Persoonia 28, 2012: 25–33 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE http://dx.doi.org/10.3767/003158512X635056 Phylogeny and taxonomy of the genus Gliocladiopsis L. Lombard1, P.W. Crous1, 2, 3 Key words Abstract Using a global set of isolates and a phylogenetic approach employing DNA sequence data from five genes ( -tubulin, histone H3, internal transcribed spacer region, 28S large subunit region and translation elonga- Gliocladiopsis β tion factor 1- ), the taxonomic status of the genus Gliocladiopsis (Glionectria) (Hypocreales, Nectriaceae) was phylogeny α re-evaluated. Gliocladiopsis sagariensis is reinstated as type species for the genus, which proved to be distinct taxonomy from its former synonym, G. tenuis. The purported teleomorph state of G. tenuis, Glionectria tenuis, is shown to be distinct based on morphological comparisons supported by phylogenetic inference, and is provided with a new name, Gliocladiopsis pseudotenuis. A further four species, mostly isolated from soil, are newly described, namely G. curvata (New Zealand, Ecuador and Indonesia), G. elghollii (USA), G. indonesiensis (Indonesia) and G. mexicana (Mexico). Although species of Gliocladiopsis are frequently isolated from roots of diseased plants or plant litter in soil, little is presently known of their ecology, or potential role as plant pathogens. Article info Received: 1 February 2012; Accepted: 29 February 2012; Published: 6 March 2012. INTRODUCTION The first phylogenetic study conducted on this generic complex was that by Schoch et al. (2000), which clearly showed that Glio- The genus Gliocladiopsis was introduced by Saksena (1954) cladiopsis was closely related to Gliocephalotrichum/Leuco- based on G. sagariensis to accommodate a fungal isolate from nectria, and removed from Cylindrocladiella, Cylindrocarpon soil that had penicillate conidiophores resembling Penicillium and Calonectria (Fig. 1). Furthermore, the genus Glionectria and Gliocladium, and cylindrical conidia similar to that of Calo- was proposed as teleomorph of Gliocladiopsis in this study, and nectria (as Cylindrocladium). Saksena (1954) distinguished defined by perithecia that are obovoid to broadly obpyriform, G. sagariensis from Penicillium and Gliocladium based on with warted, red-brown walls and dark red stromatic bases, morphological differences in conidium and conidiogenous ap- producing ellipsoidal, 1-septate ascospores. paratus morphology, and the apparent lack of chlamydospore Presently Gliocladiopsis accommodates three species which formation in culture. Agnihothrudu (1959), however, was able include G. irregularis (Crous & Peerally 1996), G. sumatrensis to observe chlamydospore formation in culture, and based on (Crous et al. 1997) and G. tenuis (Crous & Wingfield 1993), this as well as morphological similarity, synonymised G. saga- and is defined by densely penicillate conidiophores lacking a riensis under Cylindrocarpon tenue (Bugnicourt 1939). In con- stipe extension and terminal vesicle, and produce small, nar- trast, Barron (1968) considered Gliocladiopsis as a later syn- row, cylindrical, (0–)1-septate conidia held in yellow droplets, onym of Calonectria (as Cylindrocladium). and chains of globose, brown chlamydospores (Crous 2002). Crous & Wingfield (1993) resurrected the genus Gliocladiopsis Over the course of several years a collection of Gliocladiopsis to accommodate species characterised by dense, penicillate isolates have been accumulated in the culture collection of the conidiophores, which unlike Cylindrocladiella and Calonectria, CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Nether- lacked sterile stipe extensions. Based on the characteristic lands. These isolates were identified as G. tenuis based on conidiophores, the genus Cylindrocarpon was also found to be morphological comparisons only. The aim of this study was to unsuitable to accommodate these species. These observations reconsider the taxonomic status of the genus Gliocladiopsis led Crous & Wingfield (1993) to place C. tenue in Gliocladiopsis, using multigene phylogeny and morphological comparisons retaining G. sagariensis as synonym. Watanabe (1994) trans- to correctly identify these isolates. ferred G. tenuis to Cylindrocladium based on observations that isolates of Cylindrocladium and Cylindrocladiella generally lose their ability to produce stipe extensions with continuous subcul- MATERIALS AND METHODS turing, and therefore he rejected this feature as a stable charac- ter to define these genera. Various morphological studies have Isolates shown, however, that the presence of a stipe extension and the Isolates and ex-type strains of Gliocladiopsis spp. were obtained terminal vesicle shape is an important character to distinguish from the CBS-KNAW Fungal Biodiversity Centre (CBS) and species of Calonectria (Crous & Wingfield 1994, Lombard et other culture collections as indicated in Table 1. These isolates al. 2010a–c) and Cylindrocladiella (Crous & Wingfield 1993, were either isolated from plant material or baited from soil using Victor et al. 1998, van Coller et al. 2005, Lombard et al. 2012). the methods described by Crous (2002). 1 CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, Phylogeny the Netherlands; corresponding author e-mail: [email protected]. Total genomic DNA was extracted from single-conidial isolates 2 Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. grown on 2 % malt extract agar (MEA) for 7 d, using the Ultra- 3 Wageningen University and Research Centre (WUR), Laboratory of Phyto- Clean™ Microbial DNA isolation kits (Mo Bio Laboratories, pathology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. Inc., California, USA) according to the manufacturer’s protocol. © 2012 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 26 Persoonia – Volume 28, 2012 93 C. pauciramosa CMW 5683; GQ280730 C. scoparia CPC 1675; GQ280679 Calonectria 58 C. spathiphylli CBS 116168; GQ280750 C. morganii CBS 110666; GQ280748 99 C. brasiliensis CBS 230.51; GQ280746 81 X. guianense CBS 112180; AY793443 Xenocylindrocladium 86 X. guianense CBS 112179; JQ666073 C. cigneum CPC 1595; AY793431 100 C. cigneum CBS 109168; JQ666074 Curvicladium C. cigneum CBS 101411; JQ666075 L. clusiae ATCC 22228; AY489732 98 86 L. clusiae AR 2706; U17412 Gliocephalotrichum / Leuconectria 89 G. bulbilium CBS 254.82; JQ666076 G. cylindrosporum CBS 112956; JQ666077 68 G. sagariensis CBS 199.55; JQ666078 72 G. tenuis IMI 68205; JQ666079 51 G. pseudotenuis CBS 116074; JQ666080 Gliocladiopsis 93 G. sumatrensis CBS 754.97; JQ666081 75 G. irregularis CBS 755.97; JQ666082 C. gregarius CBS 101073; JQ666083 100 Cylindrocarpostylus C. gregarius CBS 101072; JQ666084 D. celtidis CBS 115994; AY793438 Dematiocladium I. liriodendri CBS 112602; HM364323 Ilyonectria N. fuckeliana CBS 125133; HM446654 87 N. neomacrospora CBS 198.62; HM364316 Neonectria 93 N. ditissima CBS 100316; HM364311 87 N. ramulariae CBS 151.29; HM364324 C. novaezelandiae CBS 486.77; JN099212 96 C. pseudohawaiiensis CBS 210.94; JN099174 66 C. camelliae CPC 234; JN0099249 Cylindrocladiella 91 C. peruviana IMUR 1843; JN099266 93 C. natalensis CBS 114943; JN099178 Nectria cinnabarina CBS 278.48; HM484729 0.005 Fig. 1 Neighbour-Joining tree (Kimara-2-parameter) using only the partial LSU sequence alignment with bootstrap values after 1 000 repetitions. Partial gene sequences were determined for β-tubulin (BT), PAUP (Phylogenetic Analysis Using Parsimony, v. 4.0b10, histone H3 (HIS3), internal transcribed spacer region (ITS), Swofford 2002) was used to analyse the DNA sequence data- 28S large subunit region (LSU) and translation elongation fac- set. Phylogenetic relationships were estimated by heuristic tor 1-α (TEF 1-α) using the primers and protocols described searches with 1 000 random addition sequences and tree by Lombard et al. (2010b). bisection-reconnection was used, with the branch swapping To ensure the integrity of the sequences, the amplicons were option set on ‘best trees’ only. All characters were weighted sequenced in both directions with the same primer pairs used equally and alignment gaps were treated as missing data. for amplification, and subsequent alignments were generated Measures calculated for parsimony included tree length (TL), using MAFFT v. 6 (Katoh & Toh 2010), and manually corrected consistency index (CI), retention index (RI) and rescaled con- where necessary. sistence index (RC). Bootstrap analysis (Hillis & Bull 1993) was Congruency of the sequence datasets for the separate loci, based on 1 000 replications. with the exception of LSU, were determined using tree topolo- A second phylogenetic analysis using a Markov Chain Monte gies of 70 % reciprocal Neighbour-Joining bootstrap trees with