A Taxonomic Revision of the Insect Biocontrol Fungus Aschersonia Aleyrodis, Its Allies with White Stromata and Their Hypocrella Sexual States

mycological research 110 (2006) 537– 554

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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 abstract

Article history: A revision of a monophyletic group of Hypocrella species and their Aschersonia anamorphs Received 4 August 2005 with white effuse stromata is presented. In addition to taxon descriptions, distributions, Received in revised form and nomenclature, a synoptic key and a molecular phylogenetic analysis are also provided. 12 January 2006 A new holomorph, Hypocrella rhombispora sp. nov., is described. Accepted 24 January 2006 This study presents a revisionary treatment of Aschersonia aleyrodis (teleomorph: Hypocrella Corresponding Editor: libera) and its allies. These fungi parasitize whiteflies and are promising candidates for Richard A. Humber whitefly biological control. Four species of Aschersonia and their Hypocrella teleomorphs are treated in detail: Aschersonia aleyrodis/Hypocrella libera; A. andropogonis/H. andropogonis, Keywords: A. placenta/H. raciborskii, and A.sp./H. rhombispora sp. nov. A synoptic key including these Ascomycota and six other morphologically similar species is presented to facilitate identification in Clavicipitaceae the field and laboratory. Phylogenetic analyses of partial DNA sequences from three genes Molecular systematics (LSU, mtSSU, and RPB2) suggest that Aschersonia species with effuse white stromata form Morphology a monophyletic group of whitefly pathogens. Phylogenetically informative characters in Phylogeny 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. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction diminished. Aschersonia species can still be found in whitefly populations in abandoned groves and backyard trees that Aschersonia aleyrodis (teleomorph: Hypocrella libera, Clavicipita- are not managed with pesticides, where we have collected ceae, Hypocreales) was among the first fungi used in the biocon- some specimens. Interest in using these fungi for the control trol of insect pests in North America. Its successful use in of pests resumed in 1960s. In Bulgaria, China, Japan and the Florida citrus groves dates from the early 1900s, when citrus USSR (Evans & Hywel-Jones 1990), A. aleyrodis has been used branches with A. aleyrodis were introduced into citrus groves against the greenhouse whitefly. The fungus has been devel- to seed epizootics in the whitefly population (Berger 1921; oped commercially by Koppert Biological Systems in Holland Fawcett 1936). With the increase in chemical pesticide use be- as a biopesticide suitable for application in greenhouses ginning in the 1940s and 1950s, the demand for biocontrols (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: [email protected]. 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.01.013 538 M. Liu et al.

seen in Azerbaijan and Jamaica to control guava whitefly and citrus whitefly, respectively (Bo¨rner 1956; McCoy, Samson & Materials and methods Boucias 1988). Recent research has revealed that A. aleyrodis has the virtues of high tolerance in low relative humidity Specimens and cultures (Fransen 1987), long persistence on leaf surfaces (Meekes et al. 2000), and compatibility with insect parasitoids (Fransen Species with white stromata include Aschersonia aleyrodis & van-Lenteren 1993) in the control of whitefly pests. (Hypocrella libera), A. placenta (H. raciborskii), A. andropogonis However, some hurdles remain: Aschersonia species are (H. andropogonis), the Aschersonia anamorph of H. rhombispora, slow-growing in culture, and not all host life stages may be and A. incrassata (H. zhongdongii)(Liu & Hodge 2005). Reference attacked (Ramakers & Samson 1984). Recent studies on spore species outside this apparently monophyletic group include production, germination, and pathogenicity have provided A. basicystis (H. phyllogena), A. cubensis, A. insperata, A. turbinata a better understanding of the biology of A. aleyrodis in order and A. viridans. Fungal material was obtained in diverse forms: to further develop this promising biocontrol agent (Meekes fresh specimens from the field, isolates on artificial media, and et al. 2002). dried herbarium specimens. Fresh specimens were collected Despite early successes with A. aleyrodis, few other Ascher- from Bolivia, Cameron, China, Costa Rica, Florida (USA), sonia species have been investigated for biological control po- Ghana, Honduras, Mexico, Panama, and Puerto Rico (Table 1). tential. Factors confronting researchers interested in For most field collections, specimens were deposited in CUP exploiting other species of the genus include: few species of (Plant Pathology Herbarium, Cornell University, Ithaca). A por- Aschersonia were available from culture collections before tion of each specimen from Costa Rica was deposited in INB our work, and had often been only tentatively identified; and (Mycological Herbarium, National Biodiversity Institute, Santo identification of newly collected cultures and specimens has Domingo, Heredia, Costa Rica), and an isotype of H. rhombispora been difficult due to the scattered and out-dated literature was deposited in UPR (Herbarium, Botanic Garden, University on the group (Evans & Hywel-Jones 1990). The primary re- of Puerto Rico). Fungal cultures were made on potato-dextrose source available for identification has been Petch’s (1921) revi- agar (PDA; Difco, Albany, NY, USA) medium following the pro- sion that needs to be updated in terms of number of species cedure described in Liu and Hodge (2005) and deposited in and terminology. ARSEF (ARS Collections of Entomopathogenic Fungi, Ithaca, Aschersonia species are found predominately in tropical New York). Additional cultures were obtained from ARSEF, and subtropical habitats and infect either whiteflies or scale BCC (BIOTEC Culture Collection, Bangkok) and CBS (Centraal- insects, presumably by the germination and direct penetra- bureau voor Schimmelcultures, Utrecht). Dried herbarium tion of conidia adhering to the host cuticle (Meekes et al. specimens including types were borrowed from B (Herbarium, 2002). We focus on a group of whitefly-pathogenic species Botanischer Garten und Botanisches Museum Berlin-Dahlem), characterized by their effuse white stromata and brightly col- BPI (US National Fungus Collections, Beltsville, MD), CUP, FH oured conidial masses. Because of their gross similarity and (Farlow Reference Library and Herbarium of Cryptogamic Bot- inadequate characterization in the literature, they are chal- any, University of Havard), HMAS (Mycological Herbarium In- lenging to identify correctly, and we suspect that some re- stitute of Microbiology, Beijing), K (Royal Botanic Gardens, cords of A. aleyrodis represent misidentifications (Liu et al. Kew), KRA (Herbarium, Institute of Botany, Krako´ w), MICH 2001; Shu 1996; Sutton 1980; Tzean et al. 1997). For example, (Herbarium, University of Michigan, Ann Arbor), PAD (Erbario the orange-spored species A. placenta (teleomorph H. racibor- Patavinum, Centro Musei Scientifici, Padova), and S (Herbar- skii) differs from A. aleyrodis mainly in the former’s paleotrop- ium, Botany Departments, Stockholm.) ical distribution. We demonstrate here that A. goldiana is merely a yellow-spored form of A. aleyrodis, as suggested by Morphological examination Mains (1959a,b). Literature records of A. goldiana include misidentifications of other yellow-spored species such as Microscopic characters were examined using optical micro- A. andropogonis, A. incrassata,orAschersonia anamorph of Hypo- scopes. For microscopic study, specimens were mounted in crella rhombispora. 85 % lactic acid–cotton blue. Digital photomicrographs were The ten species included in this study are all congeneric taken with a SPOT RT camera (Diagnostic Instruments, Ster- with the type species Hypocrella discoidea (teleomorph) or ling Heights, MI). Colours of the specimen and cultures were Aschersonia taitensis (anamorph). We revise and refine spe- recorded according to Kornerup and Wanscher (1967). Charac- cies concepts for this group of Hypocrella/Aschersonia species teristics of in vitro growth were recorded from cultures on PDA based on morphological examination of types and newly at 23 C. collected specimens and cultures. We apply phylogenetic analysis of DNA sequence data to evaluate whether mor- DNA extraction, PCR and sequencing phological species are phylogenetically discrete, and use the analysis to discuss insights about relationships and DNA was extracted from the cultures following the procedure character evolution in the group. A synoptic key is pre- of Liu et al. (2005). Regions of three genes were amplified by sented to facilitate the field and laboratory identification of PCR: nuclear LSU rDNA by primers LR0R and LR5 (Vilgalys & species. We hope that the information presented here and Hester 1990), mtSSU rDNA by MTS1f and MTS3r (Liu & Hodge the isolates collected during our investigation will spur fur- 2005), RNA polymerase unit II (RPB2) by RPB2-7f and RPB2- ther investigation into the biocontrol potential of these 11ar (Liu et al. 1999). Amplification conditions and sequencing fungi. approaches were described by Liu and Hodge (2005). Aschersonia aleyrodis and its allies 539

Table 1 – Fungal isolates, origins and GenBank accession numbers Species specimen Isolate Origin GenBank accession no.

LSU mtSSU RPB2

- ARSEF992 Japan AY518368 DQ070136 DQ070203 Hypocrella raciborskii - ARSEF4209 MalDQ070sia DQ070074 DQ070141 DQ069953 Aschersonia placenta - ARSEF2154 Indonesia DQ070070 - DQ069951 - CBS34984 Japan DQ070135 DQ070200 DQ069958 - AFR28 Ghana DQ070113 DQ070180 DQ069965 CUP67562 AFR114 Cameron DQ070119 DQ070185 DQ069964 CUP67280 ARSEF7508 Costa Rica AY932752 AY932781 AY932766 CUP67303 ARSEF7512 Costa Rica AY932761 AY932791 AY932776 CUP67310 CR11 Costa Rica DQ070080 DQ070146 DQ070209 CUP67341 CR19 Costa Rica DQ070085 DQ070151 DQ069943 CUP67342 CR20 Costa Rica DQ070086 DQ070152 DQ069941 CUP-PR-4421 ML175-1 Puerto Rico DQ0700103 DQ07070 DQ069944 CUP-PR-4421 ARSEF7393 Puerto Rico DQ0700104 DQ070171 DQ069945 CUP-PR-4421 ARSEF7394 Puerto Rico DQ0700105 DQ070172 DQ069946 CUP-PR-4421 ML175-4 Puerto Rico DQ0700106 DQ070173 DQ069947 CUP67565 ARSEF7642 Guyana - DQ070201 DQ069950 H. libera CUP67435 ARSEF7339 Florida - DQ070162 DQ069934 A. aleyrodis CUP67435 ARSEF7340 Florida DQ070096 DQ070163 DQ06935 CUP67435 ARSEF7343 Florida DQ070099 DQ070165 DQ069936 - ARSEF7641 Panama - DQ070202 DQ069940 - ARSEF7617 Costa Rica DQ070121 DQ070187 DQ069942 CUP67519 PC413-1 Honduras DQ070122 DQ070189 DQ069937 CUP67525 PC434 Mexico DQ070126 DQ070192 DQ069938 CUP67528 PC439 Mexico DQ070128 DQ070194 DQ069939 CUP67529 PC442 Mexico DQ070129 DQ070195 DQ070232 CUP67288 CR05 Costa Rica AY518370 AY932784 AY932769 CUP67313 CR12 Costa Rica AY932755 AY932785 AY932770 CUP67316 CR13 Costa Rica AY932756 AY932786 AY932771 H. zhongdongii CUP67322 CR15 Costa Rica AY932757 -- A. incrassata CUP67350 ARSEF7513 Costa Rica AY932758 AY932787 AY932772 CUP-PR-4394 ML149 Puerto Rico AY932759 AY932788 AY932773 CUP-PR-4394 ARSEF7369 Puerto Rico AY932760 AY932789 AY932774 CUP-PR-4314 ARSEF7071 Puerto Rico DQ070133 DQ070199 - - ARSEF3014 Mexico DQ070073 DQ070140 - - ARSEF4424 Brazil DQ070075 DQ070142 DQ070204 - CR03 Costa Rica DQ070076 DQ070143 DQ070205 CUP67291 ARSEF7510 Costa Rica AY932754 AY932783 AY932768 CUP67307 CR09 Costa Rica DQ07078 DQ070144 DQ070207 CUP67309 CR10 Costa Rica DQ070079 DQ070145 DQ070208 CUP67343 CR21 Costa Rica DQ070087 DQ070153 DQ070213 CUP67345 CR22 Costa Rica DQ070088 DQ070154 DQ069972 H. andropogonis CUP67436 ARSEF7341 Florida DQ070097 -- A. andropogonis CUP67436 ARSEF7342 Florida DQ070098 DQ070164 DQ070218 CUP-PR-4407 ARSEF7391 Puerto Rico DQ070102 DQ070169 DQ070221 CUP-PR-4438 ARSEF7396 Puerto Rico DQ070109 DQ070176 DQ070223 CUP-PR-4438 ARSEF7397 Puerto Rico DQ070108 DQ070175 DQ069973 CUP-PR-4438 ARSEF7398 Puerto Rico DQ070110 DQ070177 - CUP67515 PC384 Costa Rica - DQ070188 DQ070227 CUP67521 PC418 Honduras DQ070123 - DQ070228 CUP67523 PC431 Mexico DQ070124 DQ070190 DQ070229 CUP67524 PC432 Mexico DQ070125 DQ070191 DQ070230 CUP67526 PC436_1 Mexico DQ070127 DQ070193 DQ070231 CUP67531 PC451 Mexico DQ070130 DQ070196 DQ069974 CUP67532 PC452 Mexico DQ070131 DQ070197 DQ070233 CUP67533 PC455 Mexico DQ070132 DQ070198 DQ070234 CUP67556 AFR34A Ghana DQ070114 DQ070181 DQ069968 CUP67560 AFR68 Ghana DQ070115 - DQ069969 - AFR75 Cameroon DQ070116 DQ070182 DQ070226 CUP67561 AFR80 Cameroon DQ070117 DQ070183 - CUP67563 ARSEF7639 Cameroon DQ070120 DQ070186 DQ069970 CUP67283 CR04 Costa Rica DQ070077 - DQ070206 H. phyllogena CUP67340 CR17 Costa Rica DQ070083 DQ070149 DQ070212 (continued on next page) 540 M. Liu et al.

Table 1 – (continued) Species specimen Isolate Origin GenBank accession no.

LSU mtSSU RPB2

A. basicystis CUP67355 ARSEF7515 Costa Rica AY518373 AY932793 AY932778 CUP67296 ARSEF7511 Costa Rica AY518371 AY932790 AY932775 CUP CR32 Costa Rica DQ070094 DQ070160 DQ070217 H. rhombispora CUP67369 CR34 Costa Rica DQ070095 DQ070161 DQ069977 and Aschersonia anamorph CUP-PR-4406 ARSEF7390 Puerto Rico - DQ070168 DQ070220 ARSEF7395 Puerto Rico DQ070107 DQ070174 DQ070222 ARSEF7399 Puerto Rico DQ070111 DQ070178 DQ070224 ARSEF7400 Puerto Rico DQ070112 DQ070179 DQ070225 ARSEF1030 Columbia DQ070071 DQ070137 - CUP67321 CR14 Costa Rica DQ070081 DQ070147 DQ070210 CUP67339 CR18 Costa Rica DQ070084 DQ070150 - CUP67331 CR16 Costa Rica DQ070082 DQ070148 DQ070211 A. turbinata CUP67349 CR24 Costa Rica DQ070090 DQ070156 DQ070214 CUP67365 CR26 Costa Rica DQ070091 DQ070157 DQ070215 CUP67375 ARSEF7514 Costa Rica AY932762 AY932792 AY932777 CUP67375 CR29 Costa Rica DQ070093 DQ070159 DQ070216 CUP-PR-4388 ML142 Puerto Rico DQ070101 DQ070167 DQ070219 PR11 Puerto Rico DQ070134 - DQ070235 A. cubensis CUP67366 CR27 Costa Rica DQ070092 DQ070158 - A. insperata ARSEF2396 Philippines AY518374 DQ070138 DQ069976 A. viridans CUP67282 ARSEF7509 Costa Rica AY932753 AY932782 AY932767 CUP67347 CR23 Costa Rica DQ070089 DQ070155 -

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

Phylogenetic analysis AB027357, RPB2 AY932763). The distribution of key morphological characters was mapped onto the final tree topology DNA sequences of selected species (Table 1) were subjected to to investigate character evolution and to identify which char- phylogenetic analysis based on three gene regions LSU, mtSSU acters were phylogenetically informative. 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 Results the three data sets was evaluated by comparison of general tree topologies and bootstrap supports for individual clades Aligned sequences from the LSU and RPB2 genes included 889 (Mason-Gamer & Kellogg 1996). Based on the general congru- and 754 characters, respectively. For the mtSSU gene, a frag- ence, the three gene regions (LSU, mtSSU and RPB2) were ment of 60 or so bases (nt 190–255) evolved extremely fast eventually combined in a single matrix; unavailable sequences and was excluded because it could not be unambiguously for individual taxa were scored as missing data. Excluding the aligned; other parts were readily aligned, resulting in 657 char- characters that could not be aligned without ambiguity acters. Phylogenetic analyses of the three gene regions resulted in 2300 characters. To diminish the possible homo- revealed no conflict in delimiting species. For relationships plasy caused by rapid evolution at the third codon position, among the clades (species), mtSSU gives no resolution, and we down-weighted the third codon positions of the RPB2 LSU and RPB2 conflict regarding the position of A. viridans. data set as 1, while other characters (first and second codon Conflicts among the three genes were mainly found within and non-coding regions) as 2. Parsimony analyses were con- each clade (data not shown). Because there are no severe con- ducted using a heuristic search with TBR (tree bisection and flicts, the three data sets were combined into a single matrix reconnection) branch-swapping and 100 replicates of random and subjected to phylogenetic analysis by a total evidence ap- sequence addition. Most gaps in LSU and mtSSU corresponded proach. Phylogenetic analysis revealed 359,900 equally parsi- with homopolymeric repeats. We consider them to be arte- monious trees of length ¼ 2534; CI ¼ 0.573, RI ¼ 0.896 and 456 facts of sequencing, thus treat them as missing data (Sim- phylogenetically informative characters (141, 72, 243 from mons et al. 2001). No gaps were observed in the RPB2 region. LSU, mtSSU and RPB2 respectively). The trees differed from Bootstrap (BS) analysis was based on 500 replicates of a full each other mainly in the relationships within the major heuristic search, each with 10 replicates of a random addition clades. A strict consensus tree is shown in Fig 1. Seven major sequence. The trees were rooted with the outgroup Hypomyces clades were well-supported in the BS analysis, each of which aurantius (LSU AY932750, mtSSU AY932779, RPB2 AY932764), was concordant with a morphologically defined species. The Hypocrea strictipilosa (LSU AY932751, mtSSU AY932780, RPB2 relationships among these clades were clear in the strict con- AY932765), and Cordyceps militaris (LSU AB027379, mtSSU sensus but lack strong BS support. Morphological characters Aschersonia aleyrodis and its allies 541

Cordyceps militaris Hypomyces aurantius Hypocrea stritipilosa ARSEF 992 CBS 34984 ARSEF 4209 A. placenta AFR28 (H. raciborskii) 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 ARSEF 7617 A. aleyrodis 99 ARSEF 7311 (H. libera) ARSEF 7339 ARSEF 7340 B C 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 ARSEF 7396 A. andropogonis ARSEF 7397 (H. andropogonis) A 97 ARSEF 7398 CUP 67556 CUP 67560 AFR75 CUP 67561 ARSEF 7639 CUP 67515 CUP 67521 CUP 67523 CUP 67524 CUP 67526 E CUP 67531 CUP 67532 CUP 67533 CUP 67288 CUP 67313 CUP 67316 96 CUP 67322 A. incrassata ARSEF 7513 (H. zhongdongii) D CUP-PR-4394 100 CUP-PR-7369 ARSEF 7071 CUP 67283 100 CUP 67340 A. basicystis ARSEF 7515 (H. phyllogena) ARSEF 7511 CR32 F 100 CUP 67369 ARSEF 7390 H. rhombispora sp. nov. ARSEF 7395 ARSEF 7399 ARSEF 7400 ARSEF 1030 ARSEF 7514 CUP 67321 99 CUP 67339 CUP 67349 A. turbinata CUP 67331 (H. turbinata) CUP 67365 CUP 67375 CUP-PR-4388 PR11 CUP 67366 A. cubensis (H. epiphylla) ARSEF 2396 A. insperata 100 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 defined appear as strongly supported clades. Phylogenetically informative characters are indicated as vertical hashmarks: (A) indi- cates an effuse white anamorphic stroma; (B) confluent conidial masses, (C) cylindrical tubercles containing perithecia; (D) discrete conidial masses; (E) formation of a rim around conidioma orifices; (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. 542 M. Liu et al.

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

2.1 Position in stroma Taxonomy a In gregarious but well-separated tubercles..3, 5, 7, 9 b In crowded, gregarious tubercles...... 3, 4, 7, 8, 10 ... A synoptic key to treated species c Embedded in stroma, scattered ...... 4, 6 2.2 Colour of ostioles ... Taxa treated in this work, as well as superficially similar spe- a Yellow to orange yellow...... 3, 5, 7, 8, 10 ... cies, are included in the key below. Each species treated is ref- b Reddish orange...... 5 ... erenced in the key by its number. All can be identified based c Brownish yellow...... 3, 5, 6, 7, 8, 10 ... on either the anamorphic or teleomorphic stage. Two or d Red–brown ...... 4, 6 more species of Aschersonia may occur on different insects 2.3 Position of ostioles relative to the surrounding surface ... on a single leaf. a Slightly projecting...... 6 b Not projecting...... 3, 4, 5, 7, 8, 9, 10 1 A. australiensis 2 A. insperata 3 H. andropogonis/A. andropogonis Part ascospores 4 H. epiphylla/A. cubensis 5 H. libera/A. aleyrodis 6 H. phyllogena/A. basicystis 3.1 Shape .. 7 H. raciborskii/A. placenta a Cylindrical with rounded ends (Fig 2A) 3, 4, 5, 7, 10 ... 8 H. rhombispora/A. sp. (anamorph) b Fusoid (Fig 2B)...... 4, 5, 7 .. 9 H. turbinata/A. turbinata c Ventricose with rounded or acute ends (Fig 2C) .6, 8 ... 10 H. zhongdongii/A. incrassata d Ovoid (Fig 2D) ...... 7 3.2 Width ... For each character there are two or more states; each charac- a Less than 2 mm...... 3, 5, 6, 7, 8, 10 ... ter state is followed by one or more numbers that represent b 3–5 mm...... 4, 7, 9 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. Fur- Anamorphic characters ther discussion of the use of synoptic keys can be found in Korf (1972). All species are briefly discussed while detailed de- Stromata scriptions of the species listed above in bold are included in the taxonomic section following the key. 4.1 Colour a Grayish brown ...... 4 Teleomorph characters b Reddish orange...... 2, 6, 10 c Light yellow ...... 4, 9 Stromata d Yellowish white to white...... 1, 3, 5, 6, 7, 8, 10 4.2 Shape a Pulvinate base with pezizoid projections ...... 9 1.1 Colour a Reddish orange...... 6 b Yellowish orange ...... 4, 9 c Reddish brown ...... 4 d Yellowish white to white...... 3, 5, 7, 8, 10 1.2 Shape a Globose head markedly constricted at base...... 6 b Pulvinate, base slightly constricted, slightly tuberculate ...... 3, 4, 8, 10 c Pulvinate with sloping sides, ovoid or globose tubercles half-embedded ...... 3 d Pulvinate with pronounced cylindrical or ovoid tubercles...... 5, 7, 9 1.3 Surface texture a Tomentose ...... 3 b Pruinose...... 5, 7 Fig 2 – Shapes of part ascospores. (A) Cylindrical with c Smooth (minutely tomentose or pruinose) rounded ends. (B) Fusoid. (C) Ventricose with rounded ends...... 3, 4, 5, 6, 7, 8, 9, 10 (D) Ovoid. Aschersonia aleyrodis and its allies 543

b Tuberculate...... 2, 4, 9, 10 c Cylindrical...... 4, 5, 9 d Hemiglobose ...... 4, 6, 8 e Scutate (a hemispheric central region abruptly attenuating and extending to the edge) ...... 7 3, 8 f Thick pulvinate, conical pulvinate.1, 3, 4, 5, 6, 7, 8, 10 g Thin pulvinate, sometimes with pronouncedly erect tubercles...... 2, 5, 7 h Thin pulvinate with embedded tubercles...... 3 4.3 Hypothallus (a distinct thin layer of hyphae surrounding the base of the stroma and appressed to the plant surface) ... a Present...... 1, 3, 5, 6, 7, 8, 10 Fig 3 – Shapes of conidia. (A) Fusoid. (B) Fusoid with thick- ... b Absent ...... 1, 3, 4, 5, 6, 7, 8, 9, 10 ened wall at ends. (C) Ventricose. (D) Ovoid with acute ends. (E) Ovoid conidium of Hirsutella-like synanamorph.

Conidiomata

d Ovoid with acute ends (Fig 3D) ...... 4, 9 ... 5.1 Approximate number of locules e Ovoid (Hirsutella-like synanamorph) (Fig 3E)..... 2 a Fewer than ten ...... 1, 3, 4, 5, 6, 7, 8, 9, 10 b More than ten...... 1, 3, 5, 7, 8, 9, 10 Cultural characters on PDA 5.2 Shape of locules a Simple depressions of surface without distinct rims...... 1, 3, 4, 5, 6, 7, 8, 10 b With distinct rims, like half-embedded bowls..3, 10 7.1 Growth rate on PDA c Pezizoid ...... 9 a Relatively rapid, greater than 30 mm diam in three 5.3 Arrangement of ostioles on stroma weeks at 23 C ...... 4, 5, 7, 9 a Scattered ...... 1, 3, 4, 5, 7, 8, 9, 10 b Moderate, between 20 mm and 30 mm diam in three b Circular...... 5, 6, 7 weeks at 23 C ...... 2, 6, 8 5.4 Paraphyses in conidioma c Slow, smaller than 20 mm diam in three a Present...... 1, 3, 5, 7, 10 weeks at 23 C ...... 3, 10 b Absent ...... 1, 3, 4, 5, 6, 7, 8, 9, 10 7.2 Appearance and texture of the growing colony a Spreading, minutely tomentose...... 4, 5, 7, 9 Conidia b Compact, leathery...... 2, 3, 6, 8, 10 7.3 Colour of colonies a Greyish white ...... 3, 8 6.1 Colour of conidial masses b White ...... 6, 10 a Reddish brown ...... 4, 9 c Yellowish white to yellow ...... 4, 5, 7, 9 b Reddish orange...... 5, 7 d Orange yellow...... 2 c Orange ...... 4, 5, 7, 9 7.4 Colour of conidial mass d Deep yellow ...... 6, 8 a Pale yellow to yellow...... 2, 3, 10 e Pale yellow ...... 3, 6, 8, 10 b Deep yellow ...... 6, 8 6.2 Shape of conidial masses on surface of stroma c Yellowish orange ...... 5, 7 a Discrete, erumpent or contained in conidio- d Reddish orange...... 4, 5, 7, 9 mata...... 3, 4, 5, 7, 8, 9, 10 e Brownish red ...... 4, 9 b Conﬂuent, covering the centre of stroma...... 5, 7 7.5 Quantity of conidial mass c Conﬂuent, forming a ring around the stroma .....6 a Abundant ...... 2, 4, 5, 6, 7, 8, 9 d Forming an erect cirrus...... 3, 4, 5, 7, 9, 10 b Scanty...... 3, 10 6.3 Length of conidium 7.6 Hirsutella-like synanamorph a Longer than 9 mm ...... 3, 4, 5, 6, 7, 8, 9, 10 a Present...... 2 b Shorter than 9 mm ...... 1 b Absent ...... 3, 4, 5, 6, 7, 8, 9, 10 6.4 Width of conidium a 1.6–3 mm...... 1, 3, 5, 7, 10 b Wider than 3 mm ...... 4, 6, 8, 9 1. Aschersonia australiensis Henn., Hedwigia 6.5 Shape of conidium 42: 87 (1903). a Fusoid (Fig 3A) ...... 1, 2, 3, 5, 7 b Fusoid with ca 3–5 mm thickened wall at ends Note: This species resembles A. placenta in its orange–red (Fig 3B) ...... 10 conidial masses, but differs in having distinctively smaller c Ventricose (Fig 3C) ...... 6, 8 conidia (5–8 mm) (Petch 1921). It has been reported from 544 M. Liu et al.

Australasia (Hennings 1903). No teleomorph is known, and the inner surface; most conidiogenous cells arise from thick- species has not been well studied. Detailed descriptions were walled hyphae, unbranched, cylindrical, slightly narrower provided by Petch (1921). A. australiensis is further discussed near their truncate ends, unicellular, 7–16 1–1.5 mm; some under H. raciborskii. are branched (Fig 5H). Conidia fusoid, apices more or less blunt, 8–14 1.5–2 mm(Fig 5I), produced in copious slime. Paraphyses present in some specimens, ranging from 60–160 mm long 2. Aschersonia insperata Rombach et al., (Fig 5H). Mycologia 97: 251 (2005). On PDA colony grows very slowly, reaching 15–25 mm diam (Fig 3E) in five weeks at 23 C. Stromatic colonies white to greyish Note: The most distinctive character of this species is its pro- white, compact, forming a thick pulvinate structure, surface duction of both Aschersonia and Hirsutella-like synanamorphs minutely velvety, wrinkled (Fig 4A). Colonies both filamentous in young cultures and specimens, which has not been found hyphae and hyphal bodies, the latter usually 5–16 3–7 mm in any other species in this genus. The reddish orange tuber- with a thickened wall (not over 1 mm thick). Conidial masses culate stroma of this anamorphic species appears superficially usually not abundant, appear as tiny drops scattered on sur- like the perithecial stroma of H. turbinata, but only asexual face or very thin streams along wrinkles in the colony. When spores are produced. A. insperata is known only from the picked with a needle, the conidial masses appear as a solid type collections in the Philippines, and the teleomorph re- mass that is light yellow to yellow. Isolates are liable to lose mains unknown. A detailed description of this species was their ability to sporulate in serial subculture. Conidiogenous cells provided by Liu et al. (2005). 10–17 1.5–2 mm, conidia fusoid, apices acute, 8–12 1.5–2 mm. Paraphyses usually long and abundant, up to 190 mm long. 3. Hypocrella andropogonis Petch, Ann. Roy. Bot. Gard. Known distribution: Bolivia, Cameroon, China, Costa Rica, Peradeniya 7: 247 (1921). Ghana, Honduras, Mexico, Puerto Rico, Trinidad, USA. Other specimens examined: Cameroon: Korup National For- Typus: Trinidad: On leaves, R. Thaxter 18 (K(M) 120354- est, Oct. 2003, H.C. Evans & G. J. Samuels (AFR116 ¼ ARSEF holotypes). 7639 ¼ CUP67563); Costa Rica: Heredia: OTS La Selva Biological Anamorph: Aschersonia andropogonis Henn., Hedwigia 39: 139 Station, Sendero Holdridge, 18 June 2002, M. Liu (CUP 067291); (1900). Camino Cantarrana, 19 June 2002, M. Liu (CUP 067307); Beside Neotype: Puerto Rico: Mayaguez, beside Road 105, on ferns, entrance to plantation, June 20, M. Liu (CUP 067343, 067345). 15 Dec. 2003, M. Liu & Z.D. Wang (CUP-PR 4407) neotypus hic Ghana: Central Region, Jukua District, Kakum National Park, designatus). N05 210,W01230, elev. 280 m, Oct. 2003, H.C. Evans & G. J. Sam- Synonyms: Aschersonia parasitica Henn., Hedwigia 43: 149 uels, AFR34 ¼ CUP 67556, AFR68 ¼ CUP 67560. Java: Buitenzorg, (1904). Kulturgarten, on leaf of Mangifera indica, 1901, Zimmermann, Aschersonia lecanioides Henn., Hedwigia 41:145 (1902). (S F22432-type of A. lecanioides). Mexico: Veracruz: Municipio Figs 4A–C, Fig 5 Emiliano Zapata, Plan Chico, N19 26.7350,W9649.8650, elev. Teleomorphic stromata flattened pulvinate with subglo- 900 m, 11 Dec. 2003, P. Chaverri, J. Hernandez, J. Garcia-Alvarado bose tubercles (Fig 4A), some tubercles fused together PC431, (CUP 67523); behind Instituto Genetica Forestal Univer- (Fig 5A), but more often discrete, surface minutely tomentose, sidad (Veracruzana) building, 12 Dec. 2003, P. Chaverri & white or orange–white (darkened in very old specimens), 1– J. Garcia-Alvarado (PC432 ¼ CUP 67524); Catemaco, Ejido: Lopez 3 mm diam, 0.4–0.5 mm thick, edges of the stromata extend- Mateo town, project ‘Cielo, Tierra Y Selva,’ trail to mountain, ing to form a hypothallus (Fig 4B, 5B). Perithecia develop singly 200–300 m elev., 13 Dec. 2003, P. Chaverri, J. Garcia-Alvarado & in the tubercles (Fig 5C), flask-shaped, 250–450 mm deep, 160– C. Mena-Jiles (PC436 ¼ CUP 67526, PC452 ¼ CUP 67532). Philip- 300 mm diam at the widest point. Asci produced from basal pines: Laguna: Mt. Maquiling, near Los Ban˜ os, Feb. (18)94, clusters, cylindrical, 138–180 mm long, 5–8 mm wide, caps 3– C.F. Baker (FH 8); Paraguay. Cerro Coche: On leaves of Andropo- 3.5 mm thick (Fig 5D). Ascospores filiform, slight shorter than gon sp., K. Fiebrig 779 (B 70 0005658: type of A. parasitica). Puerto the perithecia, septate, dividing into part spores that are cylin- Rico: Guajataca, trail no.9, on ferns, M Liu & ZD Wang (CUP-PR drical with rounded ends, 12–15 1.5–2 mm(Fig 5E). 4438). USA: Florida: Micanopy, cross creek, Marjorie Kinnan Teleomorph and anamorph may or may not be present in Rawling’s residence, on Citrus, 22 Aug. 2003, M. Liu & Z.D. the same stromata (Figs 4B 5A–B, F). Strictly asexual stromata Wang (CUP 67436). usually pulvinate (Figs 4C, 5B, F), white to pale yellow (4A3– Notes: A. andropogonis is easily distinguished from A. aleyr- 4A5), 1–4 mm diam, 0.5–1 mm thick. Hyphae of stromata odis when the conidiomata of the former have elevated edges, forming compact textura intricata, 3–6 mm wide with a thick- but resembles a yellow-spored form of A. aleyrodis with thick ened wall, 1–2 mm wide, Conidiomata scattered in stromata, stromata in cases where the conidiomata are simply depres- 1–6 per stroma, widely open, orifice circular, 0.1–0.6 mm sions of the surface. A. andropogonis generally differs in pos- diam, rim of conidiomata sometimes distinctly elevated, con- sessing conidiomata with very wide openings, fewer idioma resembles a half-immersed bowl (Fig 4C). Conidial mass conidiomata (1–6), conidial masses that do not fuse with those yellow (4A8), yellowish orange (4B7) to orange (5A8), usually from adjacent conidiomata, and longer paraphyses; while A. contained in conidioma (Figs 4C, 5F), but sometimes erumpent aleyrodis may possess many conidiomata (1–20), smaller and or forming a column-like cirrus, never fused with conidial sometimes radially elongated orifices, and confluent conidial masses from adjacent conidiomata. In section, the conidioma masses. The colony morphology on PDA reveals more obvious is U- or V-shaped (Fig 5G). Conidioma with hymenium lining 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 fluffy, and bear abundant confluent 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 flattened pulvinate stroma, and the conidiomata produce reddish orange conidial masses. G. Culture of H. raciborskii/A. placenta: colonies are fluffy and spreading, conidial masses are abundant and confluent, 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 flattened pulvinate stroma, and confluent 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. 546 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 ﬁliform 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 A–B, F; 100 mm for Figs C, G; and 10 mm for Figs D–E, H–I.

compact, and greyish white colonies with a minutely velvety 4. Hypocrella epiphylla Sacc., Syll. Fung. 11: 368 (1895). and often wrinkled surface, while the A. aleyrodis grows faster and the white or yellowish white colonies are effuse and mi- Anamorph: Aschersonia cubensis Berk. & M. A. Curtis, J. Linn. Soc. nutely fluffy. 10:351 (1869). A. lecanioides was considered by Petch (1921) to be a syn- Notes: H. epiphylla is common in the subtropical eastern US. onym of A. placenta based on similar conidia and paraphy- The stromata typically are squat, cylindrical, and more com- ses. We examined the type specimen and observed elevated pact and slightly darker in colour than those of the A. aleyrodis edges of conidioma and other characters consistent with group. The differences are sometimes subtle, and young stro- A. andropogonis. A. andropogonis has longer paraphyses mata can be confused with those of A. aleyrodis. However, the than A. placenta. The paraphyses of A. lecanioides measured conidia of this species are wider and shorter than those of A. 70–114 mm, longer than those in A. placenta (40–70 mm). aleyrodis, a distinctive character that H. epiphylla shares with Hennings (1902) described the stromata as pale to yellow, its sister species, H. turbinata (Fig 1). The teleomorph is infre- as in A. andropogonis. The sexual state lacks cylindrical quently collected. For a detailed description, see Petch (1921). tubercles as in H. raciborskii, but forms subglobose tubercles that are half or totally embedded in the flattened stromata, 5. Hypocrella libera Syd., Ann. Mycol. 14: 85 (1916). as in H. andropogonis. We propose that A. lecanioides is conspecific with A. andropogonis. Type: Bolivia: Cobija: Rio Acre, on coccids on fallen leaves, Jan. We conclude that specimens identified by Fawcett (1908) 1912, E. Ule 3413 (W. 00939-holotypes). from Florida as A. flavocitrina are A. andropogonis. Fawcett’s de- Synonym: Hypocrella nectrioides Petch, Ann. Roy. Bot. Gard. termination was based mainly on the yellow colour of the Peradeniya 7:225 (1921). spore masses. Based on our type studies, A. flavocitrina has dis- Anamorph: Aschersonia aleyrodis Webber, Bull. USDA Div. Veg. tinctive orange discoid stromata and can be easily distin- Phy. Path. 13:21 (1897) guished from A. aleyrodis. Considering the characters of Type: USA: Florida: Manatee County, on whitefly on citrus, colonies on PDA he described in the same paper, Fawcett’s Dec. 1896, H. J. Webber (BPI 0389438–lectotypus hic designatus). specimens, which we were unable to locate, were probably Synonyms: Aschersonia goldiana Sacc. & Ellis, in Saccardo, A. andropogonis, which produces small and compact stromatal Syll. Fung. 14: 990 (1899). colonies. Aschersonia paraensis Henn., Hedwigia, 41: 17 (1902). Aschersonia aleyrodis and its allies 547

(Figs 4D–F, 6) centre of stromata usually covered with conidial masses red- Stroma white, yellowish to orange white (4A2–5A2), com- dish orange (7A6), orange (5A8–6A8), or light yellow (3A5–4A7), posed of a few to numerous gregarious tubercles arising thickened in centre. Conidiomata circularly arranged (Fig 4F) or from a pulvinate to hemispherical base (Fig 4E, 6A); sometimes scattered (Fig 6F); 3–20 per stroma; shape of conidiomatal os- surrounded by a thin hypothallus 0.7–2.0 mm wide; surface of tioles circular, radially elongated or irregular due to fusion of tubercles and base pruinose due to loosely woven, thick- adjacent ostioles; widely open, appearing as simple depres- walled hyphae that form stroma (Fig 6D). Tubercles strongly sions of stromatic surface without a differentiated rim; coni- projecting and aggregated, hemispherical, cylindrical or dioma shape in section globose (Fig 6G) or irregular. Conidial slightly narrowing apically, 0.3–0.5 mm diam, 0.3–0.7 mm in masses exuded from conidiomata as a viscous fluid, usually height; apices reddish orange (7A6) in fresh specimens, fading overflowing and confluent, sometimes forming a long erect to yellow when dry, in old specimens appearing amber cirrus. Conidiogenous cells, phialidic, 10–20 1–1.5 mm, arise sin- (brownish yellow 5C7). In most cases ostioles difficult to distin- gly or in whorls of 2–5 from compact, thick-walled hyphal tis- guish from surrounding glabrous tissue; in a few cases they sues (Fig 6I), not branched, thin-walled, smooth, cylindrical, are visible, ca 0.1 mm diam. Perithecia fully embedded slightly tapering, truncate at apices. Conidia fusiform (Fig 6K), (Fig 6B), one perithecium per tubercle, the shape of perithecia unicellular, hyaline, guttulate, ends acute but not prolonged, in section nearly globose to ovoid, 300–400 300–600 mm; stro- (9.0–) 10–16 (–18) 1.5–2.0 (–2.5) mm, produced in copious matal tissue around perithecium textura intricata (Fig 6D). Asci slime. Paraphyses abundant in the hymenium, especially in cylindrical, with a thick cap ca 5 mm long. Ascospores filiform, thick stromata, hyaline, not staining in cotton-blue, filiform, slightly shorter than perithecia, septate, sometimes helically 50–90 (–113) 1.0–1.5 mm (Fig 39). twisted in ascus, disarticulating into oblong oval or cylindrical Colonies (Fig 4D) on PDA growing relatively fast, 35 mm spores, slightly tapering towards ends, with somewhat diam in three weeks at 23 C, filamentous hyphae, tomentose, rounded ends, 13–16 2.5–3 mm(Fig 6C). white to yellowish white (4A4–4A5), sometimes with a gray- Separate from sexual stroma, anamorphic stroma thin pul- ish-yellow (4B4) peripheral circle. Conidial mass variable in col- vinate (Figs 4F, 6E–F), 1–2 mm diam, 0.1–0.3 mm thick, or scu- ouration (Fig 4D), light orange (5A4–5A5), deep orange (6A7) to tate 1–3 mm diam, 0.5–1 mm thick, white, minutely reddish orange (7A7), abundant, confluent. Most conidiogenous tomentose; hypothallus usually present in pulvinate stromata, cells, 10–19 1.0–1.5 mm, arise mononematously and laterally 0.3–1.0 mm in width, present or absent in scutate stromata; from thick-walled hyphae, or in a whorl of 2–6 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), conﬁned 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, E–F; 100 mm for Figs B, G; 10 mm for Figs C–D, H–K. 548 M. Liu et al.

of thick-walled hyphae (Fig 6I); but conidiophores with multi- of the stroma, the confluence of the conidial masses, and the ple branches also observed in culture, 35–65 mm, some phia- shape of conidia, we conclude that A. goldiana is a synonym of lides elongated to the length of paraphyses (Fig 6J). Conidia A. aleyrodis. fusoid, 9–13 1.5–2.0 mm, paraphyses not ordinarily observed The connection between H. libera and A. aleyrodis was first in all isolates, 40–123 mm when present. inferred by Petch (1925) based on two specimens from Pan- Known distribution: Bolivia, British Honduras, Costa Rica, ama. Mains (1959a) cast doubt on the connection due to the in- Cuba, Dominican Republic, Jamaica, Panama, Puerto Rico, Tri- consistency of the host: H. libera was described on coccids nidad, Venezuela (Mains 1959a,b), and Florida, Mississippi, (Sydow & Sydow 1916), whereas A. aleyrodis was described and Texas in the United States. on Aleyrodes (Webber 1897). However, our examination of the Other specimens examined: Brazil: Para´, auf lebenden Bla¨t- type specimens convinced us that H. libera is indeed the tele- tern von Psidium pomiferum, May 1901, J. Huber 50 (S F22444, omorph of A. aleyrodis, and we feel the host might have been as A. paranesis). Costa Rica: Heredia: OTS La Selva Biological misidentified by Sydow & Sydow (1916): the black oval-shaped Station, laboratory area, 17 June 2002, M. Liu (culture insect may be a whitefly nymph. Our phylogenetic analysis CR01 ¼ CUP 067280); Camino Cantarrana, 19 June 2002, M. Liu reaffirms the connection. Isolates from ascospores of fresh (CUP 067298, CUP 067303 ¼ ARSEF 7512, culture CR11 ¼ CUP H. libera collections (CR08 and CR11) grouped together with 067310); plantation beside entrance RCC, 20 June 2002, M. Liu those from A. aleyrodis conidia (Fig 1). An unidentified species (culture CR19 ¼ CUP 067341, culture CR20 ¼ CUP 067342); that is the source of GenBank sequence no. U47832 was re- Sendero Oriental 450 m, 20 June 2002, M. Liu (CUP 067325); suc- cently identified as H. nectrioides based on morphological ex- cession plots, 20 Aug. 2003, P. Chaverri PC321 ¼ ARSEF 7617. amination of the voucher specimen (Bath et al. 2005). We Guyana: Kamarang: on line to old Ayanganna Airstrip, west examined the type specimen of H. nectrioides, the general Pakaraima mountains, upper Potaro river, 20 km east of morphology of the stromata of which resembles H. libera.No Mount Ayangana, near confluence of Potaro and Alukyadong- asci were found, but, based partly on Petch’s description baru Creek, general area N5 160,W59540, approximately (Petch 1925), we consider this taxon to be a synonym of 650 m elev., 8 Jan. 2004, C. Aime, MCA 2465 ¼ ARSEF 7642. Mex- H. libera. ico: Veracruz: Catemaco, Ejido Lopez Mateo town, project H. sloaneae is similar to H. libera. The subtle differences in- ‘Cielo, Tierra Y Selva’, trail to mountain, 20–300 m elev., 13 clude: tubercles in H. sloaneae are crowded and ovoid, whereas Dec. 2003, P. Chaverri, J. Garia-Alvarado, C. Mena-Jiles, those of H. libera are separated and cylindrical; and the stro- PC434 ¼ CUP 067525 ¼ ARSEF 7706, PC439 ¼ CUP 067528. Pan- matal surface of the former is more coarse, whereas the latter ama: Furtuna: Behind field station, 14 July 2002, J. F. Bischoff, is more glabrous. We did not observe the anamorph in the JB133 (¼ARSEF 7641). Puerto Rico: Guillarte: RD 388 trail, 16 type from FH (Guadeloupe: Bois des Bains-Jaunes, 1904); how- Dec. 2003, M. Liu & Z. D. Wang (culture ML175-1, ML175-2, ever, Petch (1921) recorded anamorph characters from the ML175-3, ML175-4 ¼ CUP-PR 4421 ¼ ARSEF 7393, ARSEF 7394); type of H. amazonica (Peru: on Sterculiaceae, Iquitos, July On Psidium guajaba, 1912, (FH 360 as A. goldiana). Trinidad: 1902, E. Ule, Herb. Brasil. no. 3198), which he considered to Port of Spain, St. Ann’s Valley, on scale insect on Pentaclenthra be conspecific with H. sloaneae. According to Petch (1921), the sp., R. Thaxter, K(M)120325 (H. nectrioides); Maraval Valley, unnamed Aschersonia anamorph of this fungus produces 1912–1913, R Thaxter (FH); Anne’s valley, on leaves of Adiantum, a red–brown spore mass, and thus differs from that of A. aleyr- Feb. 1913, R. Thaxter, (FH, as A. goldiana). United States: Florida: odis. As already mentioned, colouration of conidial mass is Manatee County, on whitefly on Citrus, Dec. 1896, H. J. Webber not a constant character in A. aleyrodis/H. libera, which pro- (BPI US0389440); Mar. 1896, H. J. Webber (BPI 0389439); Mica- duces both yellowish orange and reddish orange conidial nopy, Cross Creek, Majorie Kinnan Rowlings’ residence, on Cit- masses. rus, 22 Aug. 2003, M. Liu & Z.D. Wang (CUP 067435 ¼ ARSEF 7339, ARSEF 7340, ARSEF 7343, ARSEF 7344); Lake Alfred, on cit- 6. Hypocrella phyllogena Petch, Ann. Roy. Bot. Gard. rus leaves, 9 Jan. 1980, R.S. Soper (living culture ARSEF 430); on Peradeniya 7: 228 (1921). 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, Anamorph: Aschersonia basicystis Berk. & M.A. Curtis, Jour. Linn. as A. goldiana); Gainesville, 1923, E.W. Berger (FH 6301 as Soc. Bot. 10: 352 (1869). A. goldiana). Notes: This species has a distinctive sexual stroma, which is Notes: A. aleyrodis is distinct in having a neotropical and reddish orange and composed of a globose head that is mark- subtropical distribution, orange (or sometimes yellow), nar- edly constricted at the base. Dull yellow conidial masses are row fusoid conidia, and a tuberculate teleomorph. Histori- produced at the narrowest part of the constriction, forming cally, there has been taxonomic confusion regarding the a ring around the ‘head.’ This species is variable and appears relationship between A. aleyrodis, commonly called the ‘red to be a complex of several species. In the early stages of devel- fungus,’ and the ‘yellow fungus’ A. goldiana (Fawcett 1908). opment before the perithecia are formed, this species resem- Petch (1921) and Mains (1959a, b) observed that the two spe- bles anamorph stage of H. rhombispora. Both have white cies were only distinguished by the colour of the conidial pulvinate stromata, yellow conidial masses contained in the mass, that of the former being red to reddish orange, and conidiomata, and ventricose conidia. However, the conidio- the latter yellow to orange. We mapped conidial mass colour mata of anamorph stage of H. basicystis are circularly arranged onto the phylogenetic tree shown in Fig 1 and found that it around the base of stroma, while conidiomata of A. rhombis- is not an informative character within the A. aleyrodis clade. pora are scattered on the stroma surface. This species is fur- We also examined the type of A. goldiana. Based on the shape ther discussed under H. rhombispora. Aschersonia aleyrodis and its allies 549

7. Hypocrella raciborskii Zimm., Centralblatt f. Bakt. Aschersonia javanica Penz. & Sacc., Malpighia 20: 236 (1901). 7: 875 (1901). Aschersonia tamurai Henn., Engler’s Bot. Jahrb. 31: 741 (1902). (Figs 4G–I, Fig 7) Type: Zimmermann (Centralblatt f. Bakt., 7: 875 fig 4, 1901 – Stromata white to yellowish white, 1–2 mm diam; tubercu- lectotypus hic designatus. China: Guangdong: Dinghushan, 10 late processes developing from a thin pulvinate base (Figs 4H, Aug. 2004, B. Huang DHS040810-11 (CUP CH 002621 – epitypus 7A); tubercles usually isolated from one another, rarely hic designatus). Ex-epitype living culture ¼ ARSEF 7609 ¼ CHN4). densely aggregated, occurring at margins when conidiomata Synonyms: Hypocrella warneckiana Henn., Engler’s Bot. Jahrb. 38: are present in centre. Tubercles cylindrical, ovoid or subglo- 113 (1905). bose, 0.3–0.5 mm diam, up to 0.5 mm in height. Perithecia em- Barya salaccensis Racib., Bull. Akad. Sci. Cracovie: 909 (1906). bedded singly in tubercles (Fig 7B), flask-shaped or ovoid, 300– Anamorph: Aschersonia placenta Berk., J. Linn. Soc. Bot. 14:89 420 mm deep, 200–300 mm wide with walls 20–32 mm thick. Os- (1875). tioles yellow (3A6–4A6) to orange–yellow, darkening to deep or- Type: Sri Lanka: on leaves of Loranthus sp., 1879, G. H. K. ange in old specimens; readily visible. Stromatal tissue textura Thwaites (K(M)81383-holotype). intricata around perithecium (Fig 7E). Asci cylindrical (Fig 7C), Synonyms: Aschersonia novoguineensis Henn., Engler’s Bot. 140–220 5–7 mm with 5–6 mm thick caps. Ascospores filiform, Jahrb. 25: 509 (1898). 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 conﬂuent 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, F–G; 100 mm for Fig B; and 10 mm for C–E, H–L. 550 M. Liu et al.

ascospores that are cylindrical, slightly tapering towards leaves of Ficus sp., Jan. 1924, M. Strong Clemens (4697, ends, 10–16 2.5 mm, or ovoid 8–10 3 mm(Fig 7D). K(M)128049); Zambala province, Castillejos, on leaves, Mar. 1924, Anamorph may or may not co-exist in the same stromata M. Strong Clemens (3275, K(M)128051); Laguna: Los Ban˜ os, college, on Ficus ulmifolia, 19 Dec. 1917, Torres (BPI 635849); On coccids with teleomorph. Anamorphic stromata white (Figs 4I, 7F–G) on Litsea sp., Feb. 1932, G. O. Ocfemia, (K(M)128044) San Crispin, in fresh specimens, yellowish white to orange white in old San Pablo, on coccids on Premna sp., Jan. 1932, M. S. Celino specimens; flattened pulvinate, usually very thin, 0.2–0.7 mm (K(M)128046) Cebu Island: Cebu, on leaves of Ficus sp., May 1924, thick, 1–3 mm diam, surrounded by a hyaline hypothallus up M. Strong Clemens (6210, K(M)128048); Mindanao: Davao province, to 1.5 mm wide. Stroma surface minutely tomentose, covered Mt. Apo, on leaves probably Strongylodon sp., Jan. 1924, M. Strong with confluent conidial masses that are deep yellow (4A6) to Clemens (5656, K(M)128050). Singapore: On leaves of Smilax barbata, orange yellow (4A7). Conidiomata occur as simple depressions 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 in the stroma, 3–13 per stroma, forming a ring (Fig 7G) or irreg- scale insects and whitefly, 28 Aug. 1956, E. F. Vestal, (K(M)128040) ularly (Figs 4I, 7F) arranged. Conidiogenous cells (Fig 7I), arising Northeastern Thailand (BCC1454); Western Thailand (BCC2163); Cen- singly or in a cluster of 3–5 from thick-walled hyphae, some- tral Thailand (BCC2227); Eastern Thailand (BCC2175). times branched, cylindrical, slightly tapering towards the Notes: This species closely resembles A. aleyrodis/H. libera. apex, truncate, 7–22 1–1.5 mm. Conidia fusoid (9–)11–14 Petch (1921) stated that the two could be distinguished by (–16) 1.5–2 mm(Fig 7L), produced in copious slime. Paraphyses the slightly thinner stromata and shorter paraphyses of A. pla- arising from the hymenium of the conidioma, filiform, taper- centa. In our examination, the pulvinate stromata of A. placenta ing at the apices, 40–70 mm long (Fig 7H). tended to be thinner and the tubercles (H. raciborskii) usually In culture, colonies 2–3.5 mm diam in three weeks at 23 C develop around the peripheral part of the anamorphic stro- on PDA, white to yellowish white, pulvinate, surface tomen- mata, while in H. libera the teleomorph and anamorph are tose. Colonies cultured from ascospores often form hyphal rarely formed in the same stroma. The conidiomatal paraph- tufts in early stages. Colonies usually produce abundant vis- yses reveal differences in length between two species. In cous conidial masses that become confluent, or thick cirri A. aleyrodis the paraphyses may reach 113 mm; in A. placenta that are concentrically arranged. Conidial masses pale yellow they are not longer than 80 mm. Paraphyses are not ordinarily (4A3), light yellow (4A4) to light orange (5A4) (Fig 4G). Conidio- observed in culture for either species. In addition, H. raciborskii mata evident after conidial masses washed away. Similar in produces a proportion of ovoid part spores that are shorter shape to those in natural stromata; concave, with an opening and fatter than usual, but this is not observed in H. libera.In of 0.6–1.0 mm. Conidiophores and conidiogenous cells arise from A. placenta, conidiophores arise from the ends of hyphal tufts, aggregated thick-walled hyphae (Fig 7J). Conidiophores typi- and are penicillate or verticillately branched, while in A. aleyr- cally penicillate or verticillate, branching 1–3 times, 30– odis, in addition to the aforementioned form, more conidioge- 50 mm high, forming a hymenial layer. Conidiogenous cells slen- nous cells arise singly from hyphae. der, 1.5–2 mm at widest point, 11–16 mm long, apices truncate, The type of H. warneckiana in B was probably destroyed or occasionally very long, reaching 70 mm, and resembling pa- during World War II (Burghard Hein, pers. comm.). The ori- raphyses (Fig 7K). Conidia similar in size to those derived from ginal description by Hennings (1905) agrees completely with natural specimens, 10–15 1.5– 2mm. Paraphyses occasionally H. raciborskii. found in culture, 60–114 mm long. The original description of A. novoguineenesis by Hennings Distribution: Cameroon, China, Ghana, India, Indonesia, (1898) is consistent with the characteristics of A. placenta, except Malaysia, New Guinea, the Philippines, Thailand, and that the stated width of the conidia (0.3–0.4 mm) is narrower than Vietnam. that of the A. placenta (1.5–2 mm). Our measurements of conidia Other specimens examined: Cameroon: Korup National Forest, Oct. from the type (S) were (10–)13–15 (–17) 1.5–2(–2.5) mm, which 2003, G.J. Samuels & H.C. Evans, AFR114 ¼ ARSEF 7616. Ceylon: Per- falls in the range for those of A. placenta. The stromata are small adeniya:OnSchleichera tujuga, Jan. 1919, T. Petch, (K(M)128041); (1 mm diam), had abundant paraphyses measuring 40– Salak, on Lasianthus sp., 1899, M. Raciborski (KRA-F1899-31, -32, -33) (type of Barya salaccensis). China: Guangdong: Dinghushan, 9 70 1.5 mm, and conidiogenous cells 12–17 1.5–2 mm. As these Aug. 2004, B. Huang (DHS04080907 ¼ CUP-CH 002607 ¼ ARSEF characters are consistent with A. placenta, we consider A. novo- 7607 ¼ CHN1); 10 Aug. 2004, B. Huang (DHS040810-10 ¼ CUP-CH guineenesis a later synonym of A. placenta. 002620 ¼ ARSEF 7686 ¼ CHN3). Ghana: Central Region, Jukua dis- The type of A. javanica (W 16737) is in poor condition with 0 trict, Kakum National Park, wet semideciduous forest, N05 21 , only a single stroma on the leaf. Petch (1921) observed that W01 230, elev. 280 m, 23 Oct. 2003, G.J. Samuels & H.C. Evans the morphology matched some forms of A. placenta, and we (AFR28 ¼ ARSEF 7637). Indonesia: Java: Beng Breng, on leaves of agree with Petch’s conclusion that A. javanica is a synonym of Clerodendrum sp., M. Raciborskii 26, K(M)128056; Bogor Botanical Garden, on Aleyrodidae on Aglaria odoratissima, 22 Mar. 1986, A. placenta. H. javanica is not the teleomorph of A. javanica, but M.C. Rombach (220386-1) (ARSEF 2154) Tjibodas, in foliis coriaceis of A. coffeae, which has brownish stromata, unlike A. javanica. subemortuis, 5 Feb. 1897, (Naturhistorische Museum Wein No. We examined the type of A. tamurai (S, F22458). The stroma 16737) (holotype of A. javanica). Japan: Taom:OnQuercus cuspidata, shape resembles that of A. placenta. The colour of the conidial Aug. 1901, Tamura ((S) reg. Nr. F22458) (holotype of A. tamurai). mass had faded to yellow, presumably through aging. Petch’s Malaysia: Kuala Lumpur:onAleyrodidae, 28 Mar. 1994, L.A. Lacey (1921) description of its original colour as reddish orange is (94-41, ARSEF 4209). New Guinea: Kaiser Wilhelmsland, auf der A. placenta Unterseite der Bla¨tter von Ficus sp. ((S) reg. Nr F22442) (A. novogui- consistent with . Other characters, such as the neenesis). Philippines: Luzon: Manila, San Francisco church locality size of conidia (8–12 1.5–2 mm) and the length of paraphyses or vicinity, on Anonaceae, 14 Feb. 1924, Mrs Clemens (BPI 635847); on (50 mm) are also consistent with those of A. placenta. We there- Ficus ulmifolia, Dec. 1911, P. W. Graff (BPI 635852); on Premna odor- fore consider these names to be synonyms. ata, Dec. 1911, P. W. Graft, (K(M)128043); Isabella province, on Aschersonia aleyrodis and its allies 551

Another species that resembles A. placenta is A. australien- more dense (as approaching textura epidermoidea or textura sis. A syntype specimen from B collected by Pritzel from North oblita); hyphal walls markedly thickened, 1–3 mm thick for hy- Queensland has the general shape of A. placenta with white phae 3–6 mm in diam (Fig 8H–I). Conidiomata (Fig 8H) ‘U’-shaped pulvinate stromata and circularly arranged conidiomata. or convolute in section, ostioles 50–180 mm wide. Hymenium However, as Petch (1921) pointed out, the conidia are distinc- lining inner surface of conidioma. Conidiogenous cells arising tively small, 5–8 1.5 mm, whereas in A. placenta they are typ- singly from thick-walled hyphae, narrow cylindrical, not ically over 9 mm long. A syntype collected by Diels from New branched (Fig 8I), tapering near truncate apices, 8–12 Zealand has a different stroma shape with three tubercles; it 1.5–2 mm. Conidia 9–14 2.5–3 mm, inflated at the midpoint is probably a different species. We retain A. australiensis as (2.5–3 mm wide) and tapering at both ends, forming two slender separate species. 2–4 mm ends (Fig 8K–M). Paraphyses absent. Colonies (Fig 4J) on PDA 20 mm diam after four weeks at 8. Hypocrella rhombispora Miao Liu & K. T. Hodge, 23 C, thick pulvinate, moderately compact, firm and leathery, sp. nov. greyish white (1A1–1B1) to yellowish white (3A2), surface minutely tomentose, smooth to radially wrinkled, covered with Anamorph: Aschersonia sp. deep yellow (4A8) conidial masses. Conidial masses abundant, Stromata 2–2.5 mm diametro, lutescentia vel pallide aurantiaca, formed as numerous small viscous to solid drops, some of pulvinata, subtuberculata ad basem constricta. Perithecia in stro- which fuse together. In some cases the hyphae congregate to mate dense dispersa, inclusa. Ostiola non procurrentia, brunnes- form tubercle-like tufts covered with numerous yellowish centi-flava. Asci cylindrici in apice pileati. Ascosporae filiformes, in partisporas dimorphicas abrumpentes, nunc fusoideas in white (3A2) drops of liquid exudate. Hyphae 3–6 mm wide with ambobus extremis acutas 10–14 2–3 mm, nunc cylindricas in thickened wall 0.5–1 mm thick. No conidiomata formed, conid- ambobus extremis obtusas, 7–12 1.5–2.5 mm, in toto gutulatae, ial masses directly produced from surface of colony. in medio ubi guttulae accumulatae plerumque tumidas. Anamor- Conidiogenous cells arising laterally from hyphae, single, pha in eodem stromate ac teleomorpha vel non. Stromata ana- unbranched; or in clusters. Conidiogenous cells strongly morphica alba, pulvinata vel conico-pulvinata vel scutata, in constricted at the basal septum, those arising laterally superficie minute pruinosa. Hypothallus saepe nullus. Conidioma annulatim disposita, sparsa vel in massam reticulatam, conidia- from hyphae are longer than terminal conidiogenous cells, 8– lem pallidam vel sublutescentem. Conidia rhombiformia, 9– 12(–15) 2–2.5 mm(Fig 8J). Conidia markedly inflated at the mid- 14 2.5–3 mm. Coloniae in PDA moderate auctae, compactae, point and tapering at both ends, 8.5–12 (–17) 2–3 mm; in some firmae, coriaceae, griseo-albae vel lutescenti-albae, in superficie cultures, extremely wide conidia produced, ovoid in the cen- minute tomentosae, massis conidialibus copiosis, viscosis, vivide tre, contain a single very large guttule (4–6 3–6 mm), and luteis obsitae. Forma conidiorum in cultura varior: plurima coni- have two elongated ends measuring 2–3 (–5) mm(Fig 8K–M). diorum rhombiformia vel aliquae tumidissima. Nec paraphyses Neither paraphyses nor Hirsutella-like synanamorph observed. nec synanamorpha hirsutelloidea visae. Typus: Honduras: Departamento Yojo: Los Pinos, Parque Nacional Distribution: Costa Rica, Honduras, Mexico, and Puerto Rico. Cerro Azul-Meambar, 850 m elevation, 3 Sept. 2004, P. Chaverri & Specimen and culture examined. Costa Rica: Heredia:O.T.S.La P. A. Sheikh PC691(¼CUP 067548 holotypus). Selva Biological Station, Camino Cantarrana, on Cyclanthus biparti- tus, 19 June 2002, M. Liu CR07; CUP 67296 ¼ ARSEF 7511); 5 Jan. 2004, (Figs 4J–L, 8) P. Chaverri, (PC466 ¼ CUP 067537, PC467 ¼ CUP 067538); beside en- Stromata pale yellow (4A3) to pale orange (5A3), pulvinate and trance to Plantation RCC, 20 June 2002, M. Liu ML44-3, (culture slightly tuberculate, 2–2.5 mm diam, 0.8–1.4 mm thick, slightly CR32 ¼ CUP 067346); Puntarenas, Las Cruces Biological Reserve, constricted at base, sometimes surrounded by hypothallus. Wilson Botanical Garden, large loop of jungle trail, on Guarea rho- Stromatal tissue dense textura intricata (Fig 8C). If present, palocaipa, 4 July 2002, M. Liu, ML64 (culture CR34 ¼ CUP 67369). hypothallus narrow, 0.6 mm wide, and minutely tomentose. Honduras: Yojoa: Los Pinos, Parque Nacional Cerro Azul-Meambar, Perithecia densely arranged in the stroma, embedded, ostioles 850 m elev., 3 Sep. 2004, P. Chaverri & P. A. Sheikh, (PC691 ¼ CUP 067548, PC696 ¼ CUP 067550, PC698 ¼ CUP 067551); Copan: Santa not projecting, brownish yellow (5B7); in section 300–450 mm Rita, Reserva Pen˜ a Quemada, 9 Sep. 2004, P. Chaverri & P. A. Sheikh. m Asci high, 210–300 m at widest point. subcylindrical, widest Mexico: Veracruz: Amayaga, Catemaco, 500 m elev., 14 Dec. 2003, at midpoint, slightly narrower at both ends, 148–296 mm P, Chaverri & J. Garcıá-Alvarado, (PC460 ¼ CUP 067534). USA: Puerto long, 6–14 mm at widest point, 5–8 mm at apices. Ascospores ini- Rico: Between Mayaguez and Maricao, beside road 105 15 Dec. tially filiform, slightly shorter than perithecia, 1–1.5 mm diam, 2003, M. Liu & Z.D. Wang, ML164 (CUP-PR 4406; Ex-type culture dividing into part spores (Fig 8D). Part spores fusoid, acute at ML164 ¼ ARSEF 7390; Guajataca Forest, trail no. 9, on fern, 18 both ends, 10–14 2–3 mm, or others cylindrical with blunt Dec. 2003, M. Liu & Z.D. Wang ML201-1,ML201-3, ML201-5a (CUP- PR 4437 ¼ ARSEF 7395, ARSEF 7399, ARSEF 7400). ends, 7–12 1.5–2.5 mm; guttulate, usually swollen at mid- Notes: The most distinctive characters of this species are point where guttules accumulate (Fig 8E). Paraphyses absent. the shape of the part spores and conidia, both of which are Anamorphic stromata (Figs 4L, 8F–G) white, thin pulvinate, distinctly inflated in the middle. These characters are shared Hemi-globose or scutate with a hemispheric central region by a closely-related complex of species referred to H. phyllo- abruptly attenuating and towards the edge (Fig 4L); 1–3 mm gena/A. basicystis. Hypocrella phyllogena has an orange stroma diam, 0.2–0.7 mm thick; surface minutely pruinose. Hypothal- of a globose head and a narrower neck, upon which the asex- lus, if present, 0.2–0.8 mm in width. Conidiomata 4 to numerous, ual (A. basicystis) fruiting structure usually appears. In H. rhom- arranged concentrically, scattered, or forming a reticulum on bispora, the asexual and sexual states often occur on different conical part of stroma. Conidial mass pale to light yellow (3A4– stromata. The teleomorphic stroma is white and minutely tu- 4A4), in conidiomata or accumulating as discrete drops over berculate. The conidiomata are scattered over the anamorphic the ostioles, not confluent. Stromatal tissue textura intricata, stromatic surface. near conidiomata (about 75 mm below the hymenia), becoming 552 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. (F–G) 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 ﬂask-shaped and slightly narrower at the base. (K–M) Ventricose conidia showing variation in width among several specimens. Bar [ 500 mm for Figs A, F–G; 100 mm for Fig B; 10 mm for Figs C–E, H–M.

9. Hypocrella turbinata Petch, Ann. Roy. Bot. Gard. 10. Hypocrella zhongdongii Miao Liu & K. T. Hodge, Peradeniya 5: 535 (1914). Mycol. Res. 108: 820 (2005).

Anamorph: Aschersonia turbinata Berk., Ann. Mag. Nat. Hist., (Fig 3B) ser.2 9: 192 (1852). Anamorph: Aschersonia incrassata Mains, J. Insect Pathol. 1:46 (Fig 3D) (1959). Notes: Young H. turbinata specimens produce small Note: This species most closely resembles H. andropogonis/ cylindrical stromata similar to those of A. cubensis. The A. andropogonis; both A. incrassata and A. andropogonis are com- latter two species can easily be distinguished from mon yellow-spored species in the neotropics. A. incrassata can specimens of A. aleyrodis that have scutate stromata (Fig 6E) be distinguished by the distinctive thickened walls at the ends by the shape of the conidia. In A. turbinata and A. cubensis, of conidia. Further comparison of these two species and the the conidia are wider (Fig 3D); in A. aleyrodis they are narrowly evolutionary relationships of H. zhongdongii with other species fusoid (Fig 3A). in the genus were discussed by Liu & Hodge (2005). Aschersonia aleyrodis and its allies 553

A. andropogonis and A. incrassata, etc, depending on stromatal Discussion thickness and degree of development. Thus we cannot consider conidiomatal shape useful for differentiating species in Based on morphological and phylogenetic analysis, six species this group. Conversely, for some species outside our focal with white pulvinate stromata are recognized, A. aleyrodis (tele- group (e.g. A. turbinata), the situation is different. The stromata omorph: H. libera), A. andropogonis (H. andropogonis), A. placenta do not develop from a thin layer, instead they begin as small (H. raciborskii), A. incrassata (H. zhongdongii), A. basicystis (H. phyl- masses and develop into cylindrical to complex structures. A logena), and one new species, described here as H. rhombispora vertical section of a small cylindrical A. turbinata stroma re- (with its anamorph). The stromata of H. phyllogena are orange veals that the irregularly folded hymenium develops inside and relatively dense when mature, but white and effuse during the stroma under a small orifice. Several characters that pre- early stages of development and asexual sporulation. dict phylogenetic relationships (synapomorphies) were found. Most phylogenetic species reflect Petch’s (1921, 1925) con- These include the white, effuse anamorphic stroma that is cepts , and appear as strongly supported clades. However, typical of A. aleyrodis and allies (Fig 1, character A), Other syn- the relationship between H. libera/A. aleyrodis and H. racibor- apomorphies include the arrangement of conidial masses on skii/A. placenta is not resolved. Further studies using faster the stromata or colony surfaces. This character separates all evolving genes and more intensive analyses have resolved white stroma species into two clades, i.e. a clade with conflu- their sister relationship (Liu et al., pers. obs.), supporting ent masses (A. aleyrodis and A. placenta; Fig 1, character B) and Petch’s hypothesis that these two similar fungi from the a clade in which conidial masses are discrete (A. incrassata, New World and the Old World represent different species. Ac- A. andropogonis, Aschersonia anamorph of H. rhombispora, and cordingly, we treat them here as distinct species. A. basicystis; Fig 1, character D.) The former clade is also united An examination of the phylogenetic distribution of mor- by the character of cylindrical tubercles containing perithecia phological characters within the group identified many (Fig1, character C). Ventricose conidial shape (Fig 1 character F) apomorphies. Descriptive morphological characters (autapo- defines a clade composed of A. basicystis and H. rhombispora; morphies) that characterize species include the shape of tele- the formation of a rim around the conidiomatal orifices de- omorphic stromata, shape and arrangement of tubercles fines a clade of A. andropogonis and A. incrassata. The colour containing perithecia, shape of part ascospores, shape of ana- of the conidial mass is not phylogenetically informative: taxa morphic stromata, presence/absence of a rim around the ori- producing yellow conidial masses, for example, do not form fices of conidiomata, arrangement of conidial masses on the a monophyletic group (Fig 1). The insect hosts of this genus stromata, colouration of conidial masses, shape of conidia are restricted to the families Aleyrodidae and Coccidae in the and presence/absence of paraphyses. Except for the rim order Hemiptera (Petch 1921, Mains 1959, Evans 1990, Meekes around the conidiomatal orifices, all other characters were et al. 2002). One Aschersonia species may attack a wide range used by Petch (1921) in differentiating species. It is worth not- of species within an insect genus; conversely, one insect ing that the presence or absence of conidiomatal paraphyses species in Aleyrodes or Lecanium can be attacked by many may be diagnostic, but it has been questioned as a criterion Aschersonia species (Petch 1921). Petch (1921) separated Ascher- to separate Aschersonia into two subgenera (Liu & Hodge 2005). sonia into two subgenera based on host (whitefly versus scale The shape of the conidioma (called a pycnidium by Petch insect) and the presence/absence of hymenial paraphyses. 1921) and its orifice were among the characters used by Petch These subgenera of Aschersonia were considered to correspond to differentiate species. Although the presence/absence of with the two subgenera of Hypocrella. Later authors have ques- a rim around the orifice is useful for differentiating certain tioned Petch’s subgeneric concepts (Dingley 1954, Liu & Hodge species, such as A. andropogonis and A. incrassata, the shapes 2005). Because of the difficulty in identifying the insect hosts, of the conidiomata and orifices are not constant within the ecological host range of Aschersonia species has not been each of these six species, and we consider them to be of ques- sufficiently studied. Whether or not there is host specializa- tionable use. Their morphology appears to vary with the de- tion or coevolution in Hypocrella/Aschersonia is a question gree of maturity of the conidioma. Petch (1921) observed two waiting for an answer. types of conidiomatal formation in Aschersonia. In the first type the conidiophores developed on a small actively growing area of the surface of stroma, which gradually became invag- Acknowledgements inated as it expanded. In the second type, a conidioma was formed as a locule within the stromata and later broke We thank the staff at B, BPI, CUP, FH, HMAS, K, KRA MICH, through to the surface. Aschersonia species with white pulvi- PAD, and S for kindly loaning specimens; ARSEF, BCC, and nate stromata conform to Petch’s first type of development. CBS for providing fungal isolates; the US Forest Service for per- In this group, the stromata appear to develop from a thin layer mission to collect fungi in the Caribbean National Forest (El into a thicker pulvinate one. The thinner the stroma, the wider Yunque´), and the National Biodiversity Institute (INBio) for fa- the conidiomatal orifice. On a thin stroma, conidiomata ap- cilitating collecting by ML in Costa Rica. Our thanks also go to pear as widely open shallow depressions of the surface (Figs all of the people who have provided specimens or cultures: 4F, I); as the stroma becomes thicker, the growth of the sur- Mary C. Aime, Joseph F. Bischoff, Harry C. Evans, Gary J. Sam- rounding stromatal tissue constricts the opening and forms uels, and Christopher L. Schardl; and also people who have fa- conidiomata that are V-shaped, U-shaped, globose, tubular, cilitated our collecting in different places: B. Jean Lodge, or irregularly concave (Figs 5G, 6G, 7H). Several of these Sharon Cantrell, Sandra Maldonado, Miriam Salgado in Puerto shapes might be found in a single species i.e. A. aleyrodis, Rico; Juventino Garcıá Alvarado in Mexico, Phil Arneson in 554 M. Liu et al.

Honduras, Emilia Garcıá y Diego De la Quintana in Bolivia, Luis Liu PG, Wang XH, Li ZQ, Yang ZL, Zhang DC, 2001. Notes on the Diego Go´mez in Las Cruces, CR; Clayton McCoy, Philip Stansly, bambusicolous species of the genus Hypocrella from south- Lee Mitchell, Stephan Brown, Ronald Cave, Lavern W. Timmer, western China. Mycotaxon 78: 67–74. Liu YJ, Whelen S, Hall BD, 1999. Phylogenetic relationships among and James Kimbrough in Florida; Zengzhi Li and Bo Huang in Ascomycetes: Evidence from an RNA polymerase II subunit. China. The plates were prepared by Kent Loeffler. Comments Molecular Biology and Evolution 16: 1799–1808. from two anonymous reviewers improved the manuscript. Mains EB, 1959a. North American species of Aschersonia parasitic This project was supported by the National Research Initiative on Aleyrodidae. Journal of Insect Pathology 1: 43–47. of the USDA Cooperative State Research, Education and Exten- Mains EB, 1959b. Species of Aschersonia (Sphaeropsidales). Lloydia sion Service, grant no. 2002-35316-12263, and by the National 22: 215–221. Science Foundation under Grant no. 0212719 to K.T.H. Mason-Gamer RJ, Kellogg EA, 1996. Testing for phylogenetic conflict among molecular data sets in the tribe Triticeae (Grami- neae). Systematic Biology 45: 524–545. McCoy CW, Samson RA, Boucias DG, 1988. Entomogenous fungi. references In: Ignoffo CM, Mandava NB (eds), CRC Handbook of Natural Pesticides. CRC Press, Boca Raton, pp. 151–236. Meekes ETM, Fransen JJ, van-Lenteren JC, 2002. Pathogenicity of Aschersonia spp. against whiteflies Bemisia argentifolii and Tri- Bath AK, Torres MS, Samuels GJ, White jr JF, 2005. Morphology aleurodes vaporariorum. Journal of Invertebrate Pathology 81: 1–11. and species identification of a Hypocrella species (GenBank Meekes ETM, van-Voorst S, Joosten NN, Fransen JJ, van- accession # U47832). Mycotaxon 92: 97–102. Lenteren JC, 2000. Persistence of the fungal whitefly pathogen, Berger EW, 1921. Natural enemies of scale insects and whiteflies in Aschersonia aleyrodis, on three different plant species. Myco- Florida. Florida State Plant Breading Quartely Bullatin 5: 141–154. logical Research 104: 1234–1240. Bo¨rner C, 1956. Aleyrodidae (Aleurodina). In: Sorauer CP (ed), Petch T, 1921. Studies in entomogenous fungi. II. The genera Handbuch der Pflanzenkrankheiten, Tierische Scha¨dlinge an Nutzp- Hypocrella and Aschersonia. Annals of the Royal Botanic Gardens flantzen, Teil 2, Lieferung 4. Heteroptera und Homoptera. Paul Peradeniya 7: 167–278. Parey, Berlin, pp. 331–359. Petch T, 1925. Additions to Ceylon fungi. III. Annals of the Royal Dingley JM, 1954. The Hypocreales of New Zealand. VI. The genera Botanic Gardens Peradeniya 9: 313–328. Hypocrella, Barya, Claviceps and Podonectria. Transactions of the Ramakers PMJ, Samson RA, 1984. Aschersonia aleyrodis, a fungal Royal Society of New Zealand 81: 489–499. pathogen of whitefly. II. Application as a biological insecti- Evans HC, Hywel-Jones N, 1990. Aspects of the genera Hypocrella cide in glasshouses. Zeitschrift fu¨r angewandte Entomologie 97: and Aschersonia as pathogens of coccids and whiteflies. Vth 1–8. International Colloquium on Invertebrate Pathology and Microbial Shu Z, 1996. A study of the biology of Aschersonia aleyrodis Webber. Control. Society for Invertebrate Pathology, Adelaide, 111–115. Journal of Southwest Agricultural University 18: 524–526. Fawcett HS, 1908. Fungi parasitic upon Aleyrodes citri. University of Simmons MP, Ochoterena H, Carr T-G, 2001. Incorporation, rela- Florida Special Studies 1: 1–41. tive homoplasy, and effect of gap characters in sequence- Fawcett HS, 1936. Citrus diseases and their control. McGraw-Hill, based phylogenetic analyses. Systematic Biology 50: 454–462. New York. Sutton BC, 1980. The Coelomycetes: Fungi Imperfecti with Pycnidia, Fransen JJ, 1987. Aschersonia aleyrodis as a microbial control agent of Acervuli and Stromata. Commonwealth Mycological Institute, greenhouse whitefly. Ponsen en Looijen, Wageningen. Kew. Fransen JJ, van-Lenteren JC, 1993. Host selection and survival of Swofford DL, 1998. PAUP*: Phylogenetic Analysis Using Parsimony (*and the parasitoid Encarsia formosa on greenhouse whitefly, Tria- other methods). Version 4. Sinauer Associates, Sunderland, MA. leurodes vaporariorum, in the presence of hosts infected with Sydow H, Sydow P, 1916. Fungi amzonici a cl. E. Ule lecti. Annals the fungus Aschersonia aleyrodis. Entomologia Experimentalis et Mycologici 14: 65–97. Applicata 69: 239–249. Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: im- Hennings P, 1902. Fungi javanici novi. Hedwigia 41: 140–149. proving the sensitivity of progressive multiple sequence Hennings P, 1903. Fungi australienses. Hedwigia 42: 73–88. alignment through sequence weighting, positions-specific gap Korf RP, 1972. Synoptic key to the genera of the Pezizales. Myco- penalties and weight matrix choice. Nucleic Acids Research 22: logia 64: 937–994. 4673–4680. Kornerup A, Wanscher JH, 1967. Methuen Handbook of Colour. Tzean SS, Hsieh LS, Wu WJ, 1997. Atlas of Entomopathogenic Fungi. Methuen, London. Council of Agriculture. Executive Yuan, Taiwan. Liu M, Hodge K, 2005. Hypocrella zhongdongii sp. nov., the teleo- Vilgalys R, Hester M, 1990. Rapid genetic identification and morph of Aschersonia incrassata. Mycological Research 109: 818– mapping of enzymatically amplified ribosomal DNA from 824. several Cryptococcus species. Journal of Bacteriology 172: 4238– Liu M, Rombach MC, Humber RA, Hodge KT, 2005. What’s in 4246. a name? Aschersonia insperata: a new pleoanamorphic fungus Webber HJ, 1897. Sooty mold of the orange and its treatment. with characteristics of Aschersonia and Hirsutella. Mycologia 97: Bulletin of the U.S. Department of Agriculture Division of Vegetable 249–256. Physiology and Pathology 13: 1–34.