available online at www.studiesinmycology.org StudieS in Mycology 64: 155–173. 2009. doi:10.3114/sim.2009.64.09 Molecular systematics of the marine Dothideomycetes S. Suetrong1, 2, C.L. Schoch3, J.W. Spatafora4, J. Kohlmeyer5, B. Volkmann-Kohlmeyer5, J. Sakayaroj2, S. Phongpaichit1, K. Tanaka6, K. Hirayama6 and E.B.G. Jones2* 1Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand; 2Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Paholyothin Road, Khlong 1, Khlong Luang, Pathum Thani, 12120, Thailand; 3National Center for Biothechnology Information, National Library of Medicine, National Institutes of Health, 45 Center Drive, MSC 6510, Bethesda, Maryland 20892-6510, U.S.A.; 4Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, 97331, U.S.A.; 5Institute of Marine Sciences, University of North Carolina at Chapel Hill, Morehead City, North Carolina 28557, U.S.A.; 6Faculty of Agriculture & Life Sciences, Hirosaki University, Bunkyo-cho 3, Hirosaki, Aomori 036-8561, Japan *Correspondence: E.B. Gareth Jones, [email protected] Abstract: Phylogenetic analyses of four nuclear genes, namely the large and small subunits of the nuclear ribosomal RNA, transcription elongation factor 1-alpha and the second largest RNA polymerase II subunit, established that the ecological group of marine bitunicate ascomycetes has representatives in the orders Capnodiales, Hysteriales, Jahnulales, Mytilinidiales, Patellariales and Pleosporales. Most of the fungi sequenced were intertidal mangrove taxa and belong to members of 12 families in the Pleosporales: Aigialaceae, Didymellaceae, Leptosphaeriaceae, Lenthitheciaceae, Lophiostomataceae, Massarinaceae, Montagnulaceae, Morosphaeriaceae, Phaeosphaeriaceae, Pleosporaceae, Testudinaceae and Trematosphaeriaceae. Two new families are described: Aigialaceae and Morosphaeriaceae, and three new genera proposed: Halomassarina, Morosphaeria and Rimora. Few marine species are reported from the Dothideomycetidae (e.g. Mycosphaerellaceae, Capnodiales), a group poorly studied at the molecular level. New marine lineages include the Testudinaceae and Manglicola guatemalensis in the Jahnulales. Significantly, most marine Dothideomycetes are intertidal tropical species with only a few from temperate regions on salt marsh plants (Spartina species and Juncus roemerianus), and rarely totally submerged (e.g. Halotthia posidoniae and Pontoporeia biturbinata on the seagrasses Posidonia oceanica and Cymodocea nodosum). Specific attention is given to the adaptation of theDothideomycetes to the marine milieu, new lineages of marine fungi and their host specificity. Key words: Dothideomycetes, ecology, marine fungi, multi-locus, new genera, systematics. Taxonomic novelties: Aigialaceae Suetrong, Sakayaroj, E.B.G. Jones, Kohlm., Volkm.-Kohlm. & Schoch, fam. nov., Halomassarina Suetrong, Sakayaroj, E.B.G. Jones, Kohlm., Volkm.-Kohlm. & Schoch, gen. nov., Halomassarina thalassiae (Kohlm. & Volkm.-Kohlm.), Suetrong, Sakayaroj, E.B.G. Jones, Kohlm., Volkm.-Kohlm. & Schoch, comb. nov., Suetrong, Sakayaroj, E.B.G. Jones, Kohlm., Volkm.-Kohlm.,comb. nov., Clade V. Morosphaeriaceae Suetrong, Sakayaroj, E.B.G. Jones, & Schoch, fam. nov., Morosphaeria velataspora (K.D. Hyde & Borse) Suetrong,. Sakayaroj, E.B.G. Jones & Schoch, comb. nov., Morosphaeria ramunculicola (K.D. Hyde) Suetrong,. Sakayaroj, E.B.G. Jones & Schoch, comb. nov., Rimora Kohlm., Volkm-Kohlm., Suetrong, Sakayaroj, E.B.G. Jones, gen. nov., Rimora mangrovei (Kohlm. & Vittal) Kohlm.,Volkm-Kohlm., Suetrong, Sakayaroj, E.B.G. Jones, comb. nov. INTRODUCTION Scolecosporiella typhae, Stemphylium triglochinicola and Phialophora cf. olivacea and molecular data indicates that the Most marine Dothideomycetes are intertidal, primarily from teleomorphs of Amorosia littoralis, Dendryphiella salina and D. mangrove habitats and rely on the active discharge of their arenaria may be in the Pleosporales (Mantle et al. 2006, Jones ascospores. They are frequently found as saprobes of decaying et al. 2008). This paucity of marine anamorphic fungi is in marked woody materials in the marine environment. The species that contrast to freshwater fungi and terrestrial genera of the class (Cai occur completely submerged in the sea are mostly parasites et al. 2006, Shenoy et al. 2007, Shearer et al. 2009; this volume). or symbionts of seagrasses or marine algae. It is not clear how Marine Dothideomycetes occur on a wide range of substrata: ascospore discharge occurs in these species as their hosts are mangrove wood, twigs and leaves; sea and marsh grasses often submerged for most of the time. Jones et al. (2009) list 64 (especially Spartina spp. and Juncus roemerianus) (Kohlmeyer et genera and ca. 108 species of marine Dothideomycetes that fall into al. 1995a–c, 1996, 1997a–b). Culms and leaves of sea and marsh three accepted orders (Capnodiales, Dothideales, Pleosporales), grasses are ideal substrata for saprobic fungi because they may three orders incertae sedis (Hysteriales, Patellariales, Jahnulales) remain standing for several years during and after senescence and 23 genera not assigned with confidence to any order. Most (Christian et al. 1990, Kohlmeyer & Volkmann-Kohlmeyer 2001). of these higher order taxa are represented by a single genus or Other species are found on brown and red seaweeds, e.g. Lautitia species while most are members of the Pleosporales with 25 danica and Pleospora gracilariae (Schatz 1984, Simmons & Schatz genera and 61 species (+ 13 genera, 20 species, incertae sedis). 1989), on wood associated with sand e.g. Caryospora australiensis Taxa that can not be assigned with confidence to either an order and Decaisnella formosa (Abdel-Wahab & Jones 2003) or on or family include Aigialus, Halotthia, Lautospora, Manglicola, the brackish water palm Nypa fruticans, e.g. Carinispora nypae, Mauritiana, Passeriniella, Pontoporeia, and Tirisporella. A notable Herpotrichia nypicola, Tirisporella beccariana and Helicascus feature of the marine Dothideomycetes is how few anamorphs nypae (Jones et al. 1996, Hyde & Alias 2000). Few marine are known. Examples include Amarenographium metableticum, Dothideomycetes produce elaborate appendaged ascospores, and Copyright 2009 CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 155 Suetrong et al. most possess gelatinous sheaths that swell in water when released The DNA was resuspended in 50 mL TE buffer and checked for from the asci (Massarina velataspora and Tremateia halophila). quantity and quality by 1 % agarose gel electrophoresis. Genera with appendaged ascospores, although generally The following four genes were chosen for this study: small (18S) modifications of a gelatinous sheath, include: Carinispora nypae, and large subunit (28S) of the nuclear ribosomal DNA (SSU, LSU) Decorospora gaudefroyi and Falciformispora lignatilis. plus the gene fragments from the second largest subunit of RNA The main objective of this study is to provide information on polymerase (RPB2) and the translation elongation factor 1-alpha the taxa that are unique to the marine milieu, e.g. Aigialus spp., (TEF1) gene. The rDNA was amplified with Taq DNA polymerase Manglicola guatemalensis, Halotthia posidoniae and Pontoporeia from FERMENTAS (Cat.No. MBDOEPO402) using PCR Model biturbinata and confirm the taxonomic assignment of other marine MJ Research DYAD ALD ALD 1244 thermocycler (MJ Research, ascomycetes within the context of a well sampled analysis with Waltham, MA). Primers used for amplification include the SSU, LSU, other related fungi. RPB2 and TEF1 (White et al. 1990, Bunyard et al. 1994, Liu et al. 1999, Rehner 2001, respectively). The PCR products were purified using a NucleoSpin Extraction Kit (Macherey-Nagel, Germany), MATERIAL AND METHODS following the manufacturer’s instructions. The characterisation of PCR products was performed via agarose gel electrophoresis Collection of fungi on 1 % agarose gel containing ethidium bromide as the staining Drift and attached wood, culms and leaves of marsh plants, agent. PCR products were directly sequenced by Macrogen Inc., seagrasses and seaweeds were collected from a variety of habitats Korea. The sequencing primers used for as the different regions and geographical locations, placed in clean plastic bags and are SSU: NS1, NS3, NS4, NS6 (White et al. 1990); LSU: JS1, JS8, returned to the laboratory. After washing with freshwater to remove LROR and LR7 (Bunyard et al. 1994); TEF1: 983F, 2218R, CEFF2 sediments, the samples were examined for fungi. Samples were and CEFR2 (Rehner 2001); RPB2: 5F1, 5F2, 7cR and 7R (Liu kept moist by spraying with sterilised distilled water.