Mycosphere Doi 10.5943/mycosphere/4/1/2 Detecting the variability of Hydnum ovoideisporum (Agaricomycetes, Cantharellales) on the basis of Italian collections, and H. magnorufescens sp. nov. Vizzini A1*, Picillo B2, Ercole E1 , Voyron S1 and Contu M3 1Dipartimento di Scienze della Vita e Biologia dei Sistemi, Università degli Studi di Torino, Viale P.A. Mattioli 25, 10125 Torino, Italy 2Via Roma 139, 81017 S. Angelo d’Alife (CE), Italy 3Via Marmilla 12, 07026 Olbia, Italy Vizzini A, Picillo B, Ercole E, Voyron S, Contu M 2013 – Detecting the variability of Hydnum ovoideisporum (Agaricomycetes, Cantharellales) on the basis of Italian collections, and H. magnorufescens sp. nov. Mycosphere 4(1), 32–44, Doi 10.5943 /mycosphere/4/1/2 Hydnum ovoideisporum is a species of the Hydnum rufescens complex recently described from Spain, and characterized by having a pileus with deep orange tones, ovoid to broadly ellipsoid basidiospores, and growing in Mediterranean areas on calcareous soil. In this paper, four South Italian collections found under Quercus and traditionally determined as H. rufescens, were analyzed on the basis of morphological and molecular (ITS) data; three of them were proven to be conspecific with H. ovoideisporum even though they differ from the type by globose to subglobose spores, and one has a smooth hymenophore. H. ovoideisporum f. depauperatum f. nov. is established for accommodating specimens with a smooth hymenophoral surface. Finally, H. magnorufescens sp. nov. is established for a collection with large basidiomes without strong orange-reddish tinges, and with globose to subglobose spores. Key words – Biodiversity – cantharelloid clade – Hydnaceae – ITS nrDNA phylogeny – taxonomy Article Information Received 11 December 2012 Accepted 2 January 2013 Published online 28 January 2013 *Corresponding author: Alfredo Vizzini – e-mail – [email protected] Introduction smooth, colourless, and cystidia usually absent The genus Hydnum L., typified by H. (Hall & Stuntz 1971, as Dentinum Gray; Maas repandum L., traditionally encompasses fungi Geesteranus 1971, 1974, 1975, 1976, Harrison with pileate and stipitate, usually bright & Grund 1987, Forte & Pieri 1993, Otto 1997, coloured basidiomata (whitish to yellow or Pegler et al. 1997, Arnolds 2003). They are orange-reddish) characterized by an aculeate terrestrial in deciduous and coniferous forests hymenophore, with spines paler than pileus or (Maas Geesteranus 1971, 1975), occasionally concolorous, a fleshy, brittle, homogeneous, also on decayed wood, and ectomycorrhizal not zoned monomitic context, with inflating, (Raidl & Agerer 1992, Agerer et al. 1996, thin-walled, branched, clamped hyphae, basidia Kraigher & Agerer 1996, Tedersoo et al. cylindrical-clavate, (3-)4-5(-6)-spored, stichic 2010). The genus was classified in (basidia with nuclear spindles arranged Cantharellales Gäum. by Kreisel (1969) on the longitudinally) with basal clamp-connection, basis of its stichic basidia; this placement was spores subglobose to obovoid-elliptical, corroborated by recent molecular analyses 32 Mycosphere Doi 10.5943/mycosphere/4/1/2 (Pine et al. 1999, Moncalvo et al. 2006, monophyletic clades consisting of collections Matheny et al. 2007). In particular, Pine et al. with ovoid to broadly ellipsoid basidiospores, (1999) placed it close to Craterellus Pers. and which they formally described as new species: Cantharellus Adans. ex Fr., Moncalvo et al. Hydnum ovoideisporum Olariaga, Grebenc, (2006) and Matheny et al. (2007) in the Salcedo & M.P. Martín, widespread in the cantharelloid clade as sister to Sistotrema Fr. Iberian-Mediterranean area, distinguished by Within Hydnum, species identification the pileus with deep orange tones and strong relies primarily on the observation of a rather preference for calcareous soils; Hydnum limited set of features, such as the basidioma vesterholtii Olariaga, Grebenc, Salcedo & M.P. habit, pileus color, presence of umbilicus in the Martín, from the Pyrenees, mainly pileus, spine shape and attachment to the stipe, characterized by a ochre to light ochre pileus. a central or excentric stipe, number of The purpose of the present study was to sterigmata in the basidia, and spore shape determine, using both morphological and (Maas Geesteranus 1971, 1975, Pegler et al. molecular characters (ITS region), whether 1997, Arnolds 2003, Ostrow & Beenken 2004). Italian specimens collected under Quercus in Five Hydnum species are traditionally Mediterranean areas and characterized by recognized in Europe separated by their Hydnum rufescens-like basidiomes and globose morphological differences, namely Hydnum to subglobose basidiospores represent new albidum Peck, H. repandum L., H. rufescens phyletic lines or are attributable to already Pers., H. umbilicatum Peck and H. described species or phylogenetic clades. Some ellipsosporum Ostrow & Beenken (Hrouda of these collections are distinguished by having 1999, Ostrow & Beenken 2004, Huhtinen & an entirely smooth hymenophore. Ruotsalainen 2006, Grebenc et al. 2009). Within these taxa H. rufescens is characterized Methods by having usually small and slender basidiomata with orange-reddish colours, a not Morphology umbilicate pileus, typically central stipe, free to Macromorphological features were adnate, not decurrent and not spathulate aculei, described from fresh specimens. The and globose to subglobose spores (Ostrow & micromorphological descriptions are based Beenken 2004, Huhtinen & Ruotsalainen 2006, upon study of both fresh and herbarium Grebenc et al. 2009). Agerer et al. (1996), material, the latter rehydrated in 5% KOH and using a PCR-RFLP approach, unveiled a high stained in ammoniacal Congo red or 1% variability of the ITS region of H. rufescens Phloxin. Thirty-two spores were measured in specimens from different European areas. This 5% KOH per collection. The number of high variability was later confirmed by the ITS collections measured is indicated by a “c”. The phylogenetic analysis on European Hydnum width of each basidium was measured at the species by Grebenc et al. (2009), which yielded widest part, and the length was measured from six well supported monophyletic clades (named the apex (sterigmata excluded) to the basal as RU1–RU6) for the specimens septum. The following abbreviations are used: morphologically attributable to H. rufescens; in Q = the quotient of length and width of the general, they were not able to assign specific spores in side view; Qm = average quotient. distinguishing morphological features to any Colour terms in capital letters (e.g. Mikado clade. Data from Agerer et al. (1996) and Orange, Plate III) are those of Ridgway (1912). Grebenc et al. (2009) supported the idea of Author citations follow the Index Fungorum- several new, yet undescribed phylogenetic Authors of Fungal Names species within the collections with the typical (http://www.indexfungorum.org/authorsoffung morphological features of H. rufescens alnames.htm). Herbarium abbreviations are (hereafter referred as the H. rufescens according to Thiers (2012) except that BP complex). Finally, Olariaga et al. (2012) refers to the personal herbarium of B. Picillo. recognized within the H. rufescens complex the Diagnoses of the new taxa are deposited in same six clades, RU1–RU6 (all with globose to MycoBank (http://www.mycobank.org/De- subglobose spores) and two additionally faultPage.aspx). 33 Mycosphere Doi 10.5943/mycosphere/4/1/2 DNA extraction, PCR amplification, and MCMC were run over 10 million generations. DNA sequencing Trees were sampled every 1,000 generations Genomic DNA was isolated from 1 mg resulting in an overall sampling of 10,001 of four dried herbarium specimens (TO trees. The first 2,500 trees were discarded as HG2815-TO HG2818), by using the DNeasy “burn-in” (25%). The “burn-in” value was Plant Mini Kit (Qiagen, Milan Italy) according evaluated using Tracer 1.5 (Rambaut & to the manufacturer’s instructions. Universal Drummond 2007). For the remaining trees, a primers ITS1F/ITS4 were used for the ITS majority rule consensus tree showing all region amplification (White et al. 1990, Gardes compatible partitions was computed to obtain & Bruns 1993). Amplification reactions were estimates for Bayesian Posterior Probabilities performed in a PE9700 thermal cycler (Perkin- (BPP). Branch lengths were estimated as mean Elmer, Applied Biosystems) following Vizzini values over the sampled trees. ML estimation et al. (2011). The PCR products were purified was performed through RAxML v.7.0.4 with the AMPure XP kit (Beckman) and (Stamatakis 2006) with 1,000 bootstrap sequenced by MACROGEN Inc. (Seoul, replicates (Felsenstein 1985) using the Republic of Korea). Sequences were checked GTRGAMMA algorithm to perform a tree and assembled using Geneious v5.3 inference and search for a good topology. (Drummond et al. 2010), submitted to Support values from bootstrapping runs (MLB) GenBank were mapped on the globally best tree using (http://www.ncbi.nlm.nih.gov/genbank/) and the “-f a” option of RAxML and “-x 12345” as their accession numbers are reported in Table 1 a random seed to invoke the novel rapid and Fig. 1. bootstrapping algorithm. BI and ML analyses were run on the CIPRES Science Gateway web Sequence alignment and phylogenetic server (Miller et al. 2010). Only BPP values analysis over 0.70 and MLB over 50% are reported in Sequences obtained in this study were the resulting tree (Fig. 1). Pairwise % identity compared to those available in the GenBank values of ITS sequences were calculated using