Tremella Diploschistina (Tremellales, Basidiomycota, Fungi), a New Lichenicolous Species Growing on Diploschistes
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The Lichenologist 44(3): 321–332 (2012) © British Lichen Society, 2012 doi:10.1017/S0024282911000788 Tremella diploschistina (Tremellales, Basidiomycota, Fungi), a new lichenicolous species growing on Diploschistes A. M. MILLANES, M. WESTBERG, M. WEDIN and P. DIEDERICH Abstract: Several specimens of a lichen-inhabiting Tremella, inducing the formation of pale yellow, dark brown or black galls on species of Diploschistes, have been collected in three localities in Sweden and in one locality in the USA. Morphological and molecular studies confirm that this material represents a single species, which differs from other described Tremella species in the combination of gall morphology, basidium morphology, basidium and basidiospore sizes, presence of thick-walled hyphidia, and a different host-selection. We consequently describe this fungus as Tremella diploschistina sp. nov., on the basis of morphology and phylogenetic analyses of ITS and nLSU sequences. The phylogenetic analyses reveal that the fungus clearly belongs in Tremella, although the relationships with other species in the genus remain unclear. Key words: lichens, molecular phylogeny, taxonomy, Tremellomycetes Accepted for publication 06 October 2011 Introduction have not yet been formally named (Diederich The genus Tremella Pers. (Tremellomycetes, 1996, 2007). The lichenicolous Tremella Basidiomycota, Fungi) includes predomi- species often induce the formation of rela- nantly mycoparasitic species, growing on a tively conspicuous galls on their hosts, either wide range of basidiomycete and ascomycete on the thallus or on the hymenium of the fungi (Chen 1998; Kirk et al. 2008). Each ascocarps. Some intrahymenial taxa do not particular Tremella species is, however, highly produce any external symptoms, at least not host-specific, frequently being confined to a in early stages of growth (Diederich 1996; single fungal genus or species. Many taxa Zamora et al. 2011). The phylogenetic pos- form conspicuous gelatinous basidiocarps ition of the lichen-inhabiting representatives and are well known representatives of ‘jelly had never been tested by molecular methods fungi’, such as Tremella mesenterica or until the work by Millanes et al. (2011), who Tremella foliacea. Lichenicolous species are confirmed that they were nested within the among the most poorly known representa- genus Tremella. However, Tremella as cur- tives of the genus. Fifty-one Tremella species rently circumscribed is strongly polyphyletic have been described so far, growing exclu- (Chen 1998; Fell et al. 2000; Scorzetti et al. sively on lichenized fungi (Diederich 1986, 2002; Boekhout et al. 2011; Millanes et al. 1996, 2003, 2007; Sérusiaux et al. 2003; 2011), and both the genus and the family Zamora et al. 2011), although five of these Tremellaceae are in need of a deep taxonomic revision (Boekhout et al. 2011; Millanes et al. 2011). In their study of Tremellomycetes, A. M. Millanes: Departamento de Biologı´a y Geologı´a, Millanes et al. (2011) found that the lichen- Universidad Rey Juan Carlos, E-28933 Móstoles, Spain. icolous lifestyle was not restricted to a single Email: [email protected] clade, and they distinguished three mono- M. Westberg and M. Wedin: Cryptogamic Botany, phyletic groups including mostly lichen- Swedish Museum of Natural History, P.O. Box 50007, SE-10405 Stockholm, Sweden. icolous species. P. Diederich: Musée national d’histoire naturelle, 25 rue It has been suggested that the diversity Munster, L-2160 Luxembourg of lichen-inhabiting fungi is, in general, 322 THE LICHENOLOGIST Vol. 44 probably underestimated (Lawrey & Dieder- Drawings were performed using a drawing tube and by ich 2003; Ihlen & Wedin 2008) and this is direct observation. Micrographs were taken using an Olympus BX53 microscope fitted with differential inter- also the case in Tremella in particular, where ference contrast (DIC) and an Olympus DP11 camera. the number of new descriptions of lichen- Mycological terminology follows Diederich (1996) and inhabiting taxa is certainly expected to in- Kirk et al. (2008). The apiculus was not included in crease in the future (P. Diederich, pers. obs.; basidiospore measurements. Sizes in parentheses repre- Zamora et al. 2011). Moreover, the fre- sent minimum and maximum observed values. When the number of observations is less than 30, it is indicated quency of new lichenicolous Tremella records in brackets. reported in recent years suggests that many of the species already described might have Molecular studies been largely overlooked in previous field sur- veys, and that the distribution of many Choice of additional taxa and outgroup species is possibly wider than currently con- In addition to the three specimens studied, 18 speci- sidered (Diederich 2003; Sérusiaux et al. mens representing 10 Tremella species were included in the molecular study (Table 1). The sampling included 2003; Ertz & Diederich 2008; Kukwa & the type of the genus Tremella (T. mesenterica), terminals Jabłonska 2008; Pippola & Kotiranta 2008; of the Fuciformis and Foliacea groups distinguished by Puolasmaa et al. 2008; Westberg et al. 2008; Chen (1998) and terminals representing three groups of Svensson & Westberg 2010). lichenicolous species distinguished by Millanes et al. (2011). We included two species of Filobasidiales, viz., During fieldwork on the island of Filobasidium floriforme and F. uniguttulatum,asout- Runmarö in the Stockholm archipelago group. (Sweden), the second author collected a Species names, voucher information, and GenBank specimen of the lichen Diploschistes scruposus accession numbers are given in Table 1. showing unusual dark galls on the thallus. Further microscopic observations revealed DNA extraction and amplification the presence of basidia and basidiospores of DNA was extracted directly from the three specimens the Tremella type inside these galls. The same examined (Table 1). The outer surface of the selected galls, in which most of the tremellalean hyphae and author later found two other specimens hymenial components are located, was sectioned and growing on Diploschistes scruposus in Sweden, separated with a scalpel, in order to minimize the lichen and a fourth sample, growing on Diploschistes tissue in the DNA extraction. Total DNA was extracted muscorum, had been collected previously by using the Qiagen DNeasy Plant MiniKit, according to the manufacturer’s instructions. Roger Rosentreter in the USA and sent to For PCR amplification we used general fungal Paul Diederich. No other Tremella species primers in combination with primers designed to selec- had been previously recorded on these hosts, tively amplify the DNA from tremellalean fungi (Mil- and we conclude that the four specimens lanes et al. 2011). The primers ITS1F (Gardes & Bruns correspond to a new species of lichen- 1993), BasidLSU3-3 and BasidLSU1-5 (Millanes et al. 2011), and LR5 (Vilgalys & Hester 1990) were used to inhabiting Tremella, which is described here amplify the internal transcribed spacer I, the 5.8 rDNA as Tremella diploschistina sp. nov., on the basis gene, the internal transcribed spacer II and a fragment of of morphological and molecular studies. Its approximately 1000 bp in the nLSU rDNA gene. phylogenetic relationship to other species in PCR amplifications were performed using Illustra™ Hot Start PCR beads, according to the manufacturer’s the genus is also investigated by molecular instructions, with the following settings: for the primer methods. pair ITS1F/BasidLSU3-3, we used initial denaturing at 95°C for 3 min, four cycles (95°C for 40 s, 53°C for 40 s and 72°C for 90 s), four cycles (95°C for 30 s, 50°C for 30 s and 72°C for 90 s), and finally 32 cycles (95°C for Material and Methods 30 s, 47°C for 30 s and 72°C for 90 s) with a final Morphological studies extension at 72°C for 480 s. For the primer pair BasidLSU1-5/LR5 we used initial denaturing at 95°C Macromorphological traits were observed using an for 3 min, four cycles (95°C for 40 s, 56°C for 40 s and Olympus SZX16 dissecting microscope. Microscopic 72°C for 90 s), four cycles (95°C for 30 s, 53°C for 30 s structures were studied using hand-cut sections stained and 72°C for 90 s) and finally 32 cycles (95°C for 30 s, with Phloxin (1% in water) after pre-treatment with 50°C for 30 s and 72°C for 90 s) with a final extension at KOH (5%), following the methods of Diederich (1996), 72°C for 420 s. Before sequencing, the PCR products and observed with an Olympus CX40 microscope. were purified using the PCR-M® Clean-up System of 2012 Tremella diploschistina — Millanes et al. 323 T 1. Sequences newly produced (bold) or downloaded from GenBank, with specimen data or culture references. Species names Culture Specimen data ITS nLSU references Tremella caloplacae France, Sérusiaux s. n. ( S-F102489) JN053469 JN043574 T. candelariellae Luxembourg, Diederich 12808 (S-F102492) JN053470 JN043575 T. cetrariicola-a Finland, Suija s. n. ( S-F102413) JN053490 JN043596 T. cetrariicola-b Latvia, 2005, Suija s. n. (TU) JN053491 JN043597 T. cladoniae-a France, Diederich 16031 (S-F102550) JN053478 JN043584 T. cladoniae-b Estonia, Suija 872 (TU-45019) JN053477 JN043583 T. coppinsii-a UK, Diederich 15628 (S-F102414) JN053495 JN043601 T. coppinsii-b Estonia, Suija 38a (TU-38637) JN053496 JN043602 T. diploschistina-a * Sweden, Westberg & Berglund 09-400 (S) JN790586 JN790588 T. diploschistina-b Sweden, Westberg 09-452 (S) JN790587 JN790590 T. diploschistina-c USA, Rosentreter 6836 (IMI-365462) JN790585 JN790589 T. foliacea Sweden, Wiklund 018 (S-F102409) JN053502 JN043609 T. hypogymniae-a Sweden, Wedin 6892 (UPS) JN053484 JN043590 T. hypogymniae-b Estonia, Suija s. n. (TU-39402) JN053485 JN043591 T. lobariacearum-a Madeira, Diederich 4935 (S-F102418) JN053473 JN043579 T. lobariacearum-b Canary Islands, Diederich 16468 (S-F102419) JN053474 JN043580 T. mesenterica-a Sweden, Ryman 9146 (S-F102411) JN053463 JN043568 T. mesenterica-b Sweden, Wedin 7612 (S-F102412) JN053464 JN043569 T. phaeophysciae-a Luxembourg, Diederich 12429 (S-F102505) JN053479 JN043585 T. phaeophysciae-b Estonia, Suija s. n. (TU-55041) JN053480 JN043586 Outgroup Filobasidium floriforme CBS 6241 AF190007 AF075498 F.