Karstenia 56: 27–38 A new species of Typhula with sigmoid spores: Typhula suecica IBAI OLARIAGA, GILLES CORRIOL, ISABEL SALCEDO and KAREN HANSEN OLARIAGA , I., CORRIOL, G., SALCEDO, I. & HANSEN, K. 2016: A new species of Typhula with sigmoid spores: Typhula suecica. – Karstenia 56: 27–38. HELSINKI. ISSN 0453–3402. The new species, Typhula suecica, is morphologically characterized by having partially sigmoid spores, projecting thick-walled stipe hairs, and by lacking clamp connections. The polygonal rind cells of the sclerotia are also diagnostic. Phylogenetic analyses of 58 new sequences of the LSU and tEF-1α regions show that specimens of T. suecica form a supported monophyletic group, deeply nested in Typhula. Typhula suecica appears to be a widely distributed but rare species that occurs on leaves of angiosperm trees (Acer, Alnus, Populus) in various types of damp forests. Material from South France, the Span- ish slope of the Pyrenees and Central Sweden is reported here. Key words: Typhuloid fungi, species delimitation, sclerotium Ibai Olariaga, Swedish Museum of Natural History, Department of Botany, P.O. Box 50007, SE-104 05 Stockholm, Sweden; e-mail: [email protected] Isabel Salcedo, Department of Plant Biology and Ecology (Botany), University of the Basque Country (UPV/EHU), Apdo 644, E-48080 Bilbao, Spain; e-mail: isabel.sal- [email protected] Gilles Corriol, Conservatoire botanique national des Pyrénées et de Midi-Pyrénées. Vallon de Salut, B.P. 315, F-65203 Bagnères-de-Bigorre Cedex (France); e-mail: gilles. [email protected] Karen Hansen, Swedish Museum of Natural History, Department of Botany, P.O. Box 50007, SE-104 05 Stockholm, Sweden; e-mail: [email protected] Introduction a comprehensive monograph of Typhula and al- lied genera is available (Berthier 1976), the dis- The genus Typhula (Pers.: Fr.) Fr. contains spe- tribution, host range, and morphological vari- cies with small clavarioid basidiomata, often ability of most species are poorly known. Since arising from sclerotia, and typically with amy- this monograph was published, contributions loid spores and dextrinoid hyphae. A few stud- focusing on Typhula have been very sporadic. A ies indicate that T. phacorrhiza (Reichard: Fr.) handful of new species has been described from Fr., selected type of Typhula, nests in the Agaric Asia (Hoshino et al. 2009, Ikeda et al. 2015), clade (Moncalvo et al. 2002; Dentinger et al. North America (Berthier & Redhead 1982) 2006), but the monophyly of Typhula has not and Europe (Olariaga et al. 2008, Olariaga & been tested so far. Typhuloid fungi is one of the Salcedo 2009), but many more await formal taxonomically most under-researched groups of description (Olariaga 2009), especially in the Basidiomycota, likely because its tiny basidio- Nordic countries. According to our estimate, mata are overlooked by mycologists. Although Typhula has ca 70 known species in the world, 28 OLARIAGA ET AL. TYPHULA SUECICA KARSTENIA 56 (2016) but the actual number of species is certainly DNA extraction, PCR amplification and sequencing. higher since undescribed species are found of- DNA was extracted from fresh basidiomata stored in 1% SDS extraction buffer, from living cultures or from dried ten when conducting fieldwork. A few Typhula basidiomata. DNA extractions were carried out using the species are known to be plant pathogens (Ek- DNeasy Plant Minikit (QIAGEN GmbH, Hilden, Germa- strand 1955; Matsumoto et al. 1996; Vergara et ny). The regions amplified were the partial nuclear LSU al. 2004), but most species fruit on dead culms rDNA (spanning domains D1 and D2) and the transcrip- and leaves and apparently do not affect the host tion elongation factor 1-alpha (tEF-1α) regions. The LSU was amplified employing the LR0R and LR5 primers plants. A few species produce basidiomata on (Vilgalys & Hester 1990). The tEF-1α (1200 bp) was am- twigs from mostly angiosperm trees. plified using the primers Tef1-F and 2218R (Morehouse Species recognition in Typhula has relied on et al. 2003; Rehner & Buckley 2005), or using the newly morphological characters (Berthier 1976, Olari- developed internal Typhula specific primers, 1567R-Typh (5´-ACHGTRCCRATACCDCCRATCTT-3´) and 1567F- aga 2009). Molecular data has been little used Typh (5´-AAGATYGGHGGTATYGGYACDGT-3´), to test species boundaries in Typhula, with the with Tef-1F and 2218R, respectively. Amplification con- exception of economically important pathogenic ditions follow Hansen et al. (2013). PCR products were species (Hsiang & Wu 2000, Vergara et al. 2004). purified using ExoSAP-IT® (USB, Cleveland, OH, USA) While the vast majority of Typhula species has or when multiple bands were obtained, products were gel purified using QIAquick Gel Extraction Kit (QIAGEN). never been sequenced, a few recently described Sequencing was performed by Macrogen (the Nether- species have been supported using analyses of lands). Sequences were assembled and edited with Se- the ITS region (Hoshino et al. 2009, Ikeda et quencher 4.7 software (Gene Codes Corp., Ann Arbor, al. 2015). In our experience, however, the ITS Michigan) and submitted to the EMBL/GenBank data- bases (Cochrane et al. 2010). The alignment was done in region is too variable to align unambiguously AliView (Larsson 2014) and optimized manually. across all of Typhula. No other DNA region Phylogenetic analyses. Specimens sequenced in this has previously been used for assessing species study are presented in Tab. 1. Gene congruence was eval- boundaries in Typhula. uated manually comparing supported clades among sin- This paper aims to continue to describe the di- gle-gene genealogies (Mason-Gamer & Kellogg 1996). Each locus was subjected to a Maximum Likelihood versity of Typhula. The new species T. suecica, (ML) analysis using the ‘RAxML HPC2 on XSEDE’ tool treated under the provisional name “Typhula (Stamatakis 2006) via CIPRES Science Gateway (Miller sigmoideospora” by Olariaga (2009), is here for- et al. 2010), employing a GTR-GAMMA model and start- mally described based on material from France, ing from a random tree. For branch confidence, 1000 ML bootstrap replicates were conducted using rapid boot- Spain and Sweden. After an extensive biblio- strapping. A supported clade for one marker was consid- graphical search, and examining most of the type ered to be in conflict when contradicted with significant specimens of Typhula in Europe, it was found support by another (bootstrap support > 70%). Since no not to conform to any described species. conflict was detected, the LSU and tEF-1α regions were combined into a single alignment. Macrotyphula fistu- losa (Holmsk.: Fr.) R.H. Petersen (IO.14.214) was used as outgroup, based on unpublished 6-locus-analyses of Material and methods the Agaric clade that found it is the closest sister group to Typhula. Introns in the tEF-1α region were excluded Morphological study. The macroscopic descriptions are because they were too variable to align. The alignment based on both fresh and dried material. Colour codes are was analysed with two partitions, LSU and tEF-1α, using from Munsell Color Corporation (1990). The dried mate- Bayesian and Maximum-likelihood (ML) approaches. rial was rehydrated and observed in Congo Red in 5% The Bayesian analysis was conducted in MrBayes 3.2.1. KOH. Melzer´s reagent was used to check for the amy- (Ronquist et al. 2012). The substitution models were sam- loid and dextrinoid reactions. Basidiospore measurements pled across the GTR space (Ronquist et al. 2012). Two were made in side view. Abbreviations describing basidi- parallel analyses of four MCMCMC chains were run for 5 M generations, starting from a random tree, and sam- ospore size are: Lm = mean length, Wm = mean width, pling one tree every 1000th generation. In order to check Qm = Lm/Wm; 25 basidiospores were measured per collec- tion. Basidia measurements exclude the sterigmata. The whether the chains had converged, whether the mixing terminology adopted follows Remsberg (1940), Berthier was adequate and to choose an appropriate burn-in, log- (1976) and Kirk et al. (2008). Geographic coordinates are likelihood values were plotted against the time generation given in decimal grades (WGS84) and were obtained in with Tracer 1.5 (Rambaut & Drummond 2007). Station- situ or from http://www.maps.pixelis.es/. The collections arity was assumed when the average standard deviation examined in this study are deposited in BIO, S and UPS of split frequencies fell below 0.01. A burn-in of 2500 herbaria (Thiers 2012). trees was discarded from each run. To assess branch con- KARSTENIA 56 (2016) OLARIAGA ET AL. TYPHULA SUECICA 29 Table 1. Specimens included in the molecular phylogenetic analyses, with GenBank accession numbers for LSU and tEF-1α- regions. Numbers in parentheses following the species names indicate multiple collections of a species. The GenBank accessions of sequences generated in this study are in bold. Species Voucher Country nucLSU tEF-1α specimen Macrotyphula fistulosa IO.14.214 Spain, Huesca, Lanuza KY224088 KY224117 Typhula athyrii (1) IO.14.197 France, Pyrénées atlantiques, Le Gave d´Aspe KY224089 KY224118 Typhula athyrii (2) IO.14.117 Sweden, Uppland, Rickebasta NR KY224090 KY224119 Typhula berthieri IO.15.42 Sweden, Dalarna, Särna KY224091 KY224120 Typhula capitata ss. IO.14.62 Sweden, Ångermanland, Kroktjärn KY224092 KY224121 Berthier Typhula caricina IO.14.110 Sweden, Uppland, Skärlinge KY224093 KY224122 Typhula crassipes IO.14.83 Sweden, Medelpad, Sundjöåsen NR KY224094 KY224123 Typhula culmigena IO.14.02