Elaphomyces Lilacinus Sp. Nov. (Ascomycota, Eurotiomycetes), a New Hypogeous Species from Navarre (Spain)

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Elaphomyces lilacinus sp. nov. (Ascomycota, Eurotiomycetes), a new hypogeous species from Navarre (Spain)

Julio CABERO Abstract: a new species of Elaphomyces from forests of Fagus sylvatica in the artikutza Reserve (Navarre, Pablo ALVARADO spain) is proposed, supported by diagnostic morphological features and a distinct genetic profile. images Julián ALONSO of fresh specimens, as well as observations made with an optical microscope are provided. Isabel SALCEDO Keywords: beech, Elaphomycetaceae, Fagus sylvatica, phylogeny, taxonomy. David MORENO-MATEOS Resumen: se propone una nueva especie de Elaphomyces localizado en los bosques de Fagus sylvatica en la Reserva de artikutza (Navarra, España), apoyado en características morfológicas macroscopicas, micro- Ascomycete.org, 11 (6) : 229–232 scópicas y diagnósticas con un perfil genético distinto. se proporcionan imágenes de especímenes frescos, Mise en ligne le 24/12/2019 así como observaciones hechas con un microscopio óptico. 10.25664/ART-0279 Palabras clave: haya, Elaphomycetaceae, Fagus sylvatica, filogenia, taxonomía.

Introduction 28s rdNa (n=57). The aligned data set was loaded in MRBayEs 3.2.6 (RoNquisT & HuElsENBECK, 2003) to perform a Bayesian inference (Bi) analysis (GTR+G model, two simultaneous runs, six chains, temper- The genus Elaphomyces T. Nees is typified by E. granulatus Fr. Most ature set to 0.2, sampling every 100th generation). The runs contin- European species were proposed by ViTTadiNi (1831) and TulasNE & ued until the convergence parameters were met and the standard TulasNE (1841, 1851), but recently, several new taxa were created by deviation fell below 0.01 after 0.9 M generations. The first 25% of Paz & GoNzálEz (2008), and Paz et al.. (2012, 2017). in the latter work, trees were discarded as burn in. Finally, a full search for the best- a deep review was conducted to reorganize the whole genus at scoring Ml tree was performed in RaXMl 8.2.10 (sTaMaTaKis, 2014) species and section levels with the aid of genetic data, and as a re- using the standard search algorithm (GTRCaT model, 2000 boot- sult, four new species were proposed in Elaphomyces, while another strap replications). The significance threshold was set above 0.95 Bi four were excluded from this genus. in the present work, a new for posterior probability (PP) and 70% Ml bootstrap proportions species is proposed to accommodate specimens collected in (BP). Navarre (spain), which do not fit into any known taxon. several other putatively new species from different fungal groups have been found in the same area (artikutza Reserve), and they will be Taxonomy published in parallel with the present one. Elaphomyces lilacinus J. Cabero, d. Moreno-Mateos & P. alvarado, Material and methods sp. nov. – MycoBank: MB 833189 – Pl. 2 Diagnosis: differs from the other Elaphomyces spp. because of its Morphological study. — samples were found with the aid of cottony lilac coating, as well as its large size (up to 3.5 cm), its low trained dogs. Macro images were taken with a sony dC G9 camera and soft peridium warts, and spores ornamented with a labyrinth- with an olympus 60 mm f. 2.8 objective. For light microscopy, sam- like pseudo-reticulum. ples were mounted in water, Congo Red, iKi or KoH and observed Typification: Holotype here selected aH 49155. in an olympus BHs microscope with aPo optics. Eighty spores per Etymology: lat. “lilacinus” refers to the lilac tinges of the coating specimen were arbitrarily selected and measured with Mycomètre mycelium. 2.07 (George Fannechère). samples of gleba were slightly scrapped over a slide and gold-metalized in a BalT-TEC sCd005 sputter coater Description: Ascomata irregularly globose, reniform or some- for scanning electron microscopy (sEM), which was performed in a what flattened, measuring 2–3.5 cm in diameter, sometimes pre- JEol JsM 6360lV device (15× to 290.000×, 4.5 nm resolution, 20 kV) senting a small depression, surface presenting irregularly at the university of santiago de Compostela (usC, spain). after their hemispheric warts about 0.9 mm wide and lower than 0.7 mm high, study, samples were deposited in the herbarium of the university coalescing at the base. The surface is covered with a pruinose or cot- of alcalá (aH), at alcalá de Henares (Madrid, spain). tony lilac coating where substrate and mycelium abut, odour not Phylogenetic analysis. — Total dNa was extracted from dry detected. Peridium with 1) a blackish exoperidium about 1 mm specimens using a standard CTaB method (MuRRay & THoMPsoN, thick, formed by cylindric hyphae with abundant septa forming cells 1980). PCR reactions (Mullis & FalooNa, 1987) included 35 cycles with 8–13 × 2–3 µm, occasionally intermixed with inflated elements, and an annealing temperature of 54 ºC. Primers iTs1F and iTs4 (WHiTE et 2) an endoperidium 4.0–5.0 mm thick, filamentous, lilac or slightly al.., 1990; GaRdEs & BRuNs, 1993) were employed to amplify the iTs vinaceous in color, composed of densely packed cylindrical ele- rdNa region, while lR0R and lR5 (VilGalys & HEsTER, 1990; CuBETa et ments about 1.5–1.7 µm in diameter, arranged sinuously, forming al.., 1991) were used for the 28s rdNa region. PCR products were small pockets of globose cells about 3–4.5 µm in diameter. Gleba checked in 1% agarose gels, and positive reactions were sequenced cottony, slightly granulose or pulverulent when mature, forming an with one or both PCR primers. Chromatograms were edited by hand abundant capillitium, dark grayish to blackish with subtle lilac to remove putative reading errors using MEGa 5.0 (TaMuRa et al.., tinges, formed by bifurcate and slightly nodulous septate hyphae 2011). an alignment of 28s rdNa was built. The most closely related with cell walls about 2 µm. Hymenium separated from the en- taxa were obtained from GenBank and uNiTE and added to the doperidium by a thin whitish layer. Asci hyaline, globose and sessile, alignments (sequences primarily from Paz et al.., 2017). sequences thick-walled, containing 6–8 spores. Ascospores at first subhyaline, were first aligned in MEGa 5.0 with ClusTalW followed by manual turning brownish-beige or yellowish-brown with age, and finally correction. The final alignment included 294/790 variable sites in looking dark brown or blackish. all of them are covered by a thin

229 hyaline perisporium layer that breaks and vanishes easily. spores Studied collections: sPaiN: Navarre, Goizueta, artikutza Reserve measure (19.25–) 20.0–22.0 (–23.75) µm (average 21.35 µm, orna- (N 43°13‘01’’ W 1°46’23’’), under Fagus sylvatica, in acidic soil, leg. mentation included, n=50). spores are ornamented with short and J. Cabero, d. Moreno-Mateos, 22 February 2019, aH 49155 (holotype), JC190221Ky (isotype). GenBank 28s rdNa MN595286, iTs straight aculei not exceeding 2 µm high, densely packed and some- rdNa MN595299. times coalescing into short ridges sometimes looking like a labyrinthine reticulum under sEM. Discussion Habitat. Currently known only from Fagus sylvatica forests in montane zones of Navarre (spain), but probably present also in Three sections are currently recognized within genus other mountain ranges of southern Europe, and maybe also in low- Elaphomyces: sect. Elaphomyces, sect. Ascoscleroderma (Clémencet) land forests of central Europe. Fruiting in groups of 4 to 7. Bellanger & P.-a. Moreau, and sect. Ceratogaster (Corda) Fr. (Paz et

KP115823 Eurotiomycetes 1.00/100 AF454072 TrTrichorichocoma parradoxadoxxaa 1.00/99 FJ358290 TrTrichorichocoma parradoxadoxxaa MH872089 Byssochlamys nivea 1.00/100 MH872041 Byssochlamys nivea sect KX238881 Elaphomyces atrroopurprpurereus EPITYPE

0.96/91 KX238873 Elaphomymyces mutabilliis EPITYPE . 1.00/100 NG_059246 Elaphomyces adadamizans TYPE Asc

JN168735 Pseudodotulostoma volvatum oscler KX238866 Elaphomyces foetidus EPITYPE KX238874 Elaphomyces cyanosporurus EPITYPE 1.00/98 KX238875 Elaphomyces perrssoonii EPITYPE od 0.95/75

NG_059247 Elaphomycemyces labyrrintintthhinus TYPE er 1.00/97 1.00/100 KT694149 Elaphomymyces favosus TYPE ma 0.99/87 KT694145 Elaphomyces favosus 1.00/100 KT694142 Elaphomyces iuppiitercrcellus NG_059245 Elaphomyces iupppitercrcellus TYPE 1.00/99 HM357248 Elaphomyces gguangdongensis JC190221KY Elaphomyces lilacinus TYPE KR029779 Elaphomycemyces morerettiii

0.99/90 KR029781 Elaphomycemyces leveilllei sect 1.00/100 (0.69/46) 0.99/80 KR029780 Elaphomycmyces leveeillleei KR029775 Elaphomycemyces maculatus . C

KX238868 Elaphomyces leonis er a KX238865 Elaphomycemyces spirroosporurus TYPE t og 0.98/73 KR029776 Elaphomycemyces septatus as 1.00/99 KX238871 Elaphomyces septatatus TYPE t

KR029778 Elaphomycemyces virrgrgatosporurus er 0.99/87 KR029777 Elaphomycemyces aculeatus KX238880 Elaphomycees aculeatus 1.00/99 1.00/100 KR029774 Elaphomycey s anthraracinus KR029773 Elaphomyces anthraracinus 0.99/98 1.00/100 NG_064408 Elaphomyces anthraracinus f. talosporurus KR029770 Elaphomycemyces sps . 6 0.98/83 KR029769 Elaphomyces sps . 6 1.00/99 KX238872 Elaphomyces papillatus EPITYPE KX238864 Elaphomyces sp.

1.00//75 1.00/94 KR029742 Elaphomycemyces decipiens KX238876 Elaphomycemyces dedecipiens NEOTYPE 1.00/100 KR029745 Elaphomyces sp .1 KR029747 Elaphomycemyces sp. 1

KX238879 Elaphomycmyces quercicorcicola TYPE sect 0.99/97 KR029737 Elaphomycemyces muricatricatus

1.00/9/ 9 .

0.94/96 KR029735 Elaphomycemyces muricatricatus E la 1.00/100 KR349921 Elaphomyces sp. p

KR349920 Elaphomyces sp. h o

JN713148 Elaphomycemyces digitadigitatus m

1.00/100 JN713147 Elaphomycemyces digitadigitatus y ce KR029767 Elaphomyces graranulatus s KR029765 Elaphomycey s sp. 5 KT232217 Elaphomycemyces graranulatus 1.00/9/ 3 KR029752 Elaphomycemyces sp. 2 0.60//97 KR029751 Elaphomycemyces sp. 2 KR029761 Elaphomyces sp. 4 0.83/72 KR029757 Elaphomyces sp. 3 0.99/99 KR029754 Elaphomycemyces asperrulusulus 0.02 0.86/80 KX238863 Elapphomycemycey s aspperurulus

Plate 1 – Consensus phylogram obtained in MrBayes from a 28s rdNa alignment of genus Elaphomyces. Values next to nodes represent Bayesian PP and maximum likelihood BP. only nodes supported by >0.95 Bayesian PP or >70% BP are annotated, although some non-si- gniﬁcant values are annotated in parentheses.

230 Ascomycete.org Plate 2 – Elaphomyces lilacinus (holotype) a–E: ascomata; F: Peridium; G: young spores; H: Mature spores; i–J: spores under sEM. scale bars: a = 1 cm; B = 0.5 cm; C–d= 1 cm; E = 0.5 cm; F = 20 µm; G = 20 µm; H = 10 µm; i = 5 µm; J = 2 µm.

Ascomycete.org 231 al.., 2017). The new species E. lilacinus belongs to this last one (Plate Mullis K. & FalooNa F.a. 1987. — specific synthesis of dNa in vitro 1), sect. Ceratogaster = Elaphomyces sect. Sclerodermei (Vittad.) via a polymerase-catalyzed chain reaction. Methods in Enzymol- E. Fisch., characterized by species with dark-colored spores. Paz et ogy, 155: 335–350. doi: 10.1016/0076-6879(87)55023-6 al.. (2017) accept two subsections within this clade, subsect. Sclero- MuRRay M.G. & THoMPsoN W.F. 1980. — Rapid isolation of high molec- dermei (around E. anthracinus Vittad.) with species lacking mycelial ular weight plant dNa. Nucleic Acids Research, 8 (19): 4321-4325. patches in their surface, and subsect. Maculati (around E. maculatus Paz a. & GoNzálEz J.l. 2008. — una nueva especie de ascomiceto en- Vittad.), with species presenting differently colored mycelial patches. contrado en España. Butlleti de la Associació Micològica Font i Quer, in the present analysis based on 28s rdNa data, E. lilacinus was not 8: 4–7. significantly related to either subsect. Maculati or subsect. Sclero- Paz a., laVoisE C., BaRRio l., RiCHaRd F. & MoREau P.-a. 2012. — Prop- dermei, but to E. guangdongensis B.C. zhang, forming a basal branch uesta de dos nuevas especies del género Elaphomyces, dos of sect. Ceratogaster, in the same way as E. iuppitercellus Castellano primeras citas para la Península ibérica y una clave de identifi- & T.W. Henkel. Elaphomyces morettii Vittad. and E. leveillei Tul. & C. Tul. cación de las especies del género para Europa. Boletín Micologico are probably related to subsect. Maculati, but no significant support was found in the present analysis based on 28s rdNa data (PP 0.69, de la FAMCAL, 7: 85–104. BP 46). Paz a., BEllaNGER J.-M., laVoisE C., Molia a., ŁaWRyNoWiCz M., laRssoN E., Elaphomyces lilacinus (Plate 2) is clearly different from the most olaRiaGa i., JEPPsoN M., læssøE T., sauVE M., RiCHaRd F. & MoREau P.-a. closely related species of sect. Ceratogaster. Elaphomyces guangdon- 2017. — The genus Elaphomyces (ascomycota, Eurotiales): a ribo- gensis and E. iuppitercellus have both striate spores, while E. lilacinus somal dNa-based phylogeny and revised systematics of Euro- presents spores ornamented with a pseudo-reticulum, suggesting pean ‘deer truffles’. Persoonia, 38: 197–239. doi: 10.3767/ that spore ornamentation could have varied several times in the 003158517X697309 evolutionary history of these basal lineages of sect. Ceratogaster. RoNquisT F. & HuElsENBECK J.P. 2003. — MrBayes 3: Bayesian phyloge- Elaphomyces maculatus and E. leveillei have more or less granulose netic inference under mixed models. Bioinformatics, 19 (12): 1572– spores under the light microscope, they have smaller ascomata than 1574. doi: 10.1093/bioinformatics/btg180 E. lilacinus, and a smooth peridium covered by a greenish (instead sTaMaTaKis a. 2014. — RaxMl Version 8: a tool for phylogenetic anal- of lilac) mycelium. Elaphomyces morettii has granulose spores prob- ysis and post-analysis of large phylogenies. Bioinformatics, 30 (9): ably ornamented with small isolated or coalescing spines that 1312–1313. doi: 10.1093/bioinformatics/btu033 sometimes form short ridges (Paz et al.., 2017), but the ascomata TaMuRa K., PETERsoN d., PETERsoN N., sTECHER G., NEi M. & KuMaR s. 2011. have a coarsely warted peridium, and lack a mycelium coating. — MEGa5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony Acknowledgements methods. Molecular Biology and Evolution, 28 (10): 2731–2739. TulasNE l.-R., & TulasNE C. 1841. — sur le genre Elaphomyces, et de- dr. david Moreno-Mateos has been funded for this research by scription de quelques espèces nouvelles. Annales des Sciences na- the spanish Ministry of Economy and Competiveness through so- turelles, Bot., 2e ser., 16: 5–27, pl. 1–4. cietal Challenge Program (grant CGl2015-70452-R) and María de TulasNE l.-R. & TulasNE C. 1851. — Fungi hypogaei. Histoire et mono- Maeztu excellence accreditation MdM-2017-0714. We also thank graphie des champignons hypogés. Paris, F. Klincksieck. asunción Rodríguez for her help during the collection of samples. VilGalys R. & HEsTER M. 1990. — Rapid genetic identification and map- ping of enzymatically amplified ribosomal dNa from several Cryp- References tococcus species. Journal of Bacteriology, 172 (8): 4238–4246. doi: 10.1128/jb.172.8.4238-4246.1990

CuBETa M.a., ECHaNdi E., aBERNETHy T. & VilGalys R. 1991. — Character- ViTTadiNi C. 1831. — Monographia Tuberacearum. Milano, Ty- ization of anastomosis groups of binucleate Rhizoctonia species pographia F. Rusconi. using restriction analysis of an amplified ribosomal RNa gene. WHiTE T.J., BRuNs T., lEE s. & TayloR J.W. 1990. — amplification and di- Phytopathology, 81: 1395–1400. doi: 10.1094/Phyto-81-1395 rect sequencing of fungal ribosomal RNa genes for phylogenet- GaRdEs M. & BRuNs T.d. 1993. — iTs primers with enhanced specificity ics. In: iNNis M.a., GElFaNd d.H., sNiNsKy J.J. & WHiTE T.J. (eds.). PCR for Basidiomycetes—application to the identification of mycor- Protocols: A guide to methods and applications. New york, aca- rhizae and rusts. Molecular Ecology, 2 (2): 113–118. demic Press: 315–322.

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1: J. Cabero – Barrio del Carmen 6, 49800 Toro, Zamora, Spain – [email protected] 2: P. Alvarado – La Rochela 47, 39012 Santander, Spain – [email protected] 3: J. Alonso – University of Santiago de Compostela (USC), Dept of Crop Production and Engineering Projects, Rúa Benigno Ledo, Campus Terra, 27002 Lugo, Spain – [email protected] 4: I. Salcedo – Dept. Biología Vegetal y Ecología, Facultad de Ciencia y Tecnología, UPV/EHU, Apdo. 644, 48080 Bilbao, Spain – [email protected] 5: D. Moreno-Mateos – Basque Centre for Climate Change (BC3) / Ikerbasque Foundation for Science, Parque Cientíﬁco UPV/EHU, Barrio Sarriena s/n, 48940 Leioa, Spain – [email protected]

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