Simmonsa,*, Timothy Y
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mycological research 113 (2009) 450–460 journal homepage: www.elsevier.com/locate/mycres Lobulomycetales, a new order in the Chytridiomycota D. Rabern SIMMONSa,*, Timothy Y. JAMESb, Allen F. MEYERc, Joyce E. LONGCOREa aSchool of Biology and Ecology, University of Maine, Orono, ME 04469, USA bDepartment of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109, USA cDepartment of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309, USA article info abstract Article history: The Chytridiales, one of the four orders in the Chytridiomycetes (Chytridiomycota), is polyphy- Received 13 February 2008 letic, but contains several well-supported clades. One of these clades is referred to as the Received in revised form Chytriomyces angularis clade, and the phylogenetic placement of this group within the Chy- 23 October 2008 tridiomycetes is uncertain. The morphology and zoospore ultrastructure of C. angularis have Accepted 13 November 2008 been studied using LM and were shown to differ from those of the type species of Chytrio- Published online 25 December 2008 myces, which is in the Chytridiaceae and is phylogenetically distinct from the C. angularis Corresponding Editor: clade. In this study, chytrids with morphologies or rDNA sequences similar to C. angularis, Gordon W. Beakes including two isolates of the morphologically similar C. poculatus, were isolated and their phylogenetic relationships determined using molecular sequence data. Results of Bayesian Keywords: and MP analyses of nuSSU and partial nuLSU rDNA sequences grouped the new isolates Chytridium polysiphoniae and the type isolate of C. angularis in a monophyletic clade within the Chytridiomycota Clydaea but distinct from the Chytridiaceae. Zoospores of isolates examined using TEM had ultra- Lobulomyces structural features similar to those of C. angularis. Genetic analyses, ultrastructural data, Maunachytrium and morphology support the establishment of a new order Lobulomycetales, placement of C. angularis and C. poculatus in a new genus (Lobulomyces), and description of additional taxa, which we have named Clydaea vesicula and Maunachytrium keaense. ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction placed C. angularis in the genus Chytriomyces, but noted that the zoospore ultrastructure and morphology of C. Some species of chytrids have characteristic morphologies angularis differed from C. hyalinus, the type of the genus that make them easily recognisable; Chytriomyces poculatus, (Letcher et al. 2005). Longcore (1992) hypothesised that C. a chytrid reported from three continents, is an example of angularis might represent a new genus, but waited to de- these. The C. poculatus zoosporangium is monocentric, scribe such a genus until more chytrids were isolated that operculate, elongate, and surrounded by a ‘ghostly, loose had light microscopic-level and ultrastructural characters veil’ (Sparrow & Lange 1977), or cupules (Willoughby & in common with the species. Townley 1961). Researchers noted non-cupuled forms of Analyses of sequence data (James et al. 2000; Letcher et al. C. poculatus (Booth 1971a, 1971b; Dogma 1969), but it was 2005) consistently place C. angularis by itself, within the Chytri- not until C. angularis (Longcore 1992) was brought into diomycota, but outside of phylogenetically well-supported pure culture and determined not to form cupules that these groups that coincide with groups based on ultrastructural two species were recognised as being distinct. Because of data (Barr 1980, 1990). James et al. (2006) included one GenBank its similarity to C. poculatus, Longcore (1992) provisionally submission that grouped with C. angularis, Chytridium * Corresponding author. Tel.: þ1 207 581 2954. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.11.019 Lobulomycetales, a new order in the Chytridiomycota 451 polysiphoniae. This marine chytrid isolate has since been lost (Ku¨ pper, pers. comm.) and no zoosporic ultrastructural data Materials and methods have been published. Ku¨ pper et al. (2006) sequenced C. polysi- phoniae and included five sequences of uncultured organisms Chytrid isolates, medium optimisation and temperature in a nuSSU rDNA phylogeny that grouped these isolates with maxima Chytriomyces angularis. Analyses of molecular and ultrastructural data have sup- Isolates examined in this study are listed in Table 1. Because ported major taxonomic changes in the Chytridiales, including little is known about habitat preferences of members of this the description of the Rhizophydiales from the Chytridiales group, other than that Chytriomyces poculatus has been (Letcher et al. 2006), the amendment of the Chytridiaceae to reported from acidic, peaty, or bog soils, we searched for exclude taxa not related to C. hyalinus (Letcher et al. 2005), them by baiting a variety of soil samples and brought them and the recognition of additional clades within the order into pure culture using chytrid-isolation techniques (Barr (James et al. 2006; Letcher et al. 2005). Further changes in the 1987). Stock cultures were maintained on nutrient agar slants Chytridiomycota include the elevation of the Blastocladiales in screw-topped culture tubes at 5 C and transferred every (James et al. 2006) and the Neocallimastigales (Hibbett et al. three months. 2007) to phylum level and the Monoblepharidales to class level We first isolated strains on PmTG (1 g lÀ1 peptonized milk, (Hibbett et al. 2007). 1glÀ1 tryptone, 5 g lÀ1 glucose, 10 g lÀ1 agar) nutrient agar We cultured the type isolate of C. angularis, two C. pocula- (Barr 1986). In an attempt to optimise growth of the isolates tus isolates, and six morphologically similar chytrids. We we compared growth in liquid medium without glucose compared their morphology by LM and, when possible, zoo- (PmT) or with glucose substituted with 5 g lÀ1 cellobiose or spore ultrastructure, and determined their phylogenetic rela- 5glÀ1 keratin. The PmT medium, with no added carbohydrate tionships based on analyses of the rDNA operon. C. angularis beyond that present in the peptonised milk and tryptone, pro- and C. poculatus are monophyletic as hypothesised from their vided the best growth conditions for all isolates except C. morphology, and we place them in a new genus. Our addi- angularis. Subsequently, we used PmT nutrient agar or broth tional taxon sampling provided strong molecular support for isolation and growth of all clade members except C. angu- for a clade that contains these two species plus others. We laris, which did not grow on PmT. To determine maximum describe the ‘C. angularis clade’ (James et al. 2006) as a new temperature for growth, we incubated duplicates of all iso- order, the Lobulomycetales, and name one family and three lates in 75 ml liquid medium in 125 ml flasks at 17, 20, 25, 30, genera. and 35 C for two weeks in the dark. Table 1 – Culture designations, identities, origins, habitats, and rDNA accession numbers of Lobulomycetales isolates Culture no. New taxon Origin Habitat GenBank accession no. Identification nuSSU nuLSU ITS1–5.8S–ITS2 AF011 Undescribed Peru Alpine barren soil EF432819 EF432819 EF432819 AF017 Undescribed Peru Alpine barren soil EF432821 EF432821 EF432821 AF021a,b Maunachytrium keaense Hawaii, USA Alpine barren soil EF432822 EF432822 EF432822 JEL45 Lobulomyces angularis Maine, USA Sphagnum from acidic lake AF164253 DQ273815 AY997036 JEL178a Undescribed Maine, USA Spring under mixed EF443136 EF443141 EU352772 hard woods JEL343 Lobulomyces poculatus Maine, USA Soil under pine trees EF443134 EF443139 EU352770 JEL369a,b Clydaea vesicula California, USA Soil under Eucalyptus trees EF443137 EF443142 EU352773 JEL374 Lobulomyces poculatus New Zealand Tree canopy detritus EF443135 EF443140 EU352771 PL70 Clydaea vesicula Utah, USA Creek bank EF443138 EF443143 EU352774 CCW64c Environmental PCR Massachusetts, USA Salt marsh AY180029 Chy Pyl IR 14d Chytridium polysiphoniae Shetland, UK Marine AY032608 RSC-CHU-18e Environmental PCR North Rhine-Westphalia, Rhizosphere of Zea mays AJ506000 Germany RSC-CHU-20e Environmental PCR North Rhine-Westphalia, Rhizosphere of Zea mays AJ506002 Germany RSC-CHU-23e Environmental PCR North Rhine-Westphalia, Rhizosphere of Zea mays AJ506003 Germany RSC-CHU-69e Environmental PCR North Rhine-Westphalia, Rhizosphere of Zea mays AJ506037 Germany GenBank accession numbers for species in other orders are published in James et al. (2006). a Taxa examined using LM. b Taxa examined using TEM. c Stoeck & Epstein 2003. d Ku¨ pper et al. 2006. e C. Hussels unpubl. 452 D. R. Simmons et al. Fig 1 – Single most parsimonious tree obtained from combined nuSSU and partial nuLSU rDNA sequences. Numbers to the left of the slash indicate support above 70 % in 10K replicates with parsimony analysis. Numbers to the right of the slash are Bayesian inference estimates of PPs. MP phylogeny tree length [ 2877 steps, CI [ 0.7188, RI [ 0.6902. Species of the Monoblepharidomycetes were used as outgroup taxa. Morphology and zoospore ultrastructure and LR5 and sequenced using NS1, NS2, NS4, NS6, NS7, NS8, ITS4, LR0R, and LR5 (Vilgalys & Hester 1990; White et al. 1990). We grew isolates on agar medium incubated at 17 C in the dark Our PCR products were purified with exonuclease I and to determine morphological stages using LM. During develop- shrimp alkaline phosphatase using