Mycol. Res. 109 (11): 1268–1275 (November 2005). f The British Mycological Society 1268 doi:10.1017/S0953756205003904 Printed in the United Kingdom. A new species of Hypocrella, H. macrostroma, and its phylogenetic relationships to other species with large stromata Priscila CHAVERRI1*, Joseph F. BISCHOFF2, Miao LIU1 and Kathie T. HODGE1 1 Department of Plant Pathology, Cornell University, 334 Plant Science Building, Ithaca, New York 14853, USA. 2 National Center for Biotechnology Information, National Institutes of Health, Bethesda, Maryland 20894, USA. E-mail : [email protected] Received 4 April 2005; accepted 19 July 2005. Two specimens of a new species of Hypocrella with large stromata were collected in Bolivia and Costa Rica. The morphology of the new species, H. macrostroma sp. nov., was compared with that of other species with large stromata, i.e. H. africana, H. gaertneriana, and H. schizostachyi. In addition, phylogenetic analyses of partial sequences from three genes, large subunit nuclear ribosomal DNA (LSU), translation elongation factor 1-a (EF1-a), and RNA polymerase II subunit 1 (RPB1), were conducted to determine the relationships of the new species to other species of Hypocrella/ Aschersonia. Phylogenetic analyses show that H. macrostroma belongs to a strongly supported clade that includes H. africana, H. schizostachyi, and Aschersonia insperata, whereas other Hypocrella species belong to two sister clades. Hypocrella macrostroma is described and illustrated, and a lectotype is designated for H. gaertneriana. INTRODUCTION have been linked to teleomorphs. Petch (1921) com- piled the most complete taxonomic work on Hypo- Species in the entomopathogenic genus Hypocrella crella/Aschersonia to date; he accepted 42 species. (Clavicipitaceae, Hypocreales, Ascomycota) are fre- Fungal biodiversity surveys in poorly explored geo- quently encountered in the tropics and less often in graphical regions, detailed morphological examina- the subtropics. These fungi are found growing on scale tions, and DNA sequence analyses will probably reveal insects (Coccidae, Homoptera) and whiteflies (Aleyro- many undescribed species. didae, Homoptera) that parasitize living leaves, or rarely Two specimens of an unidentified species of branches, of monocotyledonous and dicotyledonous Hypocrella with large stromata (3–22 mm diam) were plants. Hypocrella species have perithecia immersed collected in Costa Rica and Bolivia on stems of living in a brightly colored stroma and cylindrical asci that dicot vines. Generally, the stromata of Hypocrella/ contain four or eight multiseptate ascospores that Aschersonia are 2–5 mm diam, however, H. africana, disarticulate at maturity. Anamorphs of Hypocrella H. gaertneriana, H. schizostachyi (Hywel-Jones & are classified in the anamorph genus Aschersonia. The Samuels 1998), and the unidentified Hypocrella have latter state is more commonly found in the field, and markedly larger stromata that measure ca 5–30 mm in is sometimes associated with the teleomorph in diameter. These species are probably on scale insects the same stroma. Aschersonia is characterized by attached to branches of living dicotyledonous plants pycnidium-like conidiomata, phialides, sometimes (H. africana and the unidentified Hypocrella)or paraphyses, and unicellular, fusiform, hyaline conidia bamboo culms (H. gaertneriana and H. schizostachyi), that are brightly colored in mass and produced in whereas the majority of the Hypocrella/Aschersonia copious slime. species occur on scale insects or whiteflies on living Approximately 115 names in Hypocrella and 79 in leaves. The unidentified species resembles H. gaert- Aschersonia have been validly published; however, only neriana in the shape and size of the stroma; however, about 50 and 44 species, respectively, are currently the colour of the stroma and that the unidentified accepted (Petch 1921, Dingley 1954, Mains 1959a, b, species is on an insect on living dicotyledonous vines Hywel-Jones & Evans 1993). Only about 15 species suggested that it might be a new species. H. gaertneriana was described and illustrated by * Corresponding author. Present address: USDA-ARS, Systematic Botany and Mycology Laboratory, Rm. 304, B-011A, Møller (1901) based on a specimen from Brazil. 10300 Baltimore Avenue, Beltsville, Maryland 20705, USA. Specimens were deposited in the Berlin Botanical P. Chaverri and others 1269 Garden and Museum herbarium (B), but, unfortu- perithecia, asci and ascospores were characterized nately, a large part of the herbarium’s collection, by light microscopy. Colour terminology is from including Møller’s, was destroyed in 1943 (http:// Kornerup & Wanscher (1978). www.bgbm.fu-berlin.de/bgbm/research/colls/herb/). The only culture available for the unidentified H. gaertneriana was re-described and illustrated based species of Hypocrella was obtained from J.B. 115 on collections from Venezuela and French Guiana (ARSEF 7748) by isolating asci containing ascospores (Hywel-Jones & Samuels 1998); however, the authors and placing them on Difco potato dextrose agar (PDA) did not designate a neotype. In the present paper, with antibiotics. Morphological observations of the H. gaertneriana is lectotypified with the original illus- colonies and anamorph were based on cultures grown tration in Møller (1901). on PDA for four weeks in an incubator at 25 xC with Other genera in the Clavicipitaceae, such as alternating 12 h fluorescent light and 12 h darkness. Ascopolyporus, Dussiella, and Hyperdermium, also Measurements of continuous characters such as parasitize scale insects and have relatively large spore length were made using the beta 4.0.2 version stromata. In all these genera, the stromatal mass of Scion Image software (Scion, Frederick, MD). greatly exceeds that of the scale insect host. Sullivan Confidence intervals (a=0.05), minimum and maxi- et al. (2000) suggested that the large size of the stromata mum values for 10–30 anamorph and teleomorph results from a kind of secondary plant parasitism: once measurements (except where indicated) were calculated the fungus has consumed the scale insect body, the using Systat 8.0 (SPSS, Chicago, IL). fungus may continue to access plant nutrients through the insect’s stylet. Other authors have suggested that DNA extraction, PCR, and sequencing the mechanism of nutrient acquisition in species of Hypocrella with large stromata is through the living Cultures of Hypocrella sp. J.B. 115 and other scale insect that forms a bridge between the fungus Hypocrella/Aschersonia species used in the phylogenetic and the plant (Hywel-Jones & Samuels 1998). A similar analyses (Table 1) were grown on potato-dextrose mechanism has been observed in Septobasidium (Couch broth in a 6-cm-diam Petri plate for about one week. 1938). More detailed research is needed to elucidate The mycelial mat was harvested in a laminar flow this phenomenon. hood and then dried using clean, absorbent paper The main objectives of the present paper are: (1) towels. DNA was extracted with Ultra CleanTM Plant to describe a new species of Hypocrella with large DNA Isolation Kit (MO BIO Laboratories, Solana stromata; and (2) to show its phylogenetic relationships Beach, CA). To extract DNA from the herbarium to other species of Hypocrella/Aschersonia, by using specimen of Hypocrella sp. P.C. 605, the surface of the partial DNA sequences of three genes, i.e., large sub- stroma was first cleaned briefly with sterilized distilled unit nuclear ribosomal DNA (LSU), translation elon- water, then rehydrated by placing the stroma in a small gation factor 1-a (EF1-a), and RNA polymerase II Petri plate with sterilized distilled water and letting subunit 1 (RPB1). We also discuss whether Hypocrella it stand for a few minutes until the stroma became species with large stromata should be classified in a softer. Subsequently, a very thin layer of the surface separate genus. of the stroma was shaved off using a scalpel and then discarded. Pieces of the clean inner stroma, in- cluding centri, were cut out and then placed in a 1.5-mL MATERIALS AND METHODS Eppendorf tube for immediate DNA extraction with Ultra CleanTM Plant DNA Isolation Kit. Morphological examination Three partial gene regions were amplified, i.e., large Dried reference specimens were obtained from US subunit nuclear ribosomal DNA (LSU), translation National Fungus Collection (BPI; H. africana holotype elongation factor 1-a (EF1-a), and RNA polymerase II BPI 635731, and H. schizostachyi isotype BPI 635854) subunit (RPB1). The primers used were LSU: LRORf and the William and Lynda Steere Herbarium (NY; (5k-GTACCCGCTGAACTTAAGC-3k) and LR5r H. gaertneriana GJS 1776). Other specimens (Hypo- (5k-ATCCTGAGGGAAACTTC-3k) (Vilgalys & Hester crella sp. J.B. 115=CUP 67509 and P.C. 605=CUP 1990); EF1-a: 983f (5k-GCYCCYGGHCAYCGTG- 67508, and H. africana P.C. 736=CUP 67510) were AYTTYAT-3k) (Carbone & Kohn 1999) and 2218r (5k- collected during recent expeditions to Ghana, Costa ATGACACCRACRGCRACRGTYTG-3k) (Rehner Rica and Bolivia; these specimens are deposited at 2001); RPB1: cRPB1Af (5k-CAYCCWGGYTTYA- the Cornell University Plant Pathology Herbarium TCAAGAA-3k) and RPB1Cr (5k-CCNGCDATNTC- (CUP). Stromata of J.B. 115 and P.C. 605 were rehy- RTTRTCCATRTA-3k) (Castlebury et al. 2004). PCR drated briefly in distilled water with a trace of Tween1 protocols for LSU and EF1-a are described in Sung 80 (J. T. Baker Chemical, Phillipsburg, NJ). Then, et al. (2001) and Chaverri & Samuels (2003), respect- rehydrated stromata were supported by Tissue-Tek ively. PCR for RPB1 was conducted as follows: (1) O.C.T. Compound 4583 (Miles, Elkhart, IN) and 5 min at 95 x, (2) 40 cycles of denaturation at 95 x sectioned at a thickness of ca 15 mm with a freezing for 1 min, annealing at 50 x for 2 min, and extension at microtome. The characteristics of the stroma tissue, 72 x for 2 min, and (3) 72 x for 10 min. The resulting Hypocrella macrostroma sp. nov. 1270 Table 1. Isolates, geographic origin and GenBank numbers of species used in the phylogenetic analyses. GenBank accession nos Species Isolate/Specimen number Geographic origin LSU EF1-a RPB1 Aschersonia aleyrodis P.C. 445 Mexico AY986900* AY986925* DQ000326* A. andropogonis P.C. 535 Bolivia AY986901* AY986926* DQ000327* A.