Barssia hellenica sp. nov. (, ), a new hypogeous species from Greece

Vasileios KAOUNAS Summary: A new species of Barssia () is described from Greece based on morphological and Carlo AGNELLO genetic studies. Macroscopic images of fresh specimens, as well as observations made under optical micro- Pablo ALVARADO scopes, drawings and a 28S nLSU phylogenetic tree are provided. The present work is also accompanied by Monica SLAVOVA a brief discussion and a key to the known species of this genus. Keywords: hypogeous fungi, Europe, phylogeny, taxonomy, Helvellaceae.

Ascomycete.org, 7 (5) : 213-219. Août 2015 Mise en ligne le 21/08/2015

Introduction step 10 min. PCR products were checked in 1% agarose gels, and positive reactions were sequenced with primer ITS4 or LR0R. Chro- matograms were checked searching for putative reading errors, and In 2013, an unknown hypogeous was collected in Greece, these were corrected. and we soon realized we probably discovered a not yet described Phylogenetic analyses. — BLAST was used to select the most species. In 2015 we were able to collect again several specimens, to closely related sequences from INSD public databases. Sequences bring to completion the morphological study and to conduct a phy- came mainly from CARBONE et al. (2013). 28S nLSU sequences were logenetic study with the two known collections to confirm our hy- first aligned in MEGA 5.0 (TAMURA et al., 2011) with the closest pothesis. The constant and laborious field work with hypogeous matches in public databases (O’DONNELL et al., 1997; BONITO et al., fungi, often under really peculiar conditions, is sometimes rewarded: 2013; NGUYEN et al., 2013; CROUS et al., 2014). The aligned loci were Barssia hellenica is another demonstration of the special fungal di- loaded in PAUP* 4.0b10 (SWOFFORD, 2001) and subjected to MrMo- versity of Greece, encouraging us to continue researching to en- deltest 2.3 (NYLANDER, 2004). Model GTR+Γ+I was selected and im- hance our knowledge. plemented in MrBayes 3.1 (RONQUIST & HUELSENBECK, 2003), where a Bayesian analysis was performed (data partitioned, two simulta- Materials and methods neous runs, six chains, temperature set to 0.2, sampling every 100th generation) until convergence parameters were met after about Morphological study 60,000 generations, standard deviation having fell below 0.01. Si- The macroscopic study was conducted on fresh and dried speci- gnificance threshold was set above 0.90 posterior probability (PP). mens. Micromorphological observations were performed under three different trinocular microscopes with plan achromatic lenses Taxonomy (4×, 10×, 40× and 100× oil immersion). Micromorphological features were studied and measured in water mounts. The negative amyloid Barssia hellenica Kaounas, Agnello, P. Alvarado & Slavova sp. reaction was checked with Melzer’s reagent. Cotton blue was used nov. — MycoBank 813811 to highlight spore ornamentations, and Congo red was employed to stain cell walls of different elements. Finally the dimensions of the Diagnosis: Differs from other Barssia spp. by ovoid ascospores ascospores were obtained by measuring at least 50 randomly selec- (>70%), irregularly clavate or broadly ellipsoid asci and the habitat ted ascospores. under Abies cephalonica. Typification: Holotypus here selected MCVE 28663 Phylogenetic analysis Etymology: hellenica from Hellas (Greece), the country where the DNA extraction, amplification and sequencing. — Total DNA species was discovered. was extracted from dry specimens blending a portion of them with the aid of a micropestle in 600 μl CTAB buffer (CTAB 2%, NaCl 1.4 M, Description EDTA pH 8.0 20 mM, Tris-HCl pH 8.0 100 mM). The resulting mixture Ascocarps hypogeous, 0.5–2.7 × 0.5–2.0 cm in diam., spherical to was incubated for 15 min. at 65ºC. A similar volume of chloroform: subspherical, elongated and compressed, creating an irregular isoamyl alcohol (24:1) was added and carefully mixed with the sam- shape, more or less lobed, usually with an irregular apical orifice, so- ples until their emulsion. It was then centrifugated for 10 min at metimes creating folds on the inside, reddish orange to brownish 13.000 g, and the DNA in the supernatant was precipitated with a red or blackish brown. The surface is covered by irregular polygonal volume of isopropanol. After a new centrifugation of 15 min at the warts, densely packed together when young, and more isolated same speed, the pellet was washed in cold ethanol 70%, centrifu- with age, sometimes giving the appearance of a network. Peridium gated again for 2 min and dried. It was finally resuspended in 200 μl 260–420 μm, composed by a pseudoparenchymatous hyphal struc- of ddH2O. PCR amplification was performed with the primers ITS1F ture with polygonal cells measuring 20–41.5 × 15–31.0 μm, amber and ITS4 for ITS region (WHITE et al., 1990; GARDES & BRUNS, 1993), while to brownish in color and thick walled (2–5 μm) in the outer layer, co- LR0R and LR5 were used to amplify the 28S nLSU region (VILGALYS & lorless and with thinner walls in the internal layer. Presence of rare, HESTER, 1990). PCR reactions were performed under a program yellowish, light brown hairs, simple or branched, septate, thick-wal- consisting of a hot start at 95ºC for 5 min, followed by 35 cycles at led emerging from the terminal cells of the peridium with two diffe- 94 ºC, 54 ºC and 72 ºC (45, 30 and 45 s respectively) and a final 72 ºC rent external surfaces: the first smooth and/or few incrusted, the 213 second verrucose and/or incrusted, measuring up to 100 × 9 μm. Discussion Gleba mostly compact, whitish, sometimes looking a bit cottony,

with irregular sinuous, labyrinth-like and vertical straight veins, whi- The genus Barssia was first proposed by ILKEYG (1925) describing tish or yellowish, gelatinous; sterile part formed by interwoven sep- the species B. oregonensis collected in Oregon (USA) by H.P. Barss. tate hyphae, 2–5 μm in diam. Paraphyses, hyaline, cylindrical, This species has red-orange ascocarps with irregular protrusions, an simple or forked, septate, longer than the asci, 3–6(–8) μm wide. opening at the top, a solid gleba, and ellipsoid, smooth, hyaline Asci irregularly clavate or broadly ellipsoid, with a protuding hump ascospores (15 × 26 μm), lacking a mycelium tuft at the base of the at the apex and a pleurorynchous base, inamyloid, 8-spored, mea- ascoma. GILKEY (1925) classified the genusBarssia within the family Tuberaceae Dumort., but it was later transferred by TRAPPE (1979) to suring 110–185 × 36–45 μm. Ascospores ovoid, rarely ellipsoid, the family Balsamiaceae E. Fisch. However, after the ultrastructural smooth, hyaline, irregularly arranged inside the asci, measuring 21– study of KIMBROUGH et al. (1996) and phylogenetic inference analyses 27 × 16.5–20,5 μm (23.5 × 18.1 μm on average), Q (L/l) = 1.18–1.43 conducted by O’DONNELL et al. (1997) and LÆSSØE & HANSEN (2007), (Qm= 1.30), usually containing one or two large oil drops and seve- Balsamia and Barssia were shown to represent a sister clade to other ral smaller droplets. genera in the family Helvellaceae Fr., and hence considered a part of Habitat: Ascomata are found growing solitary, hypogeous, under this family. Abies cephalonica, 1040 m alt. In the protologue of the B. oregonensis, it is said to be associated with Rhamnus purshiana (literally “Cascara Sagrada”), but it has al- Studied collections: GREECE, 13.III.2013, Mainalon Arkadia, under ways been found later associated with Pseudotsuga menziesii in Abies cephalonica, leg. G. Proutzopoulos, MCVE 28664, Genbank LSU North-Western USA, finally considered as abundant especially in KT350939, ITS KT350942; 04.VI.2015, Parnitha Attica, under Abies ce- young plants (TRAPPE et al., 2007; TRAPPE et al., 2009). There is a single phalonica, leg. V. Kaounas, MCVE 28663 (holotype), Genbank LSU European collection identified asB. oregonensis (ŁAWRYNOWICZ & KT350940, ITS KT350941. SKIRGIEŁŁO, 1984) found on calcareous soil of the Carpathian Moun-

Plate 1 — Barssia hellenica ABCF: MCVE 28663 (holotype); DE: MCVE 28664. Photos V. Kaounas 214 Plate 2 — Barssia hellenica. Microscopic characters 215 Plate 3 — Barssia hellenica. Microscopic characters 216 Legend of Plate 2. A: Section with peridium and gleba in water; BC: ted that it might be a synonym of Barssia oregonensis. In our opinion, Peridium in water; DE: Peridium inside; F: Gleba in Congo red; G-O: its placement in the family Helvellaceae, probably in the genus Bars- Hairs in water and Congo red. Black scale bars =100 μm; red scale sia, seems appropriate after Mattirolo’s description (smooth, ellip- bars = 25 μm. Photos ABDFM: V. Kaounas; CEGIO: M. Slavova; HLN: soid ascospores measuring 26–29 × 15–16 μm). C. Agnello. From a molecular point of view, the new species is most closely related to the recently proposed Mediterranean species B. maroc- Legend of Plate 3. AB: Gleba in Congo red; CDF: Ascospores in cana. Both are related to the American type species of Barssia, B. ore- water; E: Ascus and ascospores in water; G: Pleurorynchous base of gonensis, but both lineages seem to be too divergent from each ascus; H: Ascus in water. Scale bars = 25 μm. Photos ABH: M. Slavova; other. The discovery of additional taxa in both lineages could help CF: C. Agnello; EG: V. Kaounas. to resolve the most suitable taxonomic status of the whole group. The subtle differences between the closely relatedBarssia and Bal- samia (MONTECCHI & SARASINI, 2000) could suggest their synonymiza- tion. However, B. hellenica displays a conspicuous apical depression, tains in Poland, under Picea abies, but the special ecological niche one of the most important features used to discriminate between suggests a closer investigation before claiming that this taxon is pre- Balsamia and Barssia (GILKEY, 1925), which is however not evident in sent also in Europe. B. maroccana, thus supporting the generic split. A second species, B. yezomontana Kobayasi, was described in Japan in the year 1937 (as B. yezo-montana): the only known collec- tion of this species was lost during the bombing of WWII (GILKEY, Conclusion 1961). The globose ascospores described in the protologue suggest it should be considered a species of uncertain placement. Recently, Barssia hellenica can be easily discriminated from other species a third species, B. maroccana G. Moreno, Manjón, Carlavilla & P. Al- due to its particular morphological, ecological and genetic features. varado, has been described from the Moroccan Atlas Cedrus atlan- Morphologically it differs by its ovoid ascospores (> 70%) of inter- tica forests. It differs by its larger, ellipsoid ascospores measuring mediate size, shape and size of the asci, and the presence of tri- 29–36 × (16–) 18–22 μm. chomes (although scarce) on the peridium. The habitat seems also MATTIROLO (1936) described Stephensia peyronelii from Piedmont to play an important role: (Italy) under Larix decidua (alt. 1400 m). Only CERUTI (1960) evoked B. oregonensis → Pseudotsuga menziesii; this species, based on the original description because he could not B. maroccana → Cedrus atlantica; locate any existing herbarium material. Later, CERUTI (1961) sugges- B. hellenica → Abies cephalonica.

Fig. 1 — Barssia hellenica. Microscopic characters A: Peridium; B: Asci; C: Ascospores. Drawing: C. Agnello 217 Fig. 2 — Consensus LSU 28S rDNA phylogram of the family Helvellaceae (using Gyromitra perlata as outgroup) obtained in MrBayes from 140 sampled trees. Only nodes supported by >0.80 Bayesian PP are annotated.

The obvious host-specific association with conifers suggests that Y., MORENO B.A., PFISTER D.H., NARA K., ZAMBONELLI A., TRAPPE J.M. & VIL- the already mentioned specimens found under Picea abies and Larix GALYS R. 2013. — Historical biogeography and diversification of decidua might well represent additional independent Barssia spe- truffles in the Tuberaceae and their newly identified southern he- cies. misphere sister lineage. PLoS ONE, 8 (1): E52765. doi:10.1371/jour- nal.pone.0052765 Acknowledgements CARBONE M., AGNELLO C. & ALVARADO P. 2013. — Phylogenenetic studies in the family Sarcosomataceae (Ascomycota, Pezizales). Ascomy- cete.org, 5 (1): 1-12. We are grateful to George Proutzopoulos (Athens, Greece) for the CERUTI A. 1960. — Elaphomycetales et Tuberales. In: BRESADOLA J. (ed.). samples of the first collection; to Milena Maione of “Biblioteca Fon- Iconografia Mycologica. Vol. 28, suppl. II. Comitato Onoranze Bre- dazione Einaudi di Torino”, Gianfranco Visentin (Rovigo, Italy) and sadoliane, Trento. Aurelia Paz Conde (Comillas, Spain) for making available rare litera- CERUTI A. 1961. — Revisione di alcune specie di Elafomicetali e di Tu- ture sources; to John Plischke (Greenburg, USA) for the review of the berali dell’America del nord. Allionia, 7: 1-25. English text and Nicolas Van Vooren (Lyon, France) for the presub- CROUS P.W., WINGFIELD M.J., SCHUMACHER R.K., SUMMERELL B.A., GIRALDO A., mission review. GENÉ J., GUARRO J., WANASINGHE D.N., HYDE K.D., CAMPORESI E., GARETH JONES E.B., THAMBUGALA K.M, MALYSHEVA E.F., MALYSHEVA V.F., ACHARYA References K., ÁLVAREZ J., ALVARADO P., ASSEFA A., BARNES C.W., BARTLETT J.S., BLAN- CHETTE R.A., BURGESS T.I., CARLAVILLA J.R., COETZEE M.P.A, DAMM U., DE- BONITO G., SMITH M.E., NOWAK M., HEALY R.A., GUEVARA G., CAZARES E., KI- COCK C.A., DEN BREEŸEN A., DE VRIES B., DUTTA A.K., HOLDOM D.G., NOSHITA A., NOUHRA E.R., DOMINGUEZ L.S., TEDERSOO L., MURAT C., WANG ROONEY-LATHAM S.,. MANJÓN J.L., MARINCOWITZ S., MIRABOLFATHY M., MO- 218 Key of the known species of Barssia based on morphological and ecological characters

1. Globose ascospores ...... B. yezomontana 2. Ellipsoid ascospores ...... 4 3. Ovoid spores...... 5 4a.Ascospores 24.0–32.0 × 12.0–17.0 μm, with cylindrical asci, under Pseudotsuga menziesii ...... B. oregonensis 4b.Larger ascospores, on average 29.0–36.0 × 16.0–22.0 μm, with ellipsoid/clavate asci, apical depression not evident, under Cedrus atlantica...... B. maroccana 5. Ascospores 21.0–27.0 × 16.0–20.5 μm, with irregularly clavate or broadly ellipsoid asci, under Abies cephalonica ...... B. hellenica

RENO G., NAKASHIMA C., PAPIZADEH M., SHAHZADEH FAZELI S.A., AMOOZEGAR O’DONNELL K., CIGELNIK E., WEBER N.S. & TRAPPE J.M. 1997. — Phyloge- M.A., ROMBERG M.K., SHIVAS R.G., STALPERS J.A., STIELOW B., STUKELY netic relationship among ascomycetous truffles and the true and M.J.C., SWART W.J., TAN Y.P., VAN DER BANK M., WOOD A.R., ZHANG Y. & false morels inferred from 18S and 28S ribosomal DNA sequence GROENEWALD J.Z. 2014. — Fungal Planet description sheets: 281– analysis. Mycologia, 89 (1): 48-65. 319. Persoonia, 33: 212-289. RONQUIST F. & HUELSENBECK J.P. 2003. — MrBayes 3: Bayesian phyloge- GARDES M. & BRUNS T.D. 1993. — ITS primers with enhanced specificity netic inference under mixed models. Bioinformatics, 19 (12): 1572– for Basidiomycetes—application to the identification of mycor- 1574. rhizae and rusts. Molecular Ecology, 2: 113-118. SWOFFORD D.L. 2001. — PAUP*4.0b10: phylogenetic analysis using GILKEY H.M. 1925. — Five new hypogaeous fungi. Mycologia, 17 (6): parsimony (and other methods). Sunderland, Sinauer Associates. 250-254. TAMURA K., PETERSON D., PETERSON N., STECHER G., NEI M. & KUMAR S. 2011. — MEGA5: Molecular evolutionary genetics analysis using GILKEY H.M. 1939. — Tuberales of North America. Oregon State Mo- nographs, 1: 1-63. maximum likelihood, evolutionary distance, and maximum par- simony methods. Molecular Biology and Evolution, 28(10): 2731- KIMBROUGH J.W., LI L.T. & WU C.G. 1996. — Ultrastructural evidence for 2739. the placement of the truffleBarssia in the Helvellaceae (Pezizales). TRAPPE J.M. 1979. — The orders, families, and genera of hypogeous Mycologia, 88 (1): 38-46. Ascomycotina (truffles and their relatives).Mycotaxon , 9 (1): 297- LÆSSØE T. & HANSEN K. 2007. — Truffle Trouble: what happened to the 340. Tuberales? Mycological Research, 111 (9): 1075-1099. TRAPPE J.M., MOLINA R., LUOMA D.L., CÁZARES E., PILZ D., SMITH J.E., CASTEL- ŁAWRYNOWICZ M. & SKIRGIEŁŁO A. 1984. — Barssia oregonensis found in LANO M.A., MILLER S.L. & TRAPPE M.J. 2009. — Diversity, ecology and the Tatra Mountains (Poland). Acta Mycologica, 20 (2): 277-280. conservation of truffle fungi in forests of the Pacific Northwest. Port- MATTIROLO O. 1936. — Funghi ipogei delle alpi occidentali. Annali land, United States Department of Agriculture. della Reale Accademia d’Agricoltura di Torino, 79: 190-191. TRAPPE M., EVANS F. & TRAPPE J.M. 2007. — Field guide to North American MONTECCHI A. & SARASINI M. 2000. — Funghi ipogei d’Europa. Trento, Truffles. Berkeley CA, Ten Speed Press. Associazione Micologica Bresadola, Fondazione Centro Studi Mi- VILGALYS R. & HESTER M. 1990. — Rapid genetic identification and map- cologici, 714 pp. ping of enzymatically amplified ribosomal DNA from several Cryp- NGUYEN N.H., LANDEROS F., GARIBAY-ORIJEL R., HANSEN K. & VELLINGA E.C. tococcus species. Journal of Bacteriology, 172: 4238–4246. 2013. — The Helvella lacunosa complex in western North America: WHITE T.J., BRUNS T., LEE S. & TAYLOR J.W. 1990. — Amplification and di- cryptic species, misapplied names and parasites. Mycologia, 105 rect sequencing of fungal ribosomal RNA genes for phylogene- (5): 1275-1286. tics. In: INNIS M.A., GELFAND D.H., SNINSKY J.J. & WHITE T.J. (eds.). PCR NYLANDER J.A.A. 2004. — MrModeltest v2. Program distributed by the Protocols: A guide to methods and applications. New York, Acade- author. Uppsala, Evolutionary Biology Centre, Uppsala University. mic Press: 315–322. ef

Vasileios Kaounas Carlo Agnello Pablo Alvarado Sokratous 40 Via A. Gramsci 11 ALVALAB, La Rochela 47 TK 19016 Artemis Attika 72023 Mesagne 39012 Santander Greece Italy Spain [email protected] [email protected] [email protected] Monica Slavova Krivolak, 4 1164 Sofia Bulgaria [email protected]

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