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Full paper Updates on Protubera (Protophallaceae, Phallales) and additional notes on P. maracuja

Larissa Trierveiler-Pereira a,*, Andre´ A.R. Meijer b, Kentaro Hosaka c, Rosa Mara B. Silveira a a Departamento de Botaˆnica, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves, 9500, Porto Alegre, Rio Grande do Sul 91501-970, Brazil b PR-405, km 9.5, Antonina, Parana´, Brazil c Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan article info abstract

Article history: Since the genus Protubera was erected in 1895 by Alfred Mo¨ller, fourteen species have been Received 7 February 2013 described in the genus, from tropical, subtropical and temperate regions of the globe. Based Received in revised form on morphological, ecological and molecular data, we concluded that only six species 3 May 2013 (P. borealis, P. jamaicensis, P. maracuja, P. nipponica, P. parvispora and P. sabulonensis) should be Accepted 8 May 2013 accepted in Protophallaceae. In this article we present an update of the genus from a Available online 10 July 2013 morphological and phylogenetic perspective, including comments on the excluded and doubtful taxa, and a key for the accepted species. Additional morphological and ecological Keywords: notes of P. maracuja, especially regarding features of peridium and substrata, as well as Kobayasia color photos, are presented. Phalloid fungi ª 2013 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Protophallus Protuberella Sequestrate fungi

1. Introduction Valley (state of Santa Catarina) and basidiomata were found throughout the year. Up to date, 14 species have been Mo¨ ller (1895) erected the curious genus Protubera based on described in the genus (Heim 1977; Kirk et al. 2008). specimens collected in coastal rainforests of southern Brazil. A revision of the genus was published by Malloch (1989) who Basidiomata resembled immature phalloids but Mo¨ ller was considered all the species described until then (seven in total) as cautious enough to follow up the ontogeny of the basidiomata belonging to the genus, and synonymized Kobayasia and Protu- and noticed that they never produce an expanded receptacle. berella e both proposed by Imai and Kawamura (1958) e with When completely mature, basidiomata open in an irregular Protubera and described P. sabulonensis Malloch as a new species. way and expose the brownish green mass of spores. During Since Malloch’s review, six new species were described or his stay in Brazil from 1890 to 1893, Mo¨ller found the type combined to Protubera, viz. P. burburiana (Rodway) Castellano & species, P. maracuja Mo¨ ller, to be abundant in forests of Itajaı´ Trappe, P. canescens G.W. Beaton & Malajczuk, P. hautuensis

* Corresponding author. Tel.: þ55 51 3308 7556; fax: þ55 51 3308 7686. E-mail address: [email protected] (L. Trierveiler-Pereira). 1340-3540/$ e see front matter ª 2013 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.myc.2013.05.001 Author's personal copy

36 mycoscience 55 (2014) 35e42

Castellano & Beever, P. nothofagi Castellano & Beever, (Swofford 2002) with a combined dataset of LSU and atp6. The P. parvispora Castellano & Beever and P. termitum R. Heim. analyses were performed under the heuristic search option Almost all of them are from Oceania (Beaton and Malajczuk (TBR and Multrees options on) and 1000 replicates of random 1986; Chang and Kantvilas 1993; Castellano and Beever addition sequence were conducted. Nodal supports were 1994), except the last one from Africa (Heim 1977). tested by bootstrapping of 1000 replicates using the heuristic Molecular phylogeny of the Phallomycetidae (Hosaka et al. search option (TBR and Multrees options on) with 10 random 2006) demonstrated that Protubera is polyphyletic, suggesting addition sequences. that many species described for the genus actually belonged to different genera and families. The aim of this study is to present an update on the species included in the genus. 3. Results and discussion Additional morphological and ecological notes on P. maracuja are also presented. 3.1. Phylogenetic analysis

The combined dataset consisted of a total of 1296 characters 2. Material and methods (LSU ¼ 624, atp6 ¼ 672), among which 119 characters (LSU ¼ 100, atp6 ¼ 19) were excluded from the analyses due to 2.1. Morphological analysis ambiguous alignment. A total of 872 characters were parsimony uninformative, and the remaining 424 characters were Field expeditions were carried out in the Southern Region of used for the analyses. MP analyses produced 24 equally Brazil, which includes the States of Parana´, Santa Catarina parsimonious trees, with Tree Length ¼ 1586, CI ¼ 0.4237, and Rio Grande do Sul. The climate is subtropical in almost the RI ¼ 0.7732, RC ¼ 0.3276. entire region, at exception of the northeast part of the region The phylogenetic tree (Fig. 1) shows representative taxa of that is tropical. Phallales and Hysterangiales within Phallomycetidae. Proto- Collected specimens were photographed in the ﬁeld and phallaceae was separated from other families in the Phallales described macro- and microscopically based on fresh and dried with high bootstrap support (100%). Within the family, the specimens. Colors were coded according to Kornerup and Neotropical (and subtropical) species [P. maracuja and Wanscher (1978). For microscopical analyses, portions of P. jamaicensis (Murrill) Zeller] were separated from the þ peridium and gleba were mounted in 5% KOH 1% aqueous temperate representatives (P. borealis S. Imai, P. nipponica solution of phloxine. For microscopical photographs, sections Kobayasi and P. sabulonensis) also with high support (100%). were made using a freezing microtome (Yamato FX-801) Other species in the genus were nested within the Lysur- equipped with an electric freezer (Yamato MC-802A) and later aceae (P. clathroidea Dring) and Clathraceae (P. canescens). Two photographed with a digital camera (Nikon Digital Sight DS-5M) species, viz. P. nothofagi and P. hautuensis, were placed within the linked to a Nomarski differential interference contrast micro- Hysterangiales. Protubera nothofagi was reported to be ectomy- scope (Olympus BX51). Vouchers are deposited at Herbaria ICN, corrhizal (Castellano and Beever 1994) as well as many other FLOR and MBM. Herbaria acronyms follow Thiers (2012). members of the Hysterangiales, however the other species in Protubera are considered saprophytic (Rinaldi et al. 2008). 2.2. Phylogenetic analysis Based on molecular and morphological evidence, it is clear that some species described within Protubera do not belong Selection of ingroup and outgroup taxa for the phylogenetic to the Protophallaceae. These species probably belong to analysis was based on the phylogeny presented by Hosaka different genera or either represent immature stages of et al. (2006), focusing on the Phallales and Hysterangiales phalloid taxa. clades were Protubera s.l. species are distributed. Members of The production of small, subcylindrical spores seems to be Geastrales were selected as outgroup. Data of taxa included in a common feature within the genus, as it happens in many the analysis along with GenBank (www.ncbi.nlm.nih.gov/ phalloid taxa. Usually the glebal mass is divided into lobes genbank/) accession numbers, are listed in Table 1. Four new or elongated plates, with the exception of P. parvispora sequences were generated for this study. (Castellano and Beever 1994), in which the gleba is unique and Nuclear large subunit ribosomal DNA (LSU) and mito- central. chondrial ATPase subunit 6 (atp6) were ampliﬁed respectively using a polymerase chain reaction (PCR) with primer combi- 3.2. Taxonomy nations LR0R/LR5 (Vilgalys and Hester 1990), and atp6-3/atp6-2 (Kretzer and Bruns 1999). DNA was extracted from glebal tis- Protubera maracuja Mo¨ ller, Bot. Mitt. Tropen 7: 10, 1895. sues of dried basidiomata following the protocol of Hosaka Figs. 2 and 3. and Castellano (2008). PCR and sequencing protocols followed Kasuya et al. (2012). Molecular LSU and atp6 datasets Lectotype (designated by Malloch 1989): Brazil, State of were aligned using Muscle v.3.6 (Edgar 2004), and manually Santa Catarina, Blumenau, 1890, A. Mo¨ ller (B). edited in BioEdit v.7.0.1 (Hall 1999). Ambiguously aligned re- Description in: Furtado and Dring (1967), Trierveiler-Pereira gions and introns were excluded from the analyses, and gaps et al. (2013). were treated as missing data. Distribution and habitat: tropical and subtropical areas of Maximum parsimony (MP) analysis was conducted under Central and South America, reported from Brazil (Mo¨ ller 1895; the equally weighted parsimony criterion using PAUP v.4.0b10 Rick 1905; Furtado and Dring 1967), Suriname (Fischer 1933), Author's personal copy

mycoscience 55 (2014) 35e42 37

Table 1 e Taxa information and GenBank accession numbers for the LSU and atp6 DNA sequences used in the phylogenetic analysis. Newly generated sequences are marked with an asterisk (*). Taxon Origin Voucher no. LSU atp6

Geastrales e outgroup Geastrum ﬂoriforme USA OSC 29328 DQ218485 DQ218769 Geastrum pectinatum Australia MEL 2096557 DQ218602 DQ218889 Radiigera fuscogleba USA OSC 58979 DQ218609 DQ218896 Hysterangiales Aroramyces gelatinosporus Australia OSC H4010 DQ218524 DQ218809 Aroramyces radiatus Zimbabwe OSC Verbeken 99-062 DQ218525 DQ218810 Austrogautieria chlorospora Australia OSC 46596 DQ218477 DQ218761 Austrogautieria manjimupana Australia OSC 55900 DQ218533 DQ218818 Chondrogaster angustisporus Australia OSC 62041 DQ218537 DQ218822 Gallacea eburnea New Zealand OSC 59601 DQ218482 DQ218766 Gallacea sp. Australia OSC 122813 DQ218543 DQ218829 Gallacea sp. New Zealand PDD REB2364 DQ218540 DQ218825 Hysterangium afﬁne Australia OSC T6884 DQ218546 DQ218831 Hysterangium crassum Germany OSC 110447 AY574687 AY574827 Hysterangium hallingii Argentina OSC Halling5741 DQ218497 DQ218781 Mesophellia clelandii Australia OSC 59292 DQ218511 DQ218795 Mesophellia trabalis Australia OSC 59282 DQ218592 DQ218880 Nothocastoreum cretaceum Australia OSC 79832 DQ218593 DQ218881 Phallogaster saccatus USA OSC T13202 DQ218595 DQ218882 Protubera hautuensis New Zealand OSC 59673 DQ218517 DQ218801 Protubera nothofagi New Zealand OSC 59699 AY574644 AY574786 Protubera sp. Australia OSC T20068 DQ218596 DQ218883 Trappea phillipsii USA OSC 56042 DQ218522 DQ218807 Trappea pinyonensis USA OSC AHF530 DQ218597 DQ218884 Phallales Anthurus archeri New Zealand PDD REB-2182 DQ218624 DQ218913 Aseroe rubra Australia OSC 122632 DQ218625 DQ218914 Ileodictyon cibarium New Zealand OSC 122734 DQ218633 DQ218922 Ileodictyon gracile Australia MEL 2024221 DQ218634 DQ218923 Ileodictyon gracile Australia MEL 2037639 DQ218635 DQ218924 Ileodictyon gracile Australia MEL 2054561 DQ218636 DQ218925 Laternea triscapa e OSC 122864 DQ218640 DQ218928 Lysurus borealis USA OSC 39531 DQ218641 DQ218929 Lysurus mokusin China CUW MB-02-012 DQ218507 DQ218791 Lysurus periphragmoides China CUW MB-02-016 DQ218521 DQ218806 Phallus costatus China CUW MB-02-040 DQ218513 DQ218797 Phallus indusiatus USA OSC 36088 DQ218627 DQ218917 Phallus ravenelli USA CUW s.n. DQ218515 DQ218799 Protubera borealis South Korea OSC OKM21898 DQ218516 DQ218800 Protubera canescens Australia MEL 2063471 DQ218644 DQ218931 Protubera canescens Australia MEL 2105035 DQ218645 DQ218932 Protubera clathroidea e BPI s.n. DQ218646 e Protubera jamaicensis e OSC T28248 DQ218647 DQ218933 Protubera maracuja Chile? OSC Garido2550-A DQ218518 DQ218802 Protubera maracuja Brazil ICN LTP220 KC808513* KC808515* Protubera maracuja Brazil ICN LTP284 KC808514* KC808516* Protubera nipponica Japan OSC 122862 DQ218638 DQ218926 Protubera nipponica Japan OSC 122863 DQ218639 DQ218927 Protubera sabulonensis USA OSC T12737 DQ218649 DQ218935

Jamaica (Dennis 1953) and Costa Rica (Trierveiler-Pereira et al. April 1992, Meijer 2237 (MBM); Guaraqueçaba, Tagaçaba, on 2013); found gregarious or solitary on soil (clayey, sandy) and humus, 6 August 2009, D. Schause, Meijer 4466 (MBM); RPPN decaying wood. Salto Morato, on decaying wood, 14 December 2011, Specimens examined: Brazil, Parana´, Curitiba, Parque L. Trierveiler-Pereira, LTP284 (ICN); Santa Catarina, Barigui, on humus, 28 August 1996, Meijer 3442 (MBM); Parque Floriano´ polis, Morro da Lagoa da Conceiçaõ, on soil, 10 May Barreirinha, on soil, 13 March 1992, Meijer 2183 (MBM); Con- 1986, J. Furlani (FLOR 10312); on soil, 19 December 2011, vento Solitude, on humus, 4 March 1995, Meijer 3032 (MBM); M. Jaeger, MJ 94 (FLOR); on soil, 16 February 2012, M. Jaeger, MJ Parque Regional do Iguaçu, on humus, 23 April 1995, Meijer 133 (FLOR); Rio Grande do Sul, Porto Alegre, Morro Santana, on 3055 (MBM); Morretes, Parque Marumbi, on humus, 7 decaying wood, 17 May 2011, L. Trierveiler-Pereira, LTP 219 September 1997, Meijer 3461 (MBM); on decaying wood, 11 (ICN); L. Trierveiler-Pereira, LTP220 (ICN 168980). Author's personal copy

38 mycoscience 55 (2014) 35e42

Fig. 1 e One of 24 equally most parsimonious trees of Phallomycetidae derived from parsimony analysis using combined LSU and atp6 dataset. Taxon names are followed by voucher numbers. Nodes supported by >70% parsimony bootstrap values are indicated by black circles, 50e70% by gray circles.

Taxonomical remarks: the most striking feature that sep- humidity and habitat conditions may have an effect on the arates P. maracuja from other Protubera species is outer overall coloration. However, we have observed whitish and peridium composed by pseudoparenchymatous hyphae brownish basidiomata growing on the same substrate at the (Fig. 3A). The presence of large crystals rosette arrangements same time (Fig. 2B). Based on these observations, we conclude in the second peridial layer (Fig. 3B) may also be considered that basidiomata coloration has just a range of variation diagnostic, as previously suggested by Malloch (1989). Basi- within the species’ population. We agree with Malloch’s diomata are usually grooved, but young ones may also have a opinion that differences in age do not account for the color nearly smooth peridial surface (Fig. 2D), resembling P. variation. jamaicensis. Ecological remarks: basidiomata of P. maracuja have always The color of P. maracuja basidiomata has been discussed in been reported to occur on soil (Fig. 2C). Mo¨ ller (1895) noticed the literature. Mo¨ ller’s description stated that young basidio- that long mycelial strands occur over decaying wood but mata are pale leather brown, while mature ones are darker. never mention the growth of basidiomata on wood. During Furtado and Dring (1967) described their specimens as whitish our ﬁeld excursions, we could conﬁrm several times that to grayish. Malloch (1989)’s opinion was that differences in P. maracuja produces basidiomata on large trunks (Fig. 2A) and Author's personal copy

mycoscience 55 (2014) 35e42 39

Fig. 2 e Basidiomata of Protubera maracuja. A: On a large fallen trunk into the woods (LTP219, ICN). B: On a small fallen branch (brownish basidiome was removed from the substrate) (LTP284, ICN). C: On sandy soil (MJ94, FLOR). D: Basidiomata with nearly smooth peridial surface (MJ133, FLOR). Photographs A and B by L. Trierveiler-Pereira; C and D by M. Jaeger. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

small tree branches (Fig. 2B) fallen on the ground. Among Protubera canescens G.W. Beaton & Malajczuk, Trans. Br. other species of Protubera considered in this study, P. jamai- Mycol. Soc. 87: 481, 1986. censis and P. parvispora have also been reported from decaying Remarks: a detailed study published by May et al. (2010) wood (Coker and Couch 1928; Castellano and Beever 1994; concluded that P. canescens corresponds to immature basi- Trierveiler-Pereira et al. 2013). diomata of Ileodictyon gracile, when the lattice arms are at a Mo¨ ller (1895) reported that usually young basidiomata of P. very early stage of development. Molecular data also maracuja are completely hypogeous, while mature basidio- confirmed that P. canescens belongs to the Clathraceae, instead mata are epigeous. During our studies we notice that P. mar- of the Protophallaceae, clustering with Ileodictyon species. acuja growing on wood also had epigeous young basidiomata. Protubera clathroidea Dring, Mycol. Pap. 98: 3, 1964. Zeller (1948) erroneously stated that P. maracuja has an Remarks: this species was described with central glebal mass, exclusively hypogeous habit. although sometimes irregular in shape, and relatively thick- Protubera species accepted in this article are saprotrophic, walled, ellipsoid to ovoid basidiospores, (3.5e)4e6 (3e) as reported by Rinaldi et al. (2008). Some studies that consider 3.5e4.5 (5) mm(Dring 1964). Dring (1964) stated that basidio- the genus ectomycorrhizal and/or related to Hysterangiales, spores of P. clathroidea are somewhat similar to those of used P. nothofagi or P. canescens as reference (Malajczuk 1987; P. africana and vastly different from other Protubera species, Giachini et al. 2010; Tedersoo et al. 2010; Comandini et al. which have minute and bacilliform spores. According to mo- 2012). As discussed above, P. nothofagi and P. canescens do not lecular data, it corresponds to an immature stage of a Lysur- belong to the Protophallaceae. Some studies have used ecto- aceae species. mycorrhizal “Protubera” specimens (probably misidentified) to Protubera termitum R. Heim, Termites et Champignons (Paris): test growth increment in Eucalyptus (Thomson et al. 1994; 178, 1977. Chen et al. 2000). Remarks: this species is based on small-sized (0.8e1.2 cm), immature basidiomata associated with termite’s nests. Basid- 3.3. Excluded taxa iospores were not described in the original publication, so it is difficult to ascertain the relationship of this fungus with any Some taxa described in Protubera actually correspond to phalloid species. Illustrations presented by Heim (1977) immature stages of different fungal species (mostly phalloid resemble Protubera species externally, but the central and species) and therefore, should not be treated as valid species. compact glebal mass is whitish, which is atypical for the genus. Author's personal copy

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Protubera burburiana (Rodway) Castellano & Trappe in Y.S. Chang & Kantvilas, Tasmanian Herb. Occas. Publ. 4: 38, 1993. Remarks: originally described within Hysterangium, Rodway (1918)’s description is brief and no illustrations are presented. Features that might indicate that the species is not a Protubera are the small-sized basidiomata (1e2 cm in diam.), rather thick and ﬂeshy peridium, and oblong basidiospores, 5 3 mm. Cunningham (1944) concluded from the type examination that it corresponds to an immature phalloid, perhaps Aseroe¨ rubra.Mo- lecular data could help to elucidate the real identity of this taxon.

3.5. Hysterangiales species

Protubera hautuensis Castellano & Beever, N.Z. Jl Bot. 32: 322, 1994. Remarks: the glebal mass ﬁlls almost the entire basidiome cavity and only a thin gelatinize layer is observed between the peridium and the gleba (Castellano and Beever 1994). Sutures are infrequent. At the molecular level, this species is related to Phallogaster. Protubera nothofagi Castellano & Beever, N.Z. Jl Bot. 32: 324, 1994. Remarks: this is an hypogeous, ectomycorrhizal species associated with Nothofagus. The glebal mass is central, not divided into elongated plates, and a thin tramal layer is observed between the peridium and the gleba. The covering layer is easily separated from the peridium and spores are large, 6e7 3e3.5 mm(Castellano and Beever 1994). According Fig. 3 e Microscopic features of Protubera maracuja. to molecular data, this species is related to Gallacea within the A: Peridial pseudoparenchymatous layer (note crystals Hysterangiales. rosette arrangements in the inner layer). B: Crystals from the inner layer. C: Basidiospores. Photographs from LTP220 (ICN 168980). Bars:A30mm; B and C 10 mm. 4. Conclusions

3.4. Doubtful taxa We accept only six species of Protubera in the Protophallaceae: P. borealis, P. jamaicensis, P. maracuja, P. nipponica, P. parvispora Some taxa were not re-collected or re-examined after original and P. sabulonensis. Protubera nipponica and P. sabulonensis are descriptions, but according to morphological characteristics morphologically very similar and additional molecular data is presented in the literature (unique and central glebal mass, needed to validate their species rank. basidiospores shape and size), we can conclude that they do In the same species, the gelatinized inner peridium may not belong to Protophallaceae. Until more collections and/or have a watery or agar-like consistency, as observed in molecular data are available, we prefer to keep the species P. maracuja and P. nipponica, so this feature should not be used separated from Protubera. to distinguish species. Protubera africana Lloyd, Mycol. Notes (Cincinnati) 64: 987, The genus Protubera is saprophytic and species are found in 1920. tropical, subtropical and temperate areas. Basidiomata are Remarks: this South African species differs from P. maracuja epigeous when mature, whitish to brownish externally, and by its single, central glebal mass, not divided into lobes or spores are small, smooth and subcylindrical. Protubera mar- ellipsoid plates (Lloyd 1920). Basidiospores of P. africana are acuja is the only species in the genus that possesses pseudo- reported to be ellipsoid-ovoid to oblong, 5.5e6.3 (8.5) 2.5e3 parenchymatous hyphae in the outer peridium. (3.5) mm(Zeller 1939). Cunningham (1944) ascertained that Lloyd’s description was based on an unexpanded basidiome of a phalloid fungus. May et al. (2010) suggested that P. africana 5. Key to the Protubera species might correspond to an immature stage of Ileodictyon, since I. gracile co-occurs with P. africana. 1. Glebal mass central and single, not divided into elon- Protubera brunnea (Zeller) Zeller, Mycologia 40: 644, 1948. gated plates. New Zealand e P. parvispora. Remarks: similar to P. africana, P. brunnea is characterized by a 10. Glebal mass divided into elongated plates arranged unique, central glebal mass, and broadly ellipsoid basidio- centripetally e 2. spores, 5e6 2.5e3 mm(Zeller 1939). The description of the 2. Outer peridium pseudoparenchymatous; inner peridium species was based on a single basidiome that has not been with large crystals forming rosette patterns. Neo- and sub- included in any molecular analyses. tropical e P. maracuja. Author's personal copy

mycoscience 55 (2014) 35e42 41

20. Outer peridium not pseudoparenchymatous, composed Chen YL, Gong MQ, Xu DP, Zhong CL, Wang FZ, Chen Y, 2000. of interwoven hyphae; large crystals forming rosette patterns Screening and inoculant efficacy of Australian absent e 3. ectomycorrhizal fungi on Eucalyptus urophylla in field. Forest Research 13: 569e576. 3. Basal mycelial strand extending into the center of the e Comandini O, Rinaldi AC, Kuyper TW, 2012. Measuring and basidiome; surface viscid. Neotropical P. jamaicensis. estimating ectomycorrhizal fungal diversity: a continuous 0 3 . Basal strand not as above; surface dry. Temperate spe- challenge. In: Pagano M (ed) Mycorrhiza: occurrence in natural cies e 4. and restored environments. Nova Science Publishers, 4. Basidiomata up to 15 cm in diam., brownish or grayish Huntington, pp 165e200. with brownish patches; glebal plates not elongated nearly to Coker WC, Couch JN, 1928. The Gasteromycetes of the Eastern United the center; gelatinous matrix present in the center at spore States and Canada. The University of North Carolina Press, Chapel Hill; http://dx.doi.org/10.5962/bhl.title.5712. maturity. Japan and South Korea e P. borealis. 0 Cunningham GH, 1944. The Gasteromycetes of Australia and New 4 . Basidiomata up to 7 cm in diam., dirty white to grayish; Zealand. McIndoe, Dunedin. glebal plates elongated nearly to the center; center hollow at Dennis RWG, 1953. Some West Indian Gasteromycetes. Kew spore maturity e 5. Bulletin 8: 307e328; http://dx.doi.org/10.2307/4115517. 5. Peridium less than 70 mm thick; gelatinous layer agar- Dring DM, 1964. Gasteromycetes of West Tropical Africa. e like; basidiospores 3.8e5 1.9e2.6 mm. Canada Mycological Papers 98: 1 60. Edgar RC, 2004. MUSCLE: multiple sequence alignment with high e P. sabulonensis. accuracy and high throughput. Nucleic Acids Research 32: 50. Peridium 1.0e1.5 mm thick; gelatinous layer not agar- 1792e1797. e e m e like; basidiospores 3.8 4.7 1.6 2.1 m. Japan P. nipponica. Fischer E, 1933. Gasteromyceteae Stahelianae. Annales Mycologici 31: 113e125. Furtado JS, Dring DM, 1967. The rediscovery of Protubera maracuja, Disclosure with additional descriptive notes. Transactions of the British Mycological Society 50: 500e502. Giachini AJ, Hosaka K, Nouhra E, Spatafora J, Trappe J, 2010. The authors declare no conflict of interest. All the experi- Phylogenetic relationships of the Gomphales based on nuc- ments undertaken in this study comply with the current laws 25S-rDNA, mit-12S-rDNA, and mit-atp6-DNA combined of Brazil and Japan. sequences. Fungal Biology 114: 224e234; http://dx.doi.org/ 10.1016/j.funbio.2010.01.002. Hall TA, 1999. BioEdit: a user-friendly biological sequence Acknowledgments alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95e98. Heim R, 1977. Termites et champignons: les champignons termitophiles The authors are grateful to Dr. Gerhard E. Overbeck for the d’Afrique noire et d’Asie meridionale. Boubeé, Paris. translation of Mo¨ller’s original description; curators from Hosaka K, Bates ST, Beever RE, Castellano ME, Colgan W, Herbaria ICN, FLOR and MBM for the loan of specimens; to the Domıńguez L, Nouhra ER, Geml J, Giachini AJ, Kenney SR, administration of Salto Morato Natural Reserve (Guaraqueçaba, Simpson NB, Spatafora JW, Trappe JM, 2006. Molecular Brazil) for collecting permits and research support; Juliano phylogenetics of the gomphoid-phalloid fungi with an M. Baltazar for assistance in collecting; Melissa Jaeger for sup- establishment of the new subclass Phallomycetidae and two e plying specimens and photographs; Yan Jie Zhao for assistance new orders. Mycologia 98: 949 959; http://dx.doi.org/10.3852/ mycologia.98.6.949. in taking the microscopical photos; Kyung-ok Nam, Kunihiko Hosaka K, Castellano ME, 2008. Molecular phylogenetics of Uno and Kazuo Nishibori for assisting the molecular work. A Geastrales with special emphasis on the position of grant from The International Association for Plant Taxonomy Sclerogaster. Bulletin of the National Museum of Nature and Science, (IAPT Research Grant2012 for thefirst author)to LTPand theJSPS Series B 34: 161e173. grants-in-aid for young scientists (A) to KH are acknowledged for Imai S, Kawamura A, 1958. On the Japanese species of Protubera. e financial support. This study is a partial result of the Ph.D. thesis Science Reports of the Yokohama National University, Section II 7: 1 6. of the first author, with a scholarship provided by the Brazilian Kasuya T, Hosaka K, Uno K, Kakishima M, 2012. Phylogenetic placement of Geastrum melanocephalum and polyphyly of Government (CAPES scholar, proceeding # 9716/11-4). Geastrum triplex. 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