View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library Mycol Progress (2007) 6:35–44 DOI 10.1007/s11557-007-0524-2 ORIGINAL ARTICLE A novel clade of sporocarp-forming species of glomeromycotan fungi in the Diversisporales lineage Dirk Redecker & Philipp Raab & Fritz Oehl & Francisco J. Camacho & Regis Courtecuisse Received: 13 October 2006 /Revised: 12 January 2007 /Accepted: 12 January 2007 / Published online: 14 February 2007 # German Mycological Society and Springer-Verlag 2007 Abstract In the early times of taxonomy of arbuscular porales. The phylogenetic divergence from other known mycorrhizal fungi (Glomeromycota), exclusively sporo- species suggests that this clade may constitute a new genus. carpic species were described. Since then the focus has These findings will have important consequences for taxon mainly shifted to species forming spores singly. For many definition in the Diversisporales. They will facilitate of the sporocarpic species, no molecular data have been identification of these fungi using rDNA sequences within made available, and their phylogenetic position has colonized roots or the environment. remained unclear. We obtained small subunit ribosomal rDNA and internal transcribed spacer data from specimens of glomeromycotan sporocarps from tropical areas that were assigned to three morphospecies. The complete Introduction sequence of the 18S small rDNA subunit sequence, internal transcribed spacers (ITS) 1 and 2 and 5.8S rDNA subunit, Arbuscular mycorrhiza is a symbiosis formed by the vast was determined from a sporocarp of Glomus fulvum. Partial majority of land plants. In this interaction, the plant partner sequences of the small subunit and the other regions were provides carbohydrates to the fungal symbiont, which obtained from Glomus pulvinatum and the newly described improves the uptake of mineral nutrients using its fine species Glomus megalocarpum. Molecular phylogenetic hyphal network. Approximately 200 morphospecies of analyses placed all species analyzed as a monophyletic arbuscular mycorrhizal fungi in ten genera have been sister group to the Diversispora spurca/Glomus versiforme described, mostly based on spore morphology. These fungi clade group (“Glomus group C”) within the Diversis- were previously placed in the family Endogonaceae (Gerdemann and Trappe 1974), later in the order Glomales of the Zygomycota (Morton and Benny 1990). Recently, a Taxonomic novelties: Glomus megalocarpum D. Redecker : : separate phylum Glomeromycota was established for them D. Redecker (*) P. Raab F. Oehl (Schüßler et al. 2001). As the spore morphology is rather Botanical Institute, University of Basel, simple in many species, molecular phylogenetic methods Hebelstrasse 1, Basel 4056, Switzerland have helped to clarify the evolutionary relationships in the e-mail: [email protected] Glomeromycota (Redecker and Raab 2006). One result of these analyses was that the largest genus Glomus, with F. J. Camacho about 70 species, is polyphyletic (Redecker et al. 2000a; Department of Environmental Science, University of California, Riverside, CA 92521, USA Schwarzott et al. 2001). Fungi falling within the previous morphological definition of Glomus have been shown to be R. Courtecuisse in at least four different monophyletic lineages. In fact, Faculté des sciences pharmaceutiques et biologiques, some dimorphic species even form glomoid spores as well Département de Botanique, B.P. 83, as other types of spores (Redecker et al. 2000a). Some of 59006 Lille Cedex, France these lineages were separated in the new genera (Morton 36 Mycol Progress (2007) 6:35–44 and Redecker 2001), partly based on molecular characters. sporocarps were collected on the Caribbean islands of One remaining clade of Glomus comprises Glomus group A Guadeloupe and Martinique in August/September 2005 by (e.g. G. intraradices, G. mosseae) and group B (e.g. G. Christophe Lécuru. Details were as follows: specimen CL/ etunicatum, G. claroideum), which are rather distantly Mart05-35: Ste. Luce, Forêt départementalo-domaniale de related but still form a monophyletic group together Montravail, on forest floor; specimen CL/Mart05-049: Case (Schwarzott et al. 2001). pilote, Crête Jean Louis (Martinique), fruiting on dead Another Glomus clade, referred to as Glomus group C, is wood; specimen CL/Guad05-51: Vieux-Fort (Guadeloupe), more closely related to the Acaulosporaceae in rDNA Ravine Blondeau, mesophile forest, on forest floor, close to phylogenies. It comprises the species G. versiforme, G. Boletellus sp.; specimen CL/Mart05-111: Forêt départa- spurcum, and G. aurantium, as well as an isolate fitting the mentalo-domaniale de La Source Berry (Martinique), morphological definition of G. etunicatum,whichis plantation of mahogany with indigenous species. otherwise thought to belong to group B. Glomus group C Slides to examine the spore morphology were obtained was recently defined as a new family Diversisporaceae with by scratching a few spores from the surface of cross- Diversispora spurca as type species of the new genus sections and embedding them into Polyvinyl alcohol (Walker and Schüßler 2004). Other species have not been lactoglycerol (PVLG, Koske and Tessier 1983). Pictures formally renamed. Because of the scarcity of morphological of the whole sporocarps or parts of them were taken using a characteristics on the light microscopic level that could aid Hewlett Packard ScanJet 5470 flatbed scanner at maximum separation from other Glomus species, signature sequences resolution (2,400 dpi). Microscopical pictures of the spores in the 18S ribosomal subunit were used for the definition of were taken with an Olympus DP70-CU CCD camera Diversispora as well as the corresponding family and order. attached to a Zeiss Axioplan compound microscope equipped Numerous Glomus species form sporocarps, which with differential interference contrast optics. For size mea- mostly are relatively undifferentiated assemblages of surements, 20 spores of each specimen were used, unless spores. An exception are some of the species previously indicated otherwise. Colors were described as a percentage of placed in the genus Sclerocystis. However, it was shown CMYK values using the INVAM color chart (http://invam. that Sclerocystis is an advanced clade within a group of caf.wvu.edu/otherinfo/articles/colorchart.htm). well-defined nonsporocarpic Glomus group A species DNA was extracted from 10 mg subsamples using a (Redecker et al. 2000b). Generally, the sporocarp-forming Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). habit is not well correlated with phylogeny, and sporocarpic Polymerase chain reaction (PCR) was conducted in two as well as nonsporocarpic representatives can be found in steps as a nested PCR using various primer combinations. different phylogenetic groups. Very often, sporocarp for- For amplifying the complete 18S small subunit of specimen mers are closely related to species forming spores singly in 1, universal primers were used (White et al. 1990). the soil, or a single species is known to form sporocarpic Preliminary experiments with universal primers had shown and nonsporocarpic spores (e.g. G. mosseae, G. corona- that the specimens belonged to the Diversisporales clade. tum). Early taxonomy of the Glomales focused very much Therefore, a primer was designed for specific amplification on sporocarpic species, a trend that has not continued of the 3′ end of the small subunit and the ITS of this lineage recently. The phylogenetic relationships of many of the (GLOC1355: TCTTAGAGGGACTATTGGCATTT). It was sporocarpic species listed in the groundbreaking study of used in the second step of the nested PCR together with Gerdemann and Trappe (1974) have not been elucidated. ITS4i (Redecker et al. 2003), as second primer. In the first Therefore, it is impossible to recognize these taxa if they PCR step primers NS5/ITS4 (White et al. 1990) were used. are detected by molecular methods within colonized roots. Cycling parameters were as described by Hijri et al. (2006). In this study, we sequenced regions of the ribosomal PCR products were ligated into pGEM-T (Promega, rDNA of some sporocarpic glomeromycotan species col- Wallisellen, Switzerland) and used to transform competent lected in tropical regions to elucidate their phylogenetic Escherichia coli DH5alpha. Clones were screened by blue/ relationships and to allow the design of specific primers for white screening and colony PCR and sequenced in both molecular detection of these species. directions using a Applied Biosystems Prism Dye Termi- nator 3.1 kit on an ABI 310 capillary sequencer (Applied Biosystems, Foster City, USA). Materials and methods Sequence Navigator (Applied Biosystems) and Bio Edit (Hall 1999) were used to proofread and assemble the Specimen AC/Pohn99-001 was collected by Francisco J. sequences. The near-complete sequence of the small Camacho in 1999 in a native forest at the ridge trail near the subunit was assembled from sequenced fragments using top of Sokehs Mountain on the pacific island of Pohnpei, variable regions for verifying that the fragments were from Federated States of Micronesia (6.97°N 158.22°E). Other the same organism. To exclude chimaera formation, the Mycol Progress (2007) 6:35–44 37 fragments were subjected separately to phylogenetic anal- Table 1 Specimens studied and sequences obtained from them ysis in PAUP* 4 (Swofford 2001). Moreover, the sequences Species Specimen Origin Sequences were tested using
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