Vous êtes sur la page 1sur 18

m y c o l o g i c a l r e s e a r c h 1 1 0 ( 2 0 0 6 ) 537 554

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/mycres

A taxonomic revision of the insect biocontrol fungus Aschersonia aleyrodis, its allies with white stromata and their Hypocrella sexual states
Miao LIU*, Priscila CHAVERRI, Kathie T. HODGE
Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA

article info
Article history: Received 4 August 2005 Received in revised form 12 January 2006 Accepted 24 January 2006 Corresponding Editor: Richard A. Humber Keywords: Ascomycota Clavicipitaceae Molecular systematics Morphology Phylogeny

A revision of a monophyletic group of Hypocrella species and their Aschersonia anamorphs with white effuse stromata is presented. In addition to taxon descriptions, distributions, and nomenclature, a synoptic key and a molecular phylogenetic analysis are also provided. A new holomorph, Hypocrella rhombispora sp. nov., is described. This study presents a revisionary treatment of Aschersonia aleyrodis (teleomorph: Hypocrella libera) and its allies. These fungi parasitize whiteies and are promising candidates for whitey biological control. Four species of Aschersonia and their Hypocrella teleomorphs are treated in detail: Aschersonia aleyrodis/Hypocrella libera; A. andropogonis/H. andropogonis, A. placenta/H. raciborskii, and A. sp./H. rhombispora sp. nov. A synoptic key including these and six other morphologically similar species is presented to facilitate identication in the eld and laboratory. Phylogenetic analyses of partial DNA sequences from three genes (LSU, mtSSU, and RPB2) suggest that Aschersonia species with effuse white stromata form a monophyletic group of whitey pathogens. Phylogenetically informative characters in the group include the colour and shape of the stromata, the arrangement of tubercles containing perithecia, the arrangement of conidial masses on the stromata, and the shape of conidia and part spores. 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Aschersonia aleyrodis (teleomorph: Hypocrella libera, Clavicipitaceae, Hypocreales) was among the rst fungi used in the biocontrol of insect pests in North America. Its successful use in Florida citrus groves dates from the early 1900s, when citrus branches with A. aleyrodis were introduced into citrus groves to seed epizootics in the whitey population (Berger 1921; Fawcett 1936). With the increase in chemical pesticide use beginning in the 1940s and 1950s, the demand for biocontrols

diminished. Aschersonia species can still be found in whitey populations in abandoned groves and backyard trees that are not managed with pesticides, where we have collected some specimens. Interest in using these fungi for the control of pests resumed in 1960s. In Bulgaria, China, Japan and the USSR (Evans & Hywel-Jones 1990), A. aleyrodis has been used against the greenhouse whitey. The fungus has been developed commercially by Koppert Biological Systems in Holland as a biopesticide suitable for application in greenhouses (Evans & Hywel-Jones 1990). Successful examples were also

* Corresponding author. Current address: 201F Plant Science Bldg., University of Kentucky, Lexington, KY 40546 USA. E-mail address: mliu4@uky.edu. 0953-7562/$ see front matter 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.01.013


M. Liu et al.

seen in Azerbaijan and Jamaica to control guava whitey and citrus whitey, respectively (Borner 1956; McCoy, Samson & Boucias 1988). Recent research has revealed that A. aleyrodis has the virtues of high tolerance in low relative humidity (Fransen 1987), long persistence on leaf surfaces (Meekes et al. 2000), and compatibility with insect parasitoids (Fransen & van-Lenteren 1993) in the control of whitey pests. However, some hurdles remain: Aschersonia species are slow-growing in culture, and not all host life stages may be attacked (Ramakers & Samson 1984). Recent studies on spore production, germination, and pathogenicity have provided a better understanding of the biology of A. aleyrodis in order to further develop this promising biocontrol agent (Meekes et al. 2002). Despite early successes with A. aleyrodis, few other Aschersonia species have been investigated for biological control potential. Factors confronting researchers interested in exploiting other species of the genus include: few species of Aschersonia were available from culture collections before our work, and had often been only tentatively identied; and identication of newly collected cultures and specimens has been difcult due to the scattered and out-dated literature on the group (Evans & Hywel-Jones 1990). The primary resource available for identication has been Petchs (1921) revision that needs to be updated in terms of number of species and terminology. Aschersonia species are found predominately in tropical and subtropical habitats and infect either whiteies or scale insects, presumably by the germination and direct penetration of conidia adhering to the host cuticle (Meekes et al. 2002). We focus on a group of whitey-pathogenic species characterized by their effuse white stromata and brightly coloured conidial masses. Because of their gross similarity and inadequate characterization in the literature, they are challenging to identify correctly, and we suspect that some records of A. aleyrodis represent misidentications (Liu et al. 2001; Shu 1996; Sutton 1980; Tzean et al. 1997). For example, the orange-spored species A. placenta (teleomorph H. raciborskii) differs from A. aleyrodis mainly in the formers paleotropical distribution. We demonstrate here that A. goldiana is merely a yellow-spored form of A. aleyrodis, as suggested by Mains (1959a,b). Literature records of A. goldiana include misidentications of other yellow-spored species such as A. andropogonis, A. incrassata, or Aschersonia anamorph of Hypocrella rhombispora. The ten species included in this study are all congeneric with the type species Hypocrella discoidea (teleomorph) or Aschersonia taitensis (anamorph). We revise and rene species concepts for this group of Hypocrella/Aschersonia species based on morphological examination of types and newly collected specimens and cultures. We apply phylogenetic analysis of DNA sequence data to evaluate whether morphological species are phylogenetically discrete, and use the analysis to discuss insights about relationships and character evolution in the group. A synoptic key is presented to facilitate the eld and laboratory identication of species. We hope that the information presented here and the isolates collected during our investigation will spur further investigation into the biocontrol potential of these fungi.

Materials and methods

Specimens and cultures
Species with white stromata include Aschersonia aleyrodis (Hypocrella libera), A. placenta (H. raciborskii), A. andropogonis (H. andropogonis), the Aschersonia anamorph of H. rhombispora, and A. incrassata (H. zhongdongii) (Liu & Hodge 2005). Reference species outside this apparently monophyletic group include A. basicystis (H. phyllogena), A. cubensis, A. insperata, A. turbinata and A. viridans. Fungal material was obtained in diverse forms: fresh specimens from the eld, isolates on articial media, and dried herbarium specimens. Fresh specimens were collected from Bolivia, Cameron, China, Costa Rica, Florida (USA), Ghana, Honduras, Mexico, Panama, and Puerto Rico (Table 1). For most eld collections, specimens were deposited in CUP (Plant Pathology Herbarium, Cornell University, Ithaca). A portion of each specimen from Costa Rica was deposited in INB (Mycological Herbarium, National Biodiversity Institute, Santo Domingo, Heredia, Costa Rica), and an isotype of H. rhombispora was deposited in UPR (Herbarium, Botanic Garden, University of Puerto Rico). Fungal cultures were made on potato-dextrose agar (PDA; Difco, Albany, NY, USA) medium following the procedure described in Liu and Hodge (2005) and deposited in ARSEF (ARS Collections of Entomopathogenic Fungi, Ithaca, New York). Additional cultures were obtained from ARSEF, BCC (BIOTEC Culture Collection, Bangkok) and CBS (Centraalbureau voor Schimmelcultures, Utrecht). Dried herbarium specimens including types were borrowed from B (Herbarium, Botanischer Garten und Botanisches Museum Berlin-Dahlem), BPI (US National Fungus Collections, Beltsville, MD), CUP, FH (Farlow Reference Library and Herbarium of Cryptogamic Botany, University of Havard), HMAS (Mycological Herbarium Institute of Microbiology, Beijing), K (Royal Botanic Gardens, Kew), KRA (Herbarium, Institute of Botany, Krakow), MICH (Herbarium, University of Michigan, Ann Arbor), PAD (Erbario Patavinum, Centro Musei Scientici, Padova), and S (Herbarium, Botany Departments, Stockholm.)

Morphological examination
Microscopic characters were examined using optical microscopes. For microscopic study, specimens were mounted in 85 % lactic acidcotton blue. Digital photomicrographs were taken with a SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI). Colours of the specimen and cultures were recorded according to Kornerup and Wanscher (1967). Characteristics of in vitro growth were recorded from cultures on PDA at 23  C.

DNA extraction, PCR and sequencing

DNA was extracted from the cultures following the procedure of Liu et al. (2005). Regions of three genes were amplied by PCR: nuclear LSU rDNA by primers LR0R and LR5 (Vilgalys & Hester 1990), mtSSU rDNA by MTS1f and MTS3r (Liu & Hodge 2005), RNA polymerase unit II (RPB2) by RPB2-7f and RPB211ar (Liu et al. 1999). Amplication conditions and sequencing approaches were described by Liu and Hodge (2005).

Aschersonia aleyrodis and its allies


Table 1 Fungal isolates, origins and GenBank accession numbers Species specimen Isolate Origin LSU
Hypocrella raciborskii Aschersonia placenta CUP67562 CUP67280 CUP67303 CUP67310 CUP67341 CUP67342 CUP-PR-4421 CUP-PR-4421 CUP-PR-4421 CUP-PR-4421 CUP67565 CUP67435 CUP67435 CUP67435 CUP67519 CUP67525 CUP67528 CUP67529 CUP67288 CUP67313 CUP67316 CUP67322 CUP67350 CUP-PR-4394 CUP-PR-4394 CUP-PR-4314 CUP67291 CUP67307 CUP67309 CUP67343 CUP67345 CUP67436 CUP67436 CUP-PR-4407 CUP-PR-4438 CUP-PR-4438 CUP-PR-4438 CUP67515 CUP67521 CUP67523 CUP67524 CUP67526 CUP67531 CUP67532 CUP67533 CUP67556 CUP67560 CUP67561 CUP67563 CUP67283 CUP67340 ARSEF992 ARSEF4209 ARSEF2154 CBS34984 AFR28 AFR114 ARSEF7508 ARSEF7512 CR11 CR19 CR20 ML175-1 ARSEF7393 ARSEF7394 ML175-4 ARSEF7642 ARSEF7339 ARSEF7340 ARSEF7343 ARSEF7641 ARSEF7617 PC413-1 PC434 PC439 PC442 CR05 CR12 CR13 CR15 ARSEF7513 ML149 ARSEF7369 ARSEF7071 ARSEF3014 ARSEF4424 CR03 ARSEF7510 CR09 CR10 CR21 CR22 ARSEF7341 ARSEF7342 ARSEF7391 ARSEF7396 ARSEF7397 ARSEF7398 PC384 PC418 PC431 PC432 PC436_1 PC451 PC452 PC455 AFR34A AFR68 AFR75 AFR80 ARSEF7639 CR04 CR17 Japan MalDQ070sia Indonesia Japan Ghana Cameron Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Puerto Rico Puerto Rico Puerto Rico Puerto Rico Guyana Florida Florida Florida Panama Costa Rica Honduras Mexico Mexico Mexico Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Puerto Rico Puerto Rico Puerto Rico Mexico Brazil Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Florida Florida Puerto Rico Puerto Rico Puerto Rico Puerto Rico Costa Rica Honduras Mexico Mexico Mexico Mexico Mexico Mexico Ghana Ghana Cameroon Cameroon Cameroon Costa Rica Costa Rica AY518368 DQ070074 DQ070070 DQ070135 DQ070113 DQ070119 AY932752 AY932761 DQ070080 DQ070085 DQ070086 DQ0700103 DQ0700104 DQ0700105 DQ0700106 DQ070096 DQ070099 DQ070121 DQ070122 DQ070126 DQ070128 DQ070129 AY518370 AY932755 AY932756 AY932757 AY932758 AY932759 AY932760 DQ070133 DQ070073 DQ070075 DQ070076 AY932754 DQ07078 DQ070079 DQ070087 DQ070088 DQ070097 DQ070098 DQ070102 DQ070109 DQ070108 DQ070110 DQ070123 DQ070124 DQ070125 DQ070127 DQ070130 DQ070131 DQ070132 DQ070114 DQ070115 DQ070116 DQ070117 DQ070120 DQ070077 DQ070083

GenBank accession no. mtSSU RPB2

H. libera A. aleyrodis

H. zhongdongii A. incrassata

H. andropogonis A. andropogonis

H. phyllogena

DQ070136 DQ070203 DQ070141 DQ069953 DQ069951 DQ070200 DQ069958 DQ070180 DQ069965 DQ070185 DQ069964 AY932781 AY932766 AY932791 AY932776 DQ070146 DQ070209 DQ070151 DQ069943 DQ070152 DQ069941 DQ07070 DQ069944 DQ070171 DQ069945 DQ070172 DQ069946 DQ070173 DQ069947 DQ070201 DQ069950 DQ070162 DQ069934 DQ070163 DQ06935 DQ070165 DQ069936 DQ070202 DQ069940 DQ070187 DQ069942 DQ070189 DQ069937 DQ070192 DQ069938 DQ070194 DQ069939 DQ070195 DQ070232 AY932784 AY932769 AY932785 AY932770 AY932786 AY932771 AY932787 AY932772 AY932788 AY932773 AY932789 AY932774 DQ070199 DQ070140 DQ070142 DQ070204 DQ070143 DQ070205 AY932783 AY932768 DQ070144 DQ070207 DQ070145 DQ070208 DQ070153 DQ070213 DQ070154 DQ069972 DQ070164 DQ070218 DQ070169 DQ070221 DQ070176 DQ070223 DQ070175 DQ069973 DQ070177 DQ070188 DQ070227 DQ070228 DQ070190 DQ070229 DQ070191 DQ070230 DQ070193 DQ070231 DQ070196 DQ069974 DQ070197 DQ070233 DQ070198 DQ070234 DQ070181 DQ069968 DQ069969 DQ070182 DQ070226 DQ070183 DQ070186 DQ069970 DQ070206 DQ070149 DQ070212 (continued on next page)


M. Liu et al.

Table 1 (continued) Species specimen Isolate Origin LSU

A. basicystis CUP67355 CUP67296 CUP CUP67369 CUP-PR-4406 ARSEF7515 ARSEF7511 CR32 CR34 ARSEF7390 ARSEF7395 ARSEF7399 ARSEF7400 ARSEF1030 CR14 CR18 CR16 CR24 CR26 ARSEF7514 CR29 ML142 PR11 CR27 ARSEF2396 ARSEF7509 CR23 Costa Costa Costa Costa Rica Rica Rica Rica AY518373 AY518371 DQ070094 DQ070095 DQ070107 DQ070111 DQ070112 DQ070071 DQ070081 DQ070084 DQ070082 DQ070090 DQ070091 AY932762 DQ070093 DQ070101 DQ070134 DQ070092 AY518374 AY932753 DQ070089

GenBank accession no. mtSSU

AY932793 AY932790 DQ070160 DQ070161 DQ070168 DQ070174 DQ070178 DQ070179 DQ070137 DQ070147 DQ070150 DQ070148 DQ070156 DQ070157 AY932792 DQ070159 DQ070167 DQ070158 DQ070138 AY932782 DQ070155

AY932778 AY932775 DQ070217 DQ069977 DQ070220 DQ070222 DQ070224 DQ070225 DQ070210 DQ070211 DQ070214 DQ070215 AY932777 DQ070216 DQ070219 DQ070235 DQ069976 AY932767 -

H. rhombispora and Aschersonia anamorph

A. turbinata

CUP67321 CUP67339 CUP67331 CUP67349 CUP67365 CUP67375 CUP67375 CUP-PR-4388 CUP67366 CUP67282 CUP67347

A. cubensis A. insperata A. viridans

Puerto Rico Puerto Rico Puerto Rico Puerto Rico Columbia Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Puerto Rico Puerto Rico Costa Rica Philippines Costa Rica Costa Rica

Genbank numbers in bold indicate the sequences generated in this study.

Phylogenetic analysis
DNA sequences of selected species (Table 1) were subjected to phylogenetic analysis based on three gene regions LSU, mtSSU and RPB2. The sequences were aligned using ClustalW (Thompson et al. 1994) with default parameters, and adjusted by eye in PAUP* 4.0b10 (Swofford 1998). Congruence among the three data sets was evaluated by comparison of general tree topologies and bootstrap supports for individual clades (Mason-Gamer & Kellogg 1996). Based on the general congruence, the three gene regions (LSU, mtSSU and RPB2) were eventually combined in a single matrix; unavailable sequences for individual taxa were scored as missing data. Excluding the characters that could not be aligned without ambiguity resulted in 2300 characters. To diminish the possible homoplasy caused by rapid evolution at the third codon position, we down-weighted the third codon positions of the RPB2 data set as 1, while other characters (rst and second codon and non-coding regions) as 2. Parsimony analyses were conducted using a heuristic search with TBR (tree bisection and reconnection) branch-swapping and 100 replicates of random sequence addition. Most gaps in LSU and mtSSU corresponded with homopolymeric repeats. We consider them to be artefacts of sequencing, thus treat them as missing data (Simmons et al. 2001). No gaps were observed in the RPB2 region. Bootstrap (BS) analysis was based on 500 replicates of a full heuristic search, each with 10 replicates of a random addition sequence. The trees were rooted with the outgroup Hypomyces aurantius (LSU AY932750, mtSSU AY932779, RPB2 AY932764), Hypocrea strictipilosa (LSU AY932751, mtSSU AY932780, RPB2 AY932765), and Cordyceps militaris (LSU AB027379, mtSSU

AB027357, RPB2 AY932763). The distribution of key morphological characters was mapped onto the nal tree topology to investigate character evolution and to identify which characters were phylogenetically informative.

Aligned sequences from the LSU and RPB2 genes included 889 and 754 characters, respectively. For the mtSSU gene, a fragment of 60 or so bases (nt 190255) evolved extremely fast and was excluded because it could not be unambiguously aligned; other parts were readily aligned, resulting in 657 characters. Phylogenetic analyses of the three gene regions revealed no conict in delimiting species. For relationships among the clades (species), mtSSU gives no resolution, and LSU and RPB2 conict regarding the position of A. viridans. Conicts among the three genes were mainly found within each clade (data not shown). Because there are no severe conicts, the three data sets were combined into a single matrix and subjected to phylogenetic analysis by a total evidence approach. Phylogenetic analysis revealed 359,900 equally parsimonious trees of length 2534; CI 0.573, RI 0.896 and 456 phylogenetically informative characters (141, 72, 243 from LSU, mtSSU and RPB2 respectively). The trees differed from each other mainly in the relationships within the major clades. A strict consensus tree is shown in Fig 1. Seven major clades were well-supported in the BS analysis, each of which was concordant with a morphologically dened species. The relationships among these clades were clear in the strict consensus but lack strong BS support. Morphological characters

Aschersonia aleyrodis and its allies


99 BC


96 100 D 100




Cordyceps militaris Hypomyces aurantius Hypocrea stritipilosa ARSEF 992 CBS 34984 A. placenta ARSEF 4209 (H. raciborskii) AFR28 CUP 67562 ARSEF 7508 ARSEF 7512 CUP 67341 CUP 67310 CUP-PR-4421-1 ARSEF 7393 ARSEF 7394 CUP-PR-4421-2 ARSEF 7642 CUP 67342 A. aleyrodis ARSEF 7617 (H. libera) ARSEF 7311 ARSEF 7339 ARSEF 7340 ARSEF 7343 CUP 67519 CUP 67525 CUP 67528 CUP 67529 ARSEF 7641 ARSEF 3014 ARSEF 4424 CR03 ARSEF 7510 CUP 67307 CUP 67343 CUP 67345 CUP 67309 ARSEF 7341 ARSEF 7342 ARSEF 7391 A. andropogonis ARSEF 7396 (H. andropogonis) ARSEF 7397 ARSEF 7398 CUP 67556 CUP 67560 AFR75 CUP 67561 ARSEF 7639 CUP 67515 CUP 67521 CUP 67523 CUP 67524 CUP 67526 CUP 67531 CUP 67532 CUP 67533 CUP 67288 CUP 67313 CUP 67316 CUP 67322 A. incrassata ARSEF 7513 (H. zhongdongii) CUP-PR-4394 CUP-PR-7369 ARSEF 7071 CUP 67283 A. basicystis CUP 67340 (H. phyllogena) ARSEF 7515 ARSEF 7511 CR32 CUP 67369 H. rhombispora sp. nov. ARSEF 7390 ARSEF 7395 ARSEF 7399 ARSEF 7400 ARSEF 1030 ARSEF 7514 CUP 67321 CUP 67339 A. turbinata CUP 67349 (H. turbinata) CUP 67331 CUP 67365 CUP 67375 CUP-PR-4388 PR11 CUP 67366 A. cubensis (H. epiphylla) ARSEF 2396 A. insperata ARSEF 7509 A. viridans CUP 67347

(H. viridans)

Fig 1 Strict consensus tree of 359,900 most parsimonious trees based on LSU rDNA, mtSSU DNA, and RNA polymerase unit II (RPB2) gene. L [ 2534, CI [ 0.573, RI [ 0.896, 456 informative characters. Species that are morphologically well dened appear as strongly supported clades. Phylogenetically informative characters are indicated as vertical hashmarks: (A) indicates an effuse white anamorphic stroma; (B) conuent conidial masses, (C) cylindrical tubercles containing perithecia; (D) discrete conidial masses; (E) formation of a rim around conidioma orices; (F) conidia ventricose. In the A. aleyrodis and A. placenta clade, taxa in bold produce yellowish orange conidial masses; others produce reddish orange conidial masses.


M. Liu et al.

were not included in the analysis; a select few were mapped a posteriori onto the trees derived from DNA sequences.


A synoptic key to treated species
Taxa treated in this work, as well as supercially similar species, are included in the key below. Each species treated is referenced in the key by its number. All can be identied based on either the anamorphic or teleomorphic stage. Two or more species of Aschersonia may occur on different insects on a single leaf. 1 2 3 4 5 6 7 8 9 10 A. australiensis A. insperata H. andropogonis/A. andropogonis H. epiphylla/A. cubensis H. libera/A. aleyrodis H. phyllogena/A. basicystis H. raciborskii/A. placenta H. rhombispora/A. sp. (anamorph) H. turbinata/A. turbinata H. zhongdongii/A. incrassata

2.1 a b c 2.2 a b c d 2.3 a b

Position in stroma In gregarious but well-separated tubercles..3, 5, 7, 9 In crowded, gregarious tubercles.........3, 4, 7, 8, 10 Embedded in stroma, scattered .........................4, 6 Colour of ostioles Yellow to orange yellow........................3, 5, 7, 8, 10 Reddish orange.........................................................5 Brownish yellow.................................3, 5, 6, 7, 8, 10 Redbrown ............................................................4, 6 Position of ostioles relative to the surrounding surface Slightly projecting....................................................6 Not projecting.................................3, 4, 5, 7, 8, 9, 10

Part ascospores

For each character there are two or more states; each character state is followed by one or more numbers that represent taxa. Underlined numbers indicate taxa exhibiting multiple possible character states; taxon numbers not present in any couplet indicate that the character is lacking or uncertain. Further discussion of the use of synoptic keys can be found in Korf (1972). All species are briey discussed while detailed descriptions of the species listed above in bold are included in the taxonomic section following the key.

3.1 a b c d 3.2 a b

Shape Cylindrical with rounded ends (Fig 2A)..3, 4, 5, 7, 10 Fusoid (Fig 2B)...................................................4, 5, 7 Ventricose with rounded or acute ends (Fig 2C)...6, 8 Ovoid (Fig 2D) ...........................................................7 Width Less than 2 mm....................................3, 5, 6, 7, 8, 10 35 mm................................................................4, 7, 9

Anamorphic characters Stromata

Teleomorph characters Stromata

1.1 a b c d 1.2 a b c d 1.3 a b c

Colour Reddish orange.........................................................6 Yellowish orange .................................................4, 9 Reddish brown .........................................................4 Yellowish white to white ......................3, 5, 7, 8, 10 Shape Globose head markedly constricted at base.........6 Pulvinate, base slightly constricted, slightly tuberculate ...............................................................3, 4, 8, 10 Pulvinate with sloping sides, ovoid or globose tubercles half-embedded .........................................................3 Pulvinate with pronounced cylindrical or ovoid tubercles .....................................................................5, 7, 9 Surface texture Tomentose ................................................................3 Pruinose................................................................. ..5, 7 Smooth (minutely tomentose or pruinose) ...................................................... ..3, 4, 5, 6, 7, 8, 9, 10

4.1 a b c d 4.2 a

Colour Grayish brown ..........................................................4 Reddish orange...............................................2, 6, 10 Light yellow ..........................................................4, 9 Yellowish white to white ..............1, 3, 5, 6, 7, 8, 10 Shape Pulvinate base with pezizoid projections .............9

Fig 2 Shapes of part ascospores. (A) Cylindrical with rounded ends. (B) Fusoid. (C) Ventricose with rounded ends. (D) Ovoid.

Aschersonia aleyrodis and its allies


b c d e f g h 4.3

a b

Tuberculate.................................................2, 4, 9, 10 Cylindrical.........................................................4, 5, 9 Hemiglobose .....................................................4, 6, 8 Scutate (a hemispheric central region abruptly attenuating and extending to the edge) ....................7 3, 8 Thick pulvinate, conical pulvinate.1, 3, 4, 5, 6, 7, 8, 10 Thin pulvinate, sometimes with pronouncedly erect tubercles............................................................2, 5, 7 Thin pulvinate with embedded tubercles.............3 Hypothallus (a distinct thin layer of hyphae surrounding the base of the stroma and appressed to the plant surface) Present.............................................1, 3, 5, 6, 7, 8, 10 Absent .....................................1, 3, 4, 5, 6, 7, 8, 9, 10

Fig 3 Shapes of conidia. (A) Fusoid. (B) Fusoid with thickened wall at ends. (C) Ventricose. (D) Ovoid with acute ends. (E) Ovoid conidium of Hirsutella-like synanamorph.

d Ovoid with acute ends (Fig 3D) ..........................4, 9 e Ovoid (Hirsutella-like synanamorph) (Fig 3E) ........2

5.1 a b 5.2 a b c 5.3 a b 5.4 a b

Approximate number of locules Fewer than ten .......................1, 3, 4, 5, 6, 7, 8, 9, 10 More than ten.................................1, 3, 5, 7, 8, 9, 10 Shape of locules Simple depressions of surface without distinct rims..............................................1, 3, 4, 5, 6, 7, 8, 10 With distinct rims, like half-embedded bowls..3, 10 Pezizoid .....................................................................9 Arrangement of ostioles on stroma Scattered .....................................1, 3, 4, 5, 7, 8, 9, 10 Circular..............................................................5, 6, 7 Paraphyses in conidioma Present.....................................................1, 3, 5, 7, 10 Absent .....................................1, 3, 4, 5, 6, 7, 8, 9, 10

Cultural characters on PDA


6.1 a b c d e 6.2 a b c d 6.3 a b 6.4 a b 6.5 a b c

Colour of conidial masses Reddish brown .....................................................4, 9 Reddish orange.....................................................5, 7 Orange ...........................................................4, 5, 7, 9 Deep yellow ..........................................................6, 8 Pale yellow ..................................................3, 6, 8, 10 Shape of conidial masses on surface of stroma Discrete, erumpent or contained in conidiomata.................................................3, 4, 5, 7, 8, 9, 10 Conuent, covering the centre of stroma.........5, 7 Conuent, forming a ring around the stroma .. ...6 Forming an erect cirrus.....................3, 4, 5, 7, 9, 10 Length of conidium Longer than 9 mm .......................3, 4, 5, 6, 7, 8, 9, 10 Shorter than 9 mm ....................................................1 Width of conidium 1.63 mm...................................................1, 3, 5, 7, 10 Wider than 3 mm ..........................................4, 6, 8, 9 Shape of conidium Fusoid (Fig 3A) ..........................................1, 2, 3, 5, 7 Fusoid with ca 35 mm thickened wall at ends (Fig 3B) .................................................................. ...10 Ventricose (Fig 3C) ...............................................6, 8

7.1 Growth rate on PDA a Relatively rapid, greater than 30 mm diam in three weeks at 23  C ..............................................4, 5, 7, 9 b Moderate, between 20 mm and 30 mm diam in three weeks at 23  C ..................................................2, 6, 8 c Slow, smaller than 20 mm diam in three weeks at 23  C ....................................................3, 10 7.2 Appearance and texture of the growing colony a Spreading, minutely tomentose.................4, 5, 7, 9 b Compact, leathery..................................2, 3, 6, 8, 10 7.3 Colour of colonies a Greyish white .......................................................3, 8 b White ...................................................................6, 10 c Yellowish white to yellow ..........................4, 5, 7, 9 d Orange yellow...........................................................2 7.4 Colour of conidial mass a Pale yellow to yellow .....................................2, 3, 10 b Deep yellow ..........................................................6, 8 c Yellowish orange .................................................5, 7 d Reddish orange.............................................4, 5, 7, 9 e Brownish red ........................................................4, 9 7.5 Quantity of conidial mass a Abundant ..........................................2, 4, 5, 6, 7, 8, 9 b Scanty ............................................................... ...3, 10 7.6 Hirsutella-like synanamorph a Present.......................................................................2 b Absent .........................................3, 4, 5, 6, 7, 8, 9, 10

1. Aschersonia australiensis Henn., Hedwigia 42: 87 (1903).

Note: This species resembles A. placenta in its orangered conidial masses, but differs in having distinctively smaller conidia (58 mm) (Petch 1921). It has been reported from


M. Liu et al.

Australasia (Hennings 1903). No teleomorph is known, and the species has not been well studied. Detailed descriptions were provided by Petch (1921). A. australiensis is further discussed under H. raciborskii.

2. Aschersonia insperata Rombach et al., Mycologia 97: 251 (2005).

(Fig 3E) Note: The most distinctive character of this species is its production of both Aschersonia and Hirsutella-like synanamorphs in young cultures and specimens, which has not been found in any other species in this genus. The reddish orange tuberculate stroma of this anamorphic species appears supercially like the perithecial stroma of H. turbinata, but only asexual spores are produced. A. insperata is known only from the type collections in the Philippines, and the teleomorph remains unknown. A detailed description of this species was provided by Liu et al. (2005).

3. Hypocrella andropogonis Petch, Ann. Roy. Bot. Gard. Peradeniya 7: 247 (1921).
Typus: Trinidad: On leaves, R. Thaxter 18 (K(M) 120354holotypes). Anamorph: Aschersonia andropogonis Henn., Hedwigia 39: 139 (1900). Neotype: Puerto Rico: Mayaguez, beside Road 105, on ferns, 15 Dec. 2003, M. Liu & Z.D. Wang (CUP-PR 4407) neotypus hic designatus). Synonyms: Aschersonia parasitica Henn., Hedwigia 43: 149 (1904). Aschersonia lecanioides Henn., Hedwigia 41:145 (1902). Figs 4AC, Fig 5 Teleomorphic stromata attened pulvinate with subglobose tubercles (Fig 4A), some tubercles fused together (Fig 5A), but more often discrete, surface minutely tomentose, white or orangewhite (darkened in very old specimens), 1 3 mm diam, 0.40.5 mm thick, edges of the stromata extending to form a hypothallus (Fig 4B, 5B). Perithecia develop singly in the tubercles (Fig 5C), ask-shaped, 250450 mm deep, 160 300 mm diam at the widest point. Asci produced from basal clusters, cylindrical, 138180 mm long, 58 mm wide, caps 3 3.5 mm thick (Fig 5D). Ascospores liform, slight shorter than the perithecia, septate, dividing into part spores that are cylindrical with rounded ends, 1215 1.52 mm (Fig 5E). Teleomorph and anamorph may or may not be present in the same stromata (Figs 4B 5AB, F). Strictly asexual stromata usually pulvinate (Figs 4C, 5B, F), white to pale yellow (4A3 4A5), 14 mm diam, 0.51 mm thick. Hyphae of stromata forming compact textura intricata, 36 mm wide with a thickened wall, 12 mm wide, Conidiomata scattered in stromata, 16 per stroma, widely open, orice circular, 0.10.6 mm diam, rim of conidiomata sometimes distinctly elevated, conidioma resembles a half-immersed bowl (Fig 4C). Conidial mass yellow (4A8), yellowish orange (4B7) to orange (5A8), usually contained in conidioma (Figs 4C, 5F), but sometimes erumpent or forming a column-like cirrus, never fused with conidial masses from adjacent conidiomata. In section, the conidioma is U- or V-shaped (Fig 5G). Conidioma with hymenium lining

inner surface; most conidiogenous cells arise from thickwalled hyphae, unbranched, cylindrical, slightly narrower near their truncate ends, unicellular, 716 11.5 mm; some are branched (Fig 5H). Conidia fusoid, apices more or less blunt, 814 1.52 mm (Fig 5I), produced in copious slime. Paraphyses present in some specimens, ranging from 60160 mm long (Fig 5H). On PDA colony grows very slowly, reaching 1525 mm diam in ve weeks at 23  C. Stromatic colonies white to greyish white, compact, forming a thick pulvinate structure, surface minutely velvety, wrinkled (Fig 4A). Colonies both lamentous hyphae and hyphal bodies, the latter usually 516 37 mm with a thickened wall (not over 1 mm thick). Conidial masses usually not abundant, appear as tiny drops scattered on surface or very thin streams along wrinkles in the colony. When picked with a needle, the conidial masses appear as a solid mass that is light yellow to yellow. Isolates are liable to lose their ability to sporulate in serial subculture. Conidiogenous cells 1017 1.52 mm, conidia fusoid, apices acute, 812 1.52 mm. Paraphyses usually long and abundant, up to 190 mm long. Known distribution: Bolivia, Cameroon, China, Costa Rica, Ghana, Honduras, Mexico, Puerto Rico, Trinidad, USA. Other specimens examined: Cameroon: Korup National Forest, Oct. 2003, H.C. Evans & G. J. Samuels (AFR116 ARSEF 7639 CUP67563); Costa Rica: Heredia: OTS La Selva Biological Station, Sendero Holdridge, 18 June 2002, M. Liu (CUP 067291); Camino Cantarrana, 19 June 2002, M. Liu (CUP 067307); Beside entrance to plantation, June 20, M. Liu (CUP 067343, 067345). Ghana: Central Region, Jukua District, Kakum National Park, N05  210 , W01  230 , elev. 280 m, Oct. 2003, H.C. Evans & G. J. Samuels, AFR34 CUP 67556, AFR68 CUP 67560. Java: Buitenzorg, Kulturgarten, on leaf of Mangifera indica, 1901, Zimmermann, (S F22432-type of A. lecanioides). Mexico: Veracruz: Municipio Emiliano Zapata, Plan Chico, N19  26.7350 , W96  49.8650 , elev. 900 m, 11 Dec. 2003, P. Chaverri, J. Hernandez, J. Garcia-Alvarado PC431, (CUP 67523); behind Instituto Genetica Forestal Universidad (Veracruzana) building, 12 Dec. 2003, P. Chaverri & J. Garcia-Alvarado (PC432 CUP 67524); Catemaco, Ejido: Lopez Mateo town, project Cielo, Tierra Y Selva, trail to mountain, 200300 m elev., 13 Dec. 2003, P. Chaverri, J. Garcia-Alvarado & C. Mena-Jiles (PC436 CUP 67526, PC452 CUP 67532). Philippines: Laguna: Mt. Maquiling, near Los Banos, Feb. (18)94, C.F. Baker (FH 8); Paraguay. Cerro Coche: On leaves of Andropogon sp., K. Fiebrig 779 (B 70 0005658: type of A. parasitica). Puerto Rico: Guajataca, trail no.9, on ferns, M Liu & ZD Wang (CUP-PR 4438). USA: Florida: Micanopy, cross creek, Marjorie Kinnan Rawlings residence, on Citrus, 22 Aug. 2003, M. Liu & Z.D. Wang (CUP 67436). Notes: A. andropogonis is easily distinguished from A. aleyrodis when the conidiomata of the former have elevated edges, but resembles a yellow-spored form of A. aleyrodis with thick stromata in cases where the conidiomata are simply depressions of the surface. A. andropogonis generally differs in possessing conidiomata with very wide openings, fewer conidiomata (16), conidial masses that do not fuse with those from adjacent conidiomata, and longer paraphyses; while A. aleyrodis may possess many conidiomata (120), smaller and sometimes radially elongated orices, and conuent conidial masses. The colony morphology on PDA reveals more obvious differences: A. andropogonis produces small, slow growing,

Fig 4 Cultural morphology on PDA and stromata of selected species. (A) Culture of Hyporella andropogonis/Aschersonia andropogonis. Compact colonies bear pale yellow conidial masses in small discrete drops. (B) H. andropogonis (CUP-PR 4438): ovoid tubercles are half-embedded in the white pulvinate stroma. (C) A. andropogonis (CUP-PR 4407): conidial masses are contained in conidiomata, and the openings of the conidiomata have distinct rims. (D) Cultures of H. libera/A. aleyrodis: the colonies are effuse, white, and uffy, and bear abundant conuent conidial masses; colouration varies from orange to reddish orange. E H. libera (MCA2335). Stromatal tubercles are cylindrical, gregarious, and crowded. F A. aleyrodis (CUP 067341) has attened pulvinate stroma, and the conidiomata produce reddish orange conidial masses. G. Culture of H. raciborskii/A. placenta: colonies are uffy and spreading, conidial masses are abundant and conuent, and colouration varies from yellowish-orange to orange. H. H. raciborskii (CUP-CH 002621) showing gregarious cylindrical tubercles in which perithecia are embedded. I. A. placenta (CUP-CH 002620) with a attened pulvinate stroma, and conuent yellowish orange conidial masses. J. Culture of H. rhombispora showing the compact colony with abundant yellow to orange conidial masses in discrete small drops. K. H. rhombispora (CUP 067548). A minutely tuberculate stroma with a slightly constricted base. L. Anamorph of H. rhombispora (CUP 067551). Scutate stroma with yellowish orange conidial masses contained in the conidiomata. Bar [ 500 mm.


M. Liu et al.

Fig 5 Hypocrella andropogonis/Aschersonia andropogonis. (A) Tuberculate stroma of sexual state (K(M) 120354-holotype). (B) Stroma with sexual (tubercles) and asexual state (holes). (C) Flask-shaped perithecium embedded in a tubercle. (D) Cylindrical asci containing liform ascospores. (E) Cylindrical part spores with rounded ends. (F) Anamorphic stroma with one conidioma in the centre; conidial mass contained in conidioma. (G) Conidioma a simple depression forming a U shape in section. (H) Long paraphyses and conidiogenous cells. (I) Fusoid conidia. Bar [ 500 mm for Figs AB, F; 100 mm for Figs C, G; and 10 mm for Figs DE, HI.

compact, and greyish white colonies with a minutely velvety and often wrinkled surface, while the A. aleyrodis grows faster and the white or yellowish white colonies are effuse and minutely uffy. A. lecanioides was considered by Petch (1921) to be a synonym of A. placenta based on similar conidia and paraphyses. We examined the type specimen and observed elevated edges of conidioma and other characters consistent with A. andropogonis. A. andropogonis has longer paraphyses than A. placenta. The paraphyses of A. lecanioides measured 70114 mm, longer than those in A. placenta (4070 mm). Hennings (1902) described the stromata as pale to yellow, as in A. andropogonis. The sexual state lacks cylindrical tubercles as in H. raciborskii, but forms subglobose tubercles that are half or totally embedded in the attened stromata, as in H. andropogonis. We propose that A. lecanioides is conspecic with A. andropogonis. We conclude that specimens identied by Fawcett (1908) from Florida as A. avocitrina are A. andropogonis. Fawcetts determination was based mainly on the yellow colour of the spore masses. Based on our type studies, A. avocitrina has distinctive orange discoid stromata and can be easily distinguished from A. aleyrodis. Considering the characters of colonies on PDA he described in the same paper, Fawcetts specimens, which we were unable to locate, were probably A. andropogonis, which produces small and compact stromatal colonies.

4. Hypocrella epiphylla Sacc., Syll. Fung. 11: 368 (1895).

Anamorph: Aschersonia cubensis Berk. & M. A. Curtis, J. Linn. Soc. 10:351 (1869). Notes: H. epiphylla is common in the subtropical eastern US. The stromata typically are squat, cylindrical, and more compact and slightly darker in colour than those of the A. aleyrodis group. The differences are sometimes subtle, and young stromata can be confused with those of A. aleyrodis. However, the conidia of this species are wider and shorter than those of A. aleyrodis, a distinctive character that H. epiphylla shares with its sister species, H. turbinata (Fig 1). The teleomorph is infrequently collected. For a detailed description, see Petch (1921).

5. Hypocrella libera Syd., Ann. Mycol. 14: 85 (1916).

Type: Bolivia: Cobija: Rio Acre, on coccids on fallen leaves, Jan. 1912, E. Ule 3413 (W. 00939-holotypes). Synonym: Hypocrella nectrioides Petch, Ann. Roy. Bot. Gard. Peradeniya 7:225 (1921). Anamorph: Aschersonia aleyrodis Webber, Bull. USDA Div. Veg. Phy. Path. 13:21 (1897) Type: USA: Florida: Manatee County, on whitey on citrus, Dec. 1896, H. J. Webber (BPI 0389438lectotypus hic designatus). Synonyms: Aschersonia goldiana Sacc. & Ellis, in Saccardo, Syll. Fung. 14: 990 (1899). Aschersonia paraensis Henn., Hedwigia, 41: 17 (1902).

Aschersonia aleyrodis and its allies


(Figs 4DF, 6) Stroma white, yellowish to orange white (4A25A2), composed of a few to numerous gregarious tubercles arising from a pulvinate to hemispherical base (Fig 4E, 6A); sometimes surrounded by a thin hypothallus 0.72.0 mm wide; surface of tubercles and base pruinose due to loosely woven, thickwalled hyphae that form stroma (Fig 6D). Tubercles strongly projecting and aggregated, hemispherical, cylindrical or slightly narrowing apically, 0.30.5 mm diam, 0.30.7 mm in height; apices reddish orange (7A6) in fresh specimens, fading to yellow when dry, in old specimens appearing amber (brownish yellow 5C7). In most cases ostioles difcult to distinguish from surrounding glabrous tissue; in a few cases they are visible, ca 0.1 mm diam. Perithecia fully embedded (Fig 6B), one perithecium per tubercle, the shape of perithecia in section nearly globose to ovoid, 300400 300600 mm; stromatal tissue around perithecium textura intricata (Fig 6D). Asci cylindrical, with a thick cap ca 5 mm long. Ascospores liform, slightly shorter than perithecia, septate, sometimes helically twisted in ascus, disarticulating into oblong oval or cylindrical spores, slightly tapering towards ends, with somewhat rounded ends, 1316 2.53 mm (Fig 6C). Separate from sexual stroma, anamorphic stroma thin pulvinate (Figs 4F, 6EF), 12 mm diam, 0.10.3 mm thick, or scutate 13 mm diam, 0.51 mm thick, white, minutely tomentose; hypothallus usually present in pulvinate stromata, 0.31.0 mm in width, present or absent in scutate stromata;

centre of stromata usually covered with conidial masses reddish orange (7A6), orange (5A86A8), or light yellow (3A54A7), thickened in centre. Conidiomata circularly arranged (Fig 4F) or scattered (Fig 6F); 320 per stroma; shape of conidiomatal ostioles circular, radially elongated or irregular due to fusion of adjacent ostioles; widely open, appearing as simple depressions of stromatic surface without a differentiated rim; conidioma shape in section globose (Fig 6G) or irregular. Conidial masses exuded from conidiomata as a viscous uid, usually overowing and conuent, sometimes forming a long erect cirrus. Conidiogenous cells, phialidic, 1020 11.5 mm, arise singly or in whorls of 25 from compact, thick-walled hyphal tissues (Fig 6I), not branched, thin-walled, smooth, cylindrical, slightly tapering, truncate at apices. Conidia fusiform (Fig 6K), unicellular, hyaline, guttulate, ends acute but not prolonged, (9.0) 1016 (18) 1.52.0 (2.5) mm, produced in copious slime. Paraphyses abundant in the hymenium, especially in thick stromata, hyaline, not staining in cotton-blue, liform, 5090 (113) 1.01.5 mm (Fig 39). Colonies (Fig 4D) on PDA growing relatively fast, 35 mm diam in three weeks at 23  C, lamentous hyphae, tomentose, white to yellowish white (4A44A5), sometimes with a grayish-yellow (4B4) peripheral circle. Conidial mass variable in colouration (Fig 4D), light orange (5A45A5), deep orange (6A7) to reddish orange (7A7), abundant, conuent. Most conidiogenous cells, 1019 1.01.5 mm, arise mononematously and laterally from thick-walled hyphae, or in a whorl of 26 from the end

Fig 6 Hypocrella libera/Aschersonia aleyrodis. (A) H. libera (CUP 067303) stroma: cylindrical tubercles arise from hemispherical base; surface pruinose. (B) Flask-shaped perithecium embedded in a tubercle; surrounding tissues are textura intricata. (C) Asci with helically twisted ascospores, which disarticulate into oblong ovoid part spores. (D) Tissues of the stromatal surface are textura intricata. (E) Stroma of A. aleyrodis (CUP-PR 4421), conned by hairs on the leaf. (F) Stroma of A. aleyrodis with scattered conidiomata (BPI 0389440). (G) Globose conidioma with hymenium, conidia and paraphyses. (H) Paraphyses extending above the hymenium. (I) Conidiogenous cells from culture arising singly from hyphae or clustered apically on hyphae. (J) Branched conidiogenous cells extending above the hymenium. (K) Fusoid conidia. Bar [ 500 mm for Figs A, EF; 100 mm for Figs B, G; 10 mm for Figs CD, HK.


M. Liu et al.

of thick-walled hyphae (Fig 6I); but conidiophores with multiple branches also observed in culture, 3565 mm, some phialides elongated to the length of paraphyses (Fig 6J). Conidia fusoid, 913 1.52.0 mm, paraphyses not ordinarily observed in all isolates, 40123 mm when present. Known distribution: Bolivia, British Honduras, Costa Rica, Cuba, Dominican Republic, Jamaica, Panama, Puerto Rico, Trinidad, Venezuela (Mains 1959a,b), and Florida, Mississippi, and Texas in the United States. Other specimens examined: Brazil: Para, auf lebenden Blat tern von Psidium pomiferum, May 1901, J. Huber 50 (S F22444, as A. paranesis). Costa Rica: Heredia: OTS La Selva Biological Station, laboratory area, 17 June 2002, M. Liu (culture CR01 CUP 067280); Camino Cantarrana, 19 June 2002, M. Liu (CUP 067298, CUP 067303 ARSEF 7512, culture CR11 CUP 067310); plantation beside entrance RCC, 20 June 2002, M. Liu (culture CR19 CUP 067341, culture CR20 CUP 067342); Sendero Oriental 450 m, 20 June 2002, M. Liu (CUP 067325); succession plots, 20 Aug. 2003, P. Chaverri PC321 ARSEF 7617. Guyana: Kamarang: on line to old Ayanganna Airstrip, west Pakaraima mountains, upper Potaro river, 20 km east of Mount Ayangana, near conuence of Potaro and Alukyadongbaru Creek, general area N5  160 , W59  540 , approximately 650 m elev., 8 Jan. 2004, C. Aime, MCA 2465 ARSEF 7642. Mexico: Veracruz: Catemaco, Ejido Lopez Mateo town, project Cielo, Tierra Y Selva, trail to mountain, 20300 m elev., 13 Dec. 2003, P. Chaverri, J. Garia-Alvarado, C. Mena-Jiles, PC434 CUP 067525 ARSEF 7706, PC439 CUP 067528. Panama: Furtuna: Behind eld station, 14 July 2002, J. F. Bischoff, JB133 (ARSEF 7641). Puerto Rico: Guillarte: RD 388 trail, 16 Dec. 2003, M. Liu & Z. D. Wang (culture ML175-1, ML175-2, ML175-3, ML175-4 CUP-PR 4421 ARSEF 7393, ARSEF 7394); On Psidium guajaba, 1912, (FH 360 as A. goldiana). Trinidad: Port of Spain, St. Anns Valley, on scale insect on Pentaclenthra sp., R. Thaxter, K(M)120325 (H. nectrioides); Maraval Valley, 19121913, R Thaxter (FH); Annes valley, on leaves of Adiantum, Feb. 1913, R. Thaxter, (FH, as A. goldiana). United States: Florida: Manatee County, on whitey on Citrus, Dec. 1896, H. J. Webber (BPI US0389440); Mar. 1896, H. J. Webber (BPI 0389439); Micanopy, Cross Creek, Majorie Kinnan Rowlings residence, on Citrus, 22 Aug. 2003, M. Liu & Z.D. Wang (CUP 067435 ARSEF 7339, ARSEF 7340, ARSEF 7343, ARSEF 7344); Lake Alfred, on citrus leaves, 9 Jan. 1980, R.S. Soper (living culture ARSEF 430); on scale insects on citrus leaves, F.A. Wolf, (F.H. gift from F.A. Wolf); Inverness, 14 Feb. 1923, E.W. Berger 34 (FH 6298, as A. goldiana); Gainesville, 1923, E.W. Berger (FH 6301 as A. goldiana). Notes: A. aleyrodis is distinct in having a neotropical and subtropical distribution, orange (or sometimes yellow), narrow fusoid conidia, and a tuberculate teleomorph. Historically, there has been taxonomic confusion regarding the relationship between A. aleyrodis, commonly called the red fungus, and the yellow fungus A. goldiana (Fawcett 1908). Petch (1921) and Mains (1959a, b) observed that the two species were only distinguished by the colour of the conidial mass, that of the former being red to reddish orange, and the latter yellow to orange. We mapped conidial mass colour onto the phylogenetic tree shown in Fig 1 and found that it is not an informative character within the A. aleyrodis clade. We also examined the type of A. goldiana. Based on the shape

of the stroma, the conuence of the conidial masses, and the shape of conidia, we conclude that A. goldiana is a synonym of A. aleyrodis. The connection between H. libera and A. aleyrodis was rst inferred by Petch (1925) based on two specimens from Panama. Mains (1959a) cast doubt on the connection due to the inconsistency of the host: H. libera was described on coccids (Sydow & Sydow 1916), whereas A. aleyrodis was described on Aleyrodes (Webber 1897). However, our examination of the type specimens convinced us that H. libera is indeed the teleomorph of A. aleyrodis, and we feel the host might have been misidentied by Sydow & Sydow (1916): the black oval-shaped insect may be a whitey nymph. Our phylogenetic analysis reafrms the connection. Isolates from ascospores of fresh H. libera collections (CR08 and CR11) grouped together with those from A. aleyrodis conidia (Fig 1). An unidentied species that is the source of GenBank sequence no. U47832 was recently identied as H. nectrioides based on morphological examination of the voucher specimen (Bath et al. 2005). We examined the type specimen of H. nectrioides, the general morphology of the stromata of which resembles H. libera. No asci were found, but, based partly on Petchs description (Petch 1925), we consider this taxon to be a synonym of H. libera. H. sloaneae is similar to H. libera. The subtle differences include: tubercles in H. sloaneae are crowded and ovoid, whereas those of H. libera are separated and cylindrical; and the stromatal surface of the former is more coarse, whereas the latter is more glabrous. We did not observe the anamorph in the type from FH (Guadeloupe: Bois des Bains-Jaunes, 1904); however, Petch (1921) recorded anamorph characters from the type of H. amazonica (Peru: on Sterculiaceae, Iquitos, July 1902, E. Ule, Herb. Brasil. no. 3198), which he considered to be conspecic with H. sloaneae. According to Petch (1921), the unnamed Aschersonia anamorph of this fungus produces a redbrown spore mass, and thus differs from that of A. aleyrodis. As already mentioned, colouration of conidial mass is not a constant character in A. aleyrodis/H. libera, which produces both yellowish orange and reddish orange conidial masses.

6. Hypocrella phyllogena Petch, Ann. Roy. Bot. Gard. Peradeniya 7: 228 (1921).
Anamorph: Aschersonia basicystis Berk. & M.A. Curtis, Jour. Linn. Soc. Bot. 10: 352 (1869). Notes: This species has a distinctive sexual stroma, which is reddish orange and composed of a globose head that is markedly constricted at the base. Dull yellow conidial masses are produced at the narrowest part of the constriction, forming a ring around the head. This species is variable and appears to be a complex of several species. In the early stages of development before the perithecia are formed, this species resembles anamorph stage of H. rhombispora. Both have white pulvinate stromata, yellow conidial masses contained in the conidiomata, and ventricose conidia. However, the conidiomata of anamorph stage of H. basicystis are circularly arranged around the base of stroma, while conidiomata of A. rhombispora are scattered on the stroma surface. This species is further discussed under H. rhombispora.

Aschersonia aleyrodis and its allies


7. Hypocrella raciborskii Zimm., Centralblatt f. Bakt. 7: 875 (1901).

Type: Zimmermann (Centralblatt f. Bakt., 7: 875 g 4, 1901 lectotypus hic designatus. China: Guangdong: Dinghushan, 10 Aug. 2004, B. Huang DHS040810-11 (CUP CH 002621 epitypus hic designatus). Ex-epitype living culture ARSEF 7609 CHN4). Synonyms: Hypocrella warneckiana Henn., Englers Bot. Jahrb. 38: 113 (1905). Barya salaccensis Racib., Bull. Akad. Sci. Cracovie: 909 (1906). Anamorph: Aschersonia placenta Berk., J. Linn. Soc. Bot. 14: 89 (1875). Type: Sri Lanka: on leaves of Loranthus sp., 1879, G. H. K. Thwaites (K(M)81383-holotype). Synonyms: Aschersonia novoguineensis Henn., Englers Bot. Jahrb. 25: 509 (1898).

Aschersonia javanica Penz. & Sacc., Malpighia 20: 236 (1901). Aschersonia tamurai Henn., Englers Bot. Jahrb. 31: 741 (1902). (Figs 4GI, Fig 7) Stromata white to yellowish white, 12 mm diam; tuberculate processes developing from a thin pulvinate base (Figs 4H, 7A); tubercles usually isolated from one another, rarely densely aggregated, occurring at margins when conidiomata are present in centre. Tubercles cylindrical, ovoid or subglobose, 0.30.5 mm diam, up to 0.5 mm in height. Perithecia embedded singly in tubercles (Fig 7B), ask-shaped or ovoid, 300 420 mm deep, 200300 mm wide with walls 2032 mm thick. Ostioles yellow (3A64A6) to orangeyellow, darkening to deep orange in old specimens; readily visible. Stromatal tissue textura intricata around perithecium (Fig 7E). Asci cylindrical (Fig 7C), 140220 57 mm with 56 mm thick caps. Ascospores liform, slightly shorter than perithecia, septate, dividing into part

Fig 7 Hypocrella raciborskii/Aschersonia placenta. (A) Sexual stroma. (B) Flask-shaped perithecium embedded in a tubercle; ostiole is not erumpent. (C) Asci. (D) Ovoid part spores. (E) Tissues of stromata, more compact near the perithecial wall (top). (F) Asexual stroma with conuent conidial masses (CUP-CH 002620). (G) Asexual stroma of the type specimen, (K(M)81383), in which conidiomata are circularly arranged. (H) Hymenium showing paraphyses and conidiogenous cells. (I) Conidiogenous cells arising singly from thick-walled hyphae (in type specimen). (J) Conidiogenous cells showing branching patterns in culture. (K) Long conidiogenous cells resembling paraphyses. (L) Fusoid conidia containing guttules. Bar [ 500 mm for Figs A, FG; 100 mm for Fig B; and 10 mm for CE, HL.


M. Liu et al.

ascospores that are cylindrical, slightly tapering towards ends, 1016 2.5 mm, or ovoid 810 3 mm (Fig 7D). Anamorph may or may not co-exist in the same stromata with teleomorph. Anamorphic stromata white (Figs 4I, 7FG) in fresh specimens, yellowish white to orange white in old specimens; attened pulvinate, usually very thin, 0.20.7 mm thick, 13 mm diam, surrounded by a hyaline hypothallus up to 1.5 mm wide. Stroma surface minutely tomentose, covered with conuent conidial masses that are deep yellow (4A6) to orange yellow (4A7). Conidiomata occur as simple depressions in the stroma, 313 per stroma, forming a ring (Fig 7G) or irregularly (Figs 4I, 7F) arranged. Conidiogenous cells (Fig 7I), arising singly or in a cluster of 35 from thick-walled hyphae, sometimes branched, cylindrical, slightly tapering towards the apex, truncate, 722 11.5 mm. Conidia fusoid (9)1114 (16) 1.52 mm (Fig 7L), produced in copious slime. Paraphyses arising from the hymenium of the conidioma, liform, tapering at the apices, 4070 mm long (Fig 7H). In culture, colonies 23.5 mm diam in three weeks at 23  C on PDA, white to yellowish white, pulvinate, surface tomentose. Colonies cultured from ascospores often form hyphal tufts in early stages. Colonies usually produce abundant viscous conidial masses that become conuent, or thick cirri that are concentrically arranged. Conidial masses pale yellow (4A3), light yellow (4A4) to light orange (5A4) (Fig 4G). Conidiomata evident after conidial masses washed away. Similar in shape to those in natural stromata; concave, with an opening of 0.61.0 mm. Conidiophores and conidiogenous cells arise from aggregated thick-walled hyphae (Fig 7J). Conidiophores typically penicillate or verticillate, branching 13 times, 30 50 mm high, forming a hymenial layer. Conidiogenous cells slender, 1.52 mm at widest point, 1116 mm long, apices truncate, or occasionally very long, reaching 70 mm, and resembling paraphyses (Fig 7K). Conidia similar in size to those derived from natural specimens, 1015 1.5 2mm. Paraphyses occasionally found in culture, 60114 mm long. Distribution: Cameroon, China, Ghana, India, Indonesia, Malaysia, New Guinea, the Philippines, Thailand, and Vietnam.
Other specimens examined: Cameroon: Korup National Forest, Oct. 2003, G.J. Samuels & H.C. Evans, AFR114 ARSEF 7616. Ceylon: Peradeniya: On Schleichera tujuga, Jan. 1919, T. Petch, (K(M)128041); Salak, on Lasianthus sp., 1899, M. Raciborski (KRA-F1899-31, -32, -33) (type of Barya salaccensis). China: Guangdong: Dinghushan, 9 Aug. 2004, B. Huang (DHS04080907 CUP-CH 002607 ARSEF 7607 CHN1); 10 Aug. 2004, B. Huang (DHS040810-10 CUP-CH 002620 ARSEF 7686 CHN3). Ghana: Central Region, Jukua district, Kakum National Park, wet semideciduous forest, N05  210 , W01  230 , elev. 280 m, 23 Oct. 2003, G.J. Samuels & H.C. Evans (AFR28 ARSEF 7637). Indonesia: Java: Beng Breng, on leaves of Clerodendrum sp., M. Raciborskii 26, K(M)128056; Bogor Botanical Garden, on Aleyrodidae on Aglaria odoratissima, 22 Mar. 1986, M.C. Rombach (220386-1) (ARSEF 2154) Tjibodas, in foliis coriaceis subemortuis, 5 Feb. 1897, (Naturhistorische Museum Wein No. 16737) (holotype of A. javanica). Japan: Taom: On Quercus cuspidata, Aug. 1901, Tamura ((S) reg. Nr. F22458) (holotype of A. tamurai). Malaysia: Kuala Lumpur: on Aleyrodidae, 28 Mar. 1994, L.A. Lacey (94-41, ARSEF 4209). New Guinea: Kaiser Wilhelmsland, auf der Unterseite der Blatter von Ficus sp. ((S) reg. Nr F22442) (A. novogui neenesis). Philippines: Luzon: Manila, San Francisco church locality or vicinity, on Anonaceae, 14 Feb. 1924, Mrs Clemens (BPI 635847); on Ficus ulmifolia, Dec. 1911, P. W. Graff (BPI 635852); on Premna odorata, Dec. 1911, P. W. Graft, (K(M)128043); Isabella province, on

leaves of Ficus sp., Jan. 1924, M. Strong Clemens (4697, K(M)128049); Zambala province, Castillejos, on leaves, Mar. 1924, M. Strong Clemens (3275, K(M)128051); Laguna: Los Banos, college, on Ficus ulmifolia, 19 Dec. 1917, Torres (BPI 635849); On coccids on Litsea sp., Feb. 1932, G. O. Ocfemia, (K(M)128044) San Crispin, San Pablo, on coccids on Premna sp., Jan. 1932, M. S. Celino (K(M)128046) Cebu Island: Cebu, on leaves of Ficus sp., May 1924, M. Strong Clemens (6210, K(M)128048); Mindanao: Davao province, Mt. Apo, on leaves probably Strongylodon sp., Jan. 1924, M. Strong Clemens (5656, K(M)128050). Singapore: On leaves of Smilax barbata, T. Petch, (K(M)128052). Sri Lanka: Nuwara Eliya, on leaves, 26 June 1927, ex herb T. Petch (R238, K(M)128055). Thailand: sine loc., on scale insects and whitey, 28 Aug. 1956, E. F. Vestal, (K(M)128040) Northeastern Thailand (BCC1454); Western Thailand (BCC2163); Central Thailand (BCC2227); Eastern Thailand (BCC2175).

Notes: This species closely resembles A. aleyrodis/H. libera. Petch (1921) stated that the two could be distinguished by the slightly thinner stromata and shorter paraphyses of A. placenta. In our examination, the pulvinate stromata of A. placenta tended to be thinner and the tubercles (H. raciborskii) usually develop around the peripheral part of the anamorphic stromata, while in H. libera the teleomorph and anamorph are rarely formed in the same stroma. The conidiomatal paraphyses reveal differences in length between two species. In A. aleyrodis the paraphyses may reach 113 mm; in A. placenta they are not longer than 80 mm. Paraphyses are not ordinarily observed in culture for either species. In addition, H. raciborskii produces a proportion of ovoid part spores that are shorter and fatter than usual, but this is not observed in H. libera. In A. placenta, conidiophores arise from the ends of hyphal tufts, and are penicillate or verticillately branched, while in A. aleyrodis, in addition to the aforementioned form, more conidiogenous cells arise singly from hyphae. The type of H. warneckiana in B was probably destroyed during World War II (Burghard Hein, pers. comm.). The original description by Hennings (1905) agrees completely with H. raciborskii. The original description of A. novoguineenesis by Hennings (1898) is consistent with the characteristics of A. placenta, except that the stated width of the conidia (0.30.4 mm) is narrower than that of the A. placenta (1.52 mm). Our measurements of conidia from the type (S) were (10)1315 (17) 1.52(2.5) mm, which falls in the range for those of A. placenta. The stromata are small (1 mm diam), had abundant paraphyses measuring 40 70 1.5 mm, and conidiogenous cells 1217 1.52 mm. As these characters are consistent with A. placenta, we consider A. novoguineenesis a later synonym of A. placenta. The type of A. javanica (W 16737) is in poor condition with only a single stroma on the leaf. Petch (1921) observed that the morphology matched some forms of A. placenta, and we agree with Petchs conclusion that A. javanica is a synonym of A. placenta. H. javanica is not the teleomorph of A. javanica, but of A. coffeae, which has brownish stromata, unlike A. javanica. We examined the type of A. tamurai (S, F22458). The stroma shape resembles that of A. placenta. The colour of the conidial mass had faded to yellow, presumably through aging. Petchs (1921) description of its original colour as reddish orange is consistent with A. placenta. Other characters, such as the size of conidia (812 1.52 mm) and the length of paraphyses (50 mm) are also consistent with those of A. placenta. We therefore consider these names to be synonyms.

Aschersonia aleyrodis and its allies


Another species that resembles A. placenta is A. australiensis. A syntype specimen from B collected by Pritzel from North Queensland has the general shape of A. placenta with white pulvinate stromata and circularly arranged conidiomata. However, as Petch (1921) pointed out, the conidia are distinctively small, 58 1.5 mm, whereas in A. placenta they are typically over 9 mm long. A syntype collected by Diels from New Zealand has a different stroma shape with three tubercles; it is probably a different species. We retain A. australiensis as separate species.

8. Hypocrella rhombispora Miao Liu & K. T. Hodge, sp. nov.

Anamorph: Aschersonia sp.
Stromata 22.5 mm diametro, lutescentia vel pallide aurantiaca, pulvinata, subtuberculata ad basem constricta. Perithecia in stromate dense dispersa, inclusa. Ostiola non procurrentia, brunnescenti-ava. Asci cylindrici in apice pileati. Ascosporae liformes, in partisporas dimorphicas abrumpentes, nunc fusoideas in ambobus extremis acutas 1014 23 mm, nunc cylindricas in ambobus extremis obtusas, 712 1.52.5 mm, in toto gutulatae, in medio ubi guttulae accumulatae plerumque tumidas. Anamorpha in eodem stromate ac teleomorpha vel non. Stromata anamorphica alba, pulvinata vel conico-pulvinata vel scutata, in supercie minute pruinosa. Hypothallus saepe nullus. Conidioma annulatim disposita, sparsa vel in massam reticulatam, conidialem pallidam vel sublutescentem. Conidia rhombiformia, 9 14 2.53 mm. Coloniae in PDA moderate auctae, compactae, rmae, coriaceae, griseo-albae vel lutescenti-albae, in supercie minute tomentosae, massis conidialibus copiosis, viscosis, vivide luteis obsitae. Forma conidiorum in cultura varior: plurima conidiorum rhombiformia vel aliquae tumidissima. Nec paraphyses nec synanamorpha hirsutelloidea visae. Typus: Honduras: Departamento Yojo: Los Pinos, Parque Nacional Cerro Azul-Meambar, 850 m elevation, 3 Sept. 2004, P. Chaverri & P. A. Sheikh PC691(CUP 067548 holotypus).

more dense (as approaching textura epidermoidea or textura oblita); hyphal walls markedly thickened, 13 mm thick for hyphae 36 mm in diam (Fig 8HI). Conidiomata (Fig 8H) U-shaped or convolute in section, ostioles 50180 mm wide. Hymenium lining inner surface of conidioma. Conidiogenous cells arising singly from thick-walled hyphae, narrow cylindrical, not branched (Fig 8I), tapering near truncate apices, 812 1.52 mm. Conidia 914 2.53 mm, inated at the midpoint (2.53 mm wide) and tapering at both ends, forming two slender 24 mm ends (Fig 8KM). Paraphyses absent. Colonies (Fig 4J) on PDA 20 mm diam after four weeks at 23  C, thick pulvinate, moderately compact, rm and leathery, greyish white (1A11B1) to yellowish white (3A2), surface minutely tomentose, smooth to radially wrinkled, covered with deep yellow (4A8) conidial masses. Conidial masses abundant, formed as numerous small viscous to solid drops, some of which fuse together. In some cases the hyphae congregate to form tubercle-like tufts covered with numerous yellowish white (3A2) drops of liquid exudate. Hyphae 36 mm wide with thickened wall 0.51 mm thick. No conidiomata formed, conidial masses directly produced from surface of colony. Conidiogenous cells arising laterally from hyphae, single, unbranched; or in clusters. Conidiogenous cells strongly constricted at the basal septum, those arising laterally from hyphae are longer than terminal conidiogenous cells, 8 12(15) 22.5 mm (Fig 8J). Conidia markedly inated at the midpoint and tapering at both ends, 8.512 (17) 23 mm; in some cultures, extremely wide conidia produced, ovoid in the centre, contain a single very large guttule (46 36 mm), and have two elongated ends measuring 23 (5) mm (Fig 8KM). Neither paraphyses nor Hirsutella-like synanamorph observed. Distribution: Costa Rica, Honduras, Mexico, and Puerto Rico.
Specimen and culture examined. Costa Rica: Heredia: O. T. S. La Selva Biological Station, Camino Cantarrana, on Cyclanthus bipartitus, 19 June 2002, M. Liu CR07; CUP 67296 ARSEF 7511); 5 Jan. 2004, P. Chaverri, (PC466 CUP 067537, PC467 CUP 067538); beside entrance to Plantation RCC, 20 June 2002, M. Liu ML44-3, (culture CR32 CUP 067346); Puntarenas, Las Cruces Biological Reserve, Wilson Botanical Garden, large loop of jungle trail, on Guarea rhopalocaipa, 4 July 2002, M. Liu, ML64 (culture CR34 CUP 67369). Honduras: Yojoa: Los Pinos, Parque Nacional Cerro Azul-Meambar, 850 m elev., 3 Sep. 2004, P. Chaverri & P. A. Sheikh, (PC691 CUP 067548, PC696 CUP 067550, PC698 CUP 067551); Copan: Santa Rita, Reserva Pena Quemada, 9 Sep. 2004, P. Chaverri & P. A. Sheikh. Mexico: Veracruz: Amayaga, Catemaco, 500 m elev., 14 Dec. 2003, P, Chaverri & J. Garca-Alvarado, (PC460 CUP 067534). USA: Puerto Rico: Between Mayaguez and Maricao, beside road 105 15 Dec. 2003, M. Liu & Z.D. Wang, ML164 (CUP-PR 4406; Ex-type culture ML164 ARSEF 7390; Guajataca Forest, trail no. 9, on fern, 18 Dec. 2003, M. Liu & Z.D. Wang ML201-1,ML201-3, ML201-5a (CUPPR 4437 ARSEF 7395, ARSEF 7399, ARSEF 7400).

(Figs 4JL, 8) Stromata pale yellow (4A3) to pale orange (5A3), pulvinate and slightly tuberculate, 22.5 mm diam, 0.81.4 mm thick, slightly constricted at base, sometimes surrounded by hypothallus. Stromatal tissue dense textura intricata (Fig 8C). If present, hypothallus narrow, 0.6 mm wide, and minutely tomentose. Perithecia densely arranged in the stroma, embedded, ostioles not projecting, brownish yellow (5B7); in section 300450 mm high, 210300 mm at widest point. Asci subcylindrical, widest at midpoint, slightly narrower at both ends, 148296 mm long, 614 mm at widest point, 58 mm at apices. Ascospores initially liform, slightly shorter than perithecia, 11.5 mm diam, dividing into part spores (Fig 8D). Part spores fusoid, acute at both ends, 1014 23 mm, or others cylindrical with blunt ends, 712 1.52.5 mm; guttulate, usually swollen at midpoint where guttules accumulate (Fig 8E). Paraphyses absent. Anamorphic stromata (Figs 4L, 8FG) white, thin pulvinate, Hemi-globose or scutate with a hemispheric central region abruptly attenuating and towards the edge (Fig 4L); 13 mm diam, 0.20.7 mm thick; surface minutely pruinose. Hypothallus, if present, 0.20.8 mm in width. Conidiomata 4 to numerous, arranged concentrically, scattered, or forming a reticulum on conical part of stroma. Conidial mass pale to light yellow (3A4 4A4), in conidiomata or accumulating as discrete drops over the ostioles, not conuent. Stromatal tissue textura intricata, near conidiomata (about 75 mm below the hymenia), becoming

Notes: The most distinctive characters of this species are the shape of the part spores and conidia, both of which are distinctly inated in the middle. These characters are shared by a closely-related complex of species referred to H. phyllogena/A. basicystis. Hypocrella phyllogena has an orange stroma of a globose head and a narrower neck, upon which the asexual (A. basicystis) fruiting structure usually appears. In H. rhombispora, the asexual and sexual states often occur on different stromata. The teleomorphic stroma is white and minutely tuberculate. The conidiomata are scattered over the anamorphic stromatic surface.


M. Liu et al.

Fig 8 Hypocrella rhombispora. (A) Minutely tuberculate teleomorphic stroma with slightly narrowed base (CUP 067548). (B) Perithecium in section, with the ostiole slightly erumpent. (C) Compact tissues around perithecium. (D) Asci containing ascospores that disarticulate to form part ascospores. (E) Ventricose part ascospores with rounded or acute ends. (FG) Anamorphic stromata (CUP-PR 4437): the conidiomata are scattered and produce discrete conidial masses. (H) Section of conidioma showing the hymenium and conidia; paraphyses are lacking, and the tissues surrounding the conidioma are compact. (I) Hymenium, showing a palisade of conidiogenous cells and compact surrounding tissues. (J) Conidiogenous cells in culture are ask-shaped and slightly narrower at the base. (KM) Ventricose conidia showing variation in width among several specimens. Bar [ 500 mm for Figs A, FG; 100 mm for Fig B; 10 mm for Figs CE, HM.

9. Hypocrella turbinata Petch, Ann. Roy. Bot. Gard. Peradeniya 5: 535 (1914).
Anamorph: Aschersonia turbinata Berk., Ann. Mag. Nat. Hist., ser.2 9: 192 (1852). (Fig 3D) Notes: Young H. turbinata specimens produce small cylindrical stromata similar to those of A. cubensis. The latter two species can easily be distinguished from specimens of A. aleyrodis that have scutate stromata (Fig 6E) by the shape of the conidia. In A. turbinata and A. cubensis, the conidia are wider (Fig 3D); in A. aleyrodis they are narrowly fusoid (Fig 3A).

10. Hypocrella zhongdongii Miao Liu & K. T. Hodge, Mycol. Res. 108: 820 (2005).
(Fig 3B) Anamorph: Aschersonia incrassata Mains, J. Insect Pathol. 1: 46 (1959). Note: This species most closely resembles H. andropogonis/ A. andropogonis; both A. incrassata and A. andropogonis are common yellow-spored species in the neotropics. A. incrassata can be distinguished by the distinctive thickened walls at the ends of conidia. Further comparison of these two species and the evolutionary relationships of H. zhongdongii with other species in the genus were discussed by Liu & Hodge (2005).

Aschersonia aleyrodis and its allies


Based on morphological and phylogenetic analysis, six species with white pulvinate stromata are recognized, A. aleyrodis (teleomorph: H. libera), A. andropogonis (H. andropogonis), A. placenta (H. raciborskii), A. incrassata (H. zhongdongii), A. basicystis (H. phyllogena), and one new species, described here as H. rhombispora (with its anamorph). The stromata of H. phyllogena are orange and relatively dense when mature, but white and effuse during early stages of development and asexual sporulation. Most phylogenetic species reect Petchs (1921, 1925) concepts , and appear as strongly supported clades. However, the relationship between H. libera/A. aleyrodis and H. raciborskii/A. placenta is not resolved. Further studies using faster evolving genes and more intensive analyses have resolved their sister relationship (Liu et al., pers. obs.), supporting Petchs hypothesis that these two similar fungi from the New World and the Old World represent different species. Accordingly, we treat them here as distinct species. An examination of the phylogenetic distribution of morphological characters within the group identied many apomorphies. Descriptive morphological characters (autapomorphies) that characterize species include the shape of teleomorphic stromata, shape and arrangement of tubercles containing perithecia, shape of part ascospores, shape of anamorphic stromata, presence/absence of a rim around the orices of conidiomata, arrangement of conidial masses on the stromata, colouration of conidial masses, shape of conidia and presence/absence of paraphyses. Except for the rim around the conidiomatal orices, all other characters were used by Petch (1921) in differentiating species. It is worth noting that the presence or absence of conidiomatal paraphyses may be diagnostic, but it has been questioned as a criterion to separate Aschersonia into two subgenera (Liu & Hodge 2005). The shape of the conidioma (called a pycnidium by Petch 1921) and its orice were among the characters used by Petch to differentiate species. Although the presence/absence of a rim around the orice is useful for differentiating certain species, such as A. andropogonis and A. incrassata, the shapes of the conidiomata and orices are not constant within each of these six species, and we consider them to be of questionable use. Their morphology appears to vary with the degree of maturity of the conidioma. Petch (1921) observed two types of conidiomatal formation in Aschersonia. In the rst type the conidiophores developed on a small actively growing area of the surface of stroma, which gradually became invaginated as it expanded. In the second type, a conidioma was formed as a locule within the stromata and later broke through to the surface. Aschersonia species with white pulvinate stromata conform to Petchs rst type of development. In this group, the stromata appear to develop from a thin layer into a thicker pulvinate one. The thinner the stroma, the wider the conidiomatal orice. On a thin stroma, conidiomata appear as widely open shallow depressions of the surface (Figs 4F, I); as the stroma becomes thicker, the growth of the surrounding stromatal tissue constricts the opening and forms conidiomata that are V-shaped, U-shaped, globose, tubular, or irregularly concave (Figs 5G, 6G, 7H). Several of these shapes might be found in a single species i.e. A. aleyrodis,

A. andropogonis and A. incrassata, etc, depending on stromatal thickness and degree of development. Thus we cannot consider conidiomatal shape useful for differentiating species in this group. Conversely, for some species outside our focal group (e.g. A. turbinata), the situation is different. The stromata do not develop from a thin layer, instead they begin as small masses and develop into cylindrical to complex structures. A vertical section of a small cylindrical A. turbinata stroma reveals that the irregularly folded hymenium develops inside the stroma under a small orice. Several characters that predict phylogenetic relationships (synapomorphies) were found. These include the white, effuse anamorphic stroma that is typical of A. aleyrodis and allies (Fig 1, character A), Other synapomorphies include the arrangement of conidial masses on the stromata or colony surfaces. This character separates all white stroma species into two clades, i.e. a clade with conuent masses (A. aleyrodis and A. placenta; Fig 1, character B) and a clade in which conidial masses are discrete (A. incrassata, A. andropogonis, Aschersonia anamorph of H. rhombispora, and A. basicystis; Fig 1, character D.) The former clade is also united by the character of cylindrical tubercles containing perithecia (Fig1, character C). Ventricose conidial shape (Fig 1 character F) denes a clade composed of A. basicystis and H. rhombispora; the formation of a rim around the conidiomatal orices denes a clade of A. andropogonis and A. incrassata. The colour of the conidial mass is not phylogenetically informative: taxa producing yellow conidial masses, for example, do not form a monophyletic group (Fig 1). The insect hosts of this genus are restricted to the families Aleyrodidae and Coccidae in the order Hemiptera (Petch 1921, Mains 1959, Evans 1990, Meekes et al. 2002). One Aschersonia species may attack a wide range of species within an insect genus; conversely, one insect species in Aleyrodes or Lecanium can be attacked by many Aschersonia species (Petch 1921). Petch (1921) separated Aschersonia into two subgenera based on host (whitey versus scale insect) and the presence/absence of hymenial paraphyses. These subgenera of Aschersonia were considered to correspond with the two subgenera of Hypocrella. Later authors have questioned Petchs subgeneric concepts (Dingley 1954, Liu & Hodge 2005). Because of the difculty in identifying the insect hosts, the ecological host range of Aschersonia species has not been sufciently studied. Whether or not there is host specialization or coevolution in Hypocrella/Aschersonia is a question waiting for an answer.

We thank the staff at B, BPI, CUP, FH, HMAS, K, KRA MICH, PAD, and S for kindly loaning specimens; ARSEF, BCC, and CBS for providing fungal isolates; the US Forest Service for permission to collect fungi in the Caribbean National Forest (El Yunque), and the National Biodiversity Institute (INBio) for facilitating collecting by ML in Costa Rica. Our thanks also go to all of the people who have provided specimens or cultures: Mary C. Aime, Joseph F. Bischoff, Harry C. Evans, Gary J. Samuels, and Christopher L. Schardl; and also people who have facilitated our collecting in different places: B. Jean Lodge, Sharon Cantrell, Sandra Maldonado, Miriam Salgado in Puerto Rico; Juventino Garca Alvarado in Mexico, Phil Arneson in


M. Liu et al.

Honduras, Emilia Garca y Diego De la Quintana in Bolivia, Luis Diego Gomez in Las Cruces, CR; Clayton McCoy, Philip Stansly, Lee Mitchell, Stephan Brown, Ronald Cave, Lavern W. Timmer, and James Kimbrough in Florida; Zengzhi Li and Bo Huang in China. The plates were prepared by Kent Loefer. Comments from two anonymous reviewers improved the manuscript. This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant no. 2002-35316-12263, and by the National Science Foundation under Grant no. 0212719 to K.T.H.


Bath AK, Torres MS, Samuels GJ, White jr JF, 2005. Morphology and species identication of a Hypocrella species (GenBank accession # U47832). Mycotaxon 92: 97102. Berger EW, 1921. Natural enemies of scale insects and whiteies in Florida. Florida State Plant Breading Quartely Bullatin 5: 141154. Borner C, 1956. Aleyrodidae (Aleurodina). In: Sorauer CP (ed), Handbuch der Panzenkrankheiten, Tierische Scha dlinge an Nutzpantzen, Teil 2, Lieferung 4. Heteroptera und Homoptera. Paul Parey, Berlin, pp. 331359. Dingley JM, 1954. The Hypocreales of New Zealand. VI. The genera Hypocrella, Barya, Claviceps and Podonectria. Transactions of the Royal Society of New Zealand 81: 489499. Evans HC, Hywel-Jones N, 1990. Aspects of the genera Hypocrella and Aschersonia as pathogens of coccids and whiteies. Vth International Colloquium on Invertebrate Pathology and Microbial Control. Society for Invertebrate Pathology, Adelaide, 111115. Fawcett HS, 1908. Fungi parasitic upon Aleyrodes citri. University of Florida Special Studies 1: 141. Fawcett HS, 1936. Citrus diseases and their control. McGraw-Hill, New York. Fransen JJ, 1987. Aschersonia aleyrodis as a microbial control agent of greenhouse whitey. Ponsen en Looijen, Wageningen. Fransen JJ, van-Lenteren JC, 1993. Host selection and survival of the parasitoid Encarsia formosa on greenhouse whitey, Trialeurodes vaporariorum, in the presence of hosts infected with the fungus Aschersonia aleyrodis. Entomologia Experimentalis et Applicata 69: 239249. Hennings P, 1902. Fungi javanici novi. Hedwigia 41: 140149. Hennings P, 1903. Fungi australienses. Hedwigia 42: 7388. Korf RP, 1972. Synoptic key to the genera of the Pezizales. Mycologia 64: 937994. Kornerup A, Wanscher JH, 1967. Methuen Handbook of Colour. Methuen, London. Liu M, Hodge K, 2005. Hypocrella zhongdongii sp. nov., the teleomorph of Aschersonia incrassata. Mycological Research 109: 818 824. Liu M, Rombach MC, Humber RA, Hodge KT, 2005. Whats in a name? Aschersonia insperata: a new pleoanamorphic fungus with characteristics of Aschersonia and Hirsutella. Mycologia 97: 249256.

Liu PG, Wang XH, Li ZQ, Yang ZL, Zhang DC, 2001. Notes on the bambusicolous species of the genus Hypocrella from southwestern China. Mycotaxon 78: 6774. Liu YJ, Whelen S, Hall BD, 1999. Phylogenetic relationships among Ascomycetes: Evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16: 17991808. Mains EB, 1959a. North American species of Aschersonia parasitic on Aleyrodidae. Journal of Insect Pathology 1: 4347. Mains EB, 1959b. Species of Aschersonia (Sphaeropsidales). Lloydia 22: 215221. Mason-Gamer RJ, Kellogg EA, 1996. Testing for phylogenetic conict among molecular data sets in the tribe Triticeae (Gramineae). Systematic Biology 45: 524545. McCoy CW, Samson RA, Boucias DG, 1988. Entomogenous fungi. In: Ignoffo CM, Mandava NB (eds), CRC Handbook of Natural Pesticides. CRC Press, Boca Raton, pp. 151236. Meekes ETM, Fransen JJ, van-Lenteren JC, 2002. Pathogenicity of Aschersonia spp. against whiteies Bemisia argentifolii and Trialeurodes vaporariorum. Journal of Invertebrate Pathology 81: 111. Meekes ETM, van-Voorst S, Joosten NN, Fransen JJ, vanLenteren JC, 2000. Persistence of the fungal whitey pathogen, Aschersonia aleyrodis, on three different plant species. Mycological Research 104: 12341240. Petch T, 1921. Studies in entomogenous fungi. II. The genera Hypocrella and Aschersonia. Annals of the Royal Botanic Gardens Peradeniya 7: 167278. Petch T, 1925. Additions to Ceylon fungi. III. Annals of the Royal Botanic Gardens Peradeniya 9: 313328. Ramakers PMJ, Samson RA, 1984. Aschersonia aleyrodis, a fungal pathogen of whitey. II. Application as a biological insecticide in glasshouses. Zeitschrift fur angewandte Entomologie 97: 18. Shu Z, 1996. A study of the biology of Aschersonia aleyrodis Webber. Journal of Southwest Agricultural University 18: 524526. Simmons MP, Ochoterena H, Carr T-G, 2001. Incorporation, relative homoplasy, and effect of gap characters in sequencebased phylogenetic analyses. Systematic Biology 50: 454462. Sutton BC, 1980. The Coelomycetes: Fungi Imperfecti with Pycnidia, Acervuli and Stromata. Commonwealth Mycological Institute, Kew. Swofford DL, 1998. PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, MA. Sydow H, Sydow P, 1916. Fungi amzonici a cl. E. Ule lecti. Annals Mycologici 14: 6597. Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specic gap penalties and weight matrix choice. Nucleic Acids Research 22: 46734680. Tzean SS, Hsieh LS, Wu WJ, 1997. Atlas of Entomopathogenic Fungi. Council of Agriculture. Executive Yuan, Taiwan. Vilgalys R, Hester M, 1990. Rapid genetic identication and mapping of enzymatically amplied ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238 4246. Webber HJ, 1897. Sooty mold of the orange and its treatment. Bulletin of the U.S. Department of Agriculture Division of Vegetable Physiology and Pathology 13: 134.