Mycological Progress 4(3): 239–250, August 2005 239 Geastrum pleosporus sp. nov., a new species of Geastraceae iden- tified by morphological and molecular phylogenetic data Clovis DOUANLA-MELI1,*, Ewald LANGER1 & Francisco D. CALONGE2 An unusual species of Geastrum was found growing on decayed wood debris and leaves of Triplochiton scleroxylon in the Mbalmayo Forest Reserve, Cameroon. The species morphologically resembles G. saccatum and G. fimbriatum in having sessile endosperidium partly enclosed by the saccate base of the exoperidium. Microscopically, it is characterized by and distinguished from all other known species of the genus, in having subsmooth, punctate to moderately verruculose, slightly thick- to distinctly thick-walled polymorphous, constricted to eight-shaped, mostly oblong, ovoid, cylindrical, elliptic to club- shaped basidiospores. G. pleosporus was studied from a collection of about fifteen basidiomata covering different stages of development. It is described as new based on morphological analyses and phylogenetic inferences made from large ribo- somal DNA sequence alignments. Phylogenetic relationship of G. pleosporus is investigated. In parsimony analyses of partial sequences of the large subunit rDNA from selected Gasteromycetes species, G. pleosporus is closely related to G. saccatum within the strongly supported clade of Geastrum species. The cluster of G. pleosporus and G. saccatum is well supported in parsimony analysis of the dataset with Geastrum species and related taxa using parsimony and maximum like- lihood analysis. Taxonomical novelty: Geastrum pleosporus Douanla-Meli Keywords: Earthstar, fibrilose-gleba, LSU rDNA, Lycoperdales, mycosclereids, peridium, polymorphous spores, subiculum he genus Geastrum was erected by PERSOON (1801) sical taxonomy. Morphologically identifying characteristics with G. coronatum Persoon as the type species. It is of Geastrum are the rays that curl back in a star-like manner, T the most widely distributed and complex genus in the this clears a space around the endoperidium which remains Puffballs and fungi with enclosed hymenophore. Species in seated in the cup thus formed, as it is the case in G. sessile (So- this genus are obviously recognised by the distinctly star-like werby) Pouzar and G. saccatum Fr., or the rays fold tightly basidiomata at maturity. The young fruitbody buried under over the spore sac and raise the latter like that in G. corona- leaf litter resembles a small, pointed, globular or dome-shaped tum Pers., and typically in G. fornicatum (Huds.) Hook., with puffball, lacking typical narrow stem and cap of other com- a peridium carried upon a pseudostem. In other species like mon fungi; the peridium is composed of not less than four G. schweinitzii (Berk. & M. A. Curtis) Zeller, the fruitbodies layers. As maturity approaches, the exoperidium splits from sit on a subiculum, a felt like mass of hyphae, which covers the apex downwards into several stellate lobes and exposes rotting wood. In some species the rays expand, but then close the endoperidium, which dehisces by a unique stoma. Within up over the endoperidium when they dry out. They are de- the family Geastraceae comprising two other genera, Myrio- scribed as hygroscopic and resemble hygroscopic species like stoma Desv. is distinguished from other earth stars by the mul- Astraeus hygrometricus (Pers.) Morgan. Significantly, how- tiple perforations on the spore sac and Radiigera Zeller is also ever, several species of Geastrum having hygroscopic rays similar to an earthstar except that it does not open. CUNNING- can be distinguished by the presence of a peristome, a colu- HAM (1979) estimated about 110 species of Geastrum, cur- mella, and generally smaller spores. Microscopically the strik- rently described are not more than 30 valid species in the genus. ing features of the genus concern the spores. They are glo- Up to 279 species have been described, basically using clas- bose, or less frequently subglobose, some shade of brown, and almost invariably verrucose or echinulate, produced on 4–8 spored basidia. Geastrum is the most common Gasteromycete genus in 1 Universität Kassel, Fachbereich 18 Naturwissenschaften, Institut für Biologie, Fachgebiet Ökologie, Heinrich-Plett-Staße. 40, 34132 Kas- Cameroon (CALONGE & DANIËLS 1998, HJORTSTAM et al., sel, Germany. 1993). In the framework of a research program aimed at the 2 Real Jardín Botánico, CSIC, Plaza de Murillo 2. E-28014 Madrid, assessment of fungal biodiversity in South Cameroon, pe- Spain. E-mail <[email protected]> * Corresponding author e-mail <[email protected]>, Tel.: (0049) 561 riodical collections were carried out. During 2002 we collected 8044215, Fax: (0049) 561 8044115 several specimens in the Mbalmayo Forest Reserve, belong- © DGfM 2005 240 DOUANLA-MELI, LANGER & CALONGE: Geastrum pleosporus spec. nov. ing to the genus Geastrum, and the study revealed well-known STONE & THOMPSON (1991) and HENRION, LE TACON & MAR- species such as G. saccatum and G. fimbriatum, representing TIN (1992). Material for DNA isolation (30–40 mg) was taken the first records for Cameroon, while other did not match with from the gleba tissue (mass of spore with capillitium) and the any of the described species (PONCE DE LEÓN 1968; CALON- exoperidium. Serial dilutions (1:100, 1:1000) were used as GE 1998; CALONGE & DEMOULIN 1975; SUNHEDE 1989; SOTO template for the polymerase chain reaction (PCR). Reaction & WRIGHT 2000, etc.). Besides the typical morphology re- for PCR amplification were performed in 50 µl mixture con- miniscent of Geastrum species, the spore patterns observed taining 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 75 mM in the whole collection were unknown in the genus. In this MgCl2, 10 mM of each of the four deoxynucleotide triphos- case an exclusively morphological study appears not suffi- phates, 25 pM of each primer, and 1 unit of Taq polymerase. ciently reliable. Therefore molecular tools for their identifi- The 5’ end domain of the nuclear DNA coding for the large cation were combined. Very limited molecular data are avail- ribosomal subunit (LSU rDNA) was amplified using the poly- able to infer phylogenetic relationships in Geastrum. In order merase chain reaction (MULLIS & FALOONA 1987; WHITE et to test the hypothesis that the fungus is related to Geastrum al. 1990) with NL1 and NL4 as primers (O’ DONNELL 1993). species, we sequenced a portion of the large subunit rDNA, The PCR reactions were performed with the TGradient Ther- and included it in two datasets of (a) Gasteromycete species mocycler 96, Biometra of Whatman Company. The cycle and (b) Geastrum species together with related taxa. Its phy- parameters were an initial denaturation at 94 °C for 3 min, fol- logenetic placement was investigated. Morphological and lowed by 35 cycles consisting of denaturation at 94 °C for 30 s, molecular studies suggested that this is a new species in Ge- annealing at 55 °C for 45 s and extension at 72 °C for 60 s, astrum. and a final elongation step of 7 min at 72 °C was included. PCR products were checked on 0.7 % agarose gel, and puri- fied with the QIAquickTM kit (QIAGEN) according to the ma- Material and methods nufacturer’s instructions. The DNA products were sequenced Basidiomata were collected and dried with a DÖRREX SIGG in both directions with the respective primers NL1 and NL4, TM 6252.10 (Switzerland). The description was made based on using the ABI PRISM Bigdye Terminator Cycle Sequenc- the examination of the collection containing fifteen fruitbo- ing Kit, Version 3.1, Applied Biosystems, and analysed using dies in all stages of development. Colour terms in parenthe- an automated DNA sequencer ABI 3100, Perkin Elmer. ses are those of KORNERUP & WANSCHER (1978). Herbaria are cited according to HOLMGREN, HOLMGREN & BARNETT Sequence alignment and phylogenetic analysis (1990). Holotype specimens are deposited at the herbarium of The contiguous nucleotide sequences were edited and assemb- Cryptogamy Laboratory, Faculty of Science, University of led using the programs EditSeq and MegAlign of Lasergene Yaoundé I, Cameroon (HUYI), an isotype at MA-Fungi. (DNASTAR 2000) software for Macintosh, and manually cor- rected with Se-Al version 2.0a11 (RAMBAUT 2002). Gaps (in- Line drawings, light and scanning electron micro- sertions/deletions) in the alignment were treated as missing scopy data. All positions were included in the final alignment. The Morphological description was based on fresh material. Micro- three sequences of Geastrum we determined have been depo- scopic structures were mainly observed on dried material. sited in Genbank with the accession numbers AY566241 and Free-hand thin sections were mounted in KOH (5 %) for more AY566242 for G. pleosporus and AY714318 for G. fimbria- detailed observations. Light microscopy studies were carried tum. Additional sequences used for comparative analyses were out at 400 and 1000 X using an Olympus BX51TF micros- obtained from GenBank (Tab. 1). We assembled two data sets cope. Line drawings were made using a grid as drawing aid of LSU sequences by selecting species of Gasteromycetes, and representing a magnification of 52000. Size ranges of basi- a selection of Geastrum species together with related species. diospores were based on at least 50 basidiospores; light pho- The aligned sequences are deposited in TreeBase in NEXUS tography was done with a Nikon COOLPIX 4500 Digital, and format, with accession numbers SN1798. processed in Photoshop (Adobe Photoshop 4.0). For the SEM Phylogenetic analysis