (ARSEF 3405). 25–27. B.sungii (ARSEF 1685). 28, 29. B. varroae (ARSEF 8257). 30–32. B. vermiconia (ARSEF 2922 5 CBS 645.74). All images at thesame magnification from material mounted in 50% acetic acid plus cotton blue.

REHNER ET AL.: BEAUVERIA PHYLOGENY, TAXONOMY 1069

conidia. Nucleotide characters are necessary to iden-tify B. pseudobassiana with confidence.

Beauveria sungii S.A. Rehner & Humber, sp.nov. FIGS. 25–27

Mycobank MB519126Similis Beauveriae brongniartii, sed conidiis latioribus,

1.5–2.5 mm, et Q minoribus, 1.2–1.8 (late ellipticis, ellipticisvel oblongis; non cylindricis) differt.

Holotypus. JAPAN. Ibaraki Prefecture: Lachnosternamorosa (Coleoptera: Scarabidae), 1984, leg. M. Shi-mazu, holotype BPI 881003 is a dried culture of ARSEF1685.

Etymology. Sungii is named in honor of Jae-Mo Sungin recognition of his contributions to knowledge of thebiodiversity and life histories of clavicipitaceous fungi.

Colony growth and appearance similar on both full-strength Sabouraud’s dextrose agar and potatodextrose agar (DifcoTM), 32–41 mm diam at 10 d at23 C, mycelium dense, cottony, often 5 mm or morethick, colony growth initially white, remaining whiteor changing to yellowish white (4A2), pale yellow(4A3) or light yellow (4A4) in older portions of thecolony. Conidia not readily observed in most cultures.Colony reverse pale yellow (4A3), light yellow (4A5) orshades of orange to deep orange (5A3–5A7). Colonyodor indistinct. Vegetative hyphae septate, branched,hyaline or translucent pale yellow, smooth-walled, 1–2 mm wide. Conidiogenous cells solitary but usually indense clusters of five or more, base subspherical toampulliform and 3–6 mm wide, apex with an indeter-minate 1 mm wide geniculate, denticulate rachis,produced laterally on aerial hyphae or from subtend-ing cells mostly 2.5–6 3 3–6 mm (FIGS. 25–27). Conidia2.5–3.5 3 1.5–2.5 mm, Q 5 1.2–1.8 (Lm 5 3 mm, Wm 5

2.1 mm, Qm 5 1.5), broadly ellipsoid, ellipsoid oroblong, sometimes with inconspicuous hilum at base,hyaline, aseptate, walls thin and smooth.

Habitat and distribution. Known from Japan andKorea. Isolated from coleopteran hosts from foresthabitats.

Additional isolates examined. (TABLE I)

Notes. B. sungii is characterized via in vitro culturesby its distinctly yellow colony pigmentation andellipsoid or oblong conidia. It is distinguished fromother species of Beauveria with ellipsoid conidia,including B. amorpha, B. asiatica, B. brongniartii andB. caledonica, by its shorter conidia and more intenseyellow pigmentation in mature colonies. All but one ofthe B. sungii strains examined here were isolated fromCordyceps collections identified either as Cordycepsstaphylinidicola Kobayasi & Shimizu or C. scarabaeicolaKobayasi, indicating the need for taxonomic studies ofthese and other yellow-pigmented Cordyceps speciesthat also produce Beauveria anamorphs.

Beauveria varroae S.A. Rehner & Humber, sp.nov. FIGS. 28, 29

Mycobank MB519127Similis Beauveriae bassianae, sed ab eo propter sequentias

Bloc dictas (Genbank HQ880733) differt.

Holotypus. FRANCE. Montardier Gard: isolatedfrom Varroa destructor (Acari: Varroidae), 29 Jun2005, leg. WG Meikle and G Mercadier, holotype BPI881004 is a dried culture of ARSEF 8257.

Etymology. Varroae refers to the host genus fromwhich this species was first isolated.

Colony growth and appearance on full-strengthSabouraud’s dextrose agar and potato dextrose agar(DifcoTM) similar to that of B. bassiana, 26–30 mmdiam at 10 d at 23 C, velutinous, closely appressed toagar surface, margin white with interior of colonybecoming yellowish white (4A2) to pale yellow (4A3).Conidia aggregating as , 0.1 mm spherical clustersand white in mass. Colony reverse uncolored to lightyellow (4A4, 4A5). Colony odor indistinct. Vegetativehyphae septate, branched, hyaline, smooth-walled, 1–2 mm wide. Conidiogenous cells solitary but usually indense clusters of five or more, base subspherical toampulliform and 3–6 mm wide, apex with an indeter-minate 1 mm wide geniculate, denticulate rachis,produced laterally on aerial hyphae or from subtend-ing cells mostly 2.5–6 3 3–6 mm, (FIGS. 28, 29). Conidia2–3.5 3 2–3 mm, Q 5 1–1.3 (Lm 5 2.5 mm, Wm 5 2.2 mm,Qm 5 1.1), globose, subglobose or broadly ellipsoid,sometimes with inconspicuous hilum at base, hyaline,aseptate, walls thin and smooth.

Habitat and distribution. Isolated from Varroadestructor mite ectoparasites of honeybee (Apis melli-fera) in France. Also infecting Larinus sp. (Coleop-tera), Switzerland.

Additional isolates examined. (TABLE I)

Notes. B. varroae is not morphologically distinguish-able from other Beauveria species that produceglobose/subglobose/broadly ellipsoid conidia. B. var-roae and B. kipukae form a sister clade to the B.bassiana complex.

Beauveria vermiconia de Hoog & V. Rao, Persoonia8:209. 1975. FIGS. 30–32Comments. See de Hoog and Rao (1975) for a

description and Rehner and Buckley (2005) foradditional remarks on this species.

DISCUSSION

Genetic diversity surveys of Beauveria in relation toinsect host, habitat and geography report theiroccurrence in a wide range of terrestrial habitats,principally as pathogens of insects and arthropodsand from soil, but also from phylloplanes and as

1070 MYCOLOGIA

endophytes (Aquino de Muro et al. 2003, 2005;Rehner et al. 2006a; Glare et al. 2008; Vega et al.2008; Meyling et al. 2009; Ghikas et al. 2010 andreferences cited therein). These studies reportedhigh genotypic diversity, however species identitiesand species boundaries often were not explicitlydefined, in part because comparable genotypicinformation from taxonomically authenticated spec-imens was not included or was unavailable. Inaddition objective comparisons among a majority ofdiversity surveys is difficult or impossible due to theuse of dissimilar types of genetic markers. To improveunderstanding of species diversity and species limitsand to develop essential data for the molecularidentification of Beauveria species we conducted ataxonomic appraisal of the genus based on a multi-locus phylogenetic analysis that included ex-typestrains for currently recognized species and newspecies lineages discovered in a globally comprehen-sive screening of Beauveria isolates.

Beauveria comprises 12 well supported terminallineages (FIG. 1), as revealed by the multilocusphylogeny. Six terminal clades correspond to previ-ously recognized species including B. amorpha, B.bassiana, B. brongniartii, B. caledonica, B. malawiensisand B. vermiconia, and six additional clades arerecognized as new species including B. asiatica, B.australis, B. kipukae, B. pseudobassiana, B. sungii andB. varroae. Morphological examinations of thesespecies, including the new species described here,serve to confirm that cultural morphology and conidialcharacteristics represent the principal visible extrinsicfeatures that vary among species of Beauveria. Howeverextensive overlap in these characters limits their utilityas diagnostic taxonomic characters for species identi-fication and only a minority of species can be positivelyidentified on the basis of phenotype alone. In contrastto the limited number and established limitations ofavailable morphological characters, each of the fourloci used here to reconstruct the phylogeny ofBeauveria are effective for accurate diagnosis of all 12species on the basis of multiple species-specificphylogenetically informative nucleotide characters.These datasets accordingly can form the basis for thefuture development of sequence-, PCR-RFLP- or SNP-based identification systems to aid accurate taxonomicassignments of Beauveria species in epidemiologicaland ecological investigations.

The majority of strains in this study were isolated asanamorphs from insects and soil. However severalstrains were isolated from Cordyceps specimens eitherfrom stromatal tissues or ascospores. These teleo-morph-derived strains were assigned phylogeneticallyeither to B. bassiana, B. brongniartii or B. sungii.Teleomorphs for B. bassiana (Li et al. 2001) and B.

brongniartii (Shimazu et al. 1988) have been de-scribed, whereas the anamorph-teleomorph connec-tion for B. sungii reported here represents a thirdBeauveria species whose life cycle is confirmed toinclude a sexual state. The teleomorph specimens forthe strains included in this study were not availablefor examination, but collection data describe all threeteleomorphs as having yellow-pigmented stromata.Additional morphological and cultural investigationsare needed to characterize these teleomorphs and todetermine whether they are synonyms of previouslydescribed Cordyceps species, such as C. staphylinidicolaKobayasi & Shimizu (1982), C. sulfurea Kobayasi &Shimizu (1983) and C. scarabaeicola Kobayasi &Shimizu (1976), and possibly additional Cordycepsspecies that produce yellow-pigmented stromata.Recent progress in methods for in vitro fruiting ofCordyceps species with Beauveria anamorphs (Sung etal. 2006; Lee et al. 2007, 2010) are promisingdevelopments with potential to inform systematicand life history studies of Beauveria and its Cordycepsteleomorphs through integrated phylogenetic, devel-opmental and mating studies.

ACKNOWLEDGMENTS

We are extremely grateful for the help of Veronica Martins,Ellen P. Buckley, Karen Hansen, Michael M. Wheeler andClare Whittaker who provided invaluable laboratory supportthroughout this investigation. In addition we thank Chris-tian Feuillet for his assistance in editing the Latin diagnoses,Genevieve Lewis-Gentry at FH for providing information onthe holotype of Isaria amorpha and Ted Schultz for adviceand assistance with the Bayesian analysis. The mention oftrade products or firm names does not imply that the U.S.Department of Agriculture recommends them over similarproducts or firms not mentioned.

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