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 f u n g a l b i o l o g y 1 1 7 ( 2 0 1 3 ) 6 6 0 e6 7 2 journal homepage: www.elsevier.com/locate/funbio The molecular phylogeny of aquatic hyphomycetes with affinity to the Leotiomycetes Christiane BASCHIENa,d,*, Clement Kin-Ming TSUIb, Vladislav GULISc,
e Ulrich SZEWZYKd, Ludmila MARVANOVA a Federal Environment Agency, Corrensplatz 1, 14195 Berlin, Germany Department of Forest and Conservation Sciences, University of British Columbia, Vancouver, BC V6T 1Z4, Canada c Department of Biology, Coastal Carolina University, Box 261954, Conway, SC 29528, USA d Department of Environmental Microbiology, Technische Universitaet Berlin, Ernst-Reuter-Platz 1, 10587 Berlin, Germany e Masaryk University, Faculty of Science, Institute of Experimental Biology, Czech Collection of Microorganisms, Tvrd
eho 14, 602 00 Brno, Czech Republic b article info abstract Article history: Aquatic hyphomycetes play a key role in decomposition of submerged organic matter and Received 14 April 2013 stream ecosystem functioning. We examined the phylogenetic relationships among vari- Received in revised form ous genera of aquatic hyphomycetes belonging to the Leotiomycetes (Ascomycota) using 14 July 2013 sequences of internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA gen- Accepted 16 July 2013 erated from 42 pure cultures including 19 ex-types. These new sequence data were ana- Available online 25 July 2013 lyzed together with additional sequences from 36 aquatic hyphomycetes and 60 related Corresponding Editor: fungi obtained from GenBank. Aquatic hyphomycetes, characterized by their tetraradiate H. Thorsten Lumbsch or sigmoid conidia, were scattered in nine supported clades within the Helotiales (Leotio- Keywords: Flagellospora are not monophyletic, with species from the same genus distributed among Biodiversity several major clades. The Gyoerffyella clade and the Hymenoscyphus clade accommodated Evolution species from eight and six different genera, respectively. Thirteen aquatic hyphomycete Molecular systematics taxa were grouped in the Leotia-Bulgaria clade while twelve species clustered within the Hy- Taxonomy menoscyphus clade along with several amphibious ascomycetes. Species of Filosporella and mycetes). Tricladium, Lemonniera, Articulospora, Anguillospora, Varicosporium, Filosporella, and some species from four other aquatic genera were placed in the Ascocoryne-Hydrocina clade. It is evident that many aquatic hyphomycetes have relatives of terrestrial origin. Adaptation to colonize the aquatic environment has evolved independently in multiple phylogenetic lineages within the Leotiomycetes. ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Aquatic hyphomycetes are defined as an ecological group of fungi that inhabit submerged leaf litters, decaying wood € rlocher 1992; Suberkropp 1992) and roots of riparian vege(Ba tation (Fisher et al. 1991; Sati & Belwal 2005), or submerged plants (Kohout et al. 2012). Studies of the biodiversity, physiology and ecology of these fungi in recent years resulted in * Corresponding author. Department of Environmental Microbiology, Technische Universitaet Berlin, Ernst-Reuter-Platz 1, 10587 Berlin, Germany. Tel.: þ49 30 89031324; fax: þ49 30 89031830. E-mail address: christiane.baschien@tu-berlin.de (C. Baschien). 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.07.004 Author's personal copy The molecular phylogeny of aquatic hyphomycetes better understanding of their critical importance in plant litter decomposition and stream ecosystem functioning (Gessner et al. 2007; Krauss et al. 2011). Aquatic hyphomycetes comprise over 300 species; most of them belong to the Ascomycota (Webster 1992; Shearer et al. 2007). The characteristic traits of most aquatic hyphomycetes are stauroconidia (e.g. tetraradiate or variously branched) or less frequently scolecoconidia (sigmoid, curved or straight) produced as morphological adaptations to survival and dispersal in aquatic habitats (Webster 1959a; Dix & Webster 1995). The current taxonomic concepts of genera are based on conidial morphology and the mode of conidiogenesis. Most aquatic hyphomycetes are holoanamorphic, however, the direct connections between sexual and asexual states through pure culture (from ascospores to conidial state or, rarely, from conidial state to ascoma) have been established only in about 10 % of all known species (Webster 1992;
1997, 2007; Sivichai & Jones 2003). Marvanova Within the Ascomycota, according to our present knowledge, aquatic hyphomycetes belong to the subphylum Pezizomycotina and they are distributed among five classes based
2007). Molecular on relationships to sexual states (Marvanova € rlocher 2005; Baschien et al. 2006; studies (Belliveau & Ba Campbell et al. 2006, 2009; Vijaykrishna et al. 2006; Shearer et al. 2009; Seena et al. 2010) have confirmed the placement of several aquatic hyphomycetes species in the same major classes: Sordariomycetes (w11 spp.), Dothideomycetes (w10 spp.), Pezizomycetes (1 sp.), Orbiliomycetes (3-5 spp.) and Leotiomycetes (>75 spp., this study). The polyphyly of aquatic hyphomycetes had been already recognized by Webster (1961) who first described the Nectria lugdunensis state (Hypocreales) of the aquatic hyphomycete Heliscus (Webster 1959b), followed by the description of a Mollisia sp. (Helotiales) as the sexual state of Anguillospora crassa. Molecular data also revealed the polyphyly of various aquatic hyphomycete gen€ rlocher 2005; Baschien et al. 2006; era (Belliveau & Ba Campbell et al. 2006, 2009). Indeed, based on the morphological studies, the two predominant spore shapes, sigmoid and tetraradiate have evolved multiple times independently in un
2007). The related taxa (Ingold 1966; Webster 1980; Marvanova contemporary system based mainly on similarity of conidia and conidiogenesis often does not reflect the phylogenetic relationships among taxa. However, for many genera, we do not yet have enough additional information inferred from phylogenetic studies to replace the taxonomic system based solely on morphology. In particular, sequences from ex-type or authentic cultures are often lacking. Helotiales is the largest order within the Leotiomycetes which represents a morphologically and ecologically diverse class of Pezizomycotina. It contains an assemblage of fungi that form apothecia with inoperculate, unitunicate asci. Members of Helotiales are saprotrophs in terrestrial and aquatic habitats (including aquatic hyphomycetes), plant pathogens, ectomycorrhizal symbionts or endophytes. Environmental sequencing studies of leaves, roots or rhizosphere often result in a high number of Leotiomycetes sequences (e.g. Kohout et al. 2012; Toju et al. 2013) of which many are from uncultured fungi. Interestingly, several aquatic hyphomycetes have been reported as plant endophytes that nested in the Helot€ rlocher 1992; Selosse et al. 2008). However, iales (Sridhar & Ba 661 the overall systematics of the Helotiales is unstable, and many of the genera are polyphyletic because currently deployed characters are insufficient to delineate taxa (Wang et al. 2006a). The whole life cycle is also poorly understood, and many helotialean fungi are known from their sexual state only. The phylogenetic relationships of the majority of aquatic hyphomycetes and the extent of convergence of morphological conidial characters that delineate the asexual genera are mostly unknown. These gaps in our understanding mostly persist due to the scarcity of sequences of ex-type or authentic cultures. In this study, we analyzed sequence data from the partial large subunit (LSU) and internal transcribed spacer (ITS) regions of ribosomal DNA from 42 pure cultures of aquatic hyphomycetes, including 19 ex-types that were combined with sequence data obtained from GenBank. The objectives of our study were (i) to provide reference (barcoding) sequence data of aquatic hyphomycetes with affinity to the Leotiomycetes using ex-type or authentic cultures from major collections, (ii) to resolve the phylogenetic placement of aquatic hyphomycetes in the Leotiomycetes, and (iii) to examine their evolutionary relationships to helotialean ascomycetes with aquatic and non-aquatic ecology. Materials and methods Taxon sampling We studied 42 isolates of aquatic hyphomycetes including 19 ex-type or ex-neotype cultures from 16 genera and 40 species (Table 1). The molecular data determined from these isolates were compared with sequences of the existing data set of aquatic hyphomycetes in GenBank (36 sequences from 35 species), and sequences of 60 helotialean fungi closely related in ecology and/or taxonomy based on Wang et al. (2006a,b; Table 1). DNA isolation, PCR, and sequencing Cultures were maintained on 2 % malt extract agar (MEA). Mycelia were harvested directly from MEA plates. Genomic DNA was extracted using the Ultra Clean Soil DNA Isolation Kit in conjunction with the Vortex Adapter for Vortex-Genie 2 (MO BIO, Carlsbad, CA, USA) or the FastDNASPIN kit for soil in conjunction with the FastPrep FP120 instrument (Qbiogene, Heidelberg, Germany) according to the manufacturer’s instructions. The ITS and the partial LSU regions of rDNA were then amplified by PCR. The ITS region was amplified with primers SR6R (http://www.biology.duke.edu/fungi/mycolab/ primers.htm) and LR1 (Vilgalys & Hester 1990), while the primer pair LROR and LR7 (Bunyard et al. 1994) was used to amplify a ca. 1400 bp fragment from the LSU region. PCR mixtures contained 10 ml PCR Mastermix M7502 (Promega, Madison, WI, USA), 20 pM of each primer, 40e200 ng of genomic DNA and 8 ml nuclease free water. The PCR was performed with an initial denaturation step for 2 min at 94  C, followed by 25e35 cycles of denaturation for 1 min at 94  C, 45 s primer annealing at 46e50  C (ITS) or 54  C (LSU) and elongation for 1 min at 72  C, final extension was for 5 min (10 min for LSU) at 72  C. The Author's personal copy 662 C. Baschien et al. Table 1 e Sources and GenBank accession numbers of species used in this study. [ Aquatic hyphomycetes,* [ type species of aquatic hyphomycete genera. Sequences indicated in bold were generated in this study or from earlier
, CB by C. Baschien and VG by V. Gulis. If investigations of CB. Strains labelled CCM F-are mostly isolated by L. Marvanova they were deposited by someone else, the depositor’s name is in parentheses. Species Strain Source GenBank ITS GenBank LSU Stream foam, CA Stream foam, GB Stream, angiosperm leaf, USA AY204590 AY204587 KC834040 e KC834018 KC834017 Stream, Fagus sylvatica leaf, CZ KC834041 e Alatospora pulchella CCM F-37194 CCM F-02383 CCM F-11302, (ex-type, ¼ ATCC 32680) CCM F-501, ex-type of Alatospora crassipes CCM F-502, ex-type KC834039 KC834019 Anguillospora crassa CCM F-15283 Stream, Athyrium filix-femina frond, CZ Sessile apothecia on angiosperm twiglet, SK Stream foam, CA Stream foam, AT N/A, CA Stream foam, SK Stream, Picea abies twiglet, CZ Stream foam, CZ Abies sp., CA N/A N/A N/A Vitis vinifera, NZ Fagus sylvatica bark, NL N/A N/A Acorn, Quercus robur, NL Diphasiastrum complanatum, FI Culture contaminant Erica tetralix root, NL N/A Submerged Pinus roxburghii needles, Kumaun, Himalaya, IN N/A Agricultural soil, under potato, NL Stream, Castanea leaf, JP Picea mariana-needles, CA N/A Stream foam, GB (E. Descals B 292-1-10) Equisetum fluviatile, BY (VG 98a) Alnus glutinosa submerged roots, GB (P.J. Fisher 7 DW) Alnus glutinosa submerged roots, GB (P.J. Fisher WF) Submerged leaf Cladrastis kentukea, USA Stream foam, CZ Submerged leaf, Crataegus monogyna, GB Stream foam, SK AY204581 e AY148104 KC834038 EU940163 KC834042 FJ000402 EU998915 U72259 AY789395 AY789345 AB190393 HM116747 GU727558 AY789352 DQ257353 AY526234 GU727553 KC834043 AY176758 AF433149 DQ202513 e e EU940086 KC834020 e EU998915 e AY789394 AY789344 AB190423 HM116758 e AY789351 DQ257352 e e e e AF433138 AY789342 U51980 AY789341 e DQ202518 AY746351 U92304 KC834044 e e AF356694 e KC834046 KC834047 e KC834021 KC834054 KC834022 KC834045 KC834024 KC834050 KC834048 KC834023 e KC834049 KC834025 KC834053 KC834051 e e Alatospora acuminata Alatospora acuminata Alatospora constricta Alatospora flagellata Cudoniella sp. Dactylaria dimorphospora CCM F-20687 CB-L16 M337 CCM F-13486, ex-type CCM F-00684 CCM F-12499 cf870061 PDD75671 ZW-Geo52-Clark IFM50530 ICMP 18084 CBS 304.74 ZW-Geo55-Clark PDD70070 CBS 655.78 CBS 731.97 CCM F-13489, ex-type, monotypic CBS110609 wz164 CBS 430.94 ex-type of Tricladium indicum ZW0068 CBS 256.70 Dimorphospora foliicola Dwayaangam colodena Fabrella tsugae Filosporella cf. annelidica CBS 221.59, ex-type, monotypic V3.13 J. Platt 256 CCM F-11702 Anguillospora filiformis Anguillospora furtiva Arachnopeziza variepilosa Arbusculina fragmentans Articulospora atra Articulospora tetracladia Ascocalyx abietina Ascocoryne cylichnium Bulgaria inquinans Cadophora finlandica Cadophora luteo-olivacea Catenulifera brachyconia Chlorencoelia sp. Chlorovibrissea sp. Ciboria batschiana Cistella spicicola Cladochasiella divergens Cryptosporiopsis rhizophila Cudonia lutea Cudoniella indica Filosporella exilis Filosporella fistucella CCM F-13097, ex-type CCM F-13091, ex-type Filosporella versimorpha CCM F-11194, ex-type Flagellospora curvula Flagellospora sp.1 Flagellospora fusarioides Flagellospora leucorhynchos Flagellospora saccata Flagellospora sp. 2 Fontanospora eccentrica Fontanospora fusiramosa Geniculospora grandis Geoglossum glabrum Gorgomyces honrubiae CB-M13 CCM F-20899 CCM F-14583 CCM F-14183 CCM F-39994 VG 31-4 CCM F-46394 CCM F-12900 UMB-176.01 OSC 60610 CCM F-12003, ex-type Stream foam, CA Submerged leaf, Rhododendron maximum, USA Stream foam, CA Stream foam, CZ Stream foam, PT N/A
n AR Stream foam, ES (A. Rolda 9761) KC834052 GQ411354 AY789318 KC834057 GQ477305 GQ477307 AY789317 KC834028 Author's personal copy The molecular phylogeny of aquatic hyphomycetes 663 Table 1 e (continued ) Species fx1* Gorgomyces hungaricus Gyoerffyella cf. craginiformis Gyoerffyella entomobryoides fx1 Gyoerffyella gemellipara Gyoerffyella rotula Gyoerffyella tricapillata Helicodendron westerdijkae Hemiphacidium longisporum Heyderia abietis Holwaya mucida Hyalodendriella betulae Hyaloscypha vitreola * Hydrocina chaetocladia Hymenoscyphus scutula Hymenoscyphus varicosporioides Hyphodiscus hymeniophilus Lachnum virgineum Lemonniera aquatica Lemonniera centrosphaera Lemonniera cornuta Lemonniera sp. Lemonniera terrestris Leohumicola minima Leohumicola verrucosa Leotia lubrica Loramyces macrosporus Margaritispora aquatica Meria laricis Microglossum olivaceum Miniancora allisoniensis Mitrula brevispora Mitrula elegans Mitrula paludosa Mollisia “rhizophila” Mollisia cinerea Mollisia dextrinospora Mollisia fusca Mollisia melaleuca Mollisia minutella Mollisia sp. Mycoarthris corallina Mycochaetophora sp. * Mycofalcella calcarata Neobulgaria pura Neofabraea alba Neofabraea malicorticis Ombrophila violacea Phialocephala helvetica Protoventuria alpina Pyrenopeziza brassicae Pyrenopeziza revincta Strain Source GenBank ITS GenBank LSU CCM F-12696, ex-type Terrestrial, on decaying leaves of € nczo € l) Carpinus betulus, HU (J. Go Liriodendron tulipifera, decaying leaves, terrestrial, NL Rosa sp., stem necrosis, NL KC834058 e KC834055 KC834026 KC834056 e Liriodendron tulipifera, decaying leaves, terrestrial, NL Stream foam, SK Rosa sp. decaying leaf in a pond, GB Aero-aquatic Pinus contorta, CA KC834060 KC834027 KC834061 KC834059 KC834029 KC834030 EF029229 AY645899 e e AY789290 DQ257357 EU040232 EU940231 KC834062 AY789289 DQ257356 e EU940155 KC834031 MBH29259 FC-2038 N/A N/A Alnus glutinosa N/A, FI Submerged alder twigs, GB (J. Webster) N/A Wood, JP AY789432 AB481291 AB481292 MUCL 9042 Betula sp., FR DQ227259 e AFTOL49 CCM F-21799 CCM F-149, ex-type Alnus cones, USA Stream foam, CZ Submerged leaf, Fagus sylvatica, SK UK, (J. Webster) Stream foam, CZ Stream foam, SK Iso€etes echinospora root, NO Soil, CA N/A Submerged Equisetum limosum, UK Submerged Alnus leaves, CZ Larix decidua, CH N/A Stream foam, CA Aero-aquatic, CN Aero-aquatic, USA aero-aquatic, Europe Aspen roots, CA Fallen log, USA Actinidia deliciosa, NZ Fagus sylvatica, CH Picea abies, DE Picea abies needles, CZ Nothofagus menziesii leaves, NZ Stream foam, GB (P.J. Fisher 91A) Gentiana scabra, JP Rotting oak twigs, GB (S. OmKalthoum-Khattab HME4405) N/A Malus domestica, NZ N/A N/A N/A, CH Arctostaphylos uva-ursi, CH N/A, UK Axenic culture, ascospores, NO DQ491485 e KC834063 AY544646 DQ267627 KC834032 e e e HQ691252 AY706323 AY789360 e DQ267629 DQ267633 DQ267634 e e AY789359 DQ470957 e DQ470954 AY789398 KC834064 AY789294 AY789331 AY789424 JN053274 DQ491498 HM116746 AY259137 AY259136 FR837920 JN225932 AF128440 AB434662 KC834065 DQ267635 DQ470954 AY789397 e AY789293 AY789330 AY789423 e DQ470942 HM116757 e e e e e AB469680 KC834037 DQ257366 AY359236 AF281386 AY789366 AY347413 EU035444 AJ305236 AJ430224 DQ257365 e e AY789365 e e e e CCM F-09367 CBS268.63, ex-type CCM F-402 CCM F-400 CBS 451.64, ex-isotype ICMP 15521 ATCC 26761 OSC60392 ZW-Geo-138CBS 261.82 M39 CCM F-10890, ex-type, monotypic CCM F-325 CCM F-19299 CCM F-11486 N086 CBS 115881 ZW-Geo59-Clark CBS 235.53 ex-type CCM F-11591 monotypic CBS 298.52 FH-DSH97-103 CCM F-30487 ex-type, monotypic ZW02-012 ZW-Geo45-Clark MBH50636 Currah lab1 AFTOL 76 ICMP 18083 CBS 234.71 CBS 589.84 ZK71/08 1.3.s.5.13 91A ex-type, monotypic MAFF 239284 CCM F-10289 ex-type CUP 063609 MM 159 DAOM 227085 WZ0024 D-ZB-40 CBS 140.83 CRB ARON3150.P (continued on next page) Author's personal copy 664 C. Baschien et al. Table 1 e (continued ) Species Strain Source GenBank ITS GenBank LSU CBS 698.79 Dactylis glomerata, CH AY140669 e BPI1843550 AY465516 AY247400 AF455526 AF433152 KC834066 e J01355 e AF433141 e EU883420 EU883420 EU883431 EU883432 AY204621 EU883431 EU883432 AY204612 Rhynchosporium orthosporum Rhytisma salicinum Saccharomyces cerevisiae Sclerotinia sclerotiorum Spathularia flavida Tetrachaetum elegans wb197 wz214 CB-M11, monotypic Tetracladium apiense CCM F-23199 Tetracladium breve Tetracladium furcatum Tetracladium marchalianum Tetracladium maxilliforme Tetracladium palmatum Tetracladium setigerum Tricladium alaskense Tricladium angulatum Tricladium attenuatum Tricladium biappendiculatum Tricladium castaneicola Tricladium caudatum Tricladium chaetocladium Tricladium curvisporum Tricladium indicum Tricladium kelleri Tricladium minutum CCM F12505 CCM F-11883 CCM F-26199 Salix scouleriana, USA N/A N/A N/A Submerged leaf Cladrastis kentukea, USA Stream, plant debris, ES (Gran Canaria) Stream, leaf cf. Frangula alnus, PT Stream foam, CZ Stream foam, CZ CCM F-14286 Stream foam, SK AF411027 e CCM F-10001 CCM F-10186 VG 69-2, ex-type CCM F-14186 CCM F-06485 CCM F-13000 PT (C. Pascoal) Stream foam, CZ Stream, Carex sp., Alaska, USA Stream foam, CZ CH (J. Rosset) Stream foam, CZ EU883424 EU883427 JQ417290 AY204611 e e EU883424 EU883427 GQ477338 GQ477311 GQ477312 GQ477314 CCM F-11296 CCM F-13498 VG 27-1 Stream foam, CZ Stream foam, CZ Stream, Acer rubrum, USA e e KC834067 GQ477316 GQ477318 e CCM F-23387 VG 112-1 VG 68-1, ex-type CCM F-10203 Stream foam, CA Foam, USA Stream, Carex sp., Alaska, USA Juncus culms, GB, (E. Descals C181-3-03) Stream foam, CZ Stream foam, CZ Juncus sp., SK Stream foam, CZ Stream, Quercus sp./Prunus sp. leaf litter, IE Stream foam, CA Litter, CA Stream foam CA Tricladium Tricladium Tricladium Tricladium Tricladium obesum patulum procerum splendens terrestre CCM F-14598, ex-type CCM F-15199 CCM F-16786, ex-type CCM-F-16599 CBS 697.73, ex-type CCM F-19494 CBS 541.92 CCM F-10987 Varicosporium delicatum Varicosporium elodeae Varicosporium giganteum Varicosporium scoparium Varicosporium trimosum Variocladium giganteum CCM F-10303, ex-type CCM F-14398 CBS 508.71, ex-type Variocladium giganteum Vibrissea albofusca Vibrissea flavovirens Vibrissea truncorum Ypsilina graminea Zalerion varium CCM F-16686 PDD 75692 MBH39316 CUP-62562 UMB-098.01, monotypic ATCC 169303 quality of PCR amplicons was checked in 1.2 % agarose gels stained with ethidium bromide under UV light using a 100 bp ladder (Promega, Madison/USA). The amplicons were purified using the Ultra Clean PCR Clean-up kit from MO BIO. Primers used for sequencing were SR6R/LR1 for ITS regions and LROR, LR3R, LR3 and LR7 (Vilgalys & Hester 1990) for partial LSU gene. Sequences were generated with an ABI 373 sequencer (Applied Biosystems, Foster City, USA) and
n 9851) River foam, ES (A. Rolda Stream foam,, CZ Submerged Crataegus monogyna leaf, GB Juncus sp., SK N/A, amphibious N/A, amphibious N/A, amphibious River foam, PT Balza wood, river, USA e JQ417288 JQ412863 GQ477322 GQ477324 GQ477337 GQ477326 KC834068 e e AY204635 DQ202519 KC834035 GQ477329 KC834034 GQ477333 JQ412864 DQ202517 e KC834036 KC834037 GQ477343 e e DQ202520 GQ477345 GQ477346 e e AY789384 AY789427 AY789403 GQ411304 AF169303 GQ477348 AY789383 e AY789402 e e analyzed with the sequence analysis software version 3.3 at SMB Dr. Martin Meixner (Berlin, Germany) or University of South Carolina, Engencore (Columbia, SC, USA). Phylogenetic analyses All sequences generated were used as queries in the GenBank sequence similarity search tool BLAST [http:// Author's personal copy The molecular phylogeny of aquatic hyphomycetes blast.ncbi.nlm.nih.gov/Blast.cgi] with default stringency. The top scoring sequences from the BLAST searches were included in the phylogenetic analyses. Additional sequences from the Leotiomycetes were also added. The full data sets were comprised of 116 ITS and 89 LSU sequences. The combined data set contained 138 taxa of which 64 had both ITS and LSU data concatenated, 55 had only ITS data and 19 had only LSU data (Table 1). Geoglossum glabrum and Saccharomyces cerevisiae were used as the outgroup taxa. Phylogenetic relationships were assessed using the ARB software package (Ludwig et al. 2004) and MrBayes version 3.2.1 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003; Ronquist et al. 2012). All sequences were aligned using Fast Aligner/ClustalW implemented in ARB V1.03. All alignments were thoroughly examined and manually optimized according to primary and secondary structure information calculated by ARB. Ambiguously aligned nucleotide characters were excluded prior to phylogenetic analyses. The alignment is available on treebase.org under the following link http:// purl.org/phylo/treebase/phylows/study/TB2:S14104. jModeltest 2.1.1 (Darriba et al. 2012) was used for the selection of the model of nucleotide substitution that best fits the sequence data employing the Akaike Information Criterion (Akaike 1974). Maximum Likelihood analyses were performed with ARB using RAxML 7.0.3 (Randomized Accelerated Maximum Likelihood, Stamatakis 2006) applying the GTR þ I þ G model of sequence evolution for the combined data set. Searches were performed with random sequence addition and 100 replicates. Branch support was tested with 1000 replications on bootstrapped data sets. Three independent Bayesian phylogenetic analyses of the combined data sets were performed using the model TIM2ef þ G (Posada 2003) revealed by jModeltest for the combined data set. Posterior probabilities for internodes were calculated with the Metropoliscoupled Markov chain Monte Carlo (MCMC) method by running four chains with 26 million generations in each of two runs with trees sampled every 1000 generations. The analyses were ended when the average standard deviation of split frequencies of the two runs was <0.05 (0.0081) and the likelihoods converged to a stable distribution. Additionally, convergence was diagnosed using AWTY (Nylander et al. 2008) and Tracer (Rambaut & Drummond 2007). Trees obtained prior to convergence were discarded as ‘burn-in’ before computing a consensus tree with TreeView version 1.6.6 (Page 1996). Posterior probability support was considered significant with PP > 0.95. For assigning families in Helotiales we used the classifications listed in Myconet (Lumbsch & Huhndorf 2010), Mycobank (http://www.mycobank.org) and The Genera of Hyphomycetes (Seifert et al. 2011). Results We generated 31 new ITS and 21 new partial LSU sequences in this study. The concatenated data set (ITS and LSU regions) comprised 138 sequences (including 78 sequences of aquatic hyphomycetes and 60 related fungi from other habitats) and 2275 nucleotide positions. After the removal of indels and 665 ambiguous flanking 50 and 30 regions, the final data set had 1741 characters. Maximum Likelihood (RAxML) analyses revealed 1089 distinct alignment patterns and the best tree had a likelihood of lnL ¼ 23438.19, while Bayesian analyses revealed a consensus tree with a likelihood of lnL ¼ 23 433.44. Both trees recovered the major clades of Leotiomycetes/Helotiales reported by Wang and co-workers (2006a). The comparison of trees inferred with individual data set (ITS or LSU data alone) revealed no significant conflicting clades (data not shown). Fourteen major clades, receiving strong bootstrap support (BS) (>95 %) and posterior probability (PP) (>0.98), were recognized (Fig 1). However, no clade corresponded well to the current circumscriptions of sexual or asexual genera of aquatic fungi that have been established based on conventional morphological characters. Seventy-eight sequences of aquatic hyphomycetes were placed in nine clades (1e5, 7e8, 11, 14) within the Helotiales (Fig 1). The polyphyly of Tricladium, Lemonniera, Anguillospora and Varicosporium was confirmed, and newly established for Articulospora, Filosporella and Flagellospora because species from the same genus were placed in several clades. Clade 1 (100 % BS and 1.0 PP support) contained eight genera of aquatic hyphomycetes including paraphyletic Gyoerffyella (five species) and polyphyletic Varicosporium (two species), Fontanospora (two species), Articulospora (tetracladia), Anguillospora ( filiformis), Tricladium (three species) as well as Tetrachaetum elegans and Cladochasiella divergens. Margaritispora aquatica forms a well supported sister branch to three Lemonniera species. In Clade 2, Pyrenopeziza brassicae, Cadophora, and Rhynchosporium formed a group with the aquatic hyphomycetes Tricladium alaskense, Tricladium kelleri, Tricladium curvisporum, and Ypsilina graminea. Clade 3 contained members of the Loramycetaceae/Vibrisseaceae including Mollisia s. str. (M. cinerea), Loramyces macrosporus, and Variocladium giganteum, while in Clade 4, Tricladium procerum and Arbusculina fragmentans were placed along with Hyaloscypha vitreola (Hyaloscyphaceae) and Cadophora finlandica. Clade 5 contained the monophyletic genus Tetracladium and other aquatic hyphomycetes (Mycoarthris corallina, Varicosporium scoparium) together with Dactylaria dimorphospora and Leohumicola spp. Mitrula species and Ascocalyx abietina formed an independent Clade 6 with strong support (1.0 PP). Albeit without statistical support, Tricladium angulatum was placed adjacent to Dimorphospora foliicola, which has a Hymenoscyphus teleomorph (Abdullah et al. 1981). The Cudoniella-Hymenoscyphus clade (Clade 7) also received strong support (100 % BS and 0.98 PP), and this included the aquatic hyphomycetes Anguillospora crassa, Anguillospora furtiva, Tricladium obesum, Tricladium splendens, Tricladium terrestre, Tricladium castaneicola, Tricladium minutum, Tricladium indicum, Mycofalcella calcarata, Filosporella annelidica, Geniculospora grandis, V. giganteum, as well as Hymenoscyphus varicosporoides, Hymenoscyphus scutula, Cudoniella sp. and Ombrophila violacea. Lachnum virgineum appeared to be the basal taxon in Clade 7. T. minutum was placed as a singleton. The Ascocoryne-Hydrocina clade (Clade 8) included species of Ascocoryne, Filosporella, Varicosporium, Articulospora, Hydrocina, Tricladium and long branched Neobulgaria pura. The Clade 14 included species of Leotiaceae and Bulgariaceae and twelve aquatic hyphomycete species in four genera. Author's personal copy 666 C. Baschien et al. Cladochasiella divergens CCM F-13489 ex-type 1 Gyoerffyella cf. craginiformis CCM F-09367 Gyoerffyella entomobryoides CBS 268.63 ex-type Gyoerffyella clade Fontanospora fusiramosa CCM F-12900 Gyoerffyella gemellipara CCM F-402 Gyoerffyella rotula CCM F-400 Gyoerffyella tricapillata CBS 451.64 ex-isotype 0.95/100 Varicosporium elodeae CBS 541.92 Varicosporium trimosum CCM F-14398 Articulospora tetracladia CCM F-12499 1.0/100 0.99/100 Anguillospora filiformis CCM F-20687 Tetrachaetum elegans CB-M11 Fontanospora eccentrica CCM F-46394 Tricladium biappendiculatum CCM F-13000 Tricladium patulum CCM F-15199 Tricladium attenuatum CCM F-06485 0.99/98 1.0/100 Lemonniera centrosphaera CCM F-149 ex-type Lemonniera cornuta CCM F-325 0.96/100 Lemonniera aquatica CCM F-21799 Lemonniera sp. CCM F-19299 0.97/100 Lemonniera terrestris CCM F-11486 Margaritispora aquatica CCM F-11591 Arachnopeziza variepilosa M33 Cadophora luteo-olivacea ICMP18084 Mycochaetophora sp. MAFF 23928 2 0.97/100 Rhynchosporium orthosporum CBS 698.79 Rhynchosporium clade Ypsilina graminea UMB-098.01 1.0/100 Pyrenopeziza brassicae CRB 0.99/100 Mollisia sp. Currahlab1 1.0/100 Tricladium kelleri VG 68-1 ex-type Tricladium alaskense VG 69-2 ex-type Protoventuria alpina CBS 140.83 Mollisia dextrinospora ICMP 18083 Tricladium curvisporum CCM F-23387 Mollisia cinerea AFTOL76 0.99/100 Variocladium giganteum CBS 508.71 ex-type 3 Mollisia fusca CBS 234.71 Vibrissea-Loramyces clade 1.0/100 Phialocephala helvetica 153-2 D-ZB-40 Mollisia sp. 11.3.s.5.13 0.95/100 0.98/99 Pyrenopeziza revincta ARON3150.P Mollisia melaleuca CBS 589.84 Mollisia minutella ZK71/08 0.99/100 Variocladium giganteum CCM F-16686 Loramyces macrosporus CBS 235.53 ex-type 0.99/100 Vibrissea flavovirens MBH39316 Vibrissea truncorum CUP-62562 0.98/96 Arbusculina fragmentans CCM F-13486 ex-type 4 Hyaloscypha clade Cadophora finlandica IFM50530 Hyaloscypha vitreola M39 Tricladium procerum CCM F-16786 ex-type 0.95/96 Tetracladium marchalianum CCM F-26199 Tetracladium apiense CCM F-23299 5 Tetracladium palmatum CCM F-10001 0.99/100 Tetracladium clade Tetracladium setigerum CCM F-10186 Tetracladium breve CCM F-12505 Tetracladium maxilliforme CCM F-529 Tetracladium furcatum CCM F-11883 1.0/100 Dactylaria dimorphospora CBS 256.70 Leohumicola verrucosa CBS 115881 0.99/100 Leohumicola minima N086 Mycoarthris corallina 91A ex-type Varicosporium scoparium CCM F-10303 ex-type 0.99/99 Mitrula paludosa MBH50636 6 1.0/100 Mitrula elegans ZW-Geo45-Clark 0.95/- 1.0/95 Mitrula brevispora ZW02-012 Mitrula clade Ascocalyx abietina cf870061 Dimorphospora foliicola CBS 221.59 ex-type Tricladium angulatum CCM F-14186 Fig 1 e MrBayes tree obtained from combined ITS and LSU rDNA sequence data. Numbers at the nodes are Bayesian posterior probabilities and ML bootstrap values. Aquatic hyphomycetes are shown in blue. Pictograms indicate major conidial shapes curved, includes sigmoid, tricladioid, variously branched (e.g. of aquatic hyphomycetes. (Tree Base Nr.: TB2:s14104), Gyoerffyella-like, tetraradiate (e.g. Lemonniera, Articulospora, Variocladium, Geniculospora, Alatospora), Varicosporium), straight, branched), dichotomously branched (e.g. Cladochasiella divergens), tetracladioid, (e.g. Gorgomyces), oval. arthroconidia, Dwayaangam colodena, tetrahedral (Margaritispora aquatica) Ypsilina (single- T-shaped (e.g. Miniancora allisoniensis), flail- shaped Author's personal copy The molecular phylogeny of aquatic hyphomycetes 667 1.0/100 1.0/100 1.0/100 Zalerion varium ATCC28878 Filosporella cf. annelidica CCM F-11702 Anguillospora furtiva CB-L16 Tricladium castaneicola CCM F-11296 1.0/100 0.98/Tricladium indicum VG 112-1 Tricladium obesum CCM F-14598 ex-type Anguillospora crassa CCM F-15283 Mycofalcella calcarata CCM F-10289 ex-type Hymenoscyphus varicosporioides FC-2038 0.99/100 Cudoniella indica CBS 430.94 ex-type Tricladium splendens CCM F-16599 0.99/100 Tricladium terrestre CBS 697.73 ex-type Geniculospora grandis UMB-176.01 Varicosporium giganteum CCM F-10987 Chlorovibrissea sp. PDD70070 0.96/95 Vibrissea albofusca PDD 75692 1.0/100 Cudoniella sp. ZW0068 0.98/100 0.98/99 Ombrophila violacea WZ0024 Hymenoscyphus scutula MBH29259 Lachnum virgineum AFTOL49 Tricladium minutum CCM F-10203 Filosporella fistucella CCM F-13091 ex-type 0.98/100 Filosporella versimorpha CCM F-11194 ex-type Filosporella exilis CCM F-13097 ex-type 0.95/Varicosporium delicatum CCM F-19494 0.99/95 Articulospora atra CCM F-00684 7 CudoniellaHymenoscyphus clade 8 AscocoryneHydrocina clade Tricladium chaetocladium VG 27-1 Hydrocina chaetocladia CCM F-10890 ex-type Dwayaangam colodena V3.13 Ascocoryne cylichnium PDD75671 Neobulgaria pura CUP 063609 Clorencoelia sp. ZW-Geo55-Clark Fabrella tsugae J.Platt 256 9 HemiphacidiumHeyderia abietis OSC60392 Meria laricis CBS 298.52 Sclerotinia clade 1.0/100 Hemiphacidium longisporum ATCC 26761 1.0/100 Sclerotinia sclerotiorum wb197 Ciboria batschiana CBS 655.78 0.99/100 Catenulifera brachyconia CBS 304.74 10 Hyphodiscus-Cistella clade Hyphodiscus hymeniophilus MUCL 9042 Cistella spicicola CBS 731.97 Hyalodendriella betulae CBS 261.82 11 Hyalodendriella clade Helicodendron websteri ICMP15521 Tricladium caudatum CCM F-13498 0.99/100 Neofabraea malicorticis DAOM 227085 12 Neofabraea clade Neofabraea alba MM 159 Cryptosporiopsis rhizophila CBS110609 1.0/100 Cudonia lutea wz164 1.0/97 Spathularia flavida wz214 13 Rhytismatales clade Rhytisma salicinum BPI1843550 1.0/100 Leotia lubrica ZW-Geo59-Clark 14 Microglossum olivaceum FH-DSH9 1.0/100 Flagellospora sp. 1 CCM F-20899 LeotiaFlagellospora curvula CB-M13 Bulgaria Flagellospora sp. 2 VG 31-4 1.0/100 clade Flagellospora fusarioides CCM F-14583 1.0/100 0.97/100 1.0/99 1.0/100 1.0/100 1.0/95 0.99/95 0.98/100 1.0/100 0.99/100 0.97/100 Gorgomyces honrubiae CCM F-12003 ex-type Flagellospora saccata CCM F-39994 Gorgomyces hungaricus CCM F-12696 ex-type Alatospora pulchella CCM F-502 ex-type Flagellospora leucorhynchos CCM F-14183 Alatospora acuminata CCM F-02383 Alatospora acuminata CCM F-37194 Alatospora constricta CCM F-11302 ex-type Alatospora flagellata CCM F-501 ex-type of A. crassipes Miniancora allisoniensis CCM F-30487 ex-type Bulgaria inquinans ZW-Geo52-Clark Holwaya mucida ZW-Geo138-Clark 1.0/100 Geoglossum glabrum OSC 60610 Saccharomyces cerevisiae 0.01 substitutions/site Fig 1 e (continued) Author's personal copy 668 However, none of the four genera is monophyletic. Alatospora appeared to be paraphyletic with Flagellospora leucorhynchos and Miniancora allisoniensis nested within the group. Gorgomyces honrubiae did not cluster with Gorgomyces hungaricus but showed a sister relationship to Flagellospora saccata. Clades 913 largely corresponded to the clades of Hemiphacidium, Sclerotinia, Dermea, Rhytismatales reported in Wang et al. (2006a). These clades did not include aquatic hyphomycetes, with the exception of Tricladium caudatum that had affinity (albeit without strong support) to the aero-aquatic fungus Helicodendron websteri and Hyalodendriella betulae in Clade 11. Discussion Molecular phylogeny of aquatic hyphomycetes We found that at least 75 species of aquatic hyphomycetes belong to the Helotiales and are distributed among nine well to moderately supported clades (Fig 1). We demonstrated that Articulospora, Filosporella, and Flagellospora are polyphyletic, in addition to other polyphyletic genera, Tricladium, Lemonniera, Anguillospora, Varicosporium discovered in previous molecular studies (Baschien et al. 2006; Campbell et al. 2006 2009). The results confirmed that morphological characters, such as conidial shape and conidiogenesis, are not always accurate in defining natural genera. Many (taxa of aquatic hyphomycetes need to be re-defined and delineated, based on molecular studies employing ex-type cultures. Frequent absence of such cultures or even type specimens in most of the larger genera of aquatic hyphomycetes, (e.g. Anguillospora, Articulospora, Flagellospora, Varicosporium, Tricladium) can be rectified by establishing lecto-, neo- or epitypes. Aquatic hyphomycetes are distributed throughout the Leotiomycetes (Fig 1). Eight clades, however, contain numerous genera and species and merit further discussion. Clade 1 represents a novel cluster discovered in this study and it contains 22 species from eight aquatic hyphomycete genera. This cluster is a sister group to Arachnopeziza variepilosa, which is a saprotrophic discomycete on wood. The prevalent genera are Gyoerffyella and Lemonniera but neither genus is monophyletic. Ingoldia craginiformis (Petersen 1962) was recombined in Gyoerf
(Marvanova
et al.1967). Later, having seen fyella by Marvanova living material showing only small differences in conidial morphology she synonymized G. craginiformis with Gyoerffyella
1975). In the present study the terrestrial rotula (Marvanova isolate CCM F-09367 (as Gyoerffyella cf. craginiformis) appears phylogenetically distant from G. rotula. Lemonniera is polyphyletic because one species (L. pseudofloscula) belongs to Pleosporaceae, Dothideomycetes (Campbell et al. 2006), even though Lemonniera is homogeneous with respect to conidiogenesis and conidial configuration. Margaritispora aquatica is very similar to Lemonniera in culture characteristics and conidiogenesis but it produces morphologically distinct conidia. However, before M. aquatica can be transferred to Lemonniera, at least the ex-neotype needs to be examined. Also the type species of Lemonniera, L. aquatica, has to be neotypified. While most members of Clade 1 are saprotrophs in aquatic environments, Articulospora tetracladia, Varicosporium elodeae (Fisher
et al. 1997) et al. 1991) and Fontanospora fusiramosa (Marvanova C. Baschien et al. were also reported as facultative endophytes in Alnus glutinosa roots growing in aquatic habitats. Furthermore, conidia of L. aquatica, L. terrestris, L. cornuta, M. aquatica, G. gemellipara, G. tricapillata, V. elodeae and T. patulum were found in the canopy (Bandoni 1981; Mackinnon 1982; Czeczuga & Or1owska 1994; € nczo € l & Re
vay 2004). Gyoerffyella. entomobryoides is described Go as terrestrial plant pathogen (Boerema & von Arx 1964). Taxonomically, this clade contained mostly asexual genera except for A. tetracladia, for which a Hymenoscyphus sexual state was described (Abdullah et al. 1981) and was later recombined in Ombrophila (Baral & Krieglsteiner 1985). However, in our study Ombrophila violacea (the type of the genus) was placed in the Hymenoscyphus-Cudoniella clade (Clade 7). Clade 2 forms a strong sister relationship to clade 3 and it accommodates three species of Tricladium and Ypsilina graminea. Interestingly, all aquatic hyphomycetes in this clade were either reported from arctic streams or are often associated with decaying sedges or grasses (Gulis et al. 2012). Some populations of these taxa may have adapted to survive in arctic or subarctic streams that lack trees in the riparian zone, but further studies are required to verify their physiological adaptations. Ypsilina graminea was also reported from tree holes in € nczo € l & Re
vay 2003) and India (Karamchand & Hungary (Go Sridhar 2008). Common plant pathogens (e.g. Rhynchosporium orthosporum, Pyrenopeziza brassicae) also belonged to this clade. Apart from aquatic hyphomycetes and plant pathogens, this clade also contained several root associated antarctic darkseptate endophytes (DSE) (Upson et al. 2009), as well as root associates Cadophora spp. which are asexual states in Dermateaceae (Harrington & McNew 2003). Clade 3 (Vibrissea-Loramyces clade sensu Wang et al. 2006b) is comprised of Vibrisseaceae, Dermateaceae, and Loramycetaceae. These families include several aquatic teleomorph species such as Vibrissea flavovirens with conidial state Anavirga dendromorpha (Hamad & Webster 1987), L. macrosporus (Ingold & Chapman 1952) and members of Mollisia. Mollisia is a polyphyletic genus because members are distributed over two clades (2 and 3). Mollisia has been reported as sexual state of Anguillospora crassa (Webster 1961; in clade 7), Filosporella sp. (Webster & Descals 1979), and Casaresia sphagnorum (Webster et al. 1993). The type species of the genus Loramyces is L. juncicola, which is linked to Anguillospora-like conidial state (Digby & Goos 1987). The sequence of Variocladium giganteum (ex-type; CBS 508.71) clustered with Mollisia fusca, while the other isolate (CCM F-16686) is placed close to L. macrosporus. Willoughby & Minshall (1975) observed a microconidial state in their isolate of V. giganteum, which they tentatively assigned to Phialocephala resembling P. dimorphospora. No such microconidial state was described in the protologue of V. giganteum by Iqbal (1971) but it was present in the CCM F-16686 isolate. Phialocephala helvetica, a cryptic species closely related to P. fortinii, appeared in the same clade as both cultures of Variocladium. Although most species in Clade 3 are saprotrophs adapted to moist or aquatic conditions, P. helvetica is not aquatic and is € nig et al. 2008). a dark-septate endophyte (Gru The grouping of Tricladium procerum with Hyaloscypha vitreola in Clade 4 is in agreement with the findings of Campbell et al. (2009). Members of the polyphyletic genus Hyaloscypha are biotrophic parasites or bryophyte symbionts (Stenroos et al. 2010). T. procerum was isolated from submerged dead Author's personal copy The molecular phylogeny of aquatic hyphomycetes
1988). Arbusculina fragmentans proJuncus stems (Marvanova duces fragmenting macroconidia and has a hyaline to pale fuscous phialidic microconidial state. Both aquatic hyphomycetes of this clade are rarely reported from ecological studies. Clade 5 (Tetracladium clade) is comprised of four genera of aquatic hyphomycetes and two Leohumicola species. Our analysis confirmed the monophyly of the genus Tetracladium € rlocher 2002; Baschien et al. 2006; (Nikolcheva & Ba Letourneau et al. 2010). Interestingly, some species of this clade were found associated with roots of terrestrial (Watanabe 1975; T. setigerum) and submerged living plants (Kohout et al. 2012; T. furcatum, T. setigerum, Tricladium sp., Leohumicola minima). Three species of the genus (T. marchalianum, T. maxilliforme, T. setigerum) were also found associated with tree leaves (Czeczuga & Or1owska 1998), and stemflow or in € nczo € l & Re
val 2004). Two Leohumicola species are ergutters (Go icoid mycorrhizae-forming fungi (Hambleton et al. 2005). Clade 7 (Hymenoscyphus-Cudoniella) is one of the largest groups containing seven genera of aquatic hyphomycetes (e.g. Abdullah et al. 1981; Descals et al. 1984; Webster et al. 1995). Several aquatic hyphomycetes from this clade were reported as root endophytes, e.g. Tricladium splendens (Fisher & Petrini 1989) and A. crassa (Sati & Belwal 2005). Conidia of T. splendens (Karamchand & Sridhar 2008) and Tricladium casta€ nczo € l & Re
vay 2003 2006) were reported from tree neicola (Go holes and from stemflow. Tricladium and Anguillopora are the two classical, albeit polyphyletic, genera of aquatic hyphomycetes, and their representatives are clustered together. The polyphyly of Anguillospora has been demonstrated earlier €rlocher 2005; Baschien et al. 2006) with species (Belliveau & Ba distributed among Dothideomycetes, Orbiliomycetes, and Leotiomycetes. In agreement with the study of Belliveau & € rlocher (2005), A. filiformis was placed in Clade 1 while two Ba other helotialean Anguillospora species were placed in Clade 7. All three Anguillospora species studied here have thalloblastic percurrent conidiogenous cells and sigmoid conidia. Tricladium is the largest genus of aquatic hyphomycetes containing 26 species with representatives in Leotiomycetes and Dothideomycetes (Campbell et al. 2009; Gulis & Baschien, unpublished). Five Tricladium species with dark colonies (T. castaneicola, T. indicum, T. obesum, T, splendens, T. terrestre) forming a cluster in the study of Campbell et al. (2009, Fig 1) received high support in our Clade 7 as well as in the study of Seena et al. (2010). In our study, fifteen Tricladium species were distributed in seven clades e an extreme example of polyphyly calling for a taxonomic revision of the genus. The teleomorph Cudoniella indica is grouped with Hymenoscyphus varicosporioides which is consistent to data of previous studies (Sivichai & Jones 2003; Campbell et al. 2009; Seena et al. 2010). Although Sivichai & Jones (2003) suggested that Hymenoscyphus varicosporoides and C. indica may be conspecific, the low resolution of this clade demonstrated the need to utilize highly discriminatory loci for delineation of genera and species. Clade 8 (Ascocoryne sensu Wang et al. 2006a) contained almost exclusively aquatic hyphomycetes with the exception of Ascocoryne cylichnium and Neobulgaria pura. Hydrocina chaetocladia is the sexual state of Tricladium chaetocladium, an aquatic hyphomycete (Webster et al. 1991). Its position here differs from that published by Wang et al. (2005, 2006a,b), who place 669 it near Mitrula, but without significant support. Even though three members of Filosporella formed a monophyletic group, the genus is polyphyletic because Filosporella annelidica nested in Clade 7, while Filosporella exilis, Filosporella fistucella and Filosporella versimorpha in clade 8. Unfortunately, no isolate of the type species of the genus, Filosporella aquatica, described from Malaysia (Nawawi 1976) was available for this study. Filosporella exilis and F. versimorpha produce at least three types of conidia (micro-, macro- and arthroconidia); the latter was isolated
& from submerged alder roots, as was F. fistucella (Marvanova
et al. 1992). The ability to produce difFisher 1991; Marvanova ferent types of conidia might be an adaptation to environmental conditions (e.g. aquatic vs. terrestrial) during different stages of life cycle. Apart from Filosporella, aquatic hyphomycetes in this clade form branched or tetraradiate conidia. Dwayaangam colodena, also isolated from roots, showed affinities to Hyaloscyphaceae (Sokolski et al. 2006). Tricladium caudatum was basal to Hyalodendriella (Clade 11; Helotiales incertae sedis, Crous et al. 2007) and Helicodendron websteri (aero-aquatic fungus), but the placement was poorly supported. The position of T. caudatum is uncertain, and it was not clearly resolved in the study of Campbell et al. (2009) when it was weakly clustered to Rhytisma acerinum. Clade 14 (Leotia-Bulgaria clade sensu Wang et al. 2006a) is comprised of four aquatic hyphomycete genera. The molecular data appeared to support the separation of Alatospora from Flagellospora except that F. leucorhynchos nested within a cluster of Alatospora species. Another species, Flagellospora saccata, grouped with Gorgomyces honrubiae. While Alatospora produces mostly branched conidia (though some isolates tend to produce almost only unbranched conidia), Flagellospora has sigmoid to arcuate conidia. Interestingly, the morphology of the phialides in F. saccata is different from all other Flagellospora species. Two other species of Flagellospora are members of the Hypocreales (Ranzoni 1956; Webster 1993). The concepts of Alatospora and Flagellospora should be revised in light of additional morphological and molecular data (Baschien et al., in preparation). Ecology and evolution of aquatic hyphomycetes No clear pattern was evident in the distribution of aquatic hyphomycetes with a particular type of conidial morphology (e.g. tetraradiate or sigmoid) among clades of the Leotiomycetes (Fig 1). Species with tetraradiate or variously branched conidia were found in all nine clades that contained aquatic hyphomycetes. In addition, we found close phylogenetic relationships between aquatic hyphomycete taxa with branched and sigmoid conidia. Moreover, there are species of aquatic hyphomycetes producing both types of conidia in nature as well as in pure culture, e.g. Alatospora acuminata, Pachycladina mutabilis, Tricladium indicum, Tricladiopsis flagelliformis. This suggests, that the two shapes may not be genetically fixed at least in some taxa. According to one hypothesis conidia are modified hyphae (Descals 1985; Kendrick 2003). In the case of aquatic hyphomycetes, we can speculate that conidia may have been evolving from simple elongate shapes to more or less branched spores. In addition to aquatic hyphomycetes, Leotiomycetes also contain aero-aquatic fungi such as Helicodendron and fungi that have amphibious lifestyles, e.g. living close by the water Author's personal copy 670 or in wet conditions (e.g. Vibrissea). The production of different conidial shapes and synanamorphs may also be an adaptation to shifts between aquatic, semi-aquatic and terrestrial habitats. The molecular data demonstrated that most aquatic hyphomycetes clustered with endophytes, mycorrhizal fungi and saprotrophs in the Helotiales, thus supporting the scenario first suggested by Shearer (1993) that aquatic hyphomycetes evolved from terrestrial plant-associated or litterassociated fungi (Selosse et al. 2008). Indeed, conidia of many aquatic hyphomycetes from the genera Alatospora, Anguillospora, Flagellospora, Gyoerffyella, Lemonniera, Tetracladium, Tricladium and Varicosporium have been reported from the canopy (tree holes, stemflow, throughfall; reviewed in € nczo € l & Re
vay Sridhar 2009). However, as pointed out by Go (2006), the group of taxa, whose stauroform or scolecoform conidia are repeatedly observed in rainwater from canopy, stemflow or throughfall (also called ‘arboreal aquatic hyphomycetes’), should have a unique, currently poorly understood ecology. Although they resemble aquatic hyphomycetes, the identifications are based on detached conidia only. To our knowledge, a few studies based on pure cultures of fungi from rainwater revealed species that are not found in typical habitats of aquatic hyphomycetes (e.g. Ando & Tubaki 1984a,b). Some species of the genera Varicosporium, Tetracladium, and Anguillospora have been reported as plant endophytes (Nemec 1969; Watanabe 1975; Sati & Belwal 2005). Many plant pathogens, endophytes, root-associated fungi (RAF) and mycorrhizal species belong to the Leotiomycetes (Selosse et al. 2008). Indeed, the endophytic lifestyle could possibly facilitate the transition from terrestrial to aquatic habitats. Endophyte and phylloplane fungi are already associated with the substrate when it enters the water (e.g. a leaf during the litter fall), which may have given such fungi a competitive advantage and eventually led to the evolution of spore shapes adapted to aquatic dispersal. Alternatively, it was hypothesized that terrestrial ancestors of the present-day aquatic plants interacted with different groups of ubiquitous RAF, both mycorrhizal and non-mycorrhizal (Kohout et al. 2013). The extant free-living aquatic hyphomycetes could have evolved from non-mycorrhizal RAF that once entered aquatic habitats together with their host plants. In fact, many aquatic hyphomycetes are capable of colonizing roots of submerged, riparian or terrestrial plants (Kohout et al. 2012 2013). Conclusions Seventy-five species of aquatic hyphomycetes and their teleomorphs are associated with the Helotiales, Leotiomycetes. We compiled the largest database of aquatic hyphomycete sequences (75 out of 300 species) and unraveled phylogenetic positions of 29 out of approximately 115 genera of aquatic hyphomycetes. Ribosomal DNA sequence data by themselves are invaluable for the purposes of barcoding and molecular microbial ecology including metagenomics. Many genera of aquatic hyphomycetes are polyphyletic suggesting that conidial adaptations to aquatic dispersal occurred independently in multiple lineages. Many genera and species of aquatic hyphomycetes require typification since type material or ex-type species are often missing. Multilocus sequencing of ex-type C. Baschien et al. strains will be necessary to better resolve phylogenetic relationships. Acknowledgements The research was partly funded by the Czech Collection of Mi
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