Mycol. Res. 104 (2): 134–150 (February 2000). Printed in the United Kingdom. 134

A taxonomic revision of based on a phylogenetic analysis of rDNA sequence data

Felice DRIVER, Richard J. MILNER and John W. H. TRUEMAN* CSIRO Entomology, GPO Box 1700, ACT 2601, Australia and *Research School of Biological Sciences, Australian National University, Canberra, ACT 2601, Australia. E-mail: richard.milner!ento.csiro.au

Received 31 October 1997; accepted 17 July 1999.

The of Metarhizium has been reassessed using sequence data and RAPD patterns from 123 isolates recognised as M. anisopliae, M. flavoviride or M. album. A high level of genetic diversity was found which was best resolved at the species\variety level by sequence data from the ITS and 28S rDNA D3 regions. RAPD patterns correlated closely with the sequence data and revealed a much greater degree of diversity useful for distinguishing strains within a variety. Ten distinct clades were revealed by the cladogram based on the combined sequence data set. Several major evolutionary lines were revealed, but the taxonomic relationships at the base of the tree are poorly resolved. The data support the monopoly of the M. anisopliae group, and recognise four clades within it. Two correspond with M. anisopliae var. anisopliae and M. anisopliae var. majus. The other two are described as new varieties based on their distinctive ITS sequence data: M. anisopliae var. lepidiotum and M. anisopliae var. acridum vars nov. M. album, M. flavoviride var. flavoviride and M. flavoviride var. minus are recognised and redefined according to ITS sequence data. Three clades represent two new varieties, M. flavoviride var. novazealandicum and M. flavoviride var. pemphigum vars nov., based on their distinct ITS sequence data. The third, with two isolates, has not been named pending further data.

morphological characters and was reviewed by Tulloch INTRODUCTION (1976), who only accepted M. flavoviride and M. anisopliae, the Metarhizium is one of the best known genera of entomo- latter with the short spored var. anisopliae and the long-spored pathogenic fungi, commonly known as ‘green muscardine var. majus. M. album, described from a leaf hopper in Sri Lanka ’. M. anisopliae, described from Russia as a pathogen of by Petch (1931), was determined by Tulloch to be an the cockchafer, austriaca (Coleoptera: Scara- immature specimen of M. anisopliae. Petch (1935) also baeidae), is used for control in many countries including described M. brunneum, from an homopteran host in the the USA, Brazil, Australia and the Philippines. Its potential in Philippines, but Latch (1965) examined authentic material of Australia for control of and was reviewed M. brunneum (IMI 14746) from wireworms (Coleoptera: by Milner (1997). The life-cycle is simple: the infectious unit Elateridae) and considered its colour to be similar to that of M. is an asexual, haploid conidiospore, formed in chains on anisopliae from Costelytra zealandica which was dark brown on phialides, which may or may not be swollen (Glare & Milner some media. Tulloch (1976) agreed with Latch (1965) that M. 1996). The conidia germinate on the cuticle of a susceptible brunneum and M. anisopliae are synonymous, and speculated insect, produce a germ tube which penetrates into the body that M. brunneum might be a naturally occurring colour cavity, where the fungus proliferates as hyphae, eventually mutant. Rombach et al. (1986) reviewed the status of M. killing the host. After death, if conditions are moist and humid, flavoviride, which is known from curculionid and the fungus grows out through the cuticle and forms new green agricultural soil. They expanded the concept of the long- and conidia aerially. It grows and sporulates well on simple media short-spored forms of M. anisopliae described by Tulloch to such as Sabouraud’s dextrose agar. No teleomorph is known accommodate Asian isolates of M. flavoviride var. minus, from Metarhizium apart from a claim that Cordyceps taii is the which were characterised by consistently producing smaller teleomorph of M. taii (Liang et al. 1991). The robust conidia spores and having been collected from plant-hoppers and leaf- can be formulated as a mycoinsecticide for pest control. hoppers (Homoptera: Delphacidae) on rice in the Philippines Registered products include BioBlast for termites in the USA and Solomon Islands. They also described M. flavoviride var. and BioGreen for pasture cockchafers in Australia (Milner minus on a (: ) from the 1998). Galapagos Islands (Evans & Samson 1982). Rombach et al. The current classification of Metarhizium is based on (1987) also described the morphological resemblance of M. F. Driver, R. J. Milner and J. W. H. Trueman 135

Table 1. List of isolates studied (the first isolate in each clade is the type, except for clade 10 where the type was not examined)

Source name (if Other different from D3 ITS RAPD FI number designation proposed)* Host and origin group group group

Clade 1. M. album MaF ARSEF 1941 — Nephotettix virescens (Homoptera), MaF MaF n.d.** Philippines 1165 ARSEF 1942 — N. virescens, Philippines MaF 1165 1165 Clade 2. M. flavoviride Type E 1173 ARSEF 2948 M. f. var. minus Homoptera, Brazil 152 1173 1173 152 — M. a. var. anisopliae Lepidiota consobrina (Coleoptera), Australia 152 152 152 Clade 3. M. flavoviride var. novazealandicum 698 F10 M. a. var. anisopliae Lepidoptera, New Zealand MaF 698 698 1124 DAT-F220 M. a. var. frigidum Soil, Australia MaF 1124 1124 1125 DAT-F368 M. a. var. frigidum Soil, Australia MaF 1125 1125 699 — — Costelytra zealandica (Coleoptera), New MaF 698 698 Zealand 702 — — Lepidoptera, New Zealand MaF 698 698 Clade 4. M. flavoviride var. pemphigum 72 — M. a. var. anisopliae Pemphigus treherni (Homoptera), Britain 405 72 72 1101 — M. a. var. anisopliae P. treherni, Britain 405 72 72 Clade 5. M. flavoviride var. minus 403 ARSEF 2037 — Niliparvata lugens (Homoptera), Philippines 405 403 403 1172 ARSEF 1764 — N. lugens, Philippines 405 1172 403 Clade 6. M. flavoviride var. flavoviride 405 ARSEF 1184 — Otiorhynchus sulcatus (Coleoptera), France 405 405 405 402 ARSEF 2024 — O. sulcatus, France 405 402 405 1170 ARSEF 2025 — Soil, Germany 405 1170 1170 38 DAT-F001 M. a. var. frigidum Adoryphorus couloni (Coleoptera), Australia 405 38 38 733, 737, — — Termite mound material, Australia 405 38 38 746–748, 758, 761, 764, 776, 777, 783, 785, 793 1120–1123, 1126 DAT-F384, F401, M. a. var. frigidum Soil, Australia 405 38 38 F234, F212, F236 1116 DAT-F496 M. a. var. frigidum Soil, Macquarie Is., Australia 405 38 38 1117–1119 DAT-F497–F499 M. a. var. frigidum Soil, Macquarie Is., Australia 405 38 38 Clade 7. M. anisopliae var. acridum 987 IMI 330189 M. f. var. minus Ornithacris cavroisi (Orthoptera), Niger 987 987 987 1216 ARSEF 2023 M. f. var. minus Acridid (Orthoptera), Galapagos Islands 987 987 987 985 ARSEF 324 M. a. var. anisopliae Austracris guttulosa (Orthoptera), Australia 985 985 985 1028 IMI 324673 M. f. var. minus Zonocerus elegans (Orthoptera), Tanzania 985 1028 1028 983, 984, 986, IIBC 191 659, 658, — Acridid, Benin 987 987 987 1067 IIBC 192 715, 701 1189–1193 — — pallens (Orthoptera), Brazil 987 987 987 Clade 8. M. anisopliae var. lepidiotum 147 — M. a. var. anisopliae Lepidiota consobrina (Coleoptera), Australia 147 147 147 375 — — Cryptotermes brevis (Isoptera), lab. infection, 147 147 147 Australia 153 — — Lepidiota frenchi (Coleoptera), Australia n.d. 147 n.d. 1042 — M. a. var. anisopliae Dermolepida albohirtum (Coleoptera), 1042 1042 1042 Australia Clade 9. M. anisopliae var. anisopliae 1029 IMI 168777ii — Schistocerca gregaria (Orthoptera), Eritrea 1029 1029 1029 610 — — Termite mound material, Australia 1029 1029 1029 25 — — Neotermes sp. (Isoptera), lab. infection, 1029 1029 1029 Australia 535, 532 — — Mastotermes darwiniensis (Isoptera), lab. n.d. 1029 1029 infection, Australia 976, 993, 487, —— Heteronyx piceus (Coleoptera), Australia 1029 1029 1029 488, 198 427, 433 — — Inopus rubriceps (Diptera), Australia n.d. 1029 1029 454 — — Staphylinid , Australia n.d. 1029 1029 1114, 1115 DAT-F494, F495 — Soil, Macquarie Is., Australia 1029 1114 1114 203 — — I. rubriceps, Australia 1029 203 203 328 — — I. rubriceps, Australia 114 328 328 1027 IIBC 191 679 — Oxya multidentata (Orthoptera), Pakistan 1029 1027 1027 322, 323 — — I. rubriceps, Australia 1029 1027 1027 Revision of Metarhizium 136

Table 1. (cont.)

Source name (if Other different from D3 ITS RAPD FI number designation proposed)* Host and origin group group group

Clade 9. (cont.) 379 — — I. rubriceps, Australia 114 379 379 1045 — — D. albohirtum, Australia 1029 1045 1045 330 — — I. rubriceps, Australia 114 1045 1045 1041 — — D. albohirtum, Australia n.d. 1045 1045 114 — — Antitrogus parvulus (Coleoptera), Australia 114 114 114 208 IIBC 191 625 — Phaulacridium vittatum (Orthoptera), 114 208 114 Australia 1033 IIBC 191 633 — Pseudosphingonotus savigni (Orthoptera), n.d. 1033 1033 Oman 1031 — — Teleogryllus sp. (Orthoptera), Oman 1029 1031 1031 1091 ARSEF 437 M. a. var. anisopliae Teleogryllus commodus (Orthoptera), 1029 1091 1091 Australia 1030 IMI 351830 — T. commodus, Australia 1029 1091 1091 1037 ARSEF 448 — T. commodus, Australia n.d. 1091 1091 1090 ARSEF 436 — T. commodus, Australia 1029 1091 1091 1092–1100 ARSEF 438 — T. commodus, Australia n.d. 1091 1091 550 — — Termite mound material, Australia 1029 1091 1091 552 — — Termite mound material, Australia n.d. 1091 1091 161 — — I. rubriceps, Australia n.d. 1091 1091 207 — — P. vittatum, Australia 1029 1091 1091 1039 IMI 89–574 — Acrotylus sp. (Orthoptera), Pakistan 1029 1091 1091 1038 IMI 91–629 — Zonocerus variegatus (Orthoptera), Benin n.d. 1091 1091 691 — — Soil, Burma 1029 1091 1091 726 — — Soil, Burma 1029 1091 1091 1011 — — Anomola sp. (Coleoptera), Burma 1029 1091 1091 1034 IIBC 191–614 — Patanga succincta (Orthoptera), Thailand 1029 1034 1034 692 — — Soil, Burma 1029 1034 1034 694 — — Soil, Burma n.d. 1034 1034 522 — — H. piceus, Australia 1029 1034 1034 903 — — H. piceus, Australia n.d. 1034 1034 700 — — Costelytra zealandica (Coleoptera), New 1029 700 n.d. Zealand 23 — — Anolamia albofasciata (), Mexico 1029 23 n.d. 1156 — — Immunocompromised patient, Australia 1029 1156 n.d. 327 — — I. rubriceps, Australia n.d. 1156 327 163 — — Kalotermes sp. (Isoptera), lab. infection, 1029 163 n.d. Australia 911 — — H. piceus, Australia 1029 163 n.d. 1036 ARSEF 727 — Tettigonid, Brazil 1029 163 1036 358 — — Heteronychus arator (Coleoptera), Australia 1029 163 n.d. Clade 10. M. anisopliae var. majus 388 ARSEF 1914 — Oryctes rhinoceros (Coleoptera), Philippines 1029 388 388 389 ARSEF 2151 — O. rhinoceros, Indonesia 1029 389 388 401 ARSEF 1946 — O. rhinoceros, Philippines 1029 389 401 Outgroups 297 — Beauveria bassiana Soil, Australia 297 297 n.d. 360 — Gliocladium sp. I. rubriceps, Australia 360 360 n.d. 442 — Gliocladium sp. I. rubriceps, Australia 360 442 n.d.

* M.a. l M. anisopliae, M. f. l M. flavoviride. ** n.d. l not done.

flavoviride var. minus to M. album. M. album, which had been Metarhizium pingshaese, M. cylindrosporae (l Nomuraea regarded as a synonym of M. anisopliae, was restored as a cylindrospora, Tzean et al. 1993), M. guizhousense (Guo et al. separate species and described as a pathogen on plant- and 1986, Shimazu 1989), and M. taii and its teleomorph C. taii leaf-hoppers from rice (Rombach et al. 1987). They considered (Liang et al. 1991) have been described from China and Japan, the primary taxonomic criteria for delimiting species to be the but none is known to be deposited in culture collections and shapes of conidia and conidiogenous cells, presence or absence so could not be included in this study. of a subhymenial zone and whether or not conidia adhere We have previously used analysis of sequence data from the laterally to form prismatic columns. They gave only secondary ITS regions and the 5.8S gene to resolve evolutionary taxonomic value to the colour of the mycelium and conidia, relationships within Metarhizium (Curran et al. 1994). Par- and suggested that conidial size is useful in delimiting species. simony analysis and tail probability (T-PTP) testing supported F. Driver, R. J. Milner and J. W. H. Trueman 137

Metarhizium as a monophyletic group and upheld the revision 1992b) and M. flavoviride (Bridge et al. 1993), FI-987 and FI- of the genus by Rombach et al. (1987). Rakotonirainy et al. 1028, as well as other isolates from west Africa and a collection (1994) used partial sequence from the 28S rDNA and isozyme of undescribed isolates from Brazil. data for phylogenetic analysis of Metarhizium. All three data M. anisopliae DAT-F001 (Rath et al. 1995) has been sets confirmed the divergence of M. flavoviride and M. commercialised in Australia for the control of the coleopteran anisopliae, as well as distinguishing var. majus and strains of pasture pest Adoryphorus couloni. This isolate has been cultured the ‘New Zealand’ type, which were shown to be bio- from FI-38 (CISRO Insect Pathogen Culture Collection), and chemically different. is one of a number of ‘cold-active’ isolates which we have In this study we attempt to redefine the phylogenetic, and examined, with distinct carbohydrate utilisation profiles of by inference taxonomic, relationships of the major mor- their strains 1 and 3 (Yip et al. 1992). Two other isolates are phological species clusters in Metarhizium. We correlated worthy of note in this group, FI-1124 and FI-1125, cold-active RAPD-PCR banding patterns with sequence data from the ITS strain 2 isolates which are ‘distinct and distant from other and 5.8S rDNA. Phylogenetic estimates were made by Metarhizium groups’ but also classified as var. frigidum (Rath parsimony and distance using the ITS, the 5.8S and the D3 et al. 1995). expansion region of the 28S rDNA (Michot et al. 1990, Nunn The most commonly isolated species, M. anisopliae,is et al. 1996). known from an enormous variety of hosts. FI-1029 was used by Veen (1968) for studies on infection in Schistocerca gregaria and is the ex-type specimen examined by Tulloch (1976). Isolates from Australian gryllids commonly used in other MATERIALS AND METHODS studies are genetically very homogeneous (Leal et al. 1994, Milner et al. 1996). Three isolates of M. anisopliae var. majus Isolates and four of the previously named ‘B-type’ variant (Curran et Most of the 126 isolates (Table 1) used in this study are al. 1994) of M. anisopliae complete the scope of isolates used. deposited in the CSIRO Insect Pathogen Culture Collection and have been isolated in Australia, but many important isolates were kindly supplied by Dr C. Prior (IIBC, Silwood DNA extraction Park, Ascot, UK), Dr P. D. Bridge (IMI, Bakeham Lane, Surrey, UK) Dr R. Humber (USDA, Ithaca, NY), Dr T. R. For DNA extraction, 50 ml of peptone\yeast extract broth in Glare (AgResearch, Lincoln, NZ) and Dr A. Rath (Biocare, a 250 ml flask was inoculated with conidia and incubated on Sydney, Australia) prior to deposition in the CSIRO collection. a shaker at 23 mC for 3 d. The mycelium was concentrated by Isolates were maintained on Sabouraud’s dextrose agar with filtration through Mira Cloth (Calbiochem) and washed with 1% yeast extract (SDYA). distilled water. The mycelial mat was squeezed dry in paper Where possible, we have cross referenced the CSIRO codes towelling and stored at k80 mC. The DNA was prepared by with the source codes, which have been used in other the methods of Curran et al. (1994). published studies on the genetic variation and molecular characterisation of Metarhizium. The first two isolates of each clade as listed in Table 1 are critical to the development of our PCR amplification and sequencing of the ITS region understanding of the taxonomy of Metarhizium and allow a direct comparison of the work described here with that of PCR (Saiki et al. 1988) was used to amplify the region of the other authors. ribosomal repeat from the 3 prime end of the 16S rDNA to The isolates of M. album F1-MaF and FI-1165, from green the 5 prime end of the 28S rDNA, spanning ITS1, the 5.8S on rice, are derived from cultures examined by rDNA and ITS2. Primer sequences and reaction conditions Rombach et al. (1987); i.e. MROX and MLAG respectively. were as described in Curran et al. (1994). PCR products were They described such cultures from cicadellids on rice in Asia purified and prepared for sequencing by electrophoresis in −" as ‘producing chains of small, brown (or rarely white), 0n8% TAE agarose gels containing 10 µgml ethidium ellipsoid conidia on clavate phialides’. bromide (Sambrook et al. 1989). Fragments were excised and Rombach et al. (1986) described various northern European transferred to a microfuge tube. The agarose slices were cultures of M. flavoviride var. flavoviride from agricultural soil mashed with 30 µl sterile distilled water using a toothpick, and (FI-1170) and beetles (FI-405 and FI-402). They distinguished incubated at 50 mC for 1 h. Samples were left at room the small spored M. flavoviride var. minus as causing natural temperature overnight to allow the DNA to elute from the epizootics of homopterans on rice in the Philippines and the gel. The samples were stored at k20 mC until required. Solomon Islands. Material they examined included FI-403, the Sequencing reactions were done in a total volume of 10 µl. ex-type isolate, and FI-1172. They also reported it on an Each reaction contained 5 µl of the eluted PCR fragment acridid host from the Galapagos Islands (FI-1216, collected by 1n6 pM of either the TW81 or AB28 PCR primers, and 4 µl Dr H. C. Evans in 1981). FI-1216 has been widely included in Prism Ready Reaction DyeDeoxy Terminator Cycle many molecular and biochemical studies on the heterogeneity Sequencing mix from Applied Biosystems Inc (ABI), Australia. of Metarhizium. We have also used other isolates from acridids Sequencing reactions were done in a thermocycler (Corbett which are well known from the literature, such as FI-985, Research, Australia) using the following programme: cycle one which is variously described as M. anisopliae (St Leger et al. 96 mC for 3 min, then 30 cycles at 96 mC for 30 s, 50 mC for Revision of Metarhizium 138

15 s and 60 mC for 4 min. The sequencing reactions were Table 2. Effect of temperature on growth of Metarhizium. Colony diam purified according to the manufacturer’s instructions and (mm) after 3 wk incubation in the dark loaded on to an ABI Model 373A Sequencer. Both strands of 10 mC15mC25mC30mC each PCR fragment were sequenced. The accuracy and repeatability of the sequence data were confirmed for selected FI-1165 M. album 0306333 M flavoviride novazealandicum isolates by comparison with previously sequenced isolates FI-699 . var. 37 66 61 57 FI-1124 M. flavoviride var. novazealandicum 52 65 68 21 using both cloned and PCR-direct manual sequencing methods FI-72 M. flavoviride var. pemphigum 39 41 73 13 (Curran et al. 1994). FI-1101 M. flavoviride var. pemphigum 40 86* 86 86 FI-38 M. flavoviride var. flavoviride 75 86 86 37 FI-1116 M. flavoviride var. flavoviride 70 85 86 26 PCR amplification and sequencing D3 expansion region FI-1117 M. flavoviride var. flavoviride 76 82 86 32 FI-985 M. anisopliae var. acridum 0406978 of 26S rDNA FI-1028 M. anisopliae var. acridum 0518080 M anisopliae anisopliae Based on the results of ITS data, selected isolates were FI-1114 . var. 78 76 86 86 FI-1115 M. anisopliae var. anisopliae 52 75 80 63 sequenced for the divergent domain three of the 26S rDNA. The D3 expansion region was amplified using the primers * Colony covering the entire plate. of Nunn et al. (1996). Primer sequences were: D3A 5h- GACC CGTCTTGAAACACACGGA-3h and D3B 5h- Cold-activity growth assays TCGGAAGGAACCAGCTACTA-3h. Amplifications were done in 50 µl reaction volumes containing 20 pM of each Isolates listed in Table 2, chosen on the basis of their position primer, 200 µ each dATP, dTTP, dCTP and dGTP, in the dendrogram, were tested for growth at low tempera- 15 m MgCl#, 50–100 ng genomic DNA, 1i supplied buffer tures. FI-1124 had been characterised by Yip et al. (1992) for and 2n5 U Taq polymerase (Bresatec, Australia) added as a growth at low temperatures and its carbohydrate utilisation ‘hot start’. Reactions were covered by a drop of light mineral patterns (Rath et al. 1995). oil and amplifications carried out in a thermocycler (Corbett All cultures sporulated and grew on SDYA. Sterile 6 mm Research, Australia) using the following programme: denatu- filter paper discs were dipped in a suspension containing about ) " ration at 94 mC for 5 min followed by the addition of the Taq 10 conidia ml− , drained and used to inoculate the centre of polymerase, then 30 cycles of 94 mC for 60 s, 55 mC for 60 s each plate. Four replicates for each temperature assayed were and 72 mC for 90 s. Final extension was carried out at 72 mC prepared for all isolates tested. The plates were sealed with for 5 min. PCR products were prepared for sequencing and Parafilm, wrapped in aluminium foil and incubated in the dark. completed as described above. Both strands of the PCR The diameter of each colony was measured after 17 d. fragments were sequenced using primers D3A and D3B.

Analysis of DNA sequences RAPD-PCR amplifications ITS and 5.8S gene sequences were aligned using Clustal W Amplifications were done in 25 µl reaction volumes containing (Thompson et al. 1994) at default settings. The alignment was 16n6m (NH%)#SO%,67m Tris HCl pH 8n8, 0n45% Triton checked visually and minor adjustments made manually. The X100, 200 µg ml-1 gelatin, 3n5m MgCl#, 200 µ each dATP, D3 sequences were aligned using Clustal W and the variable dTTP, dCTP and dGTP, 30 pM primer, 10–100 ng DNA and regions were adjusted in accordance with a model of the 2n4 U Taq polymerase (Bresatec). The reactions were covered secondary structure (Michot et al. 1990). All unique ITS by a drop of light mineral oil. Primers were those used by sequences, listed as ITS groups are deposited in GenBank. Fegan et al. (1993) from Random Primer kits H (H01 and H02) Alignments are lodged on the world wide web with the tree and F (F06, F07, F08 and F10) supplied by Operon project at Harvard (HYPERLINK http:\\herbaria.harvard.edu\ Technologies, Alameda, CA. Reactions were placed in a treebase\http:\\herbaria.harvard.edu\treebase\, Accession thermocycler (Corbett Research, Australia) and the DNA No. Tree BASE s386\No. M538). Analyses were done using amplified using the following programme: one cycle at 94 mC PAUP*4d52-59 (Swofford 1997). The partition homogeneity for 5 min, 40 mC for 2 min and 72 mC for 3 min, then 39 cycles test (Farris et al. 1995) was used to examine data for conflicting at 94 mC for 60 s, 40 mC for 90 s and 72 mC for 2 min. PCR hierarchic signals. Phylogenetic trees were estimated using products were separated by electrophoresis in 1n3% TBE parsimony and distance methods. Branch support was assessed " agarose gels containing 10 µgml− ethidium bromide using non-parametric bootstrap (Felsenstein 1985) and TPTP " (Sambrook et al. 1989). Gels were run at 7 V cm− for 2 h (Faith 1991) tests. Tree comparisons were made using Kishino- and photographed on a UV transilluminator. Hasegawa tests and Templeton tests (Templeton 1983, Kishino The presence or absence of RAPD-PCR band patterns was & Hasegawa 1989) as encoded in PAUP*. scored visually to correlate any relationships with ITS sequence data, without any attempt to produce an hierarchic ar- RESULTS rangement of clusters. RAPD groups were assigned on the basis of perfectly identical, or nearly so, banding patterns. The partition homogeneity test indicated no incongruence in Reproducibility of RAPD-PCR patterns was assured by phylogenetic signal amongst the ITS1, 5.8S, ITS2 and D3 independent replicates using a range of DNA concentrations. regions. Reported results are based on the combined data. The F. Driver, R. J. Milner and J. W. H. Trueman 139

FI-297 Beauveria bassiana 100 FI-380 Gliocladium sp. FI-442 FI-MaF Metarhizium album (Clade 1) 100 FI-152 M. flavoviride Type E (Clade 2) FI-1173 FI-1124 98 FI-1125 M. flavoviride var. novazealandicum (Clade 3) FI-698 FI-72 M. flavoviride var. pemphigum (Clade 4) 100 FI-1172 92 M. flavoviride var. minus (Clade 5) FI-403 74 0.01 FI-38 0.04 83 FI-402 89 M. flavoviride var. flavoviride (Clade 6) FI-405 FI-1170 100 FI-987 76 FI-985 M. anisopliae var. acridum (Clade 7) 0.01 FI-1028 89 FI-147 M. anisopliae var. lepidiotum (Clade 8) 99 FI-1042 0.01 FI-700 FI-1029 FI-1091 98 FI-1045 0.01 FI-1027 FI-1031 FI-1156 FI-163 90 FI-203 M. anisopliae var. anisopliae (Clade 9) 0.01 FI-23 FI-328 FI-379 FI-1033 94 FI-1034 FI-1114 54 FI-114 FI-208 81 FI-388 M. anisopliae var. majus (Clade 10) FI-389 Fig. 1. The strict consensus most parsimonious tree of the whole ITS1 and ITS2 region data sets of Metarhizium spp. and outgroups.

ITS1 region contained 71 parsimony informative sites, the unchanged. To test for possible sensitivity to tree-estimation 5.8S region seven, the ITS2 region 80 and the D3 sequence a procedure, neighbour-joining trees were constructed using the further five. Jukes–Cantor model, the Kimura 2-parameter model, and REV With alignment gaps treated as missing data and with no general time reversible models, each being applied separately differential weighting of transversions against transitions, to the ITS1 and ITS2 regions and to the whole data. So far as parsimony analysis of either the whole data set or the ITS1 relates to branches with bootstrap support " 50% the tree region identified in excess of 40000 most parsimonious (mp) topology was invariant to these manipulations. trees at length 415. Separate analysis of the ITS2 region To facilitate sampling across the large number of par- yielded a subset of those trees. The strict consensus of the simonious trees for each bootstrap, resampling of the data ITS1 and ITS2 whole data set mp trees is shown in Fig. 1. matrix on the full taxon set the analysis was based on 100 As a check for possible sensitivity of these trees to bootstrap replicates each with 10 random-addition-sequence alignment assumptions, the sequences were realigned at each starting trees, keeping % 50 trees at each replicate. Bootstrap of a wide range of gap penalties. The rDNA alignment was analysis of the reduced taxon set (Fig. 1) was based on 1000 found to vary slightly with gap parameters. As a check for bootstrap replicates each with an unlimited number of possible sensitivity to transversion: transition weighting the bootstrap trees per replicate. original alignment was analysed by parsimony with weighting The data (Fig. 1) support: (i) the monophyly of M. 2:1. Under either manipulation the consensus of mp trees was anisopliae (Clades 7–10) excluding FI-38; (ii) monophyly of a Revision of Metarhizium 140

ITS ITS

ITS group-1029 ITS group-1034 ITS group-775ITS group-114 ITS group-208 ITS group-1091 group-1027 group-1114 Clade 10 100 bp ladder FI-1029 FI-610 FI-532 FI-592 FI-976 FI-993 FI-448 FI-1034 FI-692 FI-694 FI-522 FI-903 FI-921 FI-775 FI-774 FI-788 FI-792 FI-114 FI-208 FI-1091 FI-1011 FI-161 FI-207 FI-552 FI-1027 FI-323 FI-322 FI-1114 FI-1115 FI-786 FI-799 FI-388 FI-389 FI-401

Fig. 2. RAPD-PCR banding patterns, using primer OP-F08, for ITS groups of Metarhizium clade 9 (M. anisopliae var. anisopliae) and clade 10 (M. anisopliae var. majus).

‘core’ group of M. flavoviride including FI-38 (Clades 4–6); The strict consensus for the reduced data set appears better (iii) monophyly of an acridoid group (Clade 7); and (iv) a sister resolved but the additional resolution is without bootstrap group relationship between the acridoid group (Clade 7) and support. The branch immediately below the polytomy has M. anisopliae (Clades 8–10) (excluding FI-38), rendering M. only 74% bootstrap support in the whole-taxon-set analysis. flavoviride paraphyletic. A T-PTP test indicated that monophyly of the ‘ingroup’ taxa T-PTP tests confirmed the monophyly of M. anisopliae (l all taxa except FI-297jFI-360jFI-442) is only weakly (P l 0n01), as well as monophyly of the ‘core’ M. flavoviride supported (P l 0n04) on these data. group (P l 0n01), and the acridoid group (P l 0n01). The This may also be interpreted to indicate the level at which sister relationship of the acridoid group and M. anisopliae was we might delimit species. Nucleotide divergence for the ITS examined by reducing the M. anisopliae clade to its basal node region between morphologically defined species in Metarhi- and testing for monophyly of ‘anisopliaejacridoids’.Onan zium ranges between 14–18%, whilst divergence between a priori test the relationship was confirmed (P l 0n01). recognised varieties within species does not exceed 5%. FI-38, commercially released under the name M. anisopliae, Levels of nucleotide divergence for clusters of isolates from clusters within the ‘core’ M. flavoviride group and that acridoid hosts and NZ blur the margins between species and placement is supported by bootstrap scores " 90% and varietal limits, ranging from 6n9–17n2% in paired analyses T-PTP values of 0n01 on intervening branches. To confirm that with morphologically recognised varieties. Cladistics requires an alternative placement of FI-38 within M. anisopliae can be that species represent monophyletic groups (Wiley et al. 1991) rejected on these data, a T-PTP test for the non-monophyly but there are no guide lines on taxonomic ranking (Seifert et of ‘FI-38jM. anisopliae’ was conducted. The result, P l 0n01, al. 1995). confirms that FI-38 cannot group with any member of M. anisopliae. Kishino–Hasegawa and Templeton tests comparing RAPD-PCR groups the most-parsimonious trees with FI-38, including the M. anisopliae against the most-parsimonious trees overall, also RAPD-group numbers listed in Table 1 are assigned on the confirm the incorrect identification of FI-38 as M. anisopliae basis of their corresponding ITS-group. RAPD-PCR-groups (K-H, length difference 37, ..6n89, P ! 0n0001; Templeton correlate with ITS sequence identity. Isolates which have z l 4n64, P ! 0n0001). These tests strongly suggest that FI-38 identical RAPD-PCR profiles, or exhibit only minor poly- belongs within M. flavoviride var. flavoviride. morphism with some primers, always belonged to the same Relationships within M. anisopliae var. anisopliae are almost ITS-group. Results confirmed the findings of Bidochka et al. wholly unresolved due to the lack of informative sites. At the (1993) and Fegan et al. (1993) of the greater variability of base of the tree, relationships amongst the major clades are RAPD-PCR banding patterns in M. anisopliae var. anisopliae also poorly resolved. The strict consensus of mp trees for the isolates. Small changes in ITS sequence, e.g. single point entire taxon set shows a polytomy amongst FI-album, mutations or deletions with respect to the ex-type strain of M. FI-152jFI-1173, FI-698jFI-1125jFI-1126 (NZ group), anisopliae var. anisopliae are often accompanied by dramatically ‘core’ M. flavoviride, and the ‘acridoid groupjM. anisopliae’. different RAPD-PCR patterns with most primers. Conversely, F. Driver, R. J. Milner and J. W. H. Trueman 141

FI-1124jFI-702jFI-699 which clustered on the same clades as isolates which were known to be cold-active were tested. The two M. flavoviride var. pemphigum isolates, and the FI-38 cluster, branch at nodes basal to the northern European 100 bp ladder FI-1216 FI-1189 FI-1190 FI-1191 FI-1192 FI-1193 FI-1067 FI-983 FI-984 FI-986 FI-987 FI-1028 FI-985 FI-1155 strains of M. flavoviride var. flavoviride. If cold-activity is a shared derived character state on this evolutionary line, then isolates such as FI-1170 from Germany, would be expected to show such cold-activity. FI-1114 and FI-1115 from near Antarctica were also tested for cold-activity. These two isolates, which had not been previously tested by the methods of Yip et al. (1992) and Rath et al. (1995), are embedded in the M. anisopliae var. anisopliae clade, and form a comparative group to the four other Macquarie Island isolates which share ITS sequence and RAPD-PCR identity with the FI-38 group.

Fig. 3. RAPD-PCR banding patterns, using primer OP-F06, for DESCRIPTION OF CLADES isolates of var. acridum. For details of isolate The molecular data give qualified support for the existing origin, see Table 1. morphologically based taxonomy, if we accept that species represent monophyletic groups. Consequently we have chosen 72 5 6 4 3 1 to retain the existing species and variety names and to describe those isolates that cannot be placed as new varieties, except for the Clade 2 isolates, where we regard such a move as premature. As it has been concluded that cold-activity is a 100 bp ladder FI-1216 FI-152 FI-1173 FI-403 FI-1172 FI-405 FI-402 FI-1170 FI-38 FI-72 FI-1101 FI-698 FI-699 FI-702 FI-1124 FI-1126 FI-1165 homoplasious character, the distinction of M. anisopliae var. frigidum is not supported. The polytomy at the base of the tree (Fig. 1) indicates that the discrimination of clades into species and varieties is open to question, and future taxonomic studies may provide justification for raising the level of some clades to new species. We have used molecular data as our diagnostic feature, but have deposited traditional whole organism holotypes while recognising that it has been suggested that genomic DNA material be accepted by herbaria as types (Reynolds & Taylor, 1991). Measurements are given with ... values in parentheses.

Clade 1. Metarhizium album Fig. 4. RAPD-PCR banding patterns, using primer OP-F06, for isolates of clades of Metarhizium spp. Clade nos in bold type; for The diagnostic sequences for FI-MaF are: details of isolate origin, see Table 1. ITS1 TCGAGTTTAC TACAACTCCC AAACCCCCTT GTGAACGTAT similar levels of sequence divergence in most other clades of ACCTTTCCAG TTGCTTCGGC GGGTATAGCC CCGGGGTCAG isolates which were examined, including M. anisopliae var. GTTCGCAAGA GCCTGCCCGG AACCAGGCGC CTGCCGGGGG ACCAAAACTC TTGTATTTCT GTACGATAAG GAATGTCTGA majus, were mirrored by much smaller levels of polymorphism GTGGTTTATA GAAGAAAATGAATCA in their RAPD-PCR banding patterns (Fig. 2). RAPD-PCR confirmed the high degree of genetic homogeneity of acridoid ITS2 isolates (Fig. 3) as well as the close taxonomic relationship of CAACCCTCAA GCCCCGGCGG TTTGGTGTTG GGGGCCGGCG FI-38 and its related ITS-group with other isolates of M. ATGGTGTTGG GGGCGATCTC TTCGTCCCCG CGCGCGCCGC flavoviride var. flavoviride from northern (Fig. 4). CCCCGAAATG AATTGCGGCC TCGCCGCGGC CTCCTCTGCG TAGTAACATG TTGCCCCTTC GCAACAGGAG CCCGGCGCGG CCACTGCCGT AAAAACCACC AACTTTTTTC ACAAG Cold-activity assays The only isolates identified as belonging to this clade were Parsimony analysis of the sequence data splits M. anisopliae FI-MaF and FI-1165, both of which were from infected var. frigidum isolates into two clades; strain 1 and 3 isolates leafhoppers in the Philippines (Rombach et al. 1987). Besides show sequence identity with FI-38 which forms on a clade the apparent host specificity of this clade, other characters with European strains of M. flavoviride var. flavoviride, and are the small ovoid to ellipsoid conidia measuring strain 2 isolates which form a clade containing the smaller 4–6i1n5–2n5 µm (Fig. 5), the growth of a bulging mass of spored ‘indigenous’ New Zealand strains of ‘M. aniso- hyphal bodies, rather than mycelium, prior to sporulation, and pliae’. Isolates FI-38jFI-733jFI-776jFI-1116jFI-1117j the lack of laterally adhering conidia forming prismatic Revision of Metarhizium 142

56

78

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11 12

10 lm

13 14 15

Figs 5–15. Conidia of Metarhizium spp. Fig. 5. M. album (FI-1165); Fig. 6. M. flavoviride Type E (FI-152); Figs 7, 8. M. flavoviride var. novazealandicum (FI-698 and FI-1124, respectively); Fig. 9. M. flavoviride var. pemphigum (FI-72); Fig. 10. M. flavoviride var. flavoviride (FI-38); Figs 11–13. M. anisopliae var. acridum (FI-1216, FI-985 and FI-1028, respectively); Fig. 14. M. anisopliae var. lepidiotum (FI-147); Fig. 15. M. anisopliae var. majus (FI-389). F. Driver, R. J. Milner and J. W. H. Trueman 143 columns. The pale brown colour of the spores is also The diagnostic feature is the distinctive sequence data of the characteristic, though of doubtful taxonomic significance. ITS1 and ITS2 above. Conidia elongate, often waisted, 6(p0n48)i2n4(p0n19) µm, borne in chains on clavate to Clade 2. Metarhizium flavoviride Type E cylindrical phialides 7 (p1n5)i1n8(p0n3) µm (Glare et al. 1996). Hyphae broad measuring up to 0n45 µm wide. Colonies The diagnostic sequences for FI-152 are: slow-growing on SDYA attaining 5–6 mm diam. after 7 d at ITS1 25 mC. Only known to occur on soil and in soil in CCGAGTTTAC AACTCCCAAA CCCCCAATGT GAACATATAC Australia and New Zealand. CTTTACCGTT GCTTCGGCGG GCTCGGCCCC GGGAGCAGGC This clade contains quite a large number of isolates, many TCGCCTGCCC CCCCGAGGCC TGCGCCCGCC GGGGGACTAA from New Zealand, which have been recognised as distinct by AACAAACTCT TCTGTATCTT GTATAATAAG CCTGTCTGAG several workers (Riba et al. 1990, Curran et al. 1994, Glare & TGGTATTTAA AATGAATCA Inwood 1997). Australian isolates described as Strain 2 by Yip ITS2 et al. (1992) have been included as part of a group of low CAACCCTCAA GCCCCGGCGG CTTGGTGTTG GGGACCGGCG temperature isolates under the nomen nudum M. anisopliae var. ACGGCGCTGC TCCGGCATGC GCGCGCCGCC CCCGAAATGA frigidum by Rath et al. (1995) who recognised that these were ATTGGCGGTC TCGTCGCGGC CTCCCCTGCG TAGTACCACA as distinct from M. anisopliae var. anisopliae as they were from ACCTCGCAGC GGGAGCGCGG CGCGGCCACT GCCGTAAAAC M. flavoviride, and that they may represent a new species. ACCCCAACTT CTCCAAGAG Isolates from this clade have cylindrical conidia (Figs 7 and 8) This is an unusual clade containing just two, rather diverse, and correspond to the short-spored form of M. anisopliae isolates. FI-1173 from an homopteran from Brazil was identified described from New Zealand (Glare & Inwood 1997). All as M. flavoviride var. minus by Humber (1992), but most other isolates grow well at low temperatures (! 10 mC). isolates originally identified in this paper as M. flavoviride var. minus were from leafhoppers and related insects in the Clade 4. Metarhizium flavoviride var. flavoviride Philippines and Solomon Islands (Rombach et al. 1986) and cluster in Clade 5. Morphologically they are all very similar The diagnostic sequences for FI-405 are: and come from similar hosts, so it is surprising that one isolate should be genetically so distinct. Even more surprising is that ITS1 CCGAGTTTAC AAACTCCCAA ACCCCCTGTG AACTTATACC FI-152 (Fig. 6), from a scarab in Australia, and morphologically TGTCTACCGT TGCTTCGGCG GGTTCGCCCG CCGAGGGACC resembling M. anisopliae var. anisopliae should also cluster into GACAAACAAA CTCTTGTATT TCTATCTATA GCATGTCTGA this clade. FI-152 and FI-1173 show RAPD-PCR patterns GTGGAATCAT ACACAAATGA ATCA which clearly distinguish them from other isolates of M. flavoviride var. minus. More work is needed to clarify this clade ITS2 and to determine if there are ecological and\or morphological ACAACCCTCA AGCCCCCCCG GTGCGGGAAA CGGGCTTGGT characteristics which are shared by these isolates, and to GTTGGGGACC GGCCAACTGG TGCCCTGCTG CTGCCGTAGC collect other isolates from nature which might fit into this AGGCGCCGGG CCGCCCCCGA AATGAATTGG CGGCCTCGTC clade. GCGGCCTCCC TCTGCGTAGT AGCACAAATC TCGCAGCTGG AGCGCGGCGC GGCCACTGCC GTAAAACGCA CCAACTTTTT TAAAG Clade 3. Metarhizium flavoviride var. novazealandicum Driver & Milner, var. nov. This clade contains FI-405, derived from the type material used for the original description of M. flavoviride (Gams & Haec species, ob ordinem basium distinctam in regione ITS1 (FI-698) CCGAGTTTAC AACTCCCAAA CCCCTGTGAA CTTATACCTT Roszypal 1973). It also contains other isolates described by TACTGTTGCT TCGGCGGGTC CGCCCCGGAA CAGGTTCGCG Rombach et al. (1986) as M. flavoviride var. flavoviride. These AGAGCCGGCC CGGAACCAGG CGCCCGCCGG GGGACCAAAA have large, somewhat swollen, conidia (Fig. 10) which form a CTCTTGTATT TTTTACTTTT GCATGTCTGA GTGGAATCAT pale green conidial mat in culture and come from the soil or AACAAATGAA TCA from soil-inhabiting beetles, and grow well at low tempera- tures. Rath et al. (1995) included FI-38 in M. anisopliae var. et ITS2 frigidum and this isolate, along with a number of other soil- CAACCCTCAA GCCCCAGCGG CTTGGTGTTG GGGACCGGCG derived isolates, has an ITS sequence largely identical with FI- ACCGGCGCTG CTTCGGCAGG CCCGCGCCGC CCCCGAAATG AATTGGCGGT CTCGTCGCGG CCTCCTCTGC GTAGTAGCAC 405 unequivocally identifying these isolates as M. flavoviride AACCTCGCAA CAGGAGCGCG GCGCGGCCAC TGCCGTAAAA var. flavoviride. RAPD-PCR banding patterns show a high CGCCCAACTT TTTTTAGAG degree of homogeneity and conservation with the northern European isolates, suggesting that this clade probably has a diagnoscitur. very wide geographical distribution. Rath et al. (1995) provide Holotypus: Dried culture of FI-698, originally from an a nearest-neighbour dendrogram based on their carbohydrate infected porina caterpillar (Lepidoptera: Hepialidae), deposited data which showed that FI-38 clustered nearer to M. flavoviride as DAR 74293. Paratype: 74294. than to M. anisopliae. Our data support this finding and it is proposed, therefore, that the correct identity of this isolate Other cultures examined: FI-1124 and FI-1125 from soil, (and other isolates referred to as strains 1 and 3 in Rath et al. Australia. 1995) is M. flavoviride var. flavoviride. Revision of Metarhizium 144

Clade 5. Metarhizium flavoviride var. minus representing this clade are needed and it is possible that a search for Metarhizium infection on other root aphids may The diagnostic sequences for FI-403 are: provide them. ITS1 TCGAGTTTAC TTTACAACTC CCAAACCCCC TGTGAACTTA Clade 7. Metarhizium anisopliae var. acridum Driver & TACCTGTCTA CCGTTGCCTC GGCGGGCTCG CCCGCCGCGG Milner, var. nov. GACCGACAAA CAAAACTCTT GTATTTCTAT CTTTGGCATG TCTGAGTGGA ATCACACATA AATGAATCA Haec species, ob ordinem basium distinctam in regione ITS (FI-987) CCGAGTTTAC AAAACTCCCA AACCCCTGTG AACTATACCT ITS2 GTCACTGTTG CTTCGGCGGT ACCGACCCCC CGGGAAACCG CAACCCTCAA GACCCCCCGG CGACGGGAAA CGGGCTTGGT GGACCAGGCG CCCGCCGGGG ATCCTAGTAA CATCTTGAAT GTTGGGGACC GGCCAACCGG TGCCCTGCTG CTCCTGCGGC CTTCTATATA ATATGGCATC TTCTGAGTGG TGGGAAAAAA AGGCGCCCGG CCGCCCCCGA AATGAATTGG CGGCCCCGTT ATGAATCA GCGGCCTCCC TCTGCGCAGT AGCACATGTC TCGCAGCTGG AGCGCGGCGC GGCCACTGCC GTAAAACGCA CCAACTTTCT et ITS2 TCTTTTAG CGCCCCTCAA GCCCCCTGTG GGTTTGGTGT TGGGGATCGG CGAAGCTTTT TTCAGCACGC GCCGTCCCTT AAATTTATTG In our study only two isolates FI-403 (the ex-type isolate) and GCGGTCTCGC CGTGGCCTTT CCTCTGCGCA GTAGTAACTC FI-1172, both morphologically identified as Metarhizium ACTCGCAACG GGAGCCCGGC GCGGTCCACT GCCGTAAAAC flavoviride var. minus, and obtained from infected leafhoppers CCCCAATCA CTTTGTTAC AG were included within this clade. These isolates fit the diagnoscitur. description given by Rombach and were collected in the Holotypus: Locusta migratoria (Orthoptera: Acrididae) laboratory- Philippines and the Solomon Islands suggesting a narrow host infected with FI-987 dried over silica gel. The original culture range and geographical distribution. was derived from a field-infected Ornithacris cavroisi collected in Niger, West Africa. The holotype is deposited as DAR Clade 6. Metarhizium flavoviride var. pemphigum 74297. Paratypes: DAR 74298–74301. Driver & Milner, var. nov. Other cultures examined: FI-985, FI-1028 and FI-1216. Haec species, ob ordinem basium distinctam in regione ITS1 (FI-72) CCGAGTTTAC AACTCCCAAA CCCAATGTGA ACTATACCTG TCTACCGTTG CTTCGGCGGG TTCGCCCGCC GAGGGACCGA The diagnostic feature is the distinctive sequence data of the CAAATAAACT CTTGTATTTC TATCTTTAGC ATGTCTGAGT ITS1 and ITS2 above. Conidia from the holotype are ovoid, GGAATCATAA ACAAATGAAT CA 4n5(p0n41)i2n6(p0n6) µm, borne on cylindrical to slightly swollen phialides 7n3(p1n9)i2n5(p0n4) µm. FI-985 is et ITS2 unusual in having larger conidia, 7n6(p0n78)i2n9 CAAGCCCTCA AGCCCCCCCG GCTTGGTGTT GGGGACCGGC (p0n37) µm, borne on slightly larger phialides (Glare et al. CACCGGTGCC CTGCTGCTTC GCGGCAGGCG CGCCCGGCCG 1996). Conidial mass dark yellow-green (Munsell 10Y 4\2). CCCCCGAAAT GAATTGGCGG CCCCGTCGCG GCCTCCCTCT Mycelium 2n5 µm wide. Colonies growing rapidly on SDYA, GCGTAGTAGC ACACATCTCG CAGCTGGAGC GCGGCGCGGC 30 mm diam. after 7 d at 25 mC. Only known from grass- CACTGCCGTA AAACGCACCA ACTTTTTTTT ACAG hoppers and locusts in Africa, Asia, South America and diagnoscitur. Australia. Holotypus: Laboratory infected Pemphigus bursarius dried over This clade contains all the acridoid isolates described as ‘M. silica gel and deposited as DAR 74295. Culture derived from flavoviride Group 3’ (Bridge et al. 1997). Our data supports and the type FI-72. Paratype: DAR 74296. extends those of others (Bridge et al. 1993, 1997, Cobb & Clarkson 1993, Bidochka et al. 1993) in showing that these Other culture examined: FI-1101. isolates are genetically quite uniform and are quite distinct from the type material of M. flavoviride vars flavoviride and The diagnostic feature is the distinctive sequence data of the minus. Bootstrapping and T-PTP tests give strong support for ITS1 and ITS2 above. Conidia ovoid to elongate, 5n4 the phylogenetic signal that unites these isolates to the major (p0n47)i2n4(p0n43) µm, occasionally ! 9 µm long, borne evolutionary line that gives rise to M. anisopliae rather than in chains on cylindrical phialides. Spore mass light green M. flavoviride. (Munsell greenish yellow 7n5Y 4\4). Colonies growing well Interestingly, other orthopteran hosts such as crickets are on SDYA, ca 20 mm after 7 d at 25 mC. Only known from the not susceptible and in nature are attacked (exclusively) by U.K., where it occurs on root aphids (Pemphigus trehernei). Clade 9 isolates. In our experience, grasshoppers are more This small clade of two isolates with identical collection often infected in nature by Clade 9 isolates but these isolates data except the date. Both isolates were from infected root are less virulent than the rarer Clade 7 isolates (Bateman et al. aphids in Norfolk, England (Foster 1975) and they have 1996). cylindrical green conidia morphologically resembling M. Most Clade 7 isolates produce small ovoid conidia which anisopliae var. anisopliae (Fig. 9). They grow well at low would previously have identified them as M. flavoviride var. temperatures, however, and cluster with M. flavoviride so we minus. For example, FI-1216 (Figs 11, 13) was described under regard that as the correct specific name. More isolates that name by Rombach et al. (1985). Other isolates have larger F. Driver, R. J. Milner and J. W. H. Trueman 145 spores which can be almost cylindrical (FI-985) (Fig. 12). All ITS2 isolates share some unusual characteristics such as the ability CGCCCCTCAA GTCCCCTGTG GACTTGGTGT TGGGGATCGG to sporulate internally and to grow at 37 mC or higher (Welling CGAGGCTGGT TTTCCAGCAC AGCCGTCCCT TAAATTAATT et al. 1994). GGCGGTCTCG CCGTGGCCCT CCTCTGCGCA GTAGTAAAGC ACTCGCAACA GGAGCCCGGC GCGGTCCACT GCCGTAAAAC CCCCCAACTT TTTATAG Clade 8. Metarhizium anisopliae var. lepidiotum Driver FI-1029, derived from the type material of Metarhizium & Milner, var. nov. anisopliae var. anisopliae (Tulloch 1976), forms the basis for this Haec species, ob ordinem basium distinctam in regione ITS1 (FI-147) clade. It includes the majority of isolates found in nature and CCGAGTTCTG AAAAAACTCC CAACCCCTGT GAACTATACC is genetically highly diverse. St Leger et al. (1992b) TGTAACTGTT GCTTCGGCGG GACTTGTGCC CGCCGGGGAC demonstrated the existence of clonal population structures in CCAAACCTTC TGAATTTTTT TTAAGTATCT TCTGAGTGGT M. anisopliae. Distinct groups can be identified within the AAAAAAAAAT GAATCA clade, e.g. a number of isolates from the black field cricket, Teleogryllus commodus, in Australia, share identical ITS sequence et ITS2 data and have very similar RAPD-PCR patterns (Milner et al. CGCCCCTCAA GTCCCCTGCG GACTTGGTGT TGGGGATCGG 1996). Isolates generally have green, cylindrical conidia, CGAGCGCTGT TTGCTCAGCA CGCCGTCCCC GAAATTCATT 5–7 µm long, which form in columns of chains. They normally GGCGGTCTCG CCGTGGCCCT CCTCTGCGCA GTAGTAAAAC grow poorly outside the range 15–32 mC, though we have ACTCGCAACA GGAGCCCGGT GAGGTCCACT GCCGTAAAAC found two cold temperature active isolates which came from CCCCGACTTT TTACAG soil from Macquarie Island (Dr A. C. Rath, pers. comm.), diagnoscitur. confirming that cold activity is a homoplasious character. Holotypus: Lepidiota consobrina laboratory-infected with FI-147 dried over silica gel. The original culture was derived from a Clade 10. Metarhizium anisopliae var. majus field-infected L. consobrina collected near Cairns, Queensland, Australia. The holotype is deposited as DAR 74302. The diagnostic sequence data for FI-388 (not the type) are: Paratypes: DAR 74303–74306. ITS1 CCGAGTTATC CAACTCCCAA CCCCTGTGAA TTATACCTTT Other cultures examined: FI-1042. AATTGTTGCT TCGGCGGGAC TTCGCGCTCG CCGGGGACCC AAACCTTCTG AATTTTTTAA TAAGGATCTT CTGAGTGGTT The diagnostic feature is the distinctive sequence data of the AAAAAAAAAA TGAATCA ITS1 and ITS2 region above. Conidia from the holotype are parallel-sided, 7n3–10n6i3–4n1 µm, borne on cylindrical ITS2 to slightly swollen phialides. Conidial mass dark yellow-green CGCCCCTCAA GTCCCCTGTG GACTTGGTGT TGGGGATCGG (Munsell 7n5Y 4\2). Hyphae 2 µm wide. Colonies rapid CGAGGCTGGT TTTCCAGCAC AGCCGTCCCT TAAATTGATT growing on SDYA, ca 70 mm after 14 d at 25 mC. Only known GGCGGTCTCG CCGTGGCCCT CCTTTGCGCA GTAGTAAAAC from coleoptera in Australia, New Zealand and adjacent ACTCGCAACA GGAGCCCGGC GCGGTCCACT GCCGTAAAAC ACCCCAACTT TTTATAG Pacific Islands. This is another clade with only a small number of known A typical isolate from this clade is FI-388 which conforms to isolates. All have cylindrical spores (Fig. 14) and produce a the description by Tulloch (1976). Isolates are readily identified profuse layer of green conidia in culture, and have been on the basis of the very large conidia, usually " 10 µm long isolated from scarab larvae. This cluster of isolates has (Fig. 15), rapidly growing colonies producing dark green previously been described as ‘B-type’ isolates of M. anisopliae conidia, and are most frequently found attacking dynastine (Curran et al. 1994), and probably corresponds to those of beetles in tropical countries. Our results support those of pathogenicity group 1 for Lepidiota spp.; or RAPD group A other workers (St Leger et al. 1992b, Leal et al. 1994), and (Fegan et al. 1993). Similar isolates have been reported from show that genetically this clade differs less from M. anisopliae scarab larvae in Papua New Guinea and Fiji (Dr T. R. Glare, var. anisopliae Clade 9 isolates than do other isolates such as pers. comm.). More needs to be known about this clade, but those in Clade 8 (‘B-type’), which are also currently described the sequence data clearly separate it from M. anisopliae var. as var. anisopliae. acridum and M. anisopliae var. anisopliae. DISCUSSION Clade 9. Metarhizium anisopliae var. anisopliae This study has again shown the limitations of morphological Metarhizium The diagnostic sequence data for FI-1029 are: characters in distinguishing between species of . Glare et al. (1996) tested the validity of phialide morphology, ITS1 as suggested by Rombach et al. (1986), as a useful taxonomic CCGAGTTATC CAACTCCCAA CCCCTGTGAA TCATACCTTT character by examining 11 isolates representing M. anisopliae, AATTGTTGCT TCGGCGGGAC TTCGCGCCCG CCGGGGACCC M. flavoviride, and M. album. They found that phialide AAACCTTCTG AATTTTTTAA TAAGTATCTT CTGAGTGGTT morphology of a single isolate could vary within the same AAAAAAAATG AATCA culture as well as between substrates. Furthermore they Revision of Metarhizium 146 concluded that conidial morphology was the only potentially acridoid isolates described by Cobb & Clarkson (1993), and useful morphological character. The discovery of apparently Bidochka et al. (1993) are distinguishable from M. flavoviride similar isolates from acridid hosts with spore dimensions var. flavoviride and M. flavoviride var. minus, as well as isolates intermediate between M. anisopliae and M. flavoviride of M. anisopliae also from acridids. They clearly point out the suggested that even this character was of limited value. correlation of a genotypic class with a single host in the same Other authors have combined biochemical and molecular manner that morphological and biochemical markers were approaches with morphological characters. Riba et al. (1986) used to establish the link between M. anisopliae var. majus and compared strains of M. anisopliae for their conidial size, scarabid beetles. Based on morphological classification for the virulence to European corn borer and isozyme profiles. Such acridoid isolates, Bridge et al. (1997) have tentatively proposed analysis displayed the genetic variability of strains, while three groups within M. flavoviride: Group 1 – The original recognising the relative homogeneity of M. anisopliae var. isolates of M. flavoviride var. flavoviride from northern Europe; majus strains from Oryctes spp. Yip et al. (1992) and Rath et al. Group 2 – M. flavoviride var. minus from SE Asian homopteran (1995) combined characters such as host pathogenicity, cold- hosts; and Group 3 – From acridids, isolates which have activity, conidial dimensions, sporulation colour and carbo- morphological features of both M. anisopliae and M. flavoviride. hydrate utilisation data to characterise M. anisopliae var. An anomaly of this conclusion is that the acridoid isolates in frigidum. They concluded that whilst M. anisopliae is their dendrogram cluster with the majority of the M. anisopliae heterogeneous, it could be grouped into ‘biologically relevant’ isolates. Isolates of M. anisopliae which appear to be genetically strains based on such criteria. Nearest-neighbour dendrograms more distant and both varieties of M. flavoviride are (Rath et al. 1995) showed that M. anisopliae var. frigidum was paraphyletic in their dendrogram (Bridge et al. 1997). more distant from M. anisopliae than M. flavoviride. Isolates of There are problems in interpreting the taxonomic relation- this new cold-active variety clustered together but split M. ships between clusters of isolates created by hierarchical anisopliae into two clades. The derived dendrogram er- arrangements based on RAPD-PCR, especially where genetic roneously implied that Metarhizium is paraphyletic because of distances become very large. The homology assumptions built the problems in correctly assigning species names for the into the data become dubious and bands on gels become so isolates studied. Our data suggest that these characters are completely different that nothing is shared across taxa, and more likely to be shared synapomorphies of various M. without synapomorphies there is no cladistic signal. Tree flavoviride isolates. search and clustering methods simply force a false, tree-like St Leger et al. (1992a) used isozyme analysis to detail resolution of the data in that situation. The non-hierarchic genetic variation among isolates of Metarhizium spp. and nature of methods such as ordination may be more suited to those of another entomopathogen, Beauveria spp. Similar display ‘truer’ inter-group relatedness (Bridge et al. 1997, conclusions were drawn in each study, suggesting that species Maurer et al. 1997). complexes or cryptic species are to be found. With respect to A more flexible approach to understanding the concept of Metarhizium, St Leger et al. (1992a) point out that isolates species is becoming accepted and is particularly relevant to which are displaced in the dendrogram from other isolates of the problems of fungal genetics and taxonomy. Most the same morphological species group may represent taxonomic decisions are implicitly based on the biological separate species. They identify five potentially cryptic varieties species concept of ‘groups of actually or potentially within M. anisopliae, including the long-spored var. majus. The interbreeding populations that are reproductively isolated’ data clearly indicate that M. anisopliae is distinct from other (cited by Vogler & Desalle 1994 from Mayr 1942). In practice, Metarhizium species despite the fact that isolates from acridids easily observed morphological characters are used to infer the (referred to as ‘acridoid’ isolates) such as FI-985, which they potential to interbreed (Vogler & Desalle 1994). Both recognised as M. anisopliae, clustered with FI-1216 (which was morphological and biological species concepts have been misidentified as M. flavoviride var. minus). The lack of common applied to sexually reproducing fungi such as Pleurotus spp. alleles between these isolates and M. flavoviride var. minus (Vigalys et al. 1993), Lentinula spp. (Pegler 1983, Shimomura isolates from the Philippines and Solomon Islands, which our et al. 1992), and Ascosphaera spp. (Anderson et al. 1997). Most data place on long separate branches of the tree, indicates a of these studies have resulted in disagreements over species high degree of genetic divergence. limits. Hibbett (1992) advocated the use of the phylogenetic Using PCR and RAPD, Fegan et al. (1993) and Bidochka et species concept for Lentinula spp. This species concept has al. (1993) concluded that M. anisopliae contains a number of several interpretations, all united by the notion that a cluster cryptic species. Fegan et al. (1993) and Fungaro et al. (1996) of organisms possesses a unique character, or combination of demonstrated that in some instances RAPD groupings may characters, i.e. ‘it is the smallest detectable group of organisms correlate with insect host range and the persistence of distinguishable by unique attributes’ (Vogler & Desalle 1994). particular fungal genotypes in specific locations. RAPD-PCR These characters may include genotypic, morphological, banding patterns confirmed that FI-985 was strikingly similar behavioural and ecological factors which are diagnostic to FI-1216 from the Galapagos Islands isolate and other West (Vogler & Desalle 1994). In this context, conservationists African isolates from acridids (Cobb & Clarkson 1993, have coined the term evolutionary significant units (ESUs) to Bidochka et al. 1993). define clusters of individuals identified by cladistic analysis of Further molecular characterisation of these acridoid isolates such heritable characters. Hibbett et al. (1995) used ITS has been carried out by Bridge et al. (1997) using isozyme sequence analysis and physiological characters to help resolve markers and RAPD-PCR to show that the clonal group of phylogenetic species in Lentinula spp. F. Driver, R. J. Milner and J. W. H. Trueman 147

The biological species concept of breeding populations to very divergent evolutionary lines, which are paraphyletic on delimit taxa is not directly applicable to Metarhizium spp. The all trees. By all criteria, FI-1216 and the other acridoid isolates role of the parasexual cycle in the genus and its potential for show a high degree of genetic homogeneity. Taking into genetic exchange has been demonstrated (Al-Aidroos 1980, consideration the strong bootstrap support, significant T-PTP Messias & Azevedo 1980). Using allozyme data St Leger et al. test for monophyly, sequence identity of these isolates with (1992b) were able to show considerable inter-isolate variation M. anisopliae isolates over the more conserved D3 expansion and the existence of clonal population structures within region of the 28S rDNA, isozyme and RAPD-PCR data, Metarhizium spp., which might arise as a result of heterokaryon ambiguous morphology and conidial length, and host incompatibility or other mechanisms leading to genetic association of these acridoid isolates, it is inconceivable to isolation. In a companion publication, St Leger et al. (1992a) reconcile these findings with the morphological classification describe the repeated recovery of isolates of Beauveria spp. of of these isolates as M. flavoviride var. minus. Accordingly we the same genotypic class that persist over time and space. have named them M. anisopliae var. acridum. Similar observations have been noted for Metarhizium spp. RAPD-PCR generated probes which were used for dot blot using RAPD-PCR (Fegan et al. 1993, Bridge et al. 1997), and hybridisations by Bidochka et al. (1993) emerge with some ITS sequence combined with such RAPD data. Isolates of M. potential significance in this context. They developed three anisopliae listed in Table 1 which belong to the same ITS and probes, A, B, and C. Probe B (1n6 kb) hybridises specifically to RAPD group have been recovered repeatedly from different var. acridum isolates, and is described as a species-specific continents. These homogeneous genotypes have been re- probe for M. flavoviride. If mapped on the cladogram (Fig. 1) covered from a wide range of hosts, e.g. FI-1029 group, and as a shared synapomorphy, the probe is specific for var. yet at other times homogeneity is found from isolates acridum isolates within the M. anisopliae species cluster. associated with the same host, and or set of environmental Similarly, probe C (0n63 kb) hybridises to a collection of var. factors occurring in different localities, e.g. FI-1034 group anisopliae isolates. This marker can be tentatively mapped to which is dispersed through Thailand, Burma (Myanmar) and the node on the tree which gives rise to all var. anisopliae Australia, where it has been found in association with scarabs isolates, and further testing could establish whether it can be which feed on peanut crops. placed at more basal nodes, to include M. anisopliae var. We have used nit mutants of Metarhizium with impaired lepidiotum or M. anisopliae var. majus isolates. Fragment A nitrogen metabolism as a tool to investigate the relationship (0n94 kb) is described as a Metarhizium genus-specific probe, between genotypic groups based on ITS and RAPD data, and but no ‘true’ M. flavoviride isolates were tested. If the vegetative compatibility groups (Driver & Milner, unpu- hierarchical signal in our data is correct, placing var. acridum blished), which are seen as a guide to heterokaryon on the same evolutionary line as var. anisopliae, probe A could incompatibility. All strains which gave positive results in potentially map to several different sites; i.e. at the node complementation tests had closely related genotypes, but which gives rise to all varieties of M. anisopliae, in which case exhibited minor variations in RAPD profiles, being derived it would be a species-specific probe, or at sites which may from different host and locations. A surprise in these results include all, or some of the other evolutionary significant clades has been the failure of intergenic pairings of FI-610 and FI- identified in the genus. These probes may add further weight 592, isolates from termite hosts which have identical RAPD to the present hierarchical structure of the phylogenetic tree, profiles. The implication of this is that loci which define VCGs or they may help to delimit species from the unresolved are not strictly tied to RAPD markers, although it would be evolutionary relationships between clades containing M. difficult to conceive that VCGs span genetically more unrelated flavoviride and M. album. RAPD groups. Processes such as host specialisation, and FI-403 from a brown planthopper on rice in the Philippines vegetative or nuclear incompatibility may serve to genetically is cultured from the type of M. flavoviride var. minus. FI-1172 isolate strains and make them extremely stable. from the Solomon Islands differs by a single base deletion in While our data do provide limited support for the the ITS1 and displays identical RAPD-PCR banding patterns. morphologically defined species in Metarhizium, the com- These isolates are taken to represent var. minus, and occupy a bination of molecular, biochemical and morphological markers position in the phylogenetic tree consistent with their which have been used for strain typing and identification described relationship to M. flavoviride var. flavoviride. All make a case for phylogenetically defined species or ‘evo- isolates in the clade which give rise to the species cluster M. lutionary significant’ groups. Vogler & Desalle (1994) stress flavoviride share sequence identity over the more conserved the importance of including and mapping such parameters on D3 expansion segment of the 28S rDNA. cladograms as part of a total evidence approach. In this Metarhizium flavoviride Type E context, we can identify and relate clades, or evolutionary lines in Metarhizium with other data to delimit biologically FI-152 and FI-1173 constitute a distinct evolutionary line meaningful clusters (Fig. 1). In some instances, we have taken which is phylogenetically as divergent from M. flavoviride as the additional step of naming new varieties to reflect this. each of the morphologically described species clusters in the tree are from each other. The morphological characters used M. flavoviride var. minus and M. anisopliae var. to describe M. flavoviride var. minus are homoplasious and it acridum is paraphyletic in the tree, mapping to three separate clades. The current morphologically based description of M. The resolution in the base of the tree is poor, and there is no flavoviride var. minus (Rombach et al. 1987) encompasses three strong hierarchical signal to support this clade with any of the Revision of Metarhizium 148 morphologically defined species clusters. Consequently, we to identify clades is by sequence determination of the ITS feel there is no appropriate species or subspecific designation regions, it has also been shown that these correlate strongly that can be used at present for these isolates. with RAPD patterns, in the same manner that ITS-RFLP patterns have been shown to correlate with RAPD patterns in Beauveria (Maurer et al. 1997). It is, therefore, suggested that M. flavoviride var. flavoviride and M. anisopliae var. clades be identified by using either RFLP analysis of the ITS frigidum region, or RAPD-PCR patterns established by primers such as Isolates of M. flavoviride var. flavoviride as described by Gams OP-H01, or OP-A03 which give rise to fewer polymorphic & Roszypal (1973), and Rombach et al. (1986) from coleopteran fragments against a set of ‘standard’ isolates. It is suggested hosts and soil samples in northern Europe, cluster in the tree that the first one or two isolates listed under each clade in with a large number of Australian isolates with a broad Table 1 be used as the standard or type material. These are geographic distribution from cool or temperate climatic mostly ARSEF isolates and the other isolates have been conditions. These isolates appear to be cold-active, germin- deposited in HERB-DAR. ating and sporulating at low temperatures. All Strain 1 and 3 (Yip et al. 1992) isolates which were received from Dr A. Rath share distinct carbohydrate utilisation patterns (Rath et al. ACKNOWLEDGEMENTS 1995) and in some cases are highly virulent to the pasture pest Inevitably with a large and complex project as reported here, many people A. couloni. Rath et al. (1995) invalidly described these isolates have contributed to discussions, provided isolates and reviewed various as a new variety, M. anisopliae var. frigidum, noting that they versions of this paper. The authors would particularly like to thank Dr John clustered with isolates of M. flavoviride in their nearest- Curran for providing most of the facilities needed to undertake the project and for his constant support. Drs Paul Bridge, Travis Glare and Chris Prior neighbour dendrogram. All these isolates, including a large have all contributed, directly and indirectly, and we always valued their number from the south coast of N.S.W. recovered from soil insights. Dr David Swofford kindly gave his permission to publish results samples in and around termite mounds, exhibit sequence from the pre-release version of PAUP*. identity with FI-38 over the ITS region, as well as sequence identity with all M. flavoviride isolates for the D3 region. RAPD-PCR banding patterns for all isolates from the FI-38 REFERENCES ITS group show a high degree of homogeneity and Al Aidroos, K. (1980) Demonstration of parasexual cycle in the entomopatho- conservation with the northern European isolates. genic fungus Metarhizium anisopliae. Canadian Journal of Genetics and Cold-activity appears to be a homoplasious character which Cytology 22: 309–314. can be found amongst Metarhizium isolates placed in other Anderson, D. L., Gibbs, A. J. & Gibson, N. L. (1997) Identification and phylogeny of spore-cyst fungi (Ascosphaera spp.) using ribosomal DNA clades in the tree. Strain 2 isolates, which were not pathogenic sequences. 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