This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's personal copy mycoscience 55 (2014) 35e42 Available online at www.sciencedirect.com journal homepage: www.elsevier.com/locate/myc 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 parsi- mony 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 fol- lowed 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