Basidiopycnis Hyalina and Proceropycnis Pinicola1
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Mycologia, 98(4), 2006, pp. 637–649. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897 Two new pycnidial members of the Atractiellales: Basidiopycnis hyalina and Proceropycnis pinicola1 Franz Oberwinkler atractosomes in a more or less circular arrangement Universita¨t Tu¨ bingen, Lehrstuhl Spezielle Botanik und (Weiss et al 2004). Nucleotide sequence analyses Mykologie, Auf der Morgenstelle 1, D-72076, Tu¨ bingen, confirm the monophyly of this group (Swann et al Germany 2001). Morphologically, however, the members of Roland Kirschner Atractiellales possess a high degree of divergence. Botanisches Institut, J.W. Goethe-Universita¨t, Thus Helicogloea and Saccoblastia form resupinate Siesmayerstraße 70, 60323 Frankfurt am Main, fruit bodies, whereas Atractiella and Phleogena de- Germany velop stilboid fruit bodies (Oberwinkler and Bauer Francisco Arenal 1989). In addition several anamorphic hyphomycetes, Manuel Villarreal such as Infundibura adhaerens Nag Raj & W.B. Kendr. Vı´ctor Rubio and Leucogloea compressa (Ellis & Everh.) R. Kirsch- ner, recently were ascribed to the Atractiellales Dpartamento de Proteccio´n Vegetal, Centro Ciencias Medioambientales (CCMA-CSIC), Serrano, 115bis, E- (Bandoni and Inderbitzin 2002, Kirschner 2004). 28006 Madrid, Spain Bark beetle galleries recently have been discovered as a cryptic habitat of previously unknown taxa of Dominik Begerow2 basidiomycetes (Kirschner 2001; Kirschner et al 1999, Robert Bauer 2001a, b, c). Additional collections of fungi associated Universita¨t Tu¨ bingen, Lehrstuhl Spezielle Botanik und with conifers infested by beetles revealed additional Mykologie, Auf der Morgenstelle 1, D-72076, Tu¨ bingen, Germany hitherto undescribed taxa and shed a new light on the diversity of the Atractiellales. Here we describe two new atractielloid species forming pycnidia. Abstract: Two new genera, Basidiopycnis and Procer- opycnis, and two new species, Basidiopycnis hyalina MATERIALS AND METHODS and Proceropycnis pinicola, are described. An integrat- ed analysis of morphological, ecological, ultrastruc- Fungal isolation and growth conditions.—Samples of bark with beetles and their galleries were collected in tural and molecular data indicates that the new taxa Germany, Italy and Switzerland and treated for the belong to the Atractiellales. Relevant characteristics of detection of fungi carried by the beetles by cultivating the new genera and species are discussed and methods as described in Kirschner (2001). Samples of compared with those of related taxa. bark and wood of Pinus spp. infested by beetles were Key words: Atractiellales, bark beetles, molecular collected in Spain, China and Taiwan and examined for phylogeny, symplechosomes, ultrastructure, uredi- the presence of fungi with a dissecting microscope. niomycetes Conidial masses from the apex of the pycnidia growing in situ in the bark and beetle galleries were transferred with a needle to Petri dishes containing autoclaved INTRODUCTION pieces of spruce twigs (Picea abies [L.] Karst.) embedded The Atractiellales sensu Oberwinkler and Bauer in 1.5% water agar or malt-extract agar (MEA) plates (1989) contain the teleomorphic genera Helicogloea (containing 20 g Difco agar and 20 g Merck malt extract in 1000 mL distilled water). The plates were Pat., Saccoblastia Mo¨ller, Atractiella Sacc. and Phleo- incubated at 24 C and 80% RH 14 d and preserved as gena Link. All members are phragmobasidiate and frozen agar plugs in 20% glycerol vials at 280 C. Dry characterized by having symplechosomes (Bauer and material was deposited at the herbarium of the Oberwinkler 1991, Oberwinkler and Bauer 1989). University of Alcala´ de Henares (AH), KUN, TNM and The septal pore apparatus consists of a simple pore TUB). Living cultures are deposited at CCMA-CSIC, surrounded by an organelle-free zone. Except for CIBE-MSD, TUB and CBS culture collections. Saccoblastia, the organelle-free zone is delimited by Light microscopy.—Specimens were prepared on slides with lactophenol cotton blue or 5% KOH with staining Accepted for publication 10 May 2006. with 1% aqueous phloxine and observed under a light 1 Part 227 in the series ‘‘Studies in Heterobasidiomycetes’’ from the microscope. Photographs, drawings and measurements Botanical Institute, University of Tu¨bingen. were done with light microscopy. 2 Corresponding author. E-mail: dominik.begerow@uni-tuebingen. de Electron microscopy.—For transmission electron micros- 637 Mycologia myco-98-04-06.3d 14/8/06 13:42:34 637 Cust # 05-185 638 MYCOLOGIA TABLE I. Numbers of bark beetles carrying Basidiopycnis hyalina Number of beetles Percentage of beetles Beetle species carrying B. hyalina carrying B. hyalina Total number of beetles Crypturgus cinereus 6 4.7 127 Crypturgus pusillus 17 5.1 332 Dryocoetes autographus 8 2.4 334 Hylurgops palliatus 3 0.7 437 Ips typographus 37 3.2 1128 Orthotomicus laricis 3 0.6 495 Pityogenes chalcographus 67 6.8 976 Polygraphus poligraphus 5 7.5 67 Trypodendron lineatum 1 0.7 137 Beetles were collected 1994–1998 from bark of Picea abies and Pinus sylvestris in localities of Germany, Italy and Switzerland. The fungus was detected in culture after placing beetles individually into Petri dishes with media. copy (TEM) samples were fixed overnight with 2% frequencies: pA 5 0. 2498, pC 5 0. 2069, pG 5 0. 2670, glutaraldehyde in 0.1 M sodium cacodylate buffer pT 5 0.2763; substitution rates: A/C 5 0.9884, G/T 5 (pH 7.2) at 20 C. After six transfers in 0.1 M sodium 1.0000, A/G 5 3.0591, A/T 5 1.6186, C/G 5 1.2350, cacodylate buffer samples were postfixed in 1% osmium C/T 5 5.3811; gamma shape parameter 5 0.4996; tetroxide in the same buffer 1 h in the dark, washed in percentage of invariant sites 5 0.3527). Neighbor bidistilled water and stained 1% aquaeous uranyl joining analysis was done with genetic distances accord- acetate 1 h in the dark. After five consecutive washes ing to the specified substitution model. Parsimony in bidistilled water samples were dehydrated in acetone, analysis was done in two steps, where the first with 100 using 10 min transfers at 10, 25, 50, 70, 95% acetone, 000 random additions without swapping resulted in and three times in 100% acetone. Samples were nine islands and TBR swapping confirmed these islands embedded in Spurr’s plastic and sectioned with a di- to be most parsimonious with 2,378 steps (CI 5 0.449; amond knife. Ultrathin serial sections were mounted on RI 5 0.634; RC 5 0.284). One thousand replicates were formvar-coated, single-slot copper grids, stained with used for bootstrap analyses. The ITS alignment was built lead citrate at room temperature 5 min, and washed with Muscle v3.2 (Edgar 2004) and consisted of 408 bp. with bidistilled water. The samples were studied with Pairwise distances were calculated based on the K2P- a Zeiss EM 109 transmission electron microscope model after removing five positions with gaps. The operating at 80 kV. alignments are available on request. DNA amplification and sequencing.—DNA extraction and amplification procedures were carried out as described RESULTS AND DISCUSSION in Begerow et al (1997) and Boysen et al (1996). The primers used for amplification of SSU rDNA were NS1– Fungal isolation and growth conditions.—Two species NS8 (White et al 1990) and NS17UCB to NS24UCB of fungi with superficial pycnidium-like structures (Gargas and Taylor 1992). ITS sequences of Atractiella- and affinities to the Atractiellales were found in ceae were obtained with primers ITS1 and ITS4 (White conifer samples infested by bark beetles: a pycni- et al 1990). PCR products were purified with GFXTM dial sexual species and its synnematous anamorph PCR gel band purification kit (Amersham Pharmacia in samples from central Europe and a pycnidial TM Biotech Inc., USA) or QIAquick purification kit anamorph in samples from Spain, China and (QIAGEN, Germany). All regions amplified were Taiwan. Frequencies of detection of the sexual sequenced with the ABI PRISM Dye Terminator Cycle basidiomycete from different bark beetles in Sequencing Kit, on automatic sequencer ABI PRISM 373 ABLE or ABI PRISM 3100. New sequences are deposited at central Europe are provided (T I). The GenBank (DQ198779–DQ198798). frequencies of other associated fungi were shown in Kirschner (2001). Both species developed Phylogenetic analysis.—The SSU alignment was built with pycnidial structures on autoclaved spruce twigs MAFFT 3.85 (Katoh et al 2002) with the accurate and embedded in water agar, but on conventional iterative refinement method. After trimming ends the media like MEA only reduced conidiophores alignment consisted of 1701 bp. Phylogenetic analyses were carried out with PAUP* 4.0b10 (Swofford 1998) on developed. The sexual species produced basidio- a G5 Macintosh computer. Modeltest 3.0 (Posada and mata in culture only in the presence of other fungi Crandall 1998) was carried out to determine a model of (e.g. Graphium sp., Penicillium sp., Trichoderma sp). DNA substitution that fits the data set, and GTRIG was The pycnidial wall of both species is composed of selected from the Akaike information criterion (base one or two layers of parallel, hyaline and sparsely Mycologia myco-98-04-06.3d 14/8/06 13:42:45 638 Cust # 05-185 OBERWINKLER ET AL:NEW TAXA IN THE ATRACTIELLALES 639 FIG. 1. Photographs of Basidiopycnis hyalina. A. Teleomorph. Basidiomata in culture. B. Anamorph. Synnema with conidial mass in an apical slimy drop in culture. Bars: A 5 500 mm, B 5 300 mm. branched hyphae that easily can be separated pharum et basidiorum composita. Hyphae hyali- from each other. In the pycnidium-like structures nae, laeves, septatae, efibulatae, 1.5–3 mmdiam. of one species, however, basidia developed, and Basidia cylindracea, recta vel leniter curvata, 3- conidiophores developed in the pycnidia of the septata, 48–70 3 3–5 mm, sterigmatibus minutis other species. rectis. Basidiosporae hyalinae, laeves, aseptatae, clavatae, 20–45 3 2.5–4 mm, in massa mucosa Taxonomy.—Using morphological, ultrastructural aggregatae.